Neoglycoenzyme-Gated Mesoporous Silica Nanoparticles: Toward the

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Neoglycoenzyme-Gated Mesoporous Silica Nanoparticles: Toward the Design of Nanodevices for Pulsatile Programmed Sequential Delivery Paula Díez,† Alfredo Sánchez,† Cristina de la Torre,‡,§ María Gamella,† Paloma Martínez-Ruíz,∥ Elena Aznar,‡,§ Ramón Martínez-Máñez,*,‡,§ José M. Pingarrón,*,†,⊥ and Reynaldo Villalonga*,†,⊥ †

Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain Instituto de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Centro Mixto Universidad Politécnica de Valencia, Universidad de Valencia, 46022 Valencia, Spain § Departamento de Química y CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain ∥ Department of Organic Chemistry I, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain ⊥ IMDEA Nanoscience, Cantoblanco Universitary City, 28049 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: We report herein the design of a stimulus-programmed pulsatile delivery system for sequential cargo release based on the use of a lactose-modified esterase as a capping agent in phenylboronic acid functionalized mesoporous silica nanoparticles. The dual-release mechanism was based on the distinct stability of the cyclic boronic acid esters formed with lactose residues and the long naturally occurring glycosylation chains in the modified neoglycoenzyme. Cargo delivery in succession was achieved using glucose and ethyl butyrate as triggers.

KEYWORDS: enzymes, mesoporous silica, delivery, nanoparticles, esterase, neoglycoenzyme, glucose

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delivery”.10,11 These functional nanodevices can also act as smart-delivery nanomachines by releasing their cargo in response to target chemical12−15 or physical stimuli16,17 or by exposure to specific biochemical macromolecules.18−20 Usually these nanodevices exhibit a single delivery upon the application of the triggering stimulus. However, there are certain conditions in which this release pattern is not suitable, as well as many applications in which not releasing all of the drug during the initial dosage phase is a requirement. In fact, there are a number of diseases, such as asthma, cancer, duodenal ulcers, arthritis, diabetes, neurological disorders, acute myocardial infarction, etc., and vaccination protocols21,22 in which pulsatile drug-delivery systems are preferred to conventional drug administration approaches including sustainedrelease drug-delivery systems. In this sense, programmable pulsatile release has the advantages to avoid drug tolerance and maintain drug concentration at therapeutic levels albeit

INTRODUCTION Controlled delivery technology is one of the most rapidly advancing areas in material science, with the major driving force stemming from the pharmaceutical sector. In fact, advanced release systems offer numerous pharmacological and pharmacokinetic advantages as compared to conventional dosage forms such as enhanced drug efficacy, reduced toxicity, and improved patient compliance and convenience.1 In particular, the design of advanced carriers that enable time- or stimulus-programmed drug release is of much importance and is relevant for the treatment of many diseases. This field has been traditionally dominated by the use of polymers as carriers.2−4 Recently, however, attention has been paid to mesoporous silica nanoparticles (MSN) as nanosized containers for controlled delivery due to their unique properties such as large specific volume, large loading capacity, low toxicity, and easy preparation in different forms, as well as varying morphology, size, and pore diameter.5−9 MSN can be easily functionalized to allow the rational building of stimuliresponsive molecular or supramolecular ensembles on their external surface to develop gated nanocarriers that show “zero © XXXX American Chemical Society

Received: December 25, 2015 Accepted: March 11, 2016

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DOI: 10.1021/acsami.5b12645 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Performance of Dual Stimuli-Responsive Nanodevice S3 for the Programmed and Sequential Delivery of the [Ru(bpy)3]Cl2 Complex Using Glucose and Ethyl Butyrate as Triggers

