Functionalized Mesoporous Silica Nanoparticles with Mucoadhesive

Article pubs.acs.org/Langmuir

Functionalized Mesoporous Silica Nanoparticles with Mucoadhesive and Sustained Drug Release Properties for Potential Bladder Cancer Therapy Quan Zhang,†,§ Koon Gee Neoh,*,† Liqun Xu,† Shengjie Lu,† En Tang Kang,† Ratha Mahendran,‡ and Edmund Chiong‡ †

Department of Chemical and Biomolecular Engineering and ‡Department of Surgery, National University of Singapore, Kent Ridge, Singapore 119077 § The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China S Supporting Information *

ABSTRACT: The synthesis of a series of β-cyclodextrin modified mesoporous silica nanoparticles with hydroxyl, amino, and thiol groups was described. A comparison of their mucoadhesive properties and potential as a drug delivery system for superficial bladder cancer therapy was made. The thiol-functionalized nanoparticles exhibit significantly higher mucoadhesivity on the urothelium as compared to the hydroxyl- and amino-functionalized nanoparticles. This is attributed to the formation of disulfide bonds between the thiol-functionalized nanoparticles and cysteine-rich subdomains of mucus glycoproteins. An anticancer drug, doxorubicin, was loaded into the mesopores of the thiol-functionalized nanoparticles, and sustained drug release triggered by acidic pH was achieved. The present study demonstrates that thiol-functionalized mesoporous silica nanoparticles are promising as a mucoadhesive and sustained drug delivery system for superficial bladder cancer therapy.

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INTRODUCTION

Various nanocarrier platforms, including liposomes, polymeric nanoparticles, protein nanoparticles, and dendrimers, have been developed as controlled drug release systems to increase drug residence time.9−15 These nanocarriers are generally prepared from hydrophilic polymers with abundant functional groups such as hydroxyl, carboxyl, amine, or sulfydryl that promote adhesion to the mucosal surface by means of hydrogen bonds or disulfide bridges.3 For example, chitosan’s well-known bioadhesive nature is due to the electrostatic interactions of their amino groups with negatively charged mucin in the mucus layer.16,17 Furthermore, thiolated chitosans were shown to possess further improved mucoadhesive properties by forming disulfide bonds with cysteine-rich domains of mucus glycoproteins. These covalent bonds have been confirmed to be stronger than noncovalent interactions such as hydrogen bonds, van der Waals forces, and ionic interactions of chitosans with anionic substructures of the mucus layer.18,19 Recently, Irmukhametova et al. synthesized thiolated silica nanoparticles from self-condensation of 3mercaptopropyltrimetoxysilane in dimethyl sulfoxide.20 These

Bladder cancer is one of the most commonly diagnosed malignancies and has the ninth highest incidence rate worldwide.1 The great majority of cases (70−80%) are superficial or nonmuscle-invasive carcinomas, where the tumor is confined in the urothelial lining.2,3 The current treatment of patients with superficial bladder cancer, known as transurethral resection, involves the surgical removal of tumor nodules from the bladder wall. However, there is a high rate of tumor recurrence and progression after this surgical procedure.4,5 Intravesical chemotherapy, which involves instillation of one or more chemotherapeutic agents through a catheter into the bladder following resection of the bladder tumors, has been advocated as a means to destroy residual microscopic tumor cells and to prevent reimplantation.6 Unfortunately, the major limitation of this treatment is the rapid and almost complete washout of the drugs from the bladder on first voiding of urine and the low exposure of the tumor sites to the chemotherapeutic agents.7,8 Hence, there is a need to develop mucoadhesive and sustained drug delivery systems, which can prolong the dwell time of the drug, increase drug uptake into bladder tissue, and thereby increase the efficacy of the drug treatment. © 2014 American Chemical Society

Received: September 27, 2013 Revised: May 5, 2014 Published: May 13, 2014 6151

dx.doi.org/10.1021/la500746e | Langmuir 2014, 30, 6151−6161

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Figure 1. Synthetic routes of MSNPs-CD-NH2, MSNPs-CD-OH, and MSNPs-CD-(NH2)-SH.

amidation, and the protecting groups of cysteine were removed to yield the thiol-functionalized MSNPs (MSNPs-CD-(NH2)SH). Finally, an anticancer drug, doxorubicin, was loaded into the mesopores of MSNPs-CD-(NH2)-SH, and the mucoadhesive and sustained drug release capacities of the resulting doxorubicin-loaded MSNPs-CD-(NH2)-SH were investigated.

nanoparticles showed excellent mucoadhesivity and retention on ocular surfaces. However, they were nonporous and nonswellable,15 and hence their potential as a drug carrier will be limited. Mesoporous silica nanoparticles (MSNPs) have attracted much attention for their potential use as drug delivery carriers.21−26 The unique properties of MSNPs, such as tunable nanoparticle size, uniform mesopores, porous interior amendable to drug loading, high surface area, and easily functionalizable surface, make them highly suitable as a therapeutic delivery vehicle.27−29 MSNPs-based controlled-release systems have been synthesized using different kinds of capping agents such as inorganic nanoparticles,30−32 polymer,33,34 nucleotides,35,36 antibody,37 and supramolecular assemblies.38−40 Different stimuli, such as pH,41−45 light,46,47 redox effect,48−50 temperature,51 and enzymatic action,52−55 have been applied as “triggers” for uncapping the pores and releasing the cargos from MSNPs. Encouraged by these achievements, we postulate that the introduction of thiol groups onto MSNP-based controlledrelease systems would promote the adhesion of the MSNPs to the mucous membrane of the urothelium through the formation of disulfide bonds and provide sustained release of the loaded anticancer drugs during intravesical chemotherapy. To the best of our knowledge, no study has reported on the use of MSNPs as mucoadhesive and sustained drug delivery carriers for bladder cancer therapy. Herein, we describe the design and synthesis of functionalized MSNPs as mucoadhesive and sustained drug release systems for superficial bladder cancer therapy. The structure and synthesis routes of functionalized MSNPs are illustrated in Figure 1. The surface of MSNPs was first functionalized with isocyanate groups by reacting MSNPs with 3-isocyanatopropyltriethoxysilane to form isocyanate-functionalized MSNPs (MSNPs-NCO), which were respectively reacted with perdiamino-β-cyclodextrin (β-CD-(NH2)7) and monodiamino-βcyclodextrin (β-CD-NH2) to yield the β-CD-(NH2)7 modified MSNPs (MSNPs-CD-NH2) and the β-CD-NH2-modified MSNPs (MSNPs-CD-OH). The MSNPs-CD-NH2 was further reacted with N-(tert-butoxycarbonyl)-S-trityl-L-cysteine via

