Gated Materials for On-Command Release of Guest Molecules

Review pubs.acs.org/CR

Gated Materials for On-Command Release of Guest Molecules Elena Aznar,†,∥ Mar Oroval,†,∥ Lluís Pascual,†,∥ Jose Ramón Murguía,†,‡,∥ Ramón Martínez-Máñez,*,†,§,∥ and Félix Sancenón†,§,∥ †

Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Unidad mixta Universitat Politècnica de València-Universitat de València, Camino de Vera s/n, 46022 València, Spain ‡ Departamento de Biotecnología, Universitat Politècnica de València, Camino de Vera s/n, 46022 València, Spain § Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 València, Spain ∥ CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) ABSTRACT: Multidisciplinary research at the forefront of the field of hybrid materials has paved the way to the development of endless examples of smart devices. One appealing concept in this fertile field is related to the design of gated materials. These are constructed for finely tuning the delivery of chemical or biochemical species from voids of porous supports to a solution in response to predefined stimuli. Such gated materials are composed mainly of two subunits: (i) a porous inorganic support in which a cargo is loaded and (ii) certain molecular or supramolecular entities, generally grafted onto the external surface, which can control mass transport from pores. On the basis of this concept, a large number of imaginative examples have been developed. This review intends to be a comprehensive analysis of papers published until 2014 on hybrid mesoporous gated materials. The molecules used as gates, the opening mechanisms, and controlled release behavior are detailed. We hope this review will not only help researchers who work in this field but also may open the minds of related ones to develop new advances in this fertile research area.

CONTENTS 1. Introduction 2. Light 2.1. Photodimerization 2.2. Cis−Trans photoisomerization 2.3. Photoisomerization of Spiropyrans 2.4. Photocleavage 2.5. Generation of Reactive Oxygen Species 2.6. Gold Nanostructures 2.7. Miscellaneous 3. Temperature 3.1. Poly(N-isopropylacrylamide) and Derivatives 3.2. Other Polymers 3.3. Based on DNA 3.4. Miscellaneous 4. Alternating Magnetic Field and Ultrasound 4.1. Alternating Magnetic Field 4.2. Ultrasound 5. Redox 5.1. Rotaxanes and Pseudorotaxanes 5.2. Reduction of Disulfide Bonds 5.3. Miscellaneous 6. pH 6.1. Polyamines 6.2. Metal Complexes 6.3. Macrocyles 6.3.1. Cucurbiturils 6.3.2. Cyclodextrins © 2016 American Chemical Society

6.4. Polymers 6.4.1. Chitosan 6.4.2. PEI 6.4.3. Acrylates and Methacrylates 6.4.4. Other Polymers 6.5. Layer-by-Layer 6.6. DNA and Peptide Capped 6.7. Permeation of Lipid Bilayers 6.8. Inorganic Caps 6.9. Miscellaneous 7. Molecules and Biomolecules 7.1. Triggered by Anions 7.2. Triggered by Cations 7.3. Triggered by Small Neutral Molecules 7.4. Triggered by Biomolecules 8. Enzymes 8.1. Hydrolysis of Ester and Phosphodiester Groups 8.2. Glycosidic Linkages 8.3. Hydrolysis of Amide Groups 8.4. Rupture of Azo Bonds 8.5. Hydrolysis of Phosphodiesters 8.6. Elongating DNA Sequences 9. Conclusions and Future Perspectives Author Information

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Received: August 4, 2015 Published: January 5, 2016 561

DOI: 10.1021/acs.chemrev.5b00456 Chem. Rev. 2016, 116, 561−718

Chemical Reviews Corresponding Author Author Contributions Notes Biographies Acknowledgments Abbreviations References

Review

inert under a wide range of conditions and are easily functionalized using well-known chemistries.11−13 Another key point when designing such gated nanodevices is the selection of gating entities. In line with this, imaginative molecular and supramolecular concepts have been developed to allow the controlled release of a wide range of entrapped cargos. There is a long list of clever approaches, where the driving force to switch gating mechanisms between an open state and a closed state take advantage of electrostatic or supramolecular interactions, the rupture/formation of covalent bonds, or changes in the physical properties of molecules or macromolecules.14−22 Gated nanochemistry has demonstrated the possibility of achieving new advanced predesigned functions by means of mass transport control. The most studied application of such systems is related with controlled release protocols, especially devoted to potential biomedical applications.23−26 Such gated materials have also been used to develop new sensing/recognition protocols.27 A few examples have also been reported for other applications. Nevertheless, many studies conducted to date are limited to the simple design of gated materials, and there are a number of potential applications still to be explored. The application of gated materials in the biomedical field has been boosted in the past few years because these systems allow the release of one or several drugs upon the application of external stimuli and are promising candidates for developing new therapies to improve efficacy and safety. Moreover, anchoring biological receptors (e.g., antibodies, peptides, aptamers, or carbohydrates) onto an external surface allows targeted delivery of drugs to sites such as cells and tissues that contain overexpressed receptors.28−37 In recent years, some more complicated architectures have been designed, mainly because of their possible application in the emerging area of “theranostic” devices.38−43 In these systems, nanometer-sized porous inorganic supports are loaded with a therapeutic agent, a diagnostic marker, or with an indicator molecule. Then pores are closed with stimuli-responsive caps. Moreover, certain biological receptors are grafted onto the external surface for targeting purposes. These nanodevices are capable of targeting selected cells or tissues to avoid the premature degradation of the entrapped cargo and also facilitate the transfer of the payload across the cell membrane. The presence of markers also allows the release process and the accumulation of the gated nanodevice in target tissues to be traced in real time. When dealing the application of gated material in sensing/ recognition protocols, the key point is to prepare systems capable of responding specifically to a certain target molecule which could modulate delivery of an indicator (e.g., dye or fluorophore).44 The recognition protocol which uses these “gated materials” clearly differs from the classic supramolecular “binding site-signaling subunit” paradigm because it detaches the recognition event from the signaling step.45 This makes signaling independent of the host−guest stoichiometry and sometimes allows signal amplification.46 Additionally, the approach is highly flexible given the possible selection of different porous supports, distinct selective binding sites, and a wide range of indicator molecules. Gated nanochemistry is a highly topical and rapidly developing tool. Yet a comprehensive and exhaustive review that covers all the reported porous gated materials, not only those used in controlled release processes but also those used in sensing protocols and other less common applications, is clearly lacking. Several reviews have been published which deal with the field of the controlled release of guests from several organic/inorganic

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1. INTRODUCTION Although the fields of molecular and supramolecular chemistry and inorganic materials have traditionally been poorly interrelated, research in this currently highly interdisciplinary region has recently emerged exponentially and has resulted in many novel hybrid materials with advanced functions in a surprisingly short time.1 In this context, hybrid organic− inorganic materials within the nanoscale range have attracted considerable interest due to the combination of the beneficial characteristic of organic and bioorganic chemistry and material science. Anchoring organic molecules, biomolecules, or supramolecules onto selected inorganic scaffoldings with different chemical natures, sizes, and shapes promotes the development of smart nanodevices that can be applied in certain scientific and technological fields.2−6 Among different inorganic or hybrid solids, much attention has been paid in recent years to porous materials in fundamental and applied research. One appealing concept in this fertile field is related with the design of gated materials. These gated materials are constructed for the purpose of finely tuning the movement of chemical or biochemical species from voids of porous supports to a solution, and vice versa, in response to a predefined stimulus.7,8 By taking into account this concept, several research groups have been involved in the synthesis and characterization of imaginative nanodevices in which the delivery of a certain cargo stored in a container can be triggered by applying selected external stimulus. Such gated materials are composed mainly of two subunits: (i) a porous inorganic support in which a cargo is loaded and (ii) certain molecular or supramolecular entities, generally grafted onto the external surface, which can control mass transport from pores (see Figure 1). When dealing with the inorganic support,

Figure 1. Schematic representation of a gated material for on-command controlled release.

one of the most commonly used material for preparing gated nanodevices is mesoporous solids. Mesoporous supports can be prepared in different forms that range from micrometric to nanometric and have tailor-made pores (2−10 nm in diameter) and very large specific surface areas (up to 1200 m2 g−1).9,10 Given these remarkable features, mesoporous materials have a huge loading capacity and can store not only small but also a wide variety of medium-sized molecules, such as proteins, oligonucleotides, and nucleic acids. These supports are also chemically 562


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Chart 1. Chemical Structures of the Cargos Loaded into the Inorganic Supports Used in the Preparation of Gated Materials

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Chart 2. Chemical Structures of the Cargos Loaded into the Inorganic Supports Used in the Preparation of Gated Materials

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Chart 3. Drawings and Chemical Structures of the Macrocyclic Molecules Used in the Preparation of Gated Materials by Host− Guest Interactions

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Chart 4. Chemical Structures of the Lipids Used in the Preparation of Gated Materials

2. LIGHT Light is a powerful tool for controlling open/closed protocols in mesoporous systems. Reported examples that have used light as trigger take advantage of (i) photodimerizations, cis−trans photoconversions; (ii) photocleavage of chemical bonds directly or assisted by photosensitizers; or (iii) photoinduced heating of gold nanoparticles (AuNPs) to control mass transport from pore voids to a solution. In light-driven gated systems, cargo release can, in principle, be controlled spatially and temporally by finetuning the area and time of the light stimulus. Moreover in recent years, researchers have focused their attention on developing bioapplicable systems. Light has the advantage of being applicable from outside of the patient in a noninvasive manner, and it is easily focalisable in selected areas, avoiding irradiation of the surrounding tissue. However, UV light is unable to penetrate in deep tissues. Concerned by this fact, in the last years the attention has been centered in using the more tissue-penetrating NIR irradiation as a light source thanks to the use of metal clusters or two-photon active molecules. However, most of the work in this field still is in an incipient development. In addition, researchers have not always reported the light power supply used in their work, which difficults the reproduction of the gating mechanism performance.

supports in which gated materials are included as a section or are reviewed only partially.47−53 Several other reviews have covered examples of gated materials that are opened by applying only one selected stimulus.54−65 Finally, several research groups have published reviews about the results of their own work.66−71 This review intends to be a comprehensive analysis of all the papers published until 2014 on hybrid porous gated materials based on different supports such as silica, silicon, aluminum oxide, titanium oxide, etc. The molecules used as gates, the opening mechanisms, and controlled release behavior are included. It was also our aim to include a schematic representation of the gating mechanism for clarity for most of the described examples. Moreover Chart 1 and Chart 2 show the chemical structures of the used cargos. Also Chart 3 and Chart 4 show macrocyclic molecules and lipids used in the preparation of capped materials. Tables 1 and 2 contain a summary of cell lines and organisms used in studies with gated materials, whereas Table 3 recapitulates gated materials used for sensing. The review is arranged around the stimuli used to trigger guest release and is divided into seven main sections: (i) light, (ii) temperature, (iii) alternating magnetic field (AMF) and ultrasound, (iv) redox, (v) pH, (vi) molecules and biomolecules, and (vii) enzymes. This review concludes with a section which discusses future perspectives and applications. We believe that this comprehensive review will be of interest to a larger number of researchers. We also expect this review to not only help researchers who work in this field but also may open the minds of related ones to develop new advances in this fertile research area.

2.1. Photodimerization

Photodimerization is a bimolecular photochemical process in which an electronically excited unsaturated molecule reacts with an unexcited molecule of the same species to give addition products. This simple reaction, which allows a rapid change in the size of molecules, has been used to design gated mesoporous 566


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Table 1. Cell Lines Used in Studies with Gated Materials cell line 293 293T 7402 3T3 3T3-J2 A2780 A2780Adr A2780/AD A549 B16−F10 BA BCSC BEL-7402 BxPC-3 Caco-2 CCRFCEM CHO COS-7 COLO205 DC1787 EA.hy926 FiBH H460 HCC HCT-116 HEK-293 HeLa Hep2 HEP3B HEPA-1 Hep G2 HL-60 HL-7702 HT-1080 HT-29 HuH7 HOS HUVEC KB KB-31 KB-V1 KHOS L02 L929 LF LLC-PK1

description human embryonic kidney 293 cells human embryonic kidney 293 transformed cells human hepatoma cells mouse embryonic fibroblasts cells murine fibroblasts cells human ovarian cancer cells human ovarian cancer cells human ovarian cancer cells human lung adenocarcinoma cells human melanoma cells breast adenocarcinoma cells breat cancer stem cells liver cancer cells human pancreatic cancer cells heterogeneous human epithelial colorectal adenocarcinoma cells human acute lymphoblastic leukemic cells Chinese hamster ovarian cells African green monkey fibroblasts cells human colon carcinoma cells senescemt fibroblast cells human umbilical vein cells human fibroblasts cells nonsmall lung cancer cells human hepatocellular carcinoma cells human colon cancer cells human embryonic kidney fibroblasts cells human cervix carcinoma cells human hepatocarcinoma cells human hepatocellular carcinoma cells mouse hepatocellular carcinoma cells human liver cancer cells myeloblastic leukemia cells normal human liver cells human fibrosarcoma cells human colon cancer cells human hepatocarcinoma cells human osteoblast cells human umbilical vein cells human mouth epidermal carcinoma cells human cervix carcinoma cells cervix carcinoma cells osteosarcoma cells hepatocellular carcinoma cells mouse fibroblasts cells lung fibroblast cells kidney proximal tubule cells

refs 195, 222 166, 198, 276, 277 301 112, 113 151 459 459 405 85, 100, 125, 144, 166, 191−194, 239, 275, 308, 318, 321, 357, 358, 361, 388, 398, 404, 459 189 155 342 472 227, 228, 354 448 85, 406 107, 337 210, 220 460 447 97 351 447 338 224 226, 312, 335 97, 111, 118, 119, 121, 135, 154, 155, 161, 181, 184, 185, 187, 190, 195, 201, 202, 204, 213, 214, 219, 225, 269, 275 −277, 287, 288, 292, 293, 300, 312, 314, 315, 323, 324, 330, 345, 360, 364, 375, 396, 400, 403, 431, 441, 442, 446, 458, 462, 465, 466, 468, 472−474 144 338−340 283 113, 432 208 151, 198, 110,

157, 188, 199, 200, 208, 211, 226, 266, 269, 282, 309, 320, 336, 409

317 461 111, 407

444 336 96, 100, 109, 111, 209, 302, 308 258 284 223 293, 301, 461 123, 128, 152, 162, 323 155 446 567


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Table 1. continued cell line LO2 MC3T3-E1 MCF-7 MCF-10a MDA-231 MDA-MB231 MEF MEL-5 MRC-5 NIH3T3 NLC PANC-1 PC-12 PC3 PC3M QGY7703 QSG-7701 RENCA RIN-5F SCC-7 SGC-7901 SK-N-BE(2) SKOV3 THP-1 U2O U-87 UMUC3 X-DC1774 X-DC4646 ZR75−30

description hepatocellular carcinoma cells mouse osteoblastic cells breast cancer cells breast epithelial cells cancer cells human breast carcinoma cells mouse embryonic fibroblast cells human melanoma cells human fetal lung fibroblasts cells mouse fibroblasts cells normal liver cells human pancreatic carcinoma cells human pheocromocytoma cells human prostate cancer cells human prostate cancer cells mouse hepatocellular carcinoma cells liver normal cells transformed murine renal cells rat pancreatic islet tumor cells squamous carcinoma cells human gastric cancer cells neuroblastoma cells ovarian carcinoma cells human leukemic monocyte cells human ostesarcoma U-87 MG human glioblastoma cells bladder cancer cells senescent fibroblast cells senescent fibroblast cells human breast carcinoma cells β-Gal overexpressing yeast cells human liver cells fibroblast cells normal endothelial cells skin fibroblast cells

refs 432 355, 356 82, 116, 127, 146, 153, 158, 203, 222, 240, 251, 264, 272, 273, 279, 288, 317, 319, 334, 342, 343, 346, 357, 404, 434, 441, 465, 466, 468 389, 435 226 196, 347, 389, 433, 435, 449−451 458 162 300, 361 104, 116, 224, 282, 347, 431, 434, 449, 459 204 259 400 351 299 225 472 112 395, 396 198 400 300 152 258 440 210, 332 263 447 447 274 447 98 98 187, 199 396

materials. In fact, the first example of a photoactive gated material, based on the use of the photodimerization of coumarin, was developed by Fujiwara and co-workers in 2003.72,73 This was also the first reported silica mesoporous support to contain a molecular gate. Coumarin undergoes a [2 + 2] photodimerization in which there is a cycloaddition reaction that involves carbon−carbon double bonds of two neighboring molecules to result in a cyclobutane dimer. Photodimerization is performed in the presence of light and is, in most cases, a reversible process that can be controlled by selecting the irradiation wavelength. The gate of Fujiwara’s work consisted in 7-[(3-triethoxysilyl)propoxy]coumarin groups attached to a MCM-41 support. The pores of the coumarin-functionalized hybrid material were loaded with selected cargos (phenanthrene, cholestane, and progesterone) and were then capped by irradiation with >310

nm light that triggered the photodimerization process as depicted in Figure 2 (panels A and B). Capped solids showed no cargo release; however, when the system was irradiated with a 250 nm UV-light, pore opening and cargo release was observed. Later in 2010, coumarin was also used as a gate on mesoporous bioactive glasses.74 The authors loaded the pores of mesoporous bioactive glasses with phenanthrene, and pore outlets were functionalized with a coumarin derivative (see Figure 2C). When n-hexane suspensions of the material were irradiated with light of wavelength >310 nm, negligible phenanthrene release was observed due to the formation of coumarin dimers, whereas irradiation with 250 nm light induced cargo release. In another work, Fujiwara and co-workers reported a photocontrolled storage-release nanosystem that combined the use of the coumarin gate on the outer surface and azobenzene 568


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Table 2. Organisms Used in Studies with Gated Materials organism bacteria mice

description

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E. coli athymic nude mice KB-tumor bearing nude mice H22 tumor-bearing mice Hep2-tumor bearing nude mice Ehrlich carcinoma HeLa xenografts Hep G2 xenografts A549 xenografts BxPC3 xenografts HT-29 xenografts HT1080 xenografts MCF-7 xenograft MCF-7/ADR xenograft MCF-7/MDR xenograft MCF-7/BCPR xenograft MDA-MB-231 xenograft

376, 437 272 109 275 144 127 187, 119 211 193 285 461 151 334 404 286 343 196 212 212 344 399 232 197

plant tissues protoplasts rats yeasts zebra fish

diabetic rats S. cerevisiae Danio rerio

Figure 2. (A and B) MCM-41 support or (C) mesoporous bioactive glasses loaded with (A) cholestane, (B) phenantrene, cholestane, or progesterone or (C) phenantrene and capped with cyclobutane dimers of coumarin. Upon irradiation at 250 nm, the selected cargos were released due to the regeneration of the coumarin monomers. Cargo release could be once again stopped by irradiation at 310 nm.

Table 3. Summary of Gated Materials Used for Sensing anions

cations

neutral molecules

biomolecules

analyte

refs

ATP decanoate/dodecanoate Borate CH3Hg+ Hg2+ K+ Mg2+ and Zn2+ Mg2+ and UO22+ Pb2+ cocaine glucose urea glucose or ethyl butyrate fructose or galactose TNT and tetryl DFP, DCP, and DCNP sulfathiazole finasteride TATP PbTx-2 AFB1 adenosine single-stranded DNA sequences Mycoplasma genome thrombin prostate-specific antigen telomerase biomarkers

365, 366, 370−374 367, 368 369 378 379 380, 381 382 383 384 374 390, 391 392 393 394 410−412 413 414 415 416 417, 418 419, 420 422 423, 426 424 427, 428 430 472−474 435

[(3-triethoxysilyl)propoxy]coumarin on the outer surface. The system was finally loaded with cholesterol. Release studies confirmed that coumarin moieties acted as reversible gates upon irradiation as can be observed in Figure 3. Moreover, these authors found that when the gate was opened by irradiation with 250 nm light, cholesterol release accelerated with UV/vis light irradiation (UV: λ ≈ 360 nm, Vis: λ ≈ 430 nm), which induced a reversible photoisomerization of azobenzene to create a continuous rotation-inversion movement, accompanied by

moieties anchored to inner pores. In this particular case, the cistrans isomerization of azobenzene was not used as a gate (vide infra) but as a molecular “stirrer”.75 The authors functionalized mesoporous silica nanoparticles (MSNs) with N-(3-triethoxysilyl)-propyl-4-phenylazobenzamide inside pores and with 7-

Figure 3. Mesoporous silica support functionalized with azobenzene moieties, loaded with cholesterol, and capped with dimers of coumarin. Cargo was delivered upon UV irradiation. 569


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stretch-shrink motions. In particular, 95% of cholesterol was delivered in 2 h, whereas a 30% delivery was observed without irradiation. He and co-workers developed a similar system to that described by Fujiwara and used coumarin moieties included in a copolymer.76 In particular, these authors prepared hollow MSNs functionalized with azide moieties, used to anchor a copolymer of methyl methacrylate and 7-(2-methacryloyloxy)-4methylcoumarin via a click reaction. Nanoparticles were then loaded with pyrene, which was retained in nanoparticles upon UV irradiation at 365 nm due to the formation of coumarin dimers (Figure 4). They found that when the polymer was irradiated at 264 nm, the coumarin moieties returned to the monomer form, inducing the release of entrapped pyrene.

Figure 5. MSNs loaded with Ru(bipy)32+ and capped with thymine dimers. Ru(bipy)32+ complex was released upon irradiation at 240 nm.

2.2. Cis−Trans photoisomerization

The cis−trans isomerization of azobenzene derivatives has been widely used as a mechanism for opening capped silica mesoporous supports. It is well-known that under visible light irradiation, azobenzene takes the more stable trans-state but changes its conformation to the cis form when irradiated with UV-light at ca. 365 nm. Most of the designs that contain azobenzene-gated systems involve using this light-induced trans−cis isomerization to destabilize capping structures, which resulted in cargo delivery. For instance, Stoddart, Zink, and coworkers prepared aminated MSNs loaded with rhodamine B (Rh B) and functionalized the outer surface with the (E)-4-((4(benzylcarbamoyl)phenyl)diazenyl)benzoic acid derivative. Finally, they capped pores upon the addition of β-cyclodextrins (βCDs) via the formation of inclusion complexes with the trans isomer of the grafted azobenzene derivative.78 Cargo delivery was induced by irradiation with a laser light of 351 nm as shown in Figure 6. The trans azobenzene derivative isomerized to the cis form with the subsequent dethreading of β-CD, which was unable to form inclusion complexes with the cis isomer. Later, the same authors developed a light-activated switchable nanovalve with MSNs modified with azobenzene moieties coordinated to α-CDs.79 Two different azobenzene stalks, previously coordinated to α-CDs, were attached to the surface MSNs via an amide bond (see Figure 7). Both stalks consisted of a trans-azobenzene derivative with an adamantane group that acted as a stopper and avoided α-CDs leakage when the α-CD was not coordinated to azobenzene. The difference between the stalks was that the adamantane group in one was directly attached to azobenzene by an amide bond, which resulted in a shorter stalk, whereas there was an ethylene glycol chain in the other, also connected by an amide bond, which acted as a spacer between the azobenzene and adamantane groups. While the system remained in trans-conformation, α-CD was coordinated to azobenzene close to the surface of the material blocking pores. In contrast, when the system changed to the cis conformation via UV-light irradiation, α-CD was displaced and slid away from pore openings. The mechanism was confirmed by monitoring the release of dyes of different sizes (i.e., alizarin red S, propidium iodide PI, and Hoechst 33342). In all cases, there was no dye delivery until UV-light irradiation was applied. The authors also demonstrated that the system could be reloaded and used several

Figure 4. Gated hollow MSNs loaded with pyrene and capped with a methyl methacrylate and 7-(2-methacryloyloxy)-4-methylcoumarin copolymer. Pyrene was released upon irradiation at 254 nm.

Apart from coumarin, He and Wang developed reversible light-responsive MSNs using thymine photodimerization reactions.77 In particular, it is known that thymine bases photodimerize upon irradiation at wavelengths above 270 nm and revert back to monomeric thymine upon irradiation below 270 nm. The authors functionalized MSNs with [3-(2aminoethyl)aminopropyl]-trimethoxysilane and then reacted them with thymine-1-acetic acid using 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form an amide bond. In another step, the solid was loaded with Ru(bipy)32+ in the dark, and finally the system was irradiated with a 365 nm UV-light to induce the formation of cyclobutane dimers which blocked pores. When the solid was maintained in the dark in PBS, Ru(bipy)32+ release was very poor, while irradiation with 240 nm UV-light induced a remarkable release of the fluorescent probe. The authors also demonstrated that the system could be closed and opened by alternate irradiation at 365 and 240 nm as depicted in Figure 5. The authors also explored the possibility of using the same system as a photoswitchable oxygen probe by taking advantage of the oxygen-induced quenching of the released Ru(bipy)32+ molecules. 570


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acryloxysuccinimide)-grafted mesoporous silica was derivatized with β-CDs by substituting the N-oxysuccinimide groups along the polymer with S-(2-aminoethyl)thio-2-thiopyridine and coupling thiol modified β-CDs via disulfide bond formation. Polymer chains were cross-linked to cap pores by adding a diazolinker via the formation of inclusion complexes with β-CDs as depicted in Figure 8. The hybrid material was loaded with

Figure 6. MSNs loaded with Rh B and capped with β-CD. Rh B was released upon irradiation at 351 nm.

Figure 8. MSNs loaded with calcein and capped with a copolymer based on poly(N-acryloxysuccinimide). Calcein release was triggered by DTT, light, or α-CD.

calcein.80 Capped MSNs were able to respond to three different stimuli, which induced various degrees of dye release. When the material was irradiated with UV light, weak cargo release (ca. 30%) was observed due to the conformational change (trans to cis) of the diazolinker. The addition of dithiothreitol (DTT) reduced the disulfide linkages, which resulted in β-CDs dethreading and dye delivery (ca. 70%). Finally, maximum delivery was achieved by adding α-CDs, which formed a more stable complex with the azolinker. Zhao and co-workers designed a molecular gate using MSNs and pseudorotaxanes that were capable of delivering cargo via both light and temperature (Figure 9).81 MSNs were functionalized with (3-aminopropyl)triethoxysilane (APTES) over the surface and were reacted with propargylic acid by an amidation reaction. In parallel, these authors synthesized an azobenzene derivative, which contained a preattached stopper that consisted in a naphthalene structure and a terminal azide group. α-CDs coordinated to the azobenzene derivatives and formed a pseudorotaxane. Functionalized MSNs were loaded with curcumin, and the pseudorotaxane was attached to the solid by the formation of a triazole group capping pores. When the system was heated to 37 °C or exposed to UV light (360 nm), the

Figure 7. MSNs loaded with (A) alizarin red S or (B) PI or Hoechst 33342, functionalized with different stalks and capped with α-CD. Irradiation at 403 nm triggered cargo release.

times. Moreover, for the capped MSNs that contained the shorter stalk, it was possible to control dosage by irradiation time. Feng and co-workers proposed a multiresponsive molecular gate system by combining MSNs with polymers. Poly(N571


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Figure 9. MSNs loaded with Rh B functionalized with an azo derivative and capped with α-CDs. Cargo was delivered upon UV irradiation or by temperature.

Figure 10. Two-photon fluorescent MSNs loaded with PI or CPT, functionalized with azobenzene-containing stalks and capped with βCDs. Cargo was released with irradiation at 760 nm.

conformational change of the azo group (trans to cis) caused cargo delivery, and the thermal effect was considerably stronger than light exposure. Drug delivery was studied in vivo using optically transparent zebrafish larvae. Nanoparticles were injected into the brain vesicle of 5-day-old zebrafish larvae. The intensity of fluorescence between the larvae incubated at 24 or 37 °C, and either exposed or not to light, was compared. For both experiments, the application of the studied stimuli resulted in increased fluorescence. Moreover the antioxidant properties of curcumin were also tested for heart failure treatment. The larvae treated with nanoparticles recovered heart rate values, which were near to normal for zebrafish larvae. Durand, Gary-Bobo, and Zink82 developed an azobenzenebased system that was opened via the use of a two-photon paracyclophane-based fluorophore which possesses a high twophoton absorption cross section (λem= 415 nm). In particular, the authors co-condensed paracyclophane-based fluorophores with tetraethyl orthosilicate (TEOS) and n-cetyltrimethylammonium bromide (CTAB) in basic media to obtain two-photon fluorescent MSNs. Moreover, monotriethoxysilylated azobenzene was grafted onto the external surface of nanoparticles, and PI was loaded into mesopores. Finally, the supramolecular complexation of β-CDs with azobenzene moieties blocked pores. Two-photon irradiation triggered the release of the cargo through the trans−cis photoisomerization of azobenzene moieties as shown in Figure 10. Biocompatibility and cytotoxicity studies of camptothecin (CPT)-loaded capped MSNs were performed in MCF-7 breast cancer cells. By confocal microscopy, two-photon imaging indicated that the nanomaterial was efficiently endocytosed. MTT studies revealed that nanoparticles did not induce cell death with or without irradiation in the absence of CPT. When loaded with CPT, and subsequently irradiated, cancer cell death was clearly detected. One appealing

property in this and other two-photon systems is that near-IR light achieves deeper tissue penetration, and activation could cause little, or no, tissue damage. Moreover the method offers the additional benefit of precise focal control. Li et al.83 presented another example of light-responsive azobenzene nanovalves. Hollow MSNs were first modified with alkyne groups. Subsequently, azide-modified β-CDs were bound to the surface via a click reaction. The support was loaded with ibuprofen (IBU) as a model drug. Moreover the authors prepared an azo-containing amphiphilic copolymer with a transazobenzene structure which was synthesized by radical copolymerization of 6-(4-(phenyldiazenyl)phenoxy)hexyl methacrylate and poly(ethylene glycol) methyl ether methacrylate (see Figure 11). This prepared polymer was used to cap the entrance of pores through multivalent host−guest interactions of the azo groups with β-CDs molecules. Irradiation at 365 nm transformed the azo groups from a trans to a cis conformation inducing the detachment of the copolymer from the particle’s surface, which triggered drug release. The experimental results confirmed that the system was fully controlled because drug release could be initiated by UV-light irradiation and shut off by visible light irradiation. Gao, Yang, and co-workers grafted MSNs with APTES, which was further reacted with an azobenzene derivative.84 The material was loaded with Rh B and capped by the formation of inclusion complexes between the azobenzene and β-CDs embedded in a modified poly(glycidyl methacrylate) polymer (see Figure 12). Irradiation with UV light (355 nm) induced trans-to-cis conformational changes in the azobenzene groups, which resulted in weak Rh B delivery. Furthermore, addition of 572


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Figure 12. MSNs functionalized with azobenzene moieties, loaded with Rh B and capped with β-CDs units embedded in poly(glycidyl methacrylate). Cargo was delivered upon UV irradiation.

Figure 11. Hollow MSNs loaded with IBU and capped with a 6-(4(phenyldiazenyl)phenoxy)hexyl methacrylate and poly(ethylene glycol) methyl ether methacrylate copolymer. Irradiation at 365 nm triggered IBU release.

doxorubicin (DOX)-loaded system was evaluated for both CEM and A549 cells. DOX-loaded azo-DNA MSNs of different concentrations were incubated with cells for 2 h. Without UVlight treatment, no cytotoxicity to both CEM cells and A549 cells was detected, not even at very high concentrations of nanoparticles. After UV irradiation for 30 min, particles showed dose-dependent cytotoxicity in both cell lines. Wen, Zhang, and co-workers proposed a light-driven controlled release system based on MSNs capped with AuNPs modified with DNA single strands, which contained azobenzene moieties.86 DNA-AuNPs were anchored to MSNs by the reaction of amino-ended DNA strands with carboxylic acid moieties previously created on the surface of MSNs. The system was loaded with Rh B by heating capped nanoparticles over the melting point of the DNA chain (60 °C) in a highly concentrated Rh B aqueous solution. When the solution was cooled to room temperature, azobenzene moieties were in the trans form and enabled the single-stranded DNA to form a hairpin-loop structure to close the gate. When the system was irradiated with UV light at 365 nm, azobenzene changed its conformation to the cis state destabilizing the hairpin-loop conformation and allowing Rh B delivery (Figure 14). The authors also demonstrated the reversibility of the system and studied the dependence of the cargo release rate with UV irradiation intensity. Azobenzene cis−trans photoconversion has also been widely used to develop nanoimpellers, where photosensitive units were

the competitive binding 1-adamantane molecule led to massive Rh B dye delivery. Yan et al. developed a gated system based on DNA and azobenzene units.85 MSNs were loaded with rhodamine 6G (Rh 6G) and functionalized with propyl isocyanate groups, which further reacted with amino-modified single-stranded DNA arms (arm-DNA). In a second step, azo-DNA strands, which contained azobenzene moieties and segments complementary to the arm-DNA, were used to cap pores as can be seen in Figure 13. Under visible light irradiation (λ = 450 nm), azobenzene molecules were in the trans form, and azo-DNA was hybridized with arm-DNA capping the pores. However, upon UV irradiation (λ = 365 nm), azobenzenes turned to the cis form, which induced dehybridization and pore opening with the subsequent Rh 6G release. These authors also found that by alternatively using visible and UV irradiation, the release profile showed controllable behavior attributed to the reversible dehybridization/rehybridization of DNA caps. The effect of this nanomaterial on cell proliferation was assessed in human acute lymphoblastic leukemic CCRF-CEM cells and human lung adenocarcinoma A549 cells by MTT assays. No significant cytotoxicity was detected in either cell line, which demonstrated the material’s excellent biocompatibility. Next the in vitro cytotoxicity of a 573


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Figure 14. MSNs loaded with Rh B and capped with AuNPs functionalized with azobenzene derivatized-DNA strands. Rh B was released upon irradiation at 365 nm.

Figure 13. MSNs loaded with Rh 6G or DOX and capped with an azobenzene derivatized-DNA strand. Cargo was released upon irradiation at 365 nm.

anchored to inner pore walls inducing stimulated delivery due to azobenzene movement.87−91 In line with this, Zink and coworkers compared the use of azobenzene derivatives as both inner impellers and gatekeepers, depending on where the photoresponsive molecules were located (i.e., on the inner pore surface or in pore outlets).92 The authors prepared small azobenzene units by reacting 4-phenylazoaniline with isocyanatopropyltriethoxysilane and incorporated the resulting alkoxysilane derivative into the synthesis of MSNs (see Figure 15A). In parallel, the authors prepared calcined MSNs and functionalized pore outlets with isocyanatopropyltriethoxysilane groups that were coupled to an azobenzene group modified with a G1 Frechet dendron (4-(4′-(3″,5″-bis(3‴,5‴-bis(benzyloxy)benzyloxy)phenylazo)-benzyl alcohol) (see Figure 15B). The release of coumarin 540A from the pore voids of both materials was studied upon irradiation cycles at 451 nm and darkness. The authors found that when small azobenzene units were located inside pores, it was possible to photostimulate coumarin 540A release, but if the amount of azobenzene was relatively small, the system was also leaky in the darkness. On the contrary, when bulk azobenzene units were positioned in pore entrances, the release of the entrapped cargo in the absence of light was negligible, whereas coumarin 540A molecules were released rapidly when gated MSNs were irradiated. Trans−cis isomerization of the cinnamamide group has also been used to prepare photoresponsive supramolecular nanovalves.93 The authors functionalized the external surface of MSNs with 3-aminopropyl groups. Afterward, amino modified nanoparticles were reacted with cinnamoyl chloride, and then the pores of the material were loaded with Rh B. In a final step, pores

were capped upon the addition of cucurbit[7]uril (CB[7]) due to the formation of stable inclusion complexes with the grafted cinnamamide moieties as shown in Figure 16. Irradiation with UV light with a wavelength of 300 nm of PBS suspensions of the capped material induced the release of the entrapped Rh B due to a dethreading of the inclusion complex as a result of the isomerization of the trans double bond of the cinnamamide moieties to the cis form. 2.3. Photoisomerization of Spiropyrans

Spiropyrans have also been used to design gated materials. It is well-known that spiropyran photochrome can be transformed reversibly between two forms upon the application of an external light source. When the spirocyclic molecule is kept in the dark or irradiated with UV light, it isomerizes to the merocyanine form. The merocyanine form also reverts reversibly to the closed spiropyran either thermally or by irradiation with visible light. Additionally, whereas the spirocyclic form is neutral, the merocyanine form is either positively charged at neutral pH or a zwitterion at higher pH values. Moreover, the merocyanine form is more hydrophilic than the spirocyclic form. It is precisely these light-induced changes in charge and polarity that have been used to design gated materials. The first example of gated materials using spiropyrans was ́ developed by Martinez-Má ñez et al. These authors loaded a mesoporous silica support with Ru(bipy)32+ and functionalized the external surface with a spiropyran derivative. Moreover, 1.5 poly(amidoamine) (G1.5 PAMAM) dendrimers were used to cap pores by an electrostatic interaction with the positively charged merocyanine-functionalized form.94 Aqueous (pH 7.2) 574


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Figure 16. MSNs loaded with Rh B, functionalized with cinnamamidecontaining stalks and capped with CB[7]. Rh B was released upon irradiation at λ > 380 nm.

Figure 15. MSNs loaded with Coumarin 540 or Rh 6G and functionalized with azobenzene moieties (A) on the outer surface or (B) on the inner surface. Cargo release was triggered upon continuous irradiation at 457 nm.

Figure 17. Gated MCM-41 support loaded with Ru(bipy)32+ functionalized with spiropyran derivatives and capped with G1.5 PAMAM dendrimers. Cargo release was observed upon irradiation with visible light.

suspensions irradiated with UV light in the presence of G1.5 PAMAM dendrimers showed no cargo delivery. However, as can be seen in Figure 17, through irradiation with visible light, the positively charged merocyanine isomerized to the neutral spirocyclic form, which had a lower affinity for the dendrimers with the subsequent cargo release. These authors also found that dye delivery could be achieved by changing pH; at an acidic pH, G1.5 PAMAM dendrimers did not act as stoppers because the protonation of their carboxylic groups prevented an interaction with the merocyanine isomer. Willner and co-workers functionalized aminopropylsiloxaneMSNs with N-carboxyethyl-nitrospiropyran photoisomerizable units, and Rh B was entrapped in mesopores (see Figure 18).95 The photoirradiation of nitrospiropyran with UV light yielded the positively charged merocyanine state, which induced electrostatic repulsive interactions between the nitromerocya-

nine groups and consequently unblocked pores. This uncapping mechanism was reversible upon visible light irradiation (λ > 495 nm), which regenerated the neutral nitrospiropyran state. The UV-induced change between the closed/hydrophobic and opened/hydrophilic forms of spiropyrans was used by Li et al. to prepare light-triggered delivery systems.96 The authors prepared an amphiphilic copolymer, which contain spiropyran moieties, by radical copolymerization of 1′-(2-methacryloxyethyl)-3′,3′-dimethyl-6-nitro-spiro(2H-1-benzopyran-2′,2′-indoline), Rh B (2-hydroxyethyl acrylate) ester, and aminefunctionalized poly(ethylene glycol)methacrylate (see Figure 19). Once the polymer was prepared, folic acid (FA) was linked to the final gated material through the formation of an amide in order to confer targeting abilities to cancer cells that overex575


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Figure 18. MSNs loaded with Rh B and capped with a spiropyran derivative. Rh B release was induced upon irradiation with UV-light.

pressed folate receptors. Hollow MSNs were modified by the grafting of octadecyl chains, and pores were loaded with DOX. The final capped material was prepared by coating the hydrophobic-loaded nanoparticles with the spiropyran-containing copolymer by hydrophobic van der Waals interactions. Aqueous suspensions of capped nanoparticles (PBS, pH 7.4) upon visible light irradiation showed negligible DOX release, whereas irradiation with UV light (365 nm) resulted in a marked DOX release due to the isomerization of the spiropyran to its hydrophilic conformation. This detached the polymer layer from the surface. MTT viability assays carried out with the capped nanoparticles in the dark and KB cells showed that nanoparticles were nontoxic. Yet upon irradiation with UV light, diminished cell viability was observed as a result of pore opening and DOX release. The combination of a spiropyran derivative and a perfluorohydrocarbon chain was used to prepare a light-triggered gated material.97 The external surface of MSNs was functionalized with APTES and perfluorodecyltriethoxysilane, and then a suitable spiropyran derivative was attached to the amino groups through the formation of an amide bond. Pores were loaded with fluorescein. PBS suspensions of the prepared nanoparticles at pH 7.2 showed negligible fluorescein release due to the formation of a hydrophobic layer, which protected the surface from being wet by aqueous solution (see Figure 20). However, under UV-light irradiation, spiropyran moieties isomerized to their merocyanine charged form. This changed the character of the layer around pores from hydrophobic to hydrophilic, and as a result, the surface was wetted with the subsequent dye release. In order to test the biological applicability of the solid, the same material loaded with CPT was prepared. CPT-loaded capped MSNs were internalized in HeLa and EA.hy926 cells by endocytosis and a clear loss of cell viability (51% for HeLa and 32% for EA.hy926 when using a 100 μg/mL concentration of solid) was observed upon UV-light irradiation. Cell death observed was ascribed to an efficient CPT release within the cells triggered by UV light.

Figure 19. Hollow MSNs loaded with DOX and capped with a copolymer that contained spiropyran moieties, Rh B, and FA. DOX release was induced upon irradiation at 365 nm.

gold nanoparticles with average diameter of 5 nm, which contained the photoresponsive molecule thioundecyl-tetraethylene glycolester-o-nitrobenzylethyldimethylammonium bromide. Positively charged nanoparticles were able to interact with the negative silanol groups of MSNs capping pores. The system was loaded with fluorescein.98 The authors demonstrated that the system was capped in water, yet cargo delivery was observed upon photoirradiation with a 365 nm low-power (0.49 mW/ cm2) UV lamp for 10 min (see Figure 21). This was attributed to the photocleavage of the photolabile linker, which resulted in the formation of negatively charged thioundecyl-tetraethylene glycol-carboxylate-functionalized AuNPs that detached from the silica due to charge repulsion. To validate the feasibility of the system for intracellular drug delivery, a similar capped system was loaded with hydrophobic anticancer drug paclitaxel, and the system was tested in human liver and fibroblast cells. The material was efficiently endocytosed, as determined by flow cytometry. After a 10 min UV irradiation, cell viability

2.4. Photocleavage

The external light-induced cleavage of covalently linked caps has also been thoroughly explored to develop gated materials. Lin and co-workers prepared the first photolabile gated system with 576


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ethane-1,2-diamine] and with an adamantane derivative, the latter linked to the surface by a disulfide bond as depicted in Figure 22.99 The hybrid material was loaded with Rh B or

Figure 20. MSNs loaded with fluorescein, functionalized with a perfluorohydrocarbon chain, and capped with the cyclic form of a spiropyran derivative. Irradiation with UV-light-induced fluorescein release.

Figure 22. MSNs loaded with pinacyanol iodide or Rh B, functionalized with a two-photon transducer molecule, and capped with β-CD complexed with a disulfide-containing linker. Irradiation induced cargo release. ME also induced cargo release.

pinacyanol iodide and was capped by the formation of an inclusion complex between the adamantane groups and β-CDs. The authors tested the response to UV−vis (408 nm) irradiation, which induced the photoredox cleavage of the disulfide bond that linked the adamantane groups, resulting in dye delivery. The authors also studied two-photon excitation via irradiation with a femtolaser at 800 nm, which also resulted in the delivery of Rh B. In the two experiments with light irradiation, the authors also concluded that it was necessary to inhibit the back electron transfer process by adding a sacrificial agent (i.e., ethylenediaminetetraacetic acid, EDTA) that acted as an electron donor. These authors were also able to open the gate by using mercaptoethanol (ME) due to the reduction of the disulfide bond. Li, Lu, and co-workers developed a visible light-triggered gating system using hollow MSNs coated with a photodegradable amphiphilic copolymer.100 In their work, the authors modified the external surface of nanoparticles with both octadecyltrimethoxysilane and Rh B isothiocyanate conjugated with APTES. In a second step, nanoparticles were loaded with DOX. Finally, pores were blocked with a copolymer that contained photosensitive 9,10-dialkoxyanthracene moieties and FA groups (see Figure 23). The photosensitizer eosin was also incorporated into the polymer via π−π stacking interactions. Once prepared, the authors tested the capability of the gated system to release DOX upon green light irradiation. They found poor cargo delivery (less than 5% of cargo after 100 h) when the solid suspended in simulated body fluid (SBF) was not irradiated. In contrast, eosin transformed 3O2 into 1O2 upon irradiation, which induced the conversion of 9,10-dialkoxyanthracene into 9,10-anthraquinone

Figure 21. MSNs loaded with fluorescein and capped with the AuNPs that contained photocleavable o-nitrobenzyl moieties. Fluorescein was released upon irradiation at 365 nm.

significantly decreased in both cell lines. No toxicity of the nanomaterial without paclitaxel was detected before and after UV irradiation. Zink and co-workers developed a drug delivery system that was activated by two-photon excitation. MSNs were functionalized with the phototransducer molecule [N1-(4-((1E,3 E)-4-(4(dipropylamino)phenyl)buta-1,3-dien-1-yl)phenyl)-N1-propyl577


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Figure 24. Hollow MSNs loaded with DOX and capped with a photodegradable copolymer that contained coumarin moieties and FA. DOX was released upon irradiation at 800 nm.

shifted the hydrophilic−hydrophobic balance toward the destabilization of the polymer layer. The authors clearly found that under NIR light exposure, DOX delivery increased continuously over time, while a very small amount of DOX was released without NIR irradiation. Zink and co-workers used coumarin groups and photolabile carbamate moieties to design MSNs photoactivated by two coherent NIR photons. The authors modified MSNs with a silane derivative of 7-hydroxy-4-(hydroxymethyl)-3,8a-hydro2H-chromen-2-one containing a carbamate linkage.102 Nanoparticles were loaded with Rh B and capped by the interaction of coumarin moieties with β-CDs (Figure 25). The authors demonstrated that capping moieties were photocleavable by one-photon excitation at 376 nm and by two-photon excitation at 800 nm. Kim and co-workers functionalized MSNs with an onitrobenzyl ester alkyne derivative, which was additionally coupled with mono-6-azido-β-CD via a Huisgen 1,3-dipolar cycloaddition to close pores.103 Nanoparticles were loaded with calcein. Irradiation at 350 nm UV light resulted in the photocleavage of the o-nitrobenzyl ester, which induced cargo delivery (Figure 26). These authors also used the same photoresponsive nanoparticles but formed host−guest interactions between β-CDs on the surface and a six-arm poly-

Figure 23. Hollow MSNs loaded with DOX and capped with a photodegradable copolymer that contained 9,10-dialkoxyanthracene moieties, FA, and the green light absorbing photosensitizer eosin. Irradiation at 540 nm induced DOX release.

and the subsequent degradation of the polymeric cap. In a step forward, the authors confirmed the biocompatibility and nontoxicity of the gated system and studied their uptake in the cancer cells that contained (KB) or no (A549) folate receptors by confocal microscopy. They found that after a first short period of similar endocytosis, KB cells showed a significant increase in fluorescence intensity, which was ascribed to the presence of the folate receptor that mediated endocytosis. Cell viability was not affected if cells were not irradiated, but only 40% of KB cells survived after 6 h irradiation. Following a similar approach, Li et al.101 also developed a new NIR-triggered nanovehicle for DOX delivery by coating hollow MSNs particles with a coumarin-containing photoresponsive amphiphilic polymer as depicted in Figure 24. The polymer was also functionalized with FA. 800 nm NIR light excitation caused the rupture of an ester bond, thanks to the high two-photon absorption cross section of the coumarin moiety. This resulted in the removal of coumarin groups from the block copolymers and 578


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performed, assisted by a second-generation Grubbs’ catalyst. The capped system was able to retain DOX in PBS buffer solution, while irradiation with 980 nm light induced the release of the drug as can be appreciated in Figure 27. This mechanism

Figure 25. MSNs loaded with Rh B and capped with an inclusion complex between a coumarin-containing stalk and β-CD. Rh B was released upon irradiation at 376 or 800 nm.

Figure 27. Mesoporous silica coated UCNPs loaded with DOX and functionalized with a photosensitive linker. DOX release was induced by 980 nm light.

involved absorption of light by the UCNP and the rupture of the o-nitrophenyl group of the linker. The performance of gated nanoparticles was also studied in A-498 tumor cells by confocal laser scanning microscopy (CLSM). Significant DOX red fluorescence was monitored only when cells were irradiated with NIR light. Finally, nanoparticles were derivatized with FA, and internalization in folate-rich HeLa cells was enhanced. No significant difference in uptake and performance was observed in control NIH/3T3 cells, which did not overexpress the folate receptor. Zhao and co-workers developed photoresponsive nanoparticles with amine-terminated copolymer poly(N-isopropylacrylamide-co-2-nitrobenzyl acrylate), which contained a photocleavable 2-nitrobenzyl groups (see Figure 28). The copolymer was coupled to 3-(triethoxysilyl)propyl isocyanate and was then grafted onto the surface of MSNs previously loaded with fluorescein.105 The polymer had a lower critical solution temperature (LCST) below the environmental temperature and was in a collapsed (insoluble) state, which inhibited cargo delivery. Fluorescein release was achieved by UV irradiation (310 nm), which induced the cleavage of the 2-nitrobenzyl groups. The light-induced rupture resulted in a hydrophilic acrylate polymer with LCST above the environmental temperature that allowed cargo release. Wan, Liu, and co-workers described a light-driven MSN prepared with a photodegradable protein-polyelectrolyte complex.106 In their design, the authors coated MSNs with a

Figure 26. MSNs loaded with calcein and capped with an inclusion complex between o-nitrobenzyl ester-containing stalks and β-CDs. Irradiation at 350 nm induced calcein release.

(ethylene glycol) with dodecyl end groups. The inclusion complex formed between functionalized MSNs and this poly(ethylene glycol) solution resulted in the formation of a hydrogel. In the hydrogel system, no release of calcein was observed when it was irradiated. However, addition of α-CD, which is a competitive host for the dodecyl group, induced the disassembly of the hydrogel, and the system recovered its photoresponsive gating properties. Liu, Xing, and co-workers prepared DOX-loaded aminated mesoporous silica coated upconverting nanoparticles (UCNPs) and derivatized the amino groups with a 1-(2-nitrophenyl)ethyl photoactivable oligo(ethylene)glycol linker with a vinyl group at the end.104 In another step, ring-closing methatesis of linkers was 579


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moieties, which prompted repulsion with the negatively charged BSA protein. Around 80% of the Rh B cargo was delivered in 3 h. Knežević and Lin prepared a light-activated drug delivery system using magnetic MSNs with an Fe3O4 core and capped by CdS NPs.107 The silica surface of MSNs was functionalized with isocyanate groups and loaded with anticancer drug CPT. Then mesopores were blocked by anchoring 2-nitro-5-mercaptobenzyl alcohol functionalized CdS nanoparticles via the formation of a photocleavable carbamate linkage as depicted in Figure 30. When

Figure 28. MSNs loaded with calcein and capped with a photocleavable o-nitrobenzyl-containing copolymer. Fluorescein was released upon irradiation at 310 nm.

quaternary aminated random copolymer, which was composed of oligo(ethylene glycol) monomethyl ether methacrylate, and photolabile o-nitrobenzyl-containing monomer 5-(2′(dimethylamino)ethoxy)-2-nitrobenzyl methacrylate (Figure 29). Finally, pores were capped by the complexation of bovine serum albumin (BSA) with the coating copolymer via electrostatic interactions. Irradiation of the BSA-polyelectrolyte-capped system with UV-light (360 nm, up to 3 mW cm−2) induced the cleavage of the 2-nitrobenzyl ester groups to result in carboxylate

Figure 30. MSNs loaded with CPT and capped with CdS NP using a photocleavable o-nitrobenzyl-containing linker. Irradiation at 365 nm triggered CPT release.

the hybrid material was exposed to UV light at 365 nm, high delivery of the entrapped dye in PBS buffer was achieved. These authors also evaluated the applicability of the system to treat cancer cells. Chinese hamster ovarian (CHO) cells were treated with CPT-loaded nanoparticles in the presence or absence of light irradiation at concentrations of 10 and 25 μg mL−1. With the use of low concentration of MSNs, a significant increase in the cell death of light-irradiated samples was achieved. However, at a high concentration in both the irradiated and nonirradiated experiments, a significantly enhanced cell death was found, which was more pronounced in the irradiated samples. The authors attributed this behavior to the toxicity of CdS caps. In order to confirm that enhanced cell death at a low concentration of capped MSNs was related with CPT delivery, and not by the effect of CdS caps, the same experiment was done but using unloaded MSNs. As expected, no significant differences were observed between the irradiated and nonirradiated samples when no drug was loaded inside the material. The photocleavable o-methoxybenzylamine group was used by ́ Martinez-Má ñez et al. to develop photoactivated molecular gates. They loaded MSNs with Ru(bipy)32+ fluorophore and an omethoxybenzylamine derivative that contained triethoxysilane, and two bulky t-butyl groups were grafted onto the outer silica surface (Figure 31).108 The bulky t-butyl subunits of the omethoxybenzylamine derivative precluded the release of the entrapped dye. Upon UV-light irradiation, the photocleavage of the o-methoxybenzylamine group took place with subsequently reduced steric crowding around pores, which allowed the release of the entrapped fluorophore.

Figure 29. MSNs loaded with Rh B and capped with a photocleavable onitrobenzyl-containing copolymer and BSA. Rh B was released upon irradiation at 350 nm. 580


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Figure 31. MSNs loaded with Ru(bipy)32+ and capped with an omethoxybenzylamine derivative. Ru(bipy)32+ was released upon irradiation at 254 nm.

2.5. Generation of Reactive Oxygen Species

Light can be also used to generate free radicals able to cause the rupture of chemical bonds and subsequent gate uncapping. For example, Lu and co-workers employed Mn2+-doped NaY(Mn)F4:Yb/Er UCNPs coated with a mesoporous silica shell. The silica surface was functionalized with octadecyl hydrophobic chains and with photosensitizer chlorin e6. Moreover, pores were loaded with DOX.109 The authors also prepared a hydrophobic polymer that contained 9,10-dialkoxyanthracene groups by free radical polymerization of monomers poly(ethylene glycol) methyl ester methacrylate and 3-((10-(3-(methacryloyloxy)propoxy) anthracen-9-yl)oxy) propylstearate (see Figure 32). The pores of the functionalized nanoparticles were coated with the polymer through hydrophobic wan der Waals interactions with octadecyl chains. PBS suspensions of the capped nanoparticles showed negligible DOX release without irradiation, whereas a remarkable release of the entrapped drug was observed (more than 70% after 8 h) upon NIR irradiation (with a 980 nm laser). The UCNPs absorbed the NIR light converting it into red light (660 nm), which excited the chlorin e6 photosensitizer that produced singlet oxygen. This singlet oxygen oxidized the anthracene moieties of the polymer shell to anthraquinone with the subsequent polymeric cover degradation and drug release. MTT viability studies carried out with KB cells showed that capped nanoparticles were essentially nontoxic, but significant cell death was observed upon NIR irradiation. This cell death was ascribed to a synergic effect due to DOX release and singlet oxygen generation. The nanodevice was also able to reduce tumor growth in nude mice that bore KB cells under NIR irradiation. Bräuchle et al. prepared MSNs that contained aminopropyl moieties on inner pore walls and mercaptopropyl groups on the silica’s outer surface. In a second step using the bifunctional linker 1-maleimido-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azaben-triacontan-31-oic acid, protoporphyrin-IX-bis(phenyleneaminoamide) was grafted onto the external surface.110 Then pores were loaded with different cargos (PI, Alexa fluor 647-labeled phalloidin and ATTO-594-labeled chromobodies) and capped by a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer membrane. Irradiation of proto-

Figure 32. UCNPs coated with a mesoporous layer, loaded with DOX, functionalized with photosensitizer chlorin e6 and capped with a photodegradable copolymer that contained 9,10-dialkoxyanthracene moieties. DOX was released upon irradiation at 980 nm.

porphyrin-IX with 405 nm light induced the formation of singlet oxygen, which disrupted the lipid bilayer with the subsequent cargo release as depicted in Figure 33A. To verify the release and biological effects of loaded substances on cells, cell cultures of HuH7 cells that expressed GFP were incubated with the different drug-loaded nanomaterials for 12−24 h and were exposed to photoactivation for 5−20 min. In all cases, drugs were efficiently released from nanoparticles and were able to bind to their respective cellular target structures. 581


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In a similar approach, the same authors prepared a drug delivery system based on colloidal MSNs with targeting ligands and a red-light photosensitizer.111 In this case, the external surface was functionalized with (3-cyanopropyl)triethoxysilane and cyano groups were hydrolyzed to carboxyl groups with hydrochloric acid. Then carboxylic moieties were functionalized with polyethylene glycol (PEG) with a bisamino PEG linker via EDC amidation, and the free amino groups of the PEG linker were reacted with the Al(III) phthalocyanine chloride disulfonic acid (AlPcS2a) photosensitizer via a sulfonamide bond. Inner walls of pores were functionalized with (3-mercaptopropyl)triethoxysilane (MPTS). The authors prepared two materials with different cargos: 5(6)-carboxy-X-Rh and calcein. Finally, loaded nanoparticles were covered with a lipid bilayer that consisted in a lipid mixture of DOPC and 1,2-dioleoyl-3trimethylammonium propane (DOTAP) (see Figure 33B). Irradiation of the capped material with 639 nm light induced the activation of the photosensitizer and the singlet oxygen production that reacted with the double bonds of DOPC to result in cargo delivery. In another step, two different targeting ligands [FA and epidermal growth factor (EGF)] were introduced into the materials to achieve specific uptake of nanoparticles by cancer cells. Ligands were introduced into the lipid bilayer by the diffusion of a lipid-FA or a lipid-EGF. To evaluate the functionality of the FA ligand, KB cells were used. Cells were preincubated with FA for 2 h at 37 °C, before adding the nanomaterial to cells, and then were incubated for 3 h at 37 °C. Confocal microscopy studies revealed that no nanoparticles uptake was detected in the FA preincubated cell cultures, whereas significant uptake was noted in cells without FA preincubation. This clearly indicated that particles were taken up by receptor-mediated endocytosis and that the nonspecific uptake of particles by KB cells was minor. Similarly to the FA experiments, competition experiments with EGF ligand were performed with HuH7 cells, which are known to overexpress the EGF receptor. Essentially, identical results were obtained, which thus indicates that this system could be modified efficiently to meet the specific requirements of various cancer types and to ensure a specific receptor-mediated uptake. To verify that the drug delivery mechanism works in vitro, a calcein loaded-system was incubated for 3 h with HeLa cells. In comparison to incubation with free calcein, incubation of the nanoparticles that contained this fluorescent dye as a cargo produced a significantly higher calcein concentration inside cells, with the fluorescent green-dotted pattern that is characteristic of endosomal colocalization (Figure 33B, bottom). Uptake of free calcein by KB cells was negligible and thus showed that the system efficiently delivered membrane impermeable cargos into cells. To allow endosomal escape, irradiation with 603 nm light was performed to induce the opening of the supported lipid bilayer coat. Light irradiation triggered increased calcein green fluorescence throughout the cell. In addition to calcein, other fluorescent cargos were loaded into the system with similar results. Bein and co-workers functionalized MSNs on the outer surface with thiol groups, which were further reacted with a PEG linker.112 Amino-modified photosensitizer protoporphyrin-IX (PpIX-NH2) was attached to carboxy moieties on the surface of particles via EDC-assisted amidation. The inner surface was functionalized with amino groups, which served as linkers to graft dye CysATTO633 by the EDC coupling method. A disulfide bridged dye-quencher system was then constructed in the inner particle by adding thiol-activated cysteine-modified QSY21

Figure 33. (Top) MSNs loaded with different cargoes, functionalized with photosensitizers, and capped with a lipid bilayer. Irradiation with light induced cargo release. (Bottom) Fluorescence microscopy of the nanoparticles functionalized with photosensitizer B and loaded with calcein inside HeLa cells (after 16 h incubation). (a−c) Calcein (green) and AlPcS2a (red) are colocalized (yellow) prior to photoactivation. (d) Intensity profile along the white line in the merged image for both. (e− h) 1 min, (i−l) 5 min, and (m−p) 10 min after photoactivation. Adapted from ref 111. Copyright 2013 American Chemical Society. 582


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indicated that distributions were primarily determined by endosome properties rather than by variations across nanomaterials. Qu et al.113 constructed a light-operated nanocarrier for targeted intracellular drug delivery using photosensitizerincorporated G-quadruplex DNA-capped MSNs. The authors modified the external surface of MSNs sequentially with 3chloropropyltrimethoxysilane and sodium azide. Pores were loaded with either Rh B or DOX and the meso-tetra(N-methyl-4pyridyl)porphine tetra tosylate- 5′-hexynyl-TTG GGG TTT TGG GG-3′ (TMPyP4-DNA) complex was grafted onto the surface of nanoparticles by a click reaction to cap pores (Figure 35). The oligonucleotide anchored to the outer surface self-

(CysQSY21) (Figure 34). Nanoparticles were covered with a lipid bilayer of DOPC or 1,2-dioleoyl-sn-glycero-3-phosphoe-

Figure 34. MSNs functionalized in the inner pore surface with a disulfide bridged dye-quencher system on the outer surface with protoporphyrin-IX-bis(phenyleneaminoamide) and capped with a DOPC or a DOPE lipid bilayer. In the intracellular reductive environment, irradiation at 405 nm induced bilayer rupture and recovery of the ATTO633 fluorescence.

thanolamine (DOPE). Once cells had been incubated with the material and Alexa Fluor Dextran 488, endosome lysis was induced by activating PpIX with 405 nm light. The double bonds in the lipid tails of lipid bilayers and endosomes were oxidized by the produced reactive oxygen species (ROS). Following membrane disruption, the reductive intracellular milieu cleaved the bonds between quencher CysQSY21 and ATTO633, which led to localized fluorescence at the lysis site and the simultaneous dispersion of the Alexa Fluor signal. In vitro release studies demonstrated the proposed mechanism as the addition of 10 mM glutathione (GSH) resulted in ATTO633 fluorescence. The system’s cellular uptake and individual endosome lysis studies were performed in 3T3 fibroblasts, normal, and RENCA kidney carcinoma cell lines. The activation time played a more prominent role in Renca cells than in 3T3 cells as a 2 min irradiation was enough for 3T3 fibroblasts to trigger efficient endosome lysis, which indicates some type of cell-specific effect on endosome lysis. An analysis of system load per endosome, endosome size, and uptake characteristics indicated that RENCA cells not only take up fewer NPs vs fibroblast cells but also have larger endosomes and a lighter nanoparticle load per endosome. With these data, a stochastic model to detail the steps downstream of uptake was developed to understand the extent to which factors affect lysis time distributions. The model results

Figure 35. MSNs loaded with Rh B or DOX, functionalized with FA, and capped with a DNA G-quadruplex. Porphyrin derivatives interacted with the DNA G-quadruplex. Cargo was released upon irradiation at 440 nm.

associated into the bimolecular G-quadruplex DNA structure. DNA capping was cleaved via the photosensitized production of ROS by PDT, which allowed cargo delivery. Target-directing FA molecules were covalently grafted to the surface by a click chemistry reaction to increase specificity to cancer cells. The effect of FA modification on the cellular uptake of the MSNs loaded with Hoechst dye was studied with human liver cancer cells (Hep G2) and fibroblast cells (3T3). FA modification enhanced the nanoparticle uptake in only Hep G2 cells. Particles were efficiently internalized by cells and localized to the endolysosomal compartment. Irradiation of cultures with blue excitation light for 60s triggered the release of Hoechst. Identical 583


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particular, they functionalized the external surface with N-(6aminohexyl)aminomethyltriethoxysilane stalks, loaded nanoparticles with Rh B, and finally blocked pores by adding a CB[6] ring as depicted in Figure 37.115 As the stalk-ring binding

results were obtained with DOX-loaded nanoparticles as DOX cytotoxicity was detected only after light irradiation. 2.6. Gold Nanostructures

Laser light-induced hyperthermia can be achieved using gold nanoclusters, which can absorb optical energy and generate heating. The increase in temperature using this phenomenon can be locally high enough to induce the destabilization of the capping ensemble in gated materials, which results in cargo delivery. Thanks to this principle, a number of capped systems using AuNPs and light have been described to control pore opening. In this area, the use of NIR light between 700 and 1100 nm is particularly interesting given the absence of significant absorption from either biological chromophores or water, which allows deep optical penetration into biological samples, such as tissue or whole blood. ́ Martinez-Má ñez and co-workers developed one of the first examples of gated materials that operates reversibly in response to two stimuli and which also displayed controlled release in aqueous solution.114 In their work, the authors loaded a MCM41 support with safranine O and functionalized the external surface with N-(3-triethoxysilylpropyl)gluconamide. In parallel, AuNPs functionalized with boronic acid were prepared. Then pores were capped by the formation of boroester bonds between boronic acid units in AuNP and the saccharide derivative anchored to MCM-41 pore outlets (see Figure 36). The authors

Figure 37. Au@MSNs loaded with Rh B and capped with CB[6] rings. Rh B was released upon irradiation at 514 nm.

constant decreased exponentially with increasing temperature, the authors found that at 60 °C, the complex was dethreaded and pores were unblocked. It was also observed that irradiation with an argon ion laser at wavelengths close to the plasmon resonance of the Au core (530 nm) resulted in cargo delivery. Ren et al. prepared core−shell gold nanorods (AuNRs) covered with a mesoporous silica shell.116 The authors functionalized the external surface of the mesoporous support with carboxylic groups and anchored a 12-mer oligonucleotide (DNA-1) by EDC/NHS coupling chemistry (see Figure 38). Then an oligonucleotide that contained the sequence of the AS1411 aptamer of nucleolin with a 12-base extension at the 3′ end acted as capping agent (DNA-2) via hybridization with DNA-1 and the formation of G-quadruplex structures. This capped material was unable to deliver fluorescein that was used as cargo. However, NIR irradiation at 808 nm to suspensions of capped MSNs induced cargo release. This was attributed to a photothermal effect, which induced a rapid rise in the local temperature and resulted in the dehybrization of the DNA duplex. Cellular targeting efficiency was investigated by incubating NIH3T3 (normal cell) and MCF-7 (human breast cancer) cell lines with the AS1411 aptamer-modified nanomaterial. Although similar cellular uptake was observed in both cell lines, MCF-7 cells incubated with the system displayed stronger luminescence signals than that of NIH3T3 cells, which thus indicated the efficiency of the system as a drug carrier for targeted therapy. Next the cytotoxicity of the DOX-loaded nanomaterial was tested in MCF-7 cells. As expected, the viability of the cells treated with the DOX-loaded system dramatically decreased upon NIR light irradiation, which indicated efficient intracellular DOX delivery.

