Natural Gelatin Capped Mesoporous Silica Nanoparticles for

Sep 27, 2013 - This paper proposed a natural gelatin capped mesoporous silica nanoparticles (MSN@Gelatin) based pH-responsive delivery system for ...
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Natural Gelatin Capped Mesoporous Silica Nanoparticles for Intracellular Acid-triggered Drug Delivery Zhen Zou, Dinggeng He, Xiaoxiao He, Kemin Wang, Xue Yang, and Quan Zhou Langmuir, Just Accepted Manuscript • Publication Date (Web): 27 Sep 2013 Downloaded from http://pubs.acs.org on October 1, 2013

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Natural Gelatin Capped Mesoporous Silica Nanoparticles for Intracellular Acid-triggered Drug Delivery

Zhen Zou, Dinggeng He, Xiaoxiao He*, Kemin Wang*, Xue Yang, Zhihe Qing, Quan Zhou

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University. Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, P. R. China

KEYWORDS: gelatin, mesoporous silica nanoparticles, doxorubicin, drug delivery

ABSTRACT: This paper proposed a natural gelatin capped mesoporous silica nanoparticles (MSN@Gelatin) based pH-responsive delivery system for intracellular anticancer drug controlled release. In this system, the gelatin, a proteinaceous biopolymer derived from the processing of animal collagen, was grafted onto the MSN to form capping layer via temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking, resulting in gelatin coated MSN. At neutral pH, gelatin capping layer could effectively prohibit the release of loaded drug molecules. However, the slightly acidic environment would lead to enhanced electrostatic repulsion between the gelatin and MSN, giving rise to uncapping and the subsequent controlled release of the entrapped drug. As a proof-of-the-concept, doxorubicin (DOX) was selected as the model anticancer drug. The loading and pH-responsive release experiments demonstrated that the system had excellent loading efficiency (47.3 mmol g-1 SiO2) and almost no DOX was leaked at neutral. After putting in the slightly

acidic

condition,

the

DOX

release

from

the

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DOX-loaded

MSN@Gelatin

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(DOX/MSN@Gelatin) was occurred immediately. The cellular uptake and release studies using Hep-G2 hepatoma cells indicated that the DOX/MSN@Gelatin could be endocytosed and accumulated within lysosomes. Triggered by acidic endosomal pH, the intracellular release of the loaded DOX was obviously eventuated. Further cell viability results demonstrated that DOX/MSN@Gelatin exhibited dose-dependent toxicity and high killing efficacy (IC50 = 17.27 ± 0.63 μg mL−1), whereas the MSN@Gelatin showed negligible cytotoxicity (IC50 > 100 μg mL−1). This biocompatible and effective delivery system will provide the great potential for developing delivery of cancer therapeutic agents.

INTRODUCTION Within the last decade, various novel nanomaterials such as nanocapsules,1 polymers,2 dendrimers,3 micelles,4 and inorganic nanoparticles5-7 have been proposed as smart cargo-delivery systems to improve specificity and bioavailability of cancer therapeutic agents. Among them, mesoporous silica nanoparticle (MSN), as leading candidates for next generation therapeutic carriers, have drawn a great deal of attention because of their many advantageous properties, such as facile synthesis, stable mesostructures, large surface areas, well-defined surface properties, versatile inner and outer surface chemistry, and good biocompatibility.8-10 Up to now, several sophisticated stimuli-responsive drug delivery systems based on MSN have been described. In these systems, the efficient release of guest molecules from MSN was regulated either by external stimuli,11,12 such as pH,13-15 biological triggers,16 temperature,17 enzyme,18-20 redox changes,21-23 competitive binding,24 photoirradiation,25-27 and so forth. Of the stimuli previously studied, pH-triggered approach was one of the most efficient strategies for therapies because the interstitial or extracellular environment in tumors tends to be more acidic than the normal tissues, and endosomes and lysosomes exhibit even lower pH values.28-30 Generally, pH-controlled MSN release systems were designed and fabricated using supramolecular nanovalves,31 acid-decomposable nanocoating,32 nucleic acids,33 or ACS Paragon Plus Environment