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EXPERIMENTAL SECTION

Materials. (3-Aminopropyl)triethoxysilane (APTES), cetyltrimethylammonium bromide (CTAB), β-cyclodextrin (β-CD), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin hydrochloride (Dox·HCl), fluorescein isothiocyanate (FITC), 3-isocyanatopropyltriethoxysilane, N-(tert-butoxycarbonyl)-S-trityl-Lcysteine (Boc-Cys(Trt)-OH), tetraethyl orthosilicate (TEOS), triethanolamine (TEA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich and used without further purifications. Mucin (from porcine stomach, type III, bound sialic acid 0.5−1.5%, partially purified powder) was obtained from Sigma-Aldrich. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and LysoTracker probe (Red DND-99) were purchased from Invitrogen (Carlsbad, CA). All other chemicals and solvents were purchased from commercial sources and used without further purification. Characterization. Nuclear magnetic resonance (NMR) spectroscopy was used to verify the chemical structure of the prepared samples. 1 H NMR spectra were obtained using a Bruker spectrometer operated at 400 MHz, while an ECA400 NMR spectrometer (JEOL, 400 MHz) operated with a 4 mm CD/MAS probe at room temperature was used for the collection of solid-state spectra. For transmission electron microscopy (TEM) analysis, a drop of the nanoparticle aqueous suspension was placed on a 200-mesh carbon-coated copper grid, and air-dried. The analysis was carried out using a JEOL 2100F transmission electron microscope (Tokyo, Japan) operated at 200 kV. For Fourier transform infrared (FT-IR) analysis, the samples were dispersed in KBr pellets, and the spectra were obtained with a FT-IR spectrophotometer (PerkinElmer 1760X) in transmission mode. UV− vis absorption and fluorescence measurements were performed using a Shimadzu UV3600 spectrometer and a Cary Eclipse spectrophotometer, respectively. The amount of thiol groups immobilized on the nanoparticles was determined spectrophotometrically using Ellman’s reagent as described in an earlier publication.20 L-Cysteine hydrochloride was employed to establish a calibration curve for the determination of the thiol concentration. Nitrogen adsorption/ 6152


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desorption isotherms of the nanoparticles (after degassing at 100 °C for 12 h) were obtained as described in an earlier publication.45 The specific surface area and pore size of the nanoparticles were then determined using the Brunauer−Emmett−Teller (BET) model and the Barrett−Joyner−Halenda (BJH) method, respectively. X-ray diffraction (XRD) measurements were performed using an ARL X’TRA powder diffractometer with Cu Kα radiation (λ = 1.540 562 Å). The zeta potential and hydrodynamic size of the nanoparticles in aqueous medium were determined at 37 °C using a Malvern Zetasizer NanoZS instrument. The mean hydrodynamic size was calculated from dynamic light scattering (DLS) using the Zetasizer software. A minimum of three measurements were made at each condition, and the results were reported as average ± standard derivation (SD). Synthesis of β-CD-NH2 and β-CD-(NH2)7. The synthesis procedures are described in the Supporting Information. Synthesis of MSNPs and FITC-Labeled MSNPs. CTAB (0.57 g) and TEA (50 mg) were added to distilled water (22 mL). The reaction mixture was heated to 95 °C under vigorous stirring. After the temperature was stabilized for 1 h, TEOS (1.5 mL) was added dropwise to the solution, and the mixture was stirred for another hour to form the MSNPs. For the preparation of FITC-labeled MSNPs (denoted as FMSNPs), FITC (2.7 mg) was dissolved in absolute ethanol (1 mL) containing APTES (6 μL), and the solution was gently stirred for 2 h in the dark before adding into the above-mentioned CTAB−TEOS solution. For both MSNPs and FMSNPs, the solid product formed was centrifuged (14 000 rpm, 15 min) and washed extensively with ethanol. To remove the surfactant template (CTAB), the nanoparticles were sonicated three times in a mixture solution of ethanol (15 mL) and HCl (1 mL, 37%). The nanoparticles after surfactant removal were washed extensively with ethanol and dried under reduced pressure. Synthesis of MSNPs-NCO, MSNPs-CD-NH2, and MSNPs-CDOH. MSNPs (100 mg) were suspended in anhydrous toluene (20 mL), and 3-isocyanatopropyltriethoxysilane (1 mL) was added to the solution. The mixture was stirred at room temperature under a N2 atmosphere for 12 h. The nanoparticles were collected by centrifugation and washed with toluene followed by methanol. Finally, the sample was dried under reduced pressure to yield the 3isocyanatopropyl-functionalized MSNPs (MSNPs-NCO). To prepare the MSNPs-CD-NH2, MSNPs-NCO (50 mg) and βCD-(NH2)7 (100 mg) were added to anhydrous dimethylformamide (DMF, 5 mL). The mixture was stirred for 24 h at 60 °C. The nanoparticles were then centrifuged and washed thoroughly with DMF and ethanol, respectively. MSNPs-CD-OH was prepared by simply reacting MSNPs-NCO with β-CD-NH2 using the same steps as described for the formation of MSNPs-CD-NH2. Synthesis of MSNPs-CD-(NH2)-SH. MSNPs-CD-NH2 (20 mg) was dispersed in anhydrous DMF (5 mL). Boc-Cys(Trt)-OH (200 mg, 0.43 mmol) was then added to the mixture in the presence of 2(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt). After the reaction mixture was stirred for 48 h, the resulting nanoparticles were collected by centrifugation and washed extensively with DMF. To remove the Boc and Trt protecting groups, the nanoparticles were further treated with trifluoroacetic acid/thioanisole/ethanedithiol/anisole (90/5/3/ 2). Finally, the nanoparticles were washed extensively with DMF and ethanol and denoted as MSNPs-CD-(NH2)-SH. Drug Loading and Release. MSNPs-CD-(NH2)-SH (5 mg) was added to a phosphate buffered saline (PBS, pH 7.4) solution (1 mL) containing Dox·HCl (2.5 mg mL−1). The mixture was stirred and sonicated to maximize the dispersion of the nanoparticles. After the mixture was stirred in the dark for 24 h, the Dox-loaded nanoparticles were collected by centrifugation and washed extensively with PBS buffer solution. This product is denoted as Dox@MSNPs-CD-(NH2)SH. The amount of Dox loaded into MSNPs-CD-(NH2)-SH was estimated by subtracting the amount of Dox in the collected supernatant from the initial amount of Dox in the loading solution using UV−vis absorption spectroscopy. The loading of Dox into the other types of nanoparticles was carried out in the same manner. To determine the kinetics of Dox release from these nanoparticles, the

