Morphology Evolution and Spatially Selective Functionalization of

Nov 30, 2017 - Moreover, by employing PS-b-PAA with shorter PAA block lengths as template, the morphology conversion of porous silica nanospheres from...
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Article Cite This: Chem. Mater. 2017, 29, 10377−10385

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Morphology Evolution and Spatially Selective Functionalization of Hierarchically Porous Silica Nanospheres for Improved Multidrug Delivery Nan Li,† Dechao Niu,*,† Yu Jiang,† Chuanpeng Xu,† Shan Pan,† Jianping He,† Jianzhuang Chen,† Linlin Zhang,‡ and Yongsheng Li*,† †

Lab of Low-Dimensional Materials Chemistry, Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China ‡ State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China S Supporting Information *

ABSTRACT: Hierarchically porous materials are believed one of the most promising matrix materials due to their unique multimodal pore structures and great application potentials in catalysis, separation, and biomedicine. In this article, a series of hierarchically porous silica nanospheres with adjustable morphologies and pore structures/sizes has been successfully developed by controlling the electrostatic interaction-induced interfacial self-assembly behaviors between anionic block copolymer polystyrene-b-poly(acrylic acid) (PS-b-PAA), cationic surfactant cetyltrimethylammonium bromide, and tetraethyl orthosilicate. Especially, “embedded” structured dual-mesoporous silica nanospheres (E-DMSNs) containing connected large mesopores (>10 nm) and abundant small mesopores (2−3 nm) in the large-pore framework have been prepared for the first time. Moreover, by employing PS-b-PAA with shorter PAA block lengths as template, the morphology conversion of porous silica nanospheres from core−shell structured dual-mesoporous silica nanospheres to well-defined hollow mesoporous silica nanospheres has been achieved. To endow the capability of EDMSNs as multidrug delivery vehicles, a spatially selective functionalization strategy has been adopted to obtain dualfunctionalized E-DMSNs (E-DMSNs-NH2/OH) with amino-functionalized large mesopores and hydroxyl-modified small mesopores. Thermogravimetric-differential scanning calorimetry analysis shows that the loading amount of curcumin (Cur) and doxorubicin hydrochloride (DOX) were about 3.4% and 10.0% in weight, respectively. In addition, the cytotoxicity assay and cellular uptake of DOX@Cur@E-DMSNs-NH2/OH on SMMC-7721 cells (human hepatoma cells) have been investigated. Thus, such a simple methodology to synthesize hierarchically porous silica with adjustable morphologies, pore sizes, and pore modifications provides a new pathway for the rational design of antitumor multidrug nanocarriers in further cancer treatment.

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such as hollow mesoporous silica nanospheres,10 yolk−shell nanoparticles,11,12 and dendritic mesoporous silica nanospheres,13 have been widely studied in the field of nanomedicine due to their unique loading capabilities for guest molecules/nanoparticles, excellent biocompatible features, and easily surface modification. Recently, Wang14 et al. developed brain-like mesoporous silica nanocomposites consisting of small pores in the cores and larger pores in the shells. Zhao15 et al. synthesized dual-mesoporous carbon@silica core−shell nanospheres and found that the mesoporous carbon cores have good affinity with hydrophobic drugs, while the silica shells present strong adsorption ability for hydrophilic drugs. In addition, they further prepared a kind of asymmetric single-hole mesoporous nanocages, which were used as multidrug carriers for large guest molecules in the eccentric hollow cavity and small guest molecules in the mesopores.16 Very recently, to obtain the

INTRODUCTION In past decades, hierarchically porous materials have attracted great attention due to their unique bimodal or multimodal porous structure and great application potentials in catalysis, separation, and biomedicine.1−3 Up to now, hierarchically porous materials with different components, including silica or carbon-based particles,4,5 metal−organic frameworks,6,7 organic polymers,8 have been prepared and used as imaging agents, drug delivery carriers, catalyst supports, gas uptake, and transport vehicles. As a typical example, Lee9 et al. reported the fabrication of spatially orthogonal bifunctional dual-porous silica architectures for coloading of different kinds of inorganic nanoparticles (Pt and Pd) in separated space and further demonstrated their advantages in catalytic cascade reactions. However, most of these reported hierarchically porous materials usually possess irregular particle morphologies and microscale particle sizes, which greatly limited their applications in biomedical fields, especially in drug delivery systems. To address these problems, silica-based materials with both hierarchically porous structures and nanoscale particle sizes, © 2017 American Chemical Society

Received: September 4, 2017 Revised: November 30, 2017 Published: November 30, 2017 10377