Figure 36. MCM-41 microparticles loaded with safranine O and capped with boronic acid-containing AuNPs. Safranine O delivery was controlled by laser light or pH.

found that AuNPs plasmonic heating induced by laser light caused the cleavage of boronic ester linkages, which resulted in payload release. The amount of cargo delivered by simply controlling laser irradiation was fine-tuned. The authors also discovered that cargo release was inhibited at pH 5, whereas a rapid release was observed at pH 3. That pH-controlled mechanism was associated with the reversible formation of boroesters (closed-gate), and their quick and easy hydrolysis (open-gate) at an acidic pH. Light remote triggering using AuNPs was also investigated by Zink and co-workers, which developed MSNs with a central 20 nm Au core and capped pores with a pseudorotaxane. In 584


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Following a similar approach, Cheng et al. used AuNRs covered by a mesoporous silica shell to prepare gated nanomaterials.118 The solid was loaded with the anticancer drug DOX. For the purpose of attaching covalently oligonucleotides to the silica surface, nanoparticles were first functionalized with APTES. Afterward the 4-maleimidobutyric acid Nsuccinimidyl ester was used as a linker to conjugate the amine to the thiol group of thiolated double-stranded DNA. In particular, double-stranded DNA was obtained by the hybridization of SH-5′-(CH2)6-TTT TTC CCG CGC CGG-3′ and 5′TTT TTC CGG CGC GGG-3′ in PBS at 4 °C (see Figure 40A). The final capped material showed that only 10% of DOX leaked after 10 h. In contrast when NIR light was tested, a pulsatile control of cargo release was observed. Cargo release was due to AuNRs-mediated photothermal conversion, which induced dehybridization of the DNA duplex. The authors also prepared a similar capped system where the nonthiolated strand was a GFP-interfered siRNA. The authors found that upon NIR irradiation, the released siRNAs silenced the GFP expression in GFP expressing HeLa cells. The same authors implemented the same gating mechanism in MSNs with a Fe3O4@Au trisoctahedra core (see Figure 40B).119 They found that heating the nanocarrier by NIR light irradiation induced the dehybridization of anchored double-stranded DNA (a denaturing temperature of approximately 47 °C), which allowed the cargo to escape. The magnetic Fe3O4 core also facilitated intracellular drug release by magnetic attraction. For in vitro studies, HeLa cells were selected and MTT viability assays were conducted to determine nanomaterial biocompatibility. Cells were incubated with the system at 37 °C for 24 h. Negligible cellular toxicity was detected with at least 90% of cell survival found at very high nanoparticle concentration. Cells were also incubated with the capped nanoparticles for 2 h, either with or without magnetic attraction, followed by other 24 h incubation. Then cell cultures were exposed to laser illumination. Interestingly, incubation with the DOX-loaded nanomaterial triggered a 2-fold increase in cell toxicity when compared to that of the empty nanomaterial. Finally, the studies were extended from in vitro to in vivo to investigate regression efficacy in tumor growth, which was monitored in tumor volume change terms. HeLa cells were transplanted hypodermically in the thighs of nude mice. In the control groups, mice were closely monitored for continuous tumor growth, which grew 7-fold larger on day 14 than initially (see Figure 40, bottom). In the mice intravenously injected with the system and treated with either magnet attraction or laser illumination, minor tumor growth suppression was detected (an increase of 5−6-fold after 14 days). Remarkably, in the mice injected with the system and treated with both magnet attraction + laser illumination, tumor growth was only 3-fold larger in 14 days. Most interestingly, a second injection of capped nanoparticles, plus the combination of lasermagnet therapy, fully eliminated the tumor after 14 days. Preliminary toxicological studies performed in mice tissue, such as heart, spleen, kidney, and liver, revealed no acute toxicity of the system. Given its nature, this nanomaterial was also tested for magnetic resonance imaging (MRI) performance. Mice were hypodermically injected with HeLa cells, and MRI experiments were conducted after 1 week of tumor growth with a 9.4 T animal micro MRI system. When magnetic attraction was applied after injecting nanaoprticles, an apparent contrast change in signal was found in the MR images, which confirmed that this nanomaterial was an effective MRI T2 contrast agent.

Figure 38. AuNRs@MSNs loaded with fluorescein or DOX and capped with an aptamer of nucleolin. Irradiation at 808 nm induced cargo release.

Yang and co-workers also developed a NIR-responsive nanovalve based on mesoporous silica-coated AuNRs.117 In their work, they functionalized the external surface of mesoporous silica with quaternary ammonium salts prepared by the reaction of 3-bromopropionic acid and trimethylamine. Nanoparticles were loaded with Rh B, and pores were blocked by complexation with SC[4]A moieties (Figure 39). The authors

Figure 39. AuNRs@MSNs loaded with Rh B and capped with SC[4]A. Cargo release was triggered by irradiation at 808 nm.

found that the amount of Rh B released to a PBS solution gradually increased with rising temperature in a water bath. This effect was associated with a drop in ring-stalk binding affinity. Moreover upon 808 nm NIR light irradiation (39.5 W cm−2), plasmonic heating from AuNR cores was able to disassemble the ring-stalk interaction and allowed the release of 95 wt % of Rh B in a time of 2.5 h. Finally, the authors also demonstrated that a pulsatile Rh B release could be achieved by periodic NIR laser on/off irradiation. 585


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Figure 40. (Top) MSNs with (A) AuNRs or (B) Fe3O4@Au nanotrisoctahedra as core, loaded with DOX and capped with dsDNA. DOX was released upon irradiation at 808 nm. (Bottom) Photographs of mice after treatments at different dates. Laser illumination was performed for 30 min exposure using an 808 nm diode laser at 3 W/cm2. The magnet was applied with 30 min of magnetic attraction. Adapted from ref 119. Copyright 2014 American Chemical Society).

method. This was followed by the construction of a mesoporous silica layer onto the dense silica shell via a sol−gel approach. Then the middle SiO2 layer was dissolved in a Na2CO3 solution via a cationic-surfactant-assisted selective etching route to obtain the silver nanocubes-mesoporous silica yolk−shell structure. In a further step, the AuNC was prepared by means of the galvanic replacement reaction between silver nanocubes and the HAuCl4 solution. Subsequently, the mesoporous silica surface was functionalized with APTES, which was further reacted with 2bromoisobutyryl bromide as an atom transfer radical polymerization (ATRP) initiator. Then a homogeneous poly(Nisopropylacrylamide) (PNIPAAm) coating was formed on the outer silica surface by ATRP upon the addition of Nisopropylacrylamide and N,N′-methylene bis(acrylamide). The nanocarrier was loaded with anticancer drug DOX. NIRtriggered drug release was performed in PBS solution at different pH values (7.4 and 5) as shown in Figure 42A. The slight drug

Tang and co-workers also used AuNRs coated with a mesoporous silica shell. The external surface of the nanoparticle was first functionalized with APTES, to which a single-stranded DNA was anchored by amide bonds.120 The attached DNA sequence interacted with residual free amines of the surface to cap pores as depicted in Figure 41. The authors found that an adjustable cargo delivery was achieved by irradiating with a continuous-wave NIR diode laser (808 nm) at different power densities. Irradiation resulted in a local increase of temperature all around the nanoparticle, which weakened the electrostatic interaction between aminated mesoporous silica and DNA to induce the uncapping of pores and dye leakage. The system also showed acceptable resistance to nuclease activity. Zhang, Zhao, and co-workers developed a multifunctional NIR drug release system based on gold nanocages (AuNCs) as photothermal cores.121 To build such a system, first a dense silica layer was coated on silver nanocubes by the modified Stöber 586


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nigericin in flow cytometry measurements. CPT release was accomplished by irradiation with 808 nm laser light. MTT assay was used to reveal that the CPT-loaded capped nanoparticles were unable to significantly decrease cell viability. The same result was obtained when AuNCs without CPT were irradiated. In contrast, when the CPT-loaded nanoparticles were exposed to laser light, a synergic effect between the released CPT and the photothermal effect was found, and viability decreased up to 30%. Finally, system performance was successfully validated in an orthotropic tumor model of hepatocellular carcinoma in mice. In a similar work, β-NaYF4:Yb3+/Er3+ UCNPs were coated with a mesoporous silica shell and the final material was used for the thermo/pH drug controlled release and for bioimaging applications.123 The mesoporous silica shell was functionalized with methacryloxypropyltrimethoxysilane. Then monomers Nisopropylacrylamide and methacrylic acid were copolymerized by photoinduced polymerization. This process induced the formation of P(NIPAm-co-MAA) brushes on the external surface of the mesoporous shell, which blocked pores (Figure 42B). In this case, the material was loaded with DOX. Aqueous suspensions of nanoparticles at pH 7.4 and 25 °C showed negligible DOX release (9.6% after 4 h). However, when the temperature rose to 45 °C, moderate drug release was observed (39.5% after 24 h), which was ascribed to the shrinkage of the polymer brushes around pore outlets. Irradiation with a laser light of 980 nm also induced the drug release from aqueous suspensions of the gated nanoparticles at pH 7.4 and 25 °C, which was attributed to local temperature enhancement with the subsequent shrink of polymer brushes and DOX release. The authors also found that by fixing the temperature at 25 °C, DOX delivery could also be triggered by changing pH from neutral to acidic. This pH-triggered release was related with the protonation of the methacrylic acid fragment of the P(NIPAmco-MAA) brushes. In particular, the authors suggested that at a neutral pH, when carboxylates were unprotonated, there would be electrostatic interactions with positively charged DOX, which disabled drug release. However, at an acidic pH, the electrostatic interactions were reduced due to the protonation of carboxylates, and the drug was released. For in vitro studies, L929 fibroblasts were selected and MTT viability assays indicated that the nanoparticles showed good biocompatibility. Moreover, in order to compare the pharmacological efficacy of the DOX-loaded nanospheres and free DOX, HeLa cells were incubated in culture medium in the presence of free DOX and of the DOX-loaded capped nanoparticles with various concentrations for 24 h. Then MTT cell viability assays were conducted. Free DOX exhibited slightly higher cytotoxicity than the DOX-loaded nanospheres at a lower concentration, but the result inversed when the DOX concentration went up to 12.5 μg/mL. This result suggested that free DOX cellular uptake was higher than that for capped nanoparticles as the latter had to be endocytosed prior to cargo release. The system’s efficient and time-dependent cellular uptake was confirmed by flow cytometry and confocal laser microscopy experiments. Finally, time-course up-conversion luminescence microscopy (UCLM) studies in human alveolar adenocarcinoma cells demonstrated that the nanomaterial could also be used as an excellent luminescence probe for cell imaging and monitoring the cell endocytosis process. Yavuz, Cheng, Chen, and co-workers developed a NIR lighttriggered nanocage to control the release of different cargoes.124 The authors functionalized the external surface of AuNCs with poly(N-isopropylacrylamide-co-acrylamide) with an LCST of 39 °C (Figure 43). The copolymer was prepared using a disulfide

Figure 41. AuNRs@MSNs loaded with Rh B or DOX and capped with a DNA strand. Irradiation at 808 nm triggered cargo release.

Figure 42. AuNC@MSNs or UCNPs@MSNs loaded with DOX and capped with PNIPAAm. Light irradiation triggered DOX release.

release was measured without applying NIR, while DOX release reached 32.8% and 78.9% in 8 h at pH 7.4 and 5, respectively, upon a 5 min NIR irradiation (1 W/cm2, 808 nm). These authors also tested the cytotoxicity of the nanocarrier in HeLa cells. CLSM analysis showed that DOX-loaded nanoparticles displayed lower cell viability than free DOX at various concentrations. The chemo-photothermal therapy combination was also investigated. Studies confirmed that the cell killing efficacy of the DOX-loaded capped nanoparticles with NIR irradiation (80.1%) was higher than the sum of chemotherapy (14.5%) and photothermal therapy (19.4%). In a similar work, Qu and co-workers prepared mesoporous silica coated AuNCs loaded with CPT and capped with poly(N-isopropylacrylamideco-acrylamide). 122 The system also included fluorescein isothiocyanate (FITC) anchored to the silica surface and a Rh B hydrazine derivative in the copolymer shell. Capped nanoparticles were used as a ratiometric probe that gave strong green fluorescence (518 nm), but only at a basic or neutral pH as with normal physiological conditions due to FITC, and strong red fluorescence (575 nm) due to the Rh B derivative but only at a pH below 6 as in tumor tissues. The sensory behavior of the material was confirmed in HeLa cells using H+/K+ ionophore 587


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alkaline phosphatase were also able to display an AND output upon irradiation in A549 cells. Santamariá et al. prepared MSNs functionalized with amine groups on which a porous Au shell was grown as depicted in Figure 44. The pores of the core−shell nanoparticles were loaded

Figure 44. MSNs loaded with IBU and capped with a gold porous shell. IBU was released upon irradiation at 808 nm.

Figure 43. AuNCs loaded with different cargos and capped with poly(N-isopropylacrylamide-co-acrylamide polymer). Cargo was release upon irradiation with laser light.

in this case with IBU.126 In the absence of NIR irradiation, IBU release was negligible. However, irradiation at 808 nm (using laser as the light source) induced a remarkable release of the entrapped drug. This was the result of the absorption of the NIR radiation by the Au shell, which quickly heated nanoparticles by accelerating desorption and the release of the IBU hosted on the porous network. Elbialy et al. functionalized MSNs with APTES and loaded pores with DOX. An Au shell was used to cover nanoparticles, and the nanodevice was finally coated with poly(ethylene glycol) to prevent aggregation.127 Aqueous suspensions of the goldcoated nanoparticles showed negligible dye release at pH 5.8, whereas a clear DOX delivery was observed upon NIR irradiation (see Figure 45). The sustained DOX release was ascribed to the

initiator and was anchored to the gold surface by taking advantage of the gold−thiol affinity. Below 39 °C, the polymeric shell was hydrophilic (extended conformation), and AuNCs pores were capped, whereas the polymer became hydrophobic (shrunk conformation) when temperature was above the LCST, which allowed cargo release. NIR laser irradiation on the capped AuNCs raised the local temperature and unblocked pores. Using these principles, the authors first showed the controlled release of PEG (Mn ≈5000) labeled with alizarin. In another step, the efficiency of DOX loaded and capped AuNCs was tested in breast cancer cells. Finally, lisozyme delivery was successfully controlled using a similar capped AuNC, but in this case, PNIPAAm was used as a cap instead of the copolymer to lower the LCST to 32 °C and to maintain enzyme activity. Qu and co-workers prepared a similar system but used two different AuNCs with localized surface plasmon resonance, one at 808 and one at 670 nm.125 To demonstrate that the system was able to perform an AND logic operation controlled by two different NIR lights, capped AuNC (808 nm) were loaded with enzyme alkaline phosphatase, whereas capped AuNC (670 nm) were loaded with substrate ELF97-phosphate as in Figure 43. Alkaline phosphatase converts ELF97-phosphate into green fluorescent ELF97 alcohol. An AND output (fluorescent signal) was observed only when both inputs (670 and 808 nm of laser irradiation) were simultaneously applied. The OR logic operation of the gated system was demonstrated by loading capped the AuNC(670 nm) system with enzyme acid phosphatase. In this case, ELF97 alcohol was produced in two parallel biochemical reactions to thus perform OR release behavior. Additionally, an INHIBIT gate was also designed by using L-phenylalanine as cargo of the capped AuNC(670 nm). In this case, the 670 nm laser triggered the release of Lphenylalanine, which inhibited alkaline phosphatase activity and consequently suppressed the production of fluorescent ELF97 alcohol. In addition to the in vitro release studies, the internalization experiments analyzed by CLSM demonstrated that a mixture of AuNC(670 nm) and AuNC(808 nm), loaded with fluorescein and Rh B, respectively, was taken up by A549 cells. Standard MTT assays showed that the low laser intensity used in the experiments had no effect on cell viability. The authors also found that capped AuNC(670 nm) loaded with substrate ELF97-phosphate and AuNC(808 nm) loaded with

Figure 45. MSNs loaded with DOX, capped with a gold nanoshell, and coated with PEG chains. DOX was released upon irradiation at 807 nm.

gold shell degradation after NIR light irradiation. CLSM was used to study the cellular uptake of the system in human tumor MCF-7 cells. As expected, enhanced cytotoxicity in MCF-7 cells was obtained upon the irradiation of internalized DOX-loaded nanoparticles. Flow cytometry studies of MCF-7 treated with the capped system revealed that the nanomaterial induced direct cell cycle arrest and cell death due to DOX delivery. The intravenous application of the system, followed by NIR irradiation of the tumor area, inhibited the growth of subcutaneous Ehrlich carcinoma in mice and induced a stronger anticancer effect compared to photothermal therapy or chemotherapy alone. Histopathological studies showed significantly higher cell death 588


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in mice treated with the DOX-loaded nanomaterial, which is consistent with the data obtained in cellular studies. Yang et al. prepared Na5Lu9F32:Yb/Er hollow mesoporous spheres and coated the external surface with the positively charged poly(ethylenimine) polymer.128 Then citrate-functionalized AuNPs were attached to the polymeric layer by electrostatic interactions as shown in Figure 46. The hollow

Figure 47. MSNs loaded with sulforhodamine 101 or amsacrine and capped with the Ru(bipy)2(PPh3) (GABA)+ complex. Cargo was released upon irradiation with 455 nm light.

Knežević also used the same gated material, which was in this case loaded with anticancer drug amsacrine.130 PBS (pH 7.4) solutions of the capped nanoparticles in the dark showed a very weak drug release (see Figure 47). However, upon irradiation with 455 nm light, clear amsacrine delivery was observed (72% of the total loaded amount after 120 h of irradiation). HeLa cells irradiated with visible light after nanoparticle uptake showed reduced viability (89.3%) when compared with untreated cells under dark conditions. This reduced viability was ascribed to the visible light induced drug release. Stoddart, Sauvage, and co-workers prepared MSNs capped with ruthenium(II) complexes capable of releasing an entrapped cargo upon irradiation with visible light.131 MSNs was functionalized with a molecule obtained by a reaction between (3isocyanatopropyl)triethoxysilane and 4-(aminomethyl)benzonitrile. Pores were loaded with paclitaxel, and the system was capped by the formation of a coordination complex between the grafted cyanide-containing derivative and Ru(terpy)(dppz)(H2O)2+ (see Figure 48). Aqueous suspensions of capped nanoparticles showed a negligible release of the entrapped paclitaxel in the dark. However, when suspensions were irradiated with visible light, a remarkable paclitaxel and Ru(terpy)(dppz)(H2O)2+ release was observed due to the light-induced substitution of a monodentate nitrile ligand for a water molecule. Similar materials, but loaded with other cargos (docetaxel, cyanine-5, fluorescein, calcein) instead of paclitaxel, were prepared, and their controlled release performance was studied. Cytotoxicity studies carried out with paclitaxel-loaded MSNs and MDA-MB-231 and MDA-MB-468 breast cancer cells indicated that nanoparticles were not toxic in the dark. Nevertheless, irradiation with visible light enhanced the toxicity of the material against both cancer cells due to paclitaxel release. An Annexin V assay indicated that the light activation of capped nanoparticles increased apoptosis. Wang et al. reported an original delivery system based on the use of DNA and the photoinduced formation of hydroxide ions.132 In this system, the malachite green carbinol base was immobilized into the nanochannels of MSNs as a light-induced source of hydroxide ions, whereas an i-motif quadruplex DNA was anchored to the external surface and acted as a cap. Ru(bipy)32+ molecules were also loaded into mesopores as model guests. Quadruplex DNA was attached to MSNs using azide functionalized MSNs and a click chemistry reaction,

Figure 46. Na5Lu9F32:Yb/Er hollow mesoporous spheres loaded with DOX and with an Au nanocrystals-PEG coating. Irradiation at 980 nm induced DOX release.

interior and pores were loaded with DOX. The controlled release behavior of nanoparticles was studied at different pHs and upon NIR-light irradiation. Aqueous suspensions of nanoparticles at pH 7 showed moderate DOX release (ca. 48% after 10 h), which increased to ca. 82% when pH was lowered to 4. Upon NIR-light irradiation (980 nm), a more marked DOX release was observed (96% and 59% at pH 4 and 7, respectively). The increase in cargo release upon NIR-light irradiation was ascribed to a photothermal effect induced by gold nanocrystals. The biocompatibility of the nanomaterial was tested in the L929 cell line by standard MTT cell viability assays. No significant cytotoxicity was detected in L929 cell cultures incubated with up to 250 μg/ mL of the system for 24 h. Accordingly, no hemolysis was detected at the same concentrations. Therefore, the bio- and blood compatibility of the system was excellent. Next the cytotoxicity of the DOX-loaded nanomaterial was studied in HeLa cells. When cells treated with the capped solid were irradiated with NIR light, the drug release in combination with the thermal effect resulted in a higher antitumor effect than that found for free DOX. 2.7. Miscellaneous

Knežević and co-workers reported a controlled release system that was responsive to visible light using MSNs grafted with mercaptopropyl moieties and loaded with sulforhodamine 101.129 As caps, the authors used the Ru(bipy)2(PPh3) (GABA)+ complex, which coordinated with the mercaptopropyl groups as shown in Figure 47. Kinetic delivery studies of sulforhodamine 101 and the ruthenium complex by UV/vis and fluorescence spectrometry demonstrated that no significant release took place until irradiation with visible light at 455 nm. Light irradiation lowered the activation barrier for the substitution of the sulfur moieties coordinated to ruthenium by hydroxyl ions. The authors also attempted to uncap pores by adding potassium cyanide, imidazole, or histidine, but they observed worse performance than that found upon visible light irradiation. 589


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Figure 48. MSNs loaded with different molecules and capped with a Ru(II) complex. Delivery was observed upon irradiation with visible light. Figure 49. MSNs loaded with Ru(bipy)32+, with the malachite green carbinol base embedded on the inner pore surface and capped with a DNA quadruplex. The Ru(bipy)32+ complex was released upon irradiation at 365 nm.

whereas malachite green carbinol molecules were immobilized on the pore wall via a strong interaction between malachite green carbinol molecules and silane groups. Photoirradiation with UV light (λ = 365 nm) induced malachite green carbinol molecules to dissociate into malachite green cations and OH− ions by increasing the pH value. Consequently, the i-motif quadruplex structures unfolded into single-stranded form due to the deprotonation of the C:C+ base pairs. This uncapped pores and brought about the subsequent entrapped dye release as shown in Figure 49. Cargo release was negligible in the dark, whereas a payload delivery of 83% was observed after 2 h upon 365 nm UV-light irradiation. When light was turned off, the malachite green cations recombined with the OH− ions and returned to the malachite green carbinol form. The pH value hence lowered, and the single-stranded DNA shifted back to the i-motif form capping pores again. Stoddart et al. used a very similar gated material to that described in ref 173, which was slightly modified to be opened by light irradiation. In particular, these authors included anthracenecarboxylic acid or Ru(bipy)2(bipy(CH2OH)2)2+, which were covalently anchored to the silica surface via a carbamate bond.133 When the material was irradiated with a 364 (for the anthracenecarboxylic acid-containing solid) or 488 nm laser (for the ruthenium complex-containing solid), a photoexcited electron transfer cycle reduced the 1,5-dioxynaphthalene (DNP) unit by dethreating the cyclobys(paraquat-p-phenylene) and allowing the delivery of entrapped dye (Ir(ppy)3 or Rh B) as depicted in Figure 50. To regenerate the cycle, diethanolamine was used as a sacrificial agent. Guardado-Alvarez et al.134 presented the first example of a photoacid-activated nanovalve on MSNs. In particular, 6,8dihydroxy-1,3-pyrenedisulfonic acid disodium salt (DHDS) was reacted with 3-isocyanatopropyltriethoxysilane and was then condensed onto the surface of MSNs. PI was also loaded in mesopores, and α-CDs formed inclusion complexes with two different aniline-based stalks (see Figure 51), which were anchored to the surface and blocked pores. Upon photo-

excitation of DHDS at 408 nm, α-CDs were detached due to the protonation of the stalk, which resulted in cargo release. In contrast, the proton returned to the DHDS, the aniline derivative was not protonated, and the system was closed again when irradiation was turned off. Zhang et al. combined reduced graphene oxide nanosheets coated with mesoporous silica with a thermoresponsive polymer for NIR-triggered DOX release.135 The silica surface was functionalized with aminopropyl moieties, which were further reacted with 2-bromoisobutyryl bromide. Then N-isopropylacrylamide and acrylamide were polymerized onto the external surface by ATRP (see Figure 52). The hybrid material was loaded with DOX. PBS at pH 7.4 suspensions of the capped nanosheets at 37 °C (LCST at 41 °C) showed negligible DOX release, whereas sustainable cargo delivery was observed when the temperature was raised to 50 °C. The gated nanosheet pores were also opened by irradiation with a laser at 808 nm. NIR irradiation induced an increase in the local temperature which, in turn, induced the phase transition of the polymer. Confocal microscopy and MTT studies in HeLa cells indicated that the nanocarrier demonstrated low toxicity, good dispersibility, and stability under cell culture conditions. Loaded DOX release was successfully controlled by NIR irradiation. Finally, the chemophotothermal synergistic treatments with the DOX-loaded nanomaterial exhibited site-targeted and highly efficient killing ability for HeLa cells.

3. TEMPERATURE Several examples of gated materials, which were uncapped using temperature, have been described. Most of these capped materials used thermosensitive polymers that were able to deliver cargo after a temperature-dependent phase transition. Some other interesting examples, but less explored, involve 590


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Figure 52. Reduced graphene oxide nanosheets coated with a mesoporous silica monolayer. Pores were loaded with DOX and capped with the PNIPAAm thermosensitive polymer. DOX was released upon heating up to 50 °C or upon irradiation at 808 nm.

melting double-stranded DNA sequences or also melting other organic coatings, such as paraffins. Temperature, as exogenous stimulus, has the advantage of a fine external control for tunable dose delivery. Furthermore, changes in temperature or the existence of different temperatures are a fingerprint in some diseases such as inflammation, infection, or even tumoral tissues, which can be used to specifically deliver selected cargos. However, the registered increase in temperature in such cases is no more than 4 or 5 °C, which needs highly sensitive systems working on a very narrow range of temperatures. The inability to respond in the window between 35 and 45 °C gives rise to systems not applicable in the biomedical field, while temperatures beyond 45 °C must be carefully used to avoid undesired cellular death. 3.1. Poly(N-isopropylacrylamide) and Derivatives Figure 50. MSNs loaded with different cargoes and capped with a pseudorotaxane. Cargo was released upon irradiation with laser light.

Thermo sensitive polymers have been widely used to prepare capped materials. These polymers normally show a phase transition from a swollen hydrated state to a shrunken dehydrated state in which a large part of the polymer volume is reduced. A typical thermosensitive polymer is PNIPAAm, and several examples of gated materials triggered by temperature derive from the use of this material. PNIPAAm and derivatives usually show a LCST at around human body temperatures. Given this characteristic, PNIPAAm-based polymers have been widely used in drug delivery applications. Moreover, LCST and other properties of these polymers, for instance to make them pH-sensitive, can be modulated by copolymerization, terpolymerization, or cross-linking reactions. In most reported examples that involve MSNs and PNIPAAm, the capped material was able to deliver cargo at temperatures higher than LCST when the polymer was in its shrunken dehydrated state. At a temperature lower than LCST, when the polymer was in its swollen hydrated state, cargo delivery was inhibited (see Figure 53). However, certain PNIPAAmcontaining silica mesoporous containers displayed the opposite behavior. In particular, delivery was observed at low temperature, whereas the material remained to be capped at a high temperature (vide infra) (see Figure 54). The reason why similar systems displayed the opposite behavior is not completely clear but suggests that more studies need to be done. Among possible

Figure 51. MSNs loaded with PI, functionalized with photoacid DHDS, and capped with an inclusion complex between α-CD and an aniline derived stalk. PI was released upon irradiation at 408 nm.

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reasons, the preferential formation of the PNIPAAm polymer or derivatives inside or outside the mesoporous surface, and the affinity that the payload has with the polymer brushes, may have some importance. The following examples show hybrid materials capped with polymers or copolymers based on N-isopropylacrylamide that were able to release the entrapped cargo at temperatures higher than LCST. In a pioneering paper, Lopez and co-workers described PNIPAAm as temperature-responsive cap (see Figure 53a).136 These authors used MSNs, and the PNIPAAm polymer was grown on the inner pores of the mesoporous support by ATRP of N-isopropylacrylamide. At a low temperature (below the LCST of the polymer at 32 °C), the polymer was hydrated and extended closing the pores and inhibiting dye (fluorescein and Rh 6G) delivery. In contrast at high temperatures (50 °C, above the LCST), the polymer was in a hydrophobic collapsed form, which allowed dye delivery. The same research group used a random copolymer of Nisopropylacrylamide and (3-methacryloxy)propyl trimethoxysilane, formed by free radical polymerization, to prepare another temperature-driven gated material (see Figure 53b).137 In this case, MSNs were loaded with fluorescein. The polymerfunctionalized material displayed a similar behavior to that described above (i.e., at 25 °C); the polymer was in a hydrated and expanded form and inhibited fluorescein release, whereas the polymer collapsed and allowed cargo delivery at 40 °C. The temperature dependence of permeation of crystal violet through hybrid membranes that contained PNIPAAm-functionalized silica nanoparticles was also studied. Dye permeated through the hybrid membrane at temperatures above the LCST, whereas not dye permeation took place at lower temperatures. Zhang and co-workers prepared SBA-15 mesoporous silica, having magnetic Fe3O4 nanoparticles, and containing the thermosensitive PNIPAAm polymer inside pores (see Figure 53a).138 PNIPAAm was grafted inside the pores of the support using 2,2′-azoisobutyronitrile as a free radical initiator. The system was also loaded with IBU. The authors demonstrated cargo delivery by raising the temperature above the LCST of the polymer. The same authors also implemented this gating mechanism in mesostructured cellular foam materials and observed the same behavior for IBU release.139 Yang and co-workers prepared Fe3O4 nanoparticles coated with a silica mesoporous layer which was decorated with propyl methacrylate moieties (see Figure 53c).140 Then N-isopropylacrylamide and N-hydroxymethyl acrylamide were copolymerized to yield an outer shell that acted as a temperature-controlled gate. The ratio between N-isopropylacrylamide and N-hydroxymethyl acrylamide was adjusted to render different copolymer shells with distinct transition temperatures. The pores of the materials were loaded with Zn(II) phthalocyanine tetrasulfonic acid dye, a well-known derivative used in photodynamic therapy. Controlled release experiments indicated that below the polymer’s transition temperature, pores were closed (the copolymer was in its hydrated and expanded form) and negligible dye release was observed. However, when the temperature rose above the transition temperature, a remarkable dye release took place. In a similar approach, Hanagata et al. prepared PNIPAAmmodified mesoporous SBA-15 that contained magnetic γ-Fe2O3 nanoparticles (see Figure 53a).141 This material was functionalized by a postgrafting procedure with methacryloxypropyl moieties, and then nanoparticles were coated with a PNIPAAm polymer. Pores were loaded with the antibiotic gentamicin. As in the above cases, the prepared composite showed negligible

Figure 53. MSNs capped with N-isopropylacrylamide-derivative polymers or copolymers. Temperature changes induced cargo release.

Figure 54. MSNs capped with N-isopropylacrylamide-derivative polymers or copolymers. Cargo release is induced at a temperature below LCST.

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(when the polymer was in the random coil conformation), while the material showed minimal delivery at high temperature (when the polymer adopted the globule conformation). The authors attributed this gated behavior (i.e., delivery at a low temperature) to the fact that the polymer was already preformed when grafted onto the external surface of MSNs. Li and co-workers synthesized three different PNIPAAmcoated MSNs by surface-initiated ATRP and studied the polymer phase transition at variable temperature by 1H NMR. One of these materials was selected and loaded with FITC (see Figure 54a).146 The authors performed release experiments at 20 and 40 °C and found that cargo molecules were not released at 40 °C, whereas a clear delivery occurred at 20 °C. Then they studied the behavior of the capped solid in MCF-7 cells by confocal laser microscopy and MTT assays. The authors found that gated MSNs were not toxic yet internalized into cells and colocalized with endosomes. Xiong and co-workers used magnetic Fe3O4 nanoparticles coated with a mesoporous silica shell as inorganic scaffold, and they functionalized the silica surface with 3-(trimethoxysilyl)propyl methacrylate (see Figure 54a).147 By seeded precipitation polymerization, poly(N-isopropylacrylamide) was grafted onto nanoparticles. Pores were loaded with sophoridine. The aqueous suspensions of the nanoparticles at 42 °C showed a moderate drug release, which reached ca. 26% of the amount loaded after 50 h. However, at 25 °C, the released drug increased to 64.4% after the same time period. Li and co-workers grafted (3-methacryloxy)propyl trimethoxysilane onto MSNs. Then N-isopropylacrylamide (a thermoresponsive monomer) and 4-nitrophenyl methacrylate (an electrosensitive monomer) were copolymerized on the external surface (see Figure 54b).148 IBU was loaded in mesopores by diffusion. Delivery from the capped material was tested at different temperatures (over and below LCST) and by applying, or not, an electric field in SBF. Shrinkage of the thermoresponsive units at a temperature over LCST (42 °C) favored the formation of π−π interactions of 4-nitrophenyl moieties and achieved less drug leakage than when the temperature was below LCST (25 °C). These authors also found that at a certain temperature cargo delivery was also achieved when an alternating electric field was applied. In the presence of an electric field, the rotation and reorientation of the 4-nitrophenyl moieties disrupted the π−π interactions, and a clear delivery was observed. Upon the application of both stimuli, the best performance in terms of cargo release was achieved at 42 °C and by applying an electric field of 0.5 Hz. Song and co-workers prepared hollow MSNs with AuNPs (5 nm diameter) on the inner wall of the siliceous support. After preparing the inorganic support, the external surface was functionalized with (3-methacryloxy)propyl trimethoxysilane. Finally, N-isopropylacrylamide was polymerized on the surface.149 The thermosensitive gated material was used as a catalyst to study the Au-induced reduction of 4-nitrophenol (Figure 55). Reduction of 4-nitrophenol to 4-aminophenol was achieved when aqueous suspensions of the nanoparticles were set at 30 °C (ca. 95% of conversion after 5 min), whereas the reduction reaction was inhibited (ca. 3% of conversion after 5 min) when the temperature was fixed at 50 °C. Shi and co-workers developed a temperature-driven DOX delivery system by a more complex approach. In their work, they prepared hybrid silica/polymer hollow nanoparticles with thermosensitive PNIPAAm chains located on the internal surface of the hollow nanoparticle as depicted in Figure 56.150

release features below the LCST of the polymer, whereas gentamicin was free to diffuse from inside pores to the solution at temperatures above the LCST. Several gated materials were prepared by Zhou and co-workers by the copolymerization of N-isopropylacrylamide and methacrylate derivatives (see Figure 53d).142 These authors functionalized MSNs with aminopropyl moieties and then the amino groups were reacted with 2-bromoisobutyryl bromide. Afterward, the copolymerization of 2-hydroxyethyl methacrylate and N-isopropylacrylamide by ATRP yielded the final hybrid nanoparticles. In this case, the authors loaded mesopores with aspirin as a model drug. The grafted copolymer had an LCST of 36.9 °C. The authors found that a controlled release of the entrapped aspirin was observed only when aqueous suspensions of capped material were heated above the LCST. Zink et al. developed a system based on mesoporous thin films with a polymer that acted as a thermosensitive gatekeeper (see Figure 53e).143 This is one of the few examples that used mesoporous films to prepare gated supports instead of typical MSNs. The films, synthesized by evaporation-induced selfassembly techniques, were loaded with the fluorescent dye PI, and the external surface was functionalized with APTES. Polymer poly(N-isopropylacrylamide-co-acrylamide) was grafted to the film surface using EDC as a linker. Below the LCST, the swollen polymer blocked pore outlets, while cargo release was observed when the temperature rose. Song and co-workers developed a drug delivery system which used a copolymer composed of N-isopropylacrylamide and methacrylic acid, used as cap (see Figure 53f).144 These two units conferred thermo- and pH-sensitive properties to the polymer, respectively. MSNs were selected as inorganic scaffolds, and the outer surface was functionalized with methacryloxypropyl subunits. Then N-isopropylacrylamide and methacrylic acid were copolymerized on the external surface by seeded precipitation polymerization. Finally, fluorescein was loaded in mesopores. A second capped solid was obtained by attaching FA to the free carboxylic acid moieties of the polymeric chains via amide formation to improve cellular uptake. Delivery studies demonstrated that cargo delivery was achieved when the temperature was raised over LCST and in an acidic environment. The cellular uptake of both capped solids (with or without FA) was confirmed by CLSM in Hep2 cells and also by flow cytometry. When compared to A549 cells, a targeting effect to Hep2 cells was observed when the FA-functionalized capped MSNs were employed. Finally, the authors prepared two similar capped materials but loaded with the anticancer drug cisplatin. Cell viability studies based on MTT assays were performed against Hep2 cells with free cisplatin and cisplatin-loaded solids. Only a slight effect of the cisplatin was noted when using the solid without FA moieties, but evident cell death enhancement was achieved when the FA-functionalized cisplatin-loaded nanoparticles were used. Finally, the effects on tumor growth on Hep2-tumor bearing nude mice resulted in the same tendency as that observed in cell viability studies. In contrast with the above examples, the following capped materials were also based on N-isopropylacrylamide polymers or copolymers, but in this case, cargo release was observed at temperatures below LCST. Brock and co-workers prepared thiol-functionalized MSNs, which were reacted with pyridyl disulfide-terminated PNIPAAm (see Figure 54a).145 The final PNIPAAm-functionalized MSNs were loaded with fluorescein. In this case, the authors found that the material was able to deliver the cargo at room temperature 593


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Figure 55. Hollow MSNs containing AuNPs and capped with PNIPAAm. The system was used for the temperature-controlled catalytic reduction of 4-nitrophenol to 4-aminophenol.

Figure 57. MSNs loaded with DOX and coated with a thermosensitive copolymer prepared by the copolymerization of N-isopropylacrylamide and poly(ethylene glycol)diacrylate.

58. This formed a copolymer inside the voids between the external mesoporous silica shell and the internal Gd2O3:Eu3+

Figure 56. Hollow MSNs loaded with DOX and containing PNIPAAm chains anchored to the internal surface of the material.

The system was loaded with DOX. When temperature was fixed at 25 °C (below the PNIPAAm LCST), the polymer was in its swollen form and did not block the pores. Thus, DOX was freely released to the solution. In contrast, when temperature was fixed at 37 °C (above the PNIPAAm LCST), the thermosensitive polymer turned insoluble in PBS, the channels were blocked by the collapsed PNIPAAm chains, and a slower DOX release was recorded. Bhatia et al. loaded the pores of MSNs with DOX. Then N-(3aminopropyl) methacrylamide hydrochloride was electrostatically adsorbed on the outer surface.151 The final nanoparticles were coated with a thermosensitive polymer shell obtained by radical polymerization of N-isopropylacrylamide and poly(ethylene glycol)diacrylate (see Figure 57). Below 37 °C, the release of entrapped DOX from the nanoparticles was negligible, whereas a remarkable drug release was observed above 37 °C as a result of the phase transition that the thermosensitive polymer shell underwent. Lin and co-workers prepared hollow MSNs and incorporated into the internal cavity Gd2O3:Eu3+ luminescent nanoparticles.152 After preparing the inorganic support, the hollow voids were filled with the monomers N-isopropylacrylamide and acrylic acid amide and were then subjected to polymerization by a photoinduced polymerization mechanism as depicted in Figure

Figure 58. Hollow MSNs containing Gd2O3:Eu3+ nanoparticles, a copolymer of N-isopropylacrylamide and acrylic acid amide and indomethacin.

luminescent nanoparticles. Indomethacin, a nonsteroidal antiinflammatory drug commonly used to reduce fever, pain, stiffness, and swelling, was selected to study drug release from the capped system. The release behavior of indomethacin was clearly temperature-dependent as drug release was triggered at 45 °C and stopped at 20 °C. The authors indicated that the drug release rate could be regulated by the change in temperature due to the composite nanocarrier’s unique architecture. This release was attributed to the polymer shrinking that opened the porous network with the subsequent drug release. Cell viability assays in mouse L929 fibroblasts showed that the nanomaterial presented good biocompatibility. Intracellular uptake of the fluoresceinloaded system in the ovarian carcinoma SKOV3 cell line was confirmed by flow cytometry and CLSM. The system had the 594


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addition−fragmentation chain transfer (RAFT) polymerization. The tertiary amines of the polymer brushes were functionalized with propanesulfonate moieties (see Figure 60). Nanoparticles

potential to serve as a T1-NMR contrast agent given the existence of Gd3+ ions. Cui, Wang, and co-workers prepared MSNs functionalized with APTES and loaded with DOX. Mesopores were capped with a copolymer−lipid layer. To synthesize the capping agent, the copolymer poly(N-isopropylacrylamide-methacrylic acidoctadecyl acrylate) was used to form a bilayer with the natural phospholipid soy phosphatidylcholine by the membrane evaporation method. The entrapped drug was released by increasing temperature or at an acidic pH (Figure 59). Moreover,

Figure 60. MSNs loaded with Rh B and capped with poly(2dimethylamino)ethyl methacrylate functionalized with propanesulfonate moieties.

were loaded with Rh B. Release of only ca. 23% of the loaded cargo was observed at 30 °C when the polymer was in its extended conformation. Yet when the capped manoparticles were heated at 55 °C, ca. 80% of the loaded dye was released. Moreover MTT viability assays indicated that prepared nanoparticles were essentially nontoxic for HeLa cells. Khashab and co-workers developed a thermal-activated molecular gate system using carbon nanotubes as support.155 In particular, carboxylic acid functionalized carbon nanotubes were modified with polyethylenimine (PEI) by an amide bond using oxalyl chloride in an intermediate step as presented in Figure 61. Moreover poly(vinyl alcohol) was used to form complexes with PEI via hydrogen bonding in an interaction known as “zipper effect”. This interaction is destroyed by heating. The system was loaded with DOX. In vitro delivery studies showed that the system did not allow cargo release at 25 °C, but the amount of delivered DOX increased with temperature. At 37 °C, delivery was slight; at 40 °C, delivery was significant, whereas at 70 °C, DOX delivery was remarkable. The system evidenced reversibility in on−off experiments by alternating temperatures of 25 and 40 °C. This was due to the reversible formation of hydrogen bonds between polymers. However, when the system turned back to 25 °C, the system did not recover the complete zero release. This was attributed to a possible mismatch in the formation of hydrogen bonds between the two polymers. Cell viability studies were performed in three different cell lines (i.e., lung fibroblast (LF), breast adenocarcinoma, (BA) and HeLa). The authors recorded significant reduced cell viability when cells were incubated at 40 °C, whereas the nanoparticles were almost nontoxic at 35 °C.

Figure 59. MSNs loaded with DOX and capped with a bilayer formed by soy phosphatidylcholine and poly(N-isopropylacrylamide-methacrylic acid-octadecyl acrylate).

a synergistic effect was observed when temperature and pH acidification (25 to 37 °C and 7.4 to 5.5, respectively) increased and were applied simultaneously.153 To investigate the pHresponsive release behavior, MCF-7 cells were used as a model cancer cell line. Since most tumor sites usually exhibit a more acidic environment or a higher temperature, the pH- and thermoresponsive releasing ability of this drug carrier is particularly useful and important for targeted release in the tumor region. The authors incubated the capped MSNs with MCF-7 cell cultures under different extracellular pH conditions. Confocal microscopy studies indicated that the nanocarriers were distributed mainly in the cytoplasmic region. Remarkably, DOX fluorescence intensity in cells under pH 5.5 was stronger than that found under pH 7.4 for the same incubation time. No obvious cytotoxicity to cells was detected under these experimental conditions when unloaded MSNs were employed, which proves that the nanomaterials offered good biocompatibility. 3.2. Other Polymers

Hong et al. prepared MSNs functionalized with hydroxyl moieties via the grafting of (3-glycidyloxypropyl)trimethoxysilane (GTPMS) and subsequent hydrolyzing the epoxide groups.154 Then polymer poly(2-dimethylamino)ethyl methacrylate was grafted onto the external surface by reversible

3.3. Based on DNA

The use of DNA fragments is also a procedure to prepare temperature-controlled capped materials.156 In an initial example, Bein et al. used double-stranded DNA to cap the 595


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Figure 61. Carbon nanotubes loaded with DOX and decorated with PEI and poly(vinyl alcohol) through the formation of hydrogen bonds. Cargo was delivered upon heating at 70 °C.

pores and delivery was observed when the melting of the grafted double-stranded DNA was reached. These authors used MSNs functionalized with azidopropyl moieties, which were then reacted with double-stranded DNA oligomers (containing biotin and alkyne functionalities) via a click chemistry reaction. Pores were loaded with fluorescein, and the nanoparticles were capped upon avidin protein addition (see Figure 62). The authors demonstrated that at 25 °C, the pores of the hybrid nanoparticles were tightly closed and fluorescein release was observed when the temperature went above the melting temperature for grafted double-stranded DNA. It was possible to fine-tune the temperature at which dye release occurred by selecting grafted double-stranded DNA. Ren et al. prepared MSNs functionalized with 3-cloropropyl moieties. Chloride atoms were substituted for azide groups upon the reaction with sodium azide. The pores of the nanoparticles were loaded with Rh B and capped with the self-complementary duplex DNA sequence hexynyl-GCA TGA ATT CAT GC using a CuI-catalyzed azide−alkyne reaction (Figure 63).157 The buffered suspensions, pH 7.4, of the prepared nanoparticles showed negligible dye release at 25 °C, whereas remarkable Rh B delivery was observed upon heating at 50 °C. The thermal denaturation of the duplex DNA sequence was responsible for the Rh B release observed. The authors also demonstrated that cargo delivery took place in the presence of DNase I enzyme, which was able to hydrolyze the duplex DNA sequence. An analogous capped material was prepared in this case containing CPT inside the pores instead of Rh B. This material was easily internalized by Hep G2 cells and presented remarkable cytotoxic efficacy due to the hydrolysis of the self-complementary duplex DNA by endonucleases, which resulted in entrapped cargo release. Single-stranded DNA molecules have also been used as temperature-responsive caps.158 Tang et al. functionalized MSNs

Figure 62. MSNs loaded with fluorescein and capped with a doublestranded DNA functionalized with a biotin−avidin complex. Raising the temperature above the melting point of the DNA-induced dye release.

Figure 63. MSNs loaded with Rh B and capped with a ds-DNA.

with aminopropyl moieties. Nanoparticles were loaded with Rh B dye, and some amino groups were reacted with a 15-base singlestranded DNA sequence modified with a carboxylic acid group. At room temperature, and in neutral aqueous suspensions, the pores of the nanoparticles were closed because of the adsorption of the single-stranded DNA sequence on the external silica 596


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surface due to the electrostatic interactions between the positively charged unreacted amino groups and the negatively charged bases (see Figure 64). As a result, a negligible dye release

Figure 64. MSNs loaded with Rh B and capped with a single strand DNA through electrostatic interactions with protonated amines.

Figure 65. MSNs loaded with safranin O and capped with a peptide that adopted a bulky α-helical conformation at room temperature.

was observed. However, when the temperature was set at 39 °C, more than 70% of the entrapped Rh B was released after 90 min. Gate opening was attributed to the rupture of the electrostatic interactions upon heating, which induced the DNA sequence to detach from the surface of the nanoparticles. Confocal fluorescence studies, which were carried out in MCF-7 cells incubated with the DNA-capped nanoparticles, showed that no dye release occurred when temperature was set at 37 °C. As a clear contrast, a marked emission enhancement took place upon heating at 39 °C due to the release of the entrapped Rh B. MTT viability assays demonstrated that the nanocarrier exhibited no cytotoxicity. The authors also observed that it was possible to finely tune the temperature at which the gate opened by changing the length of the anchored single-stranded DNA sequence. For instance, the gate was opened at 46 °C when a 40-base DNA sequence was used to cap the pores. 3.4. Miscellaneous

́ Martinez-Má ñez et al.159 recently reported a thermoresponsive material that was capped with a peptide. The concept was based on the use of the well-known temperature-controlled α-helix-todisordered transformation, which occurs in certain peptides. The authors used a self-aggregating 17-mer peptide (H-SAAEAYAKRIAEALAKG-OH, P), designed to adopt a high level of α-helical conformation, to cap the pores of MSNs. MSNs were loaded with safranin O and the external surface was functionalized with 3(azidopropyl)triethoxisylane moieties. Finally, the corresponding 4-pentynoic-P derivative was grafted onto the surface through a “click” chemistry reaction as presented in Figure 65. Delivery studies in phosphate buffer, pH 7, and at different temperatures were carried out. At room temperature, organization of the peptide in α-helical bundles inhibited cargo release, while the transformation to a disordered conformation at a high temperature reduced the steric crowding around the pore outlets that allowed cargo release. Kros and co-workers functionalized MSNs with APTES, which was reacted with N-succinimidyl 3-(2-pyridyldithio)propionate by an amidation reaction. Then peptide C(EIAALEK)3 was anchored to the silica surface by a disulfide bond exchange reaction as shown in Figure 66. Three solids were prepared based

Figure 66. MSNs loaded with fluorescein and capped with a heterodimeric peptide pair. Raising the temperature induced dye release.

on these materials: (i) one was loaded only with fluorescein; (ii) a second one was loaded with fluorescein and capped with peptide (KIAALKE)3 via supramolecular interactions with the anchored peptide; and (iii) a third material loaded with fluorescein and capped peptide (KIAALKE)3 was also functionalized with PEG chains.160 Leakage of fluorescein in aqueous buffer solutions over 4 h of the three solids was studied; as expected, the uncapped one presented the strongest release. However, the uncontrolled release of the other two solids was also considerable, and 23% and 15% of the cargo was released from solid two and solid three, respectively. To reduce the uncontrolled release, a second generation of similar solids was prepared using a shorter linker to anchor peptides closer to the MSNs surface. In this case, the silica surface was grafted with MPTS and was directly reacted with aldrithiol. In comparison with the first solids, these new ones 597


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achieved better zero-release performance for both capped solids, whereas significant dye leakage was still observed for the uncapped one. Capped solids released their cargo when the temperature rose to 80 °C because of the disassembling of the supramolecular motif between peptides. ́ Recently, paraffins were used by Martinez-Má ñez and coworkers to develop temperature-sensitive capped MSNs.161 The pores of the inorganic support were loaded with safranine O. Then the nanoparticles were functionalized with octadecyltrimethoxysilane to render the external surface sufficiently hydrophobic to cap the system with selected paraffins as presented in Figure 67. Different solids capped with heneicosane,

Figure 68. Maghemite nanoparticles coated with a shell of mesoporous silica and loaded with DOX. The external surface was functionalized with PEG terminated with carboxylic acid moieties, and the pores were capped with hexadecanol.

The authors found that the phase change of 1-tetradecanol was reversible and the gated material was used for pulsatile DOX delivery by simply changing temperature from 40 °C (open state) to 37 °C (closed state). Cell viability studies in mouse L929 fibroblasts and human melanoma MEL-5 cells showed that the fluorescein-loaded nanomaterial exhibited excellent biocompatibility up to a dose of 100 μg/mL. Confocal and electron microscopy studies, as well as flow cytometry studies of fluorescein-loaded nanoparticles, indicated the system showed good cellular uptake in MEL-5 cells. In vitro release of DOX in MEL-5 cell cultures revealed that DOX cytotoxicity could be strongly quenched at 37 °C; however, cell apoptosis was observed when the culture temperature rose to 40 °C as gates opened and the subsequent heat-triggered DOX release occurred.

Figure 67. MSNs loaded with safranin O and capped by the formation of a hydrophobic monolayer of heneicosane.

docosane, and tetracosane were prepared. These paraffins have melting points of 39, 42, and 49 °C, respectively. Water suspensions of the paraffin-coated MSNs at temperatures below the melting points of paraffins showed an almost “zero release” of safranine O. However, when temperature was raised above the paraffin melting point, a massive safranine O release was observed. Heneicosane-capped nanoparticles were able to release safranine O in HeLa cells when incubation was carried out at temperatures above 39 °C. Heneicosane-capped nanoparticles were also loaded with DOX and used to release this chemotherapeutic agent in HeLa cells after heating. Core−shell nanoparticles made of a maghemite core and a mesoporous silica shell were functionalized with aminopropyl moieties, and the amino groups were reacted with a PEG derivative that contained a carboxylic acid functionality. Pores were loaded with fluorescein or DOX and capped by adding 1tetradecanol as shown in Figure 68.162 Suspensions of the gated nanoparticles in PBS at 37 °C showed an almost “zero release”; less than 4% of the entrapped DOX was delivered after 96 h. At this temperature, 1-tetradecanol was in a solid state and effectively blocked the pores. However, upon heating to 40 °C, moderate DOX release was observed; 37% of the entire payload was delivered after 96 h. This release was attributed to the fact that 1-tetradecanol was in the liquid phase, which allowed the diffusion of cargo from inside pores to the bulk aqueous solution.

4. ALTERNATING MAGNETIC FIELD AND ULTRASOUND Ultrasound and magnetic fields are also physical stimuli used for opening up molecular gates with the subsequent controlled delivery of an entrapped cargo. Both techniques allow a pulsatile delivery of the cargo, are noninvasive, can penetrate depth in tissues, and can be carefully controlled by changing frequency, power, cycles, and time of application. Magnetically triggered systems have the advantage of having an extra control and possibility of guidance of the gated support, for example, to accumulate in a selected tumor area (for biomedical purposes, where also tracking by MRI is possible) or even in industrial applications where separation and recovery of the gated particles could be of crucial interest. Regarding ultrasound, this technique offers the application of a stimulus without concern of residual radiation; however, their possible genotoxic effect even at moderate doses is today under study. Several examples of capped materials that were opened upon application of these stimuli have been described. 598


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4.1. Alternating Magnetic Field

Vallet and co-workers developed a magnetically triggered system capable of releasing small molecules from the pore voids of mesoporous silica and the proteins housed in a capping polyNIPAM-block-PEI thermoresponsive copolymer at the same time.163 For this purpose, the authors incorporated superparamagnetic iron oxide nanocrystals into a mesoporous silica matrix. Nanoparticles were functionalized with (trimethoxysilyl)propyl methacrylate, and poly-NIPAM-blockPEI polymer was grafted around the nanoparticles by radical polymerization. The pores of the silica support were loaded with fluorescein, and soybean trypsin inhibitor type II−S (STI) was incorporated into the copolymer shell (see Figure 69). The Figure 70. Core−shell nanoparticles with a core of Fe3O4 and a shell of mesoporous silica loaded with DOX and capped with a diblockthermosensitive copolymer.

demonstrated the cellular uptake of the material and DOX delivery in lysosomes. Cell viability experiments resulted in enhanced cell death when cells were treated with nanoparticles and an AMF. Cheon and Zink were among the first to explore the possibility of trigger-gated materials with AMF.165 In their work, they prepared MSNs with a zinc-doped iron oxide core and attached N-(6-N-aminohexyl)aminomethyltriethoxysilane to pore outlets. Rh B or DOX was used as the cargo. Finally, pores were capped by adding CB[6], which interacted with the aminated stalks to seal pores (see Figure 71). The new gated material was able to retain its cargo until an AMF was applied, with a frequency of 500 kHz and a current amplitude of 37.4 kAm−1. Finally, these authors assessed that gated nanoparticles were endocytosed by the MDA-MB-231 breast cancer cell line. It was found that around 5% of the cells treated with DOX-loaded solid were killed without the magnetic field being applied. In the presence of an Figure 69. Superparamagnetic iron oxide nanocrystals coated with a mesoporous silica shell capped with the poly-NIPAM-block-PEI thermoresponsive copolymer.