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coordination polymers34 as pore blockers. For example, Zink et al. designed some pH-responsive supramolecular nanovalves on mesoporous materials based on the ion-dipole interactions between an aromatic amino group and a cyclodextrin cap,35 or by using the interaction between curcubit[6]uril and bis-ammonium stalks.36 With the change of pH value, the interactions were disrupted and the nanopore orifices were unblocked, resulting in the release of guest molecules. Another notable pH-responsive gate-control example reported by Lee et al. was employing an acid-decomposable absorbable calcium phosphate nanocoating to seal the pores of MSN. The calcium phosphate nanocoating could be dissolved within intracellular endosomes as nontoxic ions to initiate drug release within tumor cells.37 Recently, a novel gate-like ensemble was functionalized with i-motif DNA as a pore blocker, which could be switched “on” and “off” through a precise structural change driven by a pH change.38 Despite enormous research progress in this field, many existing pH-responsive MSN drug delivery systems still have presented disadvantages in terms of the complexity of the fabricating process, costliness of raw materials, non-natural sources of the capping agents, etc. Thence, the design of pH-responsive MSN nanocarriers using easily available and non-toxic/natural components as pore blockers remains a complex and exciting challenge. Gelatin, a natural proteinaceous biopolymer, is derived from the skin or bones of a number of species of animal and has a long history of safe use in a wide range of pharmacy, food and cosmetics because of its desirable features, such as natural origin, low-cost, low-toxicity, biodegradability, and non-immunogenicity. In addition, the basic composition of gelatin is polypeptide contained many carboxyl, amine, and amide functional groups, which could make gelatin negatively or positively charged upon the change of the pH. By taking advantage of such properties, many pH-responsive controlled drug release systems with gelation as carrier material were reported.39, 40 For instance, Li et al. recently fabricated a pH-responsive gelatin microgels copying the structure of a porous CaCO3 template. In this system, the internal charge repulsion force within gelatin microgels could increase ACS Paragon Plus Environment

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with the decrease of pH, resulting the swelling of the gelatin microgels and the release of drugs.41 Moreover, some recent investigations concluded that gelatin could adsorption onto the surface of the nanoparticles by complex interactions, such as van der Waals interactions, hydrophobic interactions, and electrostatic interactions, which make gelatin a useful candidate for the pH-sensitive coating layer.42 Despite these burgeoning achievements, the use of gelatin for designing drug release system is still an incipient area of research, and there are no report examples where the pH-responsive drug release system could be prepared by a combination of MSN as host support material and gelatin as pore-blocking agent. Herein, we integrated the advantages of gelatin and MSN to fabricate an acid-responsive intracellular drug delivery system by a simple method. In the system, the gelatin could be adsorbed on the surface of the MSN through temperature-induced gelation. Subsequently, glutaraldehyde was used to cross-link the gelatin for preventing the dissolution of adsorbed gelatin. At neutral pH, the coating layer formation directly resulted in blockage of pores and strongly inhibited the diffusion of guest molecules from pores. But in acidic environments, the increased electrostatic repulsive force between gelatin coating layer and MSN could open the pore and allow the escape of the entrapped guest molecules. The anticancer drug doxorubicin (DOX) was encapsulated into the pores to investigate the pH-responsive controlled release behavior and cytotoxicity. It was demonstrated that the DOX-loaded MSN@Gelatin system (DOX/MSN@Gelatin) was a desired candidate for intracellular drug delivery. EXPERIMENTAL SECTION Chemicals and Materials. Cow-hide gelatin, glutaraldehyde, 3-[4,5-dimethylthialzol-2-yl]-2,5diphenyltetrazo-lium bromid (MTT) was purchased from Sigma-Aldrich. Lysotracker blue DND-22 and

Heochst-33342

were

N-cetyltrimethylammonium

purchased

from

bromide

(CTAB)

Invitrogen was

Life

Technologies

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Corporation. Alfa

Aesar.

