Dual-Responsive Mesoporous Silica Nanoparticles Mediated

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Dual-Responsive Mesoporous Silica Nanoparticles Mediated Co-Delivery of Doxorubicin and Bcl-2 siRNA for Targeted Treatment of Breast Cancer Xiaojun Zhou, Liang Chen, Wei Nie, Weizhong Wang, Ming Qin, Xiumei Mo, Hongsheng Wang, and Chuanglong He J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b06759 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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The Journal of Physical Chemistry

Dual-Responsive Mesoporous Silica Nanoparticles Mediated Co-Delivery of Doxorubicin and Bcl-2 siRNA for Targeted Treatment of Breast Cancer Xiaojun Zhou, †, ‡ Liang Chen, † Wei Nie, † Weizhong Wang, † Ming Qin, † Xiumei Mo, †, ‡ Hongsheng Wang, † Chuanglong He*,†, ‡ †

College of Chemistry, Chemical Engineering and Biotechnology; State Key Laboratory for

Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, People’s Republic of China. ‡

College of Materials Science and Engineering, Donghua University, Shanghai 201620,

People’s Republic of China. *

Corresponding author:

Professor Chuanglong He

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China.

Tel. /fax: +86 21 6779 2742

Email address: [email protected] (C.L He)

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ABSTRACT: The combination of chemotherapy and gene therapy could induce the enhanced therapeutic efficacy in the cancer therapy. To achieve this goal, a new mesoporous silica nanoparticles (MSNs)-based co-delivery system was developed for targeted simultaneous delivery of doxorubicin (DOX) and Bcl-2 small interfering RNA (siRNA) into breast cancer cells. The multifunctional MSNs (MSNs-PPPFA) were prepared by modification of polyethylenimine-polylysine copolymers (PEI-PLL) via the disulfide bonds, to which a targeting ligand folate-linked polyethylene glycol (FA-PEG) was conjugated. The multifunctional MSNs-PPPFA nanocarrier has the ability to encapsulate DOX into the mesoporous channels of MSNs, while simultaneously carrying siRNA via electrostatic interaction between cationic MSNs-PPPFA and anionic siRNA. The resulting MSNs-PPPFA nanoparticles were characterized with various techniques. The drug release results reveal that DOX released from DOX-loaded MSNs-PPPFA are both pH- and redox-responsive, and the results of cell viability and hemolysis assays show that the functional nanocarrier has excellent biocompatibility. Importantly, the folate-conjugated MSNs-PPPFA showed significantly

enhanced

intracellular

uptake

in

the folate

receptor

overexpressed

MDA-MB-231 breast cancer cells than non-targeted counterparts, and thus results in more DOX and siRNA being co-delivered into the cells. Furthermore, the delivery of Bcl-2 siRNA obviously down-regulate the Bcl-2 protein expression, and thus targeted co-delivery of DOX and Bcl-2 siRNA by DOX@MSNs-PPPFA/Bcl-2 siRNA in MDA-MB-231 cells could induce remarkable cell apoptosis as indicated by the results of cell viability and cell apoptosis assays. These results indicate that the constructed DOX@MSNs-PPPFA/Bcl-2 siRNA co-delivery system is promising for targeted treatment of breast cancer. 2 ACS Paragon Plus Environment

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1. INTRODUCTION

In recent years, mesoporous silica nanoparticles (MSNs) have attracted extensive attention as nanocarriers for efficient encapsulation and protection of therapeutic drugs in biomedical applications.1 Due to the easy surface modification of MSNs, numbers of studies have focused on the development of MSNs-based controlled drug release systems. Such systems were designed to respond to internal or external stimuli, including pH,2 redox,3 enzyme,4 light5 and magnetic field6. To achieve these stimulus-responsive properties, two main strategies have been employed. One is choosing the appropriate gatekeepers attached to pore openings of drug-loaded MSNs by physically adsorption or covalently linking. Thus, the drug release from the channel of MSNs can be controlled by switching the open or close function of gatekeepers when suffering from offered stimuli. Various species were reported to be designed as the gatekeepers, such as polymers7 and inorganic nanoparticles8, 9. For example, our group has reported the construction of pH-responsive MSNs-based drug delivery systems by coating with polyelectrolytes onto the surface of MSNs via layer-by-layer (LbL) self-assembly technique.10, 11 The other strategy is that the drug was conjugated onto the pore or the surface of MSNs through a stimulus-responsive linkage.12 In cancer therapy, the desired drug delivery system was designed to accurately deliver drugs into the tumor site without any unwanted premature leakage.13 Thus, the targeted delivery of antitumor agent to tumor cells or tissues using nanoparticles is desirable to circumvent drug toxicity and improve therapeutic efficacy. To further improve the accumulation of MSN-based nanocarriers in tumor tissues, the general strategy is to conjugate these nanocarriers with targeting ligands,14 such as folate,15 hyaluronic acid,16 3 ACS Paragon Plus Environment


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Arg-Gly-Asp (RGD)13 and antibody17. Among these targeting ligands, folate has been widely selected as a targeting molecule for modification onto the MSNs due to its high affinity to folate receptors, which are often overexpressed in many human cancer cells, including breast cancer.18 The chemotherapy combined with gene therapy has received increasing attention in recent years due to its excellent therapeutic efficacy.19 By loading the anticancer drug into the mesoporous channels and complexing with therapeutic gene on the positively charged surface, MSNs can be easily developed as an efficient drug/gene co-delivery system for enhanced cancer therapy. Chen and co-workers have prepared dendrimer-modified MSNs for simultaneous delivery of doxorubicin (DOX) and Bcl-2 small interfering RNA (siRNA) into multidrug-resistant cancer cells.20 Meng et al. have reported the polyethylenimine (PEI) coated MSNs to deliver DOX and P-glycoprotein siRNA for overcoming drug resistance in cancer cells.21 Furthermore, Ma et al. have utilized folate conjugated PEI (PEI-FA) to bind with

phosphorylated

MSNs

through

electrostatic

interactions,

constructing

the

multifunctional nanoparticles for targeted co-delivery of drug and gene in cancer treatment.22 To endow MSNs with positive surface charge, cationic polymers were widely coated on the surface of MSNs through either covalent binding or electrostatic adsorption, such as dendrimer,20 cationic poly(amino acid)23 and PEI. 24 PEI is one of non-viral vectors that have been widely explored for gene delivery due to the outstanding proton buffering capacity. As compared to other cationic polymers, PEI can be further modified with targeting ligands due to its multi-amine structure. It is reported that polylysine-modified PEI (PEI-PLL) exhibits low toxicity, especially showing higher transfection efficiency and better tumor inhibition 4 ACS Paragon Plus Environment