PNIPAM polymer exhibited a phase transition at a LCST of approximately 32 °C in water. At temperatures around 30 °C, the copolymer was hydrated and blocked the release of both fluorescein and soybean trypsin inhibitor type II−S. At temperatures above LCST (around 40 °C), fluorescein and the protein were effectively released. Moreover, when the gated solid was exposed to an alternating magnetic field (AMF) of 24 kA m−1 and 100 kHz for 6 h, both the protein and fluorescein were delivered. Qu, Lin, and co-workers prepared core−shell nanoparticles using a magnetic core of Fe3O4 and a shell of mesoporous silica synthesized by the Stöber method. The outer surface was grafted with APTES and reacted with succinic anhydride to obtain carboxylic acid moieties. Afterward diblock-thermosensitive copolymer poly[(ethylene glycol)-co-(L-lactide)] was anchored via the formation of amide bonds as shown in Figure 70.164 DOX was loaded as a model drug. An equivalent second solid, with FITC attached as a marker, was also synthesized. Delivery studies demonstrated that the system was activated by heating or acidification, and that the two stimuli were cooperative. As expected, the application of an AMF also resulted in massive cargo delivery. CLSM experiments done with HeLa cells

Figure 71. MSNs with a zinc-doped iron oxide core loaded with Rh B or DOX and capped with CB[6]. 599


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AMF, the local heating caused by the magnetic cores in unloaded nanoparticles caused only 16% of cells to die. However, 37% of cells were killed when cells were treated with the DOX-loaded gated solid and AMF. ́ Bringas, Mahmoudi, Martinez-Má ñez, Stroeve, and co-workers developed a magnetically triggered system using MSNs with superparamagnetic cores of iron oxide capped with lipid bilayers.166 In their work, they loaded the mesoporous support with methylene blue (MB) and blocked pores with a lipid bilayer of DOPC as depicted in Figure 72. When an aqueous suspension

Figure 73. Fe3O4 clusters coated with a silica shell loaded with GEM and capped with hydroxypropyl cellulose.

entrapped drug release. The authors also confirmed the “on−off” behavior of the gated solid by repeated temperature-driven release cycles run at 37 and 43 °C. In vitro cell viability studies, performed in pancreatic cancer PANC-1 cells, indicated that cell growth was significantly inhibited upon the simultaneous application of an AMF and GEM delivery compared with GEM or magnetic hyperthermia alone. The delivery of injected GEM-loaded nanomaterial in PANC-1 xenografts in nude mice was viewed by both MRI and fluorescent imaging techniques. Combinations of AMF with intratumoral injections of the gated system induced a significant increase in apoptosis compared to the tumors treated with nanocarrier injections alone. Vallet-Regi ́ prepared Fe2O3 nanoparticles coated with mesoporous silica, which was also functionalized with amino groups. Nanoparticles were loaded with fluorescein, and the system was capped by a reaction of oligonucleotide sequence 5′thiol C6-TTATCGCTGATTCAA with the amino groups using sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate as a cross-linker as shown in Figure 74. The authors also functionalized Fe2O3 nanoparticles with sequence 5′-thiol C6TTGAATCAGCGATAA using a similar reaction to that described above. They used these functionalized Fe2O3 nanoparticles to cap pores via the hybridization between oligonucleotides from the silica surface and the oligonucleotides on magnetic nanoparticles.168 This hybridization had a melting point of 47 °C. The authors recorded the fluorescence of the solution at increasing temperatures and confirmed the temperature-dependent DNA dehybridization and subsequent fluorescein release. In another step, cargo delivery was also achieved using an AMF of 24 kA m−1 and 100 kHz, and the authors found that the capped system was able to heat the environment to hyperthermia level. When temperature was raised to 47 °C, the amount of loaded cargo fell within the same range as the amount released with the common thermal treatment.

Figure 72. MSNs with Fe3O4 cores capped with lipid bilayers and loaded with MB.

of capped nanoparticles was exposed to an AMF (50 Hz, 1570 G), clear entrapped dye delivery occurred, while MB was effectively retained if the field was not applied. The authors ascribed the observed effect to a combination of the lipid bilayer local warming and changes in its permeability promoted by the vibration of NPs in the presence of an AMF. The toxicity of capped nanoparticles in a collection of human cell lines, which included human nerve (A172), liver (Hep G2), heart (HCM), lung (A549), kidney (293T), colon (SW480), brain (BE(2)-C), and skin (A431), was also studied. The gated solid was not toxic at concentrations below 0.5 mg mL−1, whereas a trace of toxicity was observed for the brain, nerve, lung, and heart human cell lines at higher concentrations (from 1 to 4 mg mL−1). This agrees with the usually more sensitive behavior of these cells to alien materials. Kim and co-workers prepared ultrasmall (7 nm) superparamagnetic iron oxide nanoparticles (SPIONPs) that formed ca. 59 nm clusters with the assistance of poly(acrylic acid) (PAA). Then clusters were coated with a 18 nm silica shell with polyvinyl pyrollidone (PVPON) moieties on the external surface, and pores were loaded with gemcitabine (GEM). In another step, an etching process was run with sodium hydroxide in order to obtain pores of around 5 nm. Finally, temperature-sensitive hydroxypropyl cellulose was grafted onto the surface by hydrogen-bonding interactions with PVPON groups (see Figure 73).167 In a first step, the authors studied the release of GEM at two different temperatures (37 and 45 °C). Heating magnetic gated particles and subsequent GEM release were investigated in PBS by applying an AMF at 199 kHz (175 Oe). At both temperatures, a two-phase release pattern was monitored by UV−vis spectroscopy, with an initial fast “burst” release followed by a sustained release period. The cumulative release was significantly higher at 45 °C. At this temperature, grafted hydroxypropyl cellulose (LCST 41 °C) collapsed, which allowed

4.2. Ultrasound

Honma and co-workers developed a controlled delivery system using MSNs embedded in a poly(dimethylsiloxane) thin film, which was responsive to ultrasound stimuli.169 MSNs were loaded with IBU as model drug. Loaded MSNs were mixed with a poly(dimethylsiloxane) solution and dried to achieve the solid thin film (Figure 75). Kinetic experiments were performed by comparing the delivery behavior of the poly(dimethylsiloxane) loaded with IBU, either with or without MSNs. In the absence of ultrasound, films did not present significant cargo delivery, 600


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Figure 76. Hollow MSNs loaded with pyrene and PFH and capped with PEG-AuNPs able to release the fluorophore upon ultrasound irradiation.

liquid into small bubbles, which promoted the appearance of cavitations which, in turn, also conferred the nanomaterial with contrast-intensified ultrasound imaging properties. Ultrasound (US)-guided HIFU therapy ex vivo and in vivo with the nanomaterial was found to be highly efficient on rabbit VX2 xenograft tumor ablation due to the high thermal energy accumulation and increased mechanical/thermal effects. The system induced significant cytoclasis and enhanced drug release, served as contrast-intensified US imaging agent, and enabled efficient US-guided high-intensity focused ultrasound (HIFU) tumor ablation therapy. Therefore, this nanosystem could be used as a theranostic platform for contrast-intensified US imaging, combined chemotherapy and efficient HIFU tumor ablation. Leung et al.171 developed Fe3O4@SiO2 core−shell nanoparticles capped with a series of crown ether macrocycles for drug-controlled release. The inorganic scaffold was prepared by coating Fe3O4 nanoparticles with a silica mesoporous layer by modified hydrothermal sol−gel reactions. Then the mesoporous layer was functionalized with APTES. At the same time, three novel dibenzo-crown ethers, which contained two carboxylic groups, were synthesized and subsequently coupled to the surface of nanoparticles through an amide linkage, which was assisted by DCC/NHS. After CTAB removal, pores were loaded with DOX and then they were capped with Na+ and Cs+ ions through the formation of complexes with the grafted dibenzocrown ethers on the outer surface. Pores were uncapped by lowering pH in the presence of ultrasound and by breaking up the interaction between metal cations and dibenzo-crown ether (see Figure 77). In vitro DOX controlled release was tested in PBS at pH 4 or pH 7.4. The results showed that drug release was triggered at pH 4 in combination with noninvasive ultrasound. MTT assays confirmed that capped nanoparticles were biocompatible and relatively noncytotoxic to L929 cells (a murine aneuploidy fibrosarcoma cell line). Reasonable cellular uptake of nanoparticles in L929 cells was observed. In accordance with the MRI analysis, the system also exhibited high relaxivity. Chen et al. functionalized the external surface of mesoporous silica nanocapsules with APTES, and amines were further reacted with a maleimide PEG-NHS ester. Afterward, anticancer drug

Figure 74. Fe2O3 nanoparticles coated with a silica mesoporous shell loaded with fluorescein. Pores were capped with DNA-functionalized Fe2O3 nanoparticles.

Figure 75. MSNs coated with a poly(dimethylsiloxane) thin film capable of IBU delivery upon ultrasound application.

whereas both films exhibited drug delivery when irradiated with ultrasound. In the film with embedded MSNs, the amount of released drug was remarkably larger. The authors attributed this behavior to the generation of cavitations (formation, growth, and collapse of gas microbubbles) inside the polymeric matrix. Another remarkable achievement of the system was its pulsatile delivery capability. Chen et al. coated hollow MSNs with AuNPs through the in situ reduction of Au3+ by NaBH4. Pyrene dye, used as a model drug, and ultrasound-sensitive liquid perfluorohexane (PFH) were loaded into inner cavities. Finally, thiol-functional PEG was grafted onto the surface of the attached AuNPs as depicted in Figure 76.170 Application of ordinary ultrasound irradiation triggered the release of the loaded drug through the alteration of acoustic and thermal properties of the attached AuNPs. Irradiation of the nanocarrier with ultrasound converted PFH 601


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agent with a tumor targeting function via binding and CD44 receptor-mediated endocytosis. This, in turn, facilitated the enhanced accumulation of the system and, as a result, led to specifically intensified ultrasound imaging in the hepatocellular carcinoma Hep G2 tumor-bearing nude mice tumor area. In vivo results also showed that the system accomplished enhanced HIFU ablation efficacy of tumors by the PFH gasification in the nanocarrier.

5. REDOX Since the birth of gated materials, the possibility of controlling mass transport aided by redox processes has been widely explored. This particular stimulus is appealing since endogenous reducing agents found at the intracellular level can be used as triggers. An increase in the concentration of such redox-active species has been reported for some diseases, such as cancer, and the wide bioapplicability of redox-responsive capped systems can be easily envisioned. Numerous examples have been described in this area, and they are summarized in two groups: those based on redox-driven supramolecular interactions between macrocycles, such as cyclophanes, cyclodextrins, or cucurbiturils and wirelike moieties anchored to the surface of the porous support; those based on the rupture of disulfide bonds that bind material’s surface and capping agents, such as biomolecules, inorganic nanoparticles, or polymers, to mesoporous material or that contribute to cap pores through the formation of disulfide-crosslinked layers. In this context, thiol chemistry is widely known. Thiol-derivatized molecules and biomolecules can be easily prepared and used to obtain disulfide-linked gating mechanisms. In most cases, this fact simplifies the gated system because thiolderivatized targeting ligands can adopt a double role (i.e., as redox-responsive gating ensemble and as selective targeting agent). Most of the examples reported (vide infra) display a gating response in pure water, and some can be reoxidized to obtain reversible systems. Finally, some redox-responsive gated systems are frequently decorated with targeting ligands or agents to increase their suspensibility in biological environments. Some encouraging examples tested in in vivo models have demonstrated that gated materials loaded with cytotoxics can selectively reach a specific cell type and reduce the size of tumors and significantly decrease typical side effects.

Figure 77. Fe3O4@SiO2 core−shell nanoparticles capped with K+ or Cs+ crown ether complexes. Cargo delivery was achieved by ultrasound.

CPT11 and ultrasound-sensitive PFH were coloaded into pores. Subsequently, a thiol-functionalized hyaluronic acid (HA) was synthesized and conjugated to PEG through maleimide−thiol coupling.172 When treating the material with chloramine-T, HA was cross-linked with disulfide bonds as shown in Figure 78. The potential nanocarrier application was tested in hepatocellular carcinoma Hep G2 tumor-bearing nude mice. The results indicated that the nanomaterial could serve as a nanotheranostic

5.1. Rotaxanes and Pseudorotaxanes

As far as we know, the first example that used rotaxanes coupled with a redox reaction to design gated materials was reported by Stoddart, Zink, and co-workers. These authors prepared MSNs loaded with Ir(ppy)3 and functionalized the surface with a 1,5dioxynaphthalene (DNP) derivative (see Figure 79). Pores were capped by the formation of an inclusion complex between the DNP derivative and cyclobis(paraquat-p-phenylene) (CBPQT4+). Reduction of CBPQT4+ by NaCNBH3 resulted in the rupture of the complex and the release of the entrapped dye.173 Later, the same authors designed a more complex system in which the CBPQT4+ ring was able to move between a tetrathiafulvalene (TTF) station and a DNP moiety, both separated by an oligoethylene glycol chain that incorporated a rigid terphenylene spacer.174 In this supramolecular ensemble, CBPQT4+ has a preference for the TTF unit, but the oxidation of TTF by using Fe(ClO4)3 to give a TTF2+ dication promoted the movement of the CBPQT4+ ring to the DNP station. When ascorbic acid was added, the TTF2+ unit was reduced back to the neutral TTF, which induced the return of the CBPQT4+ ring to

Figure 78. MSNs loaded with CPT11 and PFH and capped with disulfide cross-linked HA. Application of ultrasound broke the disulfide linkages with the subsequent cargo release. 602


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Figure 79. Ir(ppy)3 delivery from pseudorotoaxane-modified MSNs triggered by NaCNBH3.

the TTF group (see Figure 80A). This bistable[2]rotaxane unit was anchored to the MSNs via the formation of a carbamate between the hydroxyl group in the [2]rotaxane and isocyanate moieties on the silica support. The material was loaded with Ir(ppy)3 or Rh B. The release kinetics of the gating system was performed in a PhMe:EtOH (1:1) solution (for the iridium complex) and in MeCN for Rh B. To complete the study, the authors synthesized another hybrid material that contained the dumbbell part of the [2]rotaxane, but without the CBPQT4+ ring. This second material was unable to control Rh B release, which was delivered to the solution immediately. These results confirmed the vital role of the CBPQT4+ ring motion to control the transport of guest molecules from the pore voids to the solution. Another double redox-active site system was also proposed by Stoddart, Zink, and co-workers.175 As in the above case, the authors functionalized MSNs with a capping system, which also consisted of a CBPQT4+ part that moved between TTF and DNP stations (see Figure 80B). In this work, the authors attached the [2]rotaxane gatekeeper to the silica surface by using linkers of different lengths. Some linkers were most likely located in the interior of pores as they were attached to nanoparticles by a cocondensation method, whereas others were most likely on the outer surface as they were attached to the silica surface by postsynthetic grafting when pores were still filled with surfactant molecules. The controlled release of certain fluorescent dyes (i.e., Rh B, Ir(ppy)3, coumarin 440, coumarin 460) from pore voids upon the addition of ascorbic acid was studied for different prepared solids. The authors concluded that nanovalves were more efficient when gatekeepers were anchored deep within pores rather than when attached more closely to pore entrances. The length of the linker between the surface and the rotaxane

Figure 80. Delivery of Ir(ppy)3, Rh B or coumarins from the rotaxanemodified MSNs triggered by reducing agents.

molecules also played an important role in determining the effectiveness of nanovalves. The same authors functionalized MSN with APTES, and the amino groups were further reacted with ferrocene dicarboxylic acid to form amide bonds. Finally, the material was loaded with Rh B and capped with β-CDs by taking advantage of host−guest inclusion complex formation as shown in Figure 81.176 This material was deposited on a Pt grid that acted as an electrode. The capped material remained closed, but if a potential of 1 V was applied, the electrochemical oxidation of ferrocene resulted in complex dethreating, which caused dye delivery. Ronconi and co-workers177 prepared an O2-triggered system, which was also based on ferrocene and β-CDs. The external surface of MSNs was functionalized with aminopropyl groups. Then, the −NH2 moieties were reacted with ferrocene carboxaldehyde, and the imines obtained were reduced with sodium borohydride. In a second step, pores were loaded with Rh B and capped upon the addition of β-CDs as shown in Figure 82. Aqueous degassed (without oxygen) suspensions (pH 6.5) of capped nanoparticles showed no Rh B release. However, O2 bubbling induced a marked release of the entrapped dye due to the oxidation of ferrocene moieties to ferrocenium and the 603


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(propyldisulfanyl)ethylamine. Finally, after loading pores with vancomycin or adenosine triphosphate (ATP), MSNs were capped with mercaptoacetic acid-coated CdS nanocrystals of ca. 2 nm via an amidation reaction (see Figure 83). Gated

Figure 81. Rh B delivery from the β-CDs-capped MSNs due to the oxidation of the ferrocene groups by the application of a potential of 1 V.

Figure 83. Delivery of vancomycin or ATP from CdS-capped MSNs due to the cleavage of disulfide bonds in the presence of reducing agents.

nanoparticles released less than 1.0% of cargo in the 10 mM PBS buffer solutions (pH 7.4) over a 12 h period. On the contrary, when dithiothreitol (DTT) or mercaptoethanol (ME) was added, the disulfide bridge was cleaved and allowed the rapid release of entrapped ATP or vancomycin. Cargo delivery was proportional to the concentration of the reducing agent in the solution. The biocompatibility and delivery efficiency of MSNs with neuroglial cells (astrocytes) were tested. Astrocytes cultured in the presence of CdS-capped MSNs with the ATP molecules encapsulated inside significantly increased in intracellular Ca2+, which indicated that the ATP molecules released from the nanomaterial reached their receptors on the cell surface and triggered the corresponding ATP receptor-mediated increase in the intracellular calcium concentration. No changes in intracellular calcium were detected in the astrocytes incubated in the presence of CdS-capped MSNs without encapsulated ATP. In a subsequent work, the same authors used G2.5 and G4.5 poly(amidoamine) (PAMAM) dendrimers as caps (see Figure 84). The gated material was prepared similarly to that followed to obtain MSNs capped with CdS (see above). In particular, the authors functionalized the external surface of MSN with a 2(propyldisulfanyl)ethylamine linker and anchored dendrimers by the formation of an amide bond. The authors conducted a comparative study of the release of ATP from the MSNs capped with dendrimers or capped with CdS (vide ante) in the presence of different reducing agents (DTT and tris(2-carboxyethyl)phosphine)). ATP real-time imaging by CLSM was monitored after the chemiluminescence generated by firefly luciferase, which correlated with the amount of ATP released.179 These authors found that CdS-capped MSN exhibited rapid ATP release from pore voids after adding the reducing agent but, in contrast, dendrimer-capped systems showed a more sustained release.

Figure 82. Rh B delivery from β-CDs-capped MSNs due to the oxidation of ferrocene groups by oxygen.

subsequent detachment of β-CDs. The release of the entrapped Rh B was also modulated by changes in the pH media when nondegassed water was used. In particular, acidification induced a more pronounced release compared to that observed in neutral water. 5.2. Reduction of Disulfide Bonds

One of the first redox-driven capped materials was described by Lin and co-workers. In their pioneering work, they used CdS nanocrystals linked with a disulfide linker to cap the pores of MSNs.178 To obtain the gated solid, the authors first prepared mercaptopropyl-modified MSNs via a co-condensation method. Then the external surface was functionalized with 2(pyridyldisulfanyl)ethylamine to obtain the linker 2604


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particles as shown in Figure 86. The iron oxide nanoparticles anchored on the surface avoided the premature release of cargo

Figure 84. Delivery of vancomycin or ATP from dendrimer-capped MSNs triggered by reducing agents.

Gaberscek et al. prepared a family of MSNs loaded with fluorescein or carboxyfluorescein and capped with poly(propyleneimine)-type dendrimers of generations 1−5, which were anchored to the surface of the nanoparticles through cleavable disulfide bonds (Figure 85).180 The release of the

Figure 86. (Top) Fluorescein delivery from Fe3O4-capped MSNs due to the cleavage of disulfide bonds by DTT. (Bottom) (a−c) Single frames of phtographs of HeLa cells with Fe3O4-capped MSNs loaded with fluorescein traveling across the cuvette, propelled by a magnet. (d−f) CLSM fluorescence of HeLa cells after 10 h incubation with Fe3O4capped MSNs loaded with fluorescein [(d) cells excited at 494 nm, (e) cells excited in the UV region, (f) pseudo-bright field image where dark aggregations of MSNs can be observed]. Adapted with permission from ref 181. Copyright 2005 Wiley-VCH. Figure 85. Delivery of fluorescein or carboxyfluorescein from dendrimer-capped MSNs due to the disulfide bond cleavage by DTT.

and conferred magnetic properties to the system, which could be used for site-specific delivery controlled by magnets. Timedependent delivery studies in the presence of dihydrolipoic acid and DTT showed selective cargo release due to the reduction of disulfide bonds. HeLa cells were incubated overnight with the capped nanomaterial. Interestingly, the application of an external magnetic field to the HeLa cell suspension triggered cells in the direction of the magnet, which indicates that the nanomaterial was indeed endocytosed. This was further confirmed by CLSM (see Figure 86, bottom). Green fluorescence was clearly detected in the intracellular vesicles of HeLa cells, which was indicative of the encapsulated fluorescein being released inside cells. The

entrapped fluorophore was triggered by the reduction of the S−S units by DTT. The experimental results showed that the largest amount of dye released, upon addition of DTT, was achieved using dendrimers of generations 1 and 2. Lin and co-workers developed a redox-active delivery system using mesoporous silica nanorods capped with SPIONPs and loaded with fluorescein molecules.181 The surface of MSNs were functionalized with 3-(propyldisulfanyl)propionic acid, which was then reacted with APTES-functionalized Fe3O4 nano605


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group at the end and a disulfide linkage) by click chemistry. The two capped systems showed zero delivery. The CB[6]-capped system was opened upon the addition of DTT, whereas ME was used to uncap the pores of the α-CD-capped nanoparticles (see Figure 88).

authors noted that as tumor cells expressed significant amounts of dihydrolipoic acid, this result was consistent with the intracellular release of fluorescein in HeLa cells. An examination of the healthy intact nuclei of grown cells by transmission microscopy suggested the good biocompatibility of the material under these conditions. In an interesting work, Fujiwara and co-workers designed MSNs with redox-active gates for the activation and deactivation of a catalytic reaction conducted in the pore voids of the material.182 For this study, silica mesoporous microparticles were modified by a postsynthesis method with Al(acac)3 in order to include Al atoms along the surface of mesopores. Aluminum atoms are highly acidic sites capable of catalyzing the dimerization of α-methylstyrene. In a second step, the mesoporous silica surface was grafted with a disilane-disulfide derivative obtained by the direct reaction of 6-hydroxy-naphthyl disulfide with 3-(triethoxysilyl)propylisocyanate (see Figure 87).

Figure 88. Rh B delivery from α-CD- or CB[6]-capped MSNs. Disulfide bond cleavage was triggered by ME or DTT.

Kros and co-workers prepared MSNs loaded with fluorescein and functionalized with different amino alkoxysilane derivatives [i.e., APTES, γ-(2-aminoethyl)aminopropyl trimethoxysilane or (3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane)], on which α-CDs were tethered (see Figure 89).184 A peptide sequence derived from the HIV-1 TAT peptide was also coupled at the terminus of the tether through a disulfide bond. The peptide acted as a part of the reducible stopper of the rotaxane and also as a targeting sequence for cell uptake. Dye release was observed in the presence of DTT, and the behavior of the materials was compared with different amino moieties. A solid modified with γ-(2-aminoethyl)aminopropyl trimethoxysilane was selected for the experiments with HeLa cells because this solid displayed the best compromise for an efficient drug delivery system with minimal leakage. The authors investigated cellular uptake and internalization in HeLa cell cultures. CLSM imaging showed that the nanomaterial was effectively internalized in cells, most likely because of the high affinity of the TAT peptide to the cell membrane. Interestingly, quantitation of cellular uptake by flow cytometry showed that 98% of cells contained TAT-functionalized nanoparticles, unlike the 13% uptake of silica nanoparticles with no peptide. An interesting approach using β-CDs to cap pores, run coupled with a Förster resonance energy transfer (FRET)-based system to monitor cargo release, was reported by Lee et al.185 This capping system comprised three parts: (i) a coumarin (donor) labeled-cysteine attached to MSNs; (ii) a FITC

Figure 87. Micrometric mesoporous silica blocked by the disulfide bond. Pore opening was achieved in the presence of DTT.

When a α-methylstyrene solution was heated at 120 °C for 2 h in the presence of gated Al-modified mesoporous silica, no remarkable dimer quantity was obtained. However, if the disilane-disulfide grafted mesoporous silica was treated with DTT, disulfide bonds were cleaved and the material recovered its catalytic activity to yield percentages of around 97% of the dimer. This process proved reversible; in particular, if the reduced material was treated with I2, the disulfide bond was recovered and the material was unable to catalyze the dimerization reaction. Zink and Stoddart reported the preparation of MSNs functionalized with rotaxanes and incorporating disulfide bonds into stalks encircled by CB[6] or α-CD rings.183 Bare MSNs were first functionalized with APTES, and then amino groups were reacted with an azide-terminated diethylene glycol monotosylate. Afterward, nanoparticles were loaded with Rh B, and an excess of either CB[6] or α-CD was added to threat the grafted stalks. Finally, the azide group at the end of the stalks was reacted with a propargyl ether (that contained a bulky adamantly 606


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environment, the close proximity between coumarin and FITC on the surface of MSNs resulted in FRET. However, in the presence of GSH, disulfide bonds were cleaved, inducing the removal of the FITC-β-CD group to thus trigger drug release and to eliminate FRET. To demonstrate that capped-MSNs responded to GSH in living cells, HeLa cell cultures were treated with modulators of intracellular GSH levels, and FRET fluorescence was monitored by CLSM. The obtained results confirmed that the opening of the gated nanoparticles correlated with changes in the GSH levels. HeLa cells were also pretreated with modulators of the internal GSH concentration and were further incubated with similar capped nanoparticles loaded with DOX. Quantification of cell viability after 24 h of treatment showed a good correlation between the FRET signal and the extent of DOX released at different GSH concentrations inside cells. Zhao and co-workers developed a redox-responsive system for siRNA and drug codelivery.186 For this purpose, the MSNs modified with MPTS by co-condensation were reacted with S-(2aminoethyl)-2-thiopyridine, which formed a disulfide bond with a terminal amino moiety (see Figure 91). This amino group was

Figure 89. MSNs capped with α-CDs and containing a TAT peptide, anchored through disulfide bonds. Cargo release was triggered by DTT.

(acceptor) attached to a mono-6-amino-β-CD via an urea bond, and (iii) a disulfide linkage connected to an adamantane group (see Figure 90). The interaction of the β-CDs with adamantane moieties allowed an effective capping. In a nonreducing

Figure 91. MSNs capped with β-CDs. Cargo and siRNA deliveries were observed in the presence of GSH.

coupled with adamantane-1-carboxylic acid via amide formation. Two different solids were obtained by loading two different cargos (Rh B or DOX) and were then capped with per-6diamino-β-CDs. Finally, siRNA was immobilized on β-CD caps via electrostatic interactions with the protonated amino groups in acidic dulbecco’s modified eagle medium (DMEM) culture media. The addition of GSH or DTT resulted in the simultaneous delivery of the drug loaded and siRNA. Another redox-responsive drug delivery system based on the use of [2]rotaxanes and disulfide bonds was reported by Zhao et al.187 The [2]rotaxane capping agent was composed of tetraethylene glycol encircled by α-CDs and ended with FA, which acted as both a stopper and a tumor-targeting agent as depicted in Figure 92. The tetraethylene glycol-α-CD-FA

Figure 90. MSNs capped with β-CDs. Cargo delivery was observed in the presence of GSH. 607


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DOX-loaded nanoparticles were better than those of free DOX and with minimal side effects. Luo et al. prepared hollow MSNs, which were grafted with adamantane moieties via a redox-cleavable disulfide bond as linker (see Figure 93). Nanoparticles were loaded with FITC or

Figure 93. MSNs capped with β-CDs that bore LA. Cargo release was observed with DTT and GSH. Figure 92. MSNs capped with α-CD and functionalized with FA. Delivery was triggered by GSH.

DOX, and the system was capped with lactobionic acid (LA)modified-β-CDs through complexation with the adamantyl group. β-CD-LA groups also acted as targeting ligands to Hep G2 cells.188 In vitro studies showed that nanoparticles were tightly capped, whereas the presence of GSH induced DOX release. The authors studied the interactions between nanoparticles and Hep G2 hepatoma cells. The uptake of the capped nanoparticles functionalized with LA was much higher than that of hollow MSNs or capped hollow MSNs without LA groups, which agrees with the targeting ability of LA. Endocytosis by Hep G2 cells of the FITC-loaded gated nanomaterials was studied. The green fluorescence of endocytosed nanoparticles was mainly located in the cytoplasm, with no permeation into the cell nucleus. These results were independently confirmed by quantitative flow cytometry analysis. When the capped nanomaterial loaded with DOX was used, an inhibitory effect on tumor cell proliferation was assessed. The characterization of the cytotoxic effect observed in the cells treated with DOX-loaded capped nanoparticles indicated that it was due to apoptosis. Finally, the authors performed in vivo experiments to investigate the delivery and distribution of the system in mice bearing Hep G2 cell xenografts. Most remarkably, tumor growth in vivo was effectively suppressed by subcutaneously injected nanoparticles. Once again, LA-functionalized hollow MSNs induced a more reduced tumor size than the controls. Zhao and co-workers studied the effect of the length, nature, and density of different anchored functional groups in the redoxtriggered release of DOX using β-CD-capped MSNs.189 These authors prepared a family of gated MSNs using MPTSfunctionalized nanoparticles. All the prepared systems contained a cleavable disulfide bond. After loading with DOX, pores were

ensemble was attached to MSNs via a linker that contained a disulfide bond. Nanoparticles were loaded with DOX. Capped MSNs remained stable over 26 h in aqueous TRIS solution, whereas DOX delivery was clearly found in the presence of GSH both at physiological and under endosome mimetic conditions (pH 5). The authors tested the interactions of this capped nanomaterial with tumor cells both in vitro and in vivo. HeLa cell cultures were treated with the system either uncapped or capped with FA, and biocompatibility and biodistribution was analyzed by TEM. Cell observation confirmed the good biocompatibility of both materials, as judged by cell and nuclear membrane intactness. Remarkably, the FA-capped nanomaterial was endocytosed more efficiently than the one uncapped by HeLa cells, which suggested that internalization was enhanced by the targeting ligand FA. Most interestingly, a 2-fold increase uptake in tumor cells was observed when the internalization of the FAcapped MSNs was quantitated in HeLa cells and normal endothelial cells. This can be explained by the fact that the amount of folate receptors on tumor cells was larger than that on normal cells, which thus favors the receptor-mediated endocytosis of the nanomaterial. DOX-loaded FA-capped nanocontainers selectively induced HeLa tumor cell death. An analysis of HeLa cells treated with the nanomaterials indicated that the mechanism of cell death was apoptosis. The effects of the DOX-loaded nanomaterial were tested in vivo by generating tumor models on nude mice via subcutaneous xenotransplants of HeLa cells. Tumor size was inhibited when treated with free DOX and DOX-loaded nanoparticles. The curative effects of 608


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capped using β-CDs via coordination with different bulky terminal moieties (see Figure 94). MSNs that contained

Figure 94. β-CDs-capped MSNs with different stalks that contained disulfide bonds. Delivery was triggered by GSH.

cyclohexyl terminated ligands of different chain lengths, and capped with β-CDs, were prepared. It was observed that the DOX release triggered by GSH was more efficient when chains of intermediate lengths were used. This effect was explained because stalk length had to be ideal to allow DOX loading in pore voids and to also retain the drug in mesopores after capping with β-CDs. The influence of the different bulky terminal groups was also studied. In particular, the authors prepared MSNs capped with β-CDs with cyclohexyl, adamantyl, and n-butyl groups. When they used redox-cleavable chains terminated with cyclohexyl groups, a high loading efficiency and a better DOX release profile were obtained. This fact was attributed to the combination of the effect of the corresponding bulky group- βCD binding constant and the steric impediment for DOX loading imposed by the cyclohexyl, adamantyl, or n-butyl group. Finally, these authors explored the influence of the amount of stalk anchored to the surface. For this purpose, the authors used the cyclohexyl-terminated stalk, which displayed better performance in previous studies. It was found that a small amount of stalks on the surface allowed GSH to easily interact with the disulfide bond to confer more efficient uncapping. This last piece of evidence was confirmed by CLSM and cytotoxicity experiments in the B16−F10 melanoma cell line, where the same tendency was found. Wu and co-workers also developed a nanocarrier based on rotaxane nanovalves that contained disulfide linkages.190 In this case, MSNs were functionalized with MPTS then thiol groups were reacted with S-(2-aminoethylthio)-2-thiopyridine to yield amino-terminated alkyl chains that contained disulfide bonds. In another step, the amino moieties were reacted with 2-((3-(2azidoethoxy)phenyloxy)methyl)-6-(bromomethyl)pyridine (see Figure 95). The pores of nanoparticles were loaded with fluorescein, and the system was capped with a tridentate macrocyclic Pd complex. Finally, alkyne-functionalized FA (for targeting purposes) was attached through a click chemistry

Figure 95. MSNs loaded with fluorescein and capped with a Pd macrocyclic-containing rotaxane. Delivery was observed in the presence of reducing agents.

reaction. PBS (pH 7.4) suspensions of capped nanoparticles showed a negligible fluorescein release, whereas a remarkable dye leakage was observed in the presence of DTT or GSH as a result of the reduction of the disulfide bond that detached the rotaxane from the surface of the nanoparticles. The same nanoparticles were prepared but loaded with DOX. The authors found that nanoparticles were endocytosed by HeLa cells and that DOX was released due to the presence of endogenous GSH. DOX delivery induced apoptosis and reduced cell viability. Kim, Park, and co-workers reported a GSH-induced delivery system that used MSNs loaded with calcein or DOX and capped with a β-CD derivative.191 These authors grafted MPTS on the surface of MSNs, which was then treated with S-(2-aminoethylthio)-2-thiopyridine to obtain amino moieties linked by a disulfide bond. The amino groups were reacted with propargyl bromide, and the stalk was further reacted with a mono-6-azidoβ-CD via click chemistry as depicted in Figure 96. Addition of biological reductive agents, such as GSH or DTT, resulted in cargo release. In order to improve the solubility of nanoparticles, isocyanate-ended PEG moieties were also attached to the β-CD derivative. The authors observed that the suspensions in PBS of these capped nanoparticles were stable for several days without 609


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delivery and intratumoral distribution of the system in mice that bore A549 cell xenografts. Accumulation of nanoparticles in the tumor was clearly detectable and, most remarkably, tumor growth in vivo was effectively suppressed by intravenously injected nanoparticles. The same authors implemented the same molecular gate described above into hollow MSNs in order to improve the loading capability of guest molecules.194 These authors found that the amount of DOX was about 4-fold larger than the amount of DOX loaded in typical MSNs. The system was also highly effective in the intracellular release of DOX in A549 cells, which are known to express high levels of GSH. The nanomaterial significantly increased cell death in a dose-dependent manner. Zhao and co-workers developed multifunctional MSNs with enhanced long-term stability under physiological conditions.195 In their work, they functionalized the external surface of MSNs with mercaptopropyl groups, which were reacted with 2carboxyethyl-2-pyridyl disulfide to obtain carboxylic acidfunctionalized MSNs. In another step, they loaded nanoparticles with DOX or calcein and capped pore entrances with per-6diamino-β-CDs via an amidation linkage. Finally, gated nanoparticles where decorated with PEG chains functionalized with an adamantane unit at one end and FA at the other. The system was capped through β-CD/adamantine interactions. Upon the addition of DTT or GSH, the disulfide bond of the linker was broken to allow the removal of the β−CD cap with the subsequent release of the entrapped DOX (see Figure 97). The authors also found that the multifunctional ensemble was sensitive to pH. The unreacted amino groups of β-CD were protonated at acidic pH, which influenced the cargo release profile due to electrostatic repulsion interactions; in other words, a rapid release of positively charged molecules, such as DOX, and a slower release for negatively charged molecules, such as calcein, were observed. After verifying the redox and pH-driven mechanism, cell targeting functionality was assessed. Fluorescein-labeled and gated nanoparticles with different amounts of folate groups were prepared and tested in folate receptor-positive HeLa cells. Increased cell uptake was recorded according to the quantity of FA groups in MSNs. Gated MSNs were also incubated with folate receptor-negative human embryonic kidney 293 cells, where no fluorescence was observed, which confirmed FA-induced MSNs uptake. The intracellular distribution of gated MSNs was investigated by CLSM. The nanoparticles colocalized with lysosomes after 3 h of incubation, but gated MSNs were also observed after 24 h in the cytoplasm due to the endosome-disrupting proton-sponge effect of the amino groups in β-CDs. Finally, the cytotoxicity of DOX-loaded capped nanoparticles was studied by CLSM and an MTS assay. The results revealed that significantly inhibited cell growth was observed in the folate-receptor expressing cells treated with DOX-loaded nanoparticles, whereas the gated nanoparticles without DOX were not toxic. The same authors, who continued with their work, prepared different sized MSNs (48−100 nm), which were loaded with DOX and capped using the same gated ensemble described above (see Figure 97).196 The presence of FA on the material surface stimulated the internalization on tumoral cells. As above, in vitro studies demonstrated that the presence of GSH caused a large delivery rupture of disulfide bonds. A drop in pH (from 7.4 to 5.5) led to certain drug delivery and was attributed to the electrostatic repulsions between the protonated amino moieties in β-CDs. A synergetic effect was observed when the two stimuli were applied at the same time. The active folate targeting ligand

Figure 96. PEG-modified β-CD-capped MSNs with a linker that contained disulfide bonds. Cargo delivery was observed in the presence of GSH.

precipitation. By CLSM, the authors investigated the intracellular cargo release for the DOX-loaded β-CD-capped nanoparticles in A549 cells, which exhibited high levels of GSH. DOX fluorescence detected within the cell indicated that DOX was released from cell-internalized nanoparticles, probably induced by high internal GSH content. To test this possibility, the authors increased intracellular GSH levels by preincubating A549 cells with the monoethyl ester of GSH (a GSH precursor). As expected, intracellular DOX fluorescence was stronger under these conditions. In another work,192 the same authors extended their study by exploring use of temperature to improve the release of DOX from the previous developed system. The in vitro release profile of DOX from the drug nanocarrier was studied in PBS, either with or without GSH, at 37 or 42 °C. No detectable drug release was observed in the absence of GSH, whereas DOX was rapidly released in the presence of GSH at both 37 and 42 °C, although, as expected, DOX release was faster at 42 °C than at 37 °C. In vitro release studies in cancer cells showed that by increasing the temperature to 42 °C, the GSH-mediated release of DOX from the drug nanocarrier was greater as the GSH level increased in cells. The same authors developed a similar capped material, but the MSNs contained a Fe3O4 core in this case.193 Pores were loaded with DOX (Figure 96). In vitro experiments showed that in the absence of GSH, no release of DOX was observed over 5 days, whereas DOX was delivered in ca. 2 h in the presence of GSH. With the A549 cells incubated with the capped material, CLSM evidenced that DOX was released as a result of the cleavage of the gatekeeper mediated by intracellular GSH. Finally, the authors performed in vivo MRI experiments to investigate the targeted 610


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MDA-MB-231 breast carcinoma cells). Consistently with the in vitro findings, mice treated with the nanomaterial exhibited significantly higher tumor shrinkage rate than those treated with free DOX. No statistically significant body weight loss was observed in any treated group, which indicated that treatment was well tolerated. An in situ histological analysis also supported the system’s excellent therapeutic effect. Few tumor cells/foci were detected in mice treated with nanoparticles. In addition, examinations of heart, lung, liver, spleen, and kidney indicated no apparent abnormality or lesions after 21 post-treatment days. These results indicated that MSNs could deliver a sufficient amount of DOX to tumors, which would result in a remarkable tumor-inhibiting effect compared with those of free DOX and nontargeted nanoparticles. Following a similar approach, Gaberscek et al. developed different redox-responsive delivery systems, all of which were based on the use of β-CDs, which were anchored to the silica surface via different linkers.197 MSNs were functionalized with APTES and then reacted with (N-succinimidyl 3-(2pyridyldithio)propionate, 4-(succinimidyloxycarbonyl)-α-(2pyridyldithio)toluene, or 4-(succinimidyloxycarbonyl)-α-methyl-α-(2-pyridyldithio)toluene to provide systems with three levels of hindrance around disulfide bonds (see Figure 98). Pores

Figure 97. (Top) FA-functionalized β-CDs-capped MSNs that contained a disulfide bond. Delivery was observed with GSH. (Bottom) CLSM images of MDA-MB-231 cancer cells incubated with FAfunctionalized β-CD-capped MSNs, β-CD-capped MSNs, and FAfunctionalized β-CDs-capped MSNs under the FA competition, respectively. The red fluorescence was from DOX, and the blue fluorescence was from DAPI used to stain the nuclei. Scale bars: 20 mm. Adapted with permission from ref 196. Copyright 2014 Wiley-VCH.

in the system was tested in the MDA-MB-231 breast cancer cell line, which is well-known for its high overexpression of folate receptors. As expected, incubating the system with MDA-MB231 cells led to improved selective uptake of nanoparticles into cells (Figure 97, bottom). After 12 h of incubation, apoptotic cell bodies were observed in cultures. Preincubation with free FA prevented nanomaterial internalization and also subsequent DOX cytotoxicity. These observations demonstrated that introducing the FA ligand can improve the specific targeting of the nanoparticle carrier toward folate receptor overexpressed MDA-MB-231 cells. The system’s reduced toxicity and selective endocytosis, when compared with free DOX, suggest that nanoparticles can effectively be used for controlling tumor growth and reducing systemic toxicity. To test this possibility, in vivo research of therapeutic efficacy was carried out in mammalian cancer-induced female nude mice (by implanting

Figure 98. β-CDs-capped MSNs that contained different linkers and disulfide bonds. Cargo delivery was observed in the presence of DTT.

were loaded with Rh B as a model drug, and the molecules monothio-β-CD or perthio-β-CD were used as capping agents through a reaction with the pyridyldithio groups of the thiolcontaining linkers, which resulted in the formation of disulfide linkages. Capped systems were tested in a nonreducing environment and showed slight leakage, whereas addition of DTT allowed clear cargo release. The delivery of trapped dye was dependent on the level of hindrance induced by the substituents around the disulfide bond. Increased hindrance caused the release rate to lower. To evaluate the teratogenicity of synthesized materials, in vivo experiments on zebra fish 611


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acid) layer was constructed by a reaction with the azide group from the hosted peptide and polymer. The gated solid was studied in SCC-7 (squamous carcinoma) and HT-29 (human colon cancer) cells, both of which are well-known for high metalloproteinase expression. The capped material was also studied in noncancerous 293T (human embryonic kidney 293 transformed) cells. Additionally, studies were performed in the presence and absence of a metalloproteinase inhibitor. The authors found that DOX-loaded capped MSNs reached tumor cells and that the poly(aspartic acid) coating was removed by metalloproteinases via the enzyme-mediated hydrolysis of the PLGVR fragment in the peptide. The RGD-exposed peptide fragment also facilitated the uptake of MSNs by cells where DOX was released due to the reduction of the disulfide linker by endogenous GSH. Cai et al. loaded MSNs with FITC as a model drug and capped the system with collagen moieties through disulfide linkers. In particular, the authors functionalized the support with APTES and then reacted the amino groups consecutively with succinic anhydride and cystamine to functionalize the external surface with organic moieties that contained disulfide bonds and terminal amines. The system was capped with collagen through the formation of amide bonds between the carboxylate groups of the biopolymer and the amines grafted on the surface as depicted in Figure 100. Finally, as a targeting element, LA was linked with

(Daniorerio) were performed. To avoid excessive animal testing, only the system that exhibited the largest amount of Rh B release (that containing the ethylene linker) was evaluated, along with two concentrations of capped MSNs. No tested sample affected hatching of larva on day 4 postfertilization in all the samples. Likewise, no effects on fish length were detected at the same time point. The system neither significantly affected embryo development and survival nor were signs of cell death detected. These data confirmed the nonteratogenicity of the nanomaterial. Zhang and co-workers also developed MSNs capped with βCDs, which were linked to the silica surface through disulfide bonds.198 In particular, these authors first functionalized MSNs with MPTS, which were further reacted with S-(2-aminoethylthio)-2-thiopyridine. In another step, the authors included an alkyne moiety by refluxing the solid with a solution that contained propargyl bromide, MeOH and HCl. Nanoparticles were loaded with DOX, and pores were blocked via a click chemistry reaction with mono-6-azido-β-CD. In another step, peptide N3-GPLGVRGRGDK-adamantane was incorporated into nanoparticles via a “host-guest” interaction between the adamantine groups and the β-CDs attached to the silica surface as shown in Figure 99. Finally, an alkyne-modified poly(aspartic

Figure 100. FITC delivery from collagen-capped mesoporous hydroxyapatite or MSNs due to disulfide bond cleavage by DTT.

grafted collagen. LA is a specific ligand that binds the asialoglycoprotein receptor onto the membrane of Hep G2 cells.199 The opening protocol was mediated by the presence of reducing agents, such as DTT, which cleaved the disulfide linker. For instance, after 2 h in the absence of DTT, the solid exhibited an FITC release of only 6.5%, whereas the solid delivered 80% of the cargo when DTT was added. The authors further investigated the endocytosis and intracellular delivery of the

Figure 99. MSNs capped with β-CDs containing a peptide and a poly(aspartic acid) coating. Cargo delivery was triggered by GSH. 612


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observed, which corroborated the simultaneous release of substrate luciferin from pores and the enzyme luciferase of the surface. Internalization studies in HeLa cells, done with a similar solid marked with FITC, corroborated the cellular uptake of the material. Cell viability experiments by Guava ViaCount assays, also in HeLa cells, resulted in good biocompatibility of capped nanoparticles. Finally, the authors assessed luciferase activity assays in the same cell line and achieved remarkably higher luminescence intensities in comparison to the same cells treated with free luciferin and luciferase, which confirmed the substrate and enzyme codelivery. Fang et al. reported MSNs capped with FA, which was anchored to the silica surface through disulfide bonds.202 In this case, MSNs were loaded with fluorescein and cargo delivery was observed in the presence of DTT and GSH as depicted in Figure 102. Cell uptake and cytotoxicity of the system was investigated

capped nanoparticles in Hep G2 and endothelial cells. Internalization in Hep G2 was at least 2-fold higher than in endothelial cells. Huang et al. applied the same system but used collagen as a capping element and LA as a targeting group (see Figure 100). Moreover the authors used mesoporous hydroxyapatite as an inorganic scaffold.200 Pores were loaded with FITC. Aqueous suspensions of the collagen-capped nanoparticles in the absence of DTT showed a 20% release of entrapped FITC after 10 h. However, when DTT was added, the dye release reached 80%. Moreover, CLSM studies indicated that collagen-capped nanoparticles were endocytosed by Hep G2 cells and released the cargo in the cytoplasm. Trewyn, Slowing, and co-workers developed MSNs loaded with luciferin and capped by AuNPs.201 Thiol-modified MSNs were obtained by a co-condensation process in the presence of MPTS. Moreover, MSNs were PEGylated by grafting 2[methoxy(polyethylenoxy)propyl] trimethoxysilane. For linking AuNPs, MSNs were treated with 2-aldrithiol and then functionalized with 3-(propyldisulfanyl)ethylamine by a disulfide bond exchange reaction. MSNs were loaded with luciferin (luciferase substrate), and carboxylic acid-terminated AuNPs with PEG linkers were attached to the amino moieties on MSNs by the formation of an amide bond (see Figure 101). The enzyme was adsorbed on the surface by incubating the luciferin-loaded and capped nanoparticles in a luciferase solution. Negligible cargo release was observed for 24 h, yet clear cargo delivery was achieved by the addition of DTT due to the reduction of disulfide bonds. The luminescence produced by enzyme activity was also

Figure 102. FA-capped MSNs that contained disulfide bonds. Delivery was observed with DTT and GSH.

in HeLa cells, which expressed high levels of the alpha folate receptor. CLSM analysis of HeLa cells treated with Rh B-loaded MSNs, both with and without FA modification, revealed that internalization was 8-fold greater for the nanoparticles that contained FA. Co-location studies of Rh B- and fluoresceinloaded FA-capped MSNs by CLSM indicated that the bioresponsive cleavage of disulfide bonds led to gate opening and to the controlled release of fluorescein. Negligible cytotoxicity was observed in HeLa cells treated with empty nanoparticles for 24 h. In contrast, the incubation of HeLa cells with DOX-loaded FA-functionalized nanoparticles resulted in a remarkable 3-fold reduction in the viability of cancer cells. Wang, Li, and co-workers developed MSNs functionalized on the outer surface with MPTS and loaded with fluorescein dye. PEG molecules were also anchored to the outer surface via a disulfide bond.203 The PEG coating was obtained by adding PEG-SS-pyridine molecules to the thiol-functionalized MSNs,

Figure 101. Luciferin delivery from MSNs capped with AuNPs. Release was observed in the presence of DTT. 613


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which resulted in an exchange reaction between the thiol end group on the outer surface of MSNs and the pyridyldisulfide groups at the end of the PEG-SS-pyridine molecules (Figure 103). Negligible dye release was observed in the absence of GSH

Figure 103. MSNs capped with PEG that contained a disulfide bond. Delivery was triggered by GSH.

over 20 h, whereas fluorescein achieved maximum delivery at 22 h in the presence of GSH. Cell uptake and cytotoxicity of capped MSNs was investigated in MCF-7 cells. CLSM demonstrated that the fluorescein-loaded capped material entered cells via endocytosis and that particles distributed mainly in the endosomal/lysosomal compartment. To test whether fluorescein was released from the cell-internalized nanomaterial by internal GSH content, MCF-7 cells were preincubated with 10 mM extracellular GSH. The intracellular fluorescein signal was significantly stronger under these conditions, which indicates that cargo release was due to intracellular GSH levels. Low cytotoxicity of the fluorescein-loaded capped material was observed in MCF-7 cells. Remarkably, the cytotoxicity of antineoplasic drug MTX (metothrexate) in MCF-7 cells was clearly enhanced 2-fold when encapsulated in the nanomaterial and further increased after preincubation with 10 mM extracellular GSH. Trewyn and co-workers developed a controlled release system using MSNs that contained phosphorylated lipid bilayers. MSNs were functionalized with MPTS, loaded with fluorescein, and further reacted with 1-thiol-2,3-dipalmitoylpropane, which resulted in the formation of a disulfide bond.204 A second layer with different phosphorylated lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid sodium salt, and cardiolipin) was assembled to obtain final capped materials. For all four capped supports, DTT induced cargo delivery as depicted in Figure 104. As expected, the phosphatidylcholine-capped solids improved cell proliferation, whereas no remarkable effects were observed using the phosphatidylethanolamine and phosphatidic acid caps. It is well-known that phosphatidic acid and ethanolamine that contains phospholipids with two palmitoyl chains are not as effective at up-regulating mitosis pathways as phospholipids that contain natural C16−C18 saturated and C18−C20 unsaturated lipid chains. In contrast,

Figure 104. Fluorescein delivery from lipid bilayer-capped MSNs due to disulfide bond cleavage by DTT.

the cardiolipin-capped material brought about enhanced cell death, which the authors attributed to the role played by cardiolipin in the apoptosis cascade in many cell types. Zhu, Bao, and co-workers proposed a redox responsive delivery system capable of releasing the entrapped cargo and a bioactive protein.205 A disulfide-containing linker unit with a carboxylic group at one end and a phenylazide moiety at the other was synthesized. MSNs were grafted with APTES and modified with the linker unit via an amidation reaction. The authors prepared a solid loaded with coumarin 460 and capped with a biocompatible Dextran polymer by a photoinduced reaction with phenylazide via a phenylnitrene intermediate. The resulting system was still capped in water until DTT was added, which resulted in huge dye delivery (Figure 105). Moreover, the authors considered that their proposed photoactivated functionalization could be a general method to immobilize a wide range of macrobiomolecules. As proof-of-concept, a second solid was synthesized, and functionalized with avidin instead of Dextran, and then conjugated with fluorescent-labeled 655-biotin. Addition of GSH resulted in protein release. 614


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MCM-41 as a solid support. It was found that in the presence of GSH, a much faster release was observed from the MCM-41based system. This fact could be attributed to the different pore size of SBA-15 and MCM-41. However, greater leaching of Rh B in the absence of a reducing agent was displayed by the MCM41-based material. Rosenholm and co-workers also used PEG-capped systems and employed them to deliver short oligonucleotides. MSNs with larger than usual pores sizes (i.e., 3.5, 4.5, and 5 nm) were prepared and loaded with a model oligo of 21 base pairs in double strand form (see Figure 107).207 The surface was grafted with

Figure 105. Coumarin 460 delivery from the dextran- or avidin−biotin capped MSNs due to disulfide bond cleavage by DTT.

Wang et al. attached a PEG derivative to mesoporous SBA-15 through an L-cystine ester with a disulfide bond linker.206 Rh B was used as a model drug. The disulfide linker was cleaved in the presence of GSH, which resulted in cargo delivery as shown in Figure 106. For comparison purposes, the authors studied the controlled release behavior of the same capping system, but used

Figure 107. MSNs loaded with double-stranded DNA and capped with PEG chains that contained a disulfide bond. Cargo was released with GSH.

PEG chains, attached with a disulfide-containing linker. The authors studied the ability of the different pore-sized MSNs to release the entrapped oligonucleotides in the presence of GSH or DTT. MSNs with a 4.5 nm pore size exhibited the best performance for both absorption and delivery of the oligonucleotide. Cai and co-workers208 used cytochrome c as a capping agent. In particular, the authors prepared MSNs functionalized with carboxylic acids by a co-condensation method. Then the carboxylic acids were amidated using cystamine, and pores were loaded with DOX or with FITC. MSNs were capped upon the addition of cytochrome c through an amidation reaction with the amine groups located on the external surface of the support. Aptamer AS1411 (HS-(CH2)6-5′-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3′) was linked with the grafted cytochrome c by using sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate as a linker (see Figure 108). PBS (pH 7.4) suspensions of the capped MSNs showed negligible entrapped cargo release. Yet in the presence of DTT, remarkable cargo release took place (ca. 79% after 3 h).

Figure 106. Mesoporous silica support capped with PEG chains. Rh B delivery was observed in the presence of GSH. 615


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the DOX-loaded-capped MSNs induced the most severe apoptosis of tumor cells among all groups. In vivo biodistribution studies of different MSNs were performed by intravenous injection of FITC-labeled MSNs or capped-MSNs in the tumorbearing mice. MSNs accumulated mainly in the lungs due to the abundant blood supply, followed by moderate accumulation in the kidney and liver. The concentration of both nanoparticles in heart, lung, and kidney gradually lowered with time. Interestingly, MSNs accumulated considerably in the tumor xenograft. Capped MSNs showed a higher concentration in tumors than MSNs due to the targeting role of the AS1411 aptamer. Multifunctional MSNs for the codelivery of a drug and a therapeutic peptide using disulfide linkages were described by Zhang et al.209 The external surface of MSNs was functionalized with mercaptopropyl moieties and the pores of the support were loaded with anticancer drug topotecan. Then peptide TPP-K(KLAKLAC)2-C (TPep), in which TPP was a triphenylphosphonium lipophilic cation and (KLAKLAC)2 an antibiotic peptide, was attached to the thiol-functionalized surface by the formation of disulfide linkages. Finally, hybrid nanoparticles were coated through electrostatic interactions with polyanion poly(ethylene glycol)-blocked-2,3-dimethylmaleic anhydride-modified poly(L-lysine). In the absence of GSH, negligible topotecan release was observed, but when GSH was added, remarkable drug release occurred due to the reduction of the disulfide linkage that detached the anchored cationic peptide from the surface as shown in Figure 109. CLSM and TEM were used to observe the system’s cellular uptake in human mouth epidermal carcinoma cells (KB cells). An appreciable difference in the cellular uptake of the nanomaterial was noted at different pHs. At pH 6.8, an enhanced intracellular red fluorescence resulting from the release of the Rh−B labeled TPep peptide was observed. Red fluorescence colocalized with mitochondria, which indicated the good mitochondrial targeting ability of TPP. No significant red fluorescence was detected at pH 7.4. These results were independently confirmed by flow cytometry analysis. The authors tested the release of Rh B labeled TPep in the presence or absence of high GSH levels. In vitro quantitation of red fluorescence revealed a very low release in the absence of GSH due to the blocking effect of the outer shielding layer. Most interestingly, after treatment with GSH 10 mM at pH 6.8 for 24 h, maximum release was detected. To investigate the mitochondrial damage/disruption by released TPP, KB cells were incubated with the system and mitochondrial dysfunction was monitored with mitochondrial fluorescence probe JC-1. As expected, incubation of nanoparticles at pH 6.8 induced a drop in JC-1 fluorescence in a time-dependent manner, which was indicative of severe mitochondrial damage. This result was also confirmed morphologically by Bio-TEM analysis of the KB cells treated with the nanomaterial. Finally, and as expected, improved cell lethality was observed in a tumor acidic microenvironment (pH 6.8) for the cells treated with topotecan-loaded nanoparticles. Cell viability was lower than the corresponding unloaded MSNs at the same concentration. The antineoplastic drug topotecan codelivered by the carrier further inhibited tumor growth with remarkable synergistic effects. Zhang and co-workers selected MSNs as inorganic scaffold and functionalized the surface consecutively with MPTS and then with 2,2′-dithiodipyridine.210 Pores were loaded with DOX, and peptide N3-RGDFFFFC (that targets several cancer cells) was grafted through a disulfide exchange reaction. The grafted peptide was decorated through a click chemistry reaction with PEG moieties that contained an acid-labile benzoic imine group.

Figure 108. Delivery of FITC or DOX from the MSNs capped with cythocrome c and bearing the AS1411 aptamer.

The authors investigated the cellular endocytosis of different FITC-loaded capped MSNs in Hep G2 and in normal HL-7702 cell cultures, respectively. CLSM and flow cytometry studies indicated that the endocytosis of capped nanoparticles was significantly greater than that of the control MSNs with no cytochrome c and aptamer. Preincubation of cells with free AS1411 aptamer eliminated this difference. TEM analysis of Hep G2 cells revealed that capped nanoparticles were located at the cytoplasm and were excluded from the nuclei. The intracellular drug delivery caused by GSH within tumor cells was studied with DOX-loaded nanoparticles. A red fluorescence signal was observed at the cell nucleus, which indicates that the DOX released from the capped MSNs system intercalated into DNA. Cytotoxicity and apoptosis induction was also tested with the DOX-loaded nanoparticles. Interestingly, the DOX-loaded capped support decreased cell viability slightly more than free DOX. Finally, the authors performed in vivo experiments to investigate the delivery and distribution of the system in mice that bore Hep G2 cell xenografts. Tumor growth in vivo was effectively suppressed by subcutaneously injected DOX-loaded capped MSNs, which proved more effective than free DOX treatment. This result could be explained by the synergic therapeutic effects of combining triplex therapy I (DOX), II (cytochrome c), and III (aptamer). No obvious difference in the body weights of the nude mice treated with different MSNs was observed, which indicated that the system was biocompatible in vivo. Quantitation of apoptosis by TUNEL assay revealed that 616


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Figure 109. Topotecan delivery from peptide-capped MSNs due to disulfide bond cleavage by GSH.

Figure 110. DOX delivery from peptide-capped MSNs due to disulfide bond cleavage by DTT.