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Tetraethylorthosilicate (TEOS, 28%), sodium hydroxide (NaOH) and dimethyl sulfoxide (DMSO) were obtained from Xilong reagent company (Guangdong, China). Doxorubicin hydrochloride was obtained from Kayon Biological Technology CO., Ltd (Shanghai, China). Nanopure water (18.2 MΩ; Millpore Co., USA) was used in all experiments and to prepare all buffers. All the chemicals were used as received without further purification. Characterization. Transmission electron microscopy (TEM) images were obtained on a JEOL 3010 microscope with an accelerating voltage of 100 kV. Small angle powder X-ray diffraction pattern of the MSN materials was obtained in a Scintag XDS-2000 powder diffractometer using CuKα irradiation (λ = 0.154 nm). N2 adsorption–desorption isotherm was obtained at 77 K on a Micromeritics ASAP 2010 sorptometer by static adsorption procedures. Samples were degassed at 373 K and 10−3 Torr for a minimum of 12 h prior to analysis. Brunauer–Emmett–Teller (BET) surface area was

calculated from the linear part of the BET plot according to

recommendations. Pore size distribution was estimated

IUPAC

from the adsorption branch of the isotherm

by the Barrett–Joyner–Halenda (BJH) method. Thermogravimetric analysis (TGA) of the grafted materials was performed using a Mettler TGA-SDTA851e Star system. The analyses were carried out under air flow with a heating rate of 15 °C min-1 and a complementary argon flow was used as a protective gas. Zeta potential experiments were performed at 25 °C using a Malvern ZetaSizer Nano instrument, equipped with a He-Ne laser (633 nm) at a fixed scattering angle of 90°. All fluorescence spectra were recorded on a Hitachi F-2500 FL Spectrophotometer in PBS buffer. The pH of solutions was measured with a Thermo-Scientific Orion 3 STAR pH meter. The Confocal laser scanning microscopy (CLSM) images were obtained on a Fluoview FV500, Olympus. The MTT assay was obtained in a Benchmark Plus, Biorad Instruments Inc, Japan. Synthesis of Mesoporous Silica Nanoparticles (MSN). The synthesis of MSN was carried out following a base-catalyzed sol-gel procedure with TEOS as the silica precursor, CTAB surfactant as ACS Paragon Plus Environment

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the template and sodium hydroxide (NaOH) as the catalyst. 43 Briefly, CTAB (1.00 g, 2.74 mmol) was first dissolved in 480 mL of deionized water. Then NaOH solution (3.5 mL, 2.00 M) was added to the CTAB solution, followed by adjusting the solution temperature to 80 °C. TEOS (5.00 mL, 2.57×10-2 mol) was then added dropwise to the surfactant solution under vigorous stirring. The mixture was allowed stirred for 2 h to give a white precipitate. The product was subsequently collected by centrifuging at 12000 rpm for 10 min, and then washed, and redispersed with deionized water and ethanol several times. Surfactant templates were removed by extraction in an ethanolic solution (80 mL) containing HCl (0.80 mL, 37.2 %) under reflux at 79 °C for 16 h. After purification, the MSN was washed with water and ethanol before being dried in vacuum. DOX Loading and Gelatin Capping. In this work, DOX was used as a model guest drug to evaluate the loading and controlled releasing behavior because of its fluorescence properties and water solubility. For the preparation of DOX-loaded MSN@Gelatin (DOX/MSN@Gelatin), MSN (10 mg) were diffusion-filled with DOX by immersing the particles in a solution of DOX (500 μL, 0.01M, pH 7.2) overnight, followed by centrifuging to remove excess DOX solution. The residual particles were gently shaken with aqueous gelatin solution (1 mL, 1%) at 50 °C for 6 h to achieve pore saturation. Then, deionized water (8 mL) at 4 °C was poured into the mixture quickly. After two centrifugation/water rinsing/redispersion cycles, 50 μL of 1% glutaraldehyde solution was added to cross-link the gelatin at 4 °C. The cross-linking reaction was continued for 8 h. Then, the samples were centrifuged, rinsed by water and redispersed for three times. The loading amount of DOX was calculated by subtracting the amount of DOX molecules remaining in the supernatant and combined washings from the amount of DOX initially added to the reaction. DOX Releasing. DOX release experiments were performed at pH values of 2.0, 4.0, 5.0, 6.0 and 7.4 respectively. A small sample of DOX/MSN@Gelatin was placed in a cuvette, which was then carefully filled with 200 μL different buffer solution with various pH values. Subsequently, the ACS Paragon Plus Environment