Dox-loaded nanoparticles (5 mg) were dispersed in 5 mL of PBS in a dialysis membrane bag (MWCO = 3500), and the bag was immersed in 50 mL of PBS (pH 7.4) or artificial urine (comprising 54 mM NaCl, 30 mM KCl, 15 mM NH4Cl, 3 mM CaCl2, 2 mM MgSO4, 2 mM NaHCO3, 9 mM Na2SO4, 3.6 mM NaH2PO4, 0.4 mM Na2HPO4, and 200 mM urea in DI water, pH 6.1)56 and shaken at 100 rpm at 37 °C. Fluorescence spectroscopy (λex = 485 nm, λem = 590 nm) was used to monitor the amount of Dox released at different time intervals over a period of 48 h. Evaluation of Mucoadhesivity Using the Mucin-Particle Method.57,58 Mucin powder was suspended in 10 mM acetate buffer solution (ABS, pH 4.5) at a concentration of 0.6% (w/v). The mucin suspension was incubated at 37 °C overnight and sonicated at 40 kHz (Branson 2510 ultrasonic bath) for 30 min until the mucin particle size was smaller than 1 μm as indicated by DLS. The mucin suspension was then centrifuged at 4000 rpm for 30 min, and the supernatant was collected. The mean hydrodynamic size of mucin particles in the supernatant was less than 200 nm. Since very little of the mucin was precipitated during centrifugation, it can be assumed that all the mucin remained in the supernatant, which was then was diluted to concentrations of 0.012%, 0.05%, 0.1%, 0.2%, and 0.4% (w/v) with 10 mM ABS, pH 4.5, before use. For evaluation of the mucoadhesivity of the nanoparticles, MSNPs-CD-OH, MSNPs-CD-NH2, and MSNPsCD-(NH2)-SH were dispersed in DI water (0.5 mg mL−1). Each nanoparticle suspension was mixed with an equal volume of the mucin suspension in ABS of different concentrations. After the mixtures were incubated at 37 °C for 30 min, the particle size was determined using the Zetasizer instrument. Bladder Tissue Preparation and Mucoadhesivity Studies. Urinary bladders, freshly excised from 6 to 10 month old pigs, were obtained from a commercial abattoir and transported on ice in Tyrode’s buffer to our laboratory within 1−1.5 h of sacrifice. Excess adipose tissue on the bladder was removed, and the bladder was cut longitudinally into left and right lateral halves and subdivided into pieces of approximately 5 cm × 5 cm. The bladder pieces were incubated with FMSNPs-CD-OH (0.5 mL, 5 mg mL−1), FMSNPsCD-NH2 (0.5 mL, 5 mg mL−1), or FMSNPs-CD-(NH2)-SH (0.5 mL, 5 mg mL−1) in artificial urine at 37 °C for 2 h. The pieces were then rinsed three times with artificial urine, and the bladder wall was observed under a Nikon confocal laser-scanning A1 microscope (10× objective, 488 nm excitation). The mucoadhesivity of Dox-loaded nanoparticles was tested using bladder pieces prepared in a similar fashion as described above. The bladder pieces were divided into two groups. For the first group, the bladder pieces were incubated with either Dox-loaded nanoparticles (0.5 mL, 5 mg mL−1) or free Dox (0.5 mL, 200 μg mL−1) in artificial urine at 37 °C for 2 h. The bladder pieces were then rinsed three times with artificial urine and subjected to confocal microscopy observation. For the second group, the bladder pieces were similarly incubated with either free Dox or Dox-loaded nanoparticles for 2 h, and after rinsing three times with artificial urine, the bladder pieces were allowed to incubate in artificial urine for another 3 h before the final washing with artificial urine and confocal microscopy observation as mentioned above. Cell Culture and Viability Studies. UMUC3 (human urothelial carcinoma) cells, purchased from American Type Culture Collection (ATCC, Manassas, VA), were cultured in DMEM containing 10% FBS, penicillin (100 U mL−1), streptomycin (100 μg mL−1), and Lglutamine (2 mM) in a humidified atmosphere with 5% CO2 at 37 °C. The cytotoxicity of the nanoparticles was evaluated using the MTT assay. UMUC3 cells were seeded into a 96-well plate at a density of 5 × 103 cells per well and maintained in growth medium (100 μL) for 24 h. The culture medium was then replaced with complete DMEM medium (100 μL) containing either free Dox or Dox-loaded nanoparticles at different Dox dosages. After 5 h, the cells were washed three times with PBS, and the medium in each well was replaced with 100 μL of fresh culture medium. After incubation for 72 h, the culture medium from each well was replaced with 100 μL of medium containing MTT solution (0.5 mg mL−1). After another 4 h of incubation, culture supernatants were removed and DMSO (100 6153


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μL) was added into each well. The plate was gently shaken for 15 min to dissolve the formazan crystals and the absorbance at 570 nm was measured using a microplate reader (Tecan GENios, Switzerland). A control experiment was carried out in a similar manner but without Dox or nanoparticles in the medium. The viability of the cells treated with Dox or nanoparticles (relative to the cells in the control experiment) was calculated from ([A]test − [A]0)/([A]control − [A]0), where [A]test, [A]control, and [A]0 are the average absorbance obtained with the test and control samples and culture medium without cells, respectively. GraphPad Prism software (version 5.01) was used to calculate the IC50 values from three independent experiments. Cellular Uptake. UMUC3 bladder cancer cells were seeded in 35 mm plastic-bottomed Ibidi μ-dishes and allowed to grow for 24 h. After incubation with Dox@FMSNPs-CD-(NH2)-SH (25 μg mL−1, loaded Dox of about 5 μg mL−1) or free Dox (5 μg mL−1) for a predetermined period of time, the cells were washed three times with PBS (pH 7.4) and then fixed with 4.0% formaldehyde at room temperature for 15 min. After washing with PBS, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg mL−1) for 15 min.59 After washing with PBS, the cells were subjected to confocal microscopy observation (100× oil objective, 405/488 nm excitation).