Aqueous suspensions of capped nanoparticles showed negligible DOX release, whereas a marked delivery of DOX and of the anchored peptide were observed upon addition of DTT (see Figure 110). The controlled release behavior of the capped nanoparticles was tested in U-87 and COS7 cells. The authors found that nanoparticles were poorly endocytosed by both cell lines at pH 7.4. However, at pH 5, a marked emission in cells was observed. This emission was ascribed to the fact that at pH 5, the benzoic imine group was hydrolyzed and the PEG fragments detached from the surface of the nanoparticles. This exposed the anchored targeting peptide, and nanoparticles were uptaken by cells. The emission observed in cancer cells U-87 was more marked than that of COS7 noncancerous cells. This internal fluorescence was ascribed to the GSH-induced rupture of the disulfide moiety that connected the peptide with the nanoparticle surface. MTT assays carried out with U-87 cells and gated nanoparticles at pH 5 indicated a marked cell viability reduction as a result of DOX release. Cai et al.211 reported redox-responsive MSNs loaded with FITC or DOX and grafted with N-deacetylated heparin on the outer surface via a linker that contained a disulfide bond. Ndeacetylated heparin provided free amino groups to also couple LA as a targeting motif (see Figure 111). Cargo delivery was observed in the presence of reducing agents. The authors studied the interactions between capped MSNs and Hep G2 hepatoma cells. The use of unloaded nanomaterials did not bring about any

alteration of cellular morphology or significant changes in cell viability. Moreover, the uptake of capped nanoparticles by Hep G2 cells was much greater than that of unfunctionalized MSNs or capped MSNs without the LA group. The endocytosis of gated MSNs was further analyzed by incubating Hep G2 cells with capped nanoparticles loaded with FITC. The green fluorescence of endocytosed nanoparticles was mainly located in the cytoplasm. Once again, the cells treated with the capped nanoparticles that contained LA moieties exhibited better internalization efficiency compared with the control nanomaterials. This was due to galactose receptor-mediated endocytosis as the preincubation of cells with free galactose significantly decreased intracellular FITC fluorescence. Next the authors encapsulated DOX, and the inhibitory effect on tumor cell viability and apoptosis was assessed by CLSM. A significantly greater apoptosis induction was observed in the capped materials functionalized with the LA groups. Finally, the authors performed in vivo experiments to investigate the delivery and distribution of the system in mice that bore Hep G2 cell xenografts. It was remarkable that tumor growth in vivo was effectively suppressed by subcutaneously injected DOXcontaining capped nanoparticles. A histological examination was performed to evaluate the damage of major mice organs. While free DOX induced typical myocardial injury in mice, those injected with the DOX-loaded nanoparticles presented a significant remission of cardiac damage. 617


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Figure 112. Delivery of plasmid DNA and fluorescein or β-oestradiol from AuNPs-capped MSNs. Release was triggered by DTT. Figure 111. Delivery of FITC or DOX from MSNs capped with deacetylated heparin due to disulfide bond cleavage by GSH.

the GFP inducer and the required cargo. These results demonstrated the interaction between the cargo inside MSNs and the DNA coated on AuNPs. Zhao and co-workers functionalized MSNs with MPTS, which were further reacted with S-(2-aminoethylthio)-2-thiopyridine. The system was loaded with FITC and capped with DNA composed of 21 random bases (21-mer) via electrostatic interactions with the partially protonated amines on MSNs. A second solid capped with 21-mer DNA labeled with Cy5 was also prepared.213 The addition of reducing agents (GSH and DTT) caused the delivery of FITC- and Cy5-labeled ssDNA as shown in Figure 113. CLSM studies confirmed the internalization and

In a complete work, Lin et al. prepared capped MSNs and studied their use for DNA and drug codelivery into plants.212 In a first attempt, the external surface of MSNs was functionalized with mercaptopropyl moieties. The resulting thiol groups were reacted with 2-(pyridyldisulfanyl) ethylamine, and then FITC was used for labeling MSNs by urea bond formation. Afterward a layer of triethylene glycol was anchored to improve the biocompatibility and suspendability of MSNs (see Figure 112). A DNA plasmid that contained a green fluorescent protein gene was used for coating the solid on the triethylene glycol layer, and its capability for gene transfection was successfully tested in protoplasts. However, the described nanoparticles proved useless for gene transfection in plant tissues by a gene gun system. Thus, in another step, a different material was prepared using MSNs grafted with MPTS and reacted with 2-(pyridyldisulfanyl) ethylamine. Instead of labeling with FITC, MSNs were loaded with fluorescein and capped with AuNPs coated with 11mercaptoundecanoic acid through the formation of amide bonds. Anchored AuNPs were coated with the DNA plasmid. These new materials were successfully tested in tissue plants by gene gun techniques. DNA plasmid internalization produced GFPexpressing foci and was viewed on tobacco cotyledons bombarded with the system. Fluorescein delivery was also observed given the reduction of disulfide bonds by DTT. Similar results were obtained when maize immature embryos were bombarded with the nanomaterial. A third solid was obtained in the same way as the previous one but was loaded with βoestradiol. This solid was tested in transgenic tobacco plants in which GFP expression could be detected only if β-oestradiol was present. As expected, the GFP signal was detected in the transgenic seeds germinated in DTT-containing media, which opened the gate by reducing the chemical linker to thus release

Figure 113. MSNs capped with DNA. Cargo delivery was triggered by GSH or DTT. 618


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triggered disulfide bond cleavage. MTT assay results also showed negligible cytotoxicity of the nanocarrier, which was not loaded with DOX. Du and co-workers prepared MSNs that were first functionalized with 3-isocyanatopropyl moieties. Then the isocyanato groups were reacted with an acridinamine derivative that contained a disulfide linkage. Pores were loaded with calcein. Finally, MSNs were capped by adding a double-stranded DNA (calf thymus DNA), which formed intercalation complexes with the grafted acridinamine moiety.215 Aqueous suspensions of the DNA-capped nanoparticles showed negligible calcein release. Yet in the presence of DTT or GSH, clear cargo delivery was observed due to the cleavage of the disulfide bond and the subsequent surface detachment of the DNA-acridinamine intercalation complex, as depicted in Figure 115. The authors

delivery of the loaded dye and Cy5-labeled ssDNA in HeLa cells. A third solid loaded with DOX instead of FITC was prepared and was also tested in HeLa cells. Clear enhanced cell death was obtained when the DOX-loaded material was used compared with free DOX or the unloaded material. Zhao and co-workers214 reported the preparation of MSNs, also capped with oligonucleotides, in which cargo delivery was triggered by reducing agents or temperature. As in the above case, MSNs were at first functionalized with MPTS and were then further reacted with S-(2-aminoethylthio)-2-thiopyridine. Afterward, functionalized MSNs were reacted with succinic anhydride to introduce a terminal carboxylic acid group. Subsequently, an amino-modified 15-mer oligonucleotide (5′TTA TCG CTG ATT CAA-3′) was grafted onto the surface of the nanoparticles through an amide bond by the EDC/NHS coupling method. After loading mespores with FITC, a long 33mer complementary ssDNA (5′-TTG AAT CAG CGA TAA TCG AAT AGC CAC TAA GTT-3′) was hybridized with the 15-mer oligonucleotide grafted onto the MSN surface and pores were capped through the formation of double-stranded DNA (see Figure114). In vitro release studies showed that a large

Figure 115. Delivery of calcein or DOX from oligonucleotide-capped MSN due to disulfide bond cleavage by DTT.

also found that it was possible to use some other stimuli to control cargo delivery. Particularly at 50 °C, remarkable calcein release was observed unlike the nearly zero delivery at 25 °C. This was the result of the dehybridation of the capping doublestranded DNA upon heating. The DNase I enzyme also induced calcein release due to the hydrolysis of the capping DNA. The DNA-capped nanoparticles loaded with DOX were also tested in A549 cells. The experiments showed a 50% viability reduction as a result of nanoparticles internalization, gate opening induced by intracellular GSH and DOX release. Feng and co-workers functionalized the external surface of MSNs with amino groups. Then they formed a poly(Nacryloxysuccinimide) layer around particles by RAFT polymerization techniques. In another step, pores were blocked by adding cystamine, which cross-linked with the N-oxysuccimide groups of the polymer chain.216 Pores were loaded with Rh B. Gated particles were opened in PBS (pH 7.4) by adding DTT, which reduced the disulfide linkers of the polymeric network (see Figure 116).

Figure 114. MSNs capped with DNA. Reduction of the disulfide bond induced cargo delivery.

amount of FITC was released from the solid in the presence of DTT. Yet when the temperature was kept at 45 °C (higher than the melting temperature of the double-stranded DNA, Tm = 40.3 °C), continuous FITC release was also observed. For the in vitro drug delivery assays, MSNs labeled with FITC were prepared and anticancer drug DOX was loaded. Effective cellular uptake of the DOX-loaded nanocarrier and successful DOX release within HeLa cells were confirmed by CLSM and flow cytometry. Intracellular drug release can be explained by intracellular GSH619


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Figure 116. MSNs capped with poly(N-acryloxysuccinimide) that contained disulfide linkers. Cargo delivery was observed in the presence of DTT.

In a similar way, Liu and co-workers prepared redoxresponsive gated MSNs by coating nanoparticles with a polymeric layer composed of N-(acryloxy)succinimide, oligo(ethylene glycol)monomethylether methacrylate, and a fluorescent methacrylamide derivative of N-(morpholinoethylamino)-4-amino-1,8-naphthalimide.217 Pores were loaded with Rh B. In another step, cystamine was used to cross-link the polymeric shell. Cargo delivery was triggered by the presence of DTT as depicted in Figure 117. Botella et al. prepared poly(lactic-co-glycolic acid) cores loaded with pyrene. These nanoparticles were also covered with an organosilica shell that contained disulfide linkages.218 PBS suspensions of the capped organosilica nanoparticles showed negligible entrapped pyrene release. However, in the presence of DTT, a marked fluorophore release was observed due to the disulfide reduction that degraded the silica shell of nanoparticles, as shown in Figure 118. Meng, Huang, and co-workers grafted biodegradable amphiphilic block polymer poly(ethylene glycol)-β-poly(εcaprolactone) (PEG−PCL) onto the outer surface of MSNs via cleavable disulfide bonds. Pores were loaded with DOX.219 Cargo was locked into the pores through the collapse of the hydrophobic polycaprolactone (PCL) chain, whereas drug release was triggered by the addition of GSH due to the cleavage of the disulfide bonds between PEG−PCL and MSNs (see Figure 119). Studies with HeLa cells showed fast endocytosis and intracellular DOX release from the capped material. Hong and co-workers functionalized the external surface of asmade MSNs with epoxide groups by grafting of GTPMS. Further treatment with HCl extracted the surfactant from pores and converted epoxide moieties into hydroxyl groups. The surface was esterified with S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate. Monomer oligo(ethylene glycol) acrylate and cross-linker N,N′-cystaminebismethacrylamide were copolymerized on the external surface of nanoparticles to yield the final capped material by RAFT (see Figure 120).220 The pores of the solid were loaded with DOX. Aqueous suspensions of loaded nanoparticles in the absence of DTT showed the release of 32% of entrapped DOX after 25 h, probably adsorbed in the copolymer layer. Yet when DTT was present, 85% of drug release was achieved after 10 h. The authors found that nanoparticles were endocytosed by COS-7 cells, and cell viability

Figure 117. MSNs capped with polymers that contained disulfide linkers. Delivery was induced by DTT.

Figure 118. Pyrene delivery from a biodegradable poly(lactic-co-glycolic acid) core coated with an organosilica shell that contained disulfide bonds. Cargo delivery was observed in the presence of DTT.

reduced as a result of entrapped DOX release due to the presence of intracellular GSH. Yang et al. prepared hollow MSNs loaded with DOX and capped with a redox and temperature-responsive copolymer of 2620


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Figure 119. DOX delivery from PEG−PCL-capped MSNs due to disulfide bond cleavage by GSH.

Figure 121. MSNs loaded with DOX and capped with a disulfide containing-copolymer of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate. DOX was released in the presence of GSH or by temperature.

(trimethoxylsilyl)propyl methacrylate to generate an MSN surface with −CC− groups. Then the mesoporous channels were superficially functionalized with carboxylic acid groups by a postsynthesis method with APTES and succinic anhydride. Then the cross-linked polymeric shell was prepared by precipitation polymerization of various monomers (N-vinylcaprolactam, methacrylic acid and N,N′-bis(acryloyl)cystamine) in the presence of functionalized MSNs as seeds. The generated −CC− superficial groups promoted the polymer network attachment to the MSNs surface by radical polymerization. Poly(ethylene glycol) methyl ether methacrylate solution was added to the mixture reaction to avoid nonspecific binding. The polymeric shell formed comprised the thermoresponsive poly(N-vinylcaprolactam) polymer and the pH-sensitive methacrylic acid molecule, both cross-linked by N,N′-bis(acryloyl)cystamine via a disulfide bond (see Figure 122). Under physiological Figure 120. DOX delivery from polymer-capped MSNs due to disulfide bond cleavage by DTT.

(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate cross-linked by disulfide bonds.221 Nanoparticles were able to retain DOX below LCST (39 °C), yet a release was observed at 41 °C. Furthermore, when temperature was adjusted to 37 °C, DOX release was triggered upon the addition of GSH, which cleaved the disulfide bonds of the copolymer, as shown in Figure 121. FITC-labeled and capped nanoparticles without DOX showed good internalization and viability in HeLa and HEK293 cells. The DOX-loaded solids showed similar toxicity to free DOX in the MTT assays, even though probably not all the encapsulated DOX was released. Yang and co-workers222 used a thermo/pH-responsive polymeric shell with a disulfide-cross-linked network as a gatekeeper in MSNs. MSNs were first functionalized with 3-

Figure 122. DOX delivery from MSNs capped with a polymer anchored with disulfide bonds. Cargo was delivered with GSH. 621


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conditions, the polymeric shell was found to be in a swollen state at neutral pH and in a collapsed state at pH 5. The swelling and shrinking temperature of poly(N-vinylcaprolactam) was near 37 °C. MSNs were loaded with DOX. The authors studied the influence of pH on the volume phase transition temperature of the polymeric shell by measuring changes in the hydrodynamic diameter by dynamic light scattering. The volume phase transition temperature values at pH 7.4, 6.5, and 5 were 40.5, 35.5, and 32.0 °C, which was above, near, and below the physiological temperature, respectively. Release studies revealed that negligible DOX was delivered below volume phase transition temperature, and a rapid release was observed above volume phase transition temperature. Faster massive drug release was observed after applying simultaneously pH 6.5 and 10 mM of GSH, which mimicked that of intracellular cytosol compartments. The DOX release from the nanocarrier after its cellular uptake by MCF-7 cells was evaluated by CLSM. The results showed that nanoparticles efficiently released DOX into the nucleus after only 0.5 h, likely due to the low pH and the presence of reducing agent GSH in lysosomes. The in vitro cytotoxicity against HEK 293 cells and MCF-7 cancer cells were tested by the MTT assay. The DOX-loaded nanocarrier displayed high cytotoxicity efficiency killing MCF-7 cancer cells. Qiao et al. used MSNs that were functionalized with GTPMS. Then low-molecular-weight PEI was grafted onto the external surface by an addition reaction between the amino moieties on the polymer and the epoxy groups on the silica surface. Pores were loaded with siRNA.223 The external surface was coated through electrostatic interactions between the negatively charged surface and the positively charged polymer upon the addition of short-branched PEI that was cross-linked with an acetaldehydecystine (AC) derivative. DMEM with 10% FBS suspensions of the capped nanoparticles in the absence of GSH showed negligible entrapped siRNA release. As a clear contrast, marked siRNA delivery was observed in the presence of GSH, which was ascribed to the reduction of disulfide bonds as depicted in Figure 123. Suspensions of coated nanoparticles at pH 7.2 also showed negligible cargo release, whereas moderate siRNA delivery was observed due to the pH-induced hydrolysis of the imine bonds in the polymer coating at pH 5. The system’s intracellular behavior was monitored in osteosarcoma KHOS cells at different time intervals by CLSM and flow cytometry. Due to AC-PEI degradation at endosomal acidic pH and cytoplasmic GSH, good internalization and controlled siRNA release were detected in KHOS cells. Wang and co-workers developed a redox-responsive drug delivery system based on MSNs modified with HA.224 Nanoparticles were functionalized with thiol groups by a cocondensation process. Thiol groups were treated with thiopyridyldisulfide and then replaced with a reaction with S-(2aminoethylthio)-2-thiopyridine hydrochloride to obtain a solid with amino moieties attached onto the silica surface via disulfide bonds. Anticancer drug 6-mercaptopurine was immobilized by disulfide bonds inside pores in a disulfide bond exchange reaction by replacing the 2-aminoethanol groups by the drug. Finally HA was grafted onto the surface by an EDC amide formation reaction with the remaining nonreplaced amino moieties to cap pores. HA is known to target CD44 receptors of the cell membrane. GSH was able to reduce the disulfide bond releasing both HA on the surface and the 6-mercaptopurine molecules inside the pores of the material (see Figure 124). The hemolytic behavior of the final capped solid was studied. Very low hemolysis was observed, especially compared with the hemolysis

Figure 123. siRNA delivery from MSNs capped with a polymer. Cargo delivery was observed in the presence of GSH and/or pH changes.

Figure 124. 6-Mercaptopurine delivery from HA-capped MSNs due to disulfide bond cleavage by GSH.

percentages obtained with initial thiol-modified MSNs. Cellular uptake was studied in the HCT-116 and NIH-3T3 cell lines. HCT-116 cells showed high expression levels of CD44 receptors, but the NIH-3T3 cells did not. In order to follow internalization, an FITC derivative was anchored to the final capped solid and to the starting thiol-modified MSNs (as control). Mean fluorescence intensity studies revealed a 1.8-fold increase in fluorescence for HCT-116 cells over NIH-3T3 using the final capped solid, while no significant differences were observed between both cell lines with thiol-modified MSNs. The experiments done with HCT cells in the presence of free HA or at a low temperature resulted in reduced fluorescence due to 622


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propylsiloxane-containing nanoparticles were modified with 2,3-dichloro-1,4-naphthoquinone to yield 2-amino-3-chloronaphthoquinone functionalized MSNs. Afterward, eosin Y dye was loaded into pores, and the system was capped by donor− acceptor interactions between eosin Y and the quinone units, which blocked pores. The reduction of the quinone groups by ascorbic acid to the chloronaphthalene hydroquinone donor groups opened the nanovalves due to the disruption of the original donor−acceptor interactions (see Figure 126) and also

the competition reaction or thermal inhibition of uptake, respectively. This demonstrated that the enhanced cellular uptake in HCT cells was related with the HA molecules anchored to the material surface. CLSM studies confirmed these results. Cytotoxicity assays demonstrated that increased cell death was achieved only in HCT-116 cells when the capped solid was used compared with treatment with free 6-mercaptopurine. 5.3. Miscellaneous

Yang, Wang, and co-workers used a mixture of polyelectrolyte multilayers and an aptamer as capping agent.225 In particular, MSNs were loaded with FITC and nanoparticles were covered by the sequential deposition of layers of PVPON and thiolated poly(methacrylic acid) (PMA). In another step, a thiolated version of cancer cell-specific DNA aptamer sgc8 was included. The aptamer was coupled to the PVPON/PMA multilayer forming a disulfide bond with free thiols from the PMA shell (see Figure 125). Negligible FITC release took place at pH 7.4 and

Figure 126. Eosin Y delivery from naphtoquinone-capped MSNs. Quinone-hydroquinone transformation was reversible and was achieved using ascorbic acid as the reducing agent and K3Fe(CN)6 as the oxidizing agent.

to the enhanced hydrophilicity of the capping valve. Subsequently, it was possible to reload pores with eosin Y, and capping was achieved by the oxidation of hydroquinone to quinone using Fe(CN)63− as an oxidizing agent. To assess the potential application of the developed nanocarrier, the authors loaded MSNs with DOX and unblocked pores by a NADH cofactor-mediated reduction of the quinone units to the hydroquinone state. Amino-functionalized MSNs were also grafted with dye FITC to confirm the effective endocytosis of capped MSNs into normal HEK-293 cells and cancer MDA-231 and Hep G2 cells. The respective cells were incubated with the labeled nanoparticles and antifluorescein antibodies were added to the cell culture to quench the fluorescence of the nanoparticles outside cells. CLSM analysis indicated that the endocytosis of the FITC-labeled capped MSNs was successful. Finally, bare MSNs, the quinone-modified MSNs and the hydroquinone-functionalized MSNs showed no cytotoxicity effect on HEK-293 or in the breast cancer MDA-231 cells when Alamar blue was employed as an imaging agent. Paramagnetic manganese oxide (Mn3O4) nanoparticles were used as stoppers in the development of a gated theranostic system.227 MSNs were first functionalized with aminopropyl moieties, and then the amino groups were reacted with succinic anhydride. The pores of the hybrid material were loaded with CPT and capped upon the addition of amino-functionalized Mn3O4 nanoparticles through the formation of amide bonds as depicted in Figure 127. PBS solutions of the gated MSNs showed negligible CPT release in the absence of any reducing agent. In contrast, marked drug delivery was observed in the presence of DTT or GSH. The observed release was ascribed to a progressive dissolution of the Mn3O4 nanoparticles, induced by the reductor to yield Mn2+. The same capped material, but loaded this time with Rh 6G, was prepared and incubated with BxPC-3 cells.

Figure 125. MSNs capped with polyelectrolyte multilayers. Cargo release was triggered by DTT.

pH 5.5 in the absence of DTT, while FITC release was clearly detected due to the disulfide linkage rupture upon the addition of the reducing agent. To validate the system as an efficient drugtargeted delivery carrier in tumor cells, HeLa cell cultures were incubated with the capped MSNs, either conjugated or not with aptamer sgc8, and cell uptake was quantified by flow cytometry. Significantly enhanced fluorescence was detected in the cells treated with the nanoparticles that contained the aptamer compared to those treated with the nanoparticles with no aptamer. This result suggested increased uptake of nanocarriers by cancer cells, favored by efficient aptamer-induced endocytosis. The authors also prepared similar capped nanoparticles, but this time loaded with DOX. Significantly increased cytotoxicity was detected in the HeLa cells treated with DOX-loaded capped MSNs that contained the sgc8 aptamer. The enhanced toxicity in HeLa cells found for the nanoparticles with the sgc8 aptamer was less evident when the materials were tested in QGY7703 hepatoma cells. Willner et al.226 developed a redox-triggered release system by using quinone units as capping agent in MSNs. Amino623


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uncapping the system and inducing cargo release in both the Rh 6G and CPT loaded solids. In order to study the therapeutic potential of these materials, cytotoxicity effects were studied by MTT viability assays on the pancreatic cell line BxPC-3 using the CPT-loaded capped solid and the corresponding unloaded solid as a model. The cell viability significantly reduced in the presence of the CPT-loaded capped nanoparticles. Lee, Kim, and co-workers229 developed MSNs, where conformational changes of peptides were used to uncap pores. MSNs were functionalized on the outer surface with APTES, which was then reacted with propargyl bromide to introduce an alkyne unit. Subsequently, calcein molecules were loaded into mesopores, and the model peptide Fmoc-CGGC-azide (Fmoc, fluorenylmethyloxycarbonyl chloride) was conjugated onto the surface by a click reaction with microwave irradiation. The model peptide blocked pores due to a turnlike conformation induced by the intramolecular disulfide bond between two cysteine units, as shown in Figure 129. Cargo release was triggered by the

Figure 127. CPT delivery from Mn3O4 nanoparticles capped MSNs due to reduction of Mn3O4 by DTT.

CLSM studies revealed that nanoparticles were uptaken by cells, and that the reducing environment in the cytosol was able to induce entrapped dye release. Zhu and co-workers also used the concept of dissolving inorganic caps in the presence of reducing agents to design gated nanoparticles. In particular, these authors prepared CeO2 nanoparticles stabilized with citric acid and then used them as caps on amine-functionalized MSNs via amide formation by EDC chemistry.228 Pores were loaded with Rh 6G or CPT. Redox active species, such as GSH or vitamin C, were used as triggers to uncap the material (see Figure 128). The presence of these species led to the dissolution of the CeO2 nanoparticles

Figure 129. Calcein delivery from peptide (CGGC)-capped MSNs due to disulfide bond cleavage by DTT.

conformational conversion of the peptide by reducing the intramolecular disulfide bond. Release experiments demonstrated that the nanocarrier exhibited a zero-release, whereas cargo delivery was clearly observed upon the addition of GSH. In the same work, the authors prepared a material capped with peptide sequence Fmoc-CGGC, which adopted a turn structure by chelation of a Zn(II) ion by the two thiol groups of CGGC. The system showed negligible cargo release in PBS. Addition of EDTA, to remove the Zn(II) ion from Fmoc-CGGC-Zn, induced a conformational change from a turn to a random structure, and guest molecules were released.

Figure 128. Delivery of CPT or Rh 6G from MSNs capped with CeO2 triggered by GSH or ascorbic acid. 624


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6. pH pH is the most popular stimulus used to develop gated materials. In the reported examples, the open/close paradigm usually relies on changes in size/shape or attraction/repulsion interactions with other charged species due to proton addition or abstraction, the use of groups that can be hydrolyzed with pH or coatings that can be dissolved upon pH changes. Hence, researchers have used a wide variety of imaginative ensembles including amines, metallic complexes, macrocycles, different polymers, supramolecular ensembles such as layer-by-layer coatings, biomolecules as DNA, proteins, lipid bilayers, and even inorganic nanoparticles to control the release of selected cargos. pHDriven gates are a promising tool to develop delivery systems for biomedicine. At the intracellular level, pH difference between the extracellular environment (pH 7) and endosomes and lysosomes (pH 5) is a useful tool to selectively release therapeutic agents directly in cells. Also, the acidic microenvironment found in many types of tumors, due to the production of lactic acid and hydrolysis of ATP, represents a pH difference between normal and tumor tissues that can be used for targeted delivery of payloads. pH-Triggered systems offer the advantage of being totally autonomous, not requiring any extra equipment, and in most cases, the closing and opening mechanism is reversible. However, in biomedicine, as the usual MSNs cell entrance pathway is via endocytosis, it is difficult to avoid, if needed, the involuntary opening of the gated system in the lysosomes. Furthermore, pH-driven systems are not restricted to the biomedical field, and a number of applications may take advantage of a selective payload delivery when a change in pH takes place.

Figure 130. Micrometric MCM-41 support functionalized with polyamines on the outer surface and with mercaptopropyl chains on the inner surface. Changes in pH media controlled squaraine access to pores.

́ Martinez-Má ñez and co-workers also developed similar pHresponsive polyamine-containing capped materials for the delivery of tebuconazol and suggested its potential application to treat candidiasis. In this case, MSNs were used as an inorganic scaffold and the external surface was also functionalized with 3[2-(2-aminoethylamino)ethylamino]propylmethoxysilane. The system was tightly capped at an acidic pH (3.7), whereas tebuconazol delivery was observed at pH 5.5 (see Figure 131 A).232 Intracellular cargo release and cell viability studies were

6.1. Polyamines

́ Martinez-Má ñez et al.230 described one of the first examples of a pH-controlled gated silica mesoporous support. This example was also the first reported capped system to work in an aqueous environment. Whereas most published examples in gated materials are used for delivery applications, this example focused on using a gated system to control the entry of a certain molecule (a squaraine dye) to the inner pores of the mesoporous scaffold. The authors used a micrometric MCM-41 support that was functionalized on the outer surface with 3-[2-(2aminoethylamino)ethylamino]propylmethoxysilane (a linear polyamine) and with mercaptopropyl chains grafted inside pores (see Figure 130). The entry of the dye into pores was indicated by the reaction of squaraine (blue) with the thiol groups placed inside pores, which gave a colorless derivative. At an acidic pH, the nitrogen atoms of the polyamine molecules were protonated and adopted a rigid-like conformation due to the Coulombic repulsions between the ammonium groups. This resulted in pore blockage and inhibited squaraine access to the thiol moieties (the solution remained blue). However, at a neutral pH, amines were partly protonated, adopted a more flexible conformation, and squaraine dye was able to enter into inner pores (the solution turned colorless). A synergic anioncontrolled outcome also resulted from the interaction between protonated amines and certain anions. For instance, the presence of large anions, such as ATP, also helped inhibit squaraine access to pores due to the formation of strong bulky ATPpolyammonium complexes. In another step, the same authors applied this molecular gate based on polyamines to develop a mesoporous silica support for the controlled release of vitamin B2.231

Figure 131. Mesoporous silica particles loaded with (A) tebuconazole, (B) garlic extract, or (C) IBU and capped with polyamines. 625


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carried out to analyze the behavior of the system by employing S. cerevisiae, a microorganism that survives in acidic environments, and perfectly suits the aperture mechanism of the nanomaterial. Clonogenic cell viability studies demonstrated the nontoxicity of the material. Moreover, the fluorescein-loaded system efficiently internalized in yeast cells and brought about the subsequent cargo delivery due to pH 5.5, as judged by fluorescence microscopy studies. Interestingly, when the system was loaded with antifungal drug tebuconazole, a 9-fold increase in tebuconazole cytotoxicity was obtained, compared with that of free tebuconazole. Therefore, the designed nanoformulation considerably improved tebuconazole efficacy and could potentially overcome the adverse side effects associated with topical therapies for vulvovaginal infection. On the basis of a similar concept, these authors also incorporated silica mesoporous particles functionalized with (2-aminoethylamino)ethylamino]propyltrimethoxysilane into polyamide nanofibrous membranes for the on-command release of garlic extracts (see Figure 131B).233 In particular, the capped material was incorporated into a nylon-6 membrane by electrospinning yielding the final composite. Aqueous suspensions of the composite at pH 2 showed negligible cargo release, while remarkable delivery took place at pH 7. By a similar approach, Gao and co-workers prepared mesoporous silica particles functionalized on the outer surface with polyamines and loaded with IBU (Figure 131C).234 Aqueous suspensions of particles at pH 4 showed a negligible entrapped IBU release, whereas cargo release was observed when changed to a neutral pH. Roik et al.235 functionalized mesoporous silica supports with (3-chloropropyl) triethoxysilane, which was further reacted with 2-aminodiphenylamine. The solid was loaded with 4-aminobenzoic acid. At a neutral pH, the N-[N′-(N′-phenyl)-2aminophenyl]-3-aminopropyl groups blocked the entry of pores, and no 4-aminobenzoic acid release was observed. However, at pH 1, repulsions of the protonated N-[N′-(N′phenyl)-2-aminophenyl]-3-aminopropyl groups allowed the release of the aromatic amino acid from the interior of pores (Figure 132). Bein and co-workers prepared MSNs, which were loaded with IBU, and the surface was functionalized with APTES. Then part of the amino moieties reacted with 4-sulphophenyl isothiocyanate. At an acidic pH, the material was capped due to the

electrostatic interactions of the protonated remaining amino moieties and the sulphonyl groups. At a neutral pH, this interaction disappeared, and the cargo was delivered (Figure 133).236

Figure 133. MSNs loaded with IBU and capped due to electrostatic interactions. Cargo release was observed at a neutral pH.

6.2. Metal Complexes

Jung et al. used metal−ligand coordination complexes as molecular caps to develop pH-triggered gated materials.237 For this work, hollow MSNs were functionalized with a phenanthroline derivative and were further loaded with curcumin. Pores were capped through the complexation of phenanthroline moieties with Cu2+ (Figure 134). The protonation of nitrogen

Figure 134. Hollow MSNs loaded with curcumin and capped with coordination complexes. Cargo release was observed at an acidic pH due to the cleavage of coordination bonds.

atoms at an acidic pH led complexes to rupture. The fluorescence emission of curcumin was negligible at pH 7, whereas the curcumin release rate increased as pH lowered. Zink and co-workers loaded MSNs with Hoechst 3342 and functionalized the external surface with chelating subunit iminodiacetate. Addition of metal cations, such as Co2+, Ni2+, and Ca2+, induced pore blockage at a neutral pH due to the formation of M2+-(iminodiacetate)2 complexes on the top of

Figure 132. Mesoporous silica support loaded with 4-aminobenzoic acid and functionalized with N-[N′-(N′-phenyl)-2-aminophenyl]-3aminopropyl groups. Cargo release was observed at an acidic pH. 626


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pores (see Figure 135).238 When adjusting pH to 5, marked delivery of the entrapped cargo was observed. This release was

Figure 135. MSNs loaded with Hoechst 3342 and capped with M2+(iminodiacetate)2 complexes. Cargo release was observed at an acidic pH or by adding 2,2′-bipyridine.

Figure 136. MSNs loaded with Ru(bipy)32+ and capped by coordination complexes. Cargo release was observed at an acidic pH.

the result of the protonation of the iminodiacetate coordinating subunit that induced the rupture of complexes. With the Co2+and Ni2+-capped solids, pores were also open when 2,2′bipyridine was added due to a displacement reaction caused by the formation of the corresponding Co2+- or Ni2+-bipyridine complexes. Du and co-workers also used metal chelating interactions to develop pH-triggered gated materials.239 These authors functionalized MSNs with different amounts of chelating ligand N-(3-trimethoxysilylpropyl)ethylenediamine triacetate trisodium salt (ADTA), and pores were loaded with Ru(bipy)32+. The system was finally capped upon the addition of Cu2+ due to the formation of complexes between this cation and ADTA. Aqueous suspensions of the capped nanoparticles at pH 7.2 showed a very poor dye release, the amount of which depended on the quantity of the chelating ADTA subunits grafted onto the outer surface of nanoparticles. In contrast, when the pH was lowered to 3, marked delivery was observed. Delivery was clearly related with the protonation of the carboxylate moieties of the chelating ADTA groups, which induced demetalation. Strikingly similar Ru(bipy)32+ delivery profiles were obtained when Cu2+ was substituted for the Ni2+ cation. In order to obtain a real “zero release” material, the authors functionalized the external surface of MSNs with a small amount of ADTA, loaded the material with Ru(bipy)32+, and then partially blocked pores upon the addition of the Cu2+ cation. Finally, addition of myoglobin, a protein with five His residues on its outer surface, completely capped pores via coordination with the Cu2+ cation on the surface (see Figure 136). Aqueous suspensions of the myoglobin-capped nanoparticles showed a zero release at pH 7.2, but remarkable cargo delivery occurred at pH 3. Once again, the release was ascribed to the protonation of the carboxylate moieties of the chelating groups, with the subsequent removal of the Cu2+ cation and myoglobin. In order to test the potential biological applications

of the myoglobin-capped nanoparticles, a similar material loaded with DOX was prepared. MTT assays carried out with these capped MSNs and A549 cells showed significantly diminished cell viability (20%) due to the internalization of nanoparticles and DOX release. Chan et al. prepared core−shell nanoparticles that contained an Fe3O4 core and a mesoporous silica shell, which was functionalized with APTES and a silanized derivative of FA. Pores were loaded with DOX and capped upon the addition of ferrocenecarboxaldehyde through the formation of acid-labile imine bonds with the amine moieties on the silica surface (see Figure 137).240 The aqueous suspensions of nanoparticles

Figure 137. Fe3O4 nanoparticles coated with a mesoporous silica shell loaded with DOX. Cargo release was observed at an acidic pH due to the hydrolysis of imine bonds. 627


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showed negligible cargo release at pH 7.4, whereas moderate DOX delivery (46% after 3 h) was observed at pH 4.5. Release was ascribed to the hydrolysis of imine bonds. MTT studies carried out with the capped nanoparticles and MCF-7 cells showed markedly reduced cell viability after the internalization of nanoparticles. Moreover, nuclear fragmentation and condensation were observed for most of the cells treated with capped nanoparticles. Both facts are indicative of nanoparticle uptake and pore opening inside MCF-7 cells. Coordination polymers have also been recently used as gatekeepers to prepare pH-triggered gated nanodevices.241 Che and co-workers used MSNs whose external surface was functionalized with aminopropyl moieties. Pores were loaded with topoisomerase I inhibitor topotecan. The system was blocked upon the addition of Zn2+ cation and 1,4-bis(imidazol-1ylmethyl) benzene due to the formation of a coordination polymer shell around nanoparticles (Figure 138). Aqueous

Figure 139. MSNs loaded with PI and capped with CB[6] by host− guest inclusion complex formation. At pH 10, Rh B was released due to the dethreading of the inclusion complex.

surface and the first amino group (ethyl or propyl spacers), they evidenced that shorter chains retained the CB[6] cap closer to the surface and prevented better premature dye release.242 In another work, the same authors functionalized MSNs with tris-ammonium stalks that ended with an aniline (Figure 140A)

Figure 138. MSNs loaded with topotecan and capped by a coordination polymer shell. Delivery was observed at an acidic pH.

suspensions of the capped MSNs showed negligible cargo release at pH 7.4, whereas moderate delivery was observed at pH 4 (40% of the loaded dye). The delivery observed at an acidic pH was ascribed to the rupture of the coordination polymer shell. Coated nanoparticles were internalized well by endocytosis in HeLa cells, and a remarkable cell growth inhibition due to the delivery of the entrapped drug took place. 6.3. Macrocyles

6.3.1. Cucurbiturils. Zink, Stoddart, and co-workers loaded MSNs with Rh B dye and functionalized the silica surface with a dialkylammine derivative that contained a 1,2,3-triazole group as a spacer. Moreover, CB[6] was used to cap pores by the formation of inclusion complexes with amine moieties. At a neutral pH, the dialkylammine groups were partially protonated and the inclusion complex with CB[6] remained stable. Conversely, when the solution was basified at pH 10, the ammonium groups deprotonated by switching off the ion− dipole interactions, dethreading the inclusion complex and allowing dye delivery (see Figure 139). When the authors used two different lengths in the alkylic spacer between the silica

Figure 140. MSNs loaded with Rh B and capped with CB[6] by host− guest inclusion complex formation. Cargo delivery was observed at both an acidic and a basic pH. The figure shows delivery at a basic pH. 628


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or p-anisidine (Figure 140B) group. Pores were loaded with PI dye and finally capped with CB[6] by the formation of inclusion complexes with the protonated amino moieties.243 When the medium was acidified, aniline or p-anisidine became protonated, which resulted in the displacement of the CB[6] molecule from the alkyl protonated nitrogens to the anilinium or p-anisodinium groups. Aniline is less basic than p-anisidine. Maximum dye delivery occurred at pH ca. 3.4 for the anilinium-ended nanoparticles, whereas maximum delivery took place at pH 5 for the p-anisidinium-containing derivative. At a pH over 10, all the amines were deprotonated, which dethreaded the inclusion complex, and cargo delivery was observed. The controlled release of coumarin 460 from the pores of the solid was also demonstrated when different bases were present (i.e., trimethylamine > N,N′-diisopropylethylamine > hexamethylphosphoroustriamide). Zink, Stoddart, and co-workers combined both previously described pH-responsive molecular gate systems and MSNs internally functionalizated with light-responsive nanoimpellers. For this purpose, MCM-41-type MSNs were functionalized with a photoresponsive azobenzene derivative (i.e., 4-phenylazoaniline coupled to 3-triethoxysylilpropyl isocyanate) over the whole inner and outer surfaces by a co-condensation procedure.244 Pores were loaded with the complex Cl−Re(CO)3-2,2′bipyridine. Finally, two different solids were prepared. The first one was functionalized with a dialkylammonium derivative that contained a 1,2,3-triazole group as a spacer.242 The second solid was functionalized with a dialkylammonium derivative with an aniline terminal group. CB[6] was used to cap pores through coordination with the bisalkylammonium groups.243 The first solid was responsive to basic conditions and the second to acidic conditions, while nanoimpellers responded to light with a conformational change (trans to cis conformation). The two solids exhibited the same behavior: dye delivery occurred only when the two stimuli acted simultaneously (change in pH, plus light irradiation at 448 nm). This system acted as an AND logic operation molecular gate. Another pH-responsive system developed by Zink and Stoddart was based on MSNs with stalks that contained a dialkyl-4,4′-bipyridinium moiety and ended with a carboxylate functional group. The pores of the inorganic scaffold were loaded with Rh B. Pores were capped by adding CB[6] due to the formation of the inclusion complexes between CB[6] and the dialkyl-4,4′-bipyridinium moieties on stalks (Figure 141).245 Aqueous suspensions of these pseudorotaxane-functionalized nanoparticles showed negligible entrapped cargo release. However, at pH 4, a remarkable cargo delivery of Rh B was observed. Delivery was due to the protonation of the carboxylate moiety, which induced the movement of the CB[6] unit from the dialkyl-4,4′-bipyridinium group to the formed carboxylic acid. Chen and Fu developed several pH-responsive gated systems and used them to prepare smart anticorrosion coatings. The corrosion process is usually accompanied by pH changes in microcathode and microanode areas.246 Bearing this idea in mind, in a preliminary work, the authors used hollow MSNs, which were functionalized on the surface with butanediamine moieties and loaded with benzotriazole (BTA), which acted as an anticorrosion agent (Figure 142). At pH 7, butanediamine moieties were partially protonated and were able to coordinate with CB[6] rings capping pores. CB[6] rings were dethreaded when the pH increased, which resulted in BTA delivery. By taking a further step, Chen and Fu implemented the basic responsive system described before and a second acid responsive

Figure 141. MSNs loaded with Rh B and capped with CB[6] by host− guest inclusion complex formation. Rh B was released at an acidic pH.

Figure 142. MSNs loaded with BTA and capped with CB[6] by host− guest inclusion complex formation. At a basic pH, BTA was released due to the detachment of CB[6].

one in an anticorrosion coating over alluminium alloys.247 The second system used hollow MSNs, which were functionalized by aniline moieties, loaded with BTA and capped with α-CDs (see Figure 147 C). As in other examples, the protonation of amine groups at an acidic pH caused the dethreading of α-CDs and the delivery of BTA (see α-CD in Figure 1a). The two capped nanoparticles were introduced into a hybrid zirconia-silica sol gel coating and deposited on an aluminum AA2024 alloy sample. Inhibition of the corrosion process was studied by electrochemical impedance spectrometry and surface electron microscopy. A remarkable decrease in corrosion was observed when the coatings that contained the pH-responsive gated nanoparticles were used. The same authors248 developed another anticorrosion coating, which consisted in pH-responsive hollow MSNs, responsive to 629


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experimental results obtained with scanning vibrating electrode techniques demonstrated that acid and alkaline dual stimuliresponsive delivery simultaneously suppressed corrosion activities in microanodic and microcathodic regions. In another work,249 these authors used the same gated nanoparticles, but loaded with GEM, which remained capped at pH 6.5 for drug deliver applications for cancer treatments. Addition of certain quantities of acid gradually increased the amount of GEM delivered, which achieved maximum release at pH 2. The addition of bases led to a more marked GEM release, with smaller increments in pH. The same authors also developed a similar system which was able to respond to acidic and basic conditions, but no anticorrosion properties were evaluated in this case. They functionalized MSNs with APTES, and the resulting material was further reacted with 1,6-dibromohexane. Subsequently, 1,6bis(pyridinium)hexane units were attached to the solid by a nucleophilic substitution reaction. The material was loaded with BTA and capped with CB[6] (Figure 144).250 The macrocycle

both basic and acidic conditions, which were deposited on aluminum alloy AA2024. In this case, caffeine was selected as the anticorrosion agent. The authors functionalized hollow MSNs with chloromethyl trimethoxysilane, which was subsequently reacted with 1,6-hexanediamine and finally coupled with ferrocene dicarboxylic acid. Caffeine molecules were loaded, and pores were capped with CB[7] (Figure 143). At a neutral

Figure 144. MSNs loaded with BTA and capped with CB[6] by host− guest inclusion complex formation. BTA was released at a basic pH. Figure 143. MSNs loaded with caffeine or GEM, and capped with CB[7] by host−guest inclusion complexes. At a basic pH, cargo was released due to the detachment of CB[7]. At an acidic pH, cargo was released due to the displacement of CB[7] to the ferrocene coordination site.

preferentially coordinated the 1,6-hexanediammonium (HDA) groups rather than the 1,6-bis(pyridinium)hexane (BPH) moieties, and pores were capped. At a basic pH, the deprotonation of the ammonium moieties weakened the affinity between CB[6] and the HAD stalk, which induced the migration of the macrocycle to the BPH group to allow cargo delivery. The higher the pH, the more rapid the BTA release. These authors also found that it was possible to restore the affinity between CB[6] and HAD by adjusting pH from alkaline to acidic. A reversible movement of CB[6] between HDA and BPH was demonstrated by the multistage pH-controlled release experiments performed at pH 4.5 and 9. The authors also proved that systems can be reloaded. Gao, Yang, and co-workers251 developed CB[7]-capped LbLcoated MSNs, which were uncapped by changes in pH or by adding competitive agents. The authors prepared two types of MSNs with different pore sizes: (i) a traditional silica mesoporous solid with a ca. 2.7 nm pore diameter and (ii) a

pH, CB[7] was coordinated to the partially protonated amines from the alkylic chain near the silica surface and pores were blocked. The more stable complex with ferrocene was unable to be formed due to the electrostatic repulsion between CB[7] and the carboxylate moiety within the ferrocene framework. When pH was acidified, the carboxylate group protonated, and CB[7] displaced to form a complex with ferrocene, which unblocked pores. Moreover when pH was basified, the amino moieties of the alkylic chain deprotonated, which induced CB[7] to leak to the solution. Corrosion experiments showed that the presence of the capped nanocontainers not only delayed the penetration rate of corrosive species but also repaired damaged aluminum oxide layers and allowed long-term anticorrosion behavior. The 630


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mesoporous support with an expanded pore of ca. 5 nm. DOXor PI-loaded MSNs were first coated with positively charged polyelectrolyte ethanediamine poly(glycerol methacrylate) (EDA-PGOHMA) via the electrostatic interactions with the negatively charged surface. Subsequently, supramolecular multilayer coatings were sequentially prepared by using CB[7]. In the LbL coating process, CB[7] acted as a molecular bridge as it held two different bis-aminated polymeric layers together by host− guest interactions between CB[7] and EDA-PGOHMA (Figure 145). At a neutral pH (7.4), the system remained capped. Upon

Figure 146. MSNs loaded with calcein and capped with α-CDs or γ-CDs by host−guest inclusion complex formation. At an acidic pH, calcein was released due to the detachment of CB[6].

protonation of the amino groups, which interacted very weakly with CDs. Zink, Liao, Stoddart, and co-workers functionalized hollow MSNs with aniline (with two different alkylic chain lengths) or panisidine moieties and loaded the supports with PI or Hoechst 33342, which resulted in six different materials. Pores were capped when α-CDs were added, which coordinated to the aniline or anisidine groups (Figure 147, panels A and B).253 While pH remained at 7, no appreciable dye release occurred in

Figure 145. MSNs loaded with DOX or PI, coated with ethanediamine poly(glycerol methacrylate), and capped by the formation of inclusion complexes with CB[7]. Cargo delivery was induced at an acidic pH.

acidification, CB[7]s formed stronger ion−dipole interactions with hydronium ions, which resulted in the dissociation of the supramolecular multilayers, pores being uncapped, and cargo release. The addition of adamantane amine hydrochloride (AH) also induced the rupture of the supramolecular polymeric coating due to the preferential coordination of CB[7] with AH. Capped MSNs were tested in PBS with buffers of increasing acidity (pH 7.4, 5, and 2). Negligible DOX release was observed at a neutral pH, but fast DOX delivery took place at a lower pH because the polymeric coating broke. Remarkably, MSNs with larger pores showed a lower specific cargo-loading capacity because of the relatively less specific internal area, despite its larger pore size. Cell viability tests of the nanomaterials in MCF-7 cells demonstrated that no significant cytotoxicity was detected when concentrations of up to 1 mg/mL were used. Moreover further studies showed that the DOX-loaded system was toxic to cells. 6.3.2. Cyclodextrins. Kim et al. reported a first example of using CDs for developing pH-responsive molecular gates. MSNs functionalized with a low-molecular-weight linear PEI were loaded with calcein dye and capped with α-CDs or γ-CDs at pH 11.252 Capping was due to the formation of the stable polypseudorotaxane complexes between CDs and PEI stalks (see Figure 146). Water suspensions of capped nanoparticles at pH 11 showed no calcein delivery, whereas remarkable cargo release was observed when pH was lowered to 5.5. This was related with the dethreading of α-CDs or γ-CDs as a result of the

Figure 147. MSNs loaded with different cargos and capped with α-CD or β-CD by host−guest inclusion complexes with superficial aniline moieties. An acidic pH induced cargo delivery, except for DB24C8 and CB[6], which were uncapped at a basic pH. 631


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complex and cargo release (see Figure 148). Hoechst and DOX release were tested in THP-1 and KB-31 cell lines before and

any of the prepared materials, whereas the addition of HCl caused the leakage of α-CDs and consequent cargo delivery. MSNs functionalized with aniline moieties showed massive delivery at pH 5, and an abrupt increase when pH was lowered from 6 to 5, while the materials functionalized with p-anisidine smoothly modulated cargo delivery, which increased within the pH 5−7 range. This behavior was explained by the fact that the pKa of p-anisidine was around one unit more basic than p-aniline. Zink and co-workers also studied the incorporation of polymers into CD-capped MSNs to improve the dispersion of particles. MSNs were functionalized with aniline moieties and methyl phosphonate groups, loaded with Hoechst 33342 and modified with the corresponding polymers (vide infra) through a reaction with the phosphonate groups.254 Finally, the system was capped with α-CDs via the formation of inclusion complexes with aniline moieties (see again Figure 147, panels A and B). Two kinds of polymers were used; first, a material was functionalized with PEI, and the second one by a copolymer formed by PEI and PEG. When nanoparticles were at a neutral pH, the system was still closed, although the inclusion complex in an acidic medium was dethreaded, which caused cargo release. Song and co-workers functionalized MSNs with p-anisidine groups.255 Pores were loaded with PI and were capped with αCDs. The gated solid was unable to release the entrapped PI in water at pH 7. However, cargo delivery was observed when pH was lowered to 5.5 (see Figure 147 B). These authors demonstrated that the uncapping mechanism also worked in a highly competitive biological medium, such as DMEM. In another work, Zink and co-workers tested five different pHresponsive mesoporous silica particles, all of which were capped with α-CD.256 In particular, the authors selected the following as inorganic scaffolds: (i) 2D hexagonal MCM-41, (ii) swollen pore MCM-41, (iii) rodlike MCM-41, (iv) hollow mesoporous nanoparticles, and (v) radial mesoporous nanoparticles. All five solids were functionalized with (N-phenylaminomethyl)triethoxysilane, loaded with Hoescht 33342, and capped by the addition of α-CDs (see Figure 147 C). The five materials showed negligible dye release at a neutral pH and different delivery profiles at an acidic pH (1 and 3) due to the dethreading of αCDs upon phenylaminomethyl protonation. The authors concluded that the uptake capacities correlated with the specific surface area of the inorganic scaffold. The radial mesoporous nanoparticles displayed the best release capacity, whereas swollen pore MCM-41 presented the worst delivery efficiency. Ha et al. functionalized hollow MSNs with N-phenylaminomethyl moieties and loaded pores with Rh B. The system was capped upon the addition of β-CDs by the formation of inclusion complexes with the grafted N-phenylaminomethyl groups.257 Aqueous suspensions of capped nanoparticles, at pH 7, showed negligible Rh B release. Yet massive dye delivery occurred when pH was lowered to 4. Additionally, the pH at which Rh B was released was tuned by selecting the capping molecule. In particular, DB24C8 and CB[6] also yielded an inclusion complex with the protonated N-phenylaminomethyl moieties at pH 7, and in this case, delivery was observed at pH 10 when the N-phenylaminomethyl groups were deprotonated (Figure 147 D). Zink, Stoddart, and co-workers prepared MSNs functionalized with benzimidazole stalks, loaded with Hoechst 33342 or DOX, and capped with β-CDs.258 At a neutral pH, β-CDs encircled benzimidazole stalks and blocked pores. In an acid environment (pH < 6, as in endosomal compartments), aromatic amines were protonated, which caused the dissociation of the inclusion

Figure 148. MSNs loaded with Hoechst 33342 or DOX, and capped with β-CDs. Cargo delivery was observed at an acidic pH.

after NH4Cl treatment. NH4Cl increased the amount of cellular uptake, but the basification of lysosomal pH to values over pH 6 avoided the release of cargos. Confocal microscopy studies demonstrated that only untreated cells clearly increased the fluorescence signal and also enhanced cell death with the DOXloaded material. Zink et al. reported one of the few known gated examples using porous silicon nanoparticles.259 These authors functionalized the external surface of porous silicon nanoparticles with 3iodopropyltrimethoxysilane, which was further reacted with benzimidazole. The pores of the silicon nanoparticles were loaded with Hoechst 33342 and capped with β-CDs via an interaction with the grafted benzimidazole units. A negligible entrapped dye release was observed when capped nanoparticles were suspended in water at pH 7. However, a remarkable dye release took place at pH 5 (see also Figure 148). The authors demonstrated that capped silicon nanoparticles also worked in complex biological media, such as DMEM containing 10% fetal bovine serum. In vitro studies, which used human pancreatic carcinoma cells PANC-1, indicated that capped silicon nanoparticles were endocytosed and the cargo was released in lysosomes because of their intrinsic acidity. In another interesting work, Zink, Stoddart, and co-workers prepared MSNs functionalized with propylmethylphosphonate groups (for biocompatibilization) and 3-isocyanatopropyltriethoxysilane. In another step, the isocyanate groups were reacted with 4-(2-hydroxyethoxy)benzaldehyde. Finally, pores were capped with per-6-diamino-β-CD rings via an imine bond formation and the subsequent reduction to amine assisted by sodium borohydride. With this procedure, the authors suggested that the entrance to nanopores were functionalized with one βCD derivative only.260 Pores were loaded with 2,6-naphthalenedisulfonic acid sodium salt and were closed by the addition of a Rh B/benzidine conjugate, which acted as a plug by the formation of a strong inclusion complex between the benzidine subunit and the grafted β-CD at pH 7. Aqueous suspensions of nanoparticles at a neutral pH nearly showed “zero release”, whereas delivery was clearly observed at pH 4. This delivery was ascribed to the dethreading of the plug from β-CD upon benzidine protonation (see Figure 149 A). The authors prepared other gated materials by loading pores with Rh B, which were 632


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capped with a β-CD derivative that contained acid-labile imine moieties. At pH 7, this gated material presented negligible Rh B release (the dye was larger in size than the β-CD cavity), whereas remarkable delivery took place at pH 5. In this case, acid media induced the rupture of imine bonds with the subsequent detachment of β-CD from the nanoparticle surface (see Figure 149B). Zink, Stoddart, and co-workers envisioned the design of gated MSNs capable of delivering different cargos sequentially.261 MSNs were functionalized with 3-isocyanatopropyltriethoxysilane and further derivatized with a propargyl ether derivative of a 2,2′-dithioethanol linker. Then Hoechst 33342 was loaded into mesopores, and the per-6-azido-β-CDs groups were anchored by a click reaction with the propargyl-containing stalk. In another step, the small p-coumaric acid molecule was loaded by diffusion though β-CDs cavities, while methyl orange was used as a stopper via the formation of an inclusion complex with β-CDs. When pH was adjusted to 3.5, the methyl orange complexation constant decreased and the molecule escaped from the CD cavity, which allowed the release of p-coumaric acid. Under these conditions, the bigger Hoechst 33342 dye was unable to diffuse through the β-CDs cavity and was retained. In a second step, when a reducing agent such as ME was added, β-CDs were removed and Hoechst 33342 was released (see Figure 150). Fu et al. functionalized the secondary side of β-CDs with 1Hbenzimidazole (pKa of 5.68) by a click chemistry reaction. Then the primary alcohols of β-CDs were transformed into iodide in order to attach mono-6-benzimidazole functionalized β-CDs to the surface of aminopropyl-functionalized MSNs.262 Pores were loaded with p-coumaric acid (see Figure 151). At pH 7, the aqueous suspensions of capped MSNs showed no dye release, yet remarkable p-coumaric acid release was observed upon acidification (76% of the cargo at pH 3) due to the protonation of the 1H-benzimidazole groups and the rupture of the 1Hbenzimidazole-β-CDs complexes. Neoh et al. functionalized MSNs with 3-isocyanatopropyltriethoxysilane and then anchored per-6-diamino-β-CDs by means of a urea bond.263 Some of the amino groups in the grafted β-CDs were further reacted with cysteine to functionalize the external surface of nanoparticles with thiol moieties. Pores were loaded with DOX. At pH 7.4, PBS suspensions of capped nanoparticles showed negligible drug release due to the presence of bulky β-CDs around pore outlets, whereas remarkable DOX release occurred when the material was suspended in artificial urine (at pH 6.1). At an acidic pH, the amino groups of β-CD around pore outlets were protonated, which gave rise to Coulombic repulsions among them. This caused mesopores to open with the subsequent DOX release (see Figure 152). To evaluate the system’s mucoadhesivity to the bladder wall, fluorescein-labeled MSNs were synthesized for fluorescent tracing purposes and were incubated with pig bladder for 2 h. The fluorescence intensity of the bladder wall increased after incubation with fluorescein-labeled nanomaterial, as judged by CLSM, which indicated that the system exhibited good mucoadhesivity. This property remained unaffected when the system was loaded with DOX. Finally, the viability of the UMUC3 bladder cancer cells after incubation with MSNs was analyzed by an MTT assay. The results revealed that the nanomaterial was not cytotoxic to cells. However, significantly inhibited cell growth was seen when bladder cancer cells were treated with free DOX or DOX-loaded nanoparticles. The cell uptake experiments also indicated that the release of DOX from

Figure 149. MSNs loaded with 2,6-naphthalenedisulfonic acid sodium salt, functionalized with a β-CDs derivative, and capped with a Rh B/ benzidine conjugate. Cargo release was observed at an acidic pH (a) by the detachment of the inclusion complex or (b) by the rupture of imine bonds. 633


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Figure 151. MSNs loaded with p-coumaric acid and capped with β-CDs derivatized with 1H-benzimidazole. Delivery was observed at an acidic pH.

Figure 150. MSNs loaded with Hoechst 33342 and p-coumaric acid and capped with β-CDs complexed with methyl orange. p-Coumaric acid was released at pH 3.5, while Hoechst 33342 was released upon the addition of ME.

the DOX-loaded system was likely to be enhanced in endosomes/lysosomes due to the acidic environment. Zink et al. developed a refined system capped with β-CDs for pH-induced cargo delivery. Two different capped materials were prepared.264 In the first one, the external surface of MSNs was functionalized with a stalk that contained a triazine heterocycle, and pores were loaded with PI or Rh B. Finally, pores were capped upon the addition of a β-CDs functionalized with uracil moieties through hydrogen bond base-pairing (see Figure 153A). The second material was prepared by functionalizing the external surface with a uracil derivative. Pores were also loaded with PI or Rh B, and the system was capped with β-CDs functionalized with adenine molecules (see Figure 153B). Capped MSNs showed negligible delivery in aqueous suspensions at pH 7, whereas clear dye release occurred at an acidic pH (lower than 5), which was more marked when the solution was more acidic. The release of dyes at a lower pH was related with the disruption of the

Figure 152. MSNs loaded with DOX and capped with β-CDs by host− guest inclusion complex formation. Cargo was delivered at an acidic pH.

hydrogen bond base-pairing interactions that detached bulky βCDs from the surface of nanoparticles. Confocal microscopy studies carried out with the four materials and MCF-7 cells showed the rapid uptake of nanoparticles. Rh B fluorescence was also observed in the cytoplasm, which indicated the uncapping of nanodevices due to the acidic environment of lysosomes. In order to test the potential applicability of these gated nanomaterials for drug delivery, both capped MSNs were loaded with DOX. When the MTT viability assay was run, the MCF-7 cells showed that both capped nanoparticles induced cell death of ca. 30%. 634


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Figure 154. MSNs loaded with DOX, grafted with PEG groups with a benzoic-imine bond linker, and capped with α-CDs. Cargo release was observed at an acidic pH.

loaded with DOX and capped with α-CDs to form a polypseudorotaxane.266 The benzoic-imine groups were highly sensitive to a drop in pH, which resulted in drug release. One important feature of this system was that even minor pH changes (from 7 to 6.8) resulted in markedly increased cargo delivery (see also Figure 154). The cellular uptake of the empty or DOXloaded system at different pHs was studied against Hep G2 using flow cytometry analysis. The obtained data indicated that the mean fluorescence intensity in the cells treated with the DOXloaded MSNs at pH 6.8 was much stronger than that at a physiological pH (7.4). The intracellular localization of free DOX and DOX-loaded MSNs against Hep G2 cells at different pHs was further investigated by confocal fluorescence microscopy. The authors concluded that the internalization mechanism of nanoparticles was endocytosis, and the carrier particles were initially located in the endosomal intracellular compartments, which released DOX in the cytosol region in a sustained manner. Finally, the cytotoxicity of free DOX and DOX-loaded MSNs was tested by MTT assays. With an equivalent drug content, free DOX exhibited a stronger killing potency than the DOX-loaded MSNs, probably due to the slow release of DOX from nanoparticles. Stoddart, Zink, and co-workers covalently modified SBA-15 MSNs with β-CDs groups and two different capping agents were used to block pores.267 The external surface of MSNs was grafted with 1-butylaldehydetriethoxysilane, and β-CDs were anchored by forming an imine bond between aldehyde moieties and the diamine derivatized β-CDs. Two different dyes were also used as cargos [i.e., fluorescein disodium salt and FITC-Dextran (3000 Da)]. The first capping agent was a 1,3,5-triphenylbenzene core, linked to three adamantane moieties through triethylene glycol chains (see Figure 155A). This agent formed an inclusion complex with β-CDs. The second capping agent was a hexahydroxytriphenylene derivative that contained six PEG chains capable of coordinating β-CDs units (see Figure 155B). When pH was acidified to below 6.5, imine linkers hydrolyzed, dethreading the large inclusion complex and causing dye leakage. Delivery studies demonstrated that the first cap was able to retain the large FITC-Dextran dye at a neutral pH but not the small

Figure 153. MSNs loaded with PI or Rh B and capped with β-CDs functionalized with (a) uracil or (b) adenine moieties coupled through hydrogen bond base-pairing with (a) triazine or (b) uracil moieties. Cargo delivery was observed at an acidic pH.