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release profiles of DOX molecules from the pores to aqueous solution were monitored via the fluorescence intensity of the dye centered at 560 nm. Time-dependent release of DOX from the MSN particles was studied for 440 min. Cellular Uptaking and Localization Investigation. To demonstrate efficient cellular uptake, Hep-G2 cells were seeded in 35 mm plastic-bottomed μ-dishes and grown in RPMI 1640 medium containing 10% fetal calf serum overnight. Then the cells were washed with D-hanks and incubated with DOX/MSN@Gelatin (40 μg mL-1) in the culture medium at 37 °C for 5 or 12 h, followed by three washes with D-hanks. The medium was replaced with D-hanks medium containing Lysotracker blue DND-22 or Heochst-33342. After incubation for 15 min, the cells were washed with fresh D-hanks medium monitored under a Fluoview confocal microscope (100 × oil objective). Cytotoxicity Assay. Hep-G2 cells were cultured in RPMI 1640 media containing 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was measured by reduction of MTT, as described previously.33 For the MTT assay, Hep-G2 cells were seeded at 104 cells per well into 96-well plates and grown overnight. Then the cells was incubated with DOX/MSN@Gelatin at concentrations ranging from 5 to 100 μg mL-1 in 200 μL growth media for 48 h. After treatment, cells were further incubated with fresh medium containing MTT (0.5 mg mL−1) for 4 h. The precipitated formazan violet crystals were dissolved in DMSO (150 μL) at 37 °C. The absorbance was measured at 490 nm by multi-detection microplate reader. The cell viability of treated cells was normalized as a percentage of untreated cells. Data are shown as the mean ± the standard error from three independent experiments performed in triplicate. RESULTS AND DISCUSSION For the design of the stated controlled release system, two components were chosen, namely a solid supporter and the pH-sensitive cap. In this work, MCM-41-type MSN was selected as a suitable inorganic material because of its advantageous features. For the capping mechanism, our ACS Paragon Plus Environment

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attention was focused on a natural proteinaceous biopolymer gelatin due to its useful properties include biodegradability, biocompatibility and pH-induced surface charge. Herein a practicable and simple method was used to fabricate the delivery system based on gelatin capped MSN. The formation of DOX/MSN@Gelatin and intracellular pH-triggered DOX release behavior was illustrated in Scheme 1. Firstly, the synthesis of MSN was carried out following a base-catalyzed sol-gel procedure.43 After getting MSN, DOX, a well-known anticancer chemotherapeutics, was loaded into the inner mesopores of the MSN by diffusion. Subsequently, the gelatin capping onto the DOX loaded MSN was performed through temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking. Briefly, DOX loaded MSN was incubated in a hot aqueous gelatin solution at 50 °C for 6 h, which allowed the gelatin to be adsorbed onto the surface of the MSN. Upon addition of cold water at 4 °C, this process was stopped due to the increase in viscosity. For the subsequent experiment, the adsorbed gelatin was cross-linked by the addition of glutaraldehyde to prevent the dissolution of gelatin in aqueous solution.44 Then, the DOX/MSN@Gelatin was obtained. At neutral pH, pores of the DOX/MSN@Gelatin were blocked to strongly inhibit the diffusion of the entrapped DOX because of the coverage of gelatin. But when DOX/MSN@Gelatin was internalized into cells by endocytosis, the intracellular slightly acidic environments such as endosomes and lysosomes could lead to the detachment of gelatin coating layer and the release of entrapped DOX.

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Scheme 1. Schematic illustration for the formation of DOX/MSN@Gelatin and intracellular pH-triggered DOX release.

