The Importance of Encapsulation Stability of Nanocarriers with High

Subscriber access provided by Kaohsiung Medical University

Article

The Importance of Encapsulation Stability of Nanocarriers with High Drug Loading Capacity for Increasing In Vivo Therapeutic Efficacy L PALANIKUMAR, Eun Seong Choi, Jun Yong Oh, Soo Ah Park, Huyeon Choi, Kibeom Kim, Chaekyu Kim, and Ja-Hyoung Ryu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00589 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The Importance of Encapsulation Stability of Nanocarriers with High Drug Loading Capacity for Increasing In Vivo Therapeutic Efficacy L. Palanikumar,† Eun Seong Choi, † Jun Yong Oh, † Soo Ah Park,‡ Huyeon Choi, † Kibeom Kim, † Chaekyu Kim, † and Ja-Hyoung Ryu, †*. †

Department of Chemistry, School of Natural Sciences, Ulsan National Institute of Science and

Technology (UNIST), Ulsan 44919, Korea ‡

In Vivo Research Centre, UNIST Central Research Facilities, Ulsan National Institute of

Science and Technology (UNIST), Ulsan 44919, Korea KEYWORDS. Mesoporous Silica Nanoparticle; Stimuli-Responsive Materials; Polymergatekeeper; Targeted delivery; Intracellular delivery

ACS Paragon Plus Environment

1

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT. Current drug delivery systems are hampered by poor delivery to tumors, in part reflecting poor encapsulation stability of nanocarriers. Although nanocarriers such as polymeric micelles have high colloidal stability and do not aggregate or precipitate in bulk solution, nanocarriers with low encapsulation stability can lose their cargo during circulation in blood, due to

interactions

with

blood

cells,

cellular

membranes,

serum

proteins,

and

other

biomacromolecules. The resulting premature drug release from carriers limits the therapeutic efficacy at target sites. Herein, we report a simple and robust technique to improve encapsulation stability of drug delivery systems. Specifically, we show that installation of disulfide crosslinked non-covalent polymer gatekeepers onto mesoporous silica nanoparticles with a high loading capacity for hydrophobic drugs enhances in vivo therapeutic efficacy by preventing premature release of cargo. Subsequent release of drug cargos was triggered by cleavage of disulfide cross-linking by glutathione, leading to improved antitumor activity of doxoroubicin in mice. These findings provide novel insights into the development of nanocarriers with high encapsulation stability and improved in vivo therapeutic efficacy.


2

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1.0 INTRODUCTION Inefficient delivery of hydrophobic anti-cancer drugs limits pharmacokinetics and drug biodistributions, and represents a major challenge in the field of drug delivery.1-2 Among supramolecular drug delivery carriers that have been developed to improve drug delivery3-4, amphiphilic block copolymers (BcP) comprising hydrophobic and hydrophilic blocks have attracted growing interest because of their high colloidal stability during storage, and their ability to ecapsultate hydrophobic anticancer drugs in hydrophobic cores.5-7 However, due to low drug delivery efficiencies, few supramolecular drug delivery systems (Genexol, Eligard, and Zinostatin stimalamer) have been assessed in clinical trials. In recent review by Chan et al. estimated median lethal efficiencies of most drug delivery systems are reportedly very low, with approximately 0.7% of the injected dose reaching the tumor.8 One of the main reasons for the low drug delivery efficiency is the low encapsulation stability of nanocarriers. Although nanocarriers have high colloidal stability and do not aggregate or precipitate in the bulk solution, nanocarriers with low encapsulation stability could lose their cargos during interactions with macromolecules of blood cells and cellular membranes, and with serum proteins.9-10 Accordingly, Park et al. investigated the effects of encapsulation stability of a drug carrier on the therapeutic efficacy, and demonstrated poor encapsulation stability of self-assembled polymeric micelles (PEG-PDLLA) with a low loading capacity of < 2%. These investigators also showed that micelles tend to release their contents before breaching plasma membranes, leading to inefficient drug delivery.10 Thus, efficient drug delivery vehicles require high encapsulation stability to remain intact until release of drugs into target tissues and cells. In recent studies, encapsulation stability was manipulated by incorporating cross-linking groups such as disulfide bonds in the cores of the polymeric micelles under physiological


3


Page 4 of 30

conditions.11-15 In particular, Thayumanavan et al. showed that a crosslinked nanogel assembly encapsulates hydrophobic cargo via disulfide bond cross-linking, offering structural stability and retention of cargo inside polymeric nanogels until arrival at target cancer cells.11-13 Park et al. also reported a shell crosslinked polymeric nanoparticle system with higher encapsulation stability than that of simple polymeric micelles.14,

15

However, their improvements in

encapsulation stability were only achieved by polymeric micellar structures with below optimal loading capacity (< 10 wt%), and loading method of hydrophobic drugs into the polymeric micelle core requires case-specific optimization by varying the drug molecules. Hence, these studies warrant further investigations of the effects of encapsulation stability on the efficacy of drug delivery nanocarriers with optimum loading capacity and simple and versatile drug loading methods. Herein, we show that non-covalent polymer gatekeepers in mesoporous silica nanoparticles (PMSNs) retain high encapsulation stability when holding large cargos, whereas block copolymer micelles have low encapsulation stability, even with low drug loads. Due to their large surface areas, mesoporous silica nanoparticles (MSNs) can hold exceptionally large quantities of drugs inside their cores, and the polymer gatekeeper by cross-linking on the nanoparticle surface acts as a stable corona to prevent premature leakage of cargo. Maintenance of high encapsulation stability remains a chalange for drug carriers with large loading capacities.6, 14 Herein, we show high encapsulation stability of the present PMSNs, and increased enhanced permeability and retention (EPR)-mediated accumulation of drugs in tumor regions. Moreover, we show greater antitumor activity of PMSNs than self-assembled block copolymer micelles in mice with SCC7 tumor xenografts (Figure. 1).