DOI: 10.1021/acs.chemmater.7b03735 Chem. Mater. 2017, 29, 10377−10385

Article

Chemistry of Materials controllable hierarchically porous structures, Zhao17 et al. proposed a heterogeneous oil−water biphase stratification reaction mechanism and successfully prepared a series of 3Ddendritic mesoporous silica nanospheres with multigenerational and hierarchical dendrimer-like center-radial mesopores channels. Alternatively, core−shell structured dual-mesoporous silica nanospheres consisted of small pores in the shell and ordered large-pore channels in the core were first synthesized by employing anionic block copolymer polystyrene-b-poly(acrylic acid) (PS-b-PAA) and cationic surfactant cetyltrimethylammonium bromide (CTAB) as cotemplates, respectively, through a well-established dual-templating route.18 Furthermore, to eradicate the small-pore shells that would hamper the utilization of the inside large pores for loading/delivery of large guest molecules, a simple self-assembly/solvothermal method was developed to synthesize monodispersed, large-pore silica nanospheres with ordered, accessible, and interconnected pore channels (up to 17 nm).19 Unfortunately, most of small pores in the shell and silica framework disappeared during the process of solvothermal treatment, which further led to the decrease of loading spaces/amounts for guest molecules. Thus, it is necessary to develop a new strategy to prepare hierarchically porous silica nanospheres with both accessible ultralarge mesopores and abundant small pores for higher loading spaces in this system and further explore their application potentials in multidrug delivery. To achieve this, we herein propose an electrostatic interaction-induced interfacial self-assembly strategy to develop a series of hierarchically porous silica nanoparticles with controllable morphologies, such as “embedded” structured dual-mesoporous silica nanospheres (E-DMSNs), core−shell structured dual-mesoporous silica nanospheres (CS-DMSNs), and hollow mesoporous silica nanospheres (HMSNs), by employing anionic block copolymer polystyrene-b-poly(acrylic acid) (PS-b-PAA) and cationic surfactant cetyltrimethylammonium bromide (CTAB) in the presence of tetraethyl orthosilicate (TEOS) at room temperature (Scheme 1). In detail, the morphology of hierarchical porous silica nanospheres can be converted from CS-DMSNs to E-DMSNs by easily

changing the CTAB concentration in the system. As a typical and unique hierarchical porous structure, E-DMSNs displayed a unique pore structure consisting of connected ultralarge mesopores and small mesopores in the large-pore framework. To further explore the application potentials of E-DMSNs in multidrug delivery, a spatially selective functionalization strategy was adopted to obtain the bifunctionalized E-DMSNs (E-DMSNs-NH2/OH) with amino-functionalized large mesopores and hydroxyl-modified small mesopores, which are able to simultaneously deliver hydrophobic and hydrophilic drugs (i.e., curcumin and doxorubicin hydrochloride, respectively), further achieving improved therapeutic effect on SMMC-7721 (human hepatoma cells) tumor cells.

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EXPERIMENTAL SECTION

Chemicals. All the chemicals were of analytical grade and used as received without further purification. Curcumin (Cur) was purchased from Sigma-Aldrich. Cetyltrimethylammonium bromide (CTAB) and 3-aminopropyl triethoxysilane (APTES) were obtained from Aladdin. Amphiphilic block copolymer, polystyrene-b-poly(acrylic acid) (PS-bPAA) with different PAA block lengths, was synthesized via sequential atomic transfer radical polymerization (ATRP) as previously reported.20 Doxorubicin hydrochloride (DOX) was purchased from Beijing Mesochem Technology Co., Ltd. Toluene, tetraethyl orthosilicate (TEOS), and N-methyl-2-pyrrolidone (NMP) were obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. Ultrapure water (18.2 MΩ cm) was used in all experiments. Synthesis of Hierarchically Porous Silica Nanospheres. In a typical synthesis, 50 mg of PS100-b-PAA16 was dissolved into 10 mL of tetrahydrofuran (THF). Then the above oil solution was poured into a mixed solution containing 40 mL of H2O, 200 mg of CTAB, and 0.5 mL of ammonia. After that, the mixed oil−water solution was diluted with 80 mL of ethanol containing 0.3 g of TEOS. After reaction for 18 h at room temperature, the as-prepared sample was collected by centrifugation (9000 rpm, 10 min) and rinsed with ultrapure water and ethanol several times. Finally, the product was obtained after air-dried and further calcined at 550 °C for 6 h to remove the surfactants (CTAB and PS-b-PAA). Other samples with various morphologies/ pore structures were prepared by varying different synthetic parameters (i.e., concentrations of PS-b-PAA/CTAB, PAA block lengths, and pH value) while keeping other parameters unchanged. To distinguish the hierarchically porous silica from different morphologies/pore structures, the definition of core−shell structured dualmesoporous silica nanospheres (CS-DMSNs) is employed to represent the sample with core−shell structure, hollow mesoporous silica nanospheres (HMSNs) is for the sample with hollow structure, and “embedded” dual-mesoporous silica nanospheres (E-DMSNs) is for the sample with large-pore channels throughout the whole sphere and small-pores filled in the silica matrix. Synthesis of Amino/Hydroxyl Bifunctionalized Hierarchically Porous Silica Nanospheres (E-DMSNs-NH2/OH). The synthesis procedure was similar to that for E-DMSNs, except that the calcination process was replaced with a further treatment with NMP. Then the as-prepared products were dispersed into 30 mL of toluene (anhydrous) and functionalized with 0.2 mL of APTES by reflux at 80 °C under nitrogen atmosphere overnight. Furthermore, the products were extracted by an ethanol-HCl solution (0.5 mL of concentrated HCl in 120 mL of ethanol) and collected by centrifugation and washed several times with ultrapure water. After dried in vacuum oven at room temperature, the samples of E-DMSNsNH2/OH were finally prepared. Synthesis of DOX/Cur Coloaded E-DMSNs-NH2/OH. Because of the presence of amino groups on the large-pores, the hydrophobic molecule of Cur can be easily adsorbed.21,22 Typically, 8 mg of Cur was dissolved in 4 mL of ethanol. Then 10 mg of E-DMSNs-NH2/OH was dispersed in the Cur solution and stirred at room temperature without light for 24 h. The products were washed quickly with ethanol and ultrapure water several times, collected by centrifugation, and