described.27 These nanoparticles were then loaded with tris(2,2′-bipyridyl)ruthenium(II) chloride ([Ru(bpy)3]Cl2) as a model drug for cargo delivery monitoring5 and further treated with (3-glycidyloxypropyl) trimethoxysilane to provide the surface of the nanoparticles with highly reactive epoxy groups. The resulting solid was then treated with 3-amino phenylboronic acid to yield solid S1. Subsequently, S1 nanoparticles were treated with native esterase to prepare the enzyme-capped control solid S2 via the formation of boronic acid cyclic esters between the naturally occurring long glycosylation chains in the enzyme and the phenylboronic acid groups on the surface of the MSN. To construct the pulsatile nanodevice, we prepared a neoglycoenzyme by the covalent anchoring of lactose to the free amino groups of lysine residues in esterase through a reductive alkylation reaction with NaBH3CN.28 The resulting glycoconjugate retained 92% of initial specific activity after the incorporation of 61 mol of lactose per mol of trimeric enzyme, as determined by the phenol-sulfuric acid method (see the Methods section), which corresponded to a modification of ca. 66% of the lysine residues in the protein.29 A low degree of modification was estimated by MALDI-TOF analysis, which revealed a molecular weight of 60858 and 64227 Da for the native and lactose-modified enzyme, respectively. This fact suggests an average of 12 mol lactose attached to each mol of monomeric enzyme in the neoglycoconjugate. This neoglycoenzyme was then employed as a capping element for S1 yielding S3, following a similar procedure to that used to prepare the control solid S2. The amount of native and lactosemodified esterase immobilized on S2 and S3 nanoparticles was estimated at 10.5 and 4.3 U/mg, respectively. All of the MSN were fully characterized. The TEM analysis revealed that the different prepared nanoparticles showed the typical porous pattern of the MCM-41 mesoporous matrix with the average size for the nanoparticles of 97 ± 15 nm (Figure S1). Moreover, TEM images of S2 and S3 displayed a diffuse thin layer that wrapped the nanoparticles, suggesting their coverage with an amorphous organic material, which could be ascribed to the esterase enzymes immobilized on the nanoparticle surfaces. All of the samples also showed the characteristic X-ray diffraction pattern of the MCM-41-type materials, with a well-defined peak at ca. 2.59° attributed to the

circadian rhythms, allowing the design of chronotherapeutic protocols for some common diseases exhibiting circadian variation.22 Nevertheless, despite the design of a number of sophisticated nanodelivery supports, very few nanodevices for sequential and controlled pulsatile cargo delivery are yet available.23,24 However, in most of enzyme-responsive MSN systems previously reported, enzymes act as triggers that hydrolyze specific molecular sequences anchored on the gated mesoporous support. As an alternative, we have recently reported that enzymes can act as caps in gated materials in which the uncapping process is triggered by the product obtained by the enzyme activity on target guests.15 Neoglycoenzymes are artificially glycosylated enzymes in which the new appended carbohydrates moieties confer novel and improved characteristics to the enzyme, such as stability, catalytic activity, and chemical and biochemical recognition properties.25,26 On the basis of our experience in the design of neoglycoenzymes,26 we envisioned that the modification of a glycoenzyme with short-chain carbohydrate residues can lead to a neoglycoconjugate being able to be used as a capping element in phenylboronic acid-grafted MSN by forming cyclic boronic acid esters through either naturally occurring long glycosylation chains or chemically attached short disaccharide chains (vide infra). In this context, we speculated that considering the different amount and geometry of the long and short glycosylation chains, as well as their different affinities and stabilities with the boronic acid groups, it might be possible to design pulsatile drug-delivery systems capable of delivering the cargo sequentially under the effect of different triggering conditions. As a proof of concept of sequential delivery with MSN using neoglycoenzymes, we report herein the preparation of solid S3 (vide infra) consisting of an MCM-41-type MSN containing phenylboronic acid residues and capped with lactose-modified pig liver esterase (see Scheme 1). This engineered nanoparticle was successfully evaluated as novel sequential and pulsatile drug-delivery system for in vitro and ex vivo experiments.

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RESULTS AND DISCUSSION To assemble the integrated delivery nanomachine, we first prepared mesoporous silica nanoparticles (S0) as previously B


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glycol). The chemistry involved in the trigger reactions is illustrated in Figure S9. The addition of ethyl butyrate caused the cargo to be completely released from both nanomaterials S2 and S3 (see Figure 1A). This behavior is related with the rapid disruption of

