Folate-Functionalized Magnetic-Mesoporous Silica Nanoparticles for

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Folate-functionalized magnetic-mesoporous silica nanoparticles for drug/gene co-delivery to potentiate the antitumor efficacy Ting Ting Li, Xue Shen, Yue Geng, Zhongyuan Chen, Li Li, Shun Li, Hong Yang, Chunhui Wu, Hongjuan Zeng, and Yi Yao Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02963 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 20, 2016

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ACS Applied Materials & Interfaces

Folate-functionalized magnetic-mesoporous silica nanoparticles for drug/gene co-delivery to potentiate the antitumor efficacy

Tingting Li 1, Xue Shen 1, Yue Geng 1, Zhongyuan Chen 1, Li Li 1, Shun Li 1, Hong Yang 1,2, Chunhui Wu 1,2, Hongjuan Zeng 1,2, Yiyao Liu 1,2 *

1

Department of Biophysics, School of Life Science and Technology,

2

Center for

Information in Medicine, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, P.R. China.

*

To whom correspondence should be addressed:

Prof. Yiyao Liu，Ph.D Department of Biophysics, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, P.R. China. Tel: +86-28-8320-3353, fax: +86-28-8320-8238, E-mail: [email protected] or [email protected]

1

ACS Paragon Plus Environment


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Abstract An appropriate co-delivery system for chemotherapeutic agents and nucleic acid drugs will provide a more efficacious approach for the treatment of cancer. Combining gene therapy with chemotherapeutics in a single delivery system is more effective than individual delivery systems carrying either gene or drug. In this work, we developed folate (FA) receptor targeted magnetic-mesoporous silica nanoparticles for the co-delivery of VEGF shRNA and doxorubicin (DOX) (denoted as M-MSN(DOX)/PEI-FA/VEGF

shRNA).

Our

data

showed

that

M-MSN(DOX)/PEI-FA could strongly condense VEGF shRNA at weight ratios of 30:1, and possesses higher stability against DNase I digestion and sodium heparin. In vitro antitumor activity assays revealed that HeLa cell growth was significantly inhibited. The intracellular accumulation of DOX by confocal microscopy and fluorescence spectrophotometry showed that M-MSN(DOX)/PEI-FA were more easily taken up than non-targeted M-MSN(DOX). Quantitative PCR and ELISA data revealed that M-MSN/PEI-FA/VEGF shRNA induced a significant decrease in VEGF expression as compared to cells treated with either the control or other complexes. The invasion and migration phenotypes of the HUVECS were significantly decrease after co-culture with MSN/PEI-FA/VEGF shRNA nanocomplexes-treated HeLa cells. The approach provides a potential strategy to treat cancer by a singular nanoparticle delivery system.

2


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Keywords: M-MSN, VEGF shRNA, DOX, co-delivery, anti-angiogenesis

Introduction Cancer is a major cause of death around the word, with a yearly rise in the number of new cases.1-2 Chemotherapy is one of the frontline strategies employed in cancer treatment, however, there are several hurdles to successful chemotherapy. As there is limited tissue specificity, chemotherapy produces serious cytotoxicity not only in cancerous but also in healthy cells.3-4 Doxorubicin (DOX), a potent anticancer drug, is effective against a wide range of human neoplasms. It has been widely applied as a chemotherapeutic agent for cancer treatment.5 However, the clinical uses of DOX are restricted largely due to limited tissue specificity and serious cardiotoxic effects.6 Another critical issue is how to deliver adequate therapeutic agents to tumor sites. The biological properties of the solid tumor, which limit the penetration of drugs into neoplastic cells distant from tumor vessels, include abnormal and heterogeneous tumor vasculature, interstitium, interstitial fluid pressure and cell density. However, even if anticancer drugs are targeted to the tumor interstitium, they also have limited efficacy as cancer cells can develop mechanisms of resistance.7-9 Thus, it is a matter of great urgency to develop more effective therapeutic regimens with enhanced antitumor efficacy and minimal adverse effects.10-12 In the recent years, the use of RNA interference (RNAi) as a tumor specific gene therapy has received extensive attention in cancer treatment.13-14 RNAi occurs post transcriptionally and involves small double stranded (ds) regulatory RNA 3