Scheme 1. Controlled Synthesis and Morphology Evolution of Hierarchically Porous Silica Nanospheres Based on Electrostatic Interaction-Induced Interfacial Self-Assembly Behaviors between PS-b-PAA, CTAB, and TEOS

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DOI: 10.1021/acs.chemmater.7b03735 Chem. Mater. 2017, 29, 10377−10385

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Chemistry of Materials

Figure 1. TEM images of hierarchically porous silica nanospheres prepared by using different amounts of CTAB: (a, d) 50 mg/CS-DMSNs-CTAB50, (b, e) 100 mg/CS-DMSNs-CTAB100, and (c, f) 200 mg/E-DMSNs-CTAB200, respectively. dried. For loading hydrophilic molecule of DOX into the pore channels, especially the small pores on the silica framework, 4 mg of DOX was dissolved in 4 mL of aqueous solution with 5% phosphate buffered saline solution (PBS, pH = 7.4). Then 10 mg of Cur@EDMSNs-NH2/OH was added into the solution, and the suspension was stirred at room temperature without light for 24 h. The products were washed quickly with PBS solution (pH = 7.4) several times, collected by centrifugation, and dried. The concentrations of the two drugs retained in the solution were determined by UV−vis spectrophotometer at 426 and 480 nm, respectively. In Vitro Release Behaviors of DOX/Cur Coloaded E-DMSNsNH2/OH. In vitro release experiments were carried out in PBS at different pH values (pH = 5.4 and 7.4). Briefly, 10 mg of DOX/Cur@ E-DMSNs-NH2/OH was suspended in 4 mL of medium, and the mixture was put into a straight shaped glass bottle. The bottle was incubated at 37 °C in an incubator shaker at 100 rpm min−1. At predetermined time intervals, the total release medium was taken out and replaced with an equal amount of fresh solution. The amount of released DOX or Cur was determined by UV−vis spectrophotometer at 480 or 426 nm. It is notable that, since the adsorption band of DOX may cover that of Cur, the release of Cur was recorded independently from Cur@E-DMSNs-NH2/OH. Preparation of FITC-Grafted E-DMSNs-NH2/OH (FITC-EDMSNs-NH2/OH). To graft the fluorescein isothiocyanate (FITC) functional groups on the E-DMSNs-NH2/OH, 30 mg of FITC was reacted with 300 μL of APTES in 3 mL of ethanol under dark conditions for 24 h. Then 100 μL of FITC-APTES solution was added into 5 mL of E-DMSNs-NH2/OH (2 mg/mL) ethanol solution with shaking and reacted under dark overnight. The as-prepared product was washed with ethanol and PBS solution several times until the fluorescein could not be detected anymore.23 Laser Scanning Confocal Microscopy (LSCM) Observation. For in vitro LSCM observation, SMMC-7721 cells (human hepatoma cells) were employed. The cell number in the LSCM-specific culture dish was 104 cells per dish, and SMMC-7721 cells were incubated with Roswell Park Memorial Institute 1640 medium (RPMI-1640) at 37 °C for 24 h. After that, the solution was replaced with fresh medium containing 50 μg/mL of FITC-E-DMSNs-NH2/OH and further incubated at 37 °C for 4 h. Then in vitro imaging of living cells was carried out after washing the cells with PBS to remove the excess materials. The cells were excited at 404 nm for DAPI and 488 nm for FITC. Cytotoxicity Assay. In vitro cytotoxicity assay of DOX/Cur coloaded E-DMSNs-NH2/OH was also evaluated on SMMC-7721 cells. For qualitative analysis, SMMC-7721 cells (5 × 103 cells per well) were incubated in 96-well plates containing RPMI-1640 medium at 37 °C for 24 h. Then different amounts of materials (0−500 μg/

mL) were added in and incubated for another 24 h. Finally, the cell viabilities were determined by MTT assay. Measurement and Characterization. Powder X-ray diffraction (XRD) data were collected on Bruker D8 Focus diffractometer equipped with Cu Kα radiation (λ= 1.5405 Å). Fourier translation infrared (FT-IR) spectra were conducted on a Nicolet 5700 Thermo FT-IR spectrometer using the KBr wafer technique. N2 sorption isotherms were measured at 77 K by using Micromeritics Tristar II 3020 system. The specific surface area and the pore size distribution were calculated by using the Brunauer−Emmett−Teller (BET) and the Barrett−Joyner−Halenda (BJH) methods, respectively. Fieldemission scanning electron microscopy (FE-SEM) images were obtained by using a Hitachi S-4800 electron microscope. Transmission electron microscopy (TEM) observations were carried out on a JEOL2100F electron microscope. The mean diameter of the samples was measured by dynamic light scattering (DLS) using a Zeta potential/ particle Sizer Nicomp TM 380 ZLS. Thermogravimetric-differential scanning calorimetry (TG-DSC) analysis was carried out on a simultaneous thermal analyzer (SDT Q600, TA Instruments) at a heating rate of 10 °C/min in air.