(100) Bragg reflection of MCM-41-type materials. The position of this peak was not significantly affected by dye loading and the different chemical modifications processes carried out in their preparation (Figure S2). Functionalization of MSN with phenylboronic acid moieties and further capping with the esterase derivatives were assessed by FT-IR (Figure S3). The assembly of the nanodevices was also confirmed by solid-state 13C NMR (Figure S4). The thermal analysis revealed that the content of the anchored boronic acid-based ligand and encapsulated [Ru(bpy)3]Cl2 dye in S1 amounted to 34 and 206 mg/gS1, respectively (Figure S5). The amount of esterase and lactose-modified esterase in S2 and S3 was estimated to be 17.3% and 32.8% by weight, respectively, according to the TG studies. This difference could be explained by the higher molecular weight showed by the lactose-modified enzyme as well as the potential higher capacity of this neoglycoconjugate to form multipoint boronic acid cyclic ester linkages at the surface of the nanoparticles. The influence of the different modification steps on the total specific surface area and pore size of the prepared MSN was also determined from the corresponding N2 adsorption−desorption isotherms (Figure S6 and Table S1). S2 and S3 showed similar specific surface area and total pore-volume values, suggesting that the different enzyme derivatives showed high capping efficiency for the mesoporous nanoparticles. The dynamic light scattering and ζ potential experiments further confirmed the successful surface functionalization of MSN. DLS data show that the starting MCM-41 material exhibited a hydrodynamic diameter of 295 nm as a result of partial aggregation in H2O. This diameter was even larger in PBS (320 nm) and cell culture medium (384 nm), also an indication aggregation of the nanoparticles in these solvents. The functionalization of the MCM-41 nanoparticles with the glycoenzymes to give S2 and S3 improved the colloidal stability of the nanoparticles, and hydrodynamic diameters of 211 and 196 were found for S2 and S3 in H2O, respectively (see the Supporting Information). This reduction in the hydrodynamic diameter could be mainly ascribed to a low degree of aggregation in the enzyme-modified nanoparticles. ζ potential measurements showed that the starting MCM-41 nanoparticles had a negative potential in water (−26 mV) that was reduced for the final capped nanoparticles S2 and S3 to −7 and −5 mV due to the functionalization of the surface and capping with the glycoenzymes. Similar results were found in PBS and cell culture medium (see Figure S7 and Table S2). Functionalization of the MCM-41 nanoparticles with the glycoenzymes was assessed by TEM after staining the nanomaterials with uranyl acetate (Figure S8). In comparison with S1, S2, and S3 showed a well-defined dark layer around the nanoparticles, demonstrating high coverage of these nanomaterials with the organic glycosylated enzyme molecules. Nanodevices S2 and S3 were tested for the on-command controlled delivery of the cargo in aqueous solutions in the presence of some potential triggers (vide infra). In a typical release assay, 10 mg of the enzyme-capped nanoparticle were suspended in 4 mL of 20 mM Na2SO4 solution at pH 7.5 and were shaken over time at 25 °C. Next, the corresponding triggering substances were added, and the mixture was incubated for 1.5 h. An aliquot was taken and centrifuged to remove the nanoparticles, and the absorbance of the released [Ru(bpy)3]Cl2 complex was measured at 454 nm. As triggers for cargo delivery, two different types of substances were tested; ethyl butyrate and 1,2- or 1,3-diols (glucose and ethylene

Figure 1. (A) Release efficiency for [Ru(bpy)3]Cl2 from S3 (a) and S2 (b) after a 90 min incubation with different trigger substances at a 200 μM concentration. (B) Dye-release efficiency from S3 (a) and S2 (b) after a 90 min incubation in the presence of glucose at different concentrations.

the boronic acid cyclic esters under acidic conditions due to the esterase-catalyzed hydrolysis of ethyl butyrate to ethanol and butyric acid (pKa = 4.82), resulting in the leakage of the immobilized enzyme from the nanosized support and cargo delivery. In contrast, and interestingly, the encapsulated [Ru(bpy)3]Cl2 complex was only partially released from S3 under the trigger effect of glucose, whereas no release was observed for S2. Delivery from S3 is related with the competition of glucose for the boronic acid residues anchored to the neoglycoenzyme on the nanoparticle surface, which results in a partial cargo release. The extent of such a delivery process in S3 increased with the sugar concentration in the incubation media, a plateau value being reached for concentrations of glucose above 200 μM (Figure 1B). Similar behavior was observed by incubation at high glucose concentration up to 5 mM. On the contrary, no significant dye release was observed for similar experiments with nanoparticles of S2, even at relatively high glucose concentrations up to 5 mM. Studies with ethylene glycol, which is able to form highly stable five-member rings with boronic C