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effects than those of PEI.25 However, to date, PEI-PLL coated onto the MSNs to construct the drug/siRNA co-delivery system is rarely reported. Therefore, we hypothesize that PEI-PLL modified MSNs plusing further conjugation of targeting ligands will be a promising multifunctional nanocarrier in cancer treatment. Herein, a new MSNs-based co-delivery system for targeted simultaneous delivery of anticancer drug and siRNA into cancer cells was developed, composed of PEI-PLL modification via cleavable disulfide bonds and further folate-linked polyethylene glycol conjugation for targeting folate receptor. Polyethylene glycol (PEG) in nanocarrier was capable of improving its biocompatibility, stability and avoiding immune stimulation.26 In addition, Bcl-2 protein, an anti-apoptotic protein, plays an important role in preventing cancer cell death, thus Bcl-2 siRNA was utilized to deliver simultaneously into the cancer cells with anticancer drug DOX to achieve increased therapeutic efficacy. The resultant multifunctional nanocarrier was characterized via different techniques. The in vitro drug release profiles were extensively studied, and the in vitro cytotoxicity and hemocompatibility of the nanoparticles were respectively evaluated by the CCK-8 and hemolysis assays. Moreover, the folate-meidated endocytosis of nanocarrier and co-delivery of DOX and siRNA into MDA-MB-231 breast cancer cells were respectively investigated by fluorescence observation and flow cytometric analysis. Most importantly, the anticancer effects of targeted co-delivery of DOX and Bcl-2 siRNA by nanocarriers were examined by the cell viability and cell apoptosis assays. The present multifunctional co-delivery system has the potential for targeted treatment of breast cancer.

2. MATERIALS AND METHODS 5 ACS Paragon Plus Environment


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2.1. Materials. Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), 3-aminopropyltriethoxysilane (APTES), fluorescein isothiocyanate (FITC), 1-ethyl-3(3-dimethylaminopropyl)

carbodiimide

N-hydroxysuccinimide

3-mercaptopropyltrimethoxysilane

(NHS),

hydrochloride

(EDC), (MPTMS),

3-mercaptopropionic acid (MPA), folic acid (FA), branched polyethylenimine (PEI, Mw = 25 kDa) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). 2,2′-dipyridyl disulfide (Py-SS-Py) and Glutathione (GSH) was obtained from Aladdin Chemistry, Co., Ltd. (Shanghai, China). NH2-PEG-COOH (Mw = 2000) was obtained from Shanghai Yanyi Biotechnology Corporation (Shanghai, China). Doxorubicin hydrochloride (DOX, Mw = 580) was obtained from the Beijing Huafeng United Technology Co., Ltd. (Beijing, China). MDA-MB-231 (human breast cancer cell line) and RAW 264.7 cells (murine macrophage cell line) were obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, USA). DMEM medium, penicillin, and streptomycin were from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). The human anti-Bcl-2 siRNA (Bcl-2 siRNA), scrambled siRNA and FAM-labeled Bcl-2 siRNA (FAM-siRNA) were purchased from GenePharma Co. Ltd. (Shanghai, China). The Bcl-2 siRNA duplex sequence consists of 5′-GUA CAU CCA UUA UAA GCU GdTdT-3′ (sense strand) and 5′-CAG CUU AUA AUG GAU GUA Cdtdt-3′ (sense strand).

The negative control siRNA (NC siRNA) with

scrambled sequence consists of 5′-UUC UCC GAA CGU GUC ACG UTT-3′ (sense strand) and 5′-ACG UGA CAC GUU CGG AGA ATT-3′ (sense strand). The Bcl-2 antibody (goat), the β-actin antibody (goat) and HRP-labeled rabbit anti-goat IgG H&L were purchased from 6 ACS Paragon Plus Environment

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Abcam (Cambridge, UK). The water used in all experiments was purified using a Milli-Q water purification system (Millipore, Bedford, MA) with a resistivity higher than 18.2 MΩ.cm. All other chemicals were of analytical grade from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2.

Synthesis

of

PEI-PLL

copolymers.

ε-Benzyloxycarbonyl

L-lysine

N-carboxyanhydride (Lys(Z)-NCA) was synthesized as reported previously.27 The PEI-PLL copolymers were synthesized according to the literature.25 Briefly, 0.408 g of PEI and 0.5 g of Lys(Z)-NCA were separately dissolved in dried chloroform. After complete dissolution, the Lys(Z)-NCA solution was added to the PEI solution and stirred for 72 h at 30 °C. Then, PEI-poly(benzyloxycarbonyl-L-lysine) [PEI-PLys(Z)] was synthesized via ring-opening polymerization (ROP) using PEI as a macro-initiator and Lys(Z)-NCA as the monomer. The mixture was concentrated and precipitated with excess diethyl ether. PEI-PLys(Z) was collected by filtration and drying under vacuum at room temperature. PEI-PLys(Z) was then dissolved in 10 mL of trifluoroacetic acid at room temperature. 3 mL of hydrobromic acid in 33% acetic acid (v/v) was added into the solution. After stirring for 2 h, the solution was added dropwise into an excess of ethyl ether. The production was collected and dried under vacuum at room temperature for 24 h. The final PEI-PLL was obtained by dialyzing (molecular weight cut-off, 14000 Da) against water (500 mL, changed four times) and freeze-dried for 24 h. 2.3. Synthesis of FA-PEG-COOH. 17.65 mg of FA was first activated with 6.9 mg of EDC and 4.14 mg of NHS in 5 mL of DMSO at room temperature for 3 h. The activated FA solution was dropwise added into 5 mL of DMSO solution containing 40 mg of 7 ACS Paragon Plus Environment