diameter of 2.7 nm as calculated from the BET and BJH analysis, respectively (Figure S1 in the Supporting Information). The BET surface area of MSNPs-CD-(NH2)-SH was measured to be 520.2 m2 g−1, which is lower than that of MSNPs, but the average pore diameter remained close to 2.7 nm (Figure S1). From powder XRD analysis, a broad peak at 2θ = 1.55° was observed for both MSNPs and MSNPs-CD(NH2)-SH (Figure S2), indicating that the as-synthesized MSNPs possess wormlike mesoporous structures, consistent with that reported by Möller et al.60 The success of the modification of the MSNPs surface by βCD-(NH2)7 was confirmed by 13C and 29Si cross-polarization magic-angle-spinning (CP-MAS) solid-state NMR spectroscopy. The 13C CP-MAS NMR spectrum (Figure 3a) of MSNPsCD-NH2 exhibits strong signals resonating at 8.7, 13.8, and 42.7 ppm, which are attributed to characteristic peaks of aliphatic carbons on the linker between the β-CD ring and the nanoparticle. Signals resonating between 70 and 110 ppm in the spectrum are attributed to characteristic carbon peaks on the β-CD skeleton, and the signal at 158.6 ppm is assigned to the carbonyl carbon of the formed urea bond. The 29Si CPMAS solid-state NMR spectrum (Figure 3c) of MSNPs-CDNH2 presents two silicon peaks around −60 and −100 ppm, corresponding to the functionalized silica (T region) and bulk silica (Q region), respectively. These observations indicate successful preparation of MSNPs-CD-NH2. The functionalization process was also followed using FT-IR spectroscopy as well as zeta-potential measurements. As shown in Figure 4, the characteristic peak at around 1100 cm−1 attributed to Si−O stretching in mesoporous silica was observed in all samples. Except for the bare MSNPs (Figure 4a), all other samples also have a peak at 2939 cm−1, which is assigned to the C−H stretching in the alkyl chains of functional groups. The appearance of a peak at 2360 cm−1 in the FT-IR spectrum of MSNPs-NCO (Figure 4b) indicates successful grafting of isocyanate groups onto the MSNPs surface. This peak disappeared after β-CD-(NH2)7 was introduced to react with isocyanate groups on the nanoparticle surface, indicating the formation of urea bonds in MSNPs-CD-NH2 (Figure 4c). Further modification of MSNPs-CD-NH2 with Boc-Cys(Trt)OH was confirmed by two new peaks at 705 and 740 cm−1, which are attributed to the benzene C−H bending vibration of Trt groups (Figure 4d). Finally, a new peak at 2560 cm−1 was observed after the removal of Boc and Trt groups with TFA treatment, indicating the formation of thiol groups in MSNPsCD-(NH2)-SH (Figure 4e). In the zeta potential measurements, MSNPs in artificial urine have a negative zeta potential (−20.0 ± 0.9 mV) attributed to the hydroxide groups on the nanoparticle surface. The surface charge was reversed to a positive value (+33.9 ± 0.5 mV) in artificial urine after the formation of MSNPs-CD-NH2 because of the amino groups in MSNPs-CD-NH2 (Figure S3 and Table S1). After the introduction of thiol groups, there was no change in the zeta potential value of MSNPs-CD-(NH2)-SH (+33.5 ± 0.6 mV), further supporting the results from the FT-IR spectra and confirming the success of the functionalization process. The mucoadhesive property of MSNPs-CD-(NH2)-SH was evaluated using the mucin-particle method reported in the literature with minor modifications.57,58 In this method, the change in the size of the particles as a result of their interaction with mucin is monitored. As shown in Figure 5a, the hydrodynamic size of MSNPs-CD-OH after mixing with different concentrations of mucin for 30 min was almost

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RESULTS AND DISCUSSION Preparation and Characterization of MSNPs-CD(NH2)-SH. The MSNPs were synthesized according to a previously reported method with minor modifications.60,61 The particle size and morphology of MSNPs-CD-(NH2)-SH were evaluated using TEM, and the images are shown in Figures 2a

Figure 2. TEM images (a, b) and histogram (c) of MSNPs-CD(NH2)-SH prepared in aqueous solution. (d) TEM images of the unmodified MSNPs.

and 2b. The nanoparticles are spherical in shape with a narrow size distribution. The average particle size calculated from the statistical results shown in Figure 2c is about 75.5 nm with a standard deviation of 3.4 nm. In comparison with unmodified MSNPs (Figure 2d), the mesopore structure on the surface of MSNPs-CD-(NH2)-SH (Figure 2b) cannot be clearly observed because of masking by the β-CD-(NH2)7. N2 adsorption/ desorption measurement shows that the unmodified MSNPs have a specific surface area of 805.0 m2 g−1 and an average pore 6154


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Figure 3. (a) 13C CP-MAS solid-state NMR spectrum of MSNPs-CD-NH2. (b) 13C CP-MAS solid-state NMR spectrum of MSNPs-NCO. (c) 29Si CP-MAS solid-state NMR spectrum of MSNPs-CD-NH2. In (b) the signal at around 50 ppm (marked by #) is attributed to the carbon peak of methanol from the purification process.

have a large number of amino groups on the particle surface, thereby resulting in electrostatic attraction between the positively charged β-CD-(NH2)7 rings and the negatively charged mucin under acidic conditions. In the case of MSNPsCD-(NH2)-SH, the presence of thiol groups on the nanoparticle surface promoted the enhancement of its mucoadhesivity due to the formation of covalent bonds between the thiol groups of MSNPs-CD-(NH2)-SH and the cysteine-rich subdomains of glycoproteins in the mucin.3,18,19 To further evaluate the mucoadhesivity of MSNPs-CD(NH2)-SH on the bladder wall, green emitting fluoresceinlabeled MSNPs-CD-(NH2)-SH (FMSNPs-CD-(NH2)-SH) was synthesized to provide a means for fluorescent tracing.62 The amount of −SH groups immobilized on FMSNPs-CD-(NH2)SH was determined to be 2.2 ± 0.4 μmol g−1. FITC-labeled MSNPs-CD-OH (FMSNPs-CD-OH) and FITC-labeled MSNPs-CD-NH2 (FMSNPs-CD-NH2) were also prepared as control samples. FMSNPs-CD-OH, FMSNPs-CD-NH2, and FMSNPs-CD-(NH2)-SH were individually incubated with pig bladder under the same conditions for 2 h. After washing three times with PBS, the bladder wall was monitored using a confocal laser scanning microscope (CLSM). Weak fluorescence was observed from the bladder wall after the incubation with FMSNPs-CD-OH (Figure 6a). The fluorescence intensity of the bladder wall increased after incubation with FMSNPsCD-NH2 (Figure 6b), and as shown in Figure 6c, very strong green fluorescence was observed after incubation with FMSNPs-CD-(NH 2 )-SH. These results confirmed that FMSNPs-CD-(NH2)-SH has highest mucoadhesivity compared with FMSNPs-CD-NH2 and FMSNPs-CD-OH, which is in line with the results obtained from mucin-particle mucoadhesivity tests (Figure 5). In order to verify the feasibility of FMSNPs-CD-(NH2)-SH acting as sustained drug release systems, Dox was loaded into the mesopores of FMSNPs-CD-(NH2)-SH in PBS (pH 7.4). The TEM image of the Dox-loaded FMSNPs-CD-(NH2)-SH (Dox@FMSNPs-CD-(NH2)-SH) (Figure S5) shows no obvious differences in particle shape and size compared with

Figure 4. FT-IR spectra of (a) bare MSNPs, (b) MSNPs-NCO, (c) MSNPs-CD-NH2, (d) MSNPs-CD-Cys(Trt)-Boc, and (e) MSNPsCD-(NH2)-SH.

unchanged. However, the hydrodynamic size of MSNPs-CDNH2 and MSNPs-CD-(NH2)-SH increased with increasing concentrations of mucin (Figure 5b,c). On the other hand, no significant size change was observed with suspensions of mucin alone after a similar period (Figure S4). The changes in mean hydrodynamic size of particle/mucin mixtures are summarized and plotted in Figure 5d. The results showed that the hydrodynamic size of the MSNPs-CD-(NH2)-SH/mucin mixture at any mucin concentration was higher than that of MSNPs-CD-NH2/mucin or MSNPs-CD-OH/mucin, indicating that the mucoadhesivity of functionalized MSNPs to mucin decreased in the following order: MSNPs-CD-(NH2)-SH > MSNPs-CD-NH2 > MSNPs-CD-OH. These results can be explained by considering the surface characteristics of these MSNPs. Compared to MSNPs-CD-OH, MSNPs-CD-NH2 6155