Shi et al.265 reacted APTES-functionalized MSNs with the methoxypoly(ethylene glycol) benzaldehyde groups. Pores were loaded with DOX, and nanoparticles were capped in the presence of α-CDs, which resulted in the formation of a polypseudorotaxane structure. The benzoic-imine bond hydrolyzed at an acidic medium but was stable at neutral and basic pH (Figure 154). DOX release from the material was tested at pH 7.4 and pH 6.8. DOX release was accelerated at pH 6.8. The same pH-responsive gated system was proposed by Shi, Kong, and co-workers, which functionalized MSNs with the PEG groups anchored via a benzoic-imine bond. Nanoparticles were 635


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fluorescein. The second complex effectively capped the system for both dyes. Zink and co-workers developed a sophisticated capped system based on a combination of porcine liver esterase (PLE) enzyme and β-CDs, which showed an amplification delivery process.268 These authors used MSNs (6.5 nm pore size) and loaded pores with the PLE enzyme. The outer surface of nanoparticles was functionalized with β-CDs that contained a pH-hydrolyzable imine moiety. The material was capped with an adamantane cluster that had a starlike structure. In order to test the proper work of the hybrid material, two substrates were selected: 4acetoxycinnamic acid (ACA) and 5-carboxyfluorescein diacetate (CFDA). Both substrates were not emissive but yielded strongly fluorescent products upon PLE-catalyzed acetate hydrolysis. When CFDA was added to an aqueous suspension of capped nanoparticles, no fluorescence was observed because this molecule was unable to enter on pores. However, when catalytic amounts of ACA were added, the emission intensity increased over time. ACA was able to enter pores and was hydrolyzed by the PLE enzyme generating acetic acid, which cleaved imine bonds with the subsequent release of the β-CDs-adamantane cluster (see Figure 156). When a small portion of pores was opened, CFDA molecules were able to diffuse and reach the entrapped enzyme, which led to the subsequent generation of a fluorescent molecule, and a more acetic acid which activated other neighboring pores, and allowed access to more enzymes. Chen and co-workers prepared MSNs which were grafted with APTES and were then sequentially reacted with 4-formylbenzoic acid and 1-adamantylamine.269 Moreover β-CDs that contained different appended molecules were synthesized. β-CDs-1 were functionalized with FITC, whereas β-CDs-2 and β-CDs-3 were functionalized with targeting agents FA and LA, respectively. The adamantane-modified MSNs were loaded with DOX and capped with two combinations of modified β-CDs (i.e., β-CDs-1 + βCDs-2 and β-CDs-1 + β-CDs-3). The authors prepared another material with no modified β-CDs. The capped systems were uncapped at an acidic pH because of the hydrolysis of imine bonds (Figure 157). The interaction of capped MSNs was studied with HeLa (with a high affinity to FA) and Hep G2 (with a high affinity to LA) cells. For HeLa cells, cell death was most enhanced with the material that was partially capped with the βCDs that contained FA. However, when free FA was present in the cell culture media, this enhancement did not take place due to the competition for the interaction with cell receptors. For Hep G2 cells, maximum cell death enhancement was observed with the MSNs partially capped with the β-CDs that contained LA. In the same line of examples described above, crown ethers have also been used to develop gated MSNs. Thus, Nguyen et al.270 developed a pH-responsive material based on MSNs and the recognition between secondary dialkylammonium ions and DB24C8. DB24C8 is a macrocyclic polyether that is large enough to form [2]-pseudorotaxanes with dialkylammonium ion centers. The authors functionalized MSNs with naphthalenecontaining dialkylammonium, loaded pores with coumarin 460 molecules and capped the material with DB24C8 (Figure 158). The unthreaded form of [2]pseudorotaxane at high pH values represented the open state, while the threading of the −CH2NH2+CH2− groups with DB24C8 closed the valve. The authors found that dethreading was achieved when they used a range of bases.

Figure 155. SBA-15 MSNs loaded with two different size cargos (FITCdextran or fluorescein) and capped with large inclusion complexes attached via imine bonds. (a) Smaller cap only retains FITC-Dextran whereas (b) larger cap is able to retain both cargoes. Cargo release was observed at acidic pH. 636


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Figure 157. MSNs loaded with DOX and functionalized with imine and adamantly containing moieties and capped with β-CD. Cargo release was observed at an acidic pH.

Figure 156. SBA-15 MSNs containing PLE inside pores and functionalized with a large inclusion complex attached via imine bonds. Entrance of ACA resulted in acetic acid formation and acidification of the pH dethreating the inclusion complex and allowing the decarboxylation of the fluorescent dye.

6.4. Polymers

6.4.1. Chitosan. Chitosan is a well-known pH-responsive polymer in which the presence of amino groups (pKa of ca. 6.5) enables this polymer to show remarkable changes in volume in response to pH variations in the medium. It is also known that the pH response in chitosan can be changed by chemically modifying the polymer, for instance, by grafting certain functional groups. In the examples below, chitosan-capped mesoporous silica supports show poor cargo release at a neutral pH because chitosan was in its unprotonated form, which collapsed onto the mesoporous surface and blocked pores. However, at an acidic pH, the chitosan polymer became positively charged through the protonation of amine moieties, and the polymer adopted a swollen conformation that resulted in pore opening. For instance, Sailor and Wu developed a pH-responsive delivery system of insulin (see Figure 159A). They used a porous silica film loaded with insulin and functionalized with a chitosanbased hydrogel through a cross-linking reaction by drop coating a

Figure 158. MSNs loaded with coumarin 460 and capped with DB24C8 by host−guest inclusion complex formation. The addition of certain bases resulted in dye release.

mixture of chitosan and GTPMS. At pH 7.4, cargo delivery was weak, while insulin was able to penetrate the swollen hydrogel layer at pH 6, which resulted in a steady release into solution.271 Xu et al. prepared hollow MSNs using chitosan as a cap, which was anchored to the silica surface via a reaction with GTPMS.272 These authors loaded pores with BSA (see Figure 159B). PBS suspensions of loaded nanoparticles at pH 7.4 showed poor BSA release (17.4% of the entrapped protein after 100 h). However, at an acidic pH (4), clear cargo delivery took place. For the in vitro cellular studies, the same nanoparticles were prepared but were 637


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assays in ZR75−30 cells were performed with (i) the chitosancapped material loaded with benzoyl peroxide, (ii) a similar material loaded with benzoyl peroxide, but uncapped, and (iii) free benzoyl peroxide. Cell death was clearly enhanced for (i) and (ii) compared with (iii). No significant differences in cytotoxicity were found when the uncapped material was used at pH 7.4 or 6.5, while clear toxicity enhancement was observed for the chitosan-capped material at an acidic pH. Fluorescence microscopy images corroborated the production of free radicals. Inverted microscopy images evidenced that the cells exposed to the benzoyl peroxide-loaded chitosan-capped material presented major cell damage. Ren, Qu, and co-workers prepared magnetic Fe3O4 nanoparticles coated with a mesoporous silica shell, which was also functionalized with aminopropyl moieties. On that aminocontaining surface, the authors anchored FITC and the TAT peptide. The dye was attached through the formation of a thiourea bond, whereas the peptide was anchored by an amidation reaction.275 Pores were loaded with CPT, and the external surface was coated by a polymer prepared by the reaction of the chitosan-FA conjugate with citraconic anhydride. The polymer was attached to the surface via electrostatic interactions between the negatively charged polymer and the positively charged anchored TAT peptide (see Figure 160). Nanoparticles efficiently accumulated in the target tissues guided by a local strong magnetic field generated by a magnet in H22 tumor-bearing mice. Nanocarriers were efficiently internalized in cancer cells via floater acceptor-mediated endocytosis. Once inside the acidic lysosomes, the citraconic amides of chitosan were hydrolyzed and chitosan was regenerated, which was detached from the nanoparticles and exposed the nucleartargeted TAT peptide groups. The regenerated chitosan ruptured lysosomes and helped nanoparticles to escape from lysosomes into the cytosol. The TAT peptide on the outer surface then guided nanoparticles to transfer from the cytosol into the cell nuclei. Thanks to this continuous 3-stage targeting process, cytotoxin CPT successfully entered the nuclei of tumor cells and induced apoptosis in HeLa but not in A549 cells, which did not express folate receptors. Yang et al.276 loaded MSNs with DOX and polymerized chitosan and PMA onto the external surface by the in situ polymerization method (see Figure 161). Aqueous suspensions of hybrid nanoparticles at pH 7.4 showed poor DOX release after 24 h (18%), whereas a clear DOX release was observed at pH 5.5 (70% after 24 h). The authors attributed this release behavior to the change in the charge of the polymeric shell around nanoparticles. At a neutral pH, the coating of nanoparticles was negatively charged, and the release of the positively charged DOX was inhibited. At an acidic pH, the negative charge of the coating was neutralized due to the protonation of carboxylates. As a result, DOX release was allowed. Finally, internalization studies carried out with HeLa cells indicated that coated nanoparticles were endocytosed and able to release entrapped DOX. Following their work, these authors used the same capped materials and suggested a new procedure to study pH-responsive delivery systems in cells in situ using CLSM.277 Two kinds of different cell lines, the tumor cell line (HeLa) and normal somatic cells (293T), were used. Zhu and co-workers coated MSNs with chitosan through simple hydrogen-bonding interactions between the silanol groups on the surface of the silica support and the amine moieties of chitosan.278 The aqueous suspensions of the chitosan-coated nanoparticles showed moderate IBU release at

Figure 159. Porous silica capped with chitosan by a cross-linking reaction with GTMPS.

loaded with TNF-α (tumor necrosis factor α) instead of BSA. Antibody ab2428 (that can bind to antigen ErbB 2) was conjugated with the chitosan polymer. After conjugating the antibody (to ErbB 2) to capped MSNs, nanocarriers exhibited good performance in delivering TNF-α to MCF-7 breast cancer cells in both in vitro and in vivo (nine 3-week-old athymic nude mice). The high affinity between the antibody and the antigen directed the system to achieve targeted delivery to cancer cells. Due to the acidic microenvironment inside solid tumors, the loaded TNF was gradually released from nanocarriers, which triggered apoptosis in tumor cells. Hu, Peng, and co-workers273 also prepared MSNs capped with chitosan through a reaction with GTPMS and loaded pores with DOX (Figure 159C). The release studies showed enhanced DOX release when the pH of the solution was lowered; for instance, at pH 4 and pH 6, DOX release reached 75.4% and 52.4% in 24 h, respectively, whereas only 16.3% of DOX was delivered over 72 h at pH 7.4. Thanks to the excellent biocompatibility of chitosan, cell viability MTT studies revealed that the system exhibited negligible cytotoxicity in MCF-7 cells. The cytotoxicity of the DOX-loaded nanomaterial against MCF7 breast cancer cells was enhanced over time. Zhu and co-workers used a pH-responsive system, based on MSNs coated with chitosan, to deliver benzoyl peroxide, a free radical precursor, for potential cancer therapy.274 Hollow MSNs were grafted with GTPMS, loaded with benzoyl peroxide and capped with chitosan (see Figure 159D). The delivery studies conducted at pH 7.4, 6.5, and 5 resulted in increased drug delivery when pH was acidified. Cytotoxicity studies using MTT 638


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Figure 162. Loaded MSNs capped with chitosan or chitosan derivatives via electrostatic interactions with the superficial (A) silanol or (B and C) amino groups. Cargo delivery was observed at an acidic pH.

162B).279 The aqueous suspensions of coated nanoparticles at pH 7.4 showed moderate entrapped DOX release (53% after 48 h), whereas DOX release was more marked at pH 4.4 (90% after the same time period). At pH 7.4, chitosan chains collapsed onto the surface of particles, whereas chitosan swelling unblocked pores and allowed cargo release at an acidic pH. FITCconjugated chitosan-coated nanoparticles were used for the cellular uptake studies. Confocal microscopy revealed that MCF7 cells uptook coated nanoparticles, which were located in the cytosol. Pourjavadi et al.280 functionalized MSNs with APTES and coated nanoparticles with a chitosan-PEG copolymer shell through electrostatic interactions. Pores were loaded with erythromycin. Cargo release was significantly enhanced at pH 5.5 compared with delivery at a neutral pH (see Figure 162C). Qiao and co-workers functionalized the surface of MSNs with 3-trihidroxysylilpropyl methylphosphonate, loaded pores with IBU, and capped the material with chitosan via EDC chemistry (see Figure 163).281 At pH 7.4, the polymer retained cargo, but clear cargo delivery occurred when the medium was acidified to pH 5 due to the hydrolysis of the phosphoramide bond that linked the chitosan layer to the solid surface. The acid hydrolysis of boronate esters combined with chitosan was used by Zhang et al. to design a pH-responsive system.282 These authors functionalized MSNs with GTPMS, and the epoxide groups were reacted with 3-aminophenyl boronic acid. Then the pores of nanoparticles were loaded with DOX and capped upon the addition of LA-functionalized chitosan through the formation of boronate esters between the grafted boronic acid moieties and the galactose moieties of LA (see Figure 164).

Figure 160. Fe3O4 NPs coated with a mesoporous silica shell, loaded with CPT, grafted with the TAT, and capped with a polymer obtained by reacting the chitosan-FA conjugate with citraconic anhydride.

Figure 161. MSNs loaded with DOX and capped with polymerized chitosan and PMA. At pH 5.5, DOX was released due to the protonation of the polymeric chains.

pH 7.4 (35% of the cargo at 24 h), whereas 65% of the cargo was delivered at 24 h at pH 6.8 (see Figure 162A) In a similar approach, Chung et al. functionalized MSNs with aminopropyl moieties and then coated nanoparticles with chitosan. The material was loaded with DOX (see Figure 639


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functionalized nanoparticles was coated with PEI (of different molecular weights ranging from 0.6 to 25 kDa). Then the plasmid (pEGFP) was adsorbed into the polymer coating (Figure 165A). The second material was similar, but siRNA was

Figure 163. MSNs loaded with IBU and capped with chitosan. Cargo delivery occurred at an acidic pH.

Figure 165. MSNs loaded with paclitaxel and coated with PEI that contained adsorbed siRNA and plasmids.

adsorbed in the polymeric layer instead of the plasmid (Figure 165B). Finally, the third material was prepared by loading the pores of the phosphonate functionalized nanoparticles with paclitaxel and then capped with PEI (Figure 165C). In order to track capped nanoparticles, Rh B isothiocyanate was grafted onto the polymeric layer of the three materials through thiourea linkages. The author found that all three capped MSNs were endocytosed by cells and were able to escape from endosomes due to PEI protonation, with the subsequent release of plasmid, siRNA, or paclitaxel. Interestingly, the siRNA-containing nanomaterial was quite effective for achieving GFP knockdown in transduced HEPA-1 cells. The facilitated cellular uptake of cationic particles enhanced the ability of nanoparticles to deliver paclitaxel to pancreatic cancer cells. The toxicity of the system was tested by injecting into mice the nanomaterial coated with PEI 25kD. No obvious toxicity was detected in vivo, as judged by the analysis of blood and major organs. Meng et al.284 developed a system based on MSNs to deliver a chemotherapeutic agent and Pgp siRNA to a drug-resistant cancer cell line. The inner and external surfaces of nanoparticles were functionalized with phosphonate groups to allow the electrostatic binding of DOX to the porous interior, from where the drug could be released by acidification of the medium (at pH 5) under abiotic and biotic conditions. Phosphonate modification also allowed exterior coating with cationic polymer PEI, which conferred the material with the ability to simultaneously bind and deliver Pgp siRNA (see Figure 166A). Hybrid nanoparticles were internalized by KB-V1 cells by endocytosis

Figure 164. MSNs loaded with DOX and capped with LA-functionalized chitosan via boronate ester bonds. Acidic hydrolysis of boroesters induced DOX release.

At pH 7.4, aqueous suspensions of capped nanoparticles showed a nearly zero release, whereas a 68.8% drug release was observed at pH 4.9. Such behavior at an acidic pH was ascribed to the hydrolysis of the boroesters that detached the bulky biopolymer from the nanoparticle surface. MTT assays carried out with the prepared nanoparticles and Hep G2 cells, which overexpressed the asialoglycoprotein receptor and could be targeted by LA, showed significantly reduced viability due to the internalization, uncapping, and release of DOX. The same tests carried out with NIH 3T3 cells indicated low toxicity. 6.4.2. PEI. Zink and co-workers used MSNs coated with PEI to deliver plasmids, siRNA, and paclitaxel into cells.283 For this purpose, three hybrid materials were prepared with MSNs functionalized with 3-trihydroxysilylpropyl methylphosphonate. In the first material, the external surface of the phosphonate 640


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transforming growth factor beta (TGF-β) receptor signaling in the xenograft. As a result of the TGF-β signaling blockage, pericyte differentiation and attachment to endothelial cells were also prevented. This allowed liposomal GEM carrier access through the open vascular fenestrations and the ability to increase GEM delivery. This two-wave approach allowed the effective shrinkage of the tumor xenografts beyond 25 days compared to the treatment only with free drug or GEM-loaded liposomes. Meng et al.286 developed an MSNs-based system capable of codelivering DOX and Pgp siRNA. The authors functionalized MSNs with a PEI−PEG copolymer. Finally, the functionalized nanoparticles were loaded with DOX, and Pgp siRNA was attached to the external surface by electrostatic interactions (Figure 166C). The controlled delivery of DOX and siRNA of MSNs was studied in saline-supplemented mouse serum at pH 7.4 and 4.5. At a neutral pH, negligible cargo delivery from MSNs was observed, whereas remarkable DOX and siRNA delivery at an acidic pH was measured. To test whether the nanomaterial provided the protected delivery of stably bound DOX and Pgp siRNA to the tumor site, a MCF-7/MDR xenograft model in nude mice was used. The system exhibited effective biodistribution and reduced endothelial reticulum uptake, thus providing 8% enhanced permeability and a retention effect at the tumor site. When compared to free DOX or the carrier loaded with either drug or siRNA alone, this dual delivery system resulted in the synergistic inhibition of tumor growth in vivo. Furthermore, significant Pgp knockdown at the heterogeneous tumor sites was observed, which corresponded to the areas in which DOX was released and triggered apoptosis. These data provide proof-of-principle testing of the use of a dual drug/ siRNA nanocarrier to overcome drug resistance in a xenograft. The pH-triggered release of hydrophobic dyes in cells was demonstrated by Lindén and co-workers.287 These authors functionalized MSNs with aminopropyl moieties. Then FITC was covalently linked to the surface via the formation of urea bonds with grafted amines, and pores were loaded with high lipophilic dyes (i.e., 1,1′-dioctadecyl-3,3,3′-3′-tetramethindocarbocyanine perchlorate and 3,3-dioctadecyloxacarbocyanine perchlorate). In a second step, PEI was grown on the surface by hyperbranching surface polymerization. Finally, FA was linked, through the formation of amide bonds to the polymer layer, to confer targeting abilities to capped nanoparticles (see Figure 167). Capped nanoparticles were endocytosed by HeLa cells, and the presence of the grafted PEI promoted both endosomal escape, due to the protonation of the polymeric layer, and the subsequent hydrophobic dye release. Zhao and co-workers used hollow MSNs for the drug/siRNA codelivery triggered by changes in pH.288 In a first step, the external surface of hollow MSNs was functionalized using (3triethoxyxilyl)propylmethylphosphonate. Then pores and the hollow interior were loaded with DOX. In a second step, pores were capped upon the addition of PEI derivatized with FA through electrostatic interactions between the negatively charged phosphates onto the surface and the partially charged amino groups of PEI. Finally, selected siRNA (Bcl-2) was adsorbed through electrostatic interactions onto the PEI coating (see Figure 168). Aqueous suspensions of the capped hollow MSNs at pH 7.4 showed negligible DOX release (less than 10%), whereas remarkable cargo delivery was observed when pH was lowered to 4.5. The pH-triggered release was ascribed to the protonation of amine moieties in PEI that induced strong Coulombic repulsions of each other and led to the dissociation of the PEI-FA layer from

Figure 166. MSNs loaded with (A and C) DOX and capped with PEI that binds (A and C) Pgp siRNA and (B) LY364947. Cargos were codelivered upon acidification.

and were localized in lysosomes. The acidic media inside lysosomes favored the codelivery of entrapped DOX and Pgp siRNA. The authors tested the utility of MSNs as a dual delivery platform in KB-V1 cells to see whether Pgp knockdown restored DOX sensitivity. KB-V1 cells exhibit multidrug resistance as a result of Pgp overexpression. As expected, Pgp siRNA codelivery increased the intracellular DOX concentrations and improved cytotoxic killing in KB-V1 cells. These results demonstrated the possibility of using the MSNs platform to effectively deliver siRNA, which knocks down the gene expression of a drug exporter to thus improve drug sensitivity to a chemotherapeutic agent. Meng et al.285 developed an engineered approach for the pancreatic ductal adenocarcinoma (PDAC) stromal barrier through the combined use of a liposome that contained GEM and MSNs coated with PEI−PEG. The authors electrostatically attached PEI to MSNs, and the polymer was also used to anchor PEG. Finally, a small molecule inhibitor of the transforming growth factor beta signaling pathway (TGF-β), LY364947, was attached to the PEI polymer through H-bonding interactions (Figure 166B). PBS suspensions of MSNs at pH 7.4 showed negligible LY364947 release, whereas delivery was marked when pH was lowered to 5.5. An optimal liposome design was achieved by using a transmembrane ammonium sulfate gradient to trap GEM inside the liposome. TGFβ delivery to the PDAC tumor site was explored in nude mice BxPC3 xenografts. These xenografts are known to elicit a dense infiltrating stroma, which surrounds nests of cancer cells and also covers tumor blood vessel fenestrations. Due to the acidic pH environment of the tumor stroma, the LY364947-coated nanoparticles were first used for the pH-dependent LY364947 delivery to inhibit the 641


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system to enhance cancer therapy. By simultaneously delivering both DOX and siRNA against the Bcl-2 protein into HeLa cells, the antiapoptotic protein Bcl-2 expression was successfully suppressed. This led to enhanced cytotoxicity in HeLa cells, which improved the therapeutic efficacy of the drug. 6.4.3. Acrylates and Methacrylates. Hong and co-workers functionalized MSNs with 5,6-epoxyhexyltriethoxysilane and then converted the epoxyhexyl groups into 5,6-dihydroxyhexyl units by adding hydrochloric acid. Subsequently, RAFT agent S1-dodecyl-S-(α,α′-dimethyl-α″-acetic acid)-trithiocarbonate was attached via esterification with hydroxyl moieties on the surface of the MSNs catalyzed by N,N′-dicyclohexylcarbodiimide.289 Then pH-sensitive PAA chains were grown by RAFT polymerization of acrylic acid using azobis(isobutyronitrile) as the initiator. The PAA shell is soluble in aqueous solution at relatively high pH values at which carboxylate groups are deprotonated (open gate). However, protonation of the carboxylate groups occurs when the pH of the solution lowers. As a result, the solubility of the polymer decreases and collapses (closed gate). In order to test the pH-sensitivity of the prepared material, fluorescein dye was loaded into the mesopores of the nanocarrier. Release experiments were carried out in PBS buffer at pH 4 and pH 8. Fluorescein delivery was observed at pH 8, whereas negligible release occurred at pH 4. (Figure 169A). Xu and co-workers developed MSNs which were coated with a copolymer formed by the free radical polymerization of methacrylic acid and vinyl triethoxysilane (Figure 169B).290 The system’s response was determined by the copolymer’s acid moieties. At an acidic pH (below pKa), the protonated polymer chains interacted via hydrogen bonding to result in a shrunken copolymer conformation that blocked pores. Conversely at a pH over pKa, polymer chains became negatively charged, the repulsion interactions expanded the polymer, and pores were unblocked. The pores of the material were loaded with IBU. At an acidic pH (4−5), no significant release of IBU was observed, but massive rapid release took place at pH 7.5. Mesoporous silica microparticles were selected by Samart et al. as an inorganic support to prepare pH-triggered materials coated with PAA.291 These authors functionalized the silica surface with benzophenone and grafted PAA onto the surface by UVirradiation polymerization. The pores of nanoparticles were loaded with indigo carmine (see Figure 169C). The aqueous suspensions of nanoparticles showed a negligible dye release at pH 1 (5.9% after 1 h), whereas a marked entrapped dye release was observed (ca. 60% after 1 h) at an almost neutral pH (8). Whereas in the above example PAA allowed cargo delivery at a neutral/basic pH, Yang et al. observed DOX delivery preferentially at an acidic pH in PAA-capped MSNs. In particular, they functionalized the external and internal surfaces with aminopropyl moieties. Then the amino groups were reacted with PAA, which led to a final material in which polymer brushes were located on the outer and inner surfaces of nanoparticles.292 Nanoparticles were loaded with DOX (Figure 169D). This molecule was positively charged at a neutral pH and interacted with the negatively charged polymer brushes through electrostatic forces. The final capped material only released 13% of the loaded DOX at pH 7.4, whereas the amount of released cargo was 70% at pH 5.6. The authors attributed the enhanced release ratio to the partial protonation of PAA brushes, which lowered the affinity toward the positively charged DOX. Finally, these authors demonstrated that the DOX-loaded PAA-capped nanoparticles were cytotoxic to HeLa cells.

Figure 167. MSNs loaded with hydrophobic dyes and capped with FAderivatized PEI.

Figure 168. Hollow MSNs loaded with DOX and capped with PEI-FA and siRNA. DOX and siRNA were released at an acidic pH due to the disruption of the PEI coating.

the nanoparticle surface. This dissociation also induced siRNA delivery. To investigate the system’s targeted drug/siRNA codelivery, HeLa cell lines with a significantly high FA receptor expression and MCF-7 cell lines with a low FA receptor expression were chosen as the positive and negative models, respectively. Upon cellular uptake, the acidic intracellular environment led to the release of siRNA and DOX from the 642


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the DOX-loaded PAA-capped MSNs. MRI measurements taken of the material in aqueous solution and in HeLa cells corroborated the possible use of this system for applications in biological systems. Poly(2-(diethylamino)ethyl methacrylate) is another pHresponsive polymer used to prepare hybrid materials for controlled release applications.294 MSNs were functionalized with aminopropyl chains. The amino groups were reacted with 2bromo-2-methylpropionyl bromide. Finally, 2-(diethylamino)ethyl methacrylate was polymerized through an ATRP method. At pH 4, the amino moieties of polymer brushes were protonated, became positively charged, and the strong chainsolvent interactions allowed free access to the pore networks. For these reasons, coated nanoparticles were loaded with Rh B at an acidic pH, and then the pH of media was raised to 8 (Figure 169F). At basic pH, the amines of polymer brushes were deprotonated and became hydrophobic. Under these conditions, polymer chain−chain interactions were strong and brushes were prone to aggregate by closing pores. In fact, negligible dye delivery was observed when coated silica nanoparticles were suspended in aqueous solutions at pH 8, whereas marked Rh B release was observed at pH 4. Capped MSNs that contained two heteropolymers, which were grafted by combining ATRP and RAFT techniques, were developed by Voit et al.295 By combining these two techniques, polymers poly(2-diethylaminoethyl methacrylate) (PDEAEMA) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) were attached to the external surface of MSNs. PDEAEMA, a pH-sensitive polymer, acted as a pH-sensitive gatekeeper, whereas PHPMA enhanced the hydrophilicity and biocompatibility of nanoparticles. In an acidic environment, positively charged PDEAEMA became hydrophilic and pores were uncapped, whereas in a basic solution, the neutralized polymer became hydrophobic and aggregated on the surface and pores were blocked. The model cancer drug DOX was loaded into mesopores. The release studies indicated that by decreasing the pH of the environment (pH 7.4, 6, and 5), the drug release rate increased (Figure 169G). Tang and co-workers functionalized hollow MSNs with 2bromoisobutyril bromide, loaded pores with the DOX drug, and capped the material with poly(N,N-dimethylaminoethyl methacrylate).296 The material was exposed to different pH conditions, and significant delivery was induced only at pH 4 (Figure 169H). This release was compared to the DOX delivery from the uncapped hollow MSNs, which led to the conclusion that capped material clearly achieved more marked release from neutral-to-acidic conditions. Sun and co-workers prepared PMA in a one-pot method by free radical copolymerization of α-methylacrylic acid with vinyl triethoxysilane. Then the polymer was grafted onto the surface of bimodal MSNs, which had small and large combined mesopore sizes. Finally, IBU was loaded inside pores (see Figure 169I).297 Different quantities of PMA were used to obtain materials with distinct amounts of polymer. The authors found that at an acidic pH, the gate remained closed and attributed to the formation of hydrogen bonds between the carboxyl moieties of the polymer. However, when the medium was basified to a neutral pH, clear IBU delivery was achieved due to the deprotonation of carboxyl groups. Comparative studies, which used different grafting ratios of the polymer, demonstrated that increasing the amount of polymer resulted in more efficient controlled release behavior. However, when excess polymer was used, less IBU was delivered due to steric impediment.

Figure 169. Mesoporous silica materials capped with different polymer derivatives. Cargo release was triggered by changes in pH.

Wu and co-workers also reported preferential DOX delivery at an acidic pH in the PAA-capped MSNs that contained a Fe3O4 core. These authors grafted APTES onto the silica surface, which was further reacted with 2-bromoisobutyryl bromide that acted as an initiator of the polymerization reaction. CuBr, N,N,N′,N″,N‴-pentamethyldiethylenetriamine and t-butyl acrylate were added to obtain poly(t-butyl acrylate), which was grafted onto MSNs.293 Finally, the system was treated with ptoluenesulfonic acid to obtain the final PAA shell. Pores were loaded with DOX (Figure 169E). The authors found that DOX delivery was observed when pH was acidified. The cell viability studies conducted by MTT assays in HeLa and L02 cells revealed no significant toxicity for the unloaded material. In contrast, enhanced cell death was achieved when cells were treated with 643


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Zhu and co-workers298 reported a dual-responsive controlled release system by modifying the external surface of MSNs with macromolecules that were responsive to both pH and electric fields. The polymeric coating was constructed onto 3methacryloxypropyltrimethoxysilane functionalized MSNs via surface-modified free radical polymerization method by mixing the 3-methacryloxypropyltrimethoxysilane-functionalized MSNs and 4-nitrophenyl methacrylate using AIBN as the initiator. IBU was then loaded into mesopores (see Figure 170). The polymeric

Figure 171. Fe3O4 nanoparticles coated with a mesoporous silica shell, whose pores were loaded with DOX and blocked with a PAA shell. DOX release was achieved at an acidic pH.

nanoparticles showed a moderate DOX release (20%) after 48 h. On the contrary at pH 5.1, 75% of entrapped DOX was released. CLSM studies revealed that coated nanoparticles were uptaken by PC3M cells by endocytosis and mainly located in the cytoplasm. MTT assays carried out with PC3M cells indicated that coated nanoparticles significantly diminished cell viability through the release of DOX into the cytoplasm and its subsequent entry into the nucleus. These authors also prepared similar capped materials but used NaYF4:Yb/Er/Gd nanorods as cores instead of Fe3O4 nanoparticles. Similar pH-controlled DOX delivery was observed. Gooding and co-workers reported another example of PAAcapped material.300 In particular, these authors prepared poreexpanded MSNs and loaded pores with Rh B or DOX. In another step, PCL was also introduced into pores by hydrophobic interactions. The resulting solids were functionalized on the external surface with amino groups and the system coated with PAA, thanks to the electrostatic interaction with the anchored protonated amines (see Figure 172). The authors found that cargo release was induced only when pH lowered and esterase was present. The drop in pH to 5.5 induced PAA coating disassembly, which allowed the diffusion of esterase to pore voids. Then the enzyme degraded the PCL framework and induced the release of the entrapped cargo. The authors studied the performance of the capped solids in SK-N-BE(2) neuroblastoma and HeLa cervical cancer cells. In their experiments, where Rh 6G was used as cargo, a time-dependent uptake by cells was observed. When the gated solid was loaded with DOX, the intracellular released payload was observed after only 1 h of incubation, as confirmed by fluorescence lifetime imaging microscopy. Finally, the authors reported no cytotoxic effect of the Rh-loaded capped material in HeLa, SK-N-BE(2), and normal MRC-5 fibroblast cells. On the contrary, they observed an 8-fold toxicity increase in SK-N-BE(2) cells when nanoparticles were loaded with DOX.

Figure 170. MSNs loaded with IBU and capped with poly(nitrophenyl methacrylate-co-methacrylic acid-co-methacryloxypropyltrimethoxysilane). IBU was released at a neutral pH and under an alternating electric field.

coating acted as a cap at a low pH due to strong hydrogen bonding and π−π interactions between the functional units of macromolecules. Conversely at a higher pH, the ionization of carboxylic units dismissed hydrogen-bonding interactions and unblocked pores. When an alternating electric field was applied, the rotation and reorientation of nitrophenyl moieties promoted drug diffusion from pores. The triggered release of IBU from the capped material was measured in both simulated gastric fluid (SGF) (pH 1.4) and SBF (pH 7.4) with the application of an electric field with different frequencies. IBU release was observed in both a basic medium and an alternating electric field. Wang and co-workers prepared Fe3O4 magnetic nanoparticles coated with a mesoporous silica shell. The silica surface was functionalized with APTES, to which FITC was covalently linked. Finally, these nanoparticles were coated with an eccentric PAA shell. Finally, DOX was incorporated into nanoparticles through electrostatic interactions between the positively charged drug and the carboxylates of the PAA polymer (see Figure 171).299 At pH 7.4, the aqueous suspensions of coated 644


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Nanoparticles were internalized by both cell types by endocytosis and delivered DOX due to the degradation of the coating in the acidic environment of endosomes/lysosomes. Xu et al. used hollow MSNs doped with YVO4:Eu3+ for cell imaging and modified the external surface with octadecyltrimethoxysilane.302 Nanoparticles were loaded with IBU, and pores were capped upon the addition of a pH-sensitive amphiphilic diblock copolymer, which was prepared by ATRP of 4-ndodecyloxybenzal acetalmethacryolyl ester and methoxypoly(ethylene glycol) 4-bromoisobutyryl ester. The interaction of the copolymer with MSNs occurred through wan der Waals forces (see Figure 174). Suspensions of capped nanoparticles in SBF at

Figure 172. pH Triggered the removal of PAA from the surface of MSNs and the esterase induced decomposition of PCL.

Li et al. developed a more complex system based on hollow MSNs functionalized with both aminopropyl moieties and ATRP initiator 2-bromoisobutyryl bromide, on which 2-phenyl-1,3dioxan-5-yl methacrylate (hydrophobic) and methyl ether methacrylate (hydrophilic) monomers were copolymerized.301 Pores were loaded with DOX (Figure 173). At pH 7.4, the

Figure 174. YVO4:Eu3+ nanoparticles coated with a mesoporous silica shell, loaded with IBU, and functionalized with octadecyl chains. Pores were capped with a diblock copolymer. Delivery was observed at an acidic pH. Figure 173. Hollow MSNs loaded with DOX and capped with an amphiphilic copolymer. Cargo release was observed at an acidic pH.

pH 7.4 showed negligible IBU release (below 5% after 150 h). However, at pH 5, a marked release took place (80% after 150 h), which was ascribed to the hydrolysis of the acetal groups in the coating polymer. CLSM studies showed that capped hollow nanoparticles were uptaken by KB cells and were located in the cytoplasm. Wang et al.303 developed a pH-responsive system for DOX release, which was based on using PAA as a cap that was anchored onto MSNs via an acid cleavable linker. In particular, these authors functionalized MSNs with (trimethoxysilylpropyl) ethylenediaminetriacetic. Subsequently, 9-bis(3-aminopropyl)2,4,8,10-tetraoxaspiro[5.5] undecane was grafted onto the

aqueous suspensions of loaded nanoparticles showed a nearly zero cargo release, whereas 65% of the entrapped drug was delivered after 10 h at pH 4.6. The fact that no cargo release took place at a neutral pH was ascribed to the shrinking of the hydrophobic part of the polymer into the pore outlet, which blocked drug delivery. Under acidic conditions, the hydrolysis of the acetal groups induced a change in the hydrophobic segment into hydrophilic, which allowed the DOX release. The internalization of capped nanoparticles in human hepatoma 7402 cells and L02 lung cancer cells was also studied. 645


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surface as the acid cleavable linker by the EDC/NHS crosslinking method. After loading with DOX, PAA was attached to the surface of MSNs by an EDC/NHS coupling procedure (see Figure 175). At pH 7 and pH 6, 10.2% and 17.3% of DOX were respectively released after 780 min, while 32.3% and 80% of DOX delivery was observed at pH 5 and at pH 4, respectively.

Figure 176. SBA-15 loaded with vancomycin and capped with PDDA. Release was observed at an acidic pH.

authors found that capped nanoparticles were able to deliver the cargo at an acidic pH due to the detachment of the PDDA layer. Rana and co-workers prepared a pH-sensitive drug delivery system using a polyamine-mediated bioinspired mineralization process.306 In their work, they prepared MSNs using poly(allylamine hydrochloride) (PAH) as the mineralizer and CTAB as the structure directing agent. Following surfactant removal by a solvent extraction process, IBU was loaded as a model drug. In this system, polyamines were not only responsible for the silicification process but also acted as pH-sensitive molecular gates (see Figure 177). The drug release profile of IBU from the

Figure 175. MSNs loaded with DOX and capped with PAA through an acid cleavable linker.

6.4.4. Other Polymers. Electrostatic repulsions between carboxylate-decorated SBA-15 and poly(dimethyldiallylammonium chloride) (PDDA) were used by Xiao and co-workers to develop a pH-triggered gate.304 In their work, they obtained a negatively charged surface on SBA-15 by anchoring 11-triethoxysilanylundecanoic acid. Then, after loading pores with vancomycin, they were capped with polycation PDDA by adjusting pH to 7.8 (see Figure 176). Only 8% of vancomycin was released at pH 6.5, while an 86% cargo release was observed at pH 2 due to the weakened electrostatic interaction between polycation PDDA and the anionic SBA-15 surface, which was related to the increasing amounts of the protonated carboxylic groups at an acidic pH. Ye and co-workers combined hydroxyapatite hollow MSNs with PDDA to develop a pH-responsive delivery system. Nanoparticles were obtained using a mixture of nonionic surfactants, Pluronic EO20PO70EO20 (P123; EO = ethylene oxide, PO = propylene oxide) and polyoxyethylene (20) sorbitan monostearate (Tween-60), as template agents and citric acid as cosurfactant.305 Two shapes of particles were obtained depending on whether citric acid was present or not. Spherical nanoparticles formed if this acid was absent. However, if citric acid was added to the reaction mixture, the resulting material took the form of nanotubes, which were covered with a citric acid layer. The nanoparticles prepared with citric acid were loaded with vancomicyn. At a neutral pH, the citric acid layer was negatively charged, which allowed the system to be capped with cationic polyelectrolyte PDDA by electrostatic interactions. The

Figure 177. MSNs that contained PAH and loaded with IBU. Cargo release was observed at an acidic pH.

nanocarrier was investigated in SGF (pH 1.2), followed by simulated intestinal fluid (SIF) (pH 6.8), to simulate the behavior of the material after oral administration. After 2 h at pH 1.2, pH changed to 6.8, and a slight jump in IBU drug release was observed. The authors attributed the material’s minor drug release capability at pH 6.8 to the electrostatic interactions between the negatively charged IBU and the positively charged ammonium groups in PAH (pKa ≥ 8). The slight increase in released IBU with the changing pH was attributed to the better solubility of IBU at a higher pH. Poly(4-vinylpyridine) has also been used as a pH-sensitive cap to prepare nanocarriers for pH-controlled release.307 These 646


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authors functionalized MSNs with bromomethyl dimethyl chlorosilane. Pores were loaded with either Ru(bipy)32+ or calcein, and nanoparticles were capped upon the addition of poly(4-vinylpyridine) through the formation of pyridininum salts with the grafted bromomethyl moieties (Figure 178). At pH

Figure 178. MSNs loaded with Ru(bipy)32+ or calcein, and capped by the covalent attachment of poly(4-vinylpyridine). Cargo was delivered at an acidic pH. Figure 179. MSNs loaded with DOX and capped by an amphiphilic polymer. Rupture of the acetal groups at an acidic pH caused DOX delivery.

7, the pyridine heterocycles were unprotonated and the polymer adopted a collapsed conformation, which inhibited Ru(bipy)32+ release. However, at an acidic pH, pyridines were protonated and the polymer backbone adopted a stretched conformation, which allowed the delivery of the entrapped cargo. Lu, Li, and co-workers designed a pH-sensitive nanocarrier using hollow MSNs combined with SPIONPs for MRI.308 For this purpose, hollow MSNs were synthesized and modified with hydrophobic octadecyl chains via the reaction with octadecyltrimethoxysilane. Then the pores of the materials were loaded with DOX. In parallel, oleic acid-stabilized SPIONPs were synthesized and then introduced into the hydrophobic layer of the outer surface of hollow MSNs. Finally a pH-sensitive amphiphilic polymer was used as capping agent. This polymer was synthesized by RAFT polymerization and included the cleavable 4-n-dodecyloxybenzalacetal groups, FA, and hydroxyethyl ester moieties. Dodecyl chains conferred hydrophobic properties to the polymer by enabling a conjugation with the hydrophobic surface of nanoparticles to block pores. FA was used to improve the uptake of nanoparticles by tumor cells, where FA receptors were overexpressed. At a weakly acidic pH (5), the acetal linkers of 4-n-dodecyloxybenzalacetal were hydrolyzed and, consequently, the polymer became hydrophilic and was detached due to the repulsion interactions with octadecyl chains on the surface of hollow MSNs, which caused cargo delivery (see Figure 179). The evaluation made of the material with no cargo revealed that it was biocompatible and nontoxic. Afterward the uptake of nanoparticles with FA, receptor-positive (KB cells) and -negative (A549) cancer cell lines was compared. After 30 min, no clear difference was observed in CLSM images, but fluorescence in KB cells clearly increased at 2 h. These results confirmed the contribution of FA receptors to the endocytosis

process. The magnetic properties of nanoparticles were evaluated and showed an evident response to an external magnetic field. Finally, good detectability for the magnetic resonance image was confirmed by T2-weighted phantom images. Wang and co-workers prepared gelatin-capped MSNs capable of releasing an entrapped cargo in response to changes in a pH environment.309 Pores were loaded with DOX, and the final capped material was prepared by coating nanoparticles with gelatin through temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking (see Figure 180). The aqueous suspensions of capped MSNs showed negligible DOX release at pH 7.4, whereas the release within the pH 2−6 interval was remarkable; the lower the pH, the greater the release. This pH-triggered delivery was the result of a detachment of the gelatin coating due to its protonation. CLSM carried out with Hep G2 cells incubated with the gelatin-coated MSNs confirmed their internalization through endocytosis and the release of entrapped DOX due to the acidic environment in lysosomes. MTT viability assays also indicated that gelatin-coated nanoparticles remarkably inhibited Hep G2 cell growth. Fu et al. prepared MSNs with a large pore diameter (4.2 nm), which were loaded with DOX and coated with polydopamine (Figure 181).310 The polydopamine coating was achieved through the oxidative self-polymerization of dopamine at a neutral pH. The authors recorded drug release at an acidic pH, but they noted that delivery was blocked at a neutral pH. DOX release at an acidic pH was ascribed to the partial detachment of the polymer from the surface of nanoparticles. 647


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Figure 180. MSNs loaded with DOX and capped by a coating with cross-linked gelatin. Protonation of gelatin induced pore opening and cargo release. Figure 182. MSNs loaded with DOX and capped with poly(glutamic acid). Payload was delivered at an acidic pH.

capped with polymer PNIPAAm-co-MAA.312 To prepare the material, the external surface of the Fe3O4 nanoparticles coated with the mesoporous shell was functionalized with (3trimethoxysilyl)propyl methacrylate. The functionalized magnetic core−shell nanoparticles were then reacted with NIPAAm and MAA to construct the external polymer layer. The pores of the capped material were loaded with DOX (see Figure 183).

Figure 181. MSNs loaded with DOX and capped with polydopamine. Cargo release was observed at an acidic pH.

Yang and co-workers used poly(glutamic acid) as a capping element to prepare pH-triggered MSNs.311 They prepared MSNs that contained aminopropyl groups. Then the outer surface of nanoparticles was functionalized with poly(γ-benzyl-Lglutamate) through the N-carboxyanhydride ring opening polymerization with the grafted amine moieties. The final poly(L-glutamic acid)-coated MSNs were obtained after the deprotection of the benzyl groups. Nanoparticles were loaded with DOX. Aqueous suspensions of capped MSNs showed a cumulative 13% DOX release after 24 h at pH 7.4. However, when pH was set at 5.5, 64% of the loaded drug was delivered after 24 h (see Figure 182). This difference in DOX release was ascribed to the electrostatic interaction between the positively charged DOX and the negatively charged polymer chains at pH 7.4, which was not operative at an acidic pH when the carboxylates of the polymer chain became protonated. HeLa viability diminished remarkably when cells were treated with the polymer-capped nanomaterial. Yang and co-workers prepared Fe3O4 nanoparticles coated with a mesoporous silica shell, which was loaded with DOX and

Figure 183. DOX-loaded Fe3 O 4 -MSNs coated with polymer PNIPAAm-co-MAA. Delivery was observed at an acidic pH or with a temperature decrease.

Aqueous solutions of the capped nanoparticles showed negligible DOX release at pH 7.4 (7.2% of the entrapped drug after 24 h), whereas a marked release occurred at pH 5 (80.2% after the same time period). At a neutral pH, the polymer shell was in a swollen state (negatively charged) and pores were capped. When pH was lowered to 5, the organic shell collapsed (neutral) and DOX was released. The same gated behavior was observed when the temperature of suspensions was changed from 38 °C (closed gate) to 33.5 °C (opened gate). MTT viability assays showed that the prepared nanoparticles were nontoxic for HEK 293 cells but were able to kill cancerous HeLa cells. Confocal microscopy 648


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studies, which were carried out with HeLa cells, showed the internalization of nanoparticles and the release of DOX in the acidic intracellular microenvironment. Trewyn and Liu developed capped nanoparticles as potential systems to deliver antidepressant venlafaxine in the intestine.313 To achieve their goal, they formed a pH-degradable cholic acidcross-linked poly(lactic acid) layer around MSNs by the polymerization of lactide monomer, and cholic acid around 5,6-dihydroxyhexyl-grafted MSNs using tin(II) 2-ethylhexanoate as a catalyst (see Figure 184). The release of venlafaxine from the

Figure 185. Magnetic Fe3O4 nanoparticles coated with a mesoporous silica shell, loaded with DOX and capped with a PEG derivative that contained pH-labile ester linkages.

Kong and co-workers prepared aminopropyl-functionalized MSNs loaded with DOX and capped with polymer PAHcitraconic acid via electrostatic interactions between the positively charged protonated amines and the carboxylates of citraconic acid at a neutral pH (see Figure 186).315 Aqueous

Figure 184. Venlafaxine loaded MSNs capped with cholic acid-crosslinked poly(lactic acid) polymer showed controlled release features in SGF.

capped system was followed in SGF (pH 1.2), which contained pepsin (pH 1.2) to mimic the stomach environment, and in SIF without pancreatin (pH 6.8) to simulate the intestinal environment. In SGF, it was found that although the poly(lactic acid) layer was degraded by the acid environment, it was possible to inhibit cargo delivery for 30 min, which proved a crucial time to avoid initial drug leakage in the stomach. After that time, the hydrolyzable layer began to decompose and the cargo release rate rose. It is noteworthy that the authors found that pepsin interacted with pore entrances, which was recorded as a delay in venlafaxine release of ca. 3 h compared with the release rate obtained in SGF, which did not contain pepsin. In a third stage, which went from 3 to 10 h, pepsin diffused into the pore voids and the release of the drug was stimulated. Magnetic Fe3O4 nanoparticles coated with a mesoporous silica shell, which was loaded with DOX and capped with a PEG derivative, were developed by Qu et al. as pH-triggered nanodevices.314 The capping macromolecule was prepared in a 2-step procedure which comprised: (i) esterification of PEG with acryloyl chloride and (ii) a Michael addition of MPTS to the esterified polymer. PBS suspensions (pH 7.4) of the magnetic core−shell nanoparticles presented a moderate DOX release because the grafted PEG chains blocked pores partially. However, at pH 5.8, remarkable drug delivery was observed, which was attributed to the hydrolysis of the ester bonds that linked the polymer to the surface of nanoparticles (see Figure 185). CLSM indicated the uptake of capped nanoparticles by HeLa cells. After a 6 h incubation, DOX was found in the nucleus.

Figure 186. MSNs loaded with DOX and capped with PAH-citraconic acid polymer. Cargo was delivered at an acidic pH.

suspensions of the polymer-capped nanoparticles at pH 7.4 showed weak drug release (less than 20% after 48 h), whereas 88% of the cargo was released after 48 h at pH 5.5. This release was ascribed to the hydrolysis of the amide bond that linked PAH with citraconic acid, which yielded a cationic polymer that detached from the surface of the positively charged nanoparticles to allow drug release. MTT assays carried out with HeLa cells showed a markedly reduced viability after the uptake of the DOX-loaded polymer-capped MSNs and, for instance, only 27% of the cells remained viable after 24 h of treatment. 649


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controlled drug delivery systems capped with polyelectrolytes.317 These authors prepared two materials: in one case, PAH and PSS were deposited on the DOX-loaded mesoporous silica nanotubes by a LbL procedure (Figure 187 B), and in the other case, fluorescein-loaded mesoporous silica nanotubes were modified with APTES to obtain positive amino moieties on the surface. Then alginate and chitosan were deposited by LbL. The first DOX-loaded material remained capped at a basic pH and delivered the drug when the pH was acidified. In contrast, the fluorescein-loaded solid remained capped at a weak acidic pH (4) and showed weak delivery when the solution was strongly acidified (pH 1.2) and massive delivery when basified (pH 8). Cytotoxicity studies using MTT assays were carried out with HT1080 and MCF-7 cell lines, with the DOX-loaded capped solid and with free DOX as a control. Nanotubes were unable to enter cells because of their large size. These authors also observed enhanced cell death when culture media were acidified to pH 6.8 in the presence of the capped solid compared with the cell cultures that remained at a neutral pH (7.4). Conversely, no significant differences were obtained by using free DOX at various pHs. Minati et al.318 described the preparation of MSNs loaded with DOX and coated by LbL with PAH and PAA (see Figure 187 C). This formed an electrostatic barrier against DOX leakage at a neutral pH. However, at pH 5, 42% of DOX was released from nanoparticles in 48 h. This effect was attributed to the electrostatic repulsion between DOX and the amino groups of the PAH layer, which were protonated at pH 5. At pH 7.4, the electrostatic interaction between DOX and the silanol groups on the silica surface prevented the drug from escaping from pores. The prepared nanocarrier was incubated with A549 cells for different times. After a 1 h incubation, CLSM analysis showed that nanoparticles were localized in the cytosol and probably retained in the endolysosomal compartment. After 2 h, stronger DOX fluorescence was observed in the cytosol, and DOX was localized in the nuclear region of cells after 17 h of incubation, which indicated certain drug release. However, a large fraction of DOX fluorescence was localized in the perinuclear region of the cell, which indicated incomplete drug release and was consistent with the in vitro drug release experiments (29% of DOX release after 17 h). Anker and co-workers prepared magnetic nanocapsules, which consisted in an iron nanocore and a mesoporous silica shell that was simultaneously loaded with DOX and T1-weighted MRI contrast agent diethylenetriaminepentaacetic acid gadolinium(III) (Gd-DTPA).319 Then nanoparticles were coated with polyL-lysine and sodium alginate by the LbL procedure (Figure 187D). When pH was lowered from 7.4 to 5, rapid DOX delivery was achieved due to the removal of layers as a result of the protonation of alginate. Cell viability studies done with MCF-7 cells demonstrated that the unloaded material was not toxic. In contrast, the DOX-loaded nanoparticles (both capped and uncapped with the polyelectrolyte layer) clearly induced enhanced cell death. Administration of DOX using an uncapped material showed a similar cytotoxicity effect to the free DOX treatment, while the DOX-loaded capped material displayed slightly increased cell death compared to the free DOX administration. The magnetic material could be potentially used for molecular resonance imaging, as confirmed by measuring their relaxivities. Different concentrations of magnetic nanoparticles with encapsulated DOX and Gd-DTPA were prepared and imaged with a 4.7 T MRI instrument. The T1weighted images became brighter with increased particle

6.5. Layer-by-Layer

Layer-by-Layer (LbL) deposition is a well-known fabrication technique in which films are formed by depositing alternating layers of oppositely charged materials. LbL offers several advantages over other deposition techniques because it is simple and usually inexpensive. On the basis of this technique, several pH-responsive gated silica mesoporous supports have been developed. Shi et al.316 reported the first example of LbL coating in a mesoporous support for controlled release applications. These authors prepared IBU-loaded hollow MSNs (c.a., 400 nm), which were coated with polyelectrolytes sodium polystyrenesulfonate (PSS) and PAH (Figure 187 A). Drug release was possible

Figure 187. MSNs loaded with different chemicals and coated with polyelectrolytes (PAH, PSS, PAA, poly-L-lysine, and alginate). Cargos were delivered at an acidic pH.

due to the pH sensitivity and salt-induced responsive properties of the PAH/PSS multilayers. In pH-driven release studies, the PAH/PSS system was found to be incompact at pH 1.4 in SGF, and the entrapped drug was released. In SIF (pH 8), the coating remained compact and no remarkable delivery occurred. Release experiments with NaCl showed that polyelectrolyte multilayers became incompact at a high salt concentration due to the lower electrostatic binding between the oppositely charged layers as a result of great ionic strength. In their interesting work, Chen, Tao, and co-workers studied the use of mesoporous silica nanotubes (6−10 μm long with a diameter that fell within the 400−600 nm range) to design pH650


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deposited. Finally, PAH was used as a third layer (Figure 189). The coating process with the three layers was repeated 3 times.

concentration, but the T2-weighted ones became darker with increased particle concentration. Ren and co-workers prepared MSNs functionalized with APTES and coated by different polyelectrolytes by LbL.320 The surface was first coated with a negatively charged layer of PSS and then with a second positively charged layer of PAH, a third layer of poly(sodium 4-styrenesulfonate), and a final positively charged and fluorescent layer of poly-[9,9-bis(6′-N,N,Ntrimethylammonium)hexyl]fluorenylene phenylene dibromide (Figure 188). Pores were loaded with DOX. At a basic pH, layers

Figure 189. MSNs loaded with drugs and capped with layers of PEI, PSS, and PAH. The disruption of polymeric layers at pH 1 induced drug release.

Aqueous suspensions of capped nanoparticles showed negligible drug release at a basic pH (8 and 9), whereas clear delivery took place at pH 1. The authors ascribed the release observed at an acidic pH to the protonation of the poly(sodium 4styrenesulfonate) layer and to the subsequent electrostatic repulsions between cationic layers. CLSM experiments carried out with A549 cells indicated the efficient internalization of coated hollow MSNs through endocytosis. Zhang and co-workers prepared MSNs functionalized with carboxylic acid moieties and loaded with Rh B. PBS suspensions of loaded nanoparticles were then coated consecutively with both 1,4-butanediamine-modified star-shaped poly(glycerol methacrylate) and PAA (Figure 190A).322 The same procedure was repeated in another cycle to prepare the final capped material. PBS suspensions of the MSNs at pH 7.4 displayed moderate dye release (35% after 50 h), but cargo delivery became greater when pH was lowered to 2 (90% after 50 h). The observed increase in dye release at pH 2 was ascribed to growth in the permeability of polymeric layers after the protonation of carboxylates in PAA and to the rupture of the electrostatic interactions with the positively charged protonated amines. Gao et al.323 prepared carboxylated MSNs (obtained by grafting the 3-isocyanatopropyltriethoxysilane and subsequent acid hydrolysis of the isocyanate moieties), aminated MSNs (functionalized with APTES), and bare hollow MSNs. The three supports were loaded with DOX and coated with different linear or star-shaped polyelectrolytes based on polyglycerol methacrylate (PGOHMAs) and PAA by LbL (Figure 190B). Changes in pH led to the protonation/deprotonation of the groups in PAA, and also in linear or star-shaped polycations PGOHMAs, which induced conformational changes that triggered or inhibited drug release. Entrapment efficiency studies revealed

Figure 188. MSNs loaded with DOX and capped with PSS, PAH, and with a fluorene-containing polymer. Cargo release was achieved at an acidic pH.

contracted and prevented guest release from the mesoporous silica channels. However, when pH was acidified, layers swelled, and DOX penetrated through layers and was released. For CLSM studies, the surface of the material was marked anchoring FITC. By monitoring images of Hep G2 cells, the authors demonstrated the internalization of nanoparticles and subsequent DOX delivery. Furthermore, FITC signals collocated with the signal of the fluorescent outer layer, which indicated that the layer was not separate from MSNs in the cellular environment. Hemolysis and cytotoxicity assays were carried out to assess the material’s biocompatibility. Negligible hemolysis of red-blood cells treated with different amounts of solid for 8 h was observed. Incubation of Hep G2 with the unloaded material did not present significant cytotoxicity. In contrast, treating cells with DOX-loaded material caused considerably increased cell death, which was higher than using an equivalent dose of free DOX. Cao et al. loaded hollow MSNs with IBU or DOX, and pores were coated with a shell of cationic polymer PEI.321 Then a second polymeric layer of the negatively charged PSS was 651


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Figure 191. MSNs loaded with DOX and capped with alginate and chitosan by LbL. DOX release was observed upon acidification.

prepared DOX-loaded capped MSNs, which were also functionalized with FITC. Functionalized MSNs exhibited improved blood compatibility in terms of low hemolytic and cytotoxic activity against human red blood cells. Cell viability studies done with HeLa cells showed that the DOX-loaded nanocarriers achieved sustained intracellular DOX release and accumulation in the nucleus, which thus resulted in prolonged therapeutic efficacy. Moreover, pharmacokinetic and biodistribution studies conducted in healthy rats showed that the DOX-loaded nanocarriers had a longer systemic circulation time and a slower plasma elimination rate than free DOX. The histological results also revealed that nanocarriers displayed good tissue compatibility. Liu and co-workers loaded MSNs with Rh B, functionalized the surface with aminopropyl moieties, which were partly protonated at a neutral pH, and capped nanoparticles with a negatively charged copolymer formed by the polymerization of N,N-dimethylacrylamide and 3,4,5,6-tetrahydrophthalic acidfunctionalized N-(3-aminopropyl)methacrylamide.325 These authors incorporated cisplatin into this layer. Then PAH was used to generate a positively charged layer (due to the presence of partly protonated amine moieties). Following this experimental procedure, nine bilayers were constructed around the mesoporous silica core. When these coated MSNs were suspended in water at pH 7.4, negligible Rh B and cisplatin release occurred (less than 10% after 12 h). However, when pH was lowered to 5, a remarkable release of both chemicals was found. Entrapped cargo release was the direct result of the disassembly of the polymeric layer. At an acidic pH, the amide groups that linked 3,4,5,6-tetrahydrophthalic acid with N-(3aminopropyl)methacrylamide were hydrolyzed, which subsequently generated positively protonated amines that yielded electrostatic repulsions with the positively charged PAH layer (see Figure 192).

Figure 190. MSNs loaded with (A) Rh B or (B) DOX and coated with PAA and with positively charged polyelectrolytes. Cargo release was observed upon acidification.

that the nanocarriers containing PGOHMAs star-shaped polymers encapsulated DOX with higher efficiency than the corresponding linear ones. Moreover carboxylated and hollow MSNs exhibited better loading capacity than aminated MSNs. The pH-triggered release behavior of carboxylated and hollow MSNs were tested further and in more detail. The DOX release from the carboxylated nanoparticles was negligible at pH 7.4 but was 60% at pH 5 and over 70% at pH 2. Coated hollow MSNs displayed the same release behavior. Cytotoxicity studies performed in L929 fibroblasts and HeLa tumor cells revealed that unloaded nanoparticles were biocompatible. Cell viability studies revealed that DOX-loaded nanoparticles exhibited greater cytotoxicity in HeLa cells than in L929 cells. He and co-workers functionalized MSNs with aminopropyl moieties, loaded pores with DOX, and coated the external surface consecutively with the negatively charged alginate and then with the positively charged chitosan polymers.324 This process was repeated until they finally obtained nanoparticles that were coated by two alginate/chitosan layers (Figure 191). Aqueous suspensions of the capped MSNs showed negligible DOX release at basic (8) or neutral (7.4 and 6.8) pH. However, upon acidification, a marked DOX release was observed (48.6 and 60.1% of delivery at pH 5.2 and 4, respectively). Cargo delivery was most likely due to the disruption of the DOX-alginate interaction upon the protonation of the carboxylate moieties of the biopolymer at an acidic pH. For cellular studies, these authors 652


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Figure 192. MSNs loaded with Rh B and coated by a negatively charged copolymer (bearing pH-labile amide groups) and positively charged PAH able to release cargo at an acidic pH. Figure 193. MSNs loaded with DOX and coated with cross-linked chitosan and dialdehyde starch layers. Cargo release occurred at an acidic pH.