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NH2-PEG-COOH under vigorous magnetic stirring at room temperature for 3 d. Then, the reaction mixture was dialyzed (molecular weight cut-off, 1000 Da) against PBS solution (500 mL, changed three times) and water (500 mL, changed three times) for 3 d, followed by freeze-drying to obtain the FA-PEG-COOH production. 2.4. Synthesis of MSNs-SH and FITC-labeled MSNs-SH. MSNs-SH was prepared by modifications of previously reported methods.28 Typically, 1.0 g of CTAB and 0.28 g of NaOH were dissolved in 480 mL of deionized water. The mixture solution was heated to 80 °C and stirred vigorously. Then, 5.0 mL of TEOS was added dropwise to the above solution, followed by addition of 0.97 mL of MPTMS, and the mixture was under vigorous stirring at 80 °C for 2 h. The products were collected by centrifugation and washed several times with water and ethanol. Subsequently, the surfactant CTAB was removed by extraction in a solution of 9 mL of HCl and 160 mL of ethanol for 24 h. The surfactant-free MSNs-SH was collected by centrifugation. The FITC-labeled MSNs-SH was synthesized using the similar method as described in our previous work.29 Briefly, 4 mg of FITC and 44 µL of APTES were reacting in 1.0 mL of ethanol under dark conditions for 24 h to obtain the FITC-conjugated APTES (FITC-APTES). Then, 0.5 mL of FITC-APTES solution was added in the synthetic process of MSNs before TEOS addition. Finally, the surfactant was removed from the resulting nanoparticles as mentioned above. 2.5. Synthesis of MSNs-COOH. 1 g of MSNs-SH and excessive 2,2′-dipyridyl disulfide (Py-SS-Py) were first dispersed in 25 mL of anhydrous ethanol containing 1 mL of acetic acid. After that, the mixture was kept at room temperature for 24 h. The resulting 8 ACS Paragon Plus Environment

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MSNs-SS-Py product were collected by centrifugation and washed with anhydrous ethanol to remove excess 2,2′-dipyridyl disulfide. To obtain MSNs-SS-COOH (MSNs-COOH), 0.8 mL of acetic acid was added to a DMF (20 mL) solution of MSNs-SS-Py under the nitrogen atmosphere, followed by addition of excessive 3-mercaptopropionic acid. The reaction was allowed to keep stirring at 40 ºC for 24 h. Finally, the production was isolated by centrifugation and purified by washing with methanol. 2.6. Synthesis of polymers conjugated MSNs. Firstly, carboxyl on the surface of MSNs-COOH (100 mg) was activated with EDC (64 mg) and NHS (38 mg) in DMSO for 2 h. 60 mg of PEI-PLL polymers was dissolved in DMSO with the aid of water and then dropwise added into the above solution. After stirring for 48 h, the polymers conjugated MSNs nanoparticles (MSNs-PP) was collected by centrifugation and purified with water. 2.7. Synthesis of FA-conjugated MSNs. 13.46 mg of FA-PEG-COOH was dissolved in DMSO containing EDC (5.18 mg) and NHS (3.11 mg) to activate the carboxyl groups. After 2 h stirring, the activated FA-PEG-COOH was dropwise added into the MSNs-PP (60 mg) solution. The mixture was further stirred for 48 h to form FA-PEG conjugated MSNs-PP (MSNs-PPPFA). Finally, the obtained MSNs-PPPFA nanoparticles were also collected by centrifugation and purified with water. 2.8. Characterization. 1H NMR spectra were collected using a Bruker AV400 nuclear magnetic resonance spectrometer. Morphology and structure of nanoparticles were observed with a JEM-2100F (Jeol Ltd., Japan) transmission electron microscope (TEM) operating at 200 kV. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was recorded on Nicolet 6700 (Thermo, USA). Small-angle X-ray diffraction (SAXRD) 9 ACS Paragon Plus Environment


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pattern was obtained by using a D/MAX-2550PC (Rigaku Inc., Japan) diffractometer with the Cu Kα radiation at 45 kV and 40 mA. The size distribution of the nanoparticles was measured by dynamic light scattering (DLS) using a BI-200SM multi-angle dynamic/static laser scattering instrument (Brookhaven, USA). Zeta potential was measured by a Zetasizer Nano ZS instrument (Malvern, UK). Thermal gravimetric analysis (TGA) were carried out using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Germany) thermogravimetric analyzer. UV-vis spectra were recorded on a Jasco V530 UV-vis spectrophotometer (Jasco, Japan). N2 adsorption–desorption isotherm measurement was operated on the V-Sorb 2800P analyzer (Gold APP, China). The Brunauer–Emmett–Teller (BET) surface area and Barret–Joyner– Halenda (BJH) pore size distribution were measured. 2.9. DOX loading and in vitro release. For DOX loading, 10 mg of MSNs-PP or MSNs-PPPFA was mixed with 2 mL of DOX solution (1 mg/mL) in PBS solution (pH 5.0). After stirred for 24 h under the dark condition and subsequent vacuum treatment, the DOX loaded nanoparticles were isolated by centrifugation. The product was further washed several times with PBS. To determine the DOX loading capacity, the supernatant solution was collected and the residual DOX amount was calculated by UV-vis measurement at the wavelength of 480 nm. The DOX loading content and loading efficiency were respectively calculated according to eqs. (1) and (2). The loading content and loading efficiency were calculated to be 9.7% and 60.2% for MSNs-PP, and 8.0% and 48.9% for MSNs-PPPFA, respectively. Loading content (%) = (weight of loaded drug/weight of drug loaded nanocarrier) × 100

(1)

Loading efficiency (%) = (weight of loaded drug/initial weight of drug) × 100

(2) 10

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To investigate the drug release behaviors, the drug loaded samples were dispersed into PBS solution (pH 5.0 or 7.4) with/without 10 M GSH, shaking at the speed of 100 rpm at 37 ºC. At predetermined time intervals, 1 mL of PBS solution was taken out and then replaced with an equal volume of fresh PBS. For pH-responsive release, the sample was first exposed to PBS solution of pH 7.4, and then transferred to PBS solution of pH 5.0 at the predetermined period. The cumulative released amount of DOX was monitored from the absorbance at 480 nm according to a standard curve of DOX in the same PBS. 2.10. Gel retardation assay. The siRNA binding ability of the MSNs-PPPFA and DOX@MSNs-PPPFA nanoparticles were determined by agarose gel electrophoresis. The complexes of nanoparticles and siRNA at various weight ratios (1:1, 5:1, 10:1, 15:1, 20:1, 40:1 and 60:1) were firstly prepared by gently vortexing the mixtures of 5 µL of siRNA solution (40 µg/mL) and 5 µL of nanoparticles solution with different concentrations, and allowing to incubate for 30 min at room temperature. The siRNA amount is 0.2 µg per cell. The complexes were then loaded in the lanes for electrophoresis. Free siRNA was used as control. Electrophoresis was conducted on a 1.5% agarose gel containing 2 µL of 0.5 µg/mL ethidium bromide in 0.5 × TBE buffer at 100 mV for 30 min. The resulting gel was imaging using a UV transilluminator (Bio-Rad, USA). 2.11. Biocompatibility assay. The cell viability assays were carried out on two types of cells, RAW 264.7 and MDA-MB-231 cells. Cells were seeded into 96-well plates at a density of 1× 104 cells per cell in 100 µL DMEM medium. After 24 h incubation, the culture medium was replaced with new medium containing MSNs-PPPFA nanoparticles at different concentrations (12.5, 25, 50, 100, 200 and 400 µg/mL). The culture medium without 11 ACS Paragon Plus Environment