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by a local decrease of pH at the microenvironment of the modified colloids. To confirm that the opening mechanism with ethyl butyrate was due to the enzyme-mediated decrease of pH after the substrate hydrolysis, we heated suspensions of S2 and S3 at pH 7.5 to 100 °C for 10 min to inactivate the enzymes, and they were further incubated with ethyl butyrate. In this case, no appreciable cargo delivery was observed from the nanodevices after 24 h of incubation. To gain insight of inside the mechanism of the sequential delivery process, we determined the activity of the enzyme immobilized on the MSN before and after the sequential incubation with the triggers. The enzymatic activity initially immobilized on S3 capped with lactose-modified esterase was estimated to be 4.3 U/mg of solid, which lowered to 2.9 U/mg of solid after 1 h of incubation with glucose, suggesting that about 32.6% of the neoglycoenzyme leaked from the solid through a monosaccharide-mediated competitive replacement. Moreover, no appreciable enzymatic activity was detected on the resulting solid after 1 h of S3 incubation with ethyl butyrate. In contrast, S2 retained full esterase activity (10.5 U/mg solid S2) after performing a similar incubation with glucose. Treatment of S2 with ethyl butyrate also caused the total reduction of immobilized enzyme activity, demonstrating that the enzyme was completely leaked from the nanoparticle surface. Taking into account these results, a mechanism for the twostep pulsatile programmed sequential delivery from S3 triggered by glucose and ethyl butyrate is proposed in Scheme 2. The obtained results indicate that native esterase is strongly attached to the nanoparticle surface in S2, probably through the formation of multipoint boronic acid cyclic ester linkages between the saccharide units at the naturally occurring glycosylation chains of the enzyme and the phenylboronic acid groups grafted on the nanoparticle surface. Regarding S3, the lactose-modified esterase is most likely linked to the boronic acid residues through two different ways: (i) by the attachment of the naturally occurring glycosylation chains of the enzyme, yielding highly stable linkages that are not displaced by incubation with glucose or ethylene glycol; and (ii) via the lactose residues in the neoglycoenzyme, which yield linkages that can be disrupted in the presence of glucose or ethylene glycol. Moreover, both kinds of linkages are prone to being hydrolyzed in acidic media, which is provoked by the enzyme-mediated hydrolysis of ethyl butyrate. In addition, the enzyme-controlled capped MSN were tested for in-cell controlled delivery applications. Therefore, after the in vitro characterization of the solid S2 and S3 (vide ante), similar nanoparticles were used for ex vivo assays by loading nanoparticles as S2 and S3 with the cytotoxic doxorubicin (Doxo) (solid S4 and S5, respectively). Solids S4 and S5 were evaluated in HeLa cells under the premise that they would be internalized by cells and would stay nearly closed until glucose or ethyl butyrate were added. In this sense, the capacity of HeLa cells to uptake 100 nm silicamodified nanoparticles has been largely documented in the literature.7,30−33 According to the in vitro S2 and S3 behavior (see panels A and B of Figure 2, respectively), the addition of D-glucose in S5 in ex vivo assays would be expected to induce partial intracellular Doxo delivery, whereas the additional presence of ethyl butyrate would result in further Doxo release. However, the presence of D-glucose in internalized S4 nanoparticles

acid residues, showed an almost complete cargo release from S3, but no noticeable effect was produced with S2 (see Figure 1A). On the basis of these results, we tested S3 for the two-step controlled cargo delivery by triggering the uncapping process through the sequential incubation with glucose and ethyl butyrate. In parallel, S2 was also tested under the same experimental conditions (see Figure 2). In the absence of

Figure 2. Kinetics of the dye release from S3 (A) and S2 (B) in 20 mM Na2SO4, pH 7.5, without (a) and with addition of ethyl butyrate (b) and glucose + ethyl butyrate (c) at a 200 μM final concentration. Triggers were added at the times indicated in the graphics.

glucose or ethyl butyrate, both nanodevices are tightly capped and showed a negligible release of [Ru(bpy)3]Cl2. Conversely, the presence of glucose resulted in the opening of some pores in S3, producing a subsequent partial cargo delivery. Further incubation of S3 with ethyl butyrate, which is transformed by the immobilized enzyme glycoconjugate in ethanol and butyric acid with a consequent drop in pH, caused the acid-mediated hydrolysis of the boronic acid cyclic esters and induced cargo delivery (see Figure 2A). On the contrary, S2 delivered the cargo only by incubation with the enzyme substrate ethyl butyrate (see Figure 2B). It should be noted that the pH of the incubating media remained unchanged after the S2 and S3 treatment with glucose but changed from 7.5 to about 5.2 after incubation with ethyl butyrate. It should be highlighted that similar release patterns were observed by performing the experiments in PBS, but a slightly lower degree of cargo delivery was achieved by using ethyl butyrate as trigger. This fact suggests the esterase-mediated release of Ru(bpy)32+ from the nanoparticles was mainly caused D