4

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 1. a) Schematic of PMSNs and BcP micelle preparation; b) higher encapsulation stability of noncovalent polymer gatekeeper MSNs (PMSNs) for in vivo therapeutics; stable encapsulation from PMSNs vs. leakage of cargo from BcP micelles; schematic of in vivo enhanced permeability and retention effect (EPR)-mediated uptake and glutathione (GSH) mediated drug release inside cancer cells. 2.0 EXPERIMENTAL METHODS 2.1 Materials, cell lines, and animals Chemicals and reagents were purchased from Sigma-Aldrich, TCI and Alfa Aesar, Korea unless otherwise specified and used without further purifications. Doxorubicin hydrochloride was obtained from Ontario Chemicals Inc, Canada. 3,3’-dioactadecyloxacarbocyanine perchlorate


5


Page 6 of 30

(DiO, donor) and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI, acceptor)

were

purchased

from

Invitrogen,

Korea.

1,1’-dioctadecyl-3,3,3’,3’-

tetramethylindocarbocyanine perchlorate analog (DiD) was purchased from Biotium, Korea. Dialysis tubing was supplied by (MWCO 3.5 kDa) Spectrum Laboratories Inc, Korea. Human cervical cancer cell line HeLa and mouse squamous cell carcinoma SCC7 was cultured in DMEM and RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Korea), respectively. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C, with the medium changed every other day. Cells were cultured on 4 well Lab-Tek glass chamber slides (Thermo Fisher Scientific, Korea). Then the cells were treated with DiI/DiO coloaded PMSN, MSNs and BCP micelle in HeLa cells at different dye loading capacity (at 5 µg/mL) and incubated for 4 h and 8 h incubation. Similarly, uptake of Dox loaded PMSNs and BCP micelle in SCC7 cancer cells after 2 h incubation. All the images were captured using the Olympus confocal laser scanning microscope model FV 1000. For cell viability analysis, cells were cultured in 96-well plate for a period of 24 h when the cell confluence reached over 80% and the fresh medium containing MSNs, PMSNs, Dox-BCP, free-Dox and Dox-PMSN at different concentration in SCC7 cancer cells (at 24 h and 48 h) using the alamar blue dye assay with excitation at 565 nm and emission at 580 nm. Normal female nude mice (average weight 19.5 ± 0.8 g) were obtained from Orientbio, Korea for the in vivo studies. All protocols for the in vivo experiments were approved by the UNISTIACUC animal ethical approval committee (document no: IACUC-1622). SCC7 cells (0.1 mL, 2.0 x 107 cells/ 100 µL in 1x PBS) were injected into flank subcutaneously of the nude mice.16 After that, all tumors were observed every day to monitor the sizes by using a digital caliper.


6

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

When the tumor size of the mice grow to 25 mm3, calculated using the formula volume = (tumor length) x (tumor width)2/2, the mice with larger or smaller size were not selected. Four representative mice were analyzed and intravenously injected with saline, PMSN, free-Dox, Dox-PMSN and Dox-BCP representatively, three times per week. The mice treated with saline (100 µL) were used as the control group. The mice treated with Dox-PMSN were injected with a dose of ~ 0.074 mg/mice (100 µL, ~ 0.75 mg/mL) each time, Dox-BCP (100 µL, ~ 10 mg/ 0.5 mL), which is equivalent to a Dox dose of ~ 0.02 mg/animal (100 µL, 0.2 mg/mL). The concentrations used for the groups treated with PMSN were same as that of Dox-PMSN. When saline and nanoparticles injected, the tumor size and body weight were analyzed using the caliper and digital weight. After treatment for 21 days, related mice were sacrificed, and the tumor tissues were removed from the bodies in order to investigate the morphology and use for further studies. Tissues recovered from the necroscopy were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E) for histological examination using standard techniques. After hematoxylin/eosin staining, the slides were observed and photos were taken using an optical microscope. 2.2 Preparation of mesoporous silica nanoparticle Mesoporous silica nanoparticles (raspberry silica) were prepared by using soft template method following the previously reported literature.17 In detail, cetyl-trimethylammonium bromide (CTAB) and triethanolamine were quickly added to the distilled water under stirring at 80 ºC for a period of 1 h. To this surfactant mixture, tetraethyl-orthosilicate (TEOS) was added and stir again for 2 h. The resulting gel mixture composed of a molar composition of 1.0 SiO2: 0.06 CTAB: 0.026 SOA: 80.0 H2O. The resulting precipitates were filtered and washed twice with DI water, and then dried at 60 ºC overnight. Finally, the dried sample was calcined at 550 ºC for 5 h