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molecules,15-16 making it possible to target and inhibit the production of specific proteins that function in tumor angiogenesis,17 drug resistance,18 or anti-apoptosis.19 Therefore, RNAi provides a strategy for effective gene therapy in tumor cells.20 The biggest hurdles to RNAi therapy are mainly related to the delivery of the siRNA.21 instance, siRNA molecules are highly susceptible to premature degradation before reach the tumor tissue, and their release into the cytoplasm after endocytosis cannot easily triggered.22 A combination of chemotherapy and gene therapy has turned out be a promising strategy for cancer treatment, which could significantly enhance anticancer activity. 23-24

There is a need for the development of co-delivery systems due to the

disadvantages mentioned above of both chemotherapy and gene therapy. Sun and co-workers designed a micelleplex system based on the assembly of a biodegradable triblock copolymer to deliver Plk1 specific siRNA (siPlk1) and paclitaxel to the same tumor cells both in vitro and in vivo. This system was able to simultaneously deliver two payloads into the same tumor cell, and can remarkably inhibit tumor growth.25 et al. fabricated a multifunctional silica-nanosystem based on layer-by-layer self-assembly for the co-delivery of DOX and VEGF siRNA, which should overcome various physiological and biological barriers by selectively delivering siRNA and to the cytosol and nucleus.26 Presently, mesoporous silica nanoparticles (MSN) have been extensively used as carriers for drug or gene delivery.22, 27 The unique properties of MSN are the large surface area, tunable pore volumes, and versatile chemistry for surface functionalization.28 For instance, the surface of MSNs can be easily tailored to 4


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load more guest molecules.29-31 Consequently, at the preclinical level, MSN have been shown to be an excellent carrier for the delivery of drugs and nucleic acids to combat diseases, such as inflammation, diabetes and cancer.22, 32 Angiogenesis is a major factor contributing to the survival, growth, migration metastasis of cancer cells.33-35 Vascular endothelial growth factor (VEGF) is a crucial regulatory cytokine during angiogenesis.

36-37

Anti-VEGF strategies have been

developed to inhibit new blood vessel growth and starve tumors of necessary oxygen and nutrients. Meanwhile, the permeability of the tumor vasculature can be enhanced by VEGF inhibitors such as bevacizumab, which leads to improved penetration of the free or liposome-encapsulated chemotherapy agents in tumors.38 It was reported that VEGF siRNA may improve the penetration and uptake of DOX in the drug-resistant tumor.39 In this study, we choose VEGF as a target gene to evaluate the effects. A novel nanocarrier consisting of magnetic mesoporous silica nanoparticles and folic acid conjugated polyethylenimine (PEI) was designed for the co-delivery of DOX and VEGF shRNA. (denoted as M-MSN(DOX)/PEI-FA/VEGF shRNA). M-MSN(DOX)/PEI-FA/VEGF

shRNA

nanocomplexes

were

endowed

with

dual-targeted functions of both folate receptor targeting and magnetic targeting. As illustrated in Figure 1, M-MSN were synthesized by sol-gel procedure. The DOX-loaded MSN was then capped by PEI-FA further adsorption of VEGF shRNA form M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes. The nanocomplexes were internalized into HeLa cells via folate receptors and released DOX and VEGF shRNA. Then, RNAi efficiency and anti-angiogenic effect were further investigated. 5



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was expected that the MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could be employed as an efficient and safe co-delivery nanoplatform with antitumor efficacy

Figure

1.