Following this design, the feasibility of gelatin coating and detachment was firstly investigated. According to the method described above, the MSN was synthesized and characterized by transmission electron microscopy (TEM), small-angle X-ray diffraction (XRD), and ZetaSizer Nano, respectively. As demonstrated in the TEM images, the prepared MSN particles possessed a typical hexagonally channel-like pore and a roughly spherical morphology with a diameter of approximately 150 nm. The XRD in the 1.6° < 2θ < 5° range both exhibited three low-angle reflections typical of hexagonal array that could be indexed as (100), (110) and (200) Bragg peaks with an a0 cell parameter of 4.1 Å, which further confirmed the structure of MSN (Figure 1a). The as-synthesized MSN was then coated with gelatin via temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking. The resulting gelatin capped MSN (MSN@Gelatin) nanoparticles with a core-shell structure could be directly observed by TEM imaging (Figure 1b and S2). The MSN

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nanoparticles were covered bright layers of gelatin with a thickness of ≈8 nm. It was the most direct evidence to prove the coating of the gelatin layer around the MSN matrix. Moreover, in contrast to MSN with the zeta potential of -25.4 mV, the value of zeta potential of MSN@Gelatin was changed into -0.226 mV, further indicating the successful coating process (Figure S1, Supporting Information). The conversion of MSN surface was also confirmed by Fourier Transform Infrared (FTIR) spectra. As illustrated in Figure 2, MSN@Gelatin showed obvious absorption band at the adsorption peak 1544 cm−1, which was assigned to the stretching vibration of N-H bending of gelatin. However, MSN had no adsorption at that wavenumber. Narrow band at 2930 cm−1 and 2750 cm-1 attributed to C-H stretching stretching vibrations. In addition, amide A tends to merge with the CH2 stretch peak expected to occur at around 2930 cm-1 when carboxylic acid groups exist in stable dimeric (intermolecular) associations. The reduction in the intensity of these peaks is anticipated as a result of the combined effects of conformational change in the protein chain of gelatin and the interaction between gelatin molecule and the silanol hydrogen. A similar phenomenon was also appeared in previous work.45,

46

Quantification of the coating layer was accomplished by

thermogravimetric analysis (TGA) with the immobilized mass percentage approximately 17.7 %. (Figure S3). Excitingly, we found that the gelatin coating layer of MSN@Gelatin could be detached from MSN due to the protonation of gelatin upon decreasing pH. TEM image clearly confirmed this acid-triggered process (Figure 1c). In addition, after the MSN@Gelatin treated in aqueous solution of pH 3.0 for 12 h and re-dispersed in water, the zeta potential was restored to -11 mV from -0.226 mV (Figure S1), indicating the re-exposure of the silanol surface. The XRD analysis showed that the well-ordered porous structures with typical MCM-41-type hexagonal arrangements were not affected by gelatin coating and detachment. These results displayed the great potential of MSN@Gelatin for acid-triggered drug delivery.

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Figure 1. Powder X-ray pattern and TEM images (insert) of a) MSN; b) MSN@Gelatin; c) MSN@Gelatin treated with an aqueous solution of pH 3.0 for 12 h.

Figure 2. FTIR spectra of the samples MSN, MSN@Gelatin, and Gelatin. ACS Paragon Plus Environment

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After confirming that the gelatin could be successfully capped onto the MSN and detached upon decreasing pH, DOX was then selected as guest molecule to fabricate DOX-loaded MSN@Gelatin (DOX/MSN@Gelatin). The N2 adsorption–desorption studies showed that the surface area (87.6 m2 g−1) and pore volume (0.24 cm3 g−1) of DOX/MSN@Gelatin had greatly reduced compared to the high surface area of 1074.4 m2 g−1 and large pore volume of 0.83 cm3 g−1 for as-synthesized MSN (Figure 3, S4 and Table S1). This result revealed that mesopores were filled with DOX and coated by gelatin. The DOX loading of MSN, estimated by UV-vis absorption measurements of the supernatant of the soaking solution, was as high as 47.3 μmol g−1. After DOX/MSN@Gelatin was treated in aqueous solution of pH 3.0 for 12 h and then washed with water, the surface area and the pore volume were reverted to 790.6 m2 g−1 and 0.65 cm3 g−1, indicating the opening of pores and the release of DOX. These results suggested that the MSN@Gelatin system could be an effective drug carrier. MSN DOX/MSN@Gelatin with acid treatment DOX/MSN@Gelatin