Huang and co-workers prepared MSNs loaded with DOX and coated consecutively with chitosan and dialdehyde starch. The coating process was repeated 3 times.326 In this case, both biopolymers were covalently linked through the formation of imine bonds (see Figure 193). Aqueous suspensions of capped nanoparticles at pH 7.4 showed weak DOX release (9% after 72 h), whereas moderate drug delivery (34% after 72 h) took place at pH 5. Drug release was ascribed to the hydrolysis of the imine bonds that linked the biopolymer shells. Assays carried out with HeLa cells indicated that the DOX-loaded nanoparticles were able to induce cell death.

tide 5′- TCCCTAACCCTAACCC TAACCCTGCAAT(CH2)6-SH-3′, which contained four stretches of the cytosine (C)-rich domain, to 3.6 nm AuNPs. MSNs were loaded with Rh B (see Figure 195). At pH 8, the C-rich DNA sequence in AuNPs adopted an extended random state, which partially hybridized with the DNA strand anchored to MSNs, and pores were capped. When pH was adjusted to 5, the C-rich domain of the AuNPs DNA strand formed a folded i-motif conformation, which induced the dehybridization of the duplex and cargo delivery. The authors also demonstrated that the gating mechanism could be switched on/off repeatedly by adjusting the pH. Jung, Jaworski, and co-workers loaded MSNs with Rh B and grafted onto the surface a thymidine derivative that contained two alkoxysilane groups. Finally, nanoparticles were capped using FAM-labeled 18-mer poly nucleotide strands via hydrogen bonding (formation of Watson−Crick base pairing) with thymidine moieties (see Figure 196).329 FAM modification was used to confirm the immobilization of strands on the surface of MSNs. At pH 7, the system showed zero release, but the delivery rate increased when pH lowered, which achieved maximum release at pH 4. Wang et al.330 developed an intracellular pH-responsive drug delivery system by using an acid-labile DNA molecular-gated switch. The working principle relied on the fact that the T− Hg2+−T base pairs in the DNA duplexes were unstable and dissociated at a slightly acidic pH, which resulted in the DNA

6.6. DNA and Peptide Capped

Ren et al. used an i-motif DNA, which had a 4-stranded DNA structure at pH 5, as a molecular cap.327 The compact quadruplex DNA structure at pH 5 changed to an open random coil conformation at pH 8. In particular, these authors functionalized MSNs with aminopropyl moieties, which were transformed into carboxylic acid groups via the reaction with succinic anhydride. The carboxylic acids were further reacted with oligonucleotide 5′-NH2-(CH2)6-CCCTAACCCTAACCCTAACCC-3′. Nanoparticles were loaded with Rh B. Clear cargo delivery took place at pH 8, but not at pH 5, due to the pH-controlled conformational change, which also allowed a pulsatile release (see Figure 194). Wen and Song developed a similar pH-driven system which, in this case, was able to release its content at an acidic pH.328 These authors anchored DNA sequence 5′-NH2-(CH2)3-ATTGCAGGGTTAGTG-3′ to MSNs following the same 3-step synthetic procedure as explained above. They also anchored oligonucleo653


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Figure 196. MSNs loaded with Rh B and capped with a polyA DNA strand coupled with thymidine moieties on the surface of the material. Cargo release took place at an acidic pH.

chloropropyltrimethoxysilane, which was then reacted with sodium azide and double-stranded DNA. Double-stranded DNA was formed by prehybridizing in Tris−HNO3 buffer (pH 7.2), which contained both Hg2+-linkers and alkyne-modified oligonucleotide 5′-alkyne-(CH2)4-ACT TTG TTC TTT GT-3′. DOX was used as a model drug and was loaded into mesopores (see Figure 197). At a neutral pH, the double-stranded DNA

Figure 194. MSNs loaded with Rh B and capped with a quadruplex DNA structure. At pH 8, Rh B was released due to the conformational change in DNA structure.

Figure 197. MSN loaded with FITC and capped with a double-stranded DNA structure formed by Hg2+-mediated T−T DNA base pairing. At pH 5, FITC was released due to the disengagement of the DNA structure.

formed by the Hg2+-mediated T−T DNA base pairing capped pores. At a pH lower than 6, the nitrogen/oxygen atoms of the thymine units in double-stranded DNA protonated and the affinity of double-stranded DNA toward Hg2+ diminished, resulting in the dehybridization of double-stranded DNA and subsequent drug release. Controlled release studies conducted in aqueous media at pH 5 and pH 7.2 were consistent with the

Figure 195. MSNs loaded with Rh B and capped with AuNPs that contained DNA. Cargo was released at an acidic pH.

conformational change from a double-stranded form to a singlestranded state. In particular, MSNs were functionalized with 3654


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proposed mechanism. The authors also demonstrated the reversibility of the DNA-gated switch by adjusting the pH value in the solution. Studies done with HeLa cells demonstrated that the DOX-loaded nanoparticles could be endocytosed and could accumulate in lysosomes by serving as a carrier for the controlled release of DOX into cell nuclei. Cell viability results showed that the inhibitory concentration (IC50) of the DOXloaded system was low, while the IC50 of bare nanoparticles was 8 times higher. Higuchi and co-workers developed an MSN-based nanocarrier using a peptide which adopted a random coil conformation at weakly acidic conditions (pH 6) and a β-sheet structure at weakly basic conditions (pH 8).331 To construct the nanocarrier, MSNs were amino-functionalized with APTES. Subsequently, 17 mer peptide Ac-(VKVS)4E-NH2 was immobilized by a condensation reaction between the amino group on the nanoparticles and the carboxyl group of the glutamic acid residue at the C-terminal of the peptide. Dye platinum octaethylporphyrin (PtOEP) was loaded into pores as a model drug (Figure 198). Payload release

Figure 199. MSNs loaded with DOX and capped with positively charged K8(RGD)2 peptide which interacted with the covalently anchored and negatively charged K8(Cit) peptide. Cargo was delivered at an acidic pH.

of citraconic amide groups), which resulted in the charge repulsion between K8 and K8(RGD)2, removal of the K8(RGD)2 cap and drug release. On the basis of this principle, the authors noted clear DOX release at pH 5 (79% within 48 h), unlike the much lower release that occurred at pH 6.5 and 7.4. In vitro cellular experiments with αvβ3-positive U87 MG cells (human glioblastoma cells) demonstrated that capped nanoparticles were efficiently taken up by RGD receptor-mediated endocytosis. In addition, rapid intracellular DOX release induced cancer cell apoptosis. pH-Induced conformational changes in lysozyme have also been used as a mechanism to construct nanodevices for controlled release purposes.333 Lysozyme is a small globular protein that changes size with pH from a hydrodynamic radius of 1.9 nm at pH 4.5 to 2.3 nm at pH 3, which has been attributed to the inner repulsive electrostatic interactions caused by protonation. The authors used two different MSNs with an expanded pore size (pore diameters of 3.5 and 3.8 nm), which were loaded with Rh B. In both MSNs, lysozyme was used as a cap via simple electrostatic interactions with the silica surface (Figure 200A). When nanoparticles were suspended in aqueous solutions at pH 2, pores were blocked. However, when pH was raised to 7, clear entrapped dye release occurred. The released pH-triggered Rh B was ascribed to lysozyme conformational changes, which induced pore opening. In a similar approach, Teng and co-workers334 developed pHresponsive MSNs by using positively charged natural protein cytochrome C (CytC) as a pore blocker. CytC was attached to the silica surface by simple electrostatic interactions with the silanol groups. Pores were loaded with DOX (Figure 200B). At pH 7.4, CytC prevented DOX release (16% in 72 h), whereas at an acidic pH (5.5), the zeta potential changes in MSNs induced

Figure 198. MSNs loaded with dye PtOEP and capped with a 17 mer peptide. Release was triggered under slight acidic conditions.

was strongly inhibited under basic conditions, whereas cargo was gradually released at pH 6. The authors found that the system could be capped−uncapped by alternately shifting pH from 8 to 6. Zhang et al. progressively functionalized MSNs with APTES and alkyne (propargyl bromide) groups before loading pores with anticancer drug DOX. Subsequently, cationic peptide K8 (octa-lysine sequence), with a terminal azide group, was conjugated to MSNs by click chemistry. The amine group in the peptide was converted into a citraconic amide by the addition of citraconic anhydride to obtain K8(Cit).332 Capping of pores was afforded at a neutral pH by the electrostatic interaction between the negatively charged K8(Cit) peptide anchored to the MSN surface and the positively charged K8(RGD)2 peptide that contained two Arg−Gly−Asp (RGD) moieties (Figure 199). The RGD peptide acted not only as a pore blocker but also as a targeting ligand to specifically bind cancerous cells. Opening of valves took place at an acidic pH. The drop in pH led to the regeneration of the positively charged K8 (due to the hydrolysis 655


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Cai and co-workers used BSA conjugated with LA as a cap to prepare pH-triggered MSNs.336 The authors functionalized the silica mesoporous scaffold with aminopropyl moieties. Then the amino groups were reacted with carboxyphenylboronic acid (through an amidation reaction), and pores were loaded with DOX. Finally, nanoparticles were capped by the addition of the BSA-LA conjugate through the formation of boronate esters between the grafted carboxyphenyl boronic acids and galactose groups of LA (Figure 202). At pH 7.4, PBS suspensions of

Figure 200. MSNs loaded with (A) Rh B or (B) DOX and capped with (A) lysozyme or (B) CytC via electrostatic interactions.

the removal of CytC caps and allowed cargo release (54% in 72 h). CLSM studies indicated that the DOX-loaded system was internalized by human breast cancer MCF-7 cells and that DOX was released from nanocarriers. Cytotoxicity and histological assays confirmed that the constructed CytC capped nanocarriers possessed lower toxicity than free DOX and unsealed drug carriers. Furthermore, the intratumoral administration of nanocarriers proved significantly more efficacious in tumor reduction than free DOX and unsealed drug carriers in xenograft MCF-7 cancer models. Qiao and co-workers prepared MCM-48 MSNs, which were functionalized with aminopropyl moieties, loaded with IBU, and capped with succinylated β-lactoglobulin through the formation of amide linkages (Figure 201).335 Aqueous suspensions of

Figure 202. MSNs loaded with DOX and capped with BSA-LA via boronate esters with superficial boronic acids. Drug release was observed at an acidic pH.

capped nanoparticles showed negligible DOX release, whereas 50% of DOX was delivered after 2 h at pH 2. The release observed at an acidic pH was the result of the detachment of BSA-LA conjugated from the nanoparticle surface due to the acid-induced hydrolysis of borate esters. Cytotoxicity studies performed in Hep G2 cells confirmed that the constructed nanomaterial exhibited good biocompatibility and had a negligible effect on cell viability. Indeed, LA enhanced internalization and endocytosis in Hep G2 cells thanks to the ASGPreceptors present in cells. Most interestingly, the DOX-loaded nanoparticles showed enhanced cytotoxicity in Hep G2 cells compared to that in human umbilical vein HUVEC cells.

Figure 201. MSNs loaded with IBU and capped with succinylated βlactoglobulin via amide linkages. Drug release was observed at a neutral pH.

6.7. Permeation of Lipid Bilayers

Brinker et al. described a system in which liposome fusion on MSNs simultaneously entrapped the cargo and created a “protocell”, which was useful for delivery applications into cells.337 Calcein was chosen as a model drug, and its uptake was achieved by incubating mesoporous cores in calcein solution to which liposomes were added. These authors prepared different materials by varying the liposome composition using DOPC, 1,2dioleoyl-sn-glycero-3-phosphoserine (DOPS), and DOTAP

nanoparticles at pH < 5 showed negligible IBU release due to the formation of a dense gel-like coating of β-lactoglobulin around the nanoparticles. However, at pH 7.4, massive IBU release occurred due to a conformational change in β-lactoglobulin, which unblocked pores. The prepared gated nanoparticles showed high biocompatibility levels in HEK 293 cells. 656


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(Figure 203). The liposomes formed with DOTAP showed the best calcein encapsulation efficiency, which was attributed to the

Figure 203. MSNs loaded with calcein and capped with a lipid bilayer formed by DOTAP.

interaction of the negatively charged drug and the positively charged liposome. Therefore, controlled calcein release was conducted from the nanocarriers prepared with the DOTAP lipid. Calcein release was faster at pH 4 compared to that at pH 8. To study the nanocarrier entry into mammalian cells, the authors prepared nanocarriers by the fusion of Texas Red-labeled DOTAP liposomes on FITC-labeled MSNs. CLSM images of Chinese hamster ovary (CHO) indicated that nanoparticles were endocytosed and calcein was released. The authors postulated that calcein release took part in the cytosol. In another work, Brinker et al. prepared MSNs loaded with several cargos (quantum dots QDs, diphteria toxin A-chain, DOX, 5-fluorouracil, or cisplatin) and also capped systems using lipid bilayers (DOPC or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) as could be seen in Figure 204. These authors studied the internalization of nanoparticles in human hepatocellular carcinoma (HCC, HEP38) cells.338 The capping lipid bilayer was functionalized with a targeting peptide (SP94), with a fusogenic peptide (H5WYG) and with PEG moieties. These hybrid nanoparticles were internalized by HCC cells and released the entrapped cargo into the cytosol upon the protonation of imidazole subunits in the fusogenic peptide, which disrupted the lipid bilayer. The system, modified with the targeting peptide, was able to bind to human hepatocellular carcinoma cells with a 10000-fold greater affinity than for hepatocytes, endothelial cells, or immune cells. Most interestingly, the system could be loaded with combinations of therapeutic (drugs, small interfering RNA, and toxins) and diagnostic (QDs) agents, and modified to promote endosomal escape and the nuclear accumulation of selected cargos. Accordingly, a nanocarrier loaded with a drug cocktail efficiently induced cytotoxicity in drug-resistant human hepatocellular carcinoma at particle:cell ratios as low as 1, which represents a 106-fold improvement over comparable liposomes. Later in a paper published in 2012, the same material was used for the pH controlled delivery of siRNA.339 The system, loaded with a cocktail of siRNAs, bound to Hep3B hepatocarcinoma cells in a manner that was dependent on the presence of the targeting peptide and promoted delivery of the silencing nucleotides to the cytoplasm via endocytosis and endosomal disruption. The expression of each gene targeted by siRNAs was repressed at the protein level and resulted in a potent induction of growth arrest and apoptosis. Incubation of the control cells, which lacked the expression of the antigen recognized by the targeting peptide with the siRNA-loaded protocells, induced neither a repression

Figure 204. MSNs loaded with different cargoes and capped by a lipid bilayer of DOPC that contained targeting (SP94) and fusogenic (H5WYG) peptides.

of protein expression nor apoptosis. This finding indicates the precise specificity of cytotoxic activity. In a following work, the authors demonstrated that their lipid bilayer-capped MSNs were able to load a 100-fold larger amount of ricin toxin A-chain than DOPC liposomes.340 When MSNs were functionalized with 3[2-(2-aminoethylamino) ethylamino]propyltrimethoxysilane, loading capacity increased one more order of magnitude. In comparison, the equivalent toxin concentration required to inhibit 50% of nascent protein synthesis and to induce apoptosis in Hep3B cells using capped MSNs was 100- and 3500-fold less than for liposomes and free toxin, respectively. These results demonstrate the better efficiency of the capped system. In an attempt to improve the stability of lipid-bilayer-coated MSNs, Rosenholm and co-workers developed a lipid coating around MSNs using hyperbranched PEI as linker between the silica surface and phospholipids.341 The authors prepared aminocontaining MSNs via a co-condensation method and then polymerized aziridine on the surface to obtain hyperbranched PEI. Next they constructed the inner leaflet of the lipid bilayer by the conjugation of the primary amines of DOPE and that of PEI using coupling agent N,N′-disuccinimidyl carbonate. In another step, the authors self-assembled another phospholipid (zwitterionic DOPC or anionic POPG) by van der Waals interactions with the anchored one using a dual solvent exchange method, which resulted in the formation of final bilayer-capped MSNs (see Figure 205). The authors demonstrated that the capped system was able to retain calcein inside pore voids for up to 1 week, even in the presence of a membrane disrupting agent (Triton X-100). Flow cytometry and cell fluorescence 657


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release of entrapped drugs (less than 1% after 48 h), whereas marked drug delivery occurred at pH 5 (100% release after 12 h) due to the disruption of the coating liposome. The authors tested whether the nanomaterial was able to target breast cancer stem cells (BCSCs) and bulk breast cancer cells. BCSCs were enriched from MCF-7 cells by a sphere formation method and were identified with the CD44+/CD24− phenotype. Cell viability studies indicated that HA promoted the uptake of the system in CD44-overexpressing MCF-7 mammospheres, which revealed the mechanism of receptor-mediated endocytosis. Free docetaxel or docetaxel-loaded nanoparticles showed enhanced cytotoxicity against MCF-7 cells compared with MCF-7 mammospheres, whereas free 8-hydroxyquinoline or 8-hydroxyquinoline-loaded nanoparticles showed enhanced cytotoxicity against MCF-7 mammospheres compared with MCF-7 cells. In the MCF-7 xenografts in mice, the combination therapy used with docetaxelloaded and 8-hydroxyquinoline-loaded nanoparticles displayed the best antitumor efficacy, with very little systemic toxicity. Li, Gan, and co-workers loaded MSNs with the antitumoral drug irinotecan and capped the system with a polymer−lipid layer, prepared by the reaction of hydroxyl groups of Pluronic 123 and the amine groups of DOPE or 1,2-distearoyl-sn-glycero3-phosphoethanolamine (DSPE) in the presence of N,N′disuccinimmidyl carbonate and trimethylamine (Figure 207).343 The system effectively retained irinotecan in SBF (pH

Figure 205. MSNs loaded with calcein and capped by a lipid bilayer formed by the PEI-DOPE conjugate and DOPC/POPC.

microscopy studies were carried out to evaluate the nanomaterial’s intracellular drug delivery ability. Good biocompatibility in the presence of the carrier was confirmed in HeLa cells, which exhibited viability over 90% at the investigated particle concentrations. Interestingly, incubation of cells with the calcein-loaded system produced a significantly stronger fluorescence signal inside cells, which implies improved calcein retention before and during the cellular internalization of the nanoparticles. pH-Triggered mesoporous nanoparticles, used to treat breast cancer cells, were prepared by Zhong and co-workers.342 MSNs were functionalized with 3-[2-(2-aminoethylamino)ethylamino]propyl trimethoxysilane, and the interior of pores was loaded with anticancer drugs (8-hydroxyquinoline or docetaxel). Finally, the system was coated with a pH-sensitive liposome shell, prepared using cholesterol, 18:0 PEG-2000 PE, DOPC, and DOPE derivatized with HA (Figure 206). PBS suspensions of the nanoparticles at pH 7.4 showed a negligible

Figure 207. MSNs loaded with irinotecan and capped by a polymer− lipid bilayer. Irinotecan was released at an acidic pH.

7.4), while DOPC liposomes or DOPC or DSPC-capped MSNs were unable to prevent drug leakage for more than 72 h. Yet when pH was adjusted to 5, rapid irinotecan release occurred. Interestingly, the system was able to inhibit breast cancer resistance protein (BCPR)-mediated irinotecan efflux in drugresistant MCF-7/BCRP breast cancer cells. Compared to free irinotecan, incubation with nanoparticles resulted in increased intracellular irinotecan levels, greater cytotoxicity, and stronger cell cycle arrest in MCF-7/BCRP cells. The irinotecan-loaded nanoparticles showed high therapeutic performance and low toxicity in xenografts of drug-resistant breast tumors in nude mice. Dengler et al. reported MSNs loaded with DNA and capped with a bilayer composed of cholesterol and positively charged DOTAP or zwitterionic DOPC for transgene brain and spinal delivery.344 In both cases, the cargo was optimally retained at pH 6, while lower pH values induced a greater cargo release. Both formulations resulted in high cellular viability. Additionally, the peri-spinal application (subarachnoid, intrathecal) of nano-

Figure 206. MSNs loaded with different anticancer drugs and capped by a lipid bilayer of DOPC-DOPE that contained HA. Release was triggered at an acidic pH. 658


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aminopropyl moieties. After extracting the structure-directing agent, amino moieties were reacted with glutaraldehyde to yield nanoparticles coated with the aldehyde groups. Then the urease enzyme was covalently conjugated to the aldehyde surface by the formation of Schiff’s bases. Pores were loaded with DOX, and the system was capped by mineralization with hydroxyapatite and urea at pH 4. This process covered the external surface of MSNs with a biocompatible calcium phosphate coating (see Figure 209). Aqueous suspensions of coated nanoparticles showed

carriers in separate groups of rats resulted in minimal sensory changes, which indicates that the systems exhibited good biocompatibility. However, the biodistribution of DOTAP:cholesterol and DOPC-capped MSNs slightly differed. Only the DOPC protocells revealed increased spread to the brain, as observed at 8 weeks. DOTAP:cholesterol protocells revealed superior transfection efficiency to naked pDNA-IL-10. The DOTAP:cholesterol nanocarriers loaded with the pDNA-IL-10 cargo were examined for their in vivo therapeutic potential as a novel nonviral gene transfer vector delivered to spinal cord to treat peripheral neuropathic pain. Remarkably, this treatment resulted in the functional suppression of pain-related behavior in rats for periods that extended up to 8 weeks. 6.8. Inorganic Caps

Luminescent carbon dots (C-dots) have been recently used as caps to prepare pH-triggered nanovehicles.345 MSNs were functionalized with aminopropyl moieties, the porous network was loaded with cytotoxic drug DOX, and pores were capped with C-dots (see Figure 208). At a neutral pH, the amine groups

Figure 209. MSNs loaded with DOX, functionalized with urease and capped with a calcium phosphate shell. DOX was released at an acidic pH.

negligible DOX release at pH 7.4, but DOX delivery was clear at pH 4.5 due to the acidic dissolution of the calcium phosphate coating. Cell viability studies were performed in human breast cancer MCF-7 cells. Before loading with DOX, the nanomaterial showed no significant cytotoxicity and was endocytosed and accumulated within lysosomes. Incubation of cells with the DOX-loaded nanoparticles induced cell death due to the gradual release of DOX into cells at the lysosomal pH level. The cell viability analysis revealed that the inhibitory concentration (IC50) of the DOX-loaded system was higher than that of free DOX because of the fast internalization of the free drug. The in vivo efficacy of the DOX-loaded system was tested by xenograft models of MCF-7 cells. A single intratumoral administration of the nanomaterial proved significantly more efficacious for tumor reduction than the control groups. Ren et al. functionalized mesoporous silica with APTES, and the anionic HA polymer was attached to the silica surface by a simple electrostatic interaction with the protonated amines. HA served as nucleation sites for calcium phosphate, which capped pores. The system was loaded with Rh B.347 These authors also found that the attachment of another HA layer to mineral surfaces by chelation interactions enhanced the colloidal stability of MSNs (see Figure 210). This procedure also conferred to the gated system the ability to target to CD44. Dye release was triggered by the disintegration of the calcium phosphate cap in acidic media. A negligible dye release was observed at pH 7.4

Figure 208. MSNs loaded with DOX and capped with C-dots via electrostatic interactions. Payload delivery was observed at an acidic pH.

on the surface were partly protonated, which yielded strong nondirectional electrostatic interactions with the carboxylate moieties located on the surface of the C-dots. The aqueous suspensions of these nanoparticles showed negligible cargo release at pH 7, whereas payload delivery was observed at pH 5 due to the protonation of carboxylate moieties. MTT viability assays showed that the C-dots-capped nanoparticles (without DOX) were essentially nontoxic for HeLa cells. Moreover, the viability of HeLa cells diminished when treated with the DOXloaded C-dots-capped nanoparticles due to the internalization via endocytosis and pore uncapping due to the acidic environment of lysosomes. Calcium phosphate was used as a biocompatible coating when preparing pH-triggered MSNs by Lee et al.346 As-synthesized MSNs were functionalized on the external surface with 659


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Figure 211. MSNs loaded with IBU and capped with a hydroxyapatite shell via an electrostatic interaction. IBU was released at an acidic pH.

Figure 210. MSNs loaded with Rh B and capped with HA and a calcium phosphate shell. DOX was released at an acidic pH.

versus pH 4.5 at which cargo delivery was fast (82% cargo released after 24 h). To confirm the system’s targeting efficiency MDA-MB-231 cells, which have a high CD44 expression, were chosen as target cancer cells, whereas NIH3T3 cells were used as a control. Fluorescence imaging revealed that after a 2 h incubation with cells, the nanomaterial concentrated in the endosomes and lysosomes of MDA-MB-231 cells, while a very poor signal was detected in NIH 3T3 cells. This result was consistent with CD44-mediated cellar uptake and endocytosis. Afterward, cargo release was triggered by the disintegration of the calcium phosphate mineral coating in the acidic subcellular compartments. Cytotoxicity studies done in the same cell lines demonstrated that the DOX-loaded nanoconjugate showed remarkably enhanced efficiency at killing MDA-MB-231 cells while sparing MDA-MB-231 cells. Zhao et al.348 developed magnetic silica mesoporous nanocomposites that were end-capped with hydroxyapatite for pHresponsive drug release. The MSNs with a Fe3O4 core were first functionalized with primary amines, which were further reacted with succinic anhydride to produce carboxyl groups. Pores were loaded with IBU. A highly crystalline hydroxyapatite coating on the silica surface was obtained through a biomimetic mineralization process by adding Ca(NO 3 ) 2·4H 2 O and (NH4)2HPO4 (see Figure 211). Dissolution of the hydroxyapatite coating in an acidic environment (pH 4.5) unblocked pores and triggered the release of the loaded drug, whereas cargo release was inhibited at a physiological pH. Additionally, nanocomposites showed field-dependent magnetism, which demonstrates that the final nanoparticles were superparamagnetic. Lin et al.349 developed a pH-responsive system based on mesoporous bioglass as an inorganic scaffold, loaded with metformin hydrochloride (MH), and coated with hydroxyapatite (Figure 212). By taking into account the spontaneous mineralization of hydroxyapatite on mesoporous bioglass in SBF, drug loading and hydroxyapatite cap growth were carried out in a one-step procedure. Drug release from pores was triggered in acid environments (pH 4), due to the degradation of the hydroxyapatite cap. After adjusting the mineralization time

Figure 212. MSNs loaded with IBU and capped with a hydroxyapatite shell via electrostatic interactions. IBU was released at an acidic pH.

and the ion concentration of the media, the authors obtained capped nanoparticles with different release kinetics at an acidic pH. When comparing the release profiles from the different mineralization times with the same ion concentration during the loading and mineralization process, they found that the release rate lowered when the mineralization time was extended. Layered double hydroxides have been recently used as caps by Guo et al.350 MSNs were loaded with Ru(bipy)32+, and pores were capped by the addition of layered double hydroxides (of Mg and Al) simply by electrostatic interactions with the surface of nanoparticles (see Figure 213). PBS suspensions at a neutral pH (7.4) of capped MSNs showed almost no Ru(bipy)32+ release. In contrast, marked cargo delivery occurred at pH 3, which was attributed to the pH-induced dissolution of the layered hydroxides. Correia and co-workers loaded MSNs with either DOX or IBU, and nanoparticles were then capped with a CaCO3 coating (Figure 214).351 PBS suspensions of capped nanoparticles at pH 7.4 showed negligible drug release (ca. 10% of cargo after 48 h). However, at pH 5.6, marked drug delivery (ca. 30% of cargo after 48 h) occurred due to the calcium carbonate coating dissolution 660


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Figure 215. MSNs loaded with calcein and capped by chelating subunits. Cargo release was observed at an acidic pH.

Figure 213. MSNs loaded with the Ru(bipy)32+complex and capped with layered double hydroxides.

in the calcein release rate profile was noted at pH 5 and 4, which was ascribed to the faster dissolving process of the nanovalve in a more acidic environment. These authors also tested the influence of ionic strength and the presence of transition metal ions and chelating anions (EDTA) on the pH-sensitive cargo release. Release experiments showed that calcein release was accelerated under acidic conditions and at high ionic strength. Tolerance of the system to 1500 ppb EDTA and 0.5 mg mL−1 of metal transition metal ions (Cu2+, Zn2+, Fe2+, and Mn2+) was observed. Zhu et al. used ZnO QDs of 3−4 nm as caps to develop pHtriggered nanoparticles.353 As-made MSNs were functionalized with APTES, which was further reacted with succinic anhydride to obtain a surface covered by the carboxylic acid groups. The surfactant was extracted, and the interior of the pores was partially functionalized with aminopropyl moieties. Finally, pores were loaded with DOX and capped with ZnO QDs functionalized with aminopropyl moieties through an amidation reaction (Figure 216). Aqueous suspensions of nanoparticles at pH 7.4 showed negligible entrapped DOX release, but remarkable cargo delivery occurred when pH was lowered to 5. This release was ascribed to the acid-induced dissolution of ZnO caps, as confirmed by TEM and ICP-OES measurements. The prepared ZnO-capped nanoparticles were internalized by HeLa cells and were able to release entrapped DOX in the acidic environment of lysosomes. Moreover, MTT assays indicated that ZnO-capped nanoparticles had a significant cytotoxic effect as a result of released DOX and the formation of Zn2+ cations upon ZnO dissolution. The same authors proposed a multidrug delivery system based on MSNs but also capped with ZnO, which was capable of supplying a combination of two different drugs at the same time.354 Capped MSNs were synthesized in the same way as described above. In this case, the pores of the MSNs were loaded by CPT, whereas the second drug used, curcumin, was anchored to the surface of the ZnO nanoparticles via the formation of complexes with Zn atoms (see also Figure 216). The capped MSNs showed no cargo delivery at a neutral pH (7.4), but when the media was acidified to pH 6, the ZnO QDs were dissolved, which resulted in a simultaneous delivery of curcumin and DOX. MTT assays were carried out with the material that contained each drug, or both (CPT, curcumin and CPT+curcumin), in Bxpc-3 cells. In all cases, solids presented clear dose-dependent anticancer activity. Enhanced cell death was obtained when the material was loaded only with CPT, while administrating both

Figure 214. MSNs loaded with DOX or IBU and capped with a calcium carbonate shell. Cargo was released at an acidic pH.

at an acidic pH. The biocompatibility of MSNs and CaCO3coated MSNs was investigated using human fibroblasts (FiBH) and prostate cancer (PC3) cells. MSNs had no significant cytotoxic effect on FibH and PC3 cells and were highly biocompatible. Interestingly, an improved cytotoxic effect on PC3 cells was achieved for MSNs coated with CaCO3 and loaded with both IBU and DOX compared with the noncoated particles. The cytotoxic effect of the dual-loaded carbonate-coated particles was similar to that of the DOX + IBU free drug administration at 72 h. Zheng, Shchukin, and co-workers developed a pH-sensitive basic cobalt carbonate nanovalve onto MSNs.352 To construct such a system, MCM-41 nanoparticles were functionalized with (trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt and further loaded with calcein as a model cargo. Subsequently, a basic cobalt carbonate precipitate was formed by the reaction between Co(NO3)2 and Na2CO3 due to the complexation between divalent cobalt ions and the carbonatehydroxide mixture. Pores were capped by both the cobalt precipitate and the chelating complexation between the loaded calcein and the precipitate since Co2+ formed a highly stable complex with the iminoacetic acid end groups of the loaded calcein (see Figure 215). Cargo release was achieved by the dissolution of the carbonate precipitate at an acidic pH and also by the dissociation of the chelating complex. The experimental results at pH 7 confirmed the effectiveness of the nanovalve to keep calcein inside the mesopores. However, a notable difference 661


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Figure 216. MSNs loaded with DOX or CPT and capped with ZnO QDs. Cargo was released at an acidic pH. Figure 217. MSNs loaded with dexamethasone and capped with Fe3O4 NPs via a boronate ester. Delivery took place at an acidic pH.

drugs in one solid resulted in a slighter effect compared with the treatment that used the material with only curcumin or CPT. These results could be attributed to an antagonist effect between drugs. CLSM images in Bxpc-3 cells also demonstrated the dissolution of ZnO fluorescent QDs (whose signal disappeared at 3 h) and the codelivery of both drugs by the colocalization of fluorescence signals of curcumin and CPT in the cytoplasmic region. Liu et al. used Fe3O4 nanoparticles as inorganic caps.355 MSNs were loaded with the hydrophobic drug dexamethasone, and the outer surface was functionalized with N-(3-triethoxysilylpropyl)gluconamide. Finally, pores were capped by the addition of Fe3O4 nanoparticles coated with boronic acid moieties through the formation of boronate esters (see Figure 217). PBS suspensions of capped nanoparticles showed negligible entrapped cargo release at pH 7. Yet at pH 3, dexamethasone delivery was remarkable (100% release after 6 h), which was ascribed to the hydrolysis of the boronate esters that detached the Fe3O4 nanoparticles from pore openings. Capped nanoparticles allowed pulsatile entrapped cargo release when pH changed from neutral to acidic. MTT viability assays also showed that the Fe3O4-capped nanoparticles were nontoxic to MC3T3E1 cells. Yuan, Liu, and co-workers prepared MSNs, loaded with dexamethasone, which were functionalized with tri(aminomethyl)ethane moieties.356 Pores were capped by the addition of carboxybenzaldehyde functionalized Fe3O4 NPs via the formation of an acid-labile 1,3,5-triazaadamantane group (Figure 218). When the capped solid was suspended in PBS at pH 7.4, no significant dexamethasone release was observed. Yet at pH 6, a substantial amount of the drug was released and nearly 80% of cargo was leaked at pH 5 within 140 min. In another step, the authors studied the biocompatibility of gated nanoparticles with MC3T3-E1 cells with MTT assays. They found that cell viability in the presence of the unloaded material was above 90%,

Figure 218. MSNs loaded with dexamethasone and capped with Fe3O4 NPs linked with a 1,3,5-triazaadamantane moiety. Dexamethasone was released at an acidic pH.

even after 3 days of incubation. They also confirmed cellular uptake by flow cytometry and confocal fluorescence microscopy using FITC-labeled nanoparticles. Finally, the DOX-loaded solid was used to study cargo release kinetics by confocal fluorescence microscopy. The authors stated that gated nanoparticles were 662


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internalized within the first 4 h, and endosomal scape took place within the first 6 h. Qu and co-workers prepared carboxyl-containing AuNCs and functionalized the surface with alendronate to obtain bisphosphonate-functionalized AuNCs.357 After loading with DOX, pores were capped with the magnetic Fe3O4 nanoparticles coated with calcium phosphate. The gated system remained closed at pH 7.4, whereas at pH 4.5, calcium phosphate was dissolved and resulted in DOX release (see Figure 219). They

Figure 220. MSNs loaded with DOX or Ru(bipy)32+ and capped with GQDs or AuNPs via an acetal linker. Cargo was released at an acidic pH.

microscopy demonstrated the cellular uptake of capped MSNs by A459 cells. Feng and co-workers prepared MSNs capped with AuNPs through an acid-labile acetal linker.359 As-made MSNs were functionalized with aminopropyl moieties, which were further converted into carboxylic acids by a reaction with succinic anhydride. Then acetal linker 3,9-bis(3-aminopropyl)-2,4,8,10tetraoxaspiro[5.5]undecane was grafted onto the external silica surface through an amidation reaction. In a second step, the template was extracted with acetone, the porous network was loaded with Ru(bipy)32+ and pores were capped with carboxylic acid functionalized AuNPs through an amidation reaction (see also Figure 220). Aqueous suspensions of capped nanoparticles showed negligible entrapped dye release at pH 7, whereas Ru(bipy)32+ delivery was observed at an acidic pH. Cargo release was related with the hydrolysis of the acetal linker in acidic media, which detached AuNPs from the surface of MSNs. Qu and co-workers also used AuNPs as caps, which were linked in this case through an acid-labile hydrazone bond. In particular, the authors selected the Fe3O4 nanoparticles that were coated with a silica mesoporous layer functionalized with aminopropyl moieties.360 The amino groups were reacted with mono methyl glutarate to obtain methyl ester groups. These were reacted with hydrazine and then with 4-mercapto-4-methyl2-pentanone to obtain a stalk that contained the acid-labile hydrazone linkage and ended in thiol moieties. In the final step, the pores of nanoparticles were loaded with DOX and capped upon the addition of AuNPs (Figure 221). Aqueous suspensions of the capped material showed very weak DOX release (10.9% after 120 h) at pH 7.4, whereas drug delivery was clearly enhanced at pH 5 (71% after 120 h). This behavior was ascribed to an acid-induced hydrolysis of the hydrazone bonds that detached AuNPs from the surface. Studies with HeLa cells

Figure 219. AuNCs loaded with DOX and capped with Fe3O4 NPs coated with a calcium phosphate layer. Delivery was observed at an acidic pH and upon NIR irradiation.

also observed that the released drug was enhanced when the nanodevice was irradiated with NIR light. The authors treated MCF-7 cells with DOX-loaded capped AuNCs and found that DOX was effectively released after 5 h of incubation, probably due to the dissolution of the calcium phosphate-containing caps in endosomes and lysosomes. In the presence of the DOX-loaded capped AuNCs, 23% of MCF-7 cells were killed. However, 47% of the cells were killed after an additional 5 min NIR irradiation, which confirmed the synergistic effect of the pH-controlled release of DOX and NIR irradiation. Similar results were obtained in A549 cells. Fu et al. functionalized MSNs with APTES, which was further reacted with succinic anhydride to obtain carboxylic moieties capable of reacting with 3,9-bis(3-aminopropyl)-2,4,8,10tetraoxaspiro[5.5]undecane to obtain acetal-modified MSNs.358 This material was loaded with DOX and was then capped with graphene quantum dots (GQDs) through an acylation reaction of the terminal amino groups of the acetal-modified MSNs (see Figure 220). The solid was capped at a neutral pH, but massive cargo delivery occurred at an acidic pH (pH 4−5). Cytotoxicity studies were carried out by MTT assays in A549 cells, and capped MSNs were used both with and without DOX. Only the DOXloaded solid was toxic due to the hydrolysis of the acetal groups in the lysosomes that induced DOX release. Fluorescence 663


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Figure 221. Fe3O4-MSNs loaded with DOX and capped with AuNPs via a hydrazone linker. Cargo was delivered at an acidic pH. Figure 222. MSNs loaded with contrast agent C6F6 and capped with AuNPs via a hydrazone linker. An acidic pH induced cargo release.

resulted in the endocytosis of the nanovehicle and the release of the entrapped DOX in the acidic environment of the endosomes, with a subsequent reduction in cell viability. Zhou and co-workers prepared MSNs functionalized with FITC, loaded with the contrast agent C6F6 for 19F MRI, and capped with AuNPs via an acid labile hydrazone linker.361 First MSNs were marked with FITC. The acid-functionalized AuNPs were treated in two steps, first with methyl-3-mercaptopropionate and then hydrazine, to obtain hydrazide groups. Parts of these hydrazide moieties on AuNPs were reacted with FA by EDC chemistry to improve cellular uptake by cancer cells. Finally, the FITC-marked MSNs were loaded with C6F6 and capped with AuNPs in methanol using acetic acid as a catalyzer (see Figure 222). The authors tested the internalization of the system in human lung cancer A549 cells and human lung MRC5 fibroblasts. It is well-known that the membranes of A549 cells have many folate receptors. After 3 h incubation, the nanomaterial was efficiently endocytosed in A549 cells, while most of it remained outside in MRC5 cell cultures. Fluorescent imaging revealed the highly selective uptake of these nanoprobes by A549 cancer cells and the release of the 19F MRI contrast agent to the cytosol, which was detected by 19F MRI.

Figure 223. Mordenite and ZSM-5 zeolite-capped with disilylbenzene moieties. When the organic cap was removed, the dimerization reaction of α-methylstyrene in pores was allowed.

bonds in the gate and resulted in pore opening. Pores were also opened through calcination. The same authors extended their studies and, in a subsequent work, they used 1,4-diboronic acid to block the pores of mordenite and ZSM-5 zeolites.363 In particular, pores were closed upon grafting benzene-1,4diboronic acid or 1,4-bis(hydroxydimethylsilyl)bezene. From the nitrogen adsorption−desorption isotherms, the authors concluded that even molecules as small as nitrogen were unable to diffuse into the interior of pores in these capped materials. Yet upon HCl treatment, porosity was recovered due to the hydrolysis of the C−B bonds in the capping molecules. Zeolite-L crystals, which are aluminosilicate materials with strictly parallel one-dimensional nanosized channels arranged in a hexagonal symmetry, have also been used as an inorganic

6.9. Miscellaneous

Fujiwara and co-workers studied the controlled accessibility of chemicals to the interior of pores in a gated mordenite zeolite.362 The pore entrances of mordenite were functionalized with disilylbenzene, which acted as a gate. The authors then studied the dimerization reaction of α-methylstyrene, with is promoted only if α-methylstyrene is able to access the pores of the mordenite zeolite (see Figure 223). Upon functionalization with disilylbenzene, the micropores of zeolites were closed and the dimerization reaction of α-methylstyrene was inhibited. However, when the capped zeolite was treated with HCl in ethanol at 80 °C, pores opened and catalytic dimerization occurred. This acidic treatment cleaved the silicon−carbon 664


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support to prepare gated materials for drug delivery into cells.364 For this purpose, the pores of the inorganic support were loaded with 4′,6-diamidino-2-phenylindole (DAPI), and the external surface was functionalized with aminopropyl moieties. Finally, the modified surface was covered with a single oligonucleotide strand labeled with Cy5 through electrostatic interactions with the positively charged protonated amines in aqueous solution (see Figure 224). Simultaneous DAPI release and the marked

Figure 225. Silica mesoporous support functionalized with polyamines in which the presence of ATP induced pore blocking.

However, the presence of ATP and, to a lesser extent ADP, allowed selective inhibition of the cargo delivery by the formation of complexes between the anchored polyamines and ATP through hydrogen bonding and electrostatic interactions. The system displayed good selectivity, and it was found that other anions such as chloride, sulfate, or GMP did not effectively cap the pores.365 In a further study, the same authors studied the behavior of a similar amine-functionalized material also loaded with Ru(bipy)32+.366 A pH-driven open/close mechanism was observed that arose from hydrogen-bonding interaction between amines at neutral pH (open gate) and Coulombic repulsions at acidic pH between closely located polyammoniums at the pore openings (closed gate). Moreover, at a certain pH cargo delivery was also modulated via an anion-controlled mechanism. Thus, for instance, several behaviors were observed from basically no action (chloride) to complete (ATP) or partial pore blockage depending on the pH (sulfate and phosphate). This anioncontrollable response was related with the formation of anion complexes with the polyamines. In another example, the same authors developed a micrometric silica mesoporous support loaded with Ru(bipy)32+ and functionalized on the external surface with imidazolium binding sites.367 The release of the entrapped cargo was studied in water at neutral pH in the presence of different linear carboxylates [CH3(CH2)nCOO−, n = 0, 2, 4, 6, 8, and 10). The authors found that short carboxylates (i.e., acetate, butanoate, hexanoate, and octanoate) were unable to induce pore blockage, whereas in the presence of larger carboxylates (i.e., decanoate and dodecanoate), cargo delivery was inhibited as shown in Figure 226. This was attributed to the electrostatic interaction of these carboxylates with imidazolium binding sites in the surface that resulted in the formation of a dense hydrophobic monolayer around pore outlets. In particular, dodecanoate completely inhibited dye delivery at millimolar level. In a similar study, the same authors368 developed micrometric silica mesoporous supports loaded with Ru(bipy)32+ and functionalized on the external surface with thiourea or urea binding sites. Again, cargo release was tested in neutral aqueous solution in the presence of the carboxylates CH3(CH2)nCOO− (n = 0, 2, 4, 6, 8, and 10). Also in this case, only the presence of decanoate and dodecanoate inhibited cargo release.

Figure 224. Zeolite inorganic support loaded with DAPI and capped with a single oligonucleotide strand labeled with Cy5 by an interaction with superficial amino moieties.

oligonucleotide were investigated by CLSM in HeLa cells. After 24-h incubation, strong DAPI fluorescence was recorded in the nucleus and a DNA signal in the cytoplasm was observed. These facts indicated nanoparticle internalization and the uncapping of pores inside cells. Cell viability studies also indicated negligible toxicity of capped nanocrystals.

7. MOLECULES AND BIOMOLECULES Mesoporous gated structures can also be governed using molecular and supramolecular interactions between certain binding sites anchored on the pore outlets with species present in the solution. In this section, a wide variety of anions, cations, neutral molecules, and biomolecules are used to trigger the release of entrapped cargos from mesoporous supports. One advantage of these molecule or biomolecule-driven systems is related with their intrinsic specificity. Only in the very restricted scenarios where the selected triggering molecule or biomolecule is present, the encapsulated cargo will be released. This opens the possibility to design precise systems for very particular biomedical applications. For the same reason, some gated systems described here have found application in sensing processes. 7.1. Triggered by Anions

́ Martinez-Má ñez et al. developed some systems that were selectively capped in the presence of certain anions and proposed their use as probes for the chromo-fluorogenic sensing of target anionic species. In a first work, the authors prepared MCM-41 mesoporous silica support, and loaded the solid with the Ru(bipy)32+ dye as reporter and functionalized the external surface with 3-[2-(2-aminoethylamino)ethylamino]propyl trimethoxysilane. At neutral pH, the gate was open and delivery of the ruthenium complex was observed as depicted in Figure 225. 665


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ATP-aptamers to develop gated materials.370 In their work, they used an amino-terminated oligonucleotide sequence that contained both an ATP aptamer and a short extra sequence which was used to induce a hairpinlike structure that blocked the pores (see Figure 228). MSNs were loaded with fluorescein and

Figure 226. Micrometric silica mesoporous support functionalized with imidazolium cations for the detection of long chain carboxylates.

A final example of the same authors for sensing applications based on an open-to-closed protocol was developed for the detection of borate based on the reaction between polyalcohols and borate anion to form boronate esters. A micrometric silica mesoporous support was loaded with Ru(bipy)32+ and functionalized with a gluconamide derivative at the external surface.369 The presence of borate inhibited dye delivery at neutral pH due to the formation of boroesters (Figure 227). Inhibition of dye

Figure 228. Aptamer-gated MSNs loaded with fluorescein and opened in the presence of ATP.

functionalized in the external surface with sulfhydryl groups. The amino functionalized aptamer was then covalently anchored to the nanoparticles using the cross-linker sulfo-N-succinimidyl 4maleimidobutyrate. The hairpin structure of the aptamer blocked the pores while the presence of ATP triggered the delivery of the entrapped dye. This was explained due to ATP binding that resulted in a conformational change from a duplex to a singlestranded DNA of the aptamer sequence that was close to the surface of the MSNs. Interestingly, similar nanoparticles capped with a mutated hairpin did not respond to ATP, and guanosine 5′-triphosphate (GTP) was also unable to induce dye release. In a further work, the same authors extended their studies and monitored the performance of aptamer-gated MSNs using circular dichroism.371 Wang and co-workers also designed ATP-selective delivery systems with MSNs, employing the same ATP aptamer used by Ozalp et al. but based on a different configuration of the gated ensemble (Figure 229).372 The authors functionalized MSNs with 3-cloropropyltrimethoxysilane and transformed the chloride atoms into azide groups by reaction with NaN3. Moreover, the ATP aptamer was hybridized with two different ssDNA sequences yielding a sandwich type DNA structure. The pores of the inorganic scaffold, functionalized with azide moieties, were

Figure 227. Borate induced controlled release of a ruthenium complex in a microporous silica support functionalized with polyalcohol moieties.

release by borate anion was very selective, and for instance other anions (i.e., carbonate, SO42−, Cl−, Br−, NO3−, and phosphate) or cations (i.e., aluminum, copper, iron, sodium, potassium, and calcium) showed no effect. A limit of detection (LOD) of ca. 70 ppb was determined for borate in water. A common characteristic of the examples above is the use of uncapped materials that were selectively capped in the presence of certain anions and that were applied for sensing protocol. In contrast, the following examples show a more common behavior in which capped materials are opened in the presence of certain anions. A procedure to achieve this was the use of aptamers. An example of this was reported by Ozalp and co-workers that used 666


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Figure 229. Aptamer-gated MSNs loaded with Ru(bipy)32+ and opened in the presence of ATP.

loaded with Ru(bipy)32+, and then the system was capped with the sandwich-type DNA structure containing the ATP aptamer sequence, using a click chemistry reaction between the azide moiety in the solid and the alkyne group in the DNA ensemble. This blocked the pores. In the presence of ATP, dye release was observed and attributed to the competitive displacement of the ATP aptamer from the sandwich-type DNA. The system was selective and cytosine 5′-triphosphate (CTP), GTP, and uridine 5′-triphosphate (UTP) at 20 mM were unable to induce dye delivery. Yang and co-workers also developed an ATP-capped material using, in this case, aptamer-containing AuNPs as caps.373 In particular, the authors functionalized with amino groups the surface of MSNs and anchored adenosine-5′-carboxylic acid moieties via an amidation reaction. The pores were loaded with FITC. Moreover the authors prepared AuNPs containing the ATP aptamer by forming an Au−S bond. The gated material was then finally prepared by capping the pores of the mesoporous silica support with the aptamer-functionalized AuNPs (Figure 230). Aqueous suspensions of the capped MSNs displayed negligible cargo delivery while in the presence of ATP, FITC release was observed which was proportional to the ATP concentration. The uncapping process was ascribed to a competitive displacement reaction of the AuNPs upon ATP coordination with the grafted aptamer. Other ATP analogues, such as CTP, GTP, and UTP, induced no cargo delivery. Tang and co-workers also designed MSNs capped with AuNPs for the detection of ATP using the same ATP aptamer used above by Ozalp, Wang, and Yang.374 The authors functionalized MSNs with aminopropyl groups and linked to the amino moieties a single-stranded oligonucleotide (DNA1) using glutaraldehyde as a linker. The pores of the MSNs were loaded with glucose. On the other hand, AuNPs coated with another single-stranded olgonucleotide (DNA2) were prepared (see Figure 231). Both DNA1 and DNA2 were complementary to adjacent areas of the ATP aptamer. In the presence of the ATP aptamer, DNA1 and DNA2 hybridized forming a three-stranded complex that resulted in pore capping. The gated nanoparticles

Figure 230. MSNs functionalized with adenosine-5-carboxylic acid and capped with AuNPs coated with an ATP-aptamer which opened in the presence of ATP.

Figure 231. MSNs loaded with glucose and capped with AuNPs containing and ATP-aptamer. The system was uncapped in the presence of ATP.

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seemed to be the dominant uptake mechanism for the MSNs, as little or no particles were detected in the cytosol of cells treated with chlorpromazine, a specific inhibitor of CDE. The in vitro release in HeLa cells of an anticancer drug from the AuNPscapped MSNs was also performed using DOX as cargo. DOXloaded MSNs showed high toxicity to HeLa cells after 24 and 48 h, suggesting that DOX was successfully released from the uptaken MSNs and killed HeLa cells. Zheng et al. developed376 sulfide and pH-responsive MSNs for applications in self-healing coatings. To achieve this goal, MSNs were first functionalized with the diamine N-(3trimethoxysilylpropyl)ethylenediamine (DiA). Moreover, the corrosion inhibitor BTA and the biocide benzalkonium chloride (TC) were loaded into the mesopores. The addition of CuSO4 induced capping of the nanoparticles by the formation of the insoluble Cu-BTA complex which was formed at the pore openings. The Cu-BTA complex dissolved at acidic pH, resulting in cargo delivery. Moreover it was found that sulfide ions were able to extract the Cu+ ions from the Cu-BTA due to the great difference between the stability constants of Cu2S and the CuBTA complex (4 × 1047 and 1.56 × 10−2, respectively). Effective release of the inhibitor and antibacterial agents was achieved when the pH was lower than 5 or [S2−] was higher than 0.02 mM (0.6 ppm). For anticorrosion and antifouling experiments, steel substrates were coated with a polyester layer incorporating the capped nanocontainer. To demonstrate corrosion protection, current density measurements were carried out in 0.1 M NaCl aqueous solution using the scanning vibrating electrode technique (SVET). In accordance with SVET current density measurements, the hybrid coating exhibited anticorrosion in response to pH decrease and the presence of S2− ions. Furthermore, the antifouling ability of the hybrid coating was demonstrated by the suppression of E. coli activity due to the release of the biocide from the capped MSNs. Feng and co-workers reported MSNs capped with AuNPs, which were opened in the presence of the cavitand C1.377 The authors prepared AuNPs functionalized with alkyltrimethylammonium groups and used these to cap the pores of MSNs through simple electrostatic interactions with the silica surface (see Figure 233). The pores of the MSNs were loaded with fluorescein. The authors found that the addition of a C1 induced the displacement of the functionalized AuNPs and cargo delivery. This was attributed to the formation of strong C1-alkyltrimethylammonium complexes.

showed no release of the entrapped glucose at neutral pH, whereas in the presence of ATP, release of glucose was detected. Glucose delivery was detected with a commercially available glucometer. Cargo delivery was attributed to the binding of ATP with the aptamer that resulted in the separation of the AuNPs from the MSNs. This material was used to sense ATP which was detected at a concentration as low as 8 μM. The presence of other nucleotides such as CTP, GTP, and UTP induced no cargo delivery. The authors also used a similar approach for the detection of cocaine, employing in this case, a cocaine aptamer. Chen et al.375 also developed capped MSNs able to be uncapped in the presence of ATP. In this case, the authors used Lcysteine-derivatized AuNPs as caps. MSNs were aminofunctionalized with APTES, and pore capping was achieved by coupling the functionalized AuNPs to the amino groups in the MSNs using Cu2+ as a bridging ion as shown in Figure 232. The

7.2. Triggered by Cations

Capped MSNs designed to be opened in the presence of cations are relatively scarce. As far as we know, the first reported example, which was used for sensing applications, was able to respond to the presence of CH3Hg+.378 The gated material consisted of a micrometric silica mesoporous support loaded with safranin O and capped with 2,4-bis(4-dialkylaminophenyl)-3-hydroxy-4alkylsulfanylcyclobut-2-one groups (APC). APC were formed by the reaction of a squaraine dye and thiol units that were previously anchored on the external surface of the mesoporous solid. When CH3Hg+ was added to a suspension of the APCcapped support in acetonitrile:toluene (4:1 v/v) release of entrapped safranin O was observed (see Figure 234). The uncapping derived from the reaction of CH3Hg+ with the thiol group on APC, resulting in the coordination of the cation to thiols, in the release of the bulky squaraine chromophore and in the delivery of the entrapped safranin O reporter. This simple chromogenic probe allowed detection of CH3Hg+ down to 0.5

Figure 232. MSNs functionalized with amino groups and capped AuNPs coated with cysteine in the presence of Cu2+. Cargo release was observed in the presence of ATP.

MSNs were loaded with fluorescein. ATP induced cargo release in water at a neutral pH, which was attributed to a preferential coordination of Cu2+ for ATP at relatively high ATP concentrations. The authors also found that the gate was also opened if the pH was lower than 5, most likely due to protonation of the amino groups. The in vitro cell cytotoxicity of the gated MSNs was tested on HeLa cells. No obvious cytotoxic effects were detected after incubation with up to 100 μg/mL of AuNPs-capped MSNs for 48 h. Next, endocytosis inhibition studies were carried out in which common chemical inhibitors of clathrin-dependent (CDE) and clathrin-independent (CIE) endocytosis were employed. Clathrin-dependent endocytosis 668


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complementary to the anchored one at the terminal parts was hybridized capping the pores of the MSNs as depicted in Figure 235. This second oligonucleotide strand was highly rich in

Figure 233. MSNs loaded with fluorescein and capped with alkyltrimethylammonium-functionalized AuNPs. Cargo delivery was achieved in the presence of the tetracarboxylate cavitand C1.

Figure 235. DNA-capped MSNs loaded with Rh 6G for the optical detection of Hg2+cation.

thymines and displayed a high affinity to Hg2+. In the presence of this cation, the strand was displaced from the surface resulting in dye delivery. The system was able to detect Hg2+ in water with a LOD of 4 ppb. In addition, no important cargo release was observed in the presence of other cations such as Ni2+, Pd2+, Fe2+, Fe3+, Ba2+, Zn2+, Ca2+, Mg2+, Cu2+, Co2+, and Cd2+. Wen, Song, and co-workers developed silica mesoporous supports capped with a K+-selective aptamer and demonstrated that cargo delivery was achieved in the presence of this cation.380 For this purpose, mesoporous silica microparticles were functionalized with aminopropyl moieties and the amino groups further reacted with succinic anhydride to prepare a carboxylate covered material. An amino-functionalized K+-selective aptamer was grafted to the silica surface by the formation of an amide bond (see Figure 236). The pores were loaded with Rh B and capped upon addition of AuNPs coated with the oligonucleotide 5′-(DTPA) TAT TTA TAC GGG TTA GGG TTA GGG TTA GGG TTT TTTTTTTTTTTTTTT TT-3′ which contained the K+ aptamer (DTPA: 1,2-dithiane-4-O-dimethoxytrityl-5-[(2cyanoethyl)- N,N-diisopropyl]-phosphoramidite). Aqueous suspensions of the capped microparticles showed nearly “zero release” in the absence of K+, whereas when this cation was present in the solution a marked delivery of the entrapped Rh B dye was observed. This release was ascribed to the fact that the single-strand DNA, which coated the AuNPs, self-assembled into a G-quadruplex structure in the presence of K+ cation with the subsequent detachment from the microparticles’ surface.

Figure 234. Micrometric silica mesoporous support capped with bulky APC moieties and loaded with safranin O for the chromo-fluorogenic detection of CH3Hg+.

ppm, whereas the use of standard fluorometric methods reduced the detection limit to less than 2 ppb. This system was successfully tested to determine CH3Hg+ in fish samples by using a simple CH3Hg+extraction procedure with toluene and CH3Hg+ detection with the APC-capped solid. Fish samples were also spiked with cations (i.e., Na+, K+, Ca2+, Mg2+, Cu2+, Ni2+, Zn2+, Ag+, Pd2+, Cd2+, Au3+, and Tl+) and various organic species such as sodium lauryl sulfate, cysteine, histamine, ethanol, heptylamine, hexanethiol, but none of those species affected the response of the capped material to CH3Hg+. In a more recent work Tan, Zhang, and co-workers designed a Hg2+ probe based on oligonucleotide-capped MSNs. The authors loaded MSNs with Rh 6G and functionalized the surface with isocyanate groups, which reacted with an amino-modified oligonucleotide.379 Moreover, a second larger oligonucleotide 669


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exchange of Cs+ as a consequence of the formation of 1:1 macrocycle- K+ complexes. Willner and co-workers reported the design of DNAzymecapped MSNs, which were opened by the cooperative effect due to the presence of the cations Mg2+ and Zn2+. The authors functionalized MSNs with two different thiolated single strands of DNA, using N-ε-maleimidicaproyl-oxosulfosuccinimide ester as a cross-linker, resulting in two different solids, one for Mg2+ and the other for Zn2+.382 Each DNA strand contained the specific sequence of basis for the respective Mg2+-dependent DNAzyme and Zn2+-dependent DNAzyme hydrolysis process. The DNAzyme hydrolyses a specific sequence of DNA only in the presence of a certain cation (Mg2+ or Zn2+ in this case) due to the complex formed by the cation and the particular region of the DNA (see Figure 238A). Then DNA-functionalized solids were loaded with MB or thionine and were capped using the corresponding complementary DNA sequence. Both system presented release of the selected dye in the presence of the corresponding cation (Mg2+ or Zn2+). The authors found this process selective, and no delivery was observed in the presence of other divalent cations. Furthermore, the inclusion in the DNA sequence of ATP-aptamers or Hg2+-depending domains was also studied. The incorporation of these foreign sequences disturbed the formation of the complexes of DNA with Mg2+ or Zn2+ due to the major flexibility of the strand. In this case, the formation of the aptamer-ATP complex or the metal ion-nucleic acid bridge (thymine-Hg2+-thymine) was a required previous step to the system to form the corresponding complexes with Mg2+ or Zn2+ and finally restore the biocatalytic activity of the DNA-depending enzyme (see Figure 238B). These result in systems that need two different and complementary stimuli (ATP and Mg2+ or Hg2+ and Zn2+) to achieve the cargo release. The authors analyzed as a model system the Mg2+-induced release of the anticancer drug DOX, and the ATP cooperative synergetic Mg2+-opening of the pores. DOX entrapped in the pores of the nanomaterial was not released by ATP alone, was inefficiently released by only Mg2+ ions, and efficiently released by the addition of ATP and Mg2+ ions. DNAzymes were also used as caps by Willner et al. for the preparation of gated MSNs that were opened in the presence of Mg2+ and UO22+ cations.383 For the preparation of the Mg2+selective capped material, MSNs were selected and were functionalized with aminopropyl moieties, and then the amino groups reacted with a cross-linker agent that yielded maleimide moieties in the external surface. Afterward, a thiolated ribonuclease, containing two complementary single-stranded DNA sequences of Mg2+-dependent DNAzyme, were linked into the external surface. In order to prepare the final material, the pores of the inorganic scaffold were loaded with MB and then capped by the addition of a Mg2+-dependent DNAzyme sequence through hybridization with the previously linked oligonucleotide (see Figure 238A). Aqueous suspensions of the capped nanoparticles showed negligible MB delivery, whereas in the presence of the Mg2+ cation, a remarkable dye release was observed. Delivery was attributed to a coordination of the Mg2+ cation with the linked DNAzyme that induced the cleavage of the caps. The optical response was selective, and the authors found that other divalent cations such as Zn2+, Pb2+, Ca2+, Sr2+, Ba2+, Cu2+, Co2+, Mn2+, Ni2+, Fe2+, and Hg2+ were unable to induce dye release. The same experimental procedure was used for the preparation of the UO22+-selective capped material but using as loading molecule thionine instead of MB and capping the pores with the corresponding UO22+-dependent DNAzyme sequence.

Figure 236. Mesoporous silica microparticles loaded with Rh B and capped with AuNPs coated with a K+-aptamer for the sensing of K+.