Schematic

illustration

showing

the

preparation

of

M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes and their delivery kinetics. (A) Schematic of the M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes; (B) Schematic illustration showing the proposed delivery of DOX and VEGF shRNA-mediated by M-MSN/PEI-FA, for a synergistic effect in vitro. The MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes were uptaken by cells via folate receptor-mediated endocytosis. DOX and VEGF shRNA were released from the nanocomplexes in cytoplasm. Then DOX entered into nucleus, and VEGF shRNA would target to degrade VEGF mRNA under the assistance of Dicer and RISC.

EXPERIMENTAL SECTION Materials. Tetraethylorthosilicate (TEOS), branched polyethyleneimine (PEI, 25 kD), N-hydroxysuccinimide (NHS), N, N´-Dicyclohexylcarbodiimide (DCC), NaN3, 6


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Nystatin, and Genistein were obtained from Sigma-Aldrich (St Louis, MO, USA). 3-Trihydroxysilylpropyl methylphosphonate was purchased from Gelest. Iron oxide nanoparticles (10 nm) were obtained from Nanjing Emperor Nano Material Co. Ltd. (Nanjing, China), Folic acid (FA) was obtained from Alexis (Los Angeles, CA, USA) and doxorubicin (DOX) was obtained from Hisun Pharmaceutical (Zhejiang, China). Chlorpromazine and cytochalasin D were from Enzo Biochem (NY, USA). RPMI cell culture medium, fetal bovine serum (FBS), 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and trypsin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Cetyltrimethylammonium bromide (CTAB), ammonium nitrate, and hexyl alcohol were purchased from Kelong Chemicals (Chengdu, China). All chemicals were used as received without further purification. Plasmid expressing small hairpin RNA against VEGF (VEGF shRNA) and scrambled shRNA (SC shRNA) were obtained from Fungenome Co., Ltd. (Guangzhou, China). The shRNA targeted VEGF sequence is “TACTGCCATCCAATCGAGA”.

Characterizations. The size and morphology of the nanoparticles were determined by scanning electron microscopy (SEM) (Helios NanoLab 650, FEI, Eindhoven, Netherlands) and high revolution transmission electron microscope (Tecnai G2 F20 S-TWIN). Zeta potentials were measured using electrophoretic mobility measurements (Malvern Instruments, Malvern, UK).

Preparation of M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes. The 7



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M-MSN were synthesized according to the previously published sol-gel procedure some modifications.40-41 Briefly, 10 ml of aqueous solution containing Fe3O4 nanocrystals and 2 g of CTAB were dissolved in 90 ml of deionized water in a 250 ml flask. Then, 3 ml of ammonia solution (28%-30%) was added into the aforementioned mixture, heated to 40ºC and stirred continuously for 2 h. Finally, 0.5 ml TEOS and 5 ethyl acetate were added and stirred for 30 min, followed by the addition of 3-trihydroxysilylpropyl methylphosphonate with continuous stirring for 6 h. The acquired product was isolated by high-speed centrifugation, and washed three times with deionized water and ethanol. The structure-templating CTAB surfactants were then removed in an ethanolic solution containing 1% NH4NO3 under reflux at 80ºC 2 h. The magnetic-mesoporous silica nanoparticles (M-MSN) were obtained after a further three washes with ethanol and deionized water. DOX loading onto M-MSN was done by mixing DOX and M-MSN at various weight ratios from 0.05 to 0.8 overnight, followed by centrifugation to remove unloaded DOX. Drug loading onto M-MSN was measured using UV-visible spectroscopy (UV-2910, Hitachi, Japan) at 480 nm. The drug payload was calculated by the equation below. Where W is the weight. Drug loading = [W(total DOX)-WDOX in supernatant)]/[W(nanoparticles)]×100%

The obtained M-MSN(DOX) were further modified with PEI-FA, a co-polymer of PEI-FA that was synthesized according to our previously published procedure.42 Folate (FA) was activated in the presence of DCC and NHS by gentle stirring in the dark for 12 h at room temperature (25ºC) before the PEI solution was added. The 8