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nanoparticles was used as the control. After incubation for another 24 h, the cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) (Beyotime, China) method which performed in accordance with the manufacturer′s instructions. The absorbance was recorded with a microplate reader (Multiskan GO, Thermo, USA) at a wavelength of 450 nm. The cell viability was expressed as a ratio of the treated group to the control group. For the hemolysis assay, the fresh blood from healthy SD rat was collected in heparin-coated tubes. To collect the red blood cells (RBCs), the serum was removed from blood by centrifugation at 3000 rpm for 10 min. The RBCs were purified by washing more than three times with sterile PBS and then diluted with PBS solution up to a concentration of 10% (v/v). 0.3 mL of the RBCs solution was mixed with 1.2 mL of MSNs-PPPFA solution at different concentrations (12.5, 25, 50, 100, 200, 400 and 800 µg/mL). Here, sterile water and PBS solution were used as positive and negative controls, respectively. After incubation for 3 h at 37 ºC, the mixtures were centrifuged at 3000 rpm for 10 min. 100 µL of the supernatant was transferred into a 96-well plate and the absorbance was recorded using the microplate reader at 541 nm. The percent hemolysis of RBCs in each sample was calculated by the formula of hemolysis% = [(sample absorbance − negative control) / (positive control − negative control)] × 100%, and the average value was obtained from four parallel samples. 2.12. Cellular uptake studies. For cellular uptake of nanoparticles, MDA-MB-231 cells were grown in a 24-well plate at a density of 4 × 104 cells per cell for 24 h. Subsequently, the culture medium was replaced by fresh medium containing MSNs-SH, MSNs-PP and MSNs-PPPFA nanoparticles at the concentration of 25 µg/mL. For FA competition experiment, the cells were cocultured with MSNs-PPPFA nanoparticles containing free FA (1 12 ACS Paragon Plus Environment

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mg/mL). After incubation for 6 h, the cells were washed with PBS solution more than three times and then fixed with 4% paraformaldehyde solution for 15 min at room temperature. The cells were further treated with 0.1% Triton X-100 in PBS for 5 min. After that, the nucleus was stained with DAPI solution (Bestbio, Shanghai) for 5 min. All the samples were observed by using an inverted fluorescence microscope (Olympus IX 71, Japan). For cellular internalization of DOX and siRNA, 1 × 105 MDA-MB-231 cells were seeded into glass bottom dishes (35 mm) and incubated for 24 h. The DOX@MSNs-PP/FAM-siRNA and DOX@MSNs-PPPFA/FAM-siRNA complexes were prepared in serum-free DMEM medium and then added into the wells. A mixture of free DOX and naked siRNA was used as a

control.

For

FA

competition

experiment,

the

cells

were

incubated

with

DOX@MSNs-PPPFA/FAM-siRNA complexes containing free FA. The equivalent concentration of DOX was 4 µg/mL and the weight ratio of nanoparticles to siRNA was fixed at 20:1. After incubation with the different formulations for 4 h, the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde solution for 15 min at room temperature. DAPI solution was used to stain the nucleus. All the samples were visualized and imaged using a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 700, Germany). To quantify the intracellular uptake of DOX and FAM-siRNA, MDA-MB-231 cells were treated with samples as mentioned above and then carried out on the flow cytometer (FCM, BD Biosciences). 2.13. Bcl-2 gene silencing by western blotting. MDA-MB-231 cells were seeded into a 6-well plate at a density of 2 × 105 cells per well for 24 h. Then, the cells were treated with 13 ACS Paragon Plus Environment


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MSNs-PPPFA/Bcl-2 siRNA at weight ratios of 5:1, 10:1 and 20:1, respectively. The concentration of Bcl-2 siRNA was 1 µg/mL and naked Bcl-2 siRNA was also carried out. After 48 h treatment, the cells were incubated with RIPA buffer supplemented with 1 mM PMSF (Beyotime, China). The proteins of each sample were collected by centrifugation at 12000 g for 5 min at 4 ºC. Total protein concentration was measured using the BCA assay kit (Beyotime, China). Finally, Western blotting was performed with equal protein content of each sample on the SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 2.14. Cytotoxicity assay.

The in vitro cytotoxicities were evaluated by the CCK-8

method against MDA-MB-231 cells. After the cells were cultured in 96-well plate for 24 h, the culture medium was replaced with various nanoparticles/siRNA complexes in complete medium. The complexes were first prepared in serum-free DMEM medium and then diluted to the desired concentrations with complete medium. For each formulation, including free DOX, DOX@MSNs-PP, DOX@MSNs-PPPFA, DOX@MSNs-PPPFA/NC siRNA and DOX@MSNs-PPPFA/Bcl-2 siRNA, the equivalent DOX concentration was from 0.5 to 4 µg/mL. After 48 h incubation, the medium was removed and the cytotoxicities were evaluated using CCK-8 assay. Finally, the absorbance was recorded and the cell viability was calculated. 2.15. Apoptosis assay. The apoptosis assay in MDA-MB-231 cells was studied by flow cytometry using the Annexin V-FITC apoptosis kit (Beyotime, China). The cells were seeded in a 6-well plate at a density of 2 × 105 per well for 24 h. Then, the cells were treated with free DOX, DOX@MSNs-PP, DOX@MSNs-PPPFA and DOX@MSNs-PPPFA/Bcl-2 siRNA at DOX concentration of 2 µg/mL. After 48 h treatment, the cells were harvested by 14 ACS Paragon Plus Environment

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trypsinization and conducted according to the manufacture′s protocol of kit. Subsequently, the stained cells were immediately analyzed by FACS Calibur flow cytometry (Becton Dickinson, USA). 2.16. Statistical analysis. All experiments were conducted at least three times, and data are presented as mean ± standard deviation (SD). Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by post hoc Tukey’s method to test all pair-wise mean comparisons. The statistical significance for all tests were considered at *P < 0.05 and **P < 0.01.

3. RESULTS AND DISCUSSION

3.1. Synthesis of PEI-PLL and FA-PEG-COOH. Prior to prepare the multifunctional MSNs-based nanocarriers, the PEI-PLL copolymers and FA-conjugated PEG were first synthesized. The 1H NMR spectrum of PEI-PLL is shown in Figure S1. The resonance peaks at 2.31–2.86 ppm are attributed to the –CH2– of PEI. After grafting with PLL, new peaks in PEI-PLL are clearly seen at 3.83 ppm and 1.23–1.86 ppm, which are assigned to the –CH2– NH2 and three –CH2– in the PLL chain, respectively. For FA-PEG-COOH conjugate, the chemical structure characterized by 1H NMR is shown in Figure S2. Compared with the 1H NMR spectra of FA and NH2-PEG-COOH monomers, the appearance of characteristic resonance peaks associated with FA at 6.63, 7.62 and 8.65 ppm in the 1H NMR spectrum of FA-PEG-COOH demonstrates the successful conjugation of FA onto the NH2-PEG-COOH. These results confirm the successful synthesis of PEI-PLL copolymers and FA-PEG-COOH.