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Scheme 2. Proposed Mechanisms for the Stimuli-Responsive Controlled Delivery from Nanocarriers S2 (A) and S3 (B)

deduced from the viability studies, the simple internalization of S4 in HeLa cells (see − /−) or the treatment with D-glucose (see ±) did not induce significant cell death, whereas ca. 60% of cells were dead or underwent cell death after the addition of ethyl butyrate (see Figure 3C). It was also found that S5 nanoparticles were not significantly toxic for HeLa cells, whereas in this case both the presence of D-glucose (see ±), ethyl butyrate (see ∓), and especially a mixture of D-glucose and ethyl butyrate (see + /+) induced an increment in dead cells and cells undergoing cell death (see Figure 3D). In particular, nearly 50% of the cells were dead or undergoing cell death when treated with both D-glucose and ethyl butyrate simultaneously (see also Figures S12 and S13). Although significantly very low, cell death caused by S4 and S5 nanoparticles in the absence of the trigger compounds could be ascribed to the unspecific release of Doxo inside the cells. This fact, which was revealed by a few leaking signals in Figure 3 (see − /−), could be caused by local acidic conditions at the microenvironment of the nanoparticles into the HeLa cells. Cell viability observations using flow cytometry were in agreement with cell-viability studies using WST-1 assays. Moreover, cell-viability studies were also in agreement with Doxo fluorescence observed in cells by confocal microscopy and determined with flow cytometry (Figures S10 and S11). Analogous results were achieved by using the sequential

should have no effect, and the addition of ethyl butyrate would induce Doxo delivery. In a typical experiment, HeLa cells were incubated for 30 min with a suspension of 75 μg/mL of S4 or S5 in PBS supplemented with 10% fetal bovine serum. Subsequently, cells were washed to remove uninternalized nanoparticles and further incubated alone or in the presence of D-glucose (input I1), ethyl butyrate (input I2), or a mixture of both (see the Methods section). The sequential addition of D-glucose and ethyl butyrate as input signals was also evaluated. Doxo delivery details obtained by confocal microscopy and cytometry are provided in Figures S10 and S11, whereas cell viability was determined by flow cytometry studies (see Figure 3) and by WST-1 assays under different conditions. Figure 3 also shows representative phase-contrast images of Doxo, Hoescht, and combined for HeLa cells first loaded with S4 or S5 and then untreated (−/−) or treated with both Dglucose and ethyl butyrate (+/+). These nanomaterials were mainly internalized into the HeLa cells, as observed from Figure 3. However, some few nanoparticles could remain outside the cells, as revealed for S5 in Figure 3B (see + /+). In flow cytometry studies, quantification of Doxo-associated cell death was performed by using 7-aminoactinomycin (7AAD) and Annexin V-FITC (Ann V) markers, which stain dead cells and cells undergoing cell death, respectively. As it can be E


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Figure 3. Internalization and release of cargo in HeLa cells. Culture were incubated with S4 (A) or S5 (B) in the presence and absence of different input I1 (D-glucose) or input I2 (ethyl butyrate) and examined for doxorubicin staining (Doxo) by confocal microscopy. Representative images at 24 h form-phase contrast (PhC), doxorubicin (Doxo), Hoescht (Hoe), and combined (Merged) are shown. Quantification of cell viability and cell death was performed by flow cytometry by means of 7-AAD and Ann V staining. The percentage of dead cells (black), cells undergoing cell death (gray), and healthy cells (white) are shown for a 50 μg/mL concentration of S4 (C) and S5 (D) in HeLa cells under different conditions at 24 h.

addition of D-glucose and ethyl butyrate to the incubation medium, showing similar release patterns than those observed in experiments involving the incubation with D-glucose and the mixture of both trigger compounds (data not shown). In addition, the intracellular distribution of S4 or S5 was evaluated by confocal laser scanning microscopy upon the addition of ethyl butyrate. A red Lysotracker probe was used to stain acidic organelles in HeLa cells. It was found that S4 and S5 were predominantly localized in Lysotracker-labeled organelles after 1.5 h of incubation. Moreover, after 12 h, a homogeneous distribution of Doxo in the cytosol and partly in the nucleus was observed (see Figure S14 and S15). Conversely, when the intracellular distribution of S4 or S5 was evaluated in the absence of ethyl butyrate, much less Doxo was released after 12 h incubation. However, Doxo fluorescence was colocalized, mostly with Lysotracker dye, strongly suggesting that the nanoparticles were internalized into the nanoparticles and remained mainly in the lysosomes without delivering the entrapped cargo (see Figure S14 and S15). To further study intracellular Doxo release in S4 and S5 for different times (2, 5, or 30 h), we took confocal images of HeLa cells in the presence or absence of ethyl butyrate. A clear Doxo fluorescence was observed for both S4 and S5 in the nucleus after 30 h when ethyl butyrate was used as the input, whereas this effect was clearly not observed in its absence (see Figure S16).