Choi et al. also reported the design of capped MSNs able to be opened in the presence of K+ but using in this case macrocycles as capping units as can be observed in Figure 237.381 MSNs were loaded with the dye curcumin, and the macrocycle 18-crown-6 was anchored on the external surface. The nanoparticles were finally capped upon addition of Cs+ cation that formed sandwich complexes with the grafted 18-crown-6 groups. The systems showed zero release until the addition of K+ cation, which induced the delivery of the entrapped curcumin due to the

Figure 237. MSNs capped with Cs+-18-crown-6 complexes and loaded with curcumin. Uncapping was achieved in the presence of K+ cation. 670


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In this case, the authors found a selective cargo release in the presence of the UO22+ cation. A similar approach was used by Tang and co-workers that also used DNAzyme-capped MSNs that were selectively opened in the presence of the Pb2+ cation.384 MSNs were selected as inorganic support, and the external surface was functionalized with GTPMS. Then, using an epoxy-amino reaction, the Pb2+DNAzyme was anchored and the pores of the support were loaded with glucose. Finally, the pores were capped by the addition of a single-stranded DNA sequence that hybridized with the grafted DNAzyme sequence (Figure 239). Aqueous

Figure 239. DNAzyme-capped MSNs loaded with glucose and opened in the presence of Pb2+cation.

suspensions of the capped nanoparticles showed negligible glucose release, whereas in the presence of Pb2+, a remarkable release of glucose was observed. Glucose delivery was measured using a personal glucometer. Cargo delivery was related to the cleavage of the caps upon Pb2+ coordination with the linked DNAzyme. The authors found that other metal cations such as Cu2+, Co2+, Cd2+, Mg2+, Zn2+, Fe3+, Ag+, and Hg2+ were unable to induce glucose release. Sun et al.385 presented a new example of pseudorotaxane nanovalves activated by the presence of methyl viologen or by pH changes. The capped material consisted of carboxylatopillar[5]arene (CP[5]A) groups that encircled positively charged pyridinium units grafted onto the surface of MSNs. The pores were loaded with Rh B, calcein, or DOX. The authors found that the addition of methyl viologen, which has a higher binding affinity with CP[5]A than pyridinium, induced an immediate cargo release as depicted in Figure 240. Moreover, the authors found that cargo release was also operated by pH changes. No cargo delivery was found at neutral pH, whereas once the pH decreased to 5, the dye was released as a result of the dethreading of the CP[5]A from the pyridinium stalks due to protonation of the carboxylate groups in CP[5]A. Stoddart et al. developed MSNs that were opened in the presence of fluorodialkylammonium salts via a competitive binding protocol. MSNs were first functionalized with isocyanatopropyltriethoxysilane that was further reacted with

Figure 238. (A) MSNs capped with DNAzymes that were opened selectively in the presence of Mg2+, Zn2+, Pb2+, and UO22+ cations. (B) MSNs capped with DNAzymes which were selectively opened in the presence of ATP and Mg2+ or Hg2+ and Mg2+. 671


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Figure 240. MSNs capped with inclusion complexes formed between pyridinium salts and CP[5]A, loaded with Rh B, calcein, or DOX. Cargo release was observed in the presence of methyl viologen.

the hydroxyl group of a Boc-protected dialkylammonium precursor stalk in order to obtain a carbamate.386 The material was treated with trifluoroacetic acid to afford the corresponding dialkylammonium ion stalk. The system was loaded with coumarin 460 or IR 140 and capped with dibenzo-[24]crown8 (DB24C8) as shown in Figure 241. The nanovalves were opened via a competitive binding mechanism using different fluorodialkylammonium salts that had more affinity for the DB24C8 than the attached dialkylammonium stalk. Moreover cargo delivery was also observed in the presence of certain metal cations that formed complexes with DB24C8. The authors found that cargo delivery depended on the metal cation employed (rate of release: Cs+ ∼ Ca2+ > K+ > Li+) and was influenced by a counterion effect (rate of release: e.g., OH− > Cl− for Li+). Moreover, cargo delivery was also found when using bases that were able to deprotonate the dialkylammonium stalks. In particular, the authors found that cargo release was governed essentially on the size as well as the basicity of the different bases; the rate of release was trimethylamine > N,N′-diisopropylethylamine > hexamethylphosphoroustriamide. Acetylcholine was used by Yang et al. as a trigger for uncapping gated MSNs.387 In order to prepare the capped solid, the authors functionalized the surface of MSNs with a pyridinium derivative and loaded the pores with Rh 6G. Finally, the pores were capped with SC[4]A or CP[5]A via the formation of inclusion complexes with the grafted pyridinium derivative (Figure 242). Water suspensions of both gated materials showed negligible cargo release, whereas upon addition of acetylcholine, a marked delivery of the dye was observed. Rh 6G release was a consequence of the formation of acetylcholine-SC[4]A and acetylcholine-CP[5]A complexes. In addition, dye release from the material capped with SC[4]A was larger than that observed for the nanoparticles capped with CP[5]A, an effect that was ascribed to the difference in binding constants. Carboxylate functionalized pillar[6]arene (CP[6]A) were used by Du et al. for the construction of nanovalves able to be opened in the presence of divalent metal cations, methylviologen, and changes in the pH.388 MSNs were functionalized with a dimethylbenzimidazolium or a bipyridinium derivative containing trialkoxysilane moieties. The pores of both nanoparticles

Figure 241. MSNs capped with nanovalves containing the macrocycle DB24C8 and loaded with coumarin 460 or IR 140. Addition of selected cations induced the release of the entrapped cargo.

Figure 242. Acetylcholine induced controlled release of Rh 6G from MSNs capped with SC[5]A or CP[5]A-pyridinium inclusion complexes.

were loaded with Ru(bipy)32+ and were capped upon addition of CP[6]A, through the formation of inclusion complexes with the anchored stalks as depicted in Figure 243. Release of the 672


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Figure 243. MSNs loaded with Ru(bipy)32+ and capped with inclusion complexes formed between dimethylbenzimidazolium or bipyridinium and CPA[6]. Cargo was released in the presence of certain divalent cations, methylviologen, or at an acidic pH.

entrapped Ru(bipy)32+ dye was triggered upon addition of divalent cations (Cu2+, Zn2+, Ni2+, Ca2+, and Mg2+) to aqueous neutral solutions of both nanoparticles. Coordination of divalent metal cations with carboxylate groups of CP[6]A induced the dethreading of the inclusion complexes with subsequent dye release. A second way to induce dye delivery was the addition of methylviologen due to its preferential coordination with CP[6]A. Moreover, the authors found that although aqueous suspensions at pH 7 of both nanoparticles showed negligible release (less than 9% after 5 h), a clear Ru(bipy)32+ delivery (ca. 83% after 5 h) was observed at pH 3. In this case, delivery was ascribed to the protonation of CP[6]A that induced its dethreading from the surface. Finally, similar CP[6]A-capped MSNs were prepared but loaded with DOX. MTT viability assays were carried out with these nanoparticles, and A549 cells showed a reduction of cell viability to ca. 20%, which was attributed to the uptake of the nanoparticles and uncapping in the acidic lysosomal environment. Willner and co-workers developed oligonucleotide-gated MSNs, taking profit of the switchable formation and dissociation of G-quadruplexes assisted by the K+ ion.389 The authors prepared a collection of capped solids. In the first one, MSNs were functionalized with amino groups to which a G-rich oligonucleotide capable of forming G-quadruplex was anchored using N-(ε-maleimidocaproyloxy)sulfosuccinimide ester as a coupling agent. The pores of the nanoparticles were loaded with Rh B. In the presence of K+ ions, the anchored oligonucleotide changed its conformation to adopt a K+-stabilized G-quadruplex structure that locked the pores (see Figure 244A). In the presence of kryptofix [2.2.2] (KP), or 18-crown-6-ether (CE), a

Figure 244. MSNs capped with oligonucleotides. The cappinguncapping mechanism was controlled by the presence of (A and B) KP or (C) K+ cation.

ligand-mediated extraction of K+ ions led to the dissociation of the G-quadruplex, resulting in the unlocking of the pores and cargo release. Moreover, the authors demonstrated that it was possible to control the process of blocking and unblocking the pores by the alternative treatment of the gated MSNs with KP and K+ ions. In a similar way, the authors prepared a second system, capping, in this case, the pores by the formation of a complex between thrombin and a thrombin-aptamer that needs K+ ions to form a G-quadruplex superstructure and strongly interact with thrombin. Again, the presence of KP or CE sequestrated the K+ ions inducing the G-quadruplex disassembling and the subsequent cargo release as shown in Figure 244B. Finally, in a third example, the authors anchored to the MSNs a small oligonucleotide and capped the pores with a second oligonucleotide that was composed of both the partially complementary sequence of the anchored oligonucleotide and the G-rich domains able to form G-quadruplex structures. In this case, the presence of K+ induced G-quadruplex formation, resulting in uncapping of the pores and Rh release (Figure 244C). Moreover, the authors demonstrated that the addition of KP or CE eliminated K+ ions, and the free strand rehybridized with the anchored strand to block the pores. The system was selective, and for instance, the ions Li+, Na+, and NH4+ ions were 673


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unable to uncap the pores. As the content of K+ ions in cells is ca. 80 mM, the author tested this DNA-capped system in a cellular environment. In this case, the nanoparticles were loaded with DOX and labeled with FITC. Interaction of these nanoparticles with breast epithelial cells (MCF-10a) and breast cancer cells (MDA-MB-231) confirmed the K+-driven uncapping mechanism. Moreover, the authors found a higher DOX fluorescence signal in cancer cells when compared to the noncancerous cells due to the enhanced endocytosis ability of the former. 7.3. Triggered by Small Neutral Molecules

Lu and co-workers developed gated MSNs that were able to deliver an entrapped cargo in the presence of glucose.390 MSNs were functionalized with prop-2-yn-1-yl(3-(triethoxysilyl)propyl)carbamate, and then the inhibitor D-(+)-glucosamine was grafted through a click chemistry reaction. The pores were loaded with Rh B and capped by the addition of the glucose oxidase (GOx) enzyme through the formation of a complex with the grafted inhibitor, as depicted in Figure 245. Aqueous

Figure 246. MSNs loaded with Ru(bipy)32+ and capped with CDfunctionalized GOx. The nanoparticles were uncapped in the presence of glucose.

authors confirmed that dye delivery was induced when glucose was present in the solution due to displacement of CD-GOx as a consequence of CD-GOx-induced oxidation of glucose to gluconic acid and the subsequent protonation of the benzimidazole group. The authors found a linear response to glucose in the 1 × 10−2−1 × 10−4 mol L−1 range and a LOD of 1.5 × 10−4 mol L−1 which was in the range of other glucose detection systems. The response to glucose was selective, and the presence of other saccharides such as mannose, fructose, galactose, maltose, and saccharose at a concentration of 1 × 10−3 mol L−1 induced no cargo delivery. In a further work,392 the same authors demonstrated that the gating mechanism and different effector ensembles can be integrated in a unique system based on the use of Janus-type nanoparticles having opposing Au and mesoporous silica faces. In particular, the porous network of the silica face was loaded with Ru(bipy)32+ and the external surface grafted with 3-(2aminoethylamino)propyltrimethoxysilane. Additionally, the gold side was functionalized with thiol modified urease enzyme as shown in Figure 247. Aqueous solution (acetate buffer pH 5) of the Janus nanoparticles showed negligible cargo release because the polyamines were protonated and the molecular gate was closed. In the presence of urea, a clear delivery of the entrapped ruthenium complex was found. This release was a consequence of the urease-catalyzed hydrolysis of urea into CO2 and NH3 that induced an increase in the pH of the local environment. With this increase, the polyamines became deprotonated and subsequently the gates were opened. Another gated system using Janus-type nanoparticles was recently described by the same authors.393 In this case, the mesoporous silica face was loaded with Ru(bipy)32+, functionalized with benzimidazole moieties, and the pores of the mesoporous structure blocked by the formation of inclusion

Figure 245. MSNs functionalized with D-(+)-glucosamine capped with GOx enzyme that were selectively uncapped in the presence of glucose.

suspensions of the capped material showed negligible cargo release, whereas the presence of glucose induced a clear dye release. The observed delivery was proportional to the amount of glucose added, and it was a consequence of a displacement reaction of the GOx due to the presence of the glucose substrate. Cargo delivery in the presence of glucose was selective, and the authors confirmed that other monosaccharides tested (i.e., fructose, mannose, and galactose) induced no payload release. Villalonga and co-workers developed MSNs in which an enzyme also acted as a cap; however, in this case, the uncapping process was triggered by the product obtained by the enzyme’s activity on glucose.391 MSNs were loaded with Ru(bipy)32+ and the external surface functionalized with 3-iodopropyltrimethoxysilane that was further transformed to 1-propyl-1-H-benzimidazole groups through a nucleophilic substitution reaction using benzimidazole. The pores were finally capped with an active CDGOx through the formation of inclusion complexes between the CDs groups in CD-GOx and the propylbenzymidazole stalks attached to the solid support (see Figure 246). In their study, the 674


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Figure 247. Janus Au-MSNs loaded with Ru(bipy)32+ and capped with polyamines. The system was uncapped in the presence of urea.

Figure 248. Janus Au-MSNs loaded with Ru(bipy)32+ and capped with β-CD-benzimidazole inclusion complexes. Uncapping of the pores was observed in the presence of glucose and ethyl butyrate.

complexes with β-CD. Moreover, thiol-modified esterase and GOx were covalently immobilized on the Au surface as depicted in Figure 248. Experimental results confirmed that the presence of either D-glucose or ethyl butyrate, or a combination of both substrates, produced a local decrease of the pH through an enzyme-catalyzed substrate conversion to gluconic acid or butyric acid which induced the opening of the β-CD-gated nanovalves and release of the entrapped dye. Moreover, the Janus ensemble was also loaded with the anticancer drug DOX and was tested in HeLa cancer cells. The authors observed a remarkable enhancement of DOX release inside the cells upon exposure to ethyl butyrate or glucose in comparison with the results obtained in the absence of these inputs. In addition, a synergistic enhanced DOX delivery was found when the cells were treated with both Dglucose and ethyl butyrate substrates simultaneously. Kim and co-workers developed MSNs that were selectively opened in the presence of fructose and galactose. MSNs were functionalized with amine moieties and coupled with phydroxyborylbenzoic acid via a NHS ester. The material was loaded with calcein, and the boronic acid units at the surface were anchored to α-, β-, or γ-CDs indistinctly through the formation of boronate esters. A clear dye delivery was observed in the presence of fructose and galactose (see Figure 249), whereas no delivery was found for mannose or glucose. Uncapping was explained due to the displacement of the CDs by competitive interaction of the monosaccharides with the boronic acid groups.394

Figure 249. MSNs capped with CDs, through the formation of boronate esters with a grafted boronic acid subunits. The nanoparticles were loaded with calcein and uncapped in the presence of galactose or fructose. 675


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capping biomolecule with the aim to overcome the common insulin release decrease of conventional glucose-responsive insulin delivery systems (Figure 251).396 In particular, MSNs

Dextran-capped magnetic MSNs that were selectively opened in the presence of glucose were prepared by Jana and coworkers.395 For this purpose, γ-Fe2O3 nanoparticles were synthesized and coated with a silica mesoporous layer. Then, the external surface was functionalized with 3-(2aminoethylamino)propyl trimetoxysilane and the grafted amines reacted with 3-carboxyphenylboronic acid using EDC-NHS coupling reaction (also folate-NHS was anchored to the surface amines for targeting purposes). Finally, the pores were loaded with drugs (CPT or tolbutamide) and capped upon addition of dextran (through the formation of boroester linkages with 1,2diol groups of the polysaccharide), as shown in Figure 250. PBS

Figure 251. Glucose induced cAMP release from MSNs capped with gluconic acid modified insulin.

functionalized with aminopropyl and phenylboronic acid groups in the pore outlets were loaded with cAMP. To block the pores, the authors prepared FITC-labeled G-Ins, which formed boroesters with the phenylboronic units. The authors tested a collection of sugars and found that the capped MSNs were opened more easily with fructose, followed by glucose. The authors studied the cAMP release at pH 7.4 and 8.5. In the absence of glucose, less than 10% of the cargo was released at both pHs. On the contrary, cAMP delivery was achieved in the presence of glucose, although delivery was higher at pH 8.5. Cell viability studies using four different cell lines (i.e., rat pancreatic islet tumor (RIN-5F), mouse liver, skin fibroblast, and HeLa cells) evidenced the nontoxicity of this material. The cellular concentration of cAMP in RIN-5F cells treated with cAMP solutions and using the capped MSNs were quantified by a Millipore cAMP HTS immunoassay evidencing that when only using the solid an elevation of intracellular cAMP concentration occurred. The controlled release of insulin in the presence of glucose was also described by Shi and co-workers.397 The authors used MSNs with a pore size of ca. 12 nm that were loaded with insulin. Then, the external surface of the particles was coated with poly(ethylenimine), through simple electrostatic interactions, in order to include amino groups at the surface. The amino groups were cross-linked with glutaraldehyde, and the obtained free aldehyde moieties reacted with GOx and catalase (CAT) enzymes to form imine bonds. PBS suspensions (pH 7.4) of the nanoparticles showed zero release of the entrapped insulin; however, when glucose was added, a remarkable insulin release, directly related with the amount of the glucose present in the media, was observed. Upon addition of glucose, a decrease in the pH of the microenvironment was produced due to its oxidation to gluconic acid induced by GOx enzyme. This acidic pH weakened the cross-linked multilayer structure by partly breaking

Figure 250. Fe2O3 nanoparticles coated with a mesoporous silica shell, loaded with selected drugs (CPT or tolbutamine) and capped by dextran. The pores were opened upon addition of glucose due to a displacement reaction that substituted the anchored dextran.

solutions (pH 7.4) of both nanoparticles (loaded with CPT or tolbutamide) showed negligible release after 12 h. However, upon addition of glucose, a remarkable drug release was observed due to the replacement of the dextran from the nanoparticle surface by the monosaccharide. CPT-loaded FA-functionalized MSNs were internalized by HeLa cells with overexpressed folate receptors and delivered the entrapped drug in the presence of the intracellular glucose. Also, the same nanoparticles were internalized by RIN-SF cells (with overexpressed glucose transporter), and again CPT was released. MTT assays carried out with CPT-loaded nanoparticles indicated no toxicity in the absence of glucose, whereas when the test was carried out in the presence of monosaccharide, the viability was reduced to 40%. The same assays showed no toxicity (in the absence or in the presence of glucose) for tolbutamide-loaded nanoparticles in RIN-SF cells. Some authors have developed a selective glucose-responsive system and have used them for the glucose-induced release of insulin which was directly related with the amount of the glucose present in the media. In this field, Lin and co-workers designed a glucose-responsive system able to stimulate insulin secretion releasing cyclic adenosine monophosphate (cAMP) and, at the same time, using gluconic acid-modified insulin (G-Ins) as a 676


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imine bonds with the subsequent enhancement of insulin release. The activity of GOx was assured by the CAT enzyme that was able to decompose the hydrogen peroxide, formed in the oxidation of glucose, into water and oxygen. Li, Zhang, and co-workers also reported the design of a capped system for insulin delivery, which was responsive to the presence of glucose and pH changes in a cooperative way. The authors functionalized MSNs with 3-(methylacryloxy)propyltrimethoxysilane and then the nanoparticles were covered with a polymeric shell formed by 3-acrylamidophenylboronic acid and N-isopropylacrylamide finally obtaining two solids; in one case, the polymer coated over the material was cross-linked by addition of dextran-maleic acid, and in the second, it was not. Phenyl boronic acids on the copolymer shell were sensitive to glucose and pH changes. The nanoparticles were loaded with insulin, and its delivery at different pHs and in the presence or absence of glucose was studied for the two polymer-coated materials. The best results were obtained using the cross-linked polymer shell in the presence of glucose at pH over 7.4. Furthermore, cell viability experiments using MTT assays demonstrated both solids were nontoxic to A549 cells.398 In a more recent work,399 the same authors used a similar synthesis procedure to prepare different materials with a copolymer shell of dextran-maleic acid bearing 3-amidophenylboronic acid moieties. Three solids containing different amounts of grafted copolymers were prepared, and their potential in vivo application as oral insulin delivery systems was studied. These studies demonstrated that a thicker layer of polymer resulted in a greater capacity of drug loading and slow delivery kinetics. The cellular uptake of the capped nanoparticles was corroborated using a similar solid loaded with insulin and capped with the same copolymer which was additionally marked with FITC. Absorption and bioavailability of protein drugs in the intestine is commonly low due to the weak mucosal membrane permeability and enzymatic degradation. Therefore, the absorption properties of capped MSNs were investigated in epitheial intestinal villi. Remarkably, strong green dots in villi were observed after incubation with capped nanoparticles, indicating good absorption of the nanomaterial. Finally, in vivo studies, via oral administration of diverse insulin-loaded capped solids having a different polymer shell thickness, in diabetic rats were compared to free insulin oral and injected administration. Oral administration of insulin-loaded nanoparticles decreased blood glucose levels at 1.5 h and remained low even at 7 h after administration. These assays confirmed that the capped support was able to reduce more efficiently blood glucose levels than oral administration of insulin. A more sustained reduction of glucose was achieved when using the capped material having the thickest polymer shell. Yang, Zhao, and co-workers400 prepared superparamagnetic mesoporous silica microspheres, capped with iron oxide, which were uncapped in the presence of glucose. The system was also opened with EDTA and sodium citrate. The particles containing a Fe3O4 core and a mesoporous shell were loaded with Rh B or paclitaxel, and the outer surface was functionalized with 3glycidoxypropyltrimethoxysilane. In parallel, iron oxide Fe3O4, nanoparticles of about 10 nm, were functionalized with iminodiacetic acid (IDA) through a coordination interaction between the carboxylic groups of IDA and the iron atoms on the surface of the nanoparticles. The pores in the silica particles were capped with the IDA-modified Fe3O4 through the reaction of the epoxy groups on the silica surface with the amino groups in the iron nanoparticles (see Figure 252). The opening protocol relies

Figure 252. Fe3O4superparamagnetic nanoparticles coated with a mesoporous silica shell, loaded with paclitaxel or with Rh B and capped with iron oxide nanoparticles. The system was uncapped upon addition of glucose, EDTA, or sodium citrate.

on a ligand-exchange strategy. The coordination between IDA and Fe3O4 is chemically labile and could be cleaved by agents which have stronger coordination ability to the iron atoms. Glucose, sodium citrate, and EDTA were used as trigger agents to release the entrapped guest in the PBS solution. Rh B-loaded nanocarrier showed good biocompatibility and was able to be endocyted in SGC-7901 and HeLa cells. Moreover, the authors found that the capped nanoparticles loaded with paclitaxel proved low cytotoxicity, good biocompatibility, and were able to be endocyted in SGC-7901, PC12, and HeLa cells. In contrast, the addition of sodium citrate induced paclitaxel delivery and reduction of cell viability. Du and co-workers prepared MCM-41 particles functionalized with 3-(chloropropyl)triethoxysilane and further reacted with 1,4-butanediamine.401 The functionalized support was loaded with calcein dye and capped with CB[7] by coordination with the protonated alkyl amines of the 1,4-butanediamine derivative at neutral pH. The formed complex was very stable, and when the material was suspended in water at pH 6.5, no dye release was observed (Figure 253). However, when pH was gradually increased, the capping efficiency was dismissed due to a lower association constant between deprotonated 1,4-butanediamine and CB[7]. Moreover, at neutral pH, CTAB and 1,6-hexanediamine were also used to displace the capping CB[7]. In their study, the authors found that the uncapping event and subsequent calcein release rate was related to both binding affinity and concentration of the competitor. In a further work, the same authors prepared MSNs containing a Fe3O4 core and the same gating mechanism. In this case, the system was opened with cadaverine or with lysine in the presence of a decarboxylase.402 When the material was suspended in the presence of lysine, no calcein release was observed. However, when decarboxylase was added, lysine was transformed into 677


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Figure 253. Mesoporous silica, loaded with calcein and capped by the formation of an inclusion complex between grafted 1,4-butanediamines and CB[7]. Changes in pH or the presence of CTAB, 1,6-hexanedimine or cadaverine induced the release of the entrapped calcein. Figure 254. MSNs loaded with Ru(bipy)32+ dye and capped with a Crich DNA sequence that hybridized in the presence of Ag+. The gate was opened in the presence of DTT.

cadaverine via decarboxylation. The protonated cadaverine at neutral pH formed more stable complexes with CB[7], resulting in cargo delivery. The authors indicated that decarboxylase and polyamines (as cadaverine) are related with cell growth and cancers and suggested that their magnetic delivery system may find applications for site-specific drug release. Wang et al. designed MSNs loaded with Ru(bipy)32+ and grafted on the pore outlets with a C-rich DNA sequence.403 In the presence of Ag+ ions, the random-coil C-rich DNA strands hybridized each other through the formation of stable C−Ag+−C base pairs blocking the pores. However, the addition of DTT induced a competitive displacement reaction of the C−Ag+−C complexes, deforming the duplex DNA into single-stranded DNA, allowing cargo delivery as shown in Figure 254. The DTTinduced DNA conformational changes were studied via staining with SYBR Green I. When Ag+ ions were present, DNA hybridized each other and SYBR inserted into the duplex structure showed a strong fluorescence, while in the presence of DTT, a poor fluorescence was observed, indicating the randomcoil state of DNA. These changes were reversible through the addition of Ag+ and DTT molecules, alternately. In addition, the same uncapping process was observed in the presence of cysteine. The cell uptake and cytotoxicity of the capped MSNs were investigated in HeLa cells. CLSM demonstrated that the system entered the cells via endocytosis, and that the particles distributed mainly in the lysosomes. At longer incubation times (5 h), Ru(bipy)32+ fluorescence was observed in whole cells, indicating that the cargo was successfully released from the nanochannels of MSN. This was attributed to the intracellular thiol-containing stimuli, such as cysteine, and GSH that induced cargo delivery. No detectable cytotoxicity of the capped MSNs was observed in HeLa cells treated with the increasing doses of the nanoparticles for 24 h. Gated materials that are opened in the presence of GSH due to its reducing ability are included in section 5. In contrast, the following gated support was opened with GSH in this case following a displacement protocol. In particular Son et al. used silica nanotubes (SNTs) as an inorganic scaffold which were

functionalized in the open end with a gold nanoring through a seed-mediated Au growth reaction.404 Then, the nanotubes were loaded with DOX and capped upon addition of 1-octadecanethiol groups that were grafted into the surface of the gold nanoring yielding a hydrophobic cap that disabled the diffusion of the entrapped DOX as depicted in Figure 255. Addition of GSH induced the nanotube opening with the subsequent release of the entrapped cargo. The substitution of 1-octadecanethiol by GSH changed the hydrophobic character around the nanotube entrance to a more hydrophilic one, allowing the release of the polar DOX. DOX release from the nanotubes was studied by

Figure 255. SNTs containing an Au nanoring in the pore outlets loaded with DOX and capped with octadecanethiol. Addition of GSH induced the displacement of the long alkyl chains and subsequent drug release. 678


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real-time live-cell fluorescence spectroscopy in the multidrugresistant human breast carcinoma MCF-7/ADR cell line. Preincubation of cells with GSH ethyl ester was used to enhance intracellular GSH concentration. Under these conditions, an increase in intracellular fluorescence intensity was found after 24 h. Similar results were independently obtained in A549 cells. To further characterize the DOX-loaded capped SNTs, an in vivo experiment was performed. The SNTs were administered via injection into MCF-7/ADR xenografted mice. Remarkably, total fluorescence intensities increased after treating mice with GSH, showing that the release of DOX appeard to increase by 2 orders of magnitude. Furthermore, an in vivo tumor growth test indicated an increased reduction using DOX-loaded capped SNTs. Minko, He, and co-workers prepared MSNs functionalized on the external surface with isocyanatopropyl moieties and loaded with DOX.405 Then pores were capped upon the addition of amine-terminated G2 PAMAM dendrimers through the formation of urea bonds. PBS suspensions of the dendrimercapped MSNs showed negligible DOX release (only 2.6% after 24 h), whereas a remarkable drug release was observed (96% after 24 h) in the presence of GSH (4.9 mM). The authors did not clarify the role played by GSH in the uncapping mechanism. In order to enhance chemotherapy in multidrug-resistant cancer cells, Bcl-2 siRNA was adsorbed through electrostatic interactions onto the capping dendrimers, obtaining a second material able to deliver DOX and Bcl-2 siRNA simultaneously. Intracellular release of siRNA and DOX in A2780/AD human ovarian cancer cells was followed by fluorescence imaging using the DOX loaded and capped material complexed with the fluorescent siGLO green siRNA transfection indicator. Red and green fluorescence images confirmed the internalization of both agents. Capability of siRNA complex to silence mRNA expression was confirmed by a quantitative reverse transcriptase-polymerase chain reaction. Furthermore, viability of cells treated with free DOX, DOX-loaded material, and DOXloaded material complexed with Bcl-2 siRNA was compared using MTT assays in A2780/AD cells. At the same dose administration, DOX-loaded material presented a significant increase of cytotoxicity compared with free DOX administration. However, the results using the material also capped with Bcl-2 siRNA showed a highly remarkable enhancement of cell death (132-fold increase compared to free DOX). Finally, the authors also demonstrated that the Bcl-2 siRNA complexed material caused a significant enhancement of apoptosis that was related to siRNA internalization. Yuan, Pang, Lu, and co-workers described MSNs capped with a protein able to be opened in the presence of vitamin H. MSNs were loaded with Rh 640 dye, and the surface of the solid was modified with amine groups in order to anchor desthiobiotin through EDC/sulfo-NHS coupling reactions. Avidin was added to cap the pores by the formation of a desthiobiotin−avidin complex. Moreover, a DNA aptamer functionalized with a biotin group at the 5′-end was attached onto the avidin moiety anchored on the surface to obtain the final capped material shown in Figure 256. This specific aptamer was shown to highly and specifically bind to the cell membrane receptor protein tyrosine kinase 7, a protein that is overexpressed on the membrane of CCRF-CEM cells, a human precursor T-cell acute lymphoblastic leukemia cell line. Dye release studies demonstrated that vitamin H (biotin) was able to displace avidin from the surface of the MSNs due to the formation of highly stable avidin-vitamine H complexes.406 To test the targeting specificity

Figure 256. MSNs loaded with Rh 640, functionalized with desthiobiotin, and capped with an aptamer-containing avidin. The dye was released upon addition of vitamin H.

of the system, nanomaterials functionalized with or without the aptamer were incubated with CEM cells. Analysis using confocal microscopy and flow cytometry revealed that CEM cultures incubated with aptamer-modified nanoparticles showed strong intracellular fluorescence, while significantly less fluorescence was detected in the CEM cells incubated with nanoparticles without aptamer. Similar experiments were performed in Ramos cells, where there is no expression of cell membrane receptor protein tyrosine kinase 7. No obvious change in intracellular fluorescence was detected in this case, showing that the aptamermodified nanoparticles have negligible binding affinity to the nontarget cells. The excellent biocompatibility of the systems was confirmed, as virtually no cell death even at the concentration of 200 μg/mL was detected in both cell lines. Finally, the in vitro cytotoxicity of DOX-loaded nanomaterials were tested through MTT assay to evaluate their anticancer potential. Aptamermodified nanoparticles exhibited a dramatic enhancement of cell death in CEM cells when compared to free DOX, or DOXloaded nanoparticles without an aptamer. A much less pronounced cytotoxicity of the system to the Ramos cells was also detected. Bein and co-workers prepared MSNs, coated with different lipid bilayers, able to release an entrapped cargo upon addition of small molecules.407 The pores of the MSNs were loaded with fluorescein or calcein and capped by the formation of a lipid 679


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solid was not toxic to Hep G2 cells even when DOX was loaded in the pore voids of the material. In contrast, when cells were incubated with DOX-loaded nanoparticles and treated with histidine, cell viability was remarkably reduced. These results were confirmed by CLSM, where nuclear condensation and deformation was observed only in cells treated with the capped material and histidine. Finally, the authors suggested that their nanosystem can be used in cell imaging techniques. Hence, when cells were treated with histidine, a bright cyan fluorescence corresponding to the released cyan fluorescent protein used as cap was observed in the cytoplasm, which confirmed the right internalization of the capped MSNs. Additionally, DOX red fluorescence was visualized in the nuclei, indicating their efficient intracellular delivery. ́ The group of Martinez-Má ñez reported several examples of capped silica mesoporous supports which were opened in the presence of nitroaromatic explosives. Moreover the designed capped materials were used for the fluorogenic detection of these explosives.410 In a first example, the authors designed a silica support loaded with Ru(bipy)32+ and functionalized in the surface with 3-(azidopropyl) triethoxysilane groups. Moreover, a suitable pyrene derivative was attached to azido moieties by using a copper(I)-catalyzed Huisgen azide/alkyne 1,3-dipolar cycloaddition click reaction leading to the formation of a 1,2,3-triazole heterocycle. In this final material, the pores were blocked due to the presence of bulky pyrene moieties on the outer surface, and dye release was inhibited in acetonitrile (see Figure 258A). The presence of the nitroaromatic explosives 2,4,6-trinitrophenylmethylnitramine (tetryl) and 2,4,6-trinitrotoluene (TNT) induced

bilayer shell using DOPC, POPC, or a mixture of DOTAP/ DOPC. Aqueous suspensions of the six prepared nanoparticles (combining the two dyes and the three lipid formulations) showed nearly a zero release of the entrapped fluorophore (more than 99.9% of the loaded dyes remained in the inner of the nanoparticles after 1 h). However, upon addition of ethanol or triton X-100 (that were able to disrupt the lipid bilayer), a marked dye release was observed as depicted in Figure 257. In

Figure 257. MSNs loaded with fluorescein, calcein, or colchicine and capped by several lipid bilayers. The cargo was delivered upon addition of methanol or triton X-100.

order to test the potential use of these nanoparticles, the authors prepared one set of the material loaded with the anticancer drug colchicine and capped with a POPC bilayer and studied its internalization into HuH7 liver cancer cells. Stroeve and co-workers studied the use of copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), also known as the trade name Pluronic, as triggers to induce cargo delivery in MSNs coated with a lipid bilayer.408 In particular, the gated system consisted of Fe3O4@MSNs core− shell nanoparticles loaded with MB and capped with unilamellar lipid vesicles of DOPC via electrostatic interactions as depicted in Figure 257. The authors used two different copolymers (i.e., pluronic L61 [with a lower content of poly(ethylene oxide)] and pluronic L64 [with a higher content of poly(ethylene oxide)]). These copolymers were able to disturb the capping lipid bilayer inducing dye delivery. The authors found that a lower concentration of pluronic 61 was needed to induce cargo delivery. This result was related to the more hydrophobic properties of pluronic L61 due to its minor content of poly(ethylene oxide). In 2013, Ren and Qu developed a His-tagged cyan fluorescent protein (HCFP)-capped magnetic MSNs for simultaneous histidine-driven drug delivery and fluorescent imaging.409 The authors functionalized the external surface of the magnetic MSNs with APTES which was further reacted with succinic anhydride to obtain carboxylic groups. Finally, the authors indicated that a niquel-nitrilotriacetate complex able to interact with the Histagged protein was anchored on the surface. In a further step, the nanoparticles were loaded with IBU and the material capped upon the addition of the protein in PBS. A marked release of IBU and HCFP was observed when the capped nanoparticles were suspended in PBS at pH 7.4 in the presence of histidine. Taking into account the perfomed experiments, the authors concluded that HCFP cap could be removed by a displacement reaction with histidine. Furthermore, the authors proved that the capped

Figure 258. Mesoporous silica microparticles functionalized with electron-rich molecules and loaded with Ru(bipy)32+ complex for the chromo-fluorogenic recognition of nitroaromatic explosives. 680


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the formation of pyrene−nitroaromatic complexes, pushing apart the bulky pyrene from the pore voids resulting in dye release. LODs of 1.4 and 11.4 ppm for tetryl and TNT, respectively, were calculated from the fluorogenic studies. The authors found that other aromatic derivatives (i.e., 2,4dinitrotoluene, N-methylaniline, 2-nitrotoluene, and nitrobenzene), nonaromatic explosives (i.e., hexahydro-1,3,5-trinitro-1,3,5-triazine and pentaerythritoltetranitrate), MB, and naphthalene were unable to uncap the pores. Moreover 2,4dinitrotoluene was also found to be able to induce a partial release of the entrapped fluorophore but to a lesser extent. In a further work,411 the same authors reported the use of TTF-capped mesoporous silica supports also for the detection of certain nitroaromatic explosives (see Figure 258B). The solid was loaded with Ru(bipy)32+, and TTF was anchored on the surface using a copper(I)-catalyzed Huisgen azide/alkyne 1,3dipolar cycloaddition “click” reaction. The presence of a dense network of TTF units around the pores inhibited cargo release. Delivery studies in acetonitrile revealed that the presence of tetryl, TNT, and to a lesser degree 1,3,5-trinitrobenzene (TNB) induced dye release. The LOD, from UV−vis titration studies, of 28 μM (8 ppm) and 66 μM (15 ppm) for tetryl and TNT, respectively, were found. By fluorescence measurements, LODs of 3.5 μM (1 ppm) and 26 μM (6 ppm) for tetryl and TNT, respectively, were observed. Moreover, the authors reported similar TTF-capped silica mesoporous supports using in this case different TTF derivatives of diverse sizes and shapes and containing different numbers of sulfur atoms in the TTF structure (see Figure 258, panels C and D).412 In all cases, the supports were loaded with Ru(bipy)32+. The slight differences in the chemical structures of the TTF capping molecules resulted in different responses of the solids. Among the explosives tested, tetryl, TNT, and TNB induced dye delivery. By using color or emission measurements, LODs in the 1−10 ppm range were found. Using one of these TTF-capped supports, the authors were able to detect low levels of tetryl in soil samples with good results. Capped silica mesoporous supports have also been used to detect nerve agent simulants.413 The nerve agents sarin, soman, and tabun are highly toxic organophosphorous derivatives that have been used in terrorist attacks. Given the high toxicity of these compounds, the stimulants diethyl chlorophosphate (DCP), diisopropyl fluorophosphate (DFP), and diethyl cyanophosphonate (DCNP), which present similar reactivity as nerve agents yet are much less toxic, were used in this work. The silica mesoporous support was loaded with Ru(bipy)32+ and capped with bis(2-hydroxyethyl)aminopropyltriethoxysilane (HET) groups which formed a hydrogen-bonding network around the mesopores in acetonitrile, inhibiting cargo delivery (see Figure 259). The authors observed that the addition of nerve agent simulants induced dye release, which was attributed to the reaction between the nerve agent simulants and the −OH groups in HET that resulted in the rupture of the hydrogen-bonding network and cargo release. Other organophosphorus compounds tested induced no response. A simple chromogenic titration allowed detection of DCP down to ca. 15 ppm, whereas the LOD for DCP using a conventional fluorimeter was as low as 0.1 ppm. Similar LODs were found for DFP and DCNP. The authors also used this material for the detection of nerve agent mimics in the gas phase with good results. The design of gated systems triggered by specific small molecules and involving biomolecules as capping agents for sensing applications has also been explored. One of the first

Figure 259. Mesoporous silica microparticles loaded with Ru(bipy)32+ complex and capped with HET groups through the formation of a dense hydrogen bond network. Addition of nerve agent simulants induced pore opening and dye release.

works in this area used a polyclonal antibody for capping the pores. In this seminal study, an antibody for sulfathiazole was selected.414 In particular, the authors loaded the pores of a silica mesoporous support with Ru(bipy)32+ and anchored the silane derivative 4-(4-aminobenzenesulfonylamino)benzoic acid on the external surface of the inorganic material. Finally, the support was capped with a polyclonal antibody for sulfathiazole that also had affinity for the anchored molecule. A release of the dye when the antigen for the capping antibody (i.e., sulfathiazole) was present in the solution was observed as depicted in Figure 260A. A LOD of ca. 10 ppb for sulfathiazole was determined. Dye delivery was not observed in the presence of a family of related compounds. Based on a similar approach, Climent et al.415 developed antibody-capped support for the selective detection of finasteride. In this case, the silica mesoporous material was loaded with Rh B and functionalized with N-(t-butyl)- 3-oxo(5a,17b)-4-aza-androst-1-ene-17-carboxamide groups, which was a similar molecule to finasteride. The addition of polyclonal antibodies for finasteride induced the capping of the pores due to the interaction with the anchored haptenlike derivative. The addition of finasteride to water suspensions of the antibodycapped material resulted in the displacement of the antibody and release of the cargo (see Figure 260B). Moreover, the amount of dye delivered was found to be proportional to finasteride concentration, and a LOD of 20 ppb was calculated. Selectivity studies showed that only finasteride, among other steroids (such as testosterone, metenolone, and 16-β-hydroxystanozolol), was able to induce a significant uncapping process. Furthermore, the material was used to detect finasteride in spiked samples of urine, and recovery ranges from 94% to 118% were observed. With the use of a similar protocol, a new antibody-gated system for the detection of peroxide-based explosive TATP was developed.416 In this work, MSNs were loaded with the dye sulforhodamine B and the external surface was functionalized with an appropriate haptenlike molecule, and the system was capped using a TATP-selective polyclonal antibody. A remarkable and highly selective uncapping of the pores with the subsequent cargo delivery in the presence of TATP was 681


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Figure 260. Mesoporous silica particles loaded with several dyes, functionalized with different haptenlike molecules, and capped with antibodies for the recognition of sulfathiazole, finasteride, and TATP.

Figure 261. Mesoporous supports loaded with MB or glucose, functionalized with brevitoxin B antibodies, and capped with polystyrene microspheres for the detection of the brevitoxin B neurotoxin.

observed in phosphate-buffered saline (pH 7.4), as depicted in Figure 260C. TATP was detected at concentrations as low as 12.5 ppb using this protocol. Moreover the authors also demonstrated that the capped-MSNs could be integrated into a lateral-flow assay. With the use of this latter procedure, a limit of detection of 15 ppb for TATP was calculated. It was also found that other common explosives such as trinitrotoluene, hexogen, nitropenta, octogen, nitroguanidine, and hexamethylenetriperoxide diamine did not induce cargo delivery. A probe also using antibodies but based on a different configuration was developed recently by Zhang et al. for the detection of the neurotoxin brevetoxin B (PbTx-2).417 The authors used MSNs that were functionalized aminopropyl moieties, and then an antibody for brevetoxin B was attached to the amino groups using glutaraldehyde as a linker. To avoid detachment of the antibody, the formed imine bonds were further reduced by the addition of sodium cyanoborohydride. The pores of the nanoparticles were loaded with MB, and the system capped upon addition of aminated polystyrene microspheres of 25 nm of diameter. This capping protocol was carried out at pH 6.5. At this pH, there were strong electrostatic interactions between the negatively charged grafted antibody and the aminated polystyrene microspheres that were positively charged (see Figure 261). Addition of brevetoxin B induced a remarkable dye delivery as consequence of the selective interaction of this toxin with the grafted antibody and the displacement of the polystyrene miscrospheres from the surface

of the nanoparticles. MB released was detected using voltammetry. The authors found a linear dependence, from 10 pg mL−1 to 3.5 ng mL−1, between the peak current and the concentration of PbTx-2. A LOD of 6 pg mL−1 for PbTx-2 was determined. The capped-system showed a high cross-reactivity for PbTx-1 and PbTx-3, which can be explained taking into account the unspecificity of the used anti-PbTx-2 antibody. The authors found that the capped material did not respond to okadaic acid, aflatoxin B1, and microcystin-LR or to the presence of several possible components in seawater, including Na+, Ca2+, Mg2+, Cl−, SO42−, HCO3−, and F−. The sensing system was used for sensing PbTx-2 in different food samples. In a following work, the same authors applied a very similar antibody-gated design to detect PbTx-2.418 In this case, the authors loaded a magnetic mesoporous NiCo2O4 nanostructure with glucose and capped the pores following a similar protocol to that shown above. In the presence of PbTx-2, the capped nanoparticles released glucose that was quantitatively determined with a glucometer. A LOD 0.01 ng mL−1 for PbTx-2 was calculated. The authors used the capped nanoparticles for the detection of PbTx-2 in real samples. Tang et al.419 used capped MSNs for the detection of the mycotoxins aflatoxin B1 (AFB1). In this case, PEI-coated MSNs were loaded with glucose and the system was capped by addition of AuNPS functionalized with antibodies for AFB1 via simple electrostatic interactions between the positively charged PEI shell and the negatively charged antibodies. Buffered suspensions at pH 7.3 of the capped nanoparticles showed no delivery, 682


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whereas a marked glucose release was observed in the presence of AFB1 (Figure 262). Glucose was detected using a personal

Figure 262. MSNs loaded with glucose, coated with PEI, and capped with AuNPs functionalized with antibodies for AFB1. The system was uncapped in the presence of AFB1.

glucometer. Cargo delivery was attributed to the coordination of AFB1 with the antibodies and subsequent detachment of the AuNPs. A linear dependence from 0.01 to 15 ppb between the concentration of AFB1 and the response of the glucometer was observed. A LOD of 5 ppt for AFB1 was calculated. The same authors designed a fluorescence immunoassay for the detection of AFB1 also based on capped MSNs.420 In order to build the probe, mannose-terminated silanes were covalently attached onto the external surface of MSNs. Subsequently, the pores were loaded with Rh B and capped with biotinylated concanavalin A (Con A) via multivalent carbohydrate−protein interactions. Moreover, the biotinylated Con A and biotinylated anti-AFB1 were conjugated with streptavidin. At the same time, AuNPs were functionalized with invertase and with a BSA-AFB1 conjugate (see Figure 263). When the target AFB1 was present, a competitive immunoreaction for the immobilized anti-AFB1 antibody on the SMS between target analyte (AFB1) and the labeled AFB1-BSA on the AuNP was carried out. Therefore, the amount functionalized AuNPs on the SMS decreased with the increasing concentration of target AFB1 in the sample. Upon the addition of sucrose, invertase on AuNP hydrolyzes the sucrose into glucose and fructose. The generated glucose competed with the mannose for Con A coordination unblocking the pores and releasing the entrapped dye. A LOD of 8 pg mL−1 (8 ppt) for AFB1 was calculated. Moreover the response of the capped material was also tested in the presence of Zn 2+, Na+, K+, Cl−, HCO3−, NO3−, collagens, mucins, thyroid-stimulating hormone, and α-fetoprotein. Inorganic ions did not interfere with the AFB1 detection; however, the presence of glycoproteins in the sample increased the fluorescence intensity to some extent.

Figure 263. Representation of a fluorescence immunoassay based on a target induced competitive displacement reaction. Coordination of glucose with Con A induced the release of the entrapped Rh B from MSNs.

Wu et al. also developed a simpler MSNs functionalized with mannose ligands and capped with Con A through multivalent carbohydrate−protein interactions as depicted in Figure 264.421 The nanoparticles were loaded with Rh 6G. The authors grafted the mannose derivative on MSNs at different surface densities. Cargo release was selectively observed by a competitive binding of glucose or in an acidic environment. Wen and co-workers prepared AuNPs-aptamer-capped MSNs for the detection of adenosine.422 The authors functionalized MSNs with 3-aminopropyl moieties, and the amino groups were transformed to carboxylic acids by reaction with succinic anhydride. Then, the single-stranded DNA sequence 3′NH2C6-TCT CTT GGA CCC CCT-5′ was grafted in the outer surface by an amidation reaction. The pores of the hybrid material were loaded with Rh B, and the system was capped with AuNPs functionalized with a complementary aptamer for adenosine as depicted in Figure 265. The capped MSNs showed negligible dye release in water, whereas the addition of adenosine induced delivery of the entrapped Rh B due to the formation of aptamer-adenosine complexes that detached the AuNPs from the 683


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Figure 265. MSNs loaded with Rh B and capped by AuNPs functionalized with an adenosine aptamer. The system was uncapped in the presence of adenosine.

Figure 264. MSNs loaded with Rh 6G and capped with Con A that delivered the entrapped cargo upon addition of glucose.

surface of the silica support. No Rh B delivery was observed upon the addition of cytidine, guanosine, and uridine. 7.4. Triggered by Biomolecules

A number of capped systems have been specifically designed to be opened in the presence of certain biomolecules. In this section, systems opened by displacement reactions are detailed, whereas examples showing cargo delivery in the presence of other biomolecules such as enzymes, by enzyme-induced hydrolysis of capping ensembles, are shown in section 8. A first example of uncapping protocol triggered by biomolecules, in particular by a certain oligonucleotide, was ́ reported by Martinez-Má ñez et al.423 The system was based on MSNs loaded with fluorescein and functionalized with APTES in the outer surface. The oligonucleotide 5′-AAT GCT AGC TAA TCA ATC GGG-3′ was used to cap the pores through electrostatic interactions with the amines at the surface that were partially protonated at neutral pH. Cargo delivery was triggered in the presence of the complementary single strand of the capping oligonucleotide as consequence of the hybridization of both single oligonucleotides and detachment from the nanoparticles’ surface as shown in Figure 266A. Dye delivery was also studied in the presence of other oligonucleotides, yet in all cases a poor cargo delivery was found. Based on an analogous design, the same authors prepared capped materials for the detection of genomic DNA.424 For this purpose, MSNs were loaded with Rh B and functionalized with APTES on the external surface. In this case, it was used as the capping oligonucleotide a sequence highly conserved in the

Figure 266. MSNs loaded with fluorescein, functionalized with aminopropyl moieties, and capped with a single-strand DNA. The pores were opened upon addition of the complementary strand.

Mycoplasma genome that corresponds to a fragment of the 16S ribosomal RNA subunit. The system displayed no dye delivery until the addition of Mycoplasma fermentans genomic DNA (Figure 266B). No cargo delivery was observed in the presence of genomic DNA from other bacteria such as Candida albicans or Legionella pneumophilla. A LOD as low as ca. 70 DNA copies 684


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μL−1 was found. The capped nanoparticles were used to detect Mycoplasma in real contaminated cell-culture media without the use of PCR techniques. Ren, Qu, and co-workers425 developed a new example of logic gates by building a system based on MSNs capped with long duplex DNA structures in a cascade manner. To construct such a system, bare MSNs were functionalized with APTES and amino moieties were further reacted with succinic anhydride to obtain carboxylated-MSNs. Then an amine-modified oligonucleotide (5′-GTGTT TATAG CGGAC CCC-NH2-3′) was grafted onto the external surface of MSNs through an amide bond by the EDC/NHS coupling method, followed by loading mesopores with dye Rh B. Finally, pores were capped by adding three different oligonucleotides, which had short complementary regions among them (5′-GAAGACTCGTAATGTGAAACCG3′, 5′-CACAT TACGAGTCTTCGTGGCATATCACTCTTGGAG-3′, 5′-GGGGTCCGCTTAAACACCT CCAAGAGTGATATGCCAC-3′) and formed a long duplex DNA structure by hybridization with the oligonucleotide grafted onto the surface of MSNs (see Figure 267). Cargo release was triggered by DNA strand displacement by adding complementary oligonucleotides to those that capped the pores in sequence. The system’s logic response was studied by adding different input combinations of

complementary DNA strands to the three capping oligonucleotides. The experimental results reflected that only the addition of the complementary oligonucleotides in sequence to the oligonucleotides that formed the long duplex DNA structure was able to open pores and release cargo. Conversely, a low dye release was observed when other input combinations were used. He, Wang, and co-workers combined the catalytic properties of platinum nanoparticles (PtNPs) and capped MSNs to design a colorimetric detection system for oligonucleotides.426 PtNPs were coated with a mesoporous silica shell, and the final gated system was prepared by functionalization of the silica shell with aminopropyl moieties and capping the pores through electrostatic interactions with a single oligonucleotide strand which contained a mutation of gene BRCA1 related with breast cancer (see Figure 268). Aqueous suspensions at pH 4.7 of the capped

Figure 268. Pt nanoparticles coated with a silica mesoporous shell in which the pores were capped with a single DNA strand. In the presence of the complementary strand, the pores were opened and TMB was oxidized by Pt yielding a colored derivative.

nanoparticles in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) remained colorless because the molecules of the indicator were unable to access the Pt core. In contrast, in the presence of the complementary strand, a clear blue color was observed due to the displacement of the capping oligonucleotide and Pt-induced catalytic oxidation of TMB. A LOD of 3 nM was calculated. Additionally, other strands with 1, 2, or 3 mismatched bases induced no color changes. ́ The groups of Martinez-Má ñez427 and Yang428 developed independently and simultaneously aptamer-gated systems which ́ were selectively opened in the presence of thrombin. MartinezMáñez et al. used MSNs loaded with the dye Rh B, while Yang’s system consisted of Fe3O4 magnetic nanoparticles coated with a mesoporous silica shell loaded with the complex Ru(bipy)32+. In both cases, the external silica surface was functionalized with APTES and a thrombin aptamer (TBA) was used as a gatekeeper. TBA was absorbed on the surface through electrostatic interactions with the positively charged amines on the surface of the silica support. In the presence of α-thrombin, TBA was displaced from the surface, due to the formation of TBA−protein

Figure 267. MSNs loaded with Rh B and capped with a long duplex DNA structure by hybridization of four different oligonucleotides with short complementary regions among them. Cargo release was observed by adding complementary oligonucleotides in a logical sequence. 685


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complexes, and the entrapped cargo was released (see Figure ́ 269). Martinez-Má ñez et al. detected the presence of thrombin

Figure 269. MSNs loaded with Rh B and capped with a TBA. In the presence of thrombin, the pores were opened and the cargo was released.

by measuring the fluorescence emission of the Rh B dye in the solution. The authors tested their capped system in simulated human blood plasma, achieving a LOD for thrombin of 2 nM and in PBS buffer with 10% of human serum reaching a LOD of 4 nM. On the other hand, Yang et al. detected the presence of thrombin by measuring the electrochemiluminescence of the Ru(bipy)32+ reporter. This allowed the authors to calculate a LOD of 0.5 pM ́ in Tris-HCl buffer. Moreover, Martinez-Má ñez et al. found that a mixture of other nonexclusive binding proteins, such as ovalbumin and BSA, were unable to induce uncapping of the pores. Yang et al. demonstrated that BSA, lysozyme, and GOx had negligible uncapping ability. Kong and co-workers developed a similar TBA-capped system that was opened in the presence of the TBA complementary strand or upon addition of thrombin.429 The authors used the claw molecule alendronate sodium trihydrate for the construction of the phosphonate−TiO2 (PTi) mesoporous nanocarrier and loaded the pores with IBU as a model drug. The positively charged amino groups in the phosphonates interacted with the negatively charged fluorescein labeled TBA resulting in closing the pores and the fluorescence quenching of fluorescein. The presence of the complementary DNA strands or the presence of thrombin resulted in the displacement of the capping TBA, cargo delivery, and restoration of dye fluorescence as depicted in Figure 270. The authors found a LOD of 2.3 nM for the detection of human thrombin. Moreover, the system was also highly selective, and a single-base mismatched oligonucleotide or other proteins (i.e., human serum albumin, collagenase, lysozyme, cytochrome c, hemoglobin, and trypsin) induced no cargo delivery. DNA-capped MSNs have also been used for the optical and electrochemical detection of the glycoprotein enzyme prostate

Figure 270. Phosphonate−TiO2 mesoporous nanocarrier loaded with IBU and capped with TBA. The pores were opened in the presence of the complementary strand or upon addition of thrombin.

specific antigen (PSA).430 MSNs were functionalized with aminopropyl groups, the pores loaded with MB and finally the pores capped upon addition of a single DNA strand through electrostatic interactions with the positively charged nanoparticle’s surface. Moreover two antibodies, able to coordinate PSA, were labeled with two single-stranded DNA fragments (i.e., DNA1 and DNA2), which were complementary to that used for capping the MSNs (see Figure 271). In the presence of both DNA-labeled antibodies and PSA, a proximate complex was formed that hybridized with the single-stranded DNA used as a cap, resulting in MB release. Cargo delivery was measured through fluorescence or differential pulse voltammetry. PSA was detected with a linear range from 0.002 to 100 ng mL−1, and a LOD of 1.3 pg mL−1 was determined. No interference was found in the detection of PSA when the carcinoembryonic antigen (CEA) was present in the solution. Min et al.431 developed silica-coated QDs able to deliver the cargo in the presence of miR-21 (one of the first mammalian microRNAs identified). In particular, the authors functionalized the silica surface with APTES, and subsequently an acid-modified short DNA strand (anchor-DNA) was attached following a EDC/NHS coupling procedure. Afterward, DOX was loaded, and the pores were capped with a DNA hybrid formed by an antimiR-21 coupled at the 3′ end with the AS1411 aptamer (Figure 272). AS1411 is guanine-rich aptamer that presents a high binding affinity to nucleolin, which is overexpressed in tumor 686


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Figure 271. MSNs loaded with MB and capped with a single DNA strand. Cargo delivery was observed in the presence of PSA.

cells. Moreover, methoxy polyethylene glycol succinimidyl propionate (mPEG-SPA) was additionally attached to the silica surface to minimize their nonspecific uptake by normal cells and improve their long-term stability in cell culture medium. To assess the target efficiency and specificity of the system, CLSM and flow cytometry analyses were performed, by determining the fluorescence of DOX when HeLa and NIH 3T3 cells were treated with the DOX-loaded capped MSNs. The fluorescence emission was stronger for HeLa cells than for NIH 3T3 cells due to the specific interaction between AS1411 and nucleolin, which is overexpressed on the membrane of HeLa cells. This was confirmed by pretreatment of HeLa cells with free AS1411 molecules to block binding of nucleolin, which led to decreased target efficiency of the nanoparticles. The traceability of the nanocarrier was investigated by CLSM during intracellular drug delivery and release. Most of the nanocarriers were trapped inside the endosome/lysosome, while some were present in the cytosol by means of the proton-sponge effect. DOX gradually diffused from the nanocarriers into the cell nucleus, thus inducing cell death. Low dosage of the nanocarriers led to a sustained lethality in HeLa cells but not in NIH 3T3 cells, indicating a high therapeutic efficacy and negligible side effects. To maximize therapeutic efficacy, the authors increased levels of miR-21 by transfecting synthesized miR-21 into HeLa cells. The expression alteration dramatically increased DOX delivery after the first 6 h, which led to an intensified cytotoxicity compared with the control group (Figure 272, bottom). Given the complexity of the regulation of miRNAs and their multiple gene targets, this miRNA-responsive drug delivery model reflects that the combination of chemotherapy and gene therapy can optimize therapeutic efficacy in cancer treatment. Xu and co-workers prepared MSNs capped with a doublestranded DNA fragment, which were selectively opened in the presence of survivin mRNA.432 MSNs were loaded with DOX,

Figure 272. (Top) Mesoporous silica-coated QDs loaded with DOX and capped with AS1411 aptamer. The cargo was released in the presence of miR-21. (Bottom) CLSM images of HeLa cells (i) without or (ii) with transfection of miR-21 before incubation with mesoporous silica-coated QDs loaded with DOX and capped with AS1411 aptamer. The apoptotic bodies of cells were marked with a circle. Adapted with permission from ref 431. Copyright 2014 Wiley-VCH.

and the external surface functionalized with aminopropyl moieties. Then, the pores were capped upon addition of a double-stranded DNA sequence through electrostatic interactions with the protonated amino groups as depicted in Figure 273. This double-stranded DNA was prepared upon hybridization of a short oligonucleotide functionalized with the dye 7amino-4-methylcoumarin (AMCA) and a long sequence functionalized with FITC which contained a recognition element to survivin mRNA. Moreover, the presence of a FRET pair (AMCA-FITC) on the capping element ensured cargo release monitoring. When the capped nanoparticles were suspended in hybridization buffer (20 mM Tris-HCl, 37.5 mM MgCl2 at pH 7.5) negligible DOX release was observed. Moreover, upon 687


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responsive drug delivery platform, the fluorescence intensity change was observed in HL-60 cells or normal liver LO2 cells after being incubated with this system for 24 h. LO2 cells were used as a control because survivin mRNA is overexpressed in many malignancies, while it is tightly controlled in terminally differentiated adult tissues, such as LO2 cells. Negligible fluorescence change in the FRET, and no fluorescence of released DOX, was observed in control cells. Interestingly, in HL-60 cells, the mean fluorescence intensity of released DOX in nucleus gradually increased with time. Simultaneously, a gradual increase in the blue emission intensity and a corresponding decrease in the green emission intensity were also detected. These results demonstrated that the system has the capability of releasing drug on-demand. Cell viability was determined after both cell lines (HL-60 and LO2 cells) were cultured with different concentrations of DOX-loaded nanoparticles. Remarkably, the system showed 2.5-fold increased lethality to just HL-60 cells. This result was confirmed by analyzing the on-demand release capability of the system in HL-60 cells treated under conditions where survivin mRNA was either up- or downregulated. As expected, more drug release was observed in HL-60 where survivin mRNA was up regulated. Decreased drug release was detected in cells downregulated for survivin mRNA. Hernandez et al. also developed AS1411 aptamer-capped MSNs able to be opened in the presence of nucleolin.433 For the preparation of the system, an amino-modified nucleolin binding aptamer (AS1411) was first prepared. Moreover, in order to conjugate this aptamer to MSNs the nanoparticles’ surface was first modified with MPTS and the pores loaded with fluorescein. Subsequently, sulfo-N-succinimidyl 4-maleimidobutyrate was attached to the sulfhydryl-modified nanoparticles. Finally, the amino-modified AS1411 aptamer was conjugated to the solid and capped the pores (see Figure 274). From the in vitro release studies, the authors found that in the presence of isolated cell membrane fragments of MDA-MB-231 cells (nucleolin

Figure 273. (Top) MSNs loaded with DOX and capped with a doublestranded DNA. Addition of surviving mRNA induced cargo release. (Bottom) CLSM fluorescence images of HL-60 cells after incubation with MSNs loaded with DOX and capped with a double-stranded DNA for 6 h at (a) 37 °C and (b) 4 °C. Channel 1 represents cell stained by Hoechst 33342, and channel 2 represents the fluorescence intensity of the FRET pair in HL-60 cells treated with MSNs, with bright-field and merged images also shown. Adapted from ref 432. Copyright 2014 American Chemical Society.

excitation at 353 nm (AMCA donor fluorophore), a dual emission with bands at 450 (from the AMCA) and 520 nm (from the FITC acceptor) was observed. Upon addition of survivin mRNA, a remarkable DOX delivery was observed. Also, a marked decrease in the emission band at 520 nm with a concomitant enhancement in the fluorescence at 450 nm was found. The observed emission changes were ascribed to a removal of FRET process due to a selective hybridization of survivin mRNA with the long DNA sequence that also resulted in cargo delivery. To investigate the intracellular uptake pathway and the intracellular localization of the system, myeloblastic leukemia (HL-60) cells were incubated with the nanoparticles at 4 and 37 °C, respectively. Confocal fluorescence microscopy analysis of HL60 cells incubated with the system at 37 °C, revealed intracellular sky blue spots attributed to FRET fluorescence (see Figure 272, bottom). This signal indicated that the nanoparticles were internalized by cells and mostly escaped from the endosome/ lysosome into the cytoplasm. Internalization was due to energydependent endocytosis, as virtually no signal was detected after incubation of the cells with the nanoparticles at 4 °C. To test whether DOX-loaded nanoparticles could serve as target-

Figure 274. MSNs loaded with fluorescein and capped with a nucleolinbinding aptamer. Addition of nucleolin induced cargo delivery. 688


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positive), fluorescein was released from the capped nanoparticles. The authors found that the interaction of the aptamer with nucleotin on the surface of cancer cells induced a conformational change of the aptamer unblocking the pores and releasing the cargo. Moreover, upon aptamer binding, the MSNs internalized via receptor-mediated endocytosis resulting in dye release. Qu, Ren, and co-workers also developed an AS1411-based molecular gate system but using in this case strontium hydroxyapatite as a support.434 Gd-doped mesoporous strontium hydroxyapatite nanorods were synthesized and then loaded with DOX. The system was capped using the AS1411 aptamer by electrostatic interaction between the positive charges induced by the strontium substitution in hydroxyapatite and the negatively charged aptamer. Dye delivery studies demonstrated that the system remains capped until the addition of nucleolin, achieving an important DOX delivery. The capped material was tested with MCF-7 cells (presenting overexpression of nucleolin) and NIH3T3 cells (as control). Remarkably, the DOX-loaded nanomaterial exhibited significantly higher cytotoxicity in MCF-7 cells than in NIH 3T3 cells, due to the specific binding and uptake of the system by the cancerous cells triggered by the AS1411 aptamer. No differences in DOX fluorescence were observed in either MCF-7 or NIH 3T3 cells incubated with random DNA-capped nanoparticles which were unresponsive to nucleolin. Cellular uptake of the solid only on MCF-7 was confirmed by fluorescence microscopy following both DOX delivery and Gd3+ presence. Moreover T1-weighted magnetic resonance images also corroborated the enhancement of cellular uptake in MCF-7 cells. Willner and co-workers prepared different DNA-capped MSNs able to be opened in the presence of certain biomarkers.435 MSNs were functionalized with aminopropyl moieties and the amino groups in the surface were used for grafting selected single-stranded DNA sequences using sulfoEMCS as a covalent cross-linker. In the first material (see Figure 275A), the DNA-1 sequence was anchored. DNA-1 contained a tailored base sequence that generated at room temperature a hairpin structure that blocked the pore entrances. DNA-1 also included a single-stranded loop for the recognition of a nucleic acid biomarker (i.e., 5′-AGT GTG CAA GGG CAG TGA AGA CTT GAT TGT-3′). The pores were loaded with Rh B. Treatment of the DNA-1-capped MSNs with the biomarker induced the opening of the hairpin forming a duplex structure that also blocked the pores inhibiting dye release. Addition of Exo III enzyme induced the hydrolysis of the 3′-end of 1, allowing the release of the entrapped Rh B and the biomarker which induced the opening of additional capped pores. The same approach was used by grafting the single-stranded DNA sequence 2 and as biomarker 5′-ATC CTC AGC TTC G-3′ (see Figure 275B). Both DNA formed a duplex structure that was able to cap the pores and inhibit dye release. In this case, the duplex included a programmed sequence for the specific nicking of one base. Upon addition of the Nb.BbvCl enzyme, the duplex was dissociated because of the nicking of the grafted DNA strand that resulted in the release of the biomarker and the entrapped Rh B. Another gated material was obtained by the grafting sequence DNA-3, which formed a hairpin structure, and that incorporated an ATPaptamer conjugated with other sequence that ensured that Exo III enzyme was unable to hydrolyze (see Figure 275C). Upon addition of ATP, the hairpin formed by DNA-3 opened and the 3′ end formed a duplex structure with the 5′ domain that was hydrolyzed by Exo III. This hydrolysis induced the release of the

Figure 275. MSNs loaded with Rh B and capped with different DNA sequences that were opened with (A and B) certain biomarkers, (C and D) ATP, and (A, B, C, and D) enzymes.

entrapped dye and ATP, which induced the opening of additional gated pores. The same methodology was used to prepare a material able to release the entrapped dye and ATP by grafting DNA-4, which contained an ATP-aptamer and a fragment able to be hydrolyzed by the Nb.BbvCl enzyme (Figure 275D). Finally, the material capped with the DNA-3 was loaded with CPT and its internalization in MDA-MB-231 (breast cancer cells) and 689


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pulse voltammetry was observed. However, upon addition of the complementary single strand, a strong current was observed due to the hybridation of the added DNA with the grafted strand. The formation of the double helix inhibited the delivery of the entrapped MB with the subsequent enhancement in the current intensity. With this assay, concentrations of DNA as low as 1 fM were detected. The second prepared material was used for thrombin detection. In the absence of this anticoalgulation protein, prepared material was unable to retain the MB into the pores and a weak current was observed. However, in the presence of thrombin, the protein binds with the grafted aptamer forming a DNA quadruplex structure that blocked the pores and inhibited MB release (with subsequent increase in current intensity). Using this material, a sensitive (LOD of 10 fM) and selective (BSA, lysozyme, and IgG were unable to close the pores) response toward thrombin was obtained. The third material prepared was used for the detection of ATP with a similar mechanism. Selective coordination of ATP with its grafted aptamer inhibited MB release with the subsequent enhancement in current intensity. Again, a highly sensitive (LOD of 10 pM) and selective (CTP, GTP, and UTP were unable to block the pores) response was observed. Finally, using the same approach, Ag+ and Hg2+ electrochemical detection was also achieved. In the presence of selected cations, the grafted DNA formed a rigid hairpin structure that blocked the pores, inhibiting redox reporter release with subsequent enhancement in intensity current. For both cations, LOD of 1 nM were measured and addition of Ni2+, Fe3+, Ca2+, Al3+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, and Zn2+ were unable to induce pore capping. ́ In 2013, Martinez-Má ñez et al.437 used mesoporous gated materials to improve the efficacy of antimicrobial drugs (ε-polyL-lysine, ε-PL) and to extend the antimicrobial spectrum of antibiotic vancomycin. This is one of the few gated materials to have been designed to target bacteria. The authors prepared MSNs, loaded with Rh B or vancomycin, and functionalized on the external surface with n-[(3 trimethoxysilyl)propyl]ethylendiaminetriacetic acid trisodium salt. Finally, pores were capped by adding cationic polymer ε-PL through electrostatic interactions with the negatively charged nanoparticles. Cargo delivery occurred in the presence of E. coli bacteria because adhesion of the positively ε-PL-capped material with the negatively charged bacterial wall weakened the interaction between ε-PL and the carboxylate-functionalized nanoparticles as depicted in Figure 277. In vitro dye release studies conducted in water at pH 7 in the absence and presence of E. coli DH5α at different cell concentrations confirmed the proposed gating mechanism. Studies were also carried out to test the interaction of ε-PL-capped nanoparticles with different Gram-negative bacteria (i.e., E. coli DH5α, E. coli 100, E. coli 405, S. typhi, and E. carotovora). The Rh B-loaded material showed significantly enhanced toxicity of ε-PL compared with free ε-PL in solution. A potent synergistic effect was also observed when ε-PL and vancomycin were used in the nanoformulation, which did not occur with the free drugs alone.