7


Page 8 of 30

in air. The characterization of synthesized MSN was performed by transmission electron microscopy (TEM), dynamic light scattering measurements (DLS) and nitrogen adsorptiondesorption isotherms. 2.3 Synthesis of PEG-PDS random co-polymer The PEG-PDS random copolymer was synthesized using the RAFT polymerization method (Figure. S1 and S2). In detail, 2-cyano-2-propyl benzodithioate (0.05 mmol), PDSEMA (3.92 mmol), poly(ethylene glycol) methacrylate (0.98 mmol) and AIBN (0.0146 mmol) were mixed together in a round bottom flask containing THF and degassed by purging with Ar under sealed condition. Under constant stirring at 70 °C for 24 h, the samples were collected, precipitated thrice in ice-cold diethyl ether and vacuum dried. The synthesized samples were analyzed by NMR spectroscopy. 1H NMR (600 MHz, CDCl3): δ 8.46, 7.68, 7.11, 4.50-4.01, 3.72-3.58, 3.38, 3.03, 2.15-1.71, 1.13-0.79. GPC (PMMA standard): Mn 9.8 kD, Mw 14.4 kD and PDI 1.47. 2.4 Synthesis of block-copolymer micelle To synthesize mPEG-b-PLA block copolymer, in-presence of mPEG-OH (MW 5 kDa), ring opening polymerization of lactide was performed (Figure. S1, S3 and S4). In detail, poly(ethylene glycol) methyl ether (500 mg, 0.1mmol) and 1,8-diazabicyloundec-7-ene (DBU) (6.7 uL, 1.0 mol% relative to lactide) were dissolved in DCM (1.0 mL). D,L-lactide (648 mg, 4.5 mmol) was dissolved in DCM (3.0 mL) with mild heating. The lactide solution was then added rapidly to the PEG/DBU solution and allowed to stir rapidly for 10 min. The reaction mixture was then quenched by addition of acetone (7.0 mL) and the PEG-b-PLA copolymer was precipitated in cold hexane, collected by decanting supernatant and drying under vacuum to yield a white amorphous polymer. 1H NMR (400 MHz, CDCl3): δ 5.17-5.15, 3.64, 3.38, 1.59-1.57.


8

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

GPC (THF): Mn (PDI) = 7.4 kD (1.06). DiI/DiO at 1wt% were co-loaded into the micelle following the previous reported methods. 18

2.5 Dye/drug loaded MSN preparation About 5 mg of MSN were dispersed well in different proportions of DiI/DiO or DiD/DiR dyes (as shown in Table S1 and S2) were dissolved in 1 mL of DMSO and stir for 24 h at RT. Then the dyes/drugs loaded MSN were collected by centrifugation and dried under high vacuum. Then the vacuum dried samples were redispersed in DI water and centrifuged to remove any DMSO. Then the dyes/drug loaded MSN were again dried using the freeze drier and used for PMSN preparation. The supernatant solution containing unloaded cargo were analyzed under UVVisible spectra using molar absorption coefficient of Dox, DiI, DiO, DiD and DiR are calculated using the following equations, Drug loading capacity (%) = Mass of the drug in MSN / Mass of MSN * 100 Entrapment efficiency (%) = Mass of the drug loaded / Initial mass of the drug * 100 2.6 Synthesis of PMSN & characterization DiI/DiO coloaded and Dox-loaded MSN were capped using the PEG-PDS copolymer following our previous report.19 About 5 mg of dye/drug loaded MSNs were dispersed in 1 mL of DI water containing 10 mg of PEG-PDS polymer and continuously stir for a period of 12 h at room temperature. To crosslink the polymer shell, partial amount of DTT was added (50 mol% against PDS group) and again stir for a period of 3 h at RT to allow in-situ crosslinking. Then the polymer capped cargo loaded MSN were collected by centrifugation, washed thrice with pH 7.4 phosphate buffers (10 mM) and DI water. The supernatant containing the released byproduct


9


Page 10 of 30

(pyridothione) from polymer crosslinking from thiol-disulfide exchange reaction and any removed drugs were measured using UV-Visible spectra. The final concentration of cargo loaded into PMSN were recalculated and shown in Table S1.

2.7 In-vitro encapsulation stability tests Encapsulation stability was measured by Förster resonance energy transfer (FRET) method. The fluorescence spectra of DiI/DiO coloaded MSN, PMSN, BCP micelle at different loading capacity (100 µg/mL) in various solvents (1x PBS, 10% FBS, 20% FBS, 30% FBS, 50% FBS and 100% FBS) were measured with excitation at 450 nm and emission scanning from 460 nm to 700 nm during incubation at room temperature. 2.8 In-vitro drug release profile analysis The release kinetics of Dox loaded micelle and PMSN in phosphate buffered saline (pH 7.4) at 37 °C were analyzed at excitation wavelengths of 480 nm at different time points (0 h to 24 h) using a Shimadzu RFPC 5430 Spectrofluorometer. In order to measure triggered drug release profile in PMSN, 5 mM GSH was added to the release media containing PMSN and analyzed using the excitation at 480 nm and emission from 490 nm to 700 nm. 2.9 In vivo and Ex vivo imaging analysis Female nude mice obtained from the Orientbio, were subcutaneously inoculated with SCC7 cells (0.1 mL, 2.0 x 107 cells/mL in 1x PBS) and when the tumor implants reached 40 mm3 in diameter, the tumor bearing mice were subjected to the in vivo imaging. In vivo fluorescence imaging was performed with an animal imaging system (Bruker in vivo Xtreme, Germany). Images were recorded in DiD channel (630 nm excitation, 690 nm emission). The exposure time was 1s for all time points. Identical standard x-ray settings were used for acquiring all images.