It should be highlighted that the design of novel multistimuli responsive nanodevices receives considerable attention due to the advantages of such systems, such as extraordinary control over drug delivery and release and the possibility to program the release sequence, leading to superior therapeutic efficacy.34 In this sense, a great variety of smart nanodevices able to respond to combination of multiple signals (pH, temperature, magnetic field, enzymes, light, guest molecules, etc.) have been reported.7,30,34−37 In our model, the advantages associated with the use of a neoglycoenzyme molecule as a sensing, signaltransforming, and capping agent allow the designing of advanced and tailor-made nanoparticulated systems for the programmed and pulsatile release of therapeutic, antimicrobial, and plant protecting and growth enhancing compounds. Despite our results, there are some aspects that could be considered in the future to improve these nanoparticles-based programmed pulsatile delivery systems. In this sense, D-glucose was here employed as model trigger, but the presence of this monosaccharide in blood should lead to the unspecific release of drugs in vivo. In this proof-of-concept, no significant delivery was observed into the HeLa cells in the absence of D-glucose, probably due to its low intracellular concentration caused by the fast metabolic consumption of this sugar by cancer cells.38,39 Although ethyl butyrate is not common in human fluids and tissues, local acidic microenvironments could also provoke unspecific drug release in vivo. In addition, our model F


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Preparation of Lactose-Modified Esterase-Capped MSN (S3). To prepare the esterase-based neoglycoconjugates, we dissolved esterase (5.7 mg) and lactose (12.5 mg, 36.5 μmol) in 1.25 mL of 50 mM sodium phosphate, pH 7.5, and stirred for 1 h at 4 °C. NaBH3CN (12.6 mg, 200 μmol) was further added, and the reaction mixture was stirred overnight at 4 °C. The solution was then exhaustively dialyzed versus 50 mM sodium phosphate buffer (pH 7.5) using Amicon Ultra-05 centrifugal filter units with Ultracel-10 membranes (Millipore) and finally concentrated to an about 20 mg/mL esterase concentration. The neoglycoenzyme gated solid S3 was prepared as described above for solid S2. Dynamic Light Scattering and ζ Potential. Dynamic Light scattering (DLS) and ζ potential were performed at 25 °C using a Malvern Zetasizer NanoZS instrument. Measurements were performed with nanoparticles S0, S2 and S3 suspended in filtered water, phosphate buffer with 10% FBS, or cell culture media DMEM with 10% FBS at a concentration of 50 μg·ml−1. Cell Culture Conditions. HeLa human cervix adenocarcinoma cells were purchased from the German Resource Centre for Biological Materials (DSMZ) and were grown in DMEM supplemented with 10% FBS. Cells were maintained at 37 °C in an atmosphere of 5% carbon dioxide and 95% air and underwent passage twice a week. WST-1 Cell Viability Assay. HeLa cells were seeded in a 24-well plate at a density of 2·104 cells/well in a 1000 μL of DMEM and were incubated 24 h in a CO2 incubator at 37 °C. Next, DMEM were replaced for PBS with 10% of fetal bovine serum, and solid S4 or S5 in DMSO were added to cells in sextuplicate at final concentrations of 75 μg·ml−1. DMSO represented 1% (v/v) of the total volume of the cell culture medium. As a control, we used cells untreated and cells treated with the same volume of DMSO as cells treated with the nanoparticles in DMSO. After 30 min, cells were washed with PBS and were incubated during 23 h in different conditions. DMEM with 10% FBS (±), DMEM with 10% FBS and ethyl butyrate (+/+), PBS with 10% FBS (∓) or PBS with 10% FBS and ethyl butyrate (∓). After this, 30 μL of WST-1 were added to each well and were incubated during 1 h; a total of 24 h of incubation was therefore studied. Before reading the plate, it was shaken for 1 min to ensure homogeneous distribution of color. Next, the absorbance was measured at a wavelength of 450 nm in VICTOR X5 PerkinElmer. Results are expressed as a promedium of the results of six independent experiments obtaining similar results. Live Confocal Microscopy. HeLa cells were seeded in a 24 mm Ø glass coverslips in 6-well plates at a seeding density of 1.8·105 cells/well. After 24 h, culture medium were replaced for PBS with 10% fetal bovine serum (FBS), and cells were treated with a suspension of solid S4 or S5 for 30 min at a final concentration of 75 μg·ml−1. Next, the medium was changed for different solutions (DMEM with 10% FBS with or without ethyl butyrate or PBS with 10% FBS with or without ethyl butyrate). After 20 h, coverslips were washed twice to eliminate compounds and were visualized under a confocal microscope employing Leica TCS SP2 AOBS (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) inverted laser scanning confocal microscope using oil objectives (63× PlanApochromat-Lambda Blue 1.4 NA). Confocal microscopy studies were performed by Confocal Microscopy Service (CIPF). The images were acquired with an excitation wavelength of 405 for Hoescht and 480 nm for Doxorubicin.

