Fabrication of Mesoporous Silica Nanoparticle with Well-Defined

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Fabrication of Mesoporous Silica Nanoparticle with Well-defined Multicompartment Structure as Efficient Drug Carrier for Cancer Therapy in vitro and in vivo Anhe Wang, Yang Yang, Yanfei Qi, Wen Qi, Jinbo Fei, Hongchao Ma, Jie Zhao, Wei Cui, and Junbai Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12031 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016

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ACS Applied Materials & Interfaces

Fabrication of Mesoporous Silica Nanoparticle with Well-defined

Multicompartment

Structure

as

Efficient Drug Carrier for Cancer Therapy in vitro and in vivo Anhe Wang, †,¶ Yang Yang, †,¶ Yanfei Qi,*, § Wen Qi,§ Jinbo Fei,‡ Hongchao Ma,‡ Jie Zhao,‡ Wei Cui,‡ and Junbai Li*,†,‡ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center

for Nanoscience and Technology, Chinese Academy of Sciences (CAS), Beijing 100190, China. ‡

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Colloid and

Interface Science, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China. §

School of Public Health, Jilin University, Changchun 130021, China.

¶

These authors contributed equally to this work.

KEYWORDS: silica particles; vaterite particles; multicompartment structure; drug carrier; cancer therapy

ACS Paragon Plus Environment

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ABSTRACT: The vaterite particles are composed of particulate CaCO3 nanoparticles, which offer an ideal platform to synthesize architectures with hierarchical structure. Herein we show that mesoporous silica particles with well-defined multicompartment structure are fabricated by employing vaterite particles as templates. The obtained silica particles inherited the structure feature of vaterite and had excellent biocompatibility and biodegradability. Moreover, the silica particles were established as an efficient anticancer drugs carrier compared with hollow silica particles, which could be applied in cancer therapy in vitro and in vivo. The silica particles obtained here offer a cheap, facile, environmentally friendly avenue to assembly of hierarchical drugs carriers.

1. Introduction Silica-based nano/micromaterials have attracted much interest because of their great potential of applications in a wide range of fields, such as catalysis, coatings and biomedicine.1-8 Particularly, those with well-defined hierarchical structure have been the most promising candidates for drug delivery due to their designable porosity and excellent biocompatibility.6 Recent years, silica materials with multicompartment structure have obtained significant attention because they can carry multiple payloads in a single carrier but release them in separated spatially manner. Compared to other counterparts with simple solid and hollow structure, the multicompartment one can satisfy the key demands better for some crucial applications (multidrug delivery, multilevel catalysis, multistimuli-responsive sensors and hierarchical microreactors, etc.).6,9-11 Up to now, there have been a lot of reports on many kinds of multicompartment architectures, such as multiple emulsions, vesosomes (liposomes-inliposomes), and polymersomes-in-polymersomes.10 In general, “Bottom-up” and “Top-down”


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strategies are the main ways to prepare multicompartment architectures.10,12-14 For the “Bottomup” one, it is needed to offer building blocks (liposomes, cubosomes, polymersomes, polymer capsules and colloidosomes, etc.) to construct multicompartment particles through the interaction among the building blocks.10 However, regarding to the “Top-down” one, particles with multicompartment structure should be prepared firstly (water-in-oil-in-water (W/O/W) and oilin-water-in-oil (O/W/O) double emulsions), which play a role of templates for multicompartment particles.12-14 Nevertheless, most of these approaches have different limitations, such as the requirement of unusual or complicated fabrication devices, time-consuming, low yields production, insufficient subcompartments.12 CaCO3 particles are used in a wide range of fields (toothpastes, cosmetics, paper industry, water treatment and biomedical therapies). Especially, the vaterite species have been intensively studied as templates or carriers because of their good biocompatibility, porous structure, large surface area, easy production and removal under mild conditions.15,16 The vaterite particles composed of particulate calcium carbonate nanoparticles are ideal templates for fabrication of architecture with well-defined multicompartment structure. Although CaCO3 particles have been taken as templates to fabricate silica particles with the advantages of involving no surfactants an easily template removal.17-19 The obtained silica particles possess hollow or fuzzy sponge-like structures because the used templates are aragonite, calcite or impure vaterite which lack the unique structure of vaterite particles.15.16 Therefore, the construction of silica particles with welldefined multicompartment structure is still a challenge. The key here is how to keep vaterite particles stable enough during the whole reaction process. To address this issue, vaterite CaCO3 particles stabilized by starch were serving as templates to fabricate multicompartment silica particles (denoted as MSP) in a controlled manner. After hydrolysis of tetraethyl orthosilicate


