Morphology Evolution and Spatially Selective Functionalization of

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Morphology evolution and spatially selective functionalization of hierarchically porous silica nanospheres for improved multi-drug delivery Nan Li, Dechao Niu, Yu Jiang, Chuanpeng Xu, Shan Pan, Jianping He, Jianzhuang Chen, Linlin Zhang, and Yongsheng Li Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b03735 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Morphology evolution and spatially selective functionalization of hierarchically porous silica nanospheres for improved multi-drug delivery Nan Lia, Dechao Niu*,a, Yu Jianga, Chuanpeng Xua, Shan Pana, Jianping Hea, Jianzhuang Chena, Linlin Zhangb, and Yongsheng Li*,a a 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 b State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China. ABSTRACT: Hierarchically porous materials are believed one of the most promising matrix materials due to their unique multi-modal 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 have 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 cetyl trimethyl ammonium bromide (CTAB) and tetraethyl orthosilicate (TEOS). 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 (CS-DMSNs) to well-defined hollow mesoporous silica nanospheres (HMSNs) has been achieved. To endow the capability of E-DMSNs as multi-drug delivery vehicles, a spatially selective functionalization strategy has been adopted to obtain dual-functionalized E-DMSNs (E-DMSNs-NH2/OH) with amino-functionalized large mesopores and hydroxyl-modified small mesopores. Thermogravimetric-differential scanning calorimetry (TG-DSC) 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 provide a new pathway for the rational design of antitumor multi-drug nanocarriers in further cancer treatment.

INTRODUCTION In the past decades, hierarchically porous materials have attracted great attentions due to their unique bi-modal or multi-modal porous structure and great application poten1-3 tials in catalysis, separation and biomedicine. Up to now, hierarchically porous materials with different components, 4, 5 including silica or carbon-based particles, metal-organic 6, 7 8 frameworks, organic polymers, have been prepared and used as imaging agents, drug delivery carriers, catalyst supports, gas uptake and transport vehicles. As a typical example, 9 Lee et al. reported the fabrication of spatially orthogonal bifunctional dual-porous silica architectures for co-loading 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 micro-scale 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 10 sizes, such as hollow mesoporous silica nanospheres, yolk11, 12 shell nanoparticles and dendritic mesoporous silica nano13 spheres, have been widely studied in the field of nanomedicine due to their unique loading capabilities for guest molecules/nanoparticles, excellent biocompatible feature and 14 easily surface modification. Recently, Wang et al. developed brain-like mesoporous silica nanocomposites consisting of 15 small pores in the cores and larger pores in the shells. Zhao

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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 hol16 low cavity and small guest molecules in the mesopores. Very recently, to obtain the controllable hierarchically po17 rous structures, Zhao et al proposed a heterogeneous oilwater bi-phase stratification reaction mechanism, and successfully prepared a series of 3D-dendritic mesoporous silica nanospheres with multi-generational 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 firstly synthesized by employing anionic block copolymer polystyrene-bpoly (acrylic acid) (PS-b-PAA) and cationic surfactant cetyl trimethyl ammonium bromide (CTAB) as co-templates, respectively, through a well-established dual-templating 18 route. Furthermore, to eradicate the small-pore shells which would hamper the utilization of the inside large pores for loading/delivery of large guest molecules, a simple selfassembly/solvothermal method was developed to synthesize monodispersed, large-pore silica nanospheres with ordered, 19 accessible and interconnected pore channels (up to 17 nm). Unfortunately, most of small pores in the shell and silica framework disappeared during the process of solvothermal treatment, 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 ultra-large mesopores and abundant small pores for higher loading spaces in this system, and further exploring their application potentials in multi-drug 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 cetyl trimethyl ammonium 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 ultra-large mesopores and small mesopores in the large-pore framework. To further explore the application potentials of E-DMSNs in multi-drug delivery, a spatially selective functionalization strategy was adopted to obtain the bifunctionalized E-DMSNs (E-DMSNs-NH2/OH) with aminofunctionalized large mesopores and hydroxyl-modified small mesopores, which is able to simultaneously deliver hydrophobic and hydrophilic drugs (i.e., curcumin and doxorubicin hydrochloride, respectively), further achieving improved

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therapeutic effect on SMMC-7721 (human hepatoma cells) tumor cells. Scheme 1. Schematic illustration for the controlled synthesis and morphology evolution of hierarchically porous silica nanospheres based on the electrostatic interaction-induced interfacial self-assembly behaviors between PS-b-PAA, CTAB and TEOS.