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Figure 5. Changes in hydrodynamic size of (a) MSNPs-CD-OH (MS1), (b) MSNPs-CD-NH2 (MS2), and (c) MSNPs-CD-(NH2)-SH (MS3) after mixing with different concentrations of mucin dispersed in ABS buffer for 30 min. (d) Effect of mucin concentration on mucin-particle interaction.

release from the nanoparticles was observed at pH 7.4 as compared with 6.1 (Figure S6). This observation is opposite to the release of positively charged Dox. With calcein, the electrostatic attraction between the positively charged β-CD rings and negatively charged calcein under acidic conditions impedes the release of the cargo.63 In a control experiment with Dox@FMSNPs-NCO, the drug release at 37 °C was observed to be independent of pH (Figure 7b), further supporting the conclusion that the β-CD-(NH2)7 rings on FMSNPs-CD(NH2)-SH can control the release of the drug molecules in the mesopores upon changes in pH. A key issue that has to be evaluated is whether the mucoadhesivity of FMSNPs-CD-(NH2)-SH is retained after loading with Dox. To investigate this issue, Dox@FMSNPsCD-(NH2)-SH (0.5 mL, 5 mg mL−1), Dox@FMSNPs-CDNH2 (0.5 mL, 5 mg mL−1), and free Dox (0.5 mL, 200 μg mL−1) were individually incubated with pig bladder in artificial urine for 2 h. From the in vitro drug release studies (Figure 7a), approximately 100 μg of free Dox was released from Dox@ FMSNPs-CD-(NH2)-SH (0.5 mL, 5 mg mL−1) in artificial urine after 2 h of incubation. Thus, for comparison, the dosage of free Dox used was selected to be 0.5 mL of artificial urine containing Dox (200 μg mL −1 ). Significant red Dox fluorescence and green FMSNPs-CD-(NH2)-SH fluorescence on the bladder wall were observed for Dox@FMSNPs-CD(NH2)-SH (Figure 8a), while Dox@FMSNPs-CD-NH2 showed much lower fluorescence from Dox as well as FMSNPs-CDNH2 (Figure 8b). The observation is in line with the results obtained with the FMSNPs-CD-NH2 and FMSNPs-CD(NH2)-SH without Dox (Figure 6). In another series of

MSNPs-CD-(NH2)-SH, which has no FITC and Dox (Figure 2b). The drug-loading capacity of FMSNPs-NCO and FMSNPs-CD-(NH2)-SH was investigated under the same conditions. The loading of Dox in FMSNPs-CD-(NH2)-SH was found to be only slightly lower (∼40% of total Dox added) compared with FMSNPs-NCO (∼43% of total Dox added). This suggests that the surface modification process did not drastically reduce the drug loading capacity of MSNPs. The amount of loaded Dox in FMSNPs-CD-(NH 2)-SH as determined by UV−vis absorption measurement is about 20% of the total weight of the Dox-loaded nanoparticles. The amount of Dox released from Dox-loaded FMSNPs-CD(NH2)-SH (Dox@FMSNPs-CD-(NH2)-SH) and Dox-loaded FMSNPs-NCO (Dox@FMSNPs-NCO) was determined quantitatively using fluorescence spectroscopy. The results shown in Figure 7a reveal a slow release of around 13% of the loaded Dox from Dox@FMSNPs-CD-(NH2)-SH after 48 h in PBS (pH 7.4). However, a much faster release of Dox was observed when Dox@FMSNPs-CD-(NH2)-SH was incubated in artificial urine (pH 6.1) and about 63% was released after 48 h. These results suggest that Dox@FMSNPs-CD-(NH2)-SH is sensitive to an acidic environment. Under acidic conditions, the unreacted amino groups of β-CD-(NH2)7 on the nanoparticles are protonated to ammonium groups, giving rise to Coulombic repulsion between the positively charged β-CD rings around the mesopores of the nanoparticles. This will cause the mesopores to open up, facilitating the release of positively charged Dox. An experiment was carried out to test this hypothesis by loading negatively charged calcein instead of positively charged Dox into FMSNPs-CD-NH2. A faster calcein 6156


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Figure 6. CLSM volume view images of pig bladder wall incubated in artificial urine (pH 6.1) containing (a) FMSNPs-CD-OH, (b) FMSNPs-CDNH2, (c) FMSNPs-CD-(NH2)-SH, and (d) PBS (control) for 2 h. The green fluorescence arises from the FITC-labeled MSNPs on the bladder wall. The results shown are representative images from three independent experiments. Scale bar: 100 μm.

Figure 7. Dox release profiles of (a) Dox@FMSNPs-CD-(NH2)-SH and (b) Dox@FMSNPs-NCO at 37 °C in artificial urine (pH 6.1) and PBS buffer solution (pH 7.4).

experiment, the bladder pieces were first incubated with free Dox or Dox-loaded nanoparticles for 2 h. After the incubation period, the pieces were washed three times with artificial urine and further incubated in artificial urine for 3 h. These incubation times were chosen to imitate the typical intravesical instillation period. For Dox@FMSNPs-CD-NH2 and free Dox, very little Dox remained on the bladder wall after the abovementioned process (Figures 9b and 9c, respectively). In contrast, the fluorescence from Dox@FMSNPs-CD-(NH2)SH remained strong on the bladder wall after 3 h (Figure 9a). These results further confirmed that the FMSNPs-CD-(NH2)SH nanoparticles can retain their mucoadhesive capacity after Dox was loaded into the mesopores of FMSNPs. Nanocarriers with minimal cytotoxicity are desirable in biological systems to avoid adverse side effects. Thus, viability of UMUC3 bladder cancer cells after incubation with MSNPs-

CD-(NH2)-SH was analyzed using the MTT assay. The results reveal that MSNPs-CD-(NH2)-SH is not cytotoxic to the cells up to 250 μg mL−1 (Figure 10a). However, significant inhibition of cell growth was observed when the bladder cancer cells were treated with free Dox or Dox-loaded nanoparticles. Dox@MSNPs-CD-(NH2)-SH demonstrated a dose-dependent cytotoxic effect on UMUC3 cells, and the IC50 of Dox@MSNPs-CD-(NH2)-SH was calculated to be 3.92 ± 1.06 μg mL−1 (based on the Dox loading of 20% in Dox@ MSNPs-CD-(NH2)-SH). As shown in Figure 10b, the IC50 of free Dox was determined to be 0.45 ± 0.05 μg mL−1. Compared to free Dox, the lower cytotoxicity of Dox@MSNPsCD-(NH2)-SH on the UMUC3 cells is probably due to the gradual release of Dox within the cells. To illustrate this effect, the red fluorescence from Dox in the nuclei of the cells after 2 h of incubation with free Dox and with Dox@FMSNPs-CD6157


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Figure 8. CLSM volume view images of pig bladder walls incubated with (a) Dox@FMSNPs-CD-(NH2)-SH (0.5 mL, 5 mg mL−1), (b) Dox@ FMSNPs-CD-NH2 (0.5 mL, 5 mg mL−1), and (c) free Dox (0.5 mL, 200 μg mL−1) for 2 h. The green fluorescence is from the mucoadhesive FITClabeled MSNPs on the bladder wall, and the red fluorescence is from released Dox as well as Dox retained within the nanoparticles. Scale bar: 200 μm.