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(TEOS) on vaterite particles and removal of the templates, MSP was obtained by copying the structure of vaterite precisely. Meanwhile, the residual starch on the surface of MSP reduced their potential toxicity in biomedical application. The obtained MSP was certified to be an efficient carrier for anticancer drugs, and further showed a good efficacy for cancer therapy in vitro and in vivo. 2. Experimental Section 2.1. Materials. Tetraethyl orthosilicate (TEOS) and 3-Aminopropyl-triethoxysilane (APTEOS) were from Acros. Na2CO3, CaCl2·2H2O, Ethylene diamine tetraacetic acid sodium (EDTA), Ammonium hydroxide, Doxorubicin (DOX), Soluble starch, Rhodamine-6G, FITC-dextran (Mw~2000), 6-CF (5(6)-carboxyfluorescein), 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Cy5 was purchased from Amresco. Cell Counting Kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. Alexa Fluor® 488 WGA and Hoechst 33342 Cell marker were obtained from Molecular Probes Inc. Hypocrellin B (HB) was obtained from Beijing Sybrisk Science & Technology Co., LTD, Beijing, China. Other reagents were obtained from Beijing Chemical Reagent Co., Beijing, China. The water used throughout the experiment was purified with a Milli-Q integral A10 system from Millipore Co., USA. 2.2. Preparation of starch doped vaterite particles. Starch doped vaterite particles with a meaning size 400 nm were synthesized by the method described by Wei et al.20 with some modifications. Firstly, soluble starch (0.25 g) was added to 100 mL water and then heated to boiling to get a transparent starch aqueous solution (0.25%, wt%). After cooling to room temperature, CaCl2 (0.07351 g) was added to the starch aqueous solution (0.005 M), then stirred for about 30 min. Finally, 100 mL Na2CO3 aqueous solution (0.005 M) was injected into CaCl2-


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starch aqueous solution under vigorous stirring, and kept stirring overnight. The obtained particles were carefully washed by water and ethanol, and then dried in vacuum. After adjusting the concentration of CaCl2 to 0.1 M or 0.0025 M, vaterite particles with a size about 1.2 µm and 300 nm were obtained, respectively. CaCO3 particles without starch (600~1000 nm) were synthesized by mixing CaCl2 and Na2CO3 solutions according to improved literature procedures.15-16 In a typical experiment, 100 mL (0.005 M) Na2CO3 was poured in to a solution of 100 mL CaCl2 (0.005 M), and the mixture was intensely agitated on a magnetic stirrer (1200 rm min-1) with stirring times of 10 min. The prepared particles were washed with deionized water thoroughly for three times and ethanol, and then dried in vacuum. 2.3. Preparation of MSP. 50 mg vaterite particles with meaning size 300 nm, 400 nm and 1.2 µm or CaCO3 without starch were incubated in 2 mL TEOS ethanol solution for 2 h. The concentration of TEOS was varied from 12.5 to 100% (12.5, 25, 50, 75, 100%, v/v%). After the adsorption of TEOS, the particles were centrifuged, and then moved to a flask with a mixture solution (ammonium hydroxide 2.5 mL (30 wt%), water 2.5 mL, ethanol 6 mL, unless otherwise noted) to allow the hydrolysis reaction to last 12 h. The effect of hydrolysis medium on the morphology of MSP was also investigated. The concentration of ethanol was varied from 0 to 75% (0, 50, 75%, v/v%, here, the volume of ammonium hydroxide was fixed at 2.5 mL, the total reaction volume was 11 mL), and then the reaction system was kept under vigorous stirring overnight. The final products were washed with water for three times, and incubated in EDTA solution (0.1 M, pH 7.4) for 1 h to remove the templates. This process was repeated for at least three times to remove the templates completely. At last, the obtained products were washed with water for three times and dried in vacuum for the following experiments. Other methods please refer to the supplementary data for further details.