nanomachines could be further improved by providing an affinity mechanism for target drug delivery to tumors in vivo.

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CONCLUSIONS In summary, here we demonstrate that the modification of a glycoenzyme by artificial glycosylation is a useful approach to manipulate the strength of the supramolecular interaction with phenylboronic acid-coated supports. Based on this concept, novel neoglycoenzyme-gated MSN, for programmed and pulsatile sequential cargo delivery, are reported. In particular, gated support S3 is able to release ca. half the cargo in the presence of glucose, whereas the remaining entrapped payload is delivered upon the addition of ethyl butyrate. Moreover, it was also demonstrated that the same control was observed with nanoparticles loaded with an anticancer drug (S5) and tested in HeLa cells. We believe that the possibility of using a wide variety of different derivatized enzymes, combined with a variety of functionalized MSN as supports, opens up new possibilities for the design of novel smart pulsatile delivery nanodevices for on-command programmed sequential delivery. These, or similar, release systems have the potential of being applied to diseases for which a pulsatile drug delivery is preferred and in which triggering can be achieved using simple nontoxic small molecules.

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METHODS Preparation of MSN (S0).27 Cetyltrimethylammonium bromide (3.0 g) was dissolved in 1.44 L of water under sonication. NaOH solution (2.0 mol/L, 10.5 mL) was then added, and the temperature of the mixture was adjusted to 80 °C. Tetraethoxysilane (15.0 mL) was added dropwise to the surfactant solution within 5 min under vigorous magnetic stirring. The mixture was allowed to react for 2 h. The resulting white solid was filtered, washed with water and methanol, and then dried in desiccator. Finally, the solid was calcined at 550 °C for 5 h to remove the organic template. Preparation of Phenylboronic Acid-Coated MSN (S1). S0 (200 mg) and 300 mg (39.6 μmol) of tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate were suspended in 25 mL of anhydrous acetonitrile inside a round-bottom flask connected to a Dean−Stark trap under Ar atmosphere. The suspension was heated at 110 °C, and about 10 mL of solvent was distilled and collected in the trap to remove the adsorbed water. After this step, the mixture was stirred for 24 h at room temperature to load the dye into the MSN face pores.15 Afterward, an excess of (3-glycidyloxypropyl)trimethoxysilane (500 μL, 2.26 mmol) was added, and the suspension was stirred for 5 h. The resulting solid was filtered off and washed two times with 10 mL of acetonitrile and then with 10 mL toluene. The solid was dispersed in 30 mL of toluene and mixed with 3aminophenyl boronic acid (308 mg, 2.26 mmol). The reaction mixture was stirred for 12 h, and the final solid (S1) was filtered off, washed with toluene, and dried at room temperature. Preparation of Esterase-Capped MSN (S2). A total of 20 mg of solid S1 were mixed with 5.7 mg of esterase in 700 μL of 50 mM sodium phosphate buffer, pH 7.5. The mixture was stirred overnight at 4 °C and then centrifuged and exhaustively washed with 20 mM Na2SO4 solution at pH 7.5 until no tris(2,2′-bipyridyl)dichlororuthenium(II) can be detected in the washed solutions. The resulting solid S2 was dried and kept at 4 °C until use. G