EXPERIMENTAL SECTION Chemicals All the chemicals were of analytical grade and used as received without further purification. Curcumin (Cur) was purchased from Sigma-Aldrich. Cetyl trimethyl ammonium bromide (CTAB) and 3-aminopropyl triethoxysilane (APTES) were obtained from Aladdin. Amphiphilic block copolymer, polystyrene-b-poly (acrylic acid) (PS-b-PAA) with different PAA block lengths, was synthesized via sequential atomic transfer radical polymerization (ATRP) as previously 20 reported. 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. Ultra-pure water (18.2MΩ 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 for several times. Finally, the product was obtained after air-dried and further calcined at 550 °C for 6 h in order 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 keep other parameters unchanged. In order to distinguish the hierarchically porous silica with different morphologies/pore structures, the definition of core-shell structured dual-mesoporous 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” dualmesoporous 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 bi-functionalized 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 cen-


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trifugation and washed several times with ultrapure water. After dried in vacuum oven at room temperature, the samples of E-DMSNs-NH2/OH were finally prepared. Synthesis of DOX/Cur co-loaded E-DMSNs-NH2/OH Because of the presence of amino groups on the large-pores, the 21, 22 hydrophobic molecule of Cur can be easily adsorbed. 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 for several times, collected by centrifugation and 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@E-DMSNs-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) for 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 co-loaded EDMSNs-NH2/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 incuba-1 tor shaker at 100 rpm min . 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 nm 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 for several times until the fluores23 cein could not be detected anymore. Laser scanning confocal microscopy (LSCM) observation For in vitro LSCM observation, SMMC-7721 cells (human hepatoma cells) were employed. The cell number in the 4 LSCM-specific culture dish was 10 cells per dish, and SMMC7721 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 co-loaded E-DMSNs-NH2/OH was also evaluated on SMMC3 7721 cells. For qualitative analysis, SMMC-7721 cells (5×10

cells per well) were incubated in 96-well plates containing RPMI-1640 medium at 37 °C for 24 h. Then different amount 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 was 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 BarrettJoyner-Halenda (BJH) methods, respectively. Field-emission scanning electron microscopy (FE-SEM) images were obtained by using a Hitachi S-4800 electron microscopy. Transmission electron microscopy (TEM) observations were carried out on a JEOL-2100F 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 o rate of 10 C/min in air.

RESULT AND DISSCUSSION Effect of the CTAB concentration on the interfacial selfassembly behaviors 18, 19

In our previous work, core-shell structured dualmesoporous 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 self-assembly behaviors between PS-b-PAA, CTAB and TEOS, further determine the morphologies and pore structures of hierarchically porous silica nanospheres. To verify the hypothesis, a series of samples have been synthesized by using different amounts of CTAB as reactant and the effects on morphologies and pore structures/sizes of porous silica nanospheres has 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 small-pore shell” structure. In addition, the shell thickness of CS-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 maintained (Figure 1b and 1e). 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 (EDMSNs-CTAB200), 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 19 with accessible large-pore channels, 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 display two capillary condensation steps at the relative pressure ranges of 0.2-0.3 and 0.7-0.9, indicating the presence of dual-mesoporous 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 18 system. Thus, it is noted that the genuine small-pore sizes of all the mentioned samples are ranged from 2~3 nm in the present work. While 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 2 structure, high specific surface area (843 m /g) and total pore 3 volume (1.09 cm /g) are obtained due to the presence of abundant small pores and uniformly distributed large pores. 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.

Figure 2. Nitrogen adsorption-desorption isotherms (a) and the corresponding BJH pore size distributions (b) of hierarchically porous silica nanospheres prepared by using different amounts of CTAB. 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-bPAA micellar aggregates and CTAB molecules or micelles. In the present study, the main driving forces for the selfassembly 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, when the amount of CTAB was relatively low (50 mg), the bi-layers 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

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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, 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-bPAA/CTAB/TEOS system, high CTAB concentrations prefer to the formation of “embedded” dual-mesoporous 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. Table 1. Structural parameters of different samples prepared by varying the amounts of CTAB. PS100-bSamples

CTAB

NH4OH

S

Dp

V

(mg)

(mL)

(m2/g)