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2.4. Characterization and instrumentation. UV-visible spectra (UV-vis) were recorded with a HITACHI U-3010 UV-visible spectrophotometer. Confocal laser scanning (CLSM) micrographs were taken with an Olympus FV1000 confocal system, which has a 60×oil-immersion objective and a numerical aperture of 1.4. The transmission electron microscopy (TEM) images were acquired by using a JEM-1011 and JEM-2011(JEOL, Japan). The scanning electron microscopy (SEM) images and energy-dispersive X-ray spectrum (EDX) pattern were obtained with an S4800 instrument with 10 kV accelerating (HITACHI, Japan). Zeta potential was documented by a dynamic light scattering technique (Zetasizer Nano, Malvern). The BET analysis was determined by a Brunauer-Emmett-Teller analyzer (BET, ASAP 2020 (M+C), Micromeritics Instrument Corporation). 3. Results and Discussion MSP was synthesized according to the procedure and as an efficient drug carrier depicted in Scheme. 1. As templates, starch doped vaterite particles with a diameter of about 400 nm were prepared by a one-pot method.20 As shown in Figure 1a, b, the vaterite particles are comprised of particulate calcium carbonate nanoparticles with a size of 10-70 nm, and the cross section of particle shows obviously the densely stacked calcium carbonate nanoparticles (Supplementary Figure S1). After adjusting the reaction condition (the concentration of CaCl2 and Na2CO3), vaterite particles with size about 300 nm and 1.2 µm were produced (Figure S2, the detail was given in supplementary data). Thermogravimetric analysis (TGA) indicates that the templates are composed of starch and CaCO3 (Supplementary Figure S3).20 Upon incubated in tetraethylorthosilicate (TEOS) ethanol solution overnight at room temperature, the pores of vaterite templates were densely filled with TEOS molecules. After hydrolysis of TEOS (at room temperature) and removal of the templates with ethylenediaminetetraacetic acid sodium (EDTA),


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Figure 2 shows a new kind of silica particles with well-defined multicompartment structure are produced at high yields. As the size of templates decreases from 1.2 µm to 300 nm, the TEM data clearly reveals that the MSPs have the same size of templates and are comprised of nanocapsules with a size of 10~70 nm which is consistent with the size of calcium carbonate nanoparticles of vaterite (Figure 2 left). SEM images (Figure 2 right) also display that the MSPs have similar size and surface morphology of vaterite. Moreover, the split single MSP shows their multicompartment structure of MSP (Figure 2 right inset), and the selected area electron diffraction (SAED) pattern verifies the crystal form of MSP was amorphous (Figure 2 left inset). We expect that MSP benefits the loading of drugs with high efficiency and release them in separated spatially manner. As a contrast, a typical one step of silica particles formation, TEOS was added in to the vaterite particle suspension in the hydrolysis medium, was performed, only a mixture of solid and hollow silica particles were obtained (Supplementary Figure S4), therefore. The pre-adsorption of TEOS by CaCO3 is a key step to form multicompartment silica nanoparticles. It should be noted that the doped starch could keep the vaterite particles stable enough during the whole experimental process. No XRD characteristic peaks assigned to aragonite or calcite were detected, further demonstrating that the vaterite particles maintained their crystal form during the hydrolysis reaction of TEOS (Supplementary Figure S5). In the absence of starch, only hollow cubic silica shells and fragments were obtained in our case (Supplementary Figure S6a). It is proposed that vaterite particles without starch transformed from vaterite to calcite with smooth surface during the reaction process (Supplementary Figure S6b), resulting in the TEOS hydrolysis occurring only on the surface of CaCO3.15,16,20 To further investigate the formation process of the MSP, the effect of experimental parameters, such as TEOS concentration, ethanol