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3.2. Synthesis and characterization of MSNs-PPPFA. To achieve simultaneous delivery of DOX and siRNA in the same nanocarrier, a new multifunctional and dual-responsive drug release system was designed and fabricated. The synthetic route for this vehicle is displayed in Scheme 1. Firstly, MSNs-SH was prepared by a facile method, and then treated with Py-SS-Py and MPA to obtain MSNs-COOH with cleavable disulfide bonds. Subsequently, the PEI-PLL copolymer was covalently grafted onto the surface of MSNs-COOH as the caps by the reaction between the amino groups of PEI-PLL and terminal carboxyl groups of MSNs-COOH via EDC/NHS chemistry. Finally, FA-PEG-COOH was conjugated onto the PEI-PLL modified MSNs also through EDC/NHS chemistry to form amide bonds between the terminal carboxyl groups of FA-PEG-COOH and amino groups on PEI-PLL, which enables the functional nanoparticles to simultaneously deliver drug and siRNA into breast cancer cells with over-expressed FA receptor.


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Scheme 1. Construction of the dual-responsive DOX@MSNs-PPPFA/Bcl-2 siRNA complexes for targeted co-delivery of drug and siRNA into cancer cells.

The morphology and structure of the prepared nanoparticles were characterized by TEM. As shown in TEM images (Figure 1A-D), the prepared MSNs-SH and MSNs-PPPFA were uniformly spherical shape with a mean diameter around 200 nm. For MSNs-SH, an ordered mesoporous network can be clearly observed in the magnified TEM image (Figure 1B). After modification, a polymeric shell coated on the surface of MSNs-SH can be seen in the MSNs-PPPFA sample (Figure 1D). The hydrodynamic diameter and size distribution of MSNs-SH and MSNs-PPPFA were obtained by DLS measurements. As shown in Figure 1E, the measured hydrodynamic diameters were 213 nm for MSNs-SH and 249 nm for 17 ACS Paragon Plus Environment


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MSNs-PPPFA with the corresponding PDI of 0.13 and 0.17, respectively. The larger hydrodynamic diameter of MSNs-PPPFA indicated that the hydrophilic polymers were successfully grafted onto the nanocarrier. In addition, the specific surface area was 929 m2/g and the pore size was predominately centered at 2.6 nm for MSNs-SH (Figure S3), which was obtained by BET and BJH analysis, respectively.

Figure 1. TEM images of (A, B) MSNs-SH and (C, D) MSNs-PPPFA. (E) Size distributions of MSNs-SH and MSNs-PPPFA.


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To further verify the successful grafting of PEI-PLL and FA-PEG on the MSNs, a series of characteristic techniques were utilized, including FTIR, TGA, zeta potential and spectral measurements. The FTIR spectra were used to confirm the step-wise synthesis of MSNs-SH, MSNs-COOH, MSNs-PP and MSNs-PPPFA, as shown in Figure 2A. In the spectrum of MSNs-COOH, a new absorption peak at 1713 cm–1 assigned to the C=O stretching vibration in the carboxyl group was appeared as compared with MSNs-SH, indicating that MPA was successfully modified on the MSNs-SH.30 For the spectrum of MSNs-PP, the absorption peak at 1713 cm–1 disappeared, instead of showing a strong absorption peak of C=O stretching vibration in the amide group at 1635 cm–1. Moreover, the peaks at 1560 and 1448 cm–1 were observed, which were assigned to the N–H bending vibration and C–N stretching vibration, confirming the successful conjugation of MSNs with PEI-PLL.31 After modification of FA-PEG, the feature peaks indexed as the symmetric stretching and bending vibration of C−H groups in FA-PEG were emerged at 2962 and 1450 cm–1,32 demonstrating that FA-PEG was linked to MSNs-PP. TGA analysis was also used to confirm the successful preparation of MSNs-PPPFA. Figure 2B presents the residual amount of each sample under the high temperature treatment. After the temperature up to 900 ºC, the weight loss of MSNs-SH, MSNs-COOH, MSNs-PP and MSNs-PPPFA were 18.22, 21.32, 26.17 and 27.53 wt.%, respectively. The results further revealed that MSNs-PPPFA was successfully fabricated, while the amounts of PEI-PLL and FA-PEG on the MSNs can be roughly estimated to be 4.81 and 1.36 wt.%, respectively. In addition, the zeta potential change was further used to demonstrate the successful modification. From Figure 2C, it can be seen that the zeta potential of MSNs-SH was -17.3 mV, and then decreased to -43.4 after modifying with MPA 19 ACS Paragon Plus Environment


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because of the strong negative charge of –COOH. However, the zeta potential of MSNs-PP was reversed to 39.7 mV after grafting with PEI-PLL copolymers due to the presence of abundant amine in PEI-PLL. After conjugation with FA-PEG, the zeta potential of MSNs-PPPFA was decreased to 26.3 mV, which was related with the decrease of amino groups by the chemical reaction and the shielding effect of polymer chain.33 It is noted that the DOX-loaded MSNs-PPPFA shows a positive charge of 22.3 mV, suggesting that DOX@MSNs-PPPFA nanoparticles can effectively bind to negatively charged siRNA through electrostatic interaction. Figure 2D shows the UV-vis spectra of the measured samples. Compared with MSNs-PP and FA-PEG-COOH, MSNs-PPPFA gives a characteristic absorption peak of the FA molecules around λ=280 nm, demonstrating the surface modification of FA-PEG.22 Taken together, these results suggested that MSNs-PPPFA nanoparticles were successfully prepared.


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Figure 2. Characterization of the synthesized nanoparticles. (A) FTIR spectra of MSNs-SH, MSNs-COOH, MSNs-PP and MSNs-PPPFA. (B) TGA curves of MSNs-SH, MSNs-COOH, MSNs-PP and MSNs-PPPFA. (C) Zeta potential measurements of MSNs-SH, MSNs-COOH, MSNs-PP, MSNs-PPPFA and DOX@MSNs-PPPFA. (D) UV-vis spectra of MSNs-PP, MSNs-PPPFA and FA-PEG-COOH.