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mixture was maintained with gentle stirring for another 12 h, then dialyzed for 48 h against deionized water to remove any unreacted components. The PEI-FA solution was added to the M-MSN(DOX) suspension (weight

ratio 5:1 of M-MSN(DOX) to

PEI-FA), which was stirred for 4 h at a speed of 180 rpm to allow PEI-FA to graft onto the surface of the M-MSN(DOX) nanoparticles. Unbound PEI-FA was removed by centrifugation at 10,000 rpm for 10 min followed by three washes with deionized water. VEGF shRNA was incubated in the M-MSN(DOX) suspension at various weight ratios (10:1, 15:1, 20:1, 30:1, and 40:1) for 30 min at room temperature to form the M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes by electrostatic absorption. The nanocomplexes were loaded on a 1% agarose gel with tris/acetate/EDTA buffer and run at 120 V for 30 min, followed by visualization by staining with ethidium bromide. Images were acquired using a UV transilluminator (Bio-Rad, Philadelphia, PA, USA)

Cytotoxicity assay. Cell viability after treatment with different nanoparticles was evaluated by CellTiter 96® AQueous One Solution cell proliferation assay (MTS) (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. Briefly, HeLa and HUVECs were seeded into 96-well plates at a density of 5×103 cells per well and incubated at 37°C in a 5% CO2 atmosphere for 24 h. The medium was then replaced with 300 µl fresh medium containing M-MSN or M-MSN/PEI-FA at various concentrations (5, 10, 20, 40, 80 µg/ml). After incubation for 72 h, cells were further incubated with 100 µL fresh medium containing 10 µl MTS. Absorbance at 490 nm 9



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was measured using a microplate reader (ELx808™, BioTek Instruments). The relative cell viability was calculated using the following equation. Where A490 is the absorbance at 490 nm wavelength. A 490 ( treated ) Re lative cell viability(%)＝ ×100% A 490 ( untreated )

Cellular uptake. Cellular uptake of DOX was confirmed by confocal laser scanning microscopy (CLSM, Leica SP5II, Germany). HeLa cells were seeded in 24-well plates at a density of 1×105 viable cells per well and incubated overnight to allow cell attachment. The medium was then replaced with fresh medium containing DOX, M-MSN(DOX) and M-MSN(DOX)/PEI-FA (DOX concentration 5 µg/ml), After incubation for 4 h, the cells were washed three times and fixed with 4% paraformaldehyde for 20 min, following which the cells were treated with DAPI for 15 min. For FA competition experiments, HeLa cells were pre-incubated with free FA (1.25 mM) for 2 h prior to the addition of M-MSN(DOX)/PEI-FA. For quantitative analysis, cells were lysed with 0.5% (w/v) SDS (pH 8.0) and the DOX fluorescence intensity was subsequently detected using a fluorospectrophotometer (Hitachi F-7000, Japan).

Gene silencing efficiency in vitro. HeLa cells were seeded in 12-well culture plates at a density of 1.5×105 per well and cultured overnight. For gene transfection, 2.5 µg VEGF shRNA was incubated with different nanocomplexes at a weight ratio of 30:1 for 30 min before adding to the plates. The cells were incubated in serum-free 10


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medium for 6 h, which was then replaced with fresh cell culture medium. After incubation for another 72 h, the supernatant from each well was collected and used to detect the concentration of secreted VEGF from the HeLa cells using a human VEGF ELISA kit (Neobioscience, Shenzhen, China) according to the manufacturer's instructions. One microgram aliquot of total mRNA was transcribed into complementary DNA using the PrimeScript™ RT Reagent Kit (Takara, Dalian, China). All qPCR was performed using the Faststart Universal SYBR Green Master mix (ROX), and the amplification threshold (Ct) of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The comparative Ct method was used to calculate fold change values. Efficiency of all primer pairs was 95%-100%.