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content in hydrolysis solution and reaction temperature were studied in detail. Figure S7a reveals the different morphologies of the MSP along with the change of ethanol content. The shells of nanocapsules (building unit of MSP) became thicker and more intact as the content of ethanol changed from 0 to 75% (0, 50, 75% (v/v%), room temperature, the concentration of TEOS in the TEOS adsorption step were fixed at 50%, (v/v%)). The reason may be that a high content of ethanol increases the solubility of TEOS in the mixture solution in our case, which supports the hydrolysis of TEOS resulting in a thick and intact MSP.21,22 Furthermore, the effect of the TEOS concentration on the morphology of the MSP has also been investigated at fixed hydrolysis condition (Room temperature, hydrolysis medium: ammonium hydroxide 2.5 mL (30 wt%), water 2.5 mL, ethanol 6 m). As shown in Figure S7b, no intact MSP could be obtained as the TEOS concentration was below 50 % (v/v%). It is reasonable to assume that TEOS with high content can cover the whole surface of vaterite particles due to their large specific surface area.15,16,20 In contrast, as the concentration of TEOS was up to 50%, perfect MSP could be produced. It has been reported that the reaction temperature had a great effect on the hydrolysis rate of TEOS, here, we performed the hydrolysis reaction at 5 oC (TEOS concentration: 50%, v/v%, hydrolysis medium: ammonium hydroxide 2.5 mL (30 wt%), water 2.5 mL, ethanol 6 mL,). As shown in Figure 3, silica particles with hollow structure were obtained (denoted as HSP), differing from the structure of MSP comprised of nanocapsules. We believe that low temperature inhibits the hydrolysis rate of TEOS, which just form the shell on the surface of vaterite particles during the hydrolysis reaction.23 The XRD pattern of vaterite particles demonstrates there was no crystal form difference before and after the hydrolysis of TEOS at 5 o

C (Supplementary Figure S8), excluding the formation of hollow structure caused by the crystal

transformation of vaterite particles during the reaction. Based on the above investigation, the


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morphology of the MSP could be regulated by the TEOS concentration, ethanol content and hydrolysis temperature.

Scheme 1. Schematic illustration of the preparation of silica particles with well-defined multicompartment structure (MSP) and their uploading with anticancer drugs.

Figure 1. Starch doped vaterite particles with a size of about 400 nm, (a) SEM and (b) TEM images of vaterite particles with low and high magnification.


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Figure 2. TEM and SEM images of the MSP with low and high magnification templated on different size of vaterite particles.(a) 1.2 µm; (b) 400 nm; (c) 300 nm. Left inset: SAED patterns of MSP. Right inset: cross section profile of a single one. (Room temperature, TEOS: 50%, v/v%, hydrolysis medium: ammonium hydroxide 2.5 mL (30 wt%), water 2.5 mL, ethanol 6 mL).

Figure 3. TEM and SEM images of the hollow silica particles (HSP) with low and high magnification templated on 400 nm vaterite particles (5 oC, TEOS: 50%, v/v%, hydrolysis medium: ammonium hydroxide 2.5 mL (30 wt%), water 2.5 mL, ethanol 6 mL). Inset: SEM image of broken HSPs.


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Energy Dispersive X-Ray Spectroscopy (EDX) of the MSP confirmed that the CaCO3 templates were removed completely (Figure 4a), and the carbon content of the MSP was about 21.59% (At%, atomic ratio). In contrast, the corresponding value of silica particles templated on CaCO3 without starch was only about 2.39% (Supplementary Figure S9). It is reasonable to believe that the extra carbon comes from the residual starch after template removal. In addition, the element mapping showed that the starch distributes over the whole MSP homogeneously (Figure 4a, insert). Furthermore, Thermogravimetric analysis (TGA) calculated that the amount of starch was about 5.0 % in weight (The mass loss at 250-350 °C was attributed to the combustion of starch,20 Supplementary Figure S10). X-ray photoelectron spectroscopy (XPS) (Figure 4b) of the MSP showed that the surface was composed of silicon, carbon and oxygen elements, and the content of carbon was about 33.3% (At%). High resolution O1s peak analysis of MSP showed a peak at 532.7 eV (Figure 4b), which was different from the O1s peak of starch and silica particles templated on CaCO3 without starch (Figure 4b, peak located at 532.5, 533, respectively). It indicates that there is a different surface chemistry between silica particles templated on CaCO3 without starch and MSP. The O1s peak obtained in the MSP could be fitted by four peaks (Figure 4b). One was at 533 eV, which could be considered as oxygen atoms in Si-OH bonds. Another peak located at 532.8 eV indicated oxygen atoms from the bond of C-O and C-O-C of starch, and the peak at 532 eV could be ascribed to oxygen atoms in the bond of Si-O-Si. The remaining peak at 530.4 resulted from the oxygen atoms in bonds of Si-OX (X=Ca, Na), but these states of oxygen were only present in negligible amounts.24-26 Based on the above results, it is believed that the surface of MSP is covered by starch, which could avoid extra steps to modify their surface and reduce the toxicity of MSP caused by Si-OH groups on their surface.27,28