3.3. Agarose gel retardation assay. The nuclear acid binding capacity of the prepared nanoparticles was evaluated by agarose gel retardation assay. As shown in Figure 3, with the increasing weight ratio of nanoparticles to siRNA from 1:1 to 60:1, the electrophoretic mobility of siRNA gradually retarded. For the MSNs-PPPFA, when the weight ratio of nanoparticles to siRNA was up to 15:1, most of the siRNA was restrained with the 21 ACS Paragon Plus Environment


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nanoparticles, while allowing to completely retaining in the electrophoretic well at weight ratio of 20:1. But for DOX@MSNs-PPPFA sample, an extremely weak free siRNA band can be seen on the gel at the same weight ratio.

Figure 3. Electrophoretic mobility of free siRNA and siRNA complexes with (A) MSNs-PPPFA and (B) DOX@MSNs-PPPFA at different weight ratios of nanoparticles to siRNA.

3.4. DOX release profiles. In this study, the caps of PEI-PLL copolymer were attached onto MSNs surface via disulfide bonds which can be removed by the cleavage of disulfide bonds in the presence of GSH. Thus, the DOX release from DOX@MSNs-PPPFA was investigated in different buffer solutions (pH 5.0 and 7.4), as well as their combination with 10 mM GSH. As shown in Figure 4A, the results show that the release rate of DOX was low in the absence of GSH at pH 5.0 and 7.4, with about 34 % and 11% of released DOX respectively after 60 h. In contrast, more DOX was released from the vector at pH 5.0 and 7.4 22 ACS Paragon Plus Environment

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buffer solution containing 10 mM GSH, with up to 58% and 45% of loaded DOX released after 60 h, respectively. It was also found that the profiles showed a rapid release of DOX in the initial time, followed by slow and sustained release over the later time, implying that the polymer caps can be efficiently removed via the cleavage of disulfide bond by addition of GSH. Apart from the effect of GSH, the release rate was significantly promoted at pH 5.0 and the cumulative release of DOX was approximately three times higher than that at pH 7.4. In this case, the result clearly shows that DOX release from the DOX@MSNs-PPPFA was pH-responsive. To further certify this property, a DOX release experiment was performed by changing the buffer solution from pH 7.4 to pH 5.0. It can be seen that the DOX release was very slow when immersed in PBS solution of pH 7.4, followed by sharply increased after transferred to PBS solution of pH 5.0, as shown in Figure 4B. This pH-responsive drug release is probably attributed to the protonation of the amine groups on the PEI-PLL polymers. At lower pH condition, the PEI-PLL is positively charged due to the protonation of the amine groups. As a result, on the one hand, the protonation may cause increased electrostatic repulsion between the DOX molecules and the ionized amino groups, thereby resulting in a rapid drug release.11 On the other hand, mutual columbic repulsion of the ionized amino groups will lead to the swelling of the modified layer from the MSNs surface, and thus allow easier leakage of DOX from the nanocarriers.22 The obtained results suggest that the prepared DOX@MSNs-PPPFA offer great potential for tumor therapy thanks to the high GSH concentration in cancer cells and acidic micro-environments of tumor tissue.



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Figure 4. (A) In vitro release of DOX from DOX@MSNs-PPPFA with/without GSH at pH 5.0 and 7.4. (B) pH-responsive release of DOX from DOX@MSNs-PPPFA.

3.5. Biocompatibility study. To be used as gene carrier, the nanoparticles were expected to have low toxicity. Thus, the cytotoxic effects of resulting MSNs-PPPFA were evaluated at different particle concentrations (12.5, 25, 50, 100, 200 and 400 µg/mL) in MDA-MB-231 cells and RAW 264.7 cells by CCK-8 assay (Figure 5). It can be seen that the MSNs-PPPFA nanoparticles show no obvious cytotoxicity at tested particle concentrations against both MDA-MB-231 and RAW 264.7 cells, which demonstrate that MSNs-PPPFA have excellent cytocompatibility. To better understand the biocompatibility of the nanoparticles, the hemocompatibility was studied by using a hemolysis assay (Figure 6). In the hemolysis assay, hemoglobin released from RBCs into the solution can be directly observed and the hemolysis percentage can be obtained by measuring the absorbance of leaked hemoglobin. As shown in Figure 6A, no obvious hemolytic effects can be observed from the optical images. The quantitative results show that MSNs-PPPFA has negligible hemolytic activity, only approximately 1.6% of hemolysis percentage measured at the concentration up to 800 µg/mL 24 ACS Paragon Plus Environment

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(Figure 6B). The results of cytotoxicity and hemolysis assay indicate that the prepared MSNs-PPPFA nanoparticles possess good biocompatibility and can be used as gene carriers.

Figure 5. Cell viability assay of (A) MDA-MB-231 and (B) RAW 264.7 cells treated with MSNs-PPPFA at different particle concentrations.

Figure 6. Hemolysis assay of RBCs incubated with MSNs-PPPFA at different particle concentrations. PBS solution and water were used as negative and positive controls, respectively. (A) Digital photo taken after centrifugation. (B) Hemolysis percentage of RBCs 25 ACS Paragon Plus Environment


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after MSNs-PPPFA samples exposure, the absorbance is proportional to the amount of hemoglobin released from RBCs.

3.6. Cellular uptake. It is noted that effective cell internalization of nanocarriers plays a critical role in the delivery of drug/gene into the target cells. The in vitro cellular uptake experiments were conducted on MDA-MB-231 breast cancer cells, known to have high expression of folate receptor.34 The uptake of nanoparticles by MDA-MB-231 cells was investigated after 6 h cell culture, as shown in Figure 7. From the fluorescence images in Figure 7A, only little green fluorescence spots can be seen in the cells after incubation with bare MSNs-SH. In contrast, the cells treated with MSNs-PP exhibit apparent green fluorescence spots, approximately 6-fold higher fluorescence intensity than that of MSNs-SH (Figure 7B). As MSNs-PP was positively charged, to some extent, it could facilitate the cellular internalization by nonspecific electrostatic interactions between the positively charged nanoparticles and the negatively charged cell membrane.35 For further modification of FA-PEG on MSNs-PP, stronger fluorescence signal in cells can be observed in the MSNs-PPPFA treated sample, with about 2-fold increase of fluorescence intensity compared to MSNs-PP treated sample. However, when the cells were incubated with MSNs-PPPFA nanoparticles containing free FA, the fluorescence intensity was dramatically decreased. It is encouraged that the FA conjugation can increase the cellular uptake of nanoparticles apart from the assistance of positively charged character. These results confirm that the cellular uptake of MSNs-PPPFA nanoparticles can be enhanced by FA receptor mediated endocytosis because of the specific affinity between FA and FA receptor. 26 ACS Paragon Plus Environment

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Figure 7. (A) Intracellular uptake of nanoparticles into MDA-MB-231 cells observed by an inverted fluorescence microscope after 6 h incubation, all scale bar = 100 µm. (B) Quantitative analysis of cellular internalization of nanoparticles into MDA-MB-231 cells through measuring mean fluorescence intensity of FITC fluorescence in the cells, which was normalized to the MSNs-SH. Samples are MSNs-SH, MSNs-PP, MSNs-PPPFA and FA+MSNs-PPPFA (cells were cocultured with free FA), respectively.