500 400

Pore Volume (cm3 g-1 nm-1 )

Volume Adsorbed (cm3 g-1 )

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0 0.0

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Relative Pressure (P/P0) Figure 3. Nitrogen sorption isotherms and pore size distributions (inset) of MSN (black circles), DOX/MSN@Gelatin (black triangles), and DOX/MSN@Gelatin with an acid treatment (open circles). ACS Paragon Plus Environment

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For the further studies of the effect of pH upon the DOX release behaviors, DOX/MSN@Gelatin samples were incubated at pH values of 2.0, 4.0, 5.0, 6.0 and 7.4, and the amount of released DOX was determined by the measurement of fluorescence intensity at 560 nm (λex = 488 nm) with reference to the standard curve. From these releasing-profile studies, we determined that the release of DOX from DOX/MSN@Gelatin was both time- and pH-dependent. As Figure 4 showed, the release amount of DOX from DOX/MSN@Gelatin suspended in solution was less than 4 % over 440 min time period because the coating layers of gelatin blocked the pore outlets of MSN and inhibited loaded DOX release at neutral pH. This finding indicated the good storage and sealing effect of the gelatin coating on MSN nanoparticles in PBS solution (pH 7.4). In contrast, satisfactory release rates of DOX showed increased sustained rates for increased acidities, with approximately 18 %, 44 %, 54 % and 83 % of the drug released within 440 min at pH 6.0, 5.0, 4.0 and 2.0, respectively, which suggested that the MSN@Gelatin system could be effective carriers. As such, these findings revealed that this pH-responsive MSN@Gelatin system had in increased release rate over the 440 min period in mimicked environments of late endosome and lysosome, where the pH values were in the range 5.0–6.0. 100

DOX release (%)

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Time (min) Figure 4. Release profiles of DOX from DOX/MSN@Gelatin system at different pH values. ACS Paragon Plus Environment

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The success in controlling the DOX release prompted us to apply this system to the intracellular drug delivery. It was reported that nanoparticles were able to be internalized into cells by endocytosis and accumulated within lysosomes.47 Confocal laser scanning microscopy (CLSM) was employed to evaluated cellular uptake and intracellular release behaviors of DOX/MSN@Gelatin. Figure 5 showed the representative images of Hep-G2 cells treated with DOX/MSN@Gelatin particles at a concentration of 40 μg mL-1. After incubation for 5 h, DOX/MSN@Gelatin internalized into the cells and localized mainly in the endosomes and lysosomes which confirmed by the clearly visible red fluorescence of DOX and co-localized with blue fluorescence of Lysotracker blue DND-22 (a special straining material for lysosomes) (Figure 5 a). It has been reported that the major mechanism of DOX toxicity to cells is believed to be the inhibition of the action of topoisomerase II or intercalation of DNA strands, leading to DNA double-strand breaks and inhibition of DNA replication and transcription.48 This means that nucleus location of DOX results in maximal drug efficacy. Thus, to prove that the released DOX from the DOX/MSN@Gelatin could be further delivered into the cell nuclei, the distribution of the released DOX in Hep-G2 cells was further investigated. The cell nucleus was labeled with blue-fluorescent Hoechst 33342 as an indicator. After 12 h of cultivation, the red fluorescence of DOX could be clearly observed in the nuclei of Hep-G2 cells (Figure 5 b), which can be ascribed to the released DOX from internalized DOX/MSN@Gelatin. The results further proved that the MSN@Gelatin as an intracellular drug delivery system was effective in holding DOX before endocytosis, and the DOX release can be facilitated within lysosomes by the dissolution of gelatin coating layer.