10

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The animal procedures were performed according to a protocol approved by the UNIST-IACUC committee. Three group of mice with SCC7 xenograft tumors were i.v. administered with DiDPMSN (2 mg/100 µL), DiD-BCP (2 mg/100 µL) and saline (100 µL).20 The mice were anesthetized with isoflurane were imaged at 2, 4, 8, 12, 24 and 48 h after injection. 2.10 Statistical analysis Values are expressed as mean ± SE, and statistical comparison between groups were made using the Student’s t-test and a P value of < 0.05, < 0.01, P < 0.001 was considered significant. 3.0 RESULTS AND DISCUSSION 3.1 Preparation and characterization of PMSNs Mesoporous silica nanoparticles (MSNs) with uniquely tunable surface areas and pore modifications can be loaded with drugs using convenient methods,21,22 and have been employed to fabricate responsive drug delivery systems in which cargo-loaded pores are sealed with inorganic nanoparticles, biomacromolecules, and organic molecules. 18,19,23-25, However, covalent surface modifications of MSNs reduces surface areas of the pores and decreases drug loading capacity. Thus, to maintain high drug loading capacity, we used non-covalent gatekeeper modifications to encapsulate large quantities of cargo without the need for multiple chemical modifications of MSN surfaces.18,19 Raspberry-type MSNs were prepared using the Stöber process;26 base-catalyzed silicate co-condensation reactions were performed under atmospheric pressure using established sol–gel techniques with cetyl-trimethylammonium bromide as a templating surfactant and small organic amines (SOAs) as mineralizing agents. The resulting raspberry-type MSNs were characterized using transmission electron microscopy (TEM; Figure. 2a, approximately 50 nm in size) and nitrogen adsorption analyses showed that the MSNs had large Brunauer–Emmett–Teller (BET) surface areas of 353 m2/g and dual pore sizes of 1.41 and


11


Page 12 of 30

1.7 nm (Figure. S5). Hydrophobic drug or dye molecules were loaded into pores of MSNs and polymer gatekeepers were then installed to stabilize cargos encapsulation via disulfide crosslinking reactions on polymer shells (Figure. 1a).19 In subsequent UV-Vis spectrometric analyzes, crosslinking densities were determined according to the release of the byproduct pyridothione, and found that pyridyl disulfide (PDS) consumption occurred at 79 mol% after the addition of substoichiometric quantities of 50 mol% dithiothreitol (DTT) (Figure. S6). Introduction of DTT at a certain concentrations leads to cleavage of PDS groups in proportion to free thiol groups, which then react with the remaining PDS to form disulfide bonds. This rapid intra/intermolecular disulfide exchange reaction forms disulfide bonds without metal-containing catalysts or solvents, and produces cross-linked polymer shells that stably hold drug cargo. TEM analyses (Figure. 2a and 2b) show clear differences in the porosity of MSNs and PMSNs, with notably less visible pores on PMSNs than on MSNs, indicating the presence of a polymer wrapping. Moreover, dynamic light scattering (DLS) experiments showed MSNs sizes of 100–140 nm after installation of the polymer gatekeeper (Figure. S6), with highly negative surface charges (−40 mV) that were neutralized (−3 mV) after introduction of the polymer gatekeeper (Figure. S6). Encapsulation of hydrophobic dyes in PMSNs at loading capacities of 6–24 wt% produced colloidal solutions (Figure. S7) of 140–160 nm with good colloidal stability at pH 7.4 in phosphate buffered saline (PBS), at pH 5.5 in sodium acetate buffer, and in serum-containing RPMI 1640 cell culture medium. No adverse changes in sizes or aggregation were noted at 72 h, indicating long-term colloidal stability in biologically-relevant fluids. Thermogravimetric analysis (TGA) were used to check the weight loss of organics in presence and absence of Dox in PMSNs. About 25 % of weight loss in absence of Dox and 35 % weight loss appeared in presence of Dox loading (Figure S8).


12

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 2. Characterization of synthesized a) mesoporous silica nanoparticles (MSNs) and b) non-covalent polymer gatekeeper nanoparticles (PMSNs) by TEM; scale bars show 50 and 20 nm, respectively. c) Fluorescent images of DiO/DiI-coloaded PMSNs at loading capacities of


13


Page 14 of 30

about 6, 13, and 24 wt %); d) schematic of Förster resonance energy transfer (FRET) experiments using DiO (FRET donor) and DiI (FRET acceptor); time-resolved FRET spectra of dye-loaded BcP micelles in e) 10%, f) 50%, and g) 100 % serum solutions at room temperature. FRET dye-loaded PMSNs in h) 10%, i) 50%, and j) 100% serum solutions at room temperature. Fluorescent intensities of DiI and DiO were determined at 570 and 510 nm, respectively.

3.2 In vitro FRET analyses of encapsulation stability and intracellular release To compare encapsulation stabilities, we performed Förster resonance energy transfer (FRET) experiments and compared FRET behaviors of PMSNs with those of monomethoxy poly(ethylene glycol)–block-poly(D,L-lactide) block copolymer (mPEG-PDLLA, BcP) micelles as a negative control (Figure. S6d and S6f). Forster resonance energy transfer (FRET) based method was adapted to check the stability of the nanoparticles. The FRET donor and acceptor pair 3,3’-dioactadecyloxacarbocyanine perchlorate [DiO] and 1,1’-dioctadecyl-3,3,3’,3’tetramethylindocarbocyanine perchlorate [DiI] were loaded into PMSNs and BcP micelles (110 nm and -10 mV), and experiments were conduced according to the FRET behaviors of the DiI/DiO co-loaded nanoparticles, as shown in the schematic in Figure. 2d. When the pair of FRET dyes are localized inside the nanoparticles, the fluorescence intensity of the donor at 508 nm is lower due to the closer proximity of acceptor. However, when the two dyes are released, the fluorescence intensity of the donor at 508 nm tend to increase, as a result of loss in the energy transfer between donor and acceptor. The DiO and DiI (1 wt% each) encapsulated BcP micelles were used to check for their stability in 10%, 50%, and 100 % serum solutions at room temperature (Figure. 2e-g). Fluorescence intensities at 508 nm increased with serum concentrations, whereas those at 570 nm decreased with time. These observations indicate that the FRET pair was released from micelles when incubated with serum, suggesting destabilization