**

P < 0.01 compared

with MSNs-SH; ##P < 0.01.

To study the co-delivery of DOX and siRNA, MDA-MB-231 cells were incubated with different formulations for 6 h and then investigated the uptake and intracellular distribution of DOX and FAM-siRNA in cells by CLSM (Figure 8). The cell nucleus was stained with DAPI and displayed the blue fluorescence. The green fluorescence and red fluorescence are from FAM-siRNA and DOX, respectively. As seen in Figure 8B, the free DOX can rapidly enter into the cells and diffuse into the cell nuclei through a passive diffusion mechanism after 6 h incubation.36 Nevertheless, no green fluorescence of FAM-siRNA was seen in the cells. The result reveals that the naked siRNA hardly can be internalized by the cells without the assistance of carrirers.37 For DOX@MSNs-PP/siRNA, both the red fluorescence spots and green fluorescence spots can be obviously observed in cells, mainly distributed in the cytoplasm (Figure 8C). As expected, the intracellular red and green fluorescence spots in MDA-MB-231 cells treated with FA-conjugated MSNs complexes were more than those treated with the former complexes (Figure 8D). According to the merged images, the overlap of red and green fluorescence indicates the simultaneous delivery of DOX and siRNA into 28 ACS Paragon Plus Environment

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cells. In addition, little amount of DOX was leaked from the carrier and accumulates in the cell nucleus, which demonstrate that DOX can release from the nanocarriers and eventually diffuse into the cell nucleus. The released DOX was possibly attributed to pH- and GSH-triggered drug release. Moreover, the green fluorescence was completely distributed in the cytoplasm, which was good for siRNA to silence the target gene within the cells. To further determine the role of FA in the co-delivery system, a competitive inhibition experiment

was

carried

out

by

incubating

the

MDA-MB-231

cells

with

DOX@MSNs-PPPFA/siRNA containing free FA. With treatment in the presence of free FA, it is clearly that the red and green fluorescence intensities of DOX and FAM-siRNA in MDA-MB-231 cells were weaker than those without the addition of free FA (Figure 8E). This result indicates that the uptake of FA-conjugated MSNs would be decreased by blocking the FA receptor mediated-endocytosis with excess free FA via competitive binding to the FA receptor on the cell surface. To further verify this, the quantitative flow cytometric analysis of cellular uptake of DOX and FAM-siRNA was carried out. According to the results of mean fluorescence intensity, the cells incubated with FA-conjugated complexes exhibit increased fluorescence intensity for both DOX and FAM-siRNA in comparison to incubating cells with non-targeted complexes (Figure S4). Similarly, in the presence of free FA, the fluorescence intensities of DOX and FAM-siRNA were relatively weaker. Consequently, our findings suggest that the as-prepared MSNs-PPPFA nanoparticles are favorable for co-delivery of anticancer drug and siRNA into breast cancer cells bearing FA receptor.



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Figure 8. Cellular uptake of DOX and FAM-siRNA in MDA-MB-231 cells treated with (A) control (without treatment), (B) a mixture of free DOX and naked FAM-siRNA, (C) DOX@MSNs-PP/FAM-siRNA,

(D)

DOX@MSNs-PPPFA/FAM-siRNA

and

(E)

DOX@MSNs-PPPFA/FAM-siRNA containing free FA. All scale bar = 20 µm.

3.7. Bcl-2 protein silencing analysis. The ability of Bcl-2 siRNA in silencing the targeted Bcl-2 protein expression in MDA-MB-231 cells was evaluated by western blot (Figure S5). To accurately reflect the silence effect of different treatments, β-Actin protein was selected as 30 ACS Paragon Plus Environment

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the internal standard. The MSNs-PPPFA/Bcl-2 siRNA complexes in different weight ratios were used to co-culture with the cells, and the naked Bcl-2 siRNA also included. Compared to the control group, no obvious silencing of the Bcl-2 protein for the naked Bcl-2 siRNA group was observed as indicated by the bands (Figure S5A), which means the naked Bcl-2 siRNA is difficult to be internalized by the cells. When the cells were treated with the MSNs-PPPFA/Bcl-2 siRNA complexes, a decreased darkness of the bands can be seen with the increased weight ratios of MSNs-PPPFA to siRNA. The Bcl-2 protein expression was effectively suppressed to 58.6% and 18.6% at the weight ratios of 10:1 and 20:1, respectively (Figure S5B). These results indicate that the used Bcl-2 siRNA has the silencing effect on Bcl-2 protein expression in MDA-MB-231 cells and the prepared MSNs-PPPFA could be a promising vector for gene delivery. 3.8. In vitro cytotoxicity. In order to evaluate the synergistic effect of co-delivered DOX and Bcl-2 siRNA by MSNs-PPPFA on the cytotoxicity against MDA-MB-231 cells, the cell viability of DOX@MSNs-PPPFA/Bcl-2 siRNA was measured by CCK-8 assay. For comparison,

the

cytotoxicities

of

DOX@MSNs-PP,

DOX@MSNs-PPPFA,

DOX@MSNs-PPPFA/NC siRNA (containing negative control siRNA) and free DOX were also investigated. The concentration of DOX was from 0.5-4 µg/mL, and the weight ratio of nanoparticles to siRNA was fixed at 20:1. As shown in Figure 9, the results show that the cytotoxicity against MDA-MB-231 cells gradually increased with increasing DOX concentration for all of the formulations after 48 h incubation. At an equivalent DOX concentration of 0.5 µg/mL, the five formulations display a minor cytotoxic effect and no significant differences among them were observed due to the low dose of DOX or Bcl-2 31 ACS Paragon Plus Environment