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Figure 5. The confocal microcopy studies of the cellular uptake and controlled release behaviors of DOX/MSN@Gelatin particles after incubation with Hep-G2 cells for (a) 5 h and (b) 12 h at a concentration of 40 μg mL-1. Lysotracker blue DND-22 was used to stain the lysosomes (a). Nuclei were stained with Hoechst 33342 (b). Cells were imaged using a 100× oil-immersion objective. To confirm that the delivery system would facilitate the cell killing activity of the drug, Hep-G2 cells were incubated with free DOX, unloaded MSN@Gelatin and DOX/MSN@Gelatin at different concentrations for 48 h. The MTT assay was used for quantitative testing of the cell viability. As Figure 6 showed, no obvious cytotoxicity was observed when cells was incubated with unloaded MSN@Gelatin nanoparticles at concentrations ranging from 5 to 100 μg mL−1. The inhibitory concentration (IC50) of MSN@Gelatin particles was much higher than 100 μg mL−1. In the same concentration range, however, the DOX/MSN@Gelatin particles remarkably inhibited Hep-G2 cell growth. The half-maximal inhibitory concentration (IC50) was calculated to be 17.27 ± 0.63 μg mL−1, suggesting a fairly high therapeutic effectiveness. As control experiments, free DOX at comparable concentrations also exhibited a high cytotoxicity towards Hep-G2 cells, which matched well with the cell viability of DOX/MSN@Gelatin. These results indicated that MSN@Gelatin was highly biocompatible and indeed served as a drug-carrier for intracellular controlled release.

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1.2 120

Concentration (μg/mL)

Figure 6. In vitro cytotoxicity assay curves for Hep-G2 cells obtained by plotting the cell viability percentage against the concentration of DOX. CONCLUSION In summary, we have developed a novel type of intracellular acid-triggered drug delivery system consisting of mesoporous silica nanoparticles capped with gelatin. In this system, antineoplastic drug DOX could be effectively loaded in MSN and non-toxic/natural gelatin was introduced as a novel pore blocker by adsorbing on MSN surface to form coating layers. The gelatin coatings could hold the encapsulated DOX at physiological conditions, whereas they can be detached from MSN to triggered release of the encapsulated DOX under slightly acidic conditions, such as endosome and lysosome. The results demonstrated that the MSN@Gelatin system had a high loading amount of drug (47.3 mmol g-1 SiO2) and good pH-triggered release behavior. In addition, the DOX/MSN@Gelatin could enter cells through endocytosis and distribute mainly in the lysosomes, and DOX could release from the pores of the MSN into the cell nuclei. The good biocompatibility and efficient intracellular drug release were confirmed via MTT assay. The results clearly indicated that MSN covered with biocompatible gelatin coatings might serve as promising specific intracellular carriers for many drugs, proteins, and imaging agents.

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ASSOCIATED CONTENT Supporting Information. The zeta potential results, thermogravimetric analysis, the data of N2 adsorption–desorption isotherms for selected materials and the IC50 values. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *E-mail: [email protected]; [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported in part by the Project of Natural Science Foundation of China (Grants 21175039, 21322509, 21221003, 21305035 and 21190044), Research Fund for the Doctoral Program of Higher Education of China (Grant 20110161110016) and the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (2012TT1003). REFERENCES (1) Xiong, M.; Bao, Y.; Yang, X.; Wang, Y.; Sun, B.; Wang, J. Lipase-Sensitive Polymeric Triple-Layered Nanogel for “On-Demand” Drug Delivery. J. Am. Chem. Soc. 2012, 134, 4355−4362. (2) Cui, W.; Lu, X.; Cui, K.; Niu, L.; Wei, Y.; Lu, Q. Dual-Responsive Controlled Drug Delivery Based on Ionically Assembled Nanoparticles. Langmuir, 2012, 28, 9413–9420. (3) Yu, T.; Liu, X.; Bolcato-Bellemin, A. L.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.; Rocchi, P.; Behr, J. P.; Peng, L. An Amphiphilic Dendrimer for Effective Delivery of Small Interfering RNA and Gene Silencing In Vitro and In Vivo. Angew. Chem. Int. Ed. 2012, 51, 8478–8484. (4) Lin, W.; Kim, D. pH-Sensitive Micelles with Cross-Linked Cores Formed from Polyaspartamide ACS Paragon Plus Environment

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