(nm)a

(cm3/g)b

50

50

0.5

707.2

1.7/16.2

1.16

50

100

0.5

688.1

1.7/12.6

1.05

50

200

0.5

843.0

1.7/10.6

1.09

PAA16 (mg)

CS-DMSNsCTAB50

CS-DMSNsCTAB100

E-DMSNsCTAB200

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

b

Total pore

Scheme 2. The proposed mechanism for the formation of large pore channels with or without small-pore walls by varying the amounts of CTAB. Effect of the PAA block length on the interfacial selfassembly behaviors 24, 25

As Eisenberg 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 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 templates while keep the PS block length unchanged (100 repeat units). From TEM images shown in Figure 3, it can be seen that monodispersed, hollow mesoporous silica nanospheres (HMSNs-PAA8) are prepared when PS100-b-PAA8 was used as template. In contrast, coreshell 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 de-


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tails, 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 18, 19, 26 our previous work. In addition, for the sample of CSDMSNs-PAA35, the diameter of large pores in the core is about 16.1 nm, 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. 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. Figure 3. TEM images of hierarchically porous silica nanospheres prepared by using PS100-b-PAA8 (a, c) and PS100-bPAA35 (b, d), respectively. Table 2. Structural parameters of different samples prepared by using PS100-b-PAA8 or PS100-b-PAA35 as templates. Samples

a

Copolymer

CTAB

NH4OH

S

Dp

V

(mg)

(mg)

(mL)

(m2/g)

(nm)a

(cm3/g)b

100

0.5

965.7

1.7

0.69

100

0.5

729.6

1.7/16.1

HMSNs-

50/

PAA8

PS100-b-PAA8

CS-DMSNs

50/

-PAA35

PS100-b-PAA35

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

0.96 b

Total pore

Effect of the PS-b-PAA concentration on the interfacial self-assembly 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 CSDMSNs-copolymer10, CS-DMSNs-copolymer25 and E-DMSNscopolymer100, respectively, were synthesized by using different amounts/concentrations of PS-b-PAA as large-pore templates. For the sample of CS-DMSNs-copolymer10, 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 and d, Movie S1). When 25 mg of PS-b-PAA was used, the large-pore 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 nm to 23 nm. For the sample of CS-DMSNs-copolymer50, the same sample with CS-DMSNs-CTAB100 (Figure 1b and e), a thinner shell thickness of about 8 nm is observed, and the small-angle XRD pattern and 3D reconstructed TEM image shown in Figure S3 and Movie S2 reveal the presence of rela-

tive 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 ultra-large 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 with the sample of E-DMSNsCTAB200 (Figure 1). More importantly, the particle diameters are found varied from 283 nm 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 nm and 1.7 nm, respectively. However, totally differences on the specific surface area and pore volume are observed in Table 3. For example, the specific surface area decreases 2 gradually from 830 to 640 m /g with the amount of PS100-bPAA16 increases from 10 to 100 mg. Meanwhile, the pore vol3 ume increases from 0.62 to 1.42 cm /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, which is different from the CTAB-induced morphological changes discussed above. 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. PS100-bSamples

CTAB

NH4OH

S

Dp

V

(mg)

(mL)

(m2/g)

(nm)a

(cm3/g)b

10

100

0.5

829.7

1.7/12.6

0.62

25

100

0.5

746.4

1.7/12.6

0.73

100

100

0.5

639.6

1.7/12.6

1.42

PAA16 (mg)

CS-DMSNscopolymer10

CS-DMSNsCopolymer25

E-DMSNscopolymer100

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

b

Total pore

Effect of the pH value (ammonia concentration) on the 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 27, 28 pH value in alkaline aqueous solution, 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 (E-DMSNs-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 dualmesoporous 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 and 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. Table 4. Structural parameters of different samples prepared by using different amounts of NH4OH. Samples

E-DMSNs -NH4OH0.1

E-DMSNs -NH4OH1.5

PS100-b-PAA16

CTAB

NH4OH

S

Dp

V

(mg)

(mg)

(mL)

(m2/g)