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siRNA. When the DOX concentration was reached to 2 µg/mL, the cell viabilities were decreased to 67.8%, 61.3%, 61.8% and 52% for DOX@MSNs-PP, DOX@MSNs-PPPFA, DOX@MSNs-PPPFA/NC siRNA and DOX@MSNs-PPPFA/Bcl-2 siRNA, respectively. The results reveal that the DOX@MSNs-PPPFA causes significantly higher cytotoxicity than that of DOX@MSNs-PP, which was most possibly ascribed to the enhanced internalization of nanoparticles via the FA receptor mediated endocytosis. Importantly, the co-delivery of DOX and Bcl-2 siRNA in DOX@MSNs-PPPFA/Bcl-2 siRNA nanocomplexes exhibit the lowest cell viability, which means that the simultaneous targeted delivery of DOX and Bcl-2 siRNA could obviously improve the chemotherapeutic efficacy of DOX compared to DOX delivery alone. The role of Bcl-2 siRNA was confirmed by replacing the Bcl-2 siRNA with the NC siRNA, where the gene sequence was scrambled. It is clear that the cells treated with DOX@MSNs-PPPFA/NC siRNA show no significant difference on cytotoxicity in comparison to that treated with DOX@MSNs-PPPFA, but killing efficiency was lower than that of DOX@MSNs-PPPFA/Bcl-2 siRNA. This result indicates that Bcl-2 siRNA could efficiently silence the Bcl-2 expression of MDA-MB-231 cells and then increase their sensitivity to DOX, resulting in facilitating the cell apoptosis.22, 37


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Figure 9. Cell viability assay of MDA-MB-231 cells treated with different samples for 48 h. The samples were (a-e) DOX@MSNs-PP, DOX@MSNs-PPPFA, DOX@MSNs-PPPFA/NC siRNA, DOX@MSNs-PPPFA/Bcl-2 siRNA and free DOX, respectively. The concentration of DOX was from 0.5-4 µg/mL, and the weight ratio of nanoparticles to siRNA was fixed at 20:1. *P < 0.05, **P < 0.01.

3.9. Apoptosis assay. To further investigate the therapeutic efficacy of simultaneous delivery of DOX and siRNA by functional MSNs, MDA-MB-231 cells were treated with different samples and then conducted on the flow cytometry to confirm the apoptotic efficiency. Both of the early apoptotic and late apoptotic cells were positive to annexin V-FITC staining,38 therefore the fluorescence intensity of stained cells reflecting the percentage of apoptosis. As shown in Figure 10, the cells treated with DOX@MSNs-PPPFA show a 22.51% of apoptotic cells, higher than that treated with DOX@MSNs-PP sample (16.35% of apoptotic cells). This enhanced efficiency could be attributed to the increased accumulation of DOX in cells which mediated by FA-modified nanocarrier compared to that of non-targeted nanocarrier. Interestingly, a much higher apoptotic rate (36.88%) was observed when the cells were treated with DOX@MSNs-PPPFA/Bcl-2 siRNA, with 14.37% of apoptotic cells enhanced as compared to that of DOX@MSNs-PPPFA, which confirm that the co-delivery of DOX and Bcl-2 siRNA offer synergistic apoptotic effects on MDA-MB-231cells. In addition, it is noted that the free DOX induces a much higher apoptotic rate (31.22%) than those of DOX@MSNs-PP and DOX@MSNs-PPPFA groups. In light of the previous results in Figure 8, this is likely because the free DOX can rapidly enter 33 ACS Paragon Plus Environment


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into the cells and accumulate in the nuclei. However, owing to the silence of Bcl-2 protein in cells by DOX@MSNs-PPPFA mediated Bcl-2 siRNA delivery resulting in high sensitivity to DOX, the apoptotic rate of the cells treated with co-delivery system was still higher than that with free DOX. As a result, more DOX was delivered into the cells by FA-conjugated nanocarrier, thus co-delivery of DOX and Bcl-2 siRNA by MSNs-PPPFA could result in robust cytotoxic effect on MDA-MB-231 cells.

Figure 10. Cell apoptosis assay of MDA-MB-231 cells treated with (A) control, (B) free DOX, (C) DOX@MSNs-PP, (D) DOX@MSNs-PPPFA and (E) DOX@MSNs-PPPFA/Bcl-2 siRNA for 48 h. The cells were stained with annexin V-FITC and then performed on the flow cytometry.

4. CONCLUSION 34 ACS Paragon Plus Environment

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In summary, we have developed a co-delivery system for simultaneous delivery of anticancer drug and siRNA to achieve enhanced efficacy of chemotherapy. The synthesized PEI-PLL copolymers as the caps were coated onto the surface of MSNs via the disulfide bonds and as the linker binds with folate-linked PEG. The as-prepared MSNs-PPPFA nanocarrier is capable of not only loading the anticancer drug DOX into the mesoporous channels of MSNs, but also simultaneously complexing Bcl-2 siRNA with its positively charged PEI-PLL. The in vitro release profiles reveal that the DOX release from the DOX@MSNs-PPPFA can be triggered by the acidic environment and the presence of GSH. The in vitro cell viability assay and hemolysis assay demonstrate a good biocompatibility of MSNs-PPPFA. After FA modification, the DOX and siRNA molecules uptake by MDA-MB-231 breast cancer cells were enhanced due to FA receptor-mediated endocytosis. Importantly, because of effective silence of Bcl-2 protein in MDA-MB-231cells, an enhanced therapeutic efficacy was obtained by simultaneous targeted delivery of DOX and Bcl-2 siRNA from functional nanocarrier, as indicated by the cytotoxicity and apoptosis assay. Therefore, our results suggest that the present multifunctional co-delivery system provides great potential for breast cancer therapy.

ASSOCIATED CONTENT

Supporting Information 1

H NMR spectra of PEI, PEI-PLL, FA, NH2-PEG-COOH and FA-PEG-COOH. Mean

fluorescence intensity of internalized DOX and FAM-siRNA in MDA-MB-231 cells by flow



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cytometry analysis. Western blot analysis of Bcl-2 protein expression in MDA-MB-231 cells. These materials are available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *

Professor Chuanglong He, college of Chemistry, Chemical Engineering and Biotechnology,

Donghua University, Shanghai 201620, China. Tel. /fax: +86 21 6779 2742. Email address: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was financially supported by the National Natural Science Foundation of China (31271028, 31570984), International Cooperation Fund of the Science and Technology Commission of Shanghai Municipality (15540723400), Open Foundation of State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (LK1416) and Chinese Universities Scientific Fund (CUSF-DH-D-2014019).

REFERENCES (1) Mekaru, H.; Lu, J.; Tamanoi, F. Development of Mesoporous Silica-Based Nanoparticles with Controlled Release Capability for Cancer Therapy. Adv. Drug Delivery Rev. 2015, 95, 40-49.


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Dual-Responsive Mesoporous Silica Nanoparticles Mediated

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