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RESULTS AND DISSCUSSION Effect of CTAB Concentration on Interfacial SelfAssembly Behaviors. In our previous work,18,19 core−shell structured dual-mesoporous silica nanospheres and large-pore silica nanospheres with ordered, accessible, and interconnected pore channels were prepared based on a well-established dualtemplating route. In the synthesis process, CTAB can not only be used as the templating agent for generation of small pores, but also as the stabilizing agent to form CTAB (molecules or micelles)-coated rod-like PS-b-PAA aggregates, which can connect with silica species (TEOS) to obtain the organic− inorganic hybrid network. Thus, it is deduced that CTAB plays a particularly important role on the regulation of interfacial selfassembly behaviors between PS-b-PAA, CTAB, and TEOS to further determine the morphologies and pore structures of hierarchically porous silica nanospheres. To verify the hypothesis, a series of samples has been synthesized by using different amounts of CTAB as reactant, and the effects on morphologies and pore structures/sizes of porous silica nanospheres have been investigated. As shown in Figure 1a and d, the sample of CS-DMSNs-CTAB50 prepared by using less CTAB (50 mg) as small-pore template displays welldefined spherical morphology with “large-pore core and smallpore shell” structure. In addition, the shell thickness of CS10379

DOI: 10.1021/acs.chemmater.7b03735 Chem. Mater. 2017, 29, 10377−10385

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Chemistry of Materials

From the above TEM images and N2 sorption results, it can be found that CTAB in the reaction system affects not only the morphological transformation, but also the large-pore sizes and distribution of small pores. These phenomena are highly attributed to the interfacial interaction between PS-b-PAA micellar aggregates and CTAB molecules or micelles. In the present study, the main driving forces for the self-assembly are interface electrostatic interactions between negatively charged PS-b-PAA, positively charged CTAB, and negatively charged silicon hydroxyl group, respectively. As shown in Scheme 2,

DMSNs-CTAB50 is estimated of about 20 nm. On increasing the amount of CTAB to 100 mg, the morphology and pore structure of CS-DMSNs-CTAB100 were found to be maintained (Figure 1b,e). However, the shell thickness decreased from 20 to 10 nm, and the small pores were formed both in the shell and the large-pore framework. Notably, with the amount of CTAB increased to 200 mg, a novel kind of “embedded” structure, dual-mesoporous silica nanospheres (E-DMSNsCTAB200), consisting of large pores with small pores in the framework, was obtained, which is evidenced by TEM and SEM observations (Figure 1c,f and Figure S1). In detail, the large pores (>10 nm) are located uniformly not only in the interior of silica nanospheres, but also on the surface of nanospheres. Meanwhile, plenty of disordered small pores (2−3 nm) are located in the large-pore frameworks. Moreover, compared to the previous reported porous silica nanospheres with accessible large-pore channels,19 E-DMSNs-CTAB200 was obtained in a simple one-pot and mild synthesis process, without further solvothermal treatment. To characterize the pore structures/ sizes of these samples, nitrogen sorption analysis was employed. As shown in Figure 2a, all the adsorption−desorption isotherms

Scheme 2. Proposed Mechanism for Formation of Large Pore Channels with or without Small-Pore Walls by Varying Amounts of CTAB

when the amount of CTAB was relatively low (50 mg), the bilayers of CTAB molecules with both sides of positive charge interact with the negative charged PAA blocks and Si−OH groups from the hydrolysis of TEOS, respectively. In this case, the large pores in the core region are generated from the sum of PS-b-PAA micellar aggregates and CTAB molecules, while the small pores in the shell corresponding to the CTAB micelles. On increasing the amount of CTAB to 100 mg, the PS-b-PAA micellar aggregates contribute the shrunken large pores due to the presence of CTAB micelles near PAA blocks. Meanwhile, both the interior pore walls and outside shells are filled with small pores. As much more CTAB (200 mg) was employed, the self-assembly of CTAB micelles and TEOS would occur inside the PAA blocks and the large pores only correspond to PS blocks, which further led to the decrease of large-pore sizes. This proposed mechanism would provide us a new pathway to control the pore sizes of the hierarchically porous materials, which is important to expand their application potentials in other fields such as nanomedicine and catalysis. Thus, in the present PS-b-PAA/CTAB/TEOS system, high CTAB concentrations prefer to the formation of “embedded” dualmesoporous structures with smaller sizes of large-pore, while relatively low CTAB concentrations would lead to the fabrication of core−shell structured dual-mesoporous silica nanospheres with larger sized large-pores. Effect of PAA Block Length on Interfacial SelfAssembly Behaviors. As Eisenberg24,25 et al. reported, various PS-b-PAA micellar aggregate morphologies such as spheres, rods, lamellae, vesicles, and large compound micelles consisting of small reverse micelles in aqueous solution could be obtained by changing the length of PS or PAA blocks. In this