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Zeta potential measurement showed that the MSP had a negative charge in aqueous solution, about -28 mV. After incubation in PBS buffer for 12 h, the distribution of MSP had no obvious change compared with those in water, indicating their excellent colloid stability in aqueous solution (DLS data, Supplementary Figure S11). Specific surface area of MSP with a meaning size 400 nm was estimated to be about 352.2 m2 g-1 from Brunauer-Emmet-Teller measurements (BET). Figure 4c, d show the corresponding nitrogen physisorption isotherms and Barret-JoynerHalenda (BJH) pore size distributions for the MSP, respectively. The isotherms displays a typical type-Ⅲ curve.29 And the pore size shows a wider distribution ranging from 2 to 80 nm. We believe that the smaller ones (2~10 nm) are assigned to pores on the nanocapsules of MSP formed during the template removal process by EDTA, and the bigger ones (10~80 nm) can be ascribed to the hollow nanocapsules of the MSP which are in agreement with TEM data showing that the MSP are comprised of nanocapsules with a size about 10~70 nm (Figure 2).

Figure 4. (a) EDX and the element mapping of MSP; (b) XPS spectrum of MSP, and O1s for pure starch, silica particles without starch and MSP, respectively; (c) BET nitrogen adsorption/desorption isotherms and (d) BJH pore size distributions of MSP, respectively. The MSP with a meaning size 400 nm.


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To assess the safety and biocompatibility of MSP in vitro, the viability of MCF-7 cells (human breast adenocarcinoma cells) was analysed with different concentration of MSP (Figure 5a, red bar). No significant in vitro cytotoxicity was observed at a concentration below 1 mg mL-1. However, the SiO2 particles without starch (Figure 5a, green bar, with a bigger size, 600-1000 nm, Figure S6a) displayed signs of cytotoxicity at a concentration below 1 mg mL-1. It is reported that silica particles with a smaller size inhibit the growth of cells more strongly than particles with a larger one.30,31 Therefore, we assume that the MSPs possess a better biocompatibility than silica particles (no starch) of similar size. Maybe the starch on the surface shields the silanol groups of silica nanostructures, which could reduce the cytotoxicity caused by silanol groups. Confocal laser scanning microscopy (CLSM) confirmed that the MSP was internalized by MCF-7 and located in the cytoplasm (Figure 5b) with a high efficiency, not causing obvious damages to the cell agreeing with the result of cell toxicity assay. Taken together, it indicates that the MSP has excellent biocompatibility and can be used as a safe drug carrier. Biodegradability is critical for materials which are used as drug carriers. To certify their biodegradability, the MSP was incubated in ɑ-amylase PBS solution (1mg mL-1) at 37 oC for 34 days. The corresponding morphological evolution of the MSP was recorded using TEM (Figure 5c). Most of the MSP remained after incubation in PBS or ɑ-amylase PBS solution for 1 h, while obvious hollow and loose features appeared in many of the MSP after 12 days for samples incubated in PBS or ɑ-amylase PBS solution. But one should note that the amount of MSP with this feature incubated in ɑ-amylase PBS was higher than those incubated in PBS solution. In the following days, the amount of such hollow and loose features of MSP incubated in ɑ-amylase PBS solution continued to increase leaving a spherical shell that became thinner after 24 days


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immersion. After 34 days, most of the MSP appeared as damaged and were completely collapsed into pieces of fragments. However, samples incubated in PBS were in the intermediate state, some decomposed into scattered fragments; others still kept structure integrity. It is proposed that MSP incubated in PBS or ɑ-amylase PBS decompose due to the enhanced solubility of silica in water at neutral pH in the beginning.32-34 However, because the existence of starch, the MSP incubated in ɑ-amylase solution has a faster decomposition rate compared with that of samples incubated in PBS, where the starch could be digested by ɑ-amylase and destroyed the structure integrity of MSP.

Figure 5. (a) In vitro viability of MCF-7 cells in the presence of SiO2 particles (no starch) and MSP with meaning size 400 nm (n=3, *P

Fabrication of Mesoporous Silica Nanoparticle with Well-Defined

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