(NH2)-SH was compared. Much stronger red Dox fluorescence was observed in the nuclei of UMUC3 cells treated with free Dox as compared with those treated with Dox@FMSNPs-CD(NH2)-SH (compare Figure S7a with Figure S7b). Thus, within the 2 h incubation period, only a very small amount of Dox was released from Dox@FMSNPs-CD-(NH2)-SH into the cell nuclei after endocytosis of the Dox-loaded nanoparticles. In another series of experiments using a longer incubation time, the cells were first incubated with FMSNPs-CD-(NH2)-SH or Dox@FMSNPs-CD-(NH2)-SH for 2 h and then washed with PBS in order to remove the excess nanoparticles outside the cells, followed by another 72 h of incubation in medium. It can be seen from Figure S8a that after the 2 h incubation period, the endocytosed nanoparticles were found in the endosomes/ lysosomes of the cells. After rinsing of the cells followed by further incubation for another 72 h, the nanoparticles remained localized in the endosomes/lysosomes of the cells (Figure S8b). However, with the longer incubation period, Dox released from the Dox@FMSNPs-CD-(NH2)-SH in the endosomes/lysosomes has penetrated into the nuclei of the cells (Figure S7c). The release of the Dox from Dox@FMSNPs-CD-(NH2)-SH is likely to be enhanced in the endosomes/lysosomes due to the acidic environment. It can be seen from Figure 9 that the strong adhesion of Dox@FMSNPs-CD-(NH2)-SH in comparison with free Dox to

the bladder wall will promote localization of the drug at the bladder wall for a prolonged period. Thus, enhanced intravesical bioavailability of the drug may reduce the dosing frequency and increase patient compliance. The high mucoadhesivity and sustained drug release capability of Dox@MSNPs-CD-(NH2)-SH make it an excellent candidate for in vivo intravesical chemotherapy, and animal model experiments are being planned as the next step.

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CONCLUSION In summary, we have synthesized MSNPs-CD-(NH2)-SH as a mucoadhesive drug delivery system for potential bladder cancer therapy. The MSNPs-CD-(NH2)-SH exhibited high mucoadhesive capacity for the mucous membrane of the urothelium compared with MSNPs-CD-NH2 and MSNPs-CD-OH due to its ability to form covalent bonds between its thiol groups and the glycoproteins in mucin. An anticancer drug, Dox, was loaded into the mesopores of MSNPs-CD-(NH2)-SH, and sustained drug release triggered by acidic pH was achieved. Dox@MSNPs-CD-(NH2)-SH can effectively induce cytotoxicity against UMUC3 bladder cancer cells with IC50 of 3.92 ± 1.06 μg Dox mL−1. The current research has demonstrated the capability of MSNPs-CD-(NH2)-SH as a mucoadhesive and sustained drug delivery system, and the potential of employing this system to deliver drugs for enhanced superficial bladder 6158


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Figure 9. CLSM volume view images of pig bladder walls incubated with (a) Dox@FMSNPs-CD-(NH2)-SH (0.5 mL, 5 mg mL−1), (b) Dox@ FMSNPs-CD-NH2 (0.5 mL, 5 mg mL−1), and (c) free Dox (0.5 mL, 200 μg mL−1) for 2 h, followed by rinsing three times with artificial urine and another 3 h incubation in artificial urine. The green fluorescence is from the mucoadhesive FITC-labeled MSNPs on the bladder wall, and the red fluorescence is from released Dox as well as Dox retained within the nanoparticles. Scale bar: 200 μm.

Figure 10. In vitro cytotoxicity profile of (a) MSNPs-CD-(NH2)-SH and Dox@MSNPs-CD-(NH2)-SH and (b) free Dox against UMUC3 bladder cancer cells. The cells were exposed to the drug or nanoparticles for 5 h and further cultured with fresh medium for 72 h.

distribution, XRD patterns, TEM image and zeta potential distribution of various MSNPs samples; hydrodynamic size of mucin dispersed in ABS buffer; pH-responsive release mechanism of FMSNPs-CD-NH2; confocal microscopy images of UMUC3 cells incubated with nanoparticles, drug and drugloaded nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

cancer therapy will be evaluated in an animal model study in the near future.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details for the synthesis of β-CD-NH2 and β-CD(NH2)7; nitrogen adsorption/desorption isotherms, BJH pore 6159