Figure 2. (a) Nitrogen adsorption−desorption isotherms and (b) corresponding BJH pore size distributions of hierarchically porous silica nanospheres prepared by using different amounts of CTAB.

display two capillary condensation steps at the relative pressure ranges of 0.2−0.3 and 0.7−0.9, indicating the presence of dualmesoporous structures. From the pore size distribution curves (Figure 2b), the small pore sizes of these samples are calculated to be about 1.7 nm derived from CTAB micelles, which is slightly smaller than that obtained from TEM observation (Figure 1). The deviation on the small pore size is attributed to the used BJH calculation method, which usually underestimate the pore size by about 1 nm in the PS-b-PAA/CTAB-templated dual-mesoporous system.18 Thus, it is noted that the genuine small-pore sizes of all the mentioned samples ranged from 2−3 nm in the present work. The large pore size increases from 10.6 to 16.2 nm along with the CTAB amount decreases from 200 to 50 mg (Table 1). In addition, for the sample of E-DMSNsCTAB200 with unique “embedded” typed dual-mesoporous structure, high specific surface area (843 m2/g) and total pore volume (1.09 cm3/g) are obtained due to the presence of abundant small pores and uniformly distributed large pores.

Table 1. Structural Parameters of Different Samples Prepared by Varying Amounts of CTAB

a

samples

PS100-b-PAA16 (mg)

CTAB (mg)

NH4OH (mL)

S (m2/g)

Dp (nm)a

V (cm3/g)b

CS-DMSNs-CTAB50 CS-DMSNs-CTAB100 E-DMSNs-CTAB200

50 50 50

50 100 200

0.5 0.5 0.5

707.2 688.1 843.0

1.7/16.2 1.7/12.6 1.7/10.6

1.16 1.05 1.09

Pore diameter calculated by BJH method on the adsorption isotherm. bTotal pore volume. 10380

DOI: 10.1021/acs.chemmater.7b03735 Chem. Mater. 2017, 29, 10377−10385

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Chemistry of Materials

Overall, it is concluded that the morphology/pore structure of hierarchically porous silica nanospheres can be tuned from core−shell structured dual-mesoporous silica nanospheres to hollow structured mesoporous silica nanospheres by decreasing the PAA block lengths of PS-b-PAA. Effect of PS-b-PAA Concentration on Interfacial SelfAssembly Behaviors. In addition to the molecular weights or block lengths of PS-b-PAA, the concentration of block copolymers also affects their self-assembly behaviors with CTAB, further regulating the morphologies/pore structures of hierarchically porous silica nanospheres. In this part, three samples, namely CS-DMSNs-copolymer10, CS-DMSNs-copolymer25, and E-DMSNs-copolymer100, respectively, were synthesized by using different amounts/concentrations of PS-b-PAA as large-pore templates. For the sample of CS-DMSNscopolymer10, monodispersed, core−shell structured nanospheres with lamellae-like large-pore channels in the core and disordered small-pores in the shell are observed (Figure 4a,d and Movie S1). When 25 mg of PS-b-PAA was used, the largepore channels of DMSNs-copolymer25 changed from laminar to 3D interconnected and relative ordered pore structures. At the same time, the thickness of small-pore shell decreases from 36 to 23 nm. For the sample of CS-DMSNs-copolymer50, the same sample with CS-DMSNs-CTAB100 (Figure 1b,e), a thinner shell thickness of about 8 nm is observed, and the small-angle XRD pattern and 3D reconstructed TEM images shown in Figure S3 and Movie S2 reveal the presence of relative ordered large pore channels and disordered small mesopores. Noticeably, the relative weak peak at around 0.5° according to the large pores can be explained by the fact that ultralarge pore channels in the relative small particle size. By further increasing the concentration of PS-b-PAA, the small-pore shells almost disappear and the corresponding proportion of large-pore channels become larger (Figure 4c,f and Figure S4). Besides, there are some small pores in the large-pore framework, similar to the sample of E-DMSNs-CTAB200 (Figure 1). More importantly, the particle diameters are found varied from 283 to 190 nm by decreasing the amount of block copolymers owing to the decrease of core regions in size, as evidenced from the DLS results (Figure S5). From the pore size distribution curves in Figure S6, all the three samples have similar large and small pore sizes of 12.6 and 1.7 nm, respectively. However, total differences on the specific surface area and pore volume are observed in Table 3. For example, the specific surface area decreases gradually from 830 to 640 m2/g as the amount of PS100-b-PAA16 increases from 10 to 100 mg. Meanwhile, the pore volume increases from 0.62 to 1.42 cm3/g. The trends in specific surface area and pore volume are attributed to the alterations in amounts of small and large pores in the silica matrix, where small pores mainly contribute to the specific surface areas and large pores contribute to pore volumes, respectively. As a result, varying the amounts of PS-b-PAA in the synthesis can not only tune the morphological/pore structural transformation in an easy manner, but also determine the particle diameter of the hierarchically porous nanospheres,

part, to investigate the effects of PAA block lengths on the interfacial self-assembly behaviors in the system, PS-b-PAA with shorter (8 repeat units) or longer (35 repeat units) PAA blocks was employed as large-pore template, while the PS block length remained unchanged (100 repeat units). From TEM images shown in Figure 3, it can be seen that monodispersed, hollow