Primer

pairs

used

were

VEGF

(forward,

5'-TTCTCAAGGACCACCGCATC-3'; reverse, 5'-AATGGGGTCGTCATCTGGT-3') and

GAPDH

(forward,

5'-GTCTCCTCTGACTTCAACAGCG-3';

reverse,

5'-ACCACCCTGTTGCTGTAGCCAA-3').

Matrigel invasion, migration, and tube formation experiments. To assess cell migration and invasion, HeLa cells were seeded in 24-well plates and treated with various nanocomplexes in a similar fashion to the qPCR and ELISA assays. After incubation for 72 h, transwells with endothelial cells (3×104 cells/well) were added to the upper compartment. For the invasion assays, the upper compartment was pre-coated with Matrigel (BD, USA). After incubation for 24 h, cells that did not migrate or invade in the upper wells were removed with cotton swabs. Cells that had 11



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passed through the membrane on the lower surface of the inserts were fixed with 4% paraformaldehyde, stained using crystal violet and quantified with Image Pro Plus 6.0 software. For the in vitro tube formation assay, HeLa cells were treated as mentioned above and the culture supernatant was collected after 72 h of treatment, followed by centrifugation at 5,000g for 5 min. Matrigel (10 µl) was used to pre-coat the bottom of a µ-Slide angiogenesis ibiTreat (Ibidi, Germany) for polymerization at 37°C for 45 min. HUVECs (6×103) were then seeded onto each Matrigel-coated slide in the culture medium collected above. After incubation for 4 h at 37°C, the cells were stained with calcein-AM solution to evaluate the formation of tubular structures and the images were taken under microscopy (Leica). Finally, the length of the vascular network was quantitatively evaluated by Wimasis image analysis.

Statistical analysis. All experiments were carried out at least in triplicate. Data are presented as the mean ± standard deviation (SD), and statistical analysis was performed using GraphPad Prism Software version 6.0 (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant for p value < 0.05.

RESULTS AND DISCUSION Synthesis and characterization of M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes. M-MSN was synthesized by the classic sol-gel method with slight modifications. SEM image in Figures 2A showed that the M-MSN had regular 12


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morphology. As shown in the high resolution TEM image (Figures 2B), the M-MSN possessed a palpable core-shell structure and clearly defined pore structure. Furthermore,

Brunauer-Emmett-Teller

(BET)

nitrogen

adsorption-desorption

isotherms were carried out to further confirm the mesoporous character of the M-MSN (Figure 2C). BET and BJH analyses demonstrated typical type-IV isotherms, consistent with a mesoporous structure with an average pore diameter of 3.4 nm and a pore volume of 0.51 cm³/g. The distribution of particle sizes (Figure 2D) was determined by dynamic light scattering (DLS), which revealed that the M-MSN were uniform with an average size of 195 nm.

Figure 2. Characterization of M-MSN. Analysis of the morphology of M-MSN by (A) scanning electron microscope (SEM) and (B) high resolution transmission electron microscopy (TEM). (C) N2 adsorption-desorption isotherms (inset: pore diameter distribution). (D) Particle size distribution of M-MSN as determined by dynamic light 13



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scattering (DLS).

SEM images demonstrated that these nanocomplexes exhibited a uniform spherical morphology. DOX loading, PEI-FA modification and VEGF shRNA complexation did not affect the morphology of the nanoparticles and resulted in a slight tendency to aggregate in solution compared with M-MSN (Figures 3A(a')-(c')), which may have been due to the increased surface charge of the nanocomplexes and VEGF

shRNA

electrostatic

interaction.

The

average

diameter

of

M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes slightly increased to 217 nm (Figure S1). Surface modification by PEI-FA lead to a high positively charged surface of the nanocomplexes (Figure 3B), which enabled loading of more negatively charged VEGF shRNA by electronic absorption. The change in zeta potential after PEI-FA modification suggests that the PEI-FA was successfully attached to the nanoparticles.