14

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

of micelles in whole serum. In contrast, no time dependent changes in FRET intensities were observed in experiments with PMSNs, indicating stable encapsulation even at higher loading capacities (24 wt%) and high serum concentrations (Figure. 2h-j and S9). These results demonstrate remarkable encapsulation stability of PMSNs containing much (24-fold) higher loads than BcP micelles, even in the presence of the highest concentration of serum proteins. The data presented above indicate that cross-linked polymer shells prevent drug leakage from the cores of PMSNs. Drug leakage from the BcP micelles (Figure. 3) is caused by changes in micelle dissociation–association equilibria, transient mixing during collisions of BcP micelles, and drug diffusion into bulk solution.6 The present cross-linked polymer gatekeeper corona prevented dissociation of the polymer shell and diffusion of drug molecules into bulk solution, thus abolishing the effects of dissociation-association equilibria and drug diffusion. In addition, mixing of hydrophobic dye molecules during collisions of PMSNs was inhibited by their presence in pores of rigid inorganic MSNs that were wrapped in polymer. These flexible corona may also dissipate the physical energy generated from collisions and can not affect the rigid silica core, leading to higher encapsulation stabilities and loading capacities than those observed previously in BcP micelles and cross-linked nanogel systems.


15


Figure

3.

Schematic

of

drug

leakage

mechanisms

Page 16 of 30

between

BcP-micelles:

a)

assembly/disassembly-based drug leakage mechanism; b) collision-exchange separation mechanism (collision-based mechanism), and c) the exit/re-entry mechanism (diffusion-based mechanism) d) stable encapsulation of drugs in PMSNs. In further experiments, we generated release profiles of PMSN-encapsulated molecules in cervical cancer HeLa cells (Figure. 4). High glutathione (GSH) concentrations in cancer cells can cleave the disulfide bonds of the present polymer gatekeeper, allowing release of cargo inside the cells. Accordingly, we observed decreased FRET intensities over 8 h incubation with cells, and FRET dyes were released more rapidly from BCP micelles than from PMSNs. Specifically, FRET donor emissions (DiO, green emission) was very quickly appeared for BcP micelles with 2 wt% loading in the cell membrane before 4 h, indicating rapid transfer of the dye into cell membranes before entry of micelles into cells. In agreement, core-loaded hydrophobic dyes were reportedly released from micelles in extracellular spaces prior to internalization by cells.10 In contrast, after loading to 6, 13, and 24 wt%, dissapearance of FRET emmisions from PMSNs was time-dependent. During the initial 4 h, emmisions were only observed from the FRET acceptor (DiI, red channel), indicating that the two-dye molecules remained in PMSN pores after


16

Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

internalization by cells. Subsequently, donor (DiO, green channel) emissions appeared after 8-h incubation, indicating that the dyes were released from PMSNs into the cytosol. In addition, FRET emmision profiles in PMSNs were prolonged in varying serum solutions, further indicating stable encapsulation.

Figure 4. Encapsulation stability analyses of DiI/DiO co-loaded BcP micelles and PMSNs at varying loading capacities in HeLa cells after 4- and 8-h incubation; scale bar shows 5 µm. 3.3 Release profile and cellular uptake analyses To determine drug encapsulation, release, and cellular uptake we performed experiments with doxorubicin (Dox)-loaded BcP micelles and PMSNs. Dox encapsulation capacity was 31 wt% in PMSNs (Table S1) and was 2 wt% in BcP micelles, and DOX release profiles were observed using fluorescence analyses under physiological conditions (1 × PBS) at 37°C. In accordance with our stability studies (Figure. 2), Dox was released from BcP micelles much more rapidly from PMSNs, despite the amount of Dox loaded in the BcP micelles being much less than that


17


Page 18 of 30

loaded in the PMSNs (Figure. 5a). Athough PMSNs did not release Dox molecules until 24 h after the start of experiments (Figure. 5b and Figure S8), release rates were significantly increased in the presence of 5-mM GSH (Figure. 5c). To assess nanocarrier biocompatibility, cell viability was evaluated using alamar blue dye assays in SCC7 squamous cell carcinoma cells incubated with varying concentrations of MSNs and PMSNs (up to 200 µg/mL). These experiments showed no significant cell death in the presence of PMSNs (Figure. 6a), and SCC7 cell viabilities after 48-h exposures to Dox-loaded BcP micelles or Dox-loaded PMSNs were slightly lower than those in the presence of equal concentrations of free Dox (Figure. 6b). These data suggest that drug molecules are released slowly into the cytosol following diffusion through silica particle nanopores. In contrast, the low toxicity observed for self-assembled BcP micelles may be attributable to poor encapsulation.