(nm)a

(cm3/g)b

50

100

0.1

616.8

1.7/12.6

1.01

50

100

1.5

650.7

1.7/13.6

a

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

1.03 b

Total pore

Multi-drug 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 multi-drug co-delivery application. In this work, to verify the multi-drug loading and delivery ability of “embedded” dual-mesoporous silica nanospheres (E-DMSNsCTAB200), hydrophobic curcumin (Cur) and hydrophilic doxorubicin hydrochloride (DOX) were chosen as two model drugs due to their combined therapy effects in multi-drug 29-32 resistance (MDR) mechanisms. Firstly, to improve the loading capability of E-DMSNs-CTAB200 for hydrophobic drugs, a spatially selective functionalization strategy was used to obtain bi-functionalized, hierarchically porous nanospheres (E-DMSNs-CTAB200-NH2/OH) with aminofunctionalized 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 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 keeping most of CTAB micelles in the silica network. As shown in Figure S9, com-

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pared to the templates-filling E-DMSNs-CTAB200, the disappearing of adsorption bands of the NMP washed samples at -1 -1 around 699 cm and 1720 cm , 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 post-grafting 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), further led to the formation of E-DMSNs-CTAB200-NH2/OH. The successful amino modification and CTAB removal are confirmed by the FT-IR spectra -1 (Figure S9), where the amino groups at around 1550 cm are -1 observed and the C-H groups at around 2800-3100 cm 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 post-grafting process, implying that these small pores in step 1 can be easily blocked. However, the residual CTABfilled small pores in Step 1 show up again after the extraction process in acidic ethanol solution (step 3), 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 nm and 9.7 nm (Figure 6b and Table S1), respectively. More importantly, the morphology, pore structure and monodispersity of the E-DMSNs-CTAB200-NH2/OH remain unchanged after the whole pore functionalization (Figure S11), which is important to further investigate its multi-drugs delivery capability. Figure 6. Synthesis and properties of E-DMSNs-CTAB200NH2/OH: (a) The 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. 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 co-loaded in the E-DMSNsCTAB200-NH2/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


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amount of about 58.6 % is obtained for DOX after 5 d in PBS (pH=5.4), while only 12.5 % is measured in neutral PBS (pH=7.4). This pH dependent release behavior of DOX is believed to attribute to the interaction between DOX mole35, 36 cules and silica matrix. 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 37 groups of Cur. Thus, it is anticipated that the prepared bifunctionalized E-DMSNs-CTAB200-NH2/OH in the present work can be applied as a kind of promising pH-dependent multi-drug nanocarriers in further tumor chemotherapy. To confirm the therapeutic effects of DOX/Cur@EDMSNs-CTAB200-NH2/OH in tumor cells, liver cancer cells SMMC-7721 were chosen as model cells. Firstly, FITC dyes were employed to label the silica network and investigate the cellular 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-CTAB200-NH2/OH was evaluated by MTT assay. As shown in Figure 6e, more than 98 % cell viability is obtained after treated with E-DMSNs-CTAB200NH2/OH at different concentrations, indicating that no obvious toxicity is found for the nanocarrier. In contrast, cell survival ratio of DOX/Cur@E-DMSNs-CTAB200-NH2/OH is 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 co-loaded 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 multi-drug including small drugs and bio-macromolecules (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.

CONCLUSION In summary, a electrostatic interaction-induced interfacial self-assembly strategy has been proposed to fabricate a series of hierarchically porous silica nanospheres with controllable morphologies for the mixture system of PS-bPAA/CTAB/TEOS under basic conditions. By investigating the effects of CTAB concentrations on the interfacial selfassembly behaviors, a kind of “embedded” structured dualmesoporous 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-b-PAA take effects on the interfacial selfassembly 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 bi-functionalized, “embedded” structured dual-mesoporous silica nanospheres for co-loading of both hydrophobic drug and hydrophilic drug. It is demonstrated that improved therapeutic effect of the Cur/DOX coloaded, hierarchically porous silica nanospheres on SMMC7721 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 multi-drug induced synergistic chemotherapy.

ASSOCIATED CONTENT Supporting Information. Supplementary figures including the structural parameters of different samples, small-angle XRD pattern, N2 sorption results, FT-IR spectra and confocal microscopy images. Two movies of CS-DMSNs-copolymer10 and CS-DMSNs-CTAB100 from various observation angles. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: +86-21-64250740. Fax: +86-21-64250740. E-mail: [email protected]; [email protected].

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.

ACKNOWLEDGMENT 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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Table of Contents


10

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Figure 1 63x33mm (300 x 300 DPI)





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Scheme 1 61x43mm (300 x 300 DPI)



Scheme 2 30x10mm (300 x 300 DPI)


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Table of contents 35x35mm (300 x 300 DPI)


Morphology Evolution and Spatially Selective Functionalization of

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