Research Article

ACS Applied Materials & Interfaces We gathered 2D pseudocolor images (255 color levels) with a size of 1024 × 1024 pixels and Airy 1 pinhole diameter. A total of three fields of each condition in two independent experiments were performed obtaining similar results. For cellular internalization observation, HeLa cells were seed in 6-well plates at a seeding density of 1.8·105 cells/well. After 24 h, culture media were replaced for PBS with 10% fetal bovine serum (FBS), and cells were treated with a suspension of solid S4 or S5 for 30 min at a final concentration of 30 μg· ml−1. Next, the medium was changed for different solutions (PBS with 10% FBS with or without ethyl butyrate). After 1 h (total of 1.5 h) or 11 h (total of 12 h), coverslips were washed, and the medium was replaced for PBS with 10% FBS preheated at 37 °C containing Lysotracker (50 nM). After 30 min, the cells were washed and treated with Hoescht for 5 min. After washing with PBS, they were fixed using 4% formaldehyde for 15 min. Confocal microscopy studies were performed by Confocal Microscopy Service (UPV). The images were acquired with an excitation wavelength of 405 for Hoescht and 480 nm for Doxorubicin. The cells were monitored under a Leica TCS SP2 laser-scanning confocal microscope (42×/62× oil objective, 405/488 excitation). For intracellular Doxo release observation, HeLa cells were seeded according to the above-mentioned description. The cells were washed three times with PBS and then incubated with S4 or S5 for 30 min and then washed again. One sample was fixed then, and the others were incubated during 2, 5, and 30 h. Cells were washed with PBS and fixed with fresh 4% formaldehyde at room temperature for 15 min. After being washed with PBS, the cells were subjected to the CLSM observation. Cytofluorometry Studies using S4 and S5. HeLa cells were seeded at 18 × 103 cells per well in a 24-well plate. After 24 h, DMEM were replaced for PBS with 10% of fetal bovine serum and solid S4 or S5 in DMSO were added to cells at final concentrations of 75 μg·ml−1. After 30 min, cells were washed with PBS and were incubated for 23 h in the different conditions (DMEM with 10% FBS, DMEM with 10% FBS and ethyl butyrate, and PBS with 10% FBS or PBS with 10% FBS and ethyl butyrate). After 24 h, media were eliminated by vacuum, and plates were washed once with PBS. Cells were detached with Trypsin/EDTA solution, centrifuged, and finally resuspended in 0.5 mL of DMEM with 10% FBS. Quantification of Doxorubicin fluorescence in the cells was performed with WinMDI program, version 2.0 in a FC500 MCL Flow Cytometer (Beckman-Coulter, CA). A total of three independent experiments containing quadruplicates were performed with similar results. For cytotoxicity assays employing flow cytometry, cells were seeded at the same conditions. After 24 h, solids S0, S4, or S5 in DMSO were added at final concentration of 50 μg· ml−1. After 30 min, cells were washed and incubated for 23 h in the different conditions. After 24 h, cells were stained with 7-AAD and Ann V-FITC according to the manufacturer’s protocol (Life Technologies). Quantification of 7-AAD-positive and AnnV-positive staining was done by the WinMDI program, version 2.9, in a FC500 MCL flow cytometer (BeckmanCoulter, CA). A total of three independent experiments were done and contained triplicates with analogous results.

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Experimental details. Figures showing TEM images, powder X-ray diffraction results, FT-IR analysis, solidstate 13C NMR spectra, TG and DTG analysis, nitrogen adsorption and desorption isotherms, pore-size and nanoparticle size distribution, trigger reactions, confocal images of HeLa cells incubated with solid S4 and S5, quantification of cell viability and cell death by flow cytometry, and CLSM images of HeLa cells. Tables showing BET-specific surface values, pore volumes, and pore sizes; and nanoparticle size and ζ potential values. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*R.V. e-mail: [email protected]. *J.M.P. e-mail: [email protected]. *R.M.M. e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.V. acknowledges to Ramón & Cajal contract from the Spanish Ministry of Science and Innovation. Financial support from the Spanish Ministry of Science and Innovation (CTQ2011-24355, CTQ2009-12650, CTQ2009-09351, and MAT2012-38429-C04-01) and Comunidad de Madrid (S2009/PPQ-1642), programme AVANSENS, is gratefully acknowledged. The Generalitat Valencia (project PROMETEOII/2014/047) is also acknowledged.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b12645. H


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Neoglycoenzyme-Gated Mesoporous Silica Nanoparticles: Toward the

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