18

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 5. Real time in vitro release profile analyses of a) Dox-BcP, b) Dox-PMSNs, and c) DoxPMSNs in the presence of 5 mM GSH. 3.4 In vivo tumor targeting To confirm that the present improvements in encapsulation efficiency of PMSNs result in greater in vivo anticancer activities, we assessed tumor targeting in nude mice bearing subcutaneous 40mm3 SCC7 cell tumors on their right shoulders. Xenograft mice were intravenously administered PMSNs containing the DiI analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-


19

Biomacromolecules

Chlorobenzenesulfonate Salt (DiD 100 µL, 1 mg/mice), or DiD-BcP micelles (5 mg/mice), and in situ particle distributions inside mice were assessed. Because BcP micelles have lower loading capacity than PMSNs, we injected 5 times more DiD-BcP than DiD-PMSNs into mice to maintain the same amount of dye between experiments. PMSN MSN

Cell Viability (%)

a 100 80 60 40 20 0 Ctrl

10

25

50

75

100

150

200

Concentration (µ µg/mL) Free-Dox Dox-PMSN Dox-BcP

b 100

Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

80 60 40 20 0 Ctrl 0.01 0.025 0.05 0.125 0.5

1

2

Dox Concentration (µ µg/mL)

Figure 6. a) Cell viability analyses of MSNs and PMSNs; b) SCC7 cell viabilities in the presence of Dox-PMSNs and Dox loaded BcP-micelles were compared with those in the presence of free-doxorubicin (Dox) after 48-h incubation. Images were then generated using whole-body fluorescent imaging analyses with an in vivo imaging system (Bruker Xtreme) at excitation and emmision wavelengths of 630 and 690 nm, respectively. These experiments showed signficant increases in DiD-PMSNs in tumor tissues during the first 12 h (Figure. 7a and S10), and comparatively minimal DiD-BcP micelle accumulation without clear tumor contrast, corresponding with relative encapsulation stabilities of PMSNs and BcP micelles. Moreover, tumor accumulation of PMSNs was confirmed in ex


20

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

vivo studies of excised organs at 48-h post-injection (Figure. 7b and S10). Specifically, fluorescent signals from PMSNs and BcP micelles were primarily observed in the liver, where DiD is metabolized by Kupffer cells prior to excretion via kidneys. The present lung signals may reflect mechanical retention of larger-sized nanoparticles in lung capillaries. Accordingly, previous studies of nanomaterial uptake into the reticuloendothelial system (RES) show that 10– 150-nm micelles are distributed in the liver, lung, and spleen,27 and these observations correspond with the present distributions of PMSNs.

Figure 7. a) Representative whole-body fluorescent images of subcutaneous SCC7 tumor-


21


Page 22 of 30

bearing mice after intravenous injections with nanoparticles; tumors are indicated by yellow dashed circles. b) Fluorescent intensities of heart, liver, tumor, lung, kidney, and spleen tissues are shown after 48-h treatments, n = 3 mice/group, mean ± SD, statistical significance was calculated by Student’s t-test, ***P < 0.001. 3.5 In vivo tumor adaptability of nanoparticles Challenges associated with targeted drug delivery of cancer chemotherapies include low delivery efficiency, often due to premature drug leakage in the circulation. Accordingly, nanocarriers that lack stability under physiological conditions fail to improve the efficiency of tumor treatments. However, consistent with our observations,

disulfide cross-linked poly(ethylene glycol)-b-

poly(L-lysine)-b-poly(L-phenylalanine), methoxypoly(ethylene glycol)-(cysteine)4-poly(D,Llactic acid) micelles with enhanced stability, reportedly prolonged circulation times and had improved encapsulation efficiency.14, 17 Insufficient loading capacity is another major obstacle to the clinical use of nanocarriers, and lowers the efficacy of tumor treatments. The present PMSNs physically entrapped hydrophobic Dox in MSNs pores with high loading capacity. Moreover, subsequent capping with disulfide functionalized polymer gatekeepers allowed spatiotemporal control of Dox release without additional chemical conjugation, and the cytotoxic properties of the anticancer drug were maintained in vivo. To assess the therapeutic efficacy of Dox-PMSNs, we generated subcutaneous SCC7 tumor xenografts in nude mice (19–20 g; Figure. 8) and allowed them to grow for one week (Figure. 8a). Body weights and tumor volumes were similar in all treatment groups, and xenograft bearing mice weights increased from 19.4 ± 0.5 g (Figure. 8b) to 22.4 ± 0.4 g. In comparisons of saline, PMSNs, free-Dox, Dox-PMSNs, and Dox-BcP treatments, tumor growth inhibition was greater in the Dox-PMSNs treatment group than in pure Dox and Dox-BcP groups (Figure. 8c, d, f), as confirmed by final tumor weights (Figure. 8e). Moreover, quantitative analysis showed increasing tumor sizes during treatments with saline, or


22

Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

PMSNs, and moderate growth inhibition in free-Dox and Dox-BcP groups. These data suggest that i) free-Dox has poor access to tumor sites in vivo, as shown previously, and ii) that after injection, Dox-BcP micelles are rapidly distributed among mouse organs and destabilized, thus limiting reductions in tumor size.

Figure 8. Enhanced antitumor effects of doxorubicin (Dox) encapsulated in non-covalent polymer gatekeeper mesoporous silica nanoparticles (PMSNs); a) schematic of 4-day dosage patterns over 21 days, b) body weight analyses, c) SCC7 tumor sizes. Dox-HCl (2 mg/kg), Dox loaded BcP, Dox-PMSNs (2-mg/kg Dox in BcP and PMSNs), and PMSNs (equivalent to DoxPMSNs) were intravenously injected at day 0, and then repeatedly at 4-day intervals. Data are expressed as means ± standard errors of the mean (SEM; n = 4). d) The image shows tumor sizes in treatment groups after 21-days treatment. Dotted circles indicate SCC7 solid tumors. e) Tumor weight analysis and f) tumors after 21-days treatment. n = 4 mice/group, mean ± SD, statistical significance was calculated by Student’s t-test, *P < 0.05.