Figure 3. (A) SEM images of M-MSN(DOX) (a'), M-MSN(DOX)/PEI-FA (b') and M-MSN(DOX)/PEI-FA/VEGF shRNA (c'). (B) Zeta potential of M-MSN and 14


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above-mentioned three nanoparticles in water (pH 7.4) at room temperature (25ºC).

In vitro DOX loading and release. Successful loading of DOX on M-MSN was demonstrated by the UV-vis absorbance spectra of the samples (Figure 4A). After loading DOX, M-MSN(DOX) displayed characteristic DOX absorption peaks at 480 nm, but the M-MSN themselves did not show the characteristic absorption peaks, suggesting that DOX molecules were successfully adsorbed onto the inner pores or the surface of M-MSN. Due to the magnetic nature of Fe3O4 included in the M-MSN, the M-MSN(DOX) were attracted toward the magnet (Figure 4B). This showed that the M-MSN could carry drugs to targeted locations under an external magnetic field. Figure S2A shows the drug payload efficiency of M-MSN was ca.13%, which agreed

with a previous report.41 According to the DOX loading efficiency of M-MSN(DOX) at weight ratio 0.4:1 of DOX to M-MSN, we calculated DOX loading efficiency of M-MSN(DOX)/PEI-FA is 12.3%. Figure S2B shows the in vitro DOX release results. The amount of DOX released from M-MSN(DOX)/PEI-FA was higher at pH 4.7 than that at pH 7.4, and was both time- and pH-dependent. A relatively fast release happened within 10 h, followed by sustained release until the end of the assay. Therefore, these particles would be of benefit to cancer chemotherapy requiring a high initial dose and sustained drug release without the frequent administration of medication. The pH-responsive release maybe attribute to the protonation of the amine groups on the PEI-FA conjugates. Under acidic condition, due to the protonation of the amine groups, PEI-FA molecules are positively charged and thus generate strong Columbic repulsion, leading to the swelling and dissociation of the 15



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PEI-FA layer on the M-MSN(DOX) surfaces. Then, DOX released from the nanocarriers.

Figure 4. In vitro payloads of DOX and VEGF shRNA and the serum stability of nanocomplexes. (A) UV-vis absorption spectra of free DOX, M-MSN and M-MSN(DOX) in water. (B) Photograph of free DOX and M-MSN(DOX) with or without magnet. (C) Agarose gel electrophoresis assay of M-MSN/PEI-FA/VEGF shRNA at various weight ratios of M-MSN/PEI-FA to VEGF shRNA. (D) DNase protection assay. Lane 1: naked VEGF shRNA; Lane 2: naked VEGF shRNA treated with

DNase

I;

Lane

3:

M-MSN(DOX)/PEI-FA/VEGF

M-MSN(DOX)/PEI-FA/VEGF shRNA

incubated

with

shRNA; heparin;

Lane

4:

Lane

5:

M-MSN(DOX)/PEI-FA/VEGF shRNA incubated with heparin, followed by treatment with DNase I; Lane 6: M-MSN(DOX)/PEI-FA/VEGF shRNA incubated with DNase I; Lane 7: M-MSN(DOX)/PEI-FA/VEGF shRNA treated with DNase I first, the subsequently separated from the mixture, thereafter, the complexes subsequent 16


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dissociation by heparin. (E) Serum stability of M-MSN/PEI-FA/VEGF shRNA.