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(17) Xu, J.; Soliman, G. M.; Barralet, J.; Cerruti, M. Mollusk Glue Inspired Mucoadhesives for Biomedical Applications. Langmuir 2012, 28, 14010−14017. (18) Bravo-Osuna, I.; Vauthier, C.; Farabollini, A.; Palmieri, G. F.; Ponchel, G. Mucoadhesion Mechanism of Chitosan and Thiolated Chitosan-Poly(isobutyl cyanoacrylate) Core-Shell Nanoparticles. Biomaterials 2007, 28, 2233−2243. (19) Yin, L.; Ding, J.; He, C.; Cui, L.; Tang, C.; Yin, C. Drug Permeability and Mucoadhesion Properties of Thiolated Trimethyl Chitosan Nanoparticles in Oral Insulin Delivery. Biomaterials 2009, 30, 5691−5700. (20) Irmukhametova, G. S.; Mun, G. A.; Khutoryanskiy, V. V. Thiolated Mucoadhesive and PEGylated Nonmucoadhesive Organosilica Nanoparticles from 3-Mercaptopropyltrimethoxysilane. Langmuir 2011, 27, 9551−9556. (21) Vivero-Escoto, J. L.; Slowing, I. I.; Trewyn, B. G.; Lin, V. S. Y. Mesoporous Silica Nanoparticles for Intracellular Controlled Drug Delivery. Small 2010, 6, 1952−1967. (22) Rosenholm, J. M.; Sahlgren, C.; Lindén, M. Towards Multifunctional, Targeted Drug Delivery Systems Using Mesoporous Silica Nanoparticles - Opportunities & Challenges. Nanoscale 2010, 2, 1870−1883. (23) Ambrogio, M. W.; Thomas, C. R.; Zhao, Y. L.; Zink, J. I.; Stoddart, J. F. Mechanized Silica Nanoparticles: A New Frontier in Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 903−913. (24) Tang, F.; Li, L.; Chen, D. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. 2012, 24, 1504−1534. (25) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2590−2605. (26) Chen, Y.; Chen, H.; Shi, J. In Vivo Bio-Safety Evaluations and Diagnostic/Therapeutic Applications of Chemically Designed Mesoporous Silica Nanoparticles. Adv. Mater. 2013, 25, 3144−3176. (27) Kecht, J.; Schlossbauer, A.; Bein, T. Selective Functionalization of the Outer and Inner Surfaces in Mesoporous Silica Nanoparticles. Chem. Mater. 2008, 20, 7207−7214. (28) He, Q.; Shi, J.; Cui, X.; Zhao, J.; Chen, Y.; Zhou, J. Rhodamine B-Co-Condensed Spherical SBA-15 Nanoparticles: Facile Co-Condensation Synthesis and Excellent Fluorescence Features. J. Mater. Chem. 2009, 19, 3395−3403. (29) Wu, S. H.; Mou, C. Y.; Lin, H. P. Synthesis of Mesoporous Silica Nanoparticles. Chem. Soc. Rev. 2013, 42, 3862−3875. (30) Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S. Y. A Mesoporous Silica Nanosphere-Based Carrier System with Chemically Removable CdS Nanoparticle Caps for Stimuli-Responsive Controlled Release of Neurotransmitters and Drug Molecules. J. Am. Chem. Soc. 2003, 125, 4451−4459. (31) Giri, S.; Trewyn, B. G.; Stellmaker, M. P.; Lin, V. S. Y. StimuliResponsive Controlled-Release Delivery System Based on Mesoporous Silica Nanorods Capped with Magnetic Nanoparticles. Angew. Chem., Int. Ed. 2005, 44, 5038−5044. (32) Aznar, E.; Marcos, M. D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Amorós, P.; Guillem, C. pH- and Photo-Switched Release of Guest Molecules from Mesoporous Silica Supports. J. Am. Chem. Soc. 2009, 131, 6833−6843. (33) Yuan, L.; Tang, Q.; Yang, D.; Zhang, J. Z.; Zhang, F.; Hu, J. Preparation of pH-Responsive Mesoporous Silica Nanoparticles and Their Application in Controlled Drug Delivery. J. Phys. Chem. C 2011, 115, 9926−9932. (34) Chang, B.; Chen, D.; Wang, Y.; Chen, Y.; Jiao, Y.; Sha, X.; Yang, W. Bioresponsive Controlled Drug Release Based on Mesoporous Silica Nanoparticles Coated with Reductively Sheddable Polymer Shell. Chem. Mater. 2013, 25, 574−585. (35) He, X.; Zhao, Y.; He, D.; Wang, K.; Xu, F.; Tang, J. ATPResponsive Controlled Release System Using Aptamer-Functionalized Mesoporous Silica Nanoparticles. Langmuir 2012, 28, 12909−12915. (36) Chen, C.; Pu, F.; Huang, Z.; Liu, Z.; Ren, J.; Qu, X. StimuliResponsive Controlled-Release System Using Quadruplex DNA-

AUTHOR INFORMATION

Corresponding Author

*Tel +65 65162176; Fax +65 67791936; e-mail chenkg@nus. edu.sg (K.G.N.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This research was supported by the National Medical Research Council of Singapore Grant IRG10nov116.

(1) Chiong, E.; Gaston, K. E.; Grossman, H. B. Urinary Markers in Screening Patients with Hematuria. World J. Urol. 2008, 26, 25−30. (2) Ploeg, M.; Aben, K. K. H.; Kiemeney, L. A. The Present and Future Burden of Urinary Bladder Cancer in the World. World J. Urol. 2009, 27, 289−293. (3) GuhaSarkar, S.; Banerjee, R. Intravesical Drug Delivery: Challenges, Current Status, Opportunities and Novel Strategies. J. Controlled Release 2010, 148, 147−159. (4) Dalbagni, G. The Management of Superficial Bladder Cancer. Nat. Clin. Pract. Urol. 2007, 4, 254−260. (5) van Rhijn, B. W.; Burger, M.; Lotan, Y.; Solsona, E.; Stief, C. G.; Sylvester, R. J.; Witjes, J. A.; Zlotta, A. R. Recurrence and Progression of Disease in Non-Muscle-Invasive Bladder Cancer: from Epidemiology to Treatment Strategy. Eur. Urol. 2009, 56, 430−442. (6) Sexton, W. J.; Wiegand, L. R.; Correa, J. J.; Politis, C.; Dickinson, S. I.; Kang, L. C. Bladder Cancer: A Review of Non-Muscle Invasive Disease. Cancer Control. 2010, 17, 256−268. (7) Tyagi, P.; Tyagi, S.; Kaufman, J.; Huang, L.; de Miguel, F. Local Drug Delivery to Bladder Using Technology Innovations. Urol. Clin. North Am. 2006, 33, 519−530. (8) Huang, C.; Neoh, K. G.; Xu, L.; Kang, E. T.; Chiong, E. Polymeric Nanoparticles with Encapsulated Superparamagnetic Iron Oxide and Conjugated Cisplatin for Potential Bladder Cancer. Biomacromolecules 2012, 13, 2513−2520. (9) Smart, J. D. The Basics and Underlying Mechanisms of Mucoadhesion. Adv. Drug Delivery Rev. 2005, 57, 1556−1568. (10) Fraser, M. O.; Chuang, Y. C.; Tyagi, P.; Yokoyama, T.; Yoshimura, N.; Huang, L.; De Groat, W. C.; Chancellor, M. B. Intravesical Liposome Administration - a Novel Treatment for Hyperactive Bladder in the Rat. Urology 2003, 61, 656−663. (11) Lu, Z.; Yeh, T. K.; Tsai, M.; Au, J. L. S.; Wientjes, M. G. Paclitaxel-Loaded Gelatin Nanoparticles for Intravesical Bladder Cancer Therapy. Clin. Cancer Res. 2004, 10, 7677−7684. (12) Mugabe, C.; Hadaschik, B. A.; Kainthan, R. K.; Brooks, D. E.; So, A. I.; Gleave, M. E.; Burt, H. M. Paclitaxel Incorporated in Hydrophobically Derivatized Hyperbranched Polyglycerols for Intravesical Bladder Cancer Therapy. BJU Int. 2009, 103, 978−986. (13) Mugabe, C.; Raven, P. A.; Fazli, L.; Baker, J. H. E.; Jackson, J. K.; Liggins, R. T.; So, A. I.; Gleave, M. E.; Minchinton, A. I.; Brooks, D. E.; Burt, H. M. Tissue Uptake of Docetaxel Loaded Hydrophobically Derivatized Hyperbranched Polyglycerols and Their Effects on the Morphology of the Bladder Urothelium. Biomaterials 2012, 33, 692− 703. (14) Barthelmes, J.; Dünnhaupt, S.; Unterhofer, S.; Perera, G.; Schlocker, W.; Bernkop-Schnürch, A. Thiolated Particles as Effective Intravesical Drug Delivery Systems for Treatment of Bladder-Related Diseases. Nanomedicine 2013, 8, 65−75. (15) Štorha, A.; Mun, E. A.; Khutoryanskiy, V. V. Synthesis of Thiolated and Acrylated Nanoparticles Using Thiol-Ene Click Chemistry: Towards Novel Mucoadhesive Materials for Drug Delivery. RSC Adv. 2013, 3, 12275−12279. (16) Sogias, I. A.; Williams, A. C.; Khutoryanskiy, V. V. Why is Chitosan Mucoadhesive? Biomacromolecules 2008, 9, 1837−1842. 6160