23


Page 24 of 30

* P < 0.05, statistically significant when compared to control saline groups. Finally, we performed histological evaluations of tumor tissues using hematoxylin and eosin (H&E) staining. H&E-stained lung, liver, spleen, and kidney samples showed no apparent abnormalities or lesions at day 21 of Dox-PMSNs treatments (Figure. S11). Toxic side effects remain a common limitation of most clinically used anticancer drugs, such as Dox. The present simple and robust intracellular redox-responsive drug delivery system offers a solution to this limitation, with targetted efficacy against tumors. After intravenous injections into mouse tumors, Dox-PMSNs selectively passed through vascular walls via EPR effects, which reflect leaky vasculature and dysfunctional lymphatic drainage near tumor sites. Dox-PMSNs then accumulated around tumor tissues and the drug was specifically internalized into tumor cells in vivo. Subsequently, Dox was released from PMSNs into the cytoplasm, presumably by GSH mediated reduction of our disulfide linked polymer. Accordingly, Dox-PMSNs increased apoptosis of tumor cells and exhibited stable encapsulation, therapeutic efficacy, and reduced side effects. Taken together, these data warrant further investigations of PMSNs as efficient redox-responsive carriers for controlled drug delivery to tumors. 4. CONCLUSIONS The present experiments show high encapsulation properties of MSNs coated in a noncovalent disufide bridged polymer, compared with those of self-assembled BcP micelles. We also showed that the FRET-based dye pair was retained in MSN pores by the non-covalently cross-linked polymer shell, and unmodified pores likely held large quantities of drug until reaching target cells. After physically loading hydrophobic FRET probes into BcP micelle cores and MSNs pores, application of polymer installation allowed spatiotemporal monitoring of


24

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

nanocarrier stability under various serum concentrations. Extrinsic environmental factors such as high serum concentrations did not impair PMSNs encapsulation, and these in vitro studies confirmed the high stability of PMSNs and their structural responses to tumor microenvironments. Subsequent mouse experiments with subcutaneous zenograft tumors showed that dye-loaded PMSNs reach the tumor site via the EPR effect and accumulate at higher concentrations than BcP micelles. Finally, significant improvements in long-term retention of Dox in tumor tissues was demonstrated, and led to enhanced antitumor activity. These findings indicate broad potential biomedical applications of PMSNs. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Results for the dye and drug loading capacity analysis for PMSNs, characterization results of the crosslinking density, size analysis and zeta potential measurements, FRET stability analysis of PMSNs, in-vivo fluorescent images of RES organs of mice treated with dye loaded BcP and PMSNs and histopathological analysis of tumor, liver, lung, spleen and kidney sections collected from mice after 21 days post treatment. AUTHOR INFORMATION Corresponding Author * Corresponding to Ja-Hyoung Ryu, email: [email protected] ACKNOWLEDGMENT This study was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science (2016-


25


Page 26 of 30

R1A5A1009405, 2017-R1A2B4003617, 2016-R1C1B1011372). We thank Sungmin Lee for helping in nitrogen adsorption desorption isotherm analysis.

REFERENCES [1] Shi, J.; Kantoff, P.W.; Wooster, R.; Farokhzad, O.C. Cancer nanomedicine: progress, challenges and opportunities, Nat. Rev. Cancer. 2017, 17, 20-37. [2] McNeil, S.E. Evaluation of nanomedicines: stick to the basics, Nat. Rev. Mater. 2016, 1, 16073. [3] Webber, M.J.; E.A. Appel, E.W. Meijer, R. Langer, Supramolecular biomaterials, Nat. Mater. 2016, 15, 13-26. [4] Cabral, H.; Nishiyama, N.; Kataoka, K. Supramolecular Nanodevices: From Design Validation to Theranostic Nanomedicine, Acc. Chem. Res. 2011, 44, 999-1008. [5] Liu, S.; Maheshwari, R.; Kiick, K.L. Polymer-Based Therapeutics, Macromolecules 2009, 42, 3-13. [6] Li, L.; Thayumanavan, S. Environment-Dependent Guest Exchange in Supramolecular Hosts, Langmuir 2014, 30, 12384-12390 [7] M. Elsabahy, K. L. Wooley, Design of polymeric nanoparticles for biomedical delivery applications, Chem. Soc. Rev. 41 (2012) 2545-2561. [8] Wilhelm, S.; Tavares, A.J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H.F.; Chan, W.C.W. Analysis of nanoparticle delivery to tumours, Nat. Rev. Mat. 2016, 1, 16014.. [9] Chacko, R.; Ventura, J.; Zhuang, J.; Thayumanavan, S. Polymer Nanogels: A Versatile Nanoscopic Drug Delivery Platform Adv. Drug Deliv. Rev. 2012, 64, 836-851.