Figure 3. TEM images of hierarchically porous silica nanospheres prepared by using (a, c) PS100-b-PAA8 and (b, d) PS100-b-PAA35, respectively.

mesoporous silica nanospheres (HMSNs-PAA8) are prepared when PS100-b-PAA8 was used as template. In contrast, core− shell structured dual-mesoporous silica nanospheres (CSDMSNs-PAA35) were formed by utilizing PS100-b-PAA35 as pore template. N2 sorption analysis results further confirmed the morphological transformation, as evidenced from the pore size distribution curves (Figure S2). The reason for this morphological transformation is attributed to the alteration of PS-b-PAA micellar aggregates in aqueous solution. In detail, when PS100-b-PAA8 with short PAA block length was used, large compound micelles containing plenty of small reverse micelles or vesicles with large sizes are formed and act as “hollow” template to obtain hollow mesoporous silica nanospheres. On the other hand, rod-like PS-b-PAA micellar aggregates were obtained by using block copolymers with long PAA lengths (PS100-b-PAA35 and PS100-b-PAA16), further resulting in the formation of large-pore structure instead of hollow structure in the core region. The similar results have been achieved by simply changing the PS block lengths in our previous work.18,19,26 In addition, for the sample of CSDMSNs-PAA35, the diameter of large pores in the core is about 16.1 nm (Table 2), which is larger than that (12.6 nm) of CSDMSNs-PAA16 (the same sample with CS-DMSNs-CTAB100). Similar to the previous synthetic mechanism of CS-DMSNsCTAB100, the enlargement on the large-pore size is mainly attributed to the increased molecular weight of PAA blocks.

Table 2. Structural Parameters of Different Samples Prepared by Using PS100-b-PAA8 or PS100-b-PAA35 as Templates

a

samples

copolymer (mg)

CTAB (mg)

NH4OH (mL)

S (m2/g)

Dp (nm)a

V (cm3/g)b

HMSNs-PAA8 CS-DMSNs -PAA35

50/PS100-b-PAA8 50/PS100-b-PAA35

100 100

0.5 0.5

965.7 729.6

1.7 1.7/16.1

0.69 0.96

Pore diameter calculated by BJH method on the adsorption isotherm. bTotal pore volume. 10381

DOI: 10.1021/acs.chemmater.7b03735 Chem. Mater. 2017, 29, 10377−10385

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Chemistry of Materials

Figure 4. TEM images of hierarchically porous silica nanospheres prepared by using different amounts of PS-b-PAA as large-pore templates: (a, d) 10 mg, (b, e) 25 mg, and (c, f) 100 mg.

Table 3. Structural Parameters of Different Samples Prepared by Using Different Amounts of PS-b-PAA as Templates

a

samples

PS100-b-PAA16 (mg)

CTAB (mg)

NH4OH (mL)

S (m2/g)

Dp (nm)a

V (cm3/g)b

CS-DMSNs-copolymer10 CS-DMSNs-copolymer25 E-DMSNs-copolymer100

10 25 100

100 100 100

0.5 0.5 0.5

829.7 746.4 639.6

1.7/12.6 1.7/12.6 1.7/12.6

0.62 0.73 1.42

Pore diameter calculated by BJH method on the adsorption isotherm. bTotal pore volume.

which is different from the CTAB-induced morphological changes discussed above. Effect of pH Value (Ammonia Concentration) on Interfacial Self-Assembly Behaviors. In this part, we try to explore the effects of pH value (ammonia concentration) on the morphologies/pore structures of hierarchically porous silica nanospheres by changing the amounts of ammonium hydroxide, which can induce the hydrolysis and condensation of silicate species. Since the hydrolysis rate of TEOS accelerates along with the increase of pH value in alkaline aqueous solution,27,28 when 0.1 mL of ammonium hydroxide was used, the silicon hydroxyl groups were produced slowly, and the silicate species only condensed around CTAB-coated PS-b-PAA micelles in the core regions, leading to the formation of “embedded” structured dual-mesoporous structures (EDMSNs-NH4OH0.1) with small pores in the large-pore framework, as shown in Figure 5a and c. For the sample of CS-DMSNs-NH4OH1.5, the morphology of nanospheres presents core−shell structured dual-mesoporous silica nanospheres due to the quick formation of abundant silicon hydroxyl groups at relative high pH value, which result in the prioritized formation of core regions and subsequent sol−gel reaction between CTAB and Si−OH in the shell regions. The shell thickness of CS-DMSNs-NH4OH1.5 is about 16 nm (Figure 5b,d). The SEM images (Figure S7) also confirm the morphological transformation induced by pH value. Besides, the large-pore size of DMSNs-NH4OH1.5 is slightly higher than that of E-DMSNs-NH4OH0.1 (Figure S8 and Table 4), indicating that the changes of pH value have a little influences on the interfacial self-assembly behaviors between PS-b-PAA, CTAB, and TEOS mentioned in Scheme 2. Consequently, the morphology/pore structure of the hierarchically porous nanospheres can also be changed from “embedded” to core−shell dual-mesoporous silica structure by varying the alkalinity of the PS-b-PAA/CTAB/TEOS system.