Langmuir

Article

Capped Silica Nanocontainers. Nucleic Acids Res. 2011, 39, 1638− 1644. (37) Climent, E.; Bernardos, A.; Martínez-Máñez, R.; Maquieira, A.; Marcos, M. D.; Pastor-Navarro, N.; Puchades, R.; Sancenón, F.; Soto, J.; Amorós, P. Controlled Delivery Systems Using Antibody-Capped Mesoporous Nanocontainers. J. Am. Chem. Soc. 2009, 131, 14075− 14080. (38) Nguyen, T. D.; Liu, Y.; Saha, S.; Leung, K. C. F.; Stoddart, J. F.; Zink, J. I. Design and Optimization of Molecular Nanovalves Based on Redox-Switchable Bistable Rotaxanes. J. Am. Chem. Soc. 2007, 129, 626−634. (39) Liu, R.; Zhang, Y.; Feng, P. Multiresponsive Supramolecular Nanogated Ensembles. J. Am. Chem. Soc. 2009, 131, 15128−15129. (40) Nguyen, T. D.; Leung, K. C. F.; Liong, M.; Liu, Y.; Stoddart, J. F.; Zink, J. I. Versatile Supramolecular Nanovalves Reconfigured for Light Activation. Adv. Funct. Mater. 2007, 17, 2101−2110. (41) Angelos, S.; Yang, Y. W.; Patel, K.; Stoddart, J. F.; Zink, J. I. pHResponsive Supramolecular Nanovalves Based on Cucurbit[6]uril Pseudorotaxanes. Angew. Chem., Int. Ed. 2008, 47, 2222−2226. (42) Park, C.; Oh, K.; Lee, S. C.; Kim, C. Controlled Release of Guest Molecules from Mesoporous Silica Particles Based on a pHResponsive Polypseudorotaxane Motif. Angew. Chem., Int. Ed. 2007, 46, 1455−1457. (43) 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. (44) Meng, H.; Xue, M.; Xia, T.; Zhao, Y. L.; Tamanoi, F.; Stoddart, J. F.; Zink, J. I.; Nel, A. E. Autonomous in Vitro Anticancer Drug Release from Mesoporous Silica Nanoparticles by pH-Sensitive Nanovalves. J. Am. Chem. Soc. 2010, 132, 12690−12697. (45) Zhang, Q.; Liu, F.; Nguyen, K. T.; Ma, X.; Wang, X.; Xing, B.; Zhao, Y. Multifunctional Mesoporous Silica Nanoparticles for CancerTargeted and Controlled Drug Delivery. Adv. Funct. Mater. 2012, 22, 5144−5156. (46) Park, C.; Lee, K.; Kim, C. Photoresponsive CyclodextrinCovered Nanocontainers and Their Sol-Gel Transition Induced by Molecular Recognition. Angew. Chem., Int. Ed. 2009, 48, 1275−1278. (47) He, D.; He, X.; Wang, K.; Cao, J.; Zhao, Y. A Light-Responsive Reversible Molecule-Gated System Using Thymine-Modified Mesoporous Silica Nanoparticles. Langmuir 2012, 28, 4003−4008. (48) Liu, R.; Zhao, X.; Wu, T.; Feng, P. Tunable Redox-Responsive Hybrid Nanogated Ensembles. J. Am. Chem. Soc. 2008, 130, 14418− 14419. (49) Wan, X.; Wang, D.; Liu, S. Fluorescent pH-Sensing Organic/ Inorganic Hybrid Mesoporous Silica Nanoparticles with Tunable Redox-Responsive Release Capability. Langmuir 2010, 26, 15574− 15579. (50) Kim, H.; Kim, S.; Park, C.; Lee, H.; Park, H. J.; Kim, C. Glutathione-Induced Intracellular Release of Guests from Mesoporous Silica Nanocontainers with Cyclodextrin Gatekeepers. Adv. Mater. 2010, 22, 4280−4283. (51) Schlossbauer, A.; Warncke, S.; Gramlich, P. M. E.; Kecht, J.; Manetto, A.; Carell, T.; Bein, T. A Programmable DNA-Based Molecular Valve for Colloidal Mesoporous Silica. Angew. Chem., Int. Ed. 2010, 49, 4734−4737. (52) Park, C.; Kim, H.; Kim, S.; Kim, C. Enzyme Responsive Nanocontainers with Cyclodextrin Gatekeepers and Synergistic Effects in Release of Guests. J. Am. Chem. Soc. 2009, 131, 16614−16615. (53) Schlossbauer, A.; Kecht, J.; Bein, T. Biotin-Avidin as a ProteaseResponsive Cap System for Controlled Guest Release from Colloidal Mesoporous Silica. Angew. Chem., Int. Ed. 2009, 48, 3092−3095. (54) Bernardos, A.; Aznar, E.; Marcos, M. D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Barat, J. M.; Amorós, P. Enzyme-Responsive Controlled Release Using Mesoporous Silica Supports Capped with Lactose. Angew. Chem., Int. Ed. 2009, 48, 5884−5887. (55) Bernardos, A.; Mondragón, L.; Aznar, E.; Marcos, M. D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Barat, J. M.; Pérez-Payá, E.; Guillem, C.; Amorós, P. Enzyme-Responsive Intracellular

Controlled Release Using Nanometric Silica Mesoporous Supports Capped with “Saccharides”. ACS Nano 2010, 4, 6353−6368. (56) Chutipongtanate, S.; Thongboonkerd, V. Systematic Comparisons of Artificial Urine Formulas for In Vitro Cellular Study. Anal. Biochem. 2010, 402, 110−112. (57) Carvalho, F. C.; Bruschi, M. L.; Evangelista, R. C.; Gremiao, M. P. D. Mucoadhesive Drug Delivery Systems. Braz. J. Pharm. Sci. 2010, 46, 1−17. (58) Thongborisute, J.; Takeuchi, H. Evaluation of Mucoadhesiveness of Polymers by BIACORE Method and Mucin-Particle Method. Int. J. Pharm. 2008, 354, 204−209. (59) Zhang, Q.; Luan, L.; Feng, S.; Yan, H.; Liu, K. Using a Bifunctional Polymer for the Functionalization of Fe3O4 Nanoparticles. React. Funct. Polym. 2012, 72, 198−205. (60) Möller, K.; Kobler, J.; Bein, T. Colloidal Suspensions of Nanometer-Sized Mesoporous Silica. Adv. Funct. Mater. 2007, 17, 605−612. (61) Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-Targeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2012, 134, 5722−5725. (62) 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. (63) Casasús, R.; Marcos, M. D.; Martínez-Máñez, R.; Ros-Lis, J. V.; Soto, J.; Villaescusa, L. A.; Amorós, P.; Beltrán, D.; Guillem, C.; Latorre, J. Toward the Development of Ionically Controlled Nanoscopic Molecular Gates. J. Am. Chem. Soc. 2004, 126, 8612− 8613.

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