Due to electrostatic interaction, the PEI-FA coated on the surface of M-MSN(DOX) could absorb VEGF shRNA to form M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes. The agarose gel electrophoresis results revealed that the VEGF shRNA was firmly combined with M-MSN(DOX)/PEI-FA when the weight ratio of M-MSN(DOX)/PEI-FA to VEGF shRNA increased to 30:1 (Figure 4C). The VEGF shRNA protective capability was further evaluated as shown in Figure 4D. Lane 3 indicates that siRNA was completely packaged within M-MSN(DOX)/PEI-FA at a weight ratio of 40:1. After incubation with heparin, a substantial amount of the VEGF shRNA was released into the solution (lane 4). No band was visible when the sample in lane 4 was further treated with DNase I (lane 5), showing that the released VEGF shRNA had been degraded (similar to the sample of naked shRNA treated with DNase I in lane 2). The sample in lane 6 was M-MSN(DOX)/PEI-FA/VEGF shRNA treated with DNase I, and no band appeared after electrophoresis. However, when the sample was mixed with heparin to re-release the shRNA, an obvious band appeared. It is well documented that our M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes effectively protect packaged VEGF shRNA from enzymatic degradation. Moreover, when naked VEGF shRNA (Figure S3(a')) and M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes (Figure S3(b')) were incubated with 10% serum at 37°C from 12 h to 72 h, naked VEGF shRNA was completely degraded and could not be seen on an agarose gel, whereas M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could

17



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still

be

seen

even

after

72-h

incubation.

This

Page 18 of 37

suggests

that

the

MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could protect VEGF shRNA from serum degradation. Aggregation induced by serum was further evaluated from the changes in turbidity. No significant aggregation was detected in various nanocomplexes suspensions after incubation in serum for 72 h, as well as 5% glucose (Figure 4E). It is essential to investigate the hemocompatibility of nanocarriers for their successful systemic administration.43 Therefore, the impact of the M-MSN/PEI-FA on RBCs was evaluated using a hemolysis assay (Figure S4). Microscopic images (Figure S4, insets of the bottom-right) of the RBCs exposed to water showed evidence of hemolysis, but M-MSN/PEI-FA exhibited no visible hemolytic effects, similar to the 0.9% NaCl control. Next, we also quantitatively evaluated the percentage of hemolysis caused by the M-MSN/PEI-FA based on the absorbance of the supernatant at 541 nm (Figure S4, the right and insets of the upper-right). Taken together, these results confirmed that the formulated nanocomplexes possess excellent biocompatibility and can be used to as efficient gene carriers.

In vitro cytotoxicity of M-MSN(DOX)/PEI-FA/VEGF shRNA. For successful drug and gene combination therapy, a safe and effective carrier system is a prerequisite to successful in vivo siRNA and drug delivery.44-45 The MTS assay was performed to measure the viability of HUVECs (normal cells) and HeLa cells (cancerous cells) incubated with M-MSN or M-MSN/PEI-FA for 72 h. As shown in Figure 5A, the control did not exhibit any detectable cytotoxicity in both HUVECs 18


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and HeLa cells even after an extended 72 h of treatment. Compared with the control, the viability of the treated groups was also above 90%. In general, the results showed that M-MSN or M-MSN/PEI-FA nanocarriers had no apparent cytotoxicity on HeLa or HUVECs. These results also demonstrated that M-MSN/PEI-FA could be a highly biocompatible delivery system for the co-delivery drugs and genes. As shown in Figure 5B, the cell viability of HeLa cells treated with M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes under external magnetic fields was only 27%, which was much lower than that of the cells treated with M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes alone. Some literature has shown that tumor cells have a greater susceptibility to DOX than siRNA.46 Raskopf and co-workers reported that VEGF siRNA efficiently inhibited intracellular signal transduction, got a good antitumor response in vivo.47 Here, we also detected that M-MSN/PEI-FA/VEGF shRNA didn’t display significant cytotoxicity on HeLa cells. The results suggest that the cellular uptake efficiency and therapeutic efficacy of DOX significantly increase via FA and external magnetic fields.

Figure 5. In vitro cytotoxic effects of various nanoparticles. (A) Cytotoxicity against 19



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HeLa cells or HUVECs after treatment with M-MSN or M-MSN/PEI-FA nanoparticles

for

72

h.

(B)

The

synergistic

therapy

efficacy

of

M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes against HeLa cells at 10 µg/ml of DOX concentration for 48 h. *p

Folate-Functionalized Magnetic-Mesoporous Silica Nanoparticles for

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