Figure 5. TEM images of hierarchically porous silica nanospheres prepared by using different amount of NH4OH: (a, c) 0.1 mL and (b, d) 1.5 mL, respectively.

Multidrug Loading and Delivery of E-DMSNs. Hierarchically porous materials can provide different loading spaces for various guest molecules/nanoparticles, showing great application potentials in variety of fields, especially in multidrug codelivery application. In this work, to verify the multidrug loading and delivery ability of “embedded” dual-mesoporous silica nanospheres (E-DMSNs-CTAB200), hydrophobic curcumin (Cur) and hydrophilic doxorubicin hydrochloride (DOX) were chosen as two model drugs due to their combined therapy effects in multidrug resistance (MDR) mechanisms.29−32 First, to improve the loading capability of E-DMSNs-CTAB200 for hydrophobic drugs, a spatially selective functionalization strategy was used to obtain bifunctionalized, hierarchically 10382

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Chemistry of Materials Table 4. Structural Parameters of Different Samples Prepared by Using Different Amounts of NH4OH

a

samples

PS100-b-PAA16 (mg)

CTAB (mg)

NH4OH (mL)

S (m2/g)

Dp (nm)a

V (cm3/g)b

E-DMSNs-NH4OH0.1 E-DMSNs-NH4OH1.5

50 50

100 100

0.1 1.5

616.8 650.7

1.7/12.6 1.7/13.6

1.01 1.03

Pore diameter calculated by BJH method on the adsorption isotherm. bTotal pore volume.

observed and the C−H groups at around 2800−3100 cm−1 decrease obviously. In addition, N2 sorption results confirm the selective functionalization process (Figure S10 and Table S1). In detail, after NMP washing process (step 1), both small pores (1.5 nm) and large pores (11.2 nm) can be obtained, which means that part of CTAB can be removed in this step. Moreover, after amino functionalization in step 2, the large pore sizes decreased from 11.2 to 9.2 nm, and the reduced pore volumes revealed the successful grafting of amino groups only in the large pore channels. Meanwhile, the small pores vanish in this postgrafting process, implying that these small pores in step 1 can be easily blocked. However, the residual CTAB-filled small pores in Step 1 showed up again after the extraction process in acidic ethanol solution (step 3), which led to the successful selective modification of E-DMSNs-CTAB200 with different functionalization in separated small and large-pore channels. The final pore sizes of E-DMSNs-CTAB200-NH2/OH are 1.5 and 9.7 nm (Figure 6b and Table S1), respectively. More importantly, the morphology, pore structure, and monodispersity of the EDMSNs-CTAB200-NH2/OH remained unchanged after the whole pore functionalization (Figure S11), which is important to further investigate its multidrugs delivery capability. By taking advantage of this unique selective modified dualmesoporous structure and considering the interactions between −NH2 groups in large-pores/−OH groups in small-pores and phenolic hydroxyl groups of Cur/amino groups of DOX,33−36 respectively, it is believed that both hydrophobic Cur and hydrophilic DOX can be coloaded in the E-DMSNs-CTAB200NH2/OH. From the thermogravimetric-differential scanning calorimetry (TG-DSC) results shown in Figure S12 and Table S2, the loading amounts of Cur and DOX were calculated to be 3.4% and 10.0% in weight, respectively. Figure 6c presents the release curves of DOX and Cur in PBS at different pH values. As expected, a cumulative release amount of about 58.6% was obtained for DOX after 5 d in PBS (pH = 5.4), while only 12.5% was measured in neutral PBS (pH = 7.4). This pH dependent release behavior of DOX is believed to attribute to the interaction between DOX molecules and silica matrix.35,36 For Cur, due to its poor solubility in aqueous solution, an extremely low value of 5.1% was released in PBS (pH = 7.4) after 5 d. In contrast, hydrophobic Cur shows better release behavior in acidic PBS (pH = 5.4) and the cumulative release amount of Cur is recorded to be 19.5% after 5 d. The significantly enhanced release can be probably explained by the fact that relatively low pH value would improve the solubility of Cur and weaken the interaction force between the amino groups in large-pores and hydroxyl groups of Cur.37 Thus, it is anticipated that the prepared bifunctionalized E-DMSNsCTAB200-NH2/OH in the present work can be applied as a kind of promising pH-dependent multidrug nanocarriers in further tumor chemotherapy. To confirm the therapeutic effects of DOX/Cur@E-DMSNsCTAB200-NH2/OH in tumor cells, liver cancer cells SMMC7721 were chosen as model cells. First, FITC dyes were employed to label the silica network and investigate the cellular

porous nanospheres (E-DMSNs-CTAB200-NH2/OH) with amino-functionalized large pores and hydroxyl-modified small pores in the silica framework, respectively. As shown in Figure 6a, the synthetic process of E-DMSNs-CTAB200-NH2/OH can