MCF-10a (normal breast cells) studied. The capped nanoparticles were internalized by endocytosis and induced a 65% cell death in MDA-MB-231 after 48 h, whereas only a cell death of 25% was observed for MCF-10a cells. These results were consistent with the fact that cancer cells have a high ATP concentration which induced an enhanced opening of the gated material. Ren and co-workers used graphene oxide nanosheets coated with a mesoporous silica shell as inorganic scaffold for the design of gated systems for the detection of certain biomolecules. In a first step, aminopropyl moieties were grafted onto the external surface of the silica shell and then reacted with the bifunctional cross-linker 4-maleimidobutyric acid NHS ester.436 This procedure yielded a hybrid material with maleimide moieties grafted onto the external mesoporous surface at which several thiol-modified DNAs were conjugated (i.e., Alzheimer’s disease DNA, Thrombin aptamer, ATP aptamer, Hg2+-selective oligonucleotide, and Ag+-selective oligonucleotide) (see Figure 276). Finally, the pores of the mesoporous shell were loaded with the redox-active molecule MB. In the materials containing the DNA of Alzheimer’s disease, the prepared support was unable to retain MB onto the pores, and negligible current in differential

8. ENZYMES Although not as popular as other stimuli, using enzymes for uncapping gated materials is quite appealing if we take into account that the use of tailor-made derivatives and specific enzymes is envisioned to have a huge potential, which may provide exquisite selectivity in the design of advanced gated devices for on-command delivery of drugs and biomolecules in realistic biological environments. For instance, enzyme over-

Figure 276. Graphene oxide nanosheets coated with mesoporous silica layer, loaded with MB, and capped with selected DNA sequences. These materials were used for the electrochemical detection of (A) DNA fragments, (B) thrombin, and (C) ATP, Hg2+, and Ag+. 690


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Figure 277. MSNs loaded with Rh B or vancomycin and capped with cationic polymer ε-PL. Cargo was delivered in the presence of bacteria.

expression in a certain disease can be used to design personalized capped systems able to be open in a specific cell type, which means a significative reduction of undesirable side effects and a large increase in the effectivity of the drug delivered. However, although certain examples that used esterases, glycosidases, peptidases, reductases, and DNases have been reported (vide infra), this subfield in the gated chemistry area has not yet been fully explored; for instance, further advances will most likely take into account that the flow of certain biological processes relies on complex biochemical networks, along with the participation of multiple enzyme-dependent steps. In relation to caps, it is important to carefully design the orientation of the gating structure, that is, cleavable groups must be accessible to the enzyme active site. Finally, the fact that synthetic molecules can be designed to contain both, fragments recognized by targetenzymes and moieties for targeting certain cells, or for cell entrycontrol, also suggests that there is still plenty of room in this research area.

Figure 278. PLE-induced controlled release of (A) Rh B and (B) CPT from MSNs capped (A) with an ester-linked adamantyl stopper or (B) with an ester-linked FA stopper.

(see Figure 278B). As in the example above, the presence of PLE induced cargo delivery. Fluorescein-loaded capped MSNs were incubated with human osteosarcoma U2Os cells to analyze cellular uptake and toxicity by confocal microscopy and flow cytometry. The system was efficiently endocytosed by up to 90% of cells, and no significant cytotoxicity was detected in cultures. Next, nanoparticles were loaded with CPT, a hydrophobic drug whose formulation and delivery is still a challenge. U2Os cells were incubated with free CPT and CPT-loaded capped MSNs. A significant increase in cell death was detected in the cells treated with the CPT-loaded nanoparticles when compared to those incubated with free CPT. ́ Inspired also by an esterase-driven mechanism, MartinezMáñez and co-workers reported a gated material composed of MSNs functionalized in pore outlets with ester-containing glycol chains.441 The capped solid was previously loaded with dye Ru(bipy)32+, and the release of the entrapped guest was studied in water at pH 8. The authors found that ruthenium complex delivery was inhibited due to the steric hindrance induced by the bulky glycol chains anchored to the pore voids of MSNs (see Figure 279). Upon addition of esterase enzyme, the hydrolysis of the ester groups enabled the entrapped cargo to be released. The authors demonstrated that no delivery was observed in the presence of other enzymes, such as amidase and urease, and with denaturated esterase, which confirmed the selective enzymatic

8.1. Hydrolysis of Ester and Phosphodiester Groups

Certain examples have taken advantage of using esterases, which are enzymes capable of catalyzing the hydrolysis of esters into an acid and an alcohol. The first enzyme-driven gated MSNs, described by Zink, Stoddart, and co-workers, used an esterase as a trigger.438 These authors encircled α-CDs within PEG stalks anchored to MSNs and further attached to stalks an ester-linked adamantly bulky stopper to retain α-CDs and to effectively block pores (see Figure 278A). The capped system was loaded with Rh B. Cargo delivery was achieved only in the presence of PLE, which was able to hydrolyze the ester group resulting in the removal of the adamantly moiety. This allowed the dethreading of α-CDs and the subsequent cargo delivery. As control experiments, the authors synthesized a similar system which contained an amide-linked adamantyl stopper that was unable to be cleaved by PLE. In another work,439 these authors used the same molecular gate in a novel hierarchically structured material consisting of micropatterned mesoporous silica films, prepared by vapor-phase infiltration by a reactive wet-stamping technique. The same authors synthesized a similar system but used FA as a stopper.440 In particular, MSNs were loaded with fluorescein or antitumoral drug CPT, PEG stalks were encircled with α-CDs, and FA was further linked to stalks by a click chemistry reaction 691


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Figure 279. Esterase-induced controlled release of a ruthenium complex or CPT from MSNs functionalized with ester-containing glycol chains.

Figure 280. MSNs functionalized with polyester polymers. Cargo delivery was triggered by esterase enzyme.

hydrolysis of the ester bond as the mechanism responsible for opening mesopores. Cell internalization and delivery studies in HeLa and MCF-7 cell lines were performed with the prepared material. Viability assays indicated that the capped nanoparticles were not toxic for cells and were found to be associated with lysosomes, as shown by colocalization studies conducted with the LAMP1-GFP lysosomal biomarker. Cell treatment with the gated nanoparticles loaded with cytotoxic drug CPT efficiently triggered cell death in HeLa cells as a result of the cell internalization of nanoparticles, the hydrolysis of capping estercontaining glycol chains in lysosomes, and CPT release. The same authors developed an enzyme-driven delivery system based on MSNs loaded with Rh B and capped with two different polyester polymers that contained side-chain azobenzene moieties (see Figure 280).442 Time-dependent experiments performed in the presence of enzyme esterase showed dye delivery, which was modulated by varying the amount of enzyme. Cell internalization and delivery studies in HeLa cells were performed with one of the capped materials. Viability assays showed that gated nanoparticles were not toxic for cells. Capping polyesters were also degraded by lysosomal enzymes, which clearly resulted in Rh B delivery. These authors also demonstrated that cell treatment with capped nanoparticles loaded with cytotoxic drug CPT efficiently triggered cell death in HeLa cells. MSNs equipped with [2]pseudorotaxanes as caps, and able to release an entrapped cargo in the presence of esterase, were prepared by Yang et al.443 The authors also prepared a second solid that could be opened in the presence of urease enzyme. For this purpose, two sets of gated MSNs were designed (see Figure 281). The first material was prepared by grafting an acetylcholine stalk onto the nanoparticles, loading the porous network with Rh B and capping pores by the formation of [2]pseudorotaxanes upon p-sulfonato-calix[4]arene (SC[4]A) addition. The second material was prepared following a similar procedure, but this time it included a stalk that contained a urea bond. Both materials showed negligible Rh B release when suspended in PBS buffer at

Figure 281. Enzyme-responsive MSNs containing SC[4]A, [2]pseudorotaxanes, and stalks with (a) urea or (b) ester moieties.

a neutral pH. Upon addition of either esterase (for the nanoparticles with the ester-containing stalk) or urease (for the nanoparticles with the urea-containing stalk), remarkable dye release was observed as a result of the ester and urea hydrolysis and the subsequent dethreading of [2]pseudorotaxane. Addition of ethanediamine to suspensions of both nanoparticles also induced gate opening and Rh B release due to the competitive binding of ethanediamine with SC[4]A. Moreover at pH 10, both 692


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the enzymatic hydrolysis of the glycosidic bond in disaccharide

nanoparticles also showed dye release due to the hydrolysis of the ester and urea bonds located in stalks. ́ Vallet-Regi ́ and Martinez-Má ñez developed a macroporous scaffold that incorporated capped MSNs, which were designed to deliver cargo in the presence of acid phosphatase.444 In their system, these authors loaded MSNs with Ru(bipy)32+ and functionalized the external surface with 3-[2-(2aminoethylamino)ethylamino]propyl-trimethoxysilane. Finally, pores were capped by treating the solid with ATP via the formation of supramolecular complexes between the negatively charged ATP anions and the positively charged ammonium groups on MSNs (see Figure 282). The authors confirmed that

lactose as depicted in Figure 283A.

Figure 282. Acid phosphatase-induced cargo release in MSNs functionalized with amines and capped with ATP.

Ru(bipy)32+ delivery was observed only in the presence of acid phosphatase in aqueous buffered solutions. In a second step, a paste was prepared by mixing capped MSNs with gelatin and using glutaraldehyde as a cross-linker, which was employed to construct a scaffold by rapid prototyping 3D printing techniques. In this scaffold, the acid phosphatase-driven release mechanism was also assessed. Finally, the biocompatibility of the scaffold was confirmed. In particular, these authors performed cell proliferation and cytotoxicity and spreading assays with a human osteoblast-like cell line. It was found that cells did not elicit any cytotoxic effect and adhered, proliferated, and spread well in the scaffold. 8.2. Glycosidic Linkages

Glycosidases catalyze the hydrolysis of glycosidic linkages by degrading oligosaccharides and glycoconjugates, which are structurally the most diverse class of biopolymers. Glycosidases are found in all domains of life and play important roles in ́ biological processes and industrial applications. Martinez-Má ñez and co-workers reported the first example of silica nanoparticles capped with “saccharide” derivatives. In this work,445 MSNs were loaded with Ru(bipy)32+ and gated with a covalently anchored lactose derivative. Dye delivery from the aqueous suspensions of the lactose-capped material was negligible due to the formation of a dense disaccharide network, in which lactose groups were most likely linked through hydrogen-bonding interactions around pore outlets. Addition of β-D-galactosidase enzyme induced progressive cargo release, which was clearly related to

Figure 283. Controlled cargo release from saccharide-capped MSNs triggered by the hydrolysis of glycosidic bonds. 693


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The utilization of “saccharides” as caps was also studied using MSNs loaded with Ru(bipy) 3 2+ and capped with the corresponding trialkoxysilane derivative of hydrolyzed starch Glucidex 47, Gludicex 39, and Glucidex 29.446 Capped solids showed zero delivery, whereas cargo release was observed in the presence of pancreatin, which contains amylases that induced the hydrolysis of the glycosidic bonds in anchored saccharides. It was found that the less the hydrolysis of the starch anchored on nanoparticles, the lower the delivery rate in the presence of pancreatin (Figure 283B). Internalization and biocompatibility studies of gated material capped with Glucidex 47 were conducted by incubation with HeLa and LLC-PK1 cell lines. Confocal microscopy detected a clear intracellular dotted signal in both cell lines, which indicated that nanoparticles were internalized. It was observed that the cellular uptake of capped MSNs was energy-dependent and most likely achieved by endocytosis. Co-localization studies showed that nanoparticles accumulated in endosomes and lysosomes. Cell viability studies performed in both cell lines indicated that MSNs were biocompatible and showed no cytotoxicity. Loading the Glucidex 47-capped MSNs with antineoplasic agent DOX significantly enhanced DOX cytotoxicity in HeLa cells. In another work, the same authors prepared silica mesoporous microparticles, which were loaded with garlic extract and capped using hydrolyzed starch, as above.233 Capped microparticles were incorporated into a nylon-6 membrane by an electrospinning technique, which yielded a final composite with a fibrous structure. This nylon-6 nanocomposite membrane showed remarkable cargo delivery in the presence of pancreatin enzyme, which was able to hydrolyze the grafted polysaccharide. In a further work, the same authors prepared MSNs loaded with Rh B and functionalized the external surface with an alkylgluconamine derivative of a galacto-oligosaccharide polymer (GOS).447 In vitro assays demonstrated that the capped material showed no cargo delivery but opened in the presence of βgalactosidase (see Figure 283C) due to the selective hydrolysis of the GOS cap. Moreover the performance of the GOS-capped nanoparticles in vitro was also studied for the potential targeting of senescent cells, in which β-galactosidase is known to be overexpressed. For instance, when GOS nanoparticles were incubated with yeast cells, Rh B staining was observed only in the β-galactosidase overexpressing strains, which indicated that the molecular gate actually worked in vitro. It was found that GOScapped MSNs were also selectively opened in human senescent fibroblasts from X-linked Dyskeratosis Congenita (X-DC) patients, in which β-galactosidase was overexpressed. Remarkably, no emission was detected in lung carcinoma H460 cells (in which β-galactosidase was not overexpressed), not even after 48 h. Cell viability remained unaffected in H460 and X-DC cells. The authors suggested that by choosing an appropriate cargo (a telomerase reactivation drug or a cytotoxic drug), the prevention and removal/replacement of senescent cells could be possible, which thus opens up new avenues for managing age-related diseases. In the same context, Lin and Qu designed MSNs loaded with Rh B using Konjac oligosaccharide (KOGC) as cap.448 Cargo delivery from gated nanoparticles was controlled by the presence of β-mannanase, an enzyme capable of degrading KOGC (see Figure 283D). The authors prepared a collection of capped solids with different amounts of KOGC. Release experiments were also carried out with a collection of enzymes, such as α-amylase, pepsin, and pancreatin at the concentrations present in human saliva, stomach, and small intestine, respectively. No significant

cargo release was detected after 48 h of incubation with the above-mentioned enzymes. In contrast, clear Rh B release took place in the presence of β-mannanase. To investigate the cellular uptake of the KOGC-capped MSNs, the authors incubated Caco-2 cells with the nanoparticles which were efficiently taken up by cells. Neither Caco-2 cell morphology nor viability was affected by addition of the capped nanoparticles. Following the concept of using “saccharides” as capping elements, Qu and co-workers prepared MSNs capped with HA.449 These authors anchored polysaccharide to MSNs through an amidation reaction and used Rh B as an encapsulated reporter. They investigated the performance of the capped nanoparticles in the presence of lysosomal enzyme hyaluronidase-1 (Hyal-1), an enzyme found in tumor microenvironments (see Figure 284). Only when the Hyal-1enzyme was added to

Figure 284. Hyal-1-induced cargo-controlled release of Rh B or DOX from HA-capped MSNs.

aqueous suspensions of the capped MSNs, the release of Rh B was observed. Apart from acting as a cap, HA was also used for targeting tumors. The principal receptor for HA is CD44, which is known to be overexpressed in several cancer cell lines. On the basis of this concept, the authors evaluated the cellular targeting efficiency of FITC-labeled HA-capped nanoparticles with the NIH3T3 (normal) and MDA-MB-231 (CD44 overexpressed) cell lines. Confocal microscopy studies detected a stronger intracellular signal in MDA-MB-231 cells than in NIH-3T3 cells, which certainly suggested that HA-capped MSNs were more easily internalized in cancer cells. This result was consistent with a specific interaction between HA in MSNs, and the CD44 receptors overexpressed in MDA-MB-231 cells. Co-localization studies performed in MDA-MB-231showed that nanoparticles accumulated in endosomes and lysosomes. The authors also found that loading HA-capped nanoparticles with antineoplasic agent DOX provided this drug with a more efficient selective cytotoxicity in MDA-MB-231 cells. The same authors prepared UCNPs (NaGdF4:Yb,Tm), which were coated with a uniform silica shell, that was additionally coated with a mesoporous TiO2 shell.450 The pores of the external TiO2 were loaded with DOX and capped with a layer of HA, which was adsorbed on the surface by simple electrostatic interactions. Buffered suspensions, at pH 4.3, of HA-capped nanoparticles showed moderate DOX release after 24 h (17%). However, DOX release rose to 95% after 24 h in the presence of 694


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with aminopropyl groups, which were further reacted with succinic anhydride. Pores were loaded with Rh B or DOX.452 Finally, nanoparticles were capped with chitosan via the formation of amide bonds. At a neutral pH, gated MSNs showed negligible cargo release, whereas the entrapped molecules were delivered in the presence of lysozyme, an enzyme capable of hydrolyzing β-1,4 glycosidic bonds as depicted in Figure 286.

Hyal-1, an enzyme that was able to hydrolyze the polysaccharide coating. In vitro studies were carried out of the internalization of HA-coated nanoparticles with cancer MDA-MB-231 cells, which overexpressed the CD44 receptor and normal NIH3T3 cells. Strong fluorescence from the NaGdF4:Yb,Tm core was observed only in MDA-MB-231 cells, which indicated preferential internalization due to the strong specific interaction between the HA and the CD44 receptor. Prepared nanoparticles were used in photodynamic therapy. Irradiation of the NaGdF4:Yb,Tm core with an NIR light at 980 nm induced an energy transfer process that activated TiO2 to generate ROS. MTT assays, which were carried out with MDA-MB-231 cells incubated with the HA-capped nanoparticles and irradiated at 980 nm, showed a marked decrease in IC50 compared with the same cells treated with the nanodevice, but with no NIR light activation. This was attributed to a synergistic effect of the ROS generated and the Hyal-1-induced DOX release. The same authors prepared AuNCs of 50 nm with pores of 5− 8 nm loaded with DOX and capped with dopamine-derivatized HA (see Figure 285).451 As in the above example, DOX release

Figure 286. Enzyme and acid-induced controlled release of Rh B or DOX from chitosan-capped MSNs.

The authors found that capped MSNs were also opened by changes in pH. A particularly marked delivery was observed at pH 4. This release at an acidic pH was ascribed to an acid-induced hydrolysis of the glycoside linkage that opened pores progressively. Kim ad co-workers prepared MSNs capped with β-CDs, which were able to deliver an entrapped cargo in the presence of αamylase.453 In particular, these authors prepared two different supports, both containing calcein as cargo, which were capped with β-CDs using a click chemistry reaction. One of the solids contained a stalk with an amino group (Figure 287A), whereas the other support contained a stalk with an o-nitrobenzyl ester moiety (Figure 287B). In the absence of α-amylase enzyme in PBS (pH 7.4) both capped MSNs showed negligible dye release due to the presence of bulky β-CDs that blocked the pores. However, in the presence of α-amylase, clear calcein delivery was observed due to the enzyme-induced hydrolysis of β-CDs gatekeepers. Additionally, the material with the stalks that contained o-nitrobenzyl ester moieties was also opened upon addition of lipase enzyme, which was able to hydrolyze the ester moieties with the subsequent detachment of β-CDs. This material was also opened using UV-light irradiation. When both lipase and UV irradiation were used as stimuli, calcein release accelerated. ́ In 2014, Martinez-Má ñez et al.454 presented the first proof of concept of a hierarchically organized family of nanoparticles that can communicate through chemical messengers. This concept is based on using capped mesoporous silica supports in which the chemical messenger delivered by a first type of gated nanoparticle upon an external stimulus was used to open a second type of nanoparticle, which delivered another messenger that opened a third group of gated system as shown in Figure 288. In the designed communication chain, the first gated solid was an

Figure 285. AuNCs loaded with DOX and capped HA. DOX was released in the presence of hyal or by NIR irradiation.

was controlled by the presence of a Hyal enzyme and was enhanced by NIR light. By going one step further, the authors confirmed the preferential internalization of GFP-labeled HAcapped AuNCs in CD44 overexpressed MDA-MB-231 cells. Likewise, the colocalization of particles with lysosomes and endosomes was confirmed. Hyal induced DOX release, and a subsequent decrease in cell viability was assessed by confocal microscopy, MTT, and flow cytometry assays. In the same experiments, irradiation with NIR light demonstrated the enhanced effectiveness of the capped system. This synergism was also found in MDA-MB-231 tumor-bearing nude mice treated intratumorally with DOX-loaded capped AuNCs. In these experiments, solid tumors were totally eliminated by the DOX release and NIR irradiation combination. Natural chitosan has also been used as a cap to design enzymedriven gated materials. Lou et al. prepared MSNs functionalized 695


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Figure 287. α-Amylase and lipase-induced controlled release of calcein from two different β-CDs-capped MSNs.

enzyme-triggered MSN loaded with reducing agent tris(2carboxyethyl)phosphine (TCEP), the first messenger. The second nanoparticulated system was capped with PEG chains attached by disulfide linkages to the silica surface and contained in pores dodecyltrimethylammonium bromide (DTAB), the second messenger. Finally, a third system hosted a capping lipid bilayer (prepared from DOPC) and was loaded with dye safranin O. In an aqueous suspension that contained the three solids described above, the communication chain was activated by the hydrolysis of grafted polysaccharide in the first solid in the presence of pancreatin, with the subsequent delivery of the first messenger (TCEP). In the second step, TCEP triggered the delivery of DTAB from the second solid by the rupture of redoxlabile disulfide bonds. Lastly, DTAB molecules disrupted the lipid bilayer around the third solid, which allowed entrapped dye (safranin O) delivery. To summarize, the final dye release was related to the information that three different nanoparticles previously shared via two different chemical messengers. The authors corroborated the proposed paradigm by release studies conducted with the three solids in water at pH 7. Although negligible dye release was observed in the absence of the enzyme, remarkable fluorophore release occurred in the presence of pancreatin, which was ascribed to chemical interparticle communication.

Figure 288. MSNs loaded with different cargos and capped with different molecules in such a way that the final dye release was ascribed to the information that three different nanoparticles had previously shared via chemical messengers.

8.3. Hydrolysis of Amide Groups

Peptidases are enzymes capable of inducing the hydrolysis of peptide bonds that link amino acids in a polypeptide chain. Several gated examples have been reported in which delivery was regulated using different peptidase enzymes. In a seminal report, Bein et al.455 prepared capped colloidal MSNs via the interaction of avidin caps on biotinylated MSNs. Attachment of avidin moieties to biotinylated MSNs led to the formation of the avidin−biotin complexes, which resulted in tight pore closure as depicted in Figure 289. The material was loaded with fluorescein. Protease trypsin addition resulted in the hydrolysis of the attached protein avidin and the release of the entrapped guest. Heise et al.456 also described one of the first examples of an enzyme-mediated release mechanism which involved peptidemodified silica particles. In their work, amine-functionalized silica microparticles were loaded with FITC-functionalized dextran (4 kDa). Two different peptide sequences, which contained the Fmoc protecting group and enzyme-cleavable Ala ∼ Ala bonds 696


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Figure 289. Trypsin-induced cargo release from MSNs functionalized with biotin and capped with avidin. Figure 290. Protease-mediated FITC-dextran release from the pores of peptide-modified silica microparticles.

[i.e., Fmoc-Asp(−)-Ala-Ala-Arg(+) and Fmoc-Glu(−)-Ala-AlaArg(+)], were directly coupled to the surface of microparticles by stepwise Fmoc solid-phase peptide synthesis (see Figure 290). The release properties of the two different materials were tested in the presence of a specific enzyme that possessed the correct specificity to cleave the Ala ∼ Ala bond of the grafted peptide sequence: thermolysin for the Fmoc-Asp(−)-Ala-Ala-Arg(+)capped material and elastase for the Fmoc-Glu(−)-Ala-AlaArg(+)-capped material. The results demonstrated that successful enzymatic hydrolysis endowed cargo release. In addition, the specificity of the mechanism was demonstrated by anchoring a peptide whose sequence did not possess an elastase cleavable Ala ∼ Ala bond. Another example in this area involved attaching a designed peptide sequence to MSNs, which was hydrolyzed in the presence of target proteases. In particular, the modular peptide H-GGD EVD GGD EVD GGD EVD-OH (P1) was employed as a molecular gate (see Figure 291A).457 This peptide was designed to act as a substrate of the proteolytic enzymes obtained from Streptomyces griseus (PESG), which are able to perform cleavage at the C terminus of the amide bond from negatively charged aspartic (D) and glutamic (E) amino acids. The peptide, which was attached to azide-functionalized MSNs through a click chemistry reaction, was able to hinder the release of Ru(bipy)32+ dye, whereas delivery was clearly triggered in the presence of PESG. ́ Martinez-Má ñez and co-workers also designed capped MSNs, which were opened in the presence of cathepsin B.458 Cathepsin B is a lysosomal enzyme that is highly associated with cancer development and metastasis. These authors loaded MSNs with safranine O dye or DOX and functionalized the external surface

with 3-(azidopropyl)triethoxysilane. The final gated material was obtained through the reaction of azidopropyl groups on the surface with an alkynyl-derivatized peptide (i.e., alkynyl-GIV RAK EAE GIV RAK-OH) via a click chemistry reaction (see Figure 291B). In vitro studies with the capped solid loaded with safranin O evidenced that cargo release was achieved only in the presence of lysosomal extracts that contained cathepsin B. Cell internalization and delivery studies in HeLa cells and primary culture cells of mouse embryonic fibroblasts were performed with the safranin-loaded peptide-capped nanomaterial. Proper system internalization was confirmed by flow cytometry. No significant cytotoxicity was detected by incubating cells with up to 200 μg/mL of the capped nanoparticles. CLSM of the cultures revealed that safranine O-associated fluorescence was present in cell cultures, which indicated proper internalization and cargo release. Delivery was due to the action of cathepsin B because preincubation of cultures with an inhibitor of the lysosomal enzyme remarkably diminished the safranine O-associated signal. Essentially similar results were obtained by incubation of MEFs primary cells deficient for cathepsin B with gated MSNs. Interestingly cell treatment with the capped nanoparticles loaded with cytotoxic drug DOX efficiently triggered cell death in HeLa and MEF cells. Cytotoxicity of nanomaterial significantly reduced by either preincubation with the cathepsin B inhibitor or by incubation of the MEFs primary cells deficient for cathepsin B. QDs coated with a mesoporous silica shell were used as inorganic scaffold to prepare cathepsin B-responsive capped 697


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Figure 291. Controlled cargo release induced by peptidase or cathepsin B from MSNs capped with a peptide.

Figure 292. Cathepsin B-responsive peptide-capped QD coated with a mesoporous silica layer loaded with DOX and capped with a selected peptide. Cargo delivery was observed in the presence of cathepsin B.

nanodevices.459 The external silica surface was functionalized with aminopropyl moieties, which were further reacted with a maleimide-PEG2-succinimidyl ester cross-linker. The threedomain peptide CRRRQRRKKR-PGFK-EEEEEE was then anchored to the silica surface (see Figure 292). In this peptide, CRRRQRRKKR is a cationic TAT peptide; PGFK is a linker that is able to be selectively hydrolyzed by cathepsin B enzyme, and EEEEEE is an oligo-anionic sequence used to neutralize the charge of the whole peptide. The authors loaded the nanoparticles with DOX. When the capped nanoparticles were suspended in PBS at pH 5.5, a negligible DOX release was observed. However, remarkable drug delivery occurred in the presence of cathepsin B. Cell imaging and cytotoxicity studies in A549 cells (with high levels of cathepsin B) and NIH 3T3 cells (with no cathepsin B activity) confirmed that the enzyme responsive DOX-loaded nanomaterial selectively released DOX only in A549 cells, with high cathepsin B expression, and facilitated the nuclear accumulation of DOX, thus enhancing the antitumor activity of DOX. In contrast, limited nuclear-targeted drug accumulation and lower cytotoxicity of the nanomaterial was observed in NIH 3T3 cells, with no expression of cathepsin B. Next, the authors tested the antitumor activity of the DOXloaded system in A2780 and A2780Adr human ovarian carcinoma cells by confocal microscopy and MTT viability

studies. A2780Adr is an antitumor drug-resistant clone which derives from the A2780 cell line. Remarkably, significant nuclear DOX accumulation and enhanced cytotoxicity of the drug were observed in A2780Adr cells. Raichur and co-workers designed a trypsin-driven drug delivery system prepared from MSNs and the FDA-approved peptide drug protamine.460 To synthesize the gated nanoparticles, the amino-functionalized MSNs were capped with protamine by a simple amine-aldehyde cross-linking reaction in which glutaraldehyde was used as a linker (see Figure 293A). As a model drug, diclofenac was encapsulated in pores. In release experiments, the authors found that gated MSNs exhibited minimal premature release in the absence of the trigger, whereas selective diclofenac release was observed in the presence of trypsin. These authors also investigated the system’s efficacy as a carrier for the delivery of hydrophobic anticancer drugs to trypsin-overexpressing cells. In particular, trypsin and trypsin-like enzymes were seen to be overexpressed under conditions such as inflammation and cancer. Capped MSNs were loaded with antineoplasic drug curcumin and incubated with COLO205 cells at different time points. Nanoparticles were internalized after 1 h of incubation, and intense curcumin fluorescence was observed 698


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xenografted mice was delayed when DOX-loaded gelatin-capped nanoparticles were injected into the xenograft. ́ Martinez-Má ñez and co-workers prepared MSNs loaded with Ru(bipy)32+and capped with ε-PL.462 Two different anchoring strategies to attach ε-PL to the silica surface were used. One approach involved the random formation of urea bonds between isocyanate-functionalized MSNs and lysine amino groups located on the ε-PL backbone. The second strategy involved specific attachment through the carboxyl terminus of the ε-PL polypeptide with azidopropyl-functionalized MSNs (see Figure 294). In both cases, the authors observed selective cargo delivery

Figure 293. MMP-9- or trypsin-induced controlled cargo delivery from protamine or gelatin-capped MSNs.

inside cells. No significant cell fluorescence was detected in the cells incubated with curcumin and suspended in water. The ability of curcumin-loaded nanoparticles to induce cell death in COLO205 cells was also evaluated. It was noteworthy that encapsulated curcumin exhibited significantly greater anticancer activity than free curcumin. Wang and co-workers employed gelatin as a coating to prepare enzyme-responsive gated nanoparticles, which were uncapped using metalloproteinase-9 (MMP-9).461 As-made MSNs were functionalized with aminopropyl moieties. Then the structure directing agent was removed from pores, and amino moieties were reacted with glutaraldehyde. The pores of the aldehyde decorated material were loaded with DOX and capped upon gelatin addition through the formation of imine bonds between the −NH2 groups of the biopolymer and the −CHO groups on the silica surface (see Figure 293B). PBS suspensions of the capped nanoparticles, at pH 7.4, showed a negligible DOX release (3% after 24 h), whereas a moderate drug release (8% after 24 h) was noted in the presence of MMP-9. Release of DOX was ascribed to the hydrolysis of gelatin coating by the MMP-9 enzyme. The intracellular uptake and in vitro cytotoxicity of the prepared gelatin-capped nanoparticles were studied using the LO2 (MMP-9 negative) and cancer HT-29 (MMP-9 positive) cell lines. The results showed that gelatin-capped nanoparticles were only efficiently taken up by HT-29 cells. Presence of MMP9 enzyme in the HT-29 line induced pore opening with the subsequent DOX release and also significantly reduced cell viability. Finally, in vivo studies showed that tumor growth in

Figure 294. MSNs capped with ε-PL. Delivery was observed in the presence of amidase enzyme.

in the presence of amidase due to the hydrolysis of the amide bonds in the peptide. A faster release was also observed for the solid with ε-PL attached randomly. Cell internalization and delivery studies in HeLa cells were performed with the two gated materials. Viability assays indicated that unloaded silica nanoparticles were not toxic for cells. Moreover, cell treatment with the ε-PL-capped nanoparticles loaded with cytotoxic drug CPT efficiently triggered cell death in HeLa cells. Bathia and co-workers prepared polymer-coated nanoparticles, which were capable of releasing entrapped DOX in the presence of proteases.151 DOX-loaded MSNs were coated with a polymer shell composed of poly(ethylene glycol) diacrylate and a peptide macromere that contained MMP (matrix metalloproteinases) substrate polypeptide CGPQGIWGQGCR. The protease-induced controlled release of DOX was studied by incubating coated nanoparticles with 3T3-J2 fibroblasts. These nanoparticles presented high cytotoxicity levels due to DOX release as a result of the cleavage of the polymer coating because of MMP enzymes. Finally, in vivo studies were conducted in subcutaneous xenograft mouse models to test the protease-triggered release of DOX from the polymer-coated nanoparticles. Human sarcoma HT-1080 cells, known to have elevated levels of MMPs, were injected subcutaneously into flanks of immune-compromised mice. 699


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shown in Figure 296. The authors found that upon αchymotrypsin addition, the poly(L-lysine) polymer was de-

Two weeks later, the DOX-loaded nanomaterial and its respective controls were normalized to a drug concentration of 2 mg/kg and injected into well-defined tumors. Analysis of DOXinduced apoptosis indicated that the material coated with an MMP-sensitive shell exhibited greater DOX-induced cytotoxicity than those with a non MMP-sensitive coating due to the efficient release of DOX triggered by MMPs in vivo. In this area, Ren et al. prepared MSNs, which were covalently functionalized with cofactor flavin adenine dinucleotide (FAD).463 Deflavined GOx was then immobilized on the surface via interaction with FAD. Nanoparticles were loaded with Rh B. In the presence of protease K, clear dye delivery was achieved due to the proteolytic digestion of GOx caps as depicted in Figure 295. Concentration-dependent studies demonstrated that the

Figure 296. α-Chymotrypsin-induced controlled release of fluorescein from MSNs coated with cytosine-phosphodiester-guanine oligodeoxynucleotide and poly(L-lysine) polymers.

graded, which led to the disassembly of the capping unit and induced the release of the entrapped dye and oligodeoxynucleotide. MSNs loaded with safranin O and capped with N-(3́ triethoxysilylpropyl)gluconamide were reported by MartinezMáñez et al. to be a relatively simple gated system that opened in the presence of amidase or pronase enzymes.465 At a high concentration, N-(3-triethoxysilylpropyl)gluconamide formed a polymerized gluconamide layer that avoided cargo release (see Figure 297). Upon the addition of amidase and pronase enzymes, the amide bond in the anchored gluconamide derivative was hydrolyzed, which resulted in the subsequent reduction in size of the capping molecule and cargo release. These authors also found Figure 295. Protease-induced controlled release of Rh B from deflavined GOx-capped MSNs.

release rate was related with the amount of added protease and was, therefore, associated with enzyme activity. Two more proteases (trypsin and chymotripsin) were also studied by the authors, and the results correlated with the expected activity due to the accessible cleavage sites in GOx for each protease. Dye delivery was also studied in the presence of protease inhibitors phenymethyl-sulfonylfluoride and Hg2+, and no release was observed. The authors suggested that this protocol could be applied as a simple fast probe to evaluate protease activity and to screen its inhibitors. Zhu and co-workers developed nanoparticles in which delivery was triggered by α-chymotrypsin.464 The authors selected hollow MSNs, which were loaded with fluorescein. The surface was functionalized with 3-aminopropyl units, which were partially protonated at a neutral pH to confer a positively charged surface. Then particles were covered with a LbL coating composed of negatively charged cytosine-phosphodiester-guanine oligodeoxynucleotide and positively charged poly(L-lysine) polymers as

Figure 297. Amidase/Pronase-induced controlled release of safranin O from the pores of N-(3-triethoxysilylpropyl)gluconamide-capped MSNs. 700


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gated nanodevice in HeLa and MCF-7 cell lines. Confocal images were consistent with the intracellular localization of the nanoparticles in the lysosomal compartment. The solid was biocompatible at the tested concentrations, as no significant reduction in cell viability was observed. To further demonstrate a possible therapeutic application, the capped system was loaded with antitumor drug CPT and the nanomaterial was tested in HeLa cells. Cell viability significantly reduced when treated with the CPT-loaded nanoparticles, whereas no detectable cell death was observed in the cells exposed to the nanomaterial with no cargo. Yu and co-workers467 specifically designed MSNs for targeted cargo delivery in the gastrointestinal tract. In their work, they functionalized MCM-48 type nanoparticles with APTES and loaded the sulfasalazine (SZ) pro-drug into pores. Pore blocking was achieved by grafting succinylated soy protein isolate (SSPI) onto the particle’s surface through an amide bond as depicted in Figure 299. The resulting nanoparticles were programmed to

that CPT-loaded nanoparticles were efficiently taken up by HeLa and MCF-7 tumoral cells. The molecular gate was degraded by lysosomal enzymes with the subsequent cargo release, which caused cell death. The same authors reported one of the few examples that studied the possibility of including different enzyme-hydrolyzable groups, located at predefined positions, in the capping molecule as a method to control cargo delivery profiles by using defined combinations of enzymes.466 In this work, MSNs were loaded with Ru(bipy)32+dye. Then pores were capped using a bulky molecule that contained amide and urea linkages (Figure 298). In the absence of enzymes, a negligible cargo release was

Figure 299. Pancreatin-induced SZ release from MSNs capped with SSPI.

release their cargo according to their location in the gastrointestinal tract. The release profile of SZ was studied at pH 1.2 (simulated stomach, 2 h), pH 5.5 (simulated duodenum, 1 h) and pH 7.4 (simulated small intestine, >8 h) in the absence and presence of pepsin and pancreatin. The SSPI coating prevented SZ release in the simulated stomach and duodenum, whereas pro-drug SZ was released in the simulated small intestine in the presence of pancreatin enzyme via SSPI hydrolysis. Finally, SZ was broken down by the azo-reductase enzyme (simulated bacterial medium) into its active metabolite 5-aminosalycilic acid, which is an effective drug for treating colon diseases.

Figure 298. Amidase- and urease-induced controlled release of a ruthenium complex or CPT from MSNs capped with bulky molecules that contained amide and urea linkages.

detected. When amidase was present, a first relatively quick delivery took place, but only up to 40% of the cargo was released. Conversely in the presence of urease, the delivery profile was slower, but ca. 80% of the dye was delivered. The simultaneous treatment with both enzymes displayed a synergistic effect with a delivery profile that showed fast complete payload release. The different enzyme-dependent delivery rates were ascribed to the relative position of the hydrolyzable groups on the molecule. Amidase induced the hydrolysis of the amide bonds located far away from the surface and reduced steric crowding around pore outlets but hampered complete dye release. In contrast, presence of urease induced the hydrolysis of the urea bond, which was located more deeply inside the molecule structure and drastically reduced the thread size that caused a high degree, but quite slow, of cargo release. The authors explored the feasibility of using this

8.4. Rupture of Azo Bonds

In the following two examples, capped nanoparticles were designed to show “zero” delivery and to display cargo release in the presence of reductases, which are enzymes usually found in the presence of indigenous intestinal microflora. In a first work, the authors reported gated MSNs loaded with Rh B and capped with an azopyridine derivative that was attached to the surface through pyridinium salt formation as depicted in Figure 300.468 Cargo delivery from capped nanoparticles in the presence of reductase and esterase enzymes was observed. A detailed characterization indicated that the release was induced by the hydrolysis of the NN bond in the presence of the reductase 701


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Figure 301. Azo-reductase-induced controlled release of IBU from MSNs capped with azo-containing derivatives.

phosphodiester-guanine oligodeoxynucleotide was attached to the material surface via simple electrostatic interactions to inhibit dye release as shown in Figure 302A. Cargo delivery was achieved via the hydrolysis of the cap in the presence of DNase I.

Figure 300. Reductase-induced controlled cargo release from azopyridinium-functionalized MSNs.

enzyme and by pyridinium salt rupture in the presence of esterase. The capped solid was unable to release the payload in the presence of the stomach-resident enzyme pepsin. These results evidenced specific delivery to defined environments, which was achieved with these gated nanoparticles. Reductases/ esterases, which trigger cargo release, are usually present in the colon, mainly due to intestinal microflora. The authors suggested that these materials might be suitable to be used as a smart delivery system of drugs in colonic diseases. Viability assays with HeLa and MCF-7 cell lines indicated that nanoparticles were not toxic. An intracellular pattern was observed, which was consistent with the endocytosis of nanoparticles and further localization in lysosomes. Treating cells with capped MSNs, loaded with cytotoxic drug CPT, efficiently triggered cell death in HeLa cells. Similarly, Sun et al. reported the preparation of MSNs loaded with IBU and capped with an azo derivative capable of being opened only in the presence of colon azo-reductases.469 These authors synthesized the capping moiety by linking APTES with azobenzene-4,4′-dicarboxylic acid via amide formation (see Figure 301). Capped MSNs were maintained for 1 h in SGF, 4 h in SIF, and 24 h in simulated colonic fluid (SCF). Cargo release was not observed in the SIF and SGF stages, whereas a massive IBU release (up to 83%) was seen in the SCF step due to the selective reductive cleavage of the aromatic azo bond. In ref 467, Yu et al. also used azoreductase to break down sulfasalazine by the azo-reductase enzyme in its active metabolite 5-aminosalycilic acid.

Figure 302. DNase I-induced controlled release of fluorescein or colchicine from oligodeoxynucleotide or ssDNA-capped MSNs.

Wu et al. also developed capped nanoparticles, which were uncapped using enzyme DNase I.471 These authors encapsulated the drug colchicine used in cancer treatments on the pores of MSNs, which were modified with APTES. Capping of pores was carried out in SBF and was achieved through electrostatic interactions between the protonated amines on the nanoparticle’s surface and several negatively charged single-stranded DNAs of edible plants Solanum lycopersicum and Triticum aestivum (see Figure 302B). Cargo release was accomplished upon the addition of DNase I enzyme to SBF. These authors found that the cargo release rate lowered with an increasing molecular weight of capping DNA.

8.5. Hydrolysis of Phosphodiesters

A few reports have used DNases (particularly DNase I) as triggers to open capped materials. DNase I is a nuclease that cleaves DNA preferentially at the phosphodiester linkages that are adjacent to a pyrimidine nucleotide. Zhu and co-workers reported an oligodeoxynucleotide capped material using hollow MSNs, which was opened in the presence of DNase I.470 Fluorescein was selected as the entrapped cargo, and the surfaces of nanoparticles were functionalized APTES. At a neutral pH, amino moieties are partially protonated and confer a positive charge to the particles. A negatively charged cytosine-

8.6. Elongating DNA Sequences

An exotic trigger reported to uncap pores was the reverse transcriptase called telomerase. Telomerase is a ribonucleoprotein that adds DNA sequence repeats (particularly TTAGGG 702


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telomerase-induced cargo release. These authors also performed an extended in vitro study on the gated material using HeLa cells and concluded that fluorescein release was activated by the action of intracellular telomerase. They suggested that these nanoparticles could be used for tracking intracellular telomerase activity. Moreover telomerase activity was proved using different model drugs, such as telomerase sense oligodeoxynucleotide, telomerase antisense oligodeoxynucleotide, and epigallocatechingallate. The authors also performed different assays with other cell lines, such as BEL-7402 cells (liver cancer cells) and QSG-7701 cells (liver normal cells), which confirmed that the designed strategy can be applied to distinguish cancer cells from normal cells. With a similar approach, Lu and co-workers used the same gated design, but they loaded MSNs with glucose to monitor telomerase activity with a glucometer.473 In this case, the linear dependence between the glucometer readout and the number of HeLa cells used in the experiment was achieved within the 100− 5000 cells range. The limit of detection was set at 80 HeLa cells mL−1. Cui and co-workers also prepared a similar system, but in this case, they used Au@Ag nanorods coated with mesoporous silica and loaded with DOX.474 These authors also demonstrated the telomerase-induced release of the loaded drug, and they traced the uptake of nanoparticles in HeLa cells by SERS.

fragments) to the 3′ end of DNA in the telomere region, which are found at the ends of eukaryotic chromosomes. In the examples below, telomerase extended the length of the capping DNA sequences until interaction with the porous surface was inefficient and cargo delivery was observed. Ju and co-workers reported a telomerase-responsive gated system for sensitive in situ tracking of telomerase activity in living cells, which can be used to distinguish cancer cells from normal cells.472 In their work, the authors prepared MSNs, which were functionalized with a fluorescence quencher in inner pores which were additionally loaded with fluorescein (see Figure 303).

9. CONCLUSIONS AND FUTURE PERSPECTIVES Multidisciplinary research at the forefront of the field of hybrid materials has paved the way to the development of endless examples of stimuli-responsive devices,475 hydrogels,476,477 nanocapsules,478 or liposomes.479,480 We have herein reviewed the use of porous supports with gating functionalities for the controlled delivery of target guests at will. The design of such gated mesoporous materials, capable of controlling on-command the release of species in the presence of a predefined stimulus, have proved very fruitful. In particular, these materials are a promising starting point for applying the versatility of molecularbased ideas to design smart solids with controlled release features, and a way of studying factors that can influence the design of molecular gating functions based on molecular/ biomolecular/supramolecular concepts. With the use of a wide range of varied interactions and different external stimuli, such as light, changes in pH, changes in redox potential, temperature, ultrasound, alternating magnetic fields, enzymes or presence of small ions, molecules or biomolecules, among others, a relatively large number of existing ingenious capping systems and uncapping protocols to date capable of triggering mass delivery have been described. Whereas the first reports centered on the design of different gating systems, today gated nanochemistry focuses more on their use on practical applications. In this sense, the most reported gated materials seek applications, such as drug delivery carriers at the cell level with still relatively few examples on in vivo models. Other interesting applications, which have been barely studied, have been devoted to the design of capped material for sensing, controlling catalytic processes, and for use in anticorrosion coatings. Moreover, other unforeseen applications will be found in the near future. Despite the exponential development of this field, drawbacks still need to be overcome. It must be recognized that in most published works, triggered delivered molecules are dyes, and there are fewer examples designed for real applications. Despite scientific interest, many reported systems respond to stimuli that are actually hard to apply in real systems. Moreover, MSNs bioaccumulation when used in vivo is a clear problem that must be addressed. Several

Figure 303. Telomerase-induced controlled release of fluorescein from ssDNA-capped MSNs functionalized with a fluorescence quencher in inner walls.

Nanoparticles were functionalized with amino groups on the external surface, and the system was capped with a DNA sequence that contained a telomerase primer. In the presence of telomerase and deoxy-nucleoside triphosphate (dNTPs) monomers, the oligonucleotide sequence was extended, and it formed a rigid hairpin-like DNA structure that moved away from the silica surface to allow the release of the entrapped cargo. The telomerase-driven response was studied by incubating the enzyme, dNTPs, and capped MSNs, and the fluorescein release by fluorescence and UV−vis spectroscopy was monitored. A gradual increase in fluorescein intensity with increasing incubation times was observed, which demonstrated the 703


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new advances in the gated nanochemistry field are anticipated in the near future.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Lluiś Pascual graduated in Chemistry at the University of Valencia (UV) in 2011, he received his Master’s Degree (Master in sensors for industrial applications) at the Politechnic University of Valencia (UPV) in 2012. He is a Ph.D. student from the Politechnic University of Valencia (UPV), and his main area of interest is the development of chemical sensors and probes.

Biographies

Elena Aznar was born in 1982 in Valencia, Spain, and graduated in chemistry in 2005. She gained her Ph.D. degree in 2011 with Professor ́ R. Martinez-Má ñez in the Polytechnic University of Valencia on the development of gated materials for advanced applications. After this, she ́ joined R. Martinez-Má ñ ez’s group of the Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) and has worked since then on the development of smart nanomaterials for controlled release and sensing applications. She has been a CIBER permanent researcher since 2012. Her actual research interest is related to the design of gating mechanisms for advanced applications such as targeted drug delivery and sensing in the biomedical field.

José Ramón Murguiá was born in 1966 in Vitoria, Spain. He received his Ph.D. in Biochemistry and Molecular Biology from the Autonomous University of Madrid in 1993 and was an EMBO postdoctoral fellow at the Imperial Cancer Research Fund, U.K. Presently, he is lecturer of biomedicine in the Department of Biotechnology at the Politechnic University of Valencia (UPV). He also is the Deputy director of Biotechnology at the School of Agricultural Engineering and Environment at the UPV. He is the coauthor of 40 research publications and inventor of 2 patents. He is a member of the European Association of Cancer Research. He is deeply interested in exploring the interface between hybrid organic−inorganic nanostructured gated materials and biological systems (cells, tissues, and animal models).

Mar Oroval graduated in Chemistry at the University of Valencia (UV) in 2010, she received her Master’s Degree (Master in sensors for industrial applications) at the Politechnic University of Valencia (UPV) in 2012. She is a Ph.D. student from the Politechnic University of Valencia (UPV), and her main area of interest is the development of chemical sensors and probes based on the use of gated nanomaterials. 704


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́ Ramón Martinez-Má ñez was born in Valencia, Spain. He received his Ph.D. in Chemistry from the University of Valencia in 1986 and was a postdoctoral fellowship at Cambridge University, U.K. Presently, he is a full professor in the Department of Chemistry at the Polytechnic University of Valencia and also is the director of the IDM Research Institute at the Polytechnic University of Valencia and of the Biomedical Research Networking center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). He is the coauthor of more than 300 research publications and nine patents. He is a member of the American Chemical Society. His current research interest involves designing gated hybrid materials for on-command delivery applications. He is also involved in developing new sensing methods for different chemicals of interest, including anions, cations, and neutral species, such as explosives and chemical warfare agents.

DAPI DCNP DCP DFP DMEM DNP DOPC DOPE DOPS DOTAP DOX DPPC DSPE DTT EDC EDTA FA FITC Fmoc FRET GEM GOx GSH GTP GTPMS HA IBU LA LCST MB ME MPTS MRI MSNs NHS PAA PAH PCL PEG PEI PFH PI PLE PMA PNIPAAm PtOEP PVPON QDs RAFT Rh ROS SBF SCF SGF SIF SC[x]A SPIONPs TEOS TMB TNB TTF UCNPs UTP

Félix Sancenón was born in 1968 in Manises, Valencia, Spain, and graduated in Chemistry in 1991. He received his Ph.D. degree in 2003. Afterward, he worked with Professor L. Fabbrizzi at the Universitá di Pavia on the synthesis of chromogenic receptors for ion pairs. Then, he joined the Department of Chemistry at the Polytechnic University of Valencia with a Ramón y Cajal contract. He became a lecturer in 2006. He is the coauthor of more than 180 research publications. His current research interest comprises the use of hybrid materials for the development of sensors and for the construction of molecular gates.

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the Spanish Government (Project MAT2012-38429-C04-01) and the Generalitat Valenciana (Project PROMETEOII/2014/047). Ll.P. and M.O. also thank the Universidad Politécnica de Valencia for their predoctoral grants. ABBREVIATIONS AMF alternating magnetic field APTES (3-aminopropyl)triethoxysilane ATP adenosine triphosphate ATRP atom transfer radical polymerization AuNCs gold nanocages AuNPs gold nanoparticles BSA bovine serum albumin BTA benzotriazole cAMP cyclic adenosine monophosphate CB cucurbituril CBPQT cyclobis(paraquat-p-phenylene) CD cyclodextrin CLSM confocal laser scanning microscopy CPT camptothecin CTAB n-cetyltrimethylammonium bromide 705

4′,6-diamidino-2-phenylindole diethyl cyanophosphonate diethyl chlorophosphate diisopropyl fluorophosphate dulbecco’s modified eagle medium 1,5-dioxynaphthalene 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-phosphoserine 1,2-dioleoyl-3-trimethylammonium propane doxorubicin 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1,2-distearoyl-sn-glycero-3-phosphoethanolamine dithiothreitol 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ethylenediaminetetraacetic acid folic acid fluorescein isothiocyanate fluorenylmethyloxycarbonyl chloride Förster resonance energy transfer gemcitabine glucose oxidase glutathione guanosine triphosphate (3-glycidyloxypropyl)trimethoxysilane hyaluronic acid ibuprofen lactobionic acid lower critical solution temperature methylene blue mercaptoethanol (3-mercaptopropyl)triethoxysilane magnetic resonance imaging mesoporous silica nanoparticles N-hydroxysuccinimide poly(acrylic acid) poly(allylamine hydrochloride) polycaprolactone polyethylene glycol polyethylenimine perfluorohexane propidium iodide porcine liver esterase poly(methacrylic acid) poly(N-isopropylacrylamide) platinum octaethylporphyrin poly(vinylpyrrolidone) quantum dots reversible addition−fragmentation chain transfer rhodamine reactive oxygen species simulated body fluid simulated colonic fluid simulated gastric fluid simulated intestinal fluid sulfonatocalix[x]arene superparamagnetic iron oxide nanoparticles tetraethyl orthosilicate tetramethylbenzidine 1,3,5-trinitrobenzene tetrathiafulvalene upconverting nanoparticles uridine triphosphate DOI: 10.1021/acs.chemrev.5b00456 Chem. Rev. 2016, 116, 561−718

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