26

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

[10] Chen, H.; Kim, S.; Li, L.; Wang, S.; Park, K.; Cheng, J.-X. Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging, Proc. Nat. Acad. Sci. 2008, 105, 6596-6601. [11] Ryu, J.-H.; Chacko, R.T.; Jiwpanich, S.; Bickerton, S.; Babu, R.P.; Thayumanavan, S. Selfcrosslinked polymer nanogels: a novel platform for intracellular delivery vehicles, J. Am. Chem. Soc. 2010, 132, 17227-17235. [12] Liu, B.; Thayumanavan, S. Substituent Effects on the pH Sensitivity of Acetals and Ketals and Their Correlation with Encapsulation Stability in Polymeric Nanogels, J. Am. Chem. Soc. 2017, 139, 2306-2317. [13] Jiwpanich, S.; Ryu, J.-H.; Bickerton, S. Thayumanavan, S. Noncovalent Encapsulation Stabilities in Supramolecular Nanoassemblies, J. Am. Chem. Soc. 2010, 132, 10683-10685. [14] Lee, S.-Y.; Kim, S.; Tyler, J.Y.; Park, K.; Cheng, J.-X. Blood-stable, tumor-adaptable disulfide bonded mPEG-(Cys)4-PDLLA micelles for chemotherapy, Biomaterials 2013, 34, 552-561. [15] Lee, S.Y.; Tyler, J.Y.; Kim, S.; Park, K.; Cheng, J.X. FRET imaging reveals different cellular entry routes of self-assembled and disulfide bonded polymeric micelles, Mol. Pharm. 2013, 10, 3497–3506. [16] Lu, J.; Liong, M.; Zink, J. I.; Tamanoi, F., Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs. Small 2007, 3, 1341-1346. [17] Argyo, C.; Weiss, V.; Bräuchle, C.; Bein, T., Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. Chem. Mater. 2014, 26, 435-451.8. [18] Palanikumar, L.; Choi, E.S.; Cheon, J.Y.; Joo, S.H.; Ryu, J.-H. Noncovalent PolymerGatekeeper in Mesoporous Silica Nanoparticles as a Targeted Drug Delivery Platform, Adv.


27


Page 28 of 30

Funct. Mat. 2015, 25, 957-965. [19] Palanikumar, L.; Kim, H.Y.; Oh, J.Y.; Thomas, A.P.; Choi, E.S.; Jeena, M.T.; Joo, S.H.; Ryu, J.-H. Noncovalent Surface Locking of Mesoporous Silica Nanoparticles for Exceptionally High Hydrophobic Drug Loading and Enhanced Colloidal Stability, Biomacromolecules 2015, 16, 2701-2714. [20] Luo, Z.; Ding, X.; Hu, Y.; Wu, S.; Xiang, Y.; Zeng, Y.; Zhang, B.; Yan, H.; Zhang, H.; Zhu, L.; Liu, J.; Li, J.; Cai, K.; Zhao, Y. L. Engineering a hollow nanocontainer platform with multifunctional molecular machines for tumor-targeted therapy in vitro and in vivo. ACS Nano 2013, 7, 10271-10284. [21] Zhao, Y.-L.; Li, Z.; Kabehie, S.; Botros, Y.Y.; Stoddart, J.F.; Zink, J.I. pH-Operated Nanopistons on the Surfaces of Mesoporous Silica Nanoparticles, J. Am. Chem. Soc. 2010, 132, 13016-13025. [22] Thomas, C.R.; Ferris, D.P.; Lee, J.-H.; Choi, E.; Cho, M.H.; Kim, E.S.; Stoddart, J.F.; Shin, J.-S.; Cheon, J.; Zink, J.I. Noninvasive Remote-Controlled Release of Drug Molecules in Vitro Using Magnetic Actuation of Mechanized Nanoparticles, J. Am.Chem. Soc. 2010, 132, 10623-10625. [23] Zhang, K.; Xu, L.-L.; Jiang, J.-G.; Calin, N.; Lam, K.-F.; Zhang, S.-J.; Wu, H.-H.; Wu, G.D.; Albela, B.; Bonneviot, L.; Wu, P. Facile Large-Scale Synthesis of Monodisperse Mesoporous Silica Nanospheres with Tunable Pore Structure, J. Am. Chem. Soc. 2013, 135, 2427-2430. [24] Bickerton, S.; Jiwpanich, S.; Thayumanavan, S. Interconnected Roles of Scaffold Hydrophobicity, Drug Loading, and Encapsulation Stability in Polymeric Nanocarriers, Mol. Pharm.2012, 9, 3569-3578.


28

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

[25] Erogbogbo, F.; Yong, K.-T.; Roy, I.; Hu, R.; Law, W.-C.; Zhao, W.; Ding, H.; Wu, F.; Kumar, R.; Swihart, M.T.; Prasad, P.N. In Vivo Targeted Cancer Imaging, Sentinel Lymph Node Mapping and Multi-Channel Imaging with Biocompatible Silicon Nanocrystals, ACS Nano 2011, 5, 413-423. [26] Zhang, K.; Xu, L.L; Jiang, J.G.; Calin, N.; Lam, K.F.; Zhang, S-J.; Wu, H-H.; Wu, G-D.; Albela, B.; Bonneviot, L.; Wu, P. Facile Large-Scale Synthesis of Monodisperse Mesoporous Silica Nanospheres with Tunable Pore Structure. [27] Erogbogbo, F.; Yong, K.-T.; Roy, I.; Hu, R.; Law, W.-C.; Zhao, W.; Ding, H.; Wu, F.; Kumar, R.; Swihart, M.T.; Prasad, P.N. In Vivo Targeted Cancer Imaging, Sentinel Lymph Node Mapping and Multi-Channel Imaging with Biocompatible Silicon Nanocrystals, ACS Nano 2011, 5, 413-423.


29


Page 30 of 30

Table of Contents Graphic


30

The Importance of Encapsulation Stability of Nanocarriers with High

Recommend Documents