Figure 6. Synthesis and properties of E-DMSNs-CTAB200-NH2/OH: (a) synthetic illustration; (b) N2 sorption isotherm and the corresponding BJH pore size distribution (insert); (c) drug release profiles of Cur and DOX in PBS (pH = 5.4, 7.4); (d) LSCM image of SMMC-7721 cells treated with E-DMSNs-CTAB200-NH2/OH grafted with FITC groups (green fluorescence of FITC and blue fluorescence of DAPI were employed for labeling the cells); (e) SMMC-7721 cell viabilities after 24 h of incubation with different materials under a series of concentrations.

be divided into three steps. In Step 1, for the sample of templates-filling E-DMSNs-CTAB200, N-methyl-2-pyrrolidone (NMP), a good solvent for PS polymer was first chosen to remove the block copolymers, while most of CTAB micelles were kept in the silica network. As shown in Figure S9, compared to the templates-filling E-DMSNs-CTAB200, the disappearing of adsorption bands of the NMP washed samples at around 699 and 1720 cm−1, which represent the PS and PAA blocks of PS-b-PAA, respectively, indicates the complete removal of PS-b-PAA. In Step 2, the exposed large-pore channels were modified with amino groups by traditional postgrafting method, while the small pores were protected with the filling of CTAB. Then the small pores with hydroxyl groups were obtained via an extraction approach in EtOH/HCl solution (Step 3), which further led to the formation of EDMSNs-CTAB200-NH2/OH. The successful amino modification and CTAB removal are confirmed by the FT-IR spectra (Figure S9), where the amino groups at around 1550 cm−1 are 10383

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Chemistry of Materials uptake of DOX/Cur@E-DMSNs-CTAB200-NH2/OH. The LSCM images in Figure 6d and Figure S13 display remarkable intracellular green luminescence from the cytoplasm and blue light from DAPI in the cell nucleus, respectively, implying the successful cellular uptake of nanoparticles. Furthermore, the in vitro antitumor activity of DOX/Cur@E-DMSNs-CTAB200NH2/OH was evaluated by MTT assay. As shown in Figure 6e, more than 98% cell viability was obtained after treated with EDMSNs-CTAB200-NH2/OH at different concentrations, indicating that no obvious toxicity was found for the nanocarrier. In contrast, cell survival ratio of DOX/Cur@E-DMSNsCTAB200-NH2/OH was lower than that of either Cur or DOX-loaded samples at the same concentration, suggesting that the therapeutic effect can be substantially improved by employing the dual-drug coloaded hierarchically porous nanospheres as an advanced nanocarrier platform. In fact, considering the practical application of this nanocarrier in cancer therapy, it is necessary to investigate the synergistic effects of multidrug including small drugs and biomacromolecules (genes or proteins) by employing this kind of hierarchically porous silica nanospheres as nanocarriers in animal models, which will be done in our further work.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-21-64250740. Fax: +86-21-64250740. *E-mail: [email protected]. ORCID

Yongsheng Li: 0000-0003-0896-339X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (Grant No. 2016YFA0203700); Shanghai Rising-Star Program (16QA1401300); NSFC (Grant Nos. 51572083, 51572084, 51461165202, 51472085); the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, 111 Project (B14018); The Fundamental Research Funds for the Central Universities.

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CONCLUSION In summary, a electrostatic interaction-induced interfacial selfassembly strategy has been proposed to fabricate a series of hierarchically porous silica nanospheres with controllable morphologies for the mixture system of PS-b-PAA/CTAB/ TEOS under basic conditions. By investigating the effects of CTAB concentrations on the interfacial self-assembly behaviors, a kind of “embedded” structured dual-mesoporous silica nanospheres with abundant small mesopores only located into the large-pore framework have been synthesized for the first time. Besides, the concentration or block lengths of PS-bPAA take effect on the interfacial self-assembly behaviors, further resulting in the controlled transformation of various morphologies/pore structures, such as core−shell structured dual-mesoporous silica nanospheres consisting of large pores in the core and small pores in the shell and traditional hollow mesoporous silica nanospheres. Furthermore, a spatially selective functionalization strategy was adopted to prepare bifunctionalized, “embedded” structured dual-mesoporous silica nanospheres for coloading of both hydrophobic drug and hydrophilic drug. It is demonstrated that improved therapeutic effect of the Cur/DOX coloaded, hierarchically porous silica nanospheres on SMMC-7721 cells has been achieved. Overall, this unique and interesting “embedded” structured hierarchically porous silica nanostructure and its selective spatial functionalization in separated pore spaces will provide us a new pathway in developing excellent nanocarrier platform for multidrug induced synergistic chemotherapy.

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Movie of CS-DMSNs-CTAB100 from various observation angles (MPG)

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b03735. Structural parameters of different samples, small-angle XRD pattern, N2 sorption results, FT-IR spectra, and confocal microscopy images (PDF) Movie of CS-DMSNs-copolymer10 from various observation angles (MPG) 10384

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Chemistry of Materials

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