Folate-Functionalized Magnetic-Mesoporous Silica ... - ACS Publications

Research Article www.acsami.org

Folate-Functionalized Magnetic-Mesoporous Silica Nanoparticles for Drug/Gene Codelivery To Potentiate the Antitumor Efficacy Tingting Li,† Xue Shen,† Yue Geng,† Zhongyuan Chen,† Li Li,† Shun Li,† Hong Yang,†,‡ Chunhui Wu,†,‡ Hongjuan Zeng,†,‡ and Yiyao Liu*,†,‡ †

Department of Biophysics, School of Life Science and Technology, ‡Center for Information in Medicine, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, People’s Republic of China S Supporting Information *

ABSTRACT: An appropriate codelivery 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 codelivery of VEGF shRNA and doxorubicin (DOX) (denoted as MMSN(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 MMSN(DOX)/PEI-FA were more easily taken up than nontargeted 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 coculture 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. KEYWORDS: M-MSN, VEGF shRNA, DOX, codelivery, antiangiogenesis

■

INTRODUCTION

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 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 antiapoptosis.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 For instance, siRNA molecules are highly susceptible to premature degradation before they reach the tumor tissue, and their release into the cytoplasm after endocytosis cannot be 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 codelivery 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

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 © 2016 American Chemical Society

Received: March 9, 2016 Accepted: May 18, 2016 Published: May 18, 2016 13748

DOI: 10.1021/acsami.6b02963 ACS Appl. Mater. Interfaces 2016, 8, 13748−13758

Research Article

ACS Applied Materials & Interfaces

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.

codelivery of DOX and VEGF shRNA. (denoted as MMSN(DOX)/PEI-FA/VEGF shRNA). M-MSN(DOX)/PEIFA/VEGF shRNA nanocomplexes were endowed with dualtargeted 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 to 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 antiangiogenic effects were further investigated. It was expected that the MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could be employed as an efficient and safe codelivery nanoplatform with antitumor efficacy

specific siRNA (siPlk1) and paclitaxel to the same tumor cells both in vitro and in vivo. This system was able to simultaneously deliver the two payloads into the same tumor cell, and can remarkably inhibit tumor growth.25 Han et al. fabricated a multifunctional silica-nanosystem based on layer-by-layer selfassembly for the codelivery of DOX and VEGF siRNA, which should overcome various physiological and biological barriers by selectively delivering siRNA and DOX to the cytosol and nucleus.26 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 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, and 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 drugresistant tumor.39 In this study, we choose VEGF as a target gene to evaluate the antiangiogenic effects. A novel nanocarrier consisting of magnetic mesoporous silica nanoparticles and folic acid conjugated polyethylenimine (PEI) was designed for the

■

EXPERIMENTAL SECTION

Materials. Tetraethylorthosilicate (TEOS), branched polyethylenimine (PEI, 25 kDa), N-hydroxysuccinimide (NHS), N,N′dicyclohexylcarbodiimide (DCC), NaN3, 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 1640 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 13749


Research Article

ACS Applied Materials & Interfaces 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). The ζpotentials were measured using electrophoretic mobility measurements (Malvern Instruments, Malvern, UK). Preparation of M-MSN(DOX)/PEI-FA/VEGF shRNA Nanocomplexes. The M-MSN were synthesized according to the previously published sol−gel procedure with some modifications.40,41 Briefly, 10 mL of aqueous solution containing Fe3O4 nanocrystals and 0.2 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 40 °C and stirred continuously for 2 h. Finally, 0.5 mL of TEOS and 5 mL of 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 80 °C for 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 following equation:

relative cell viability (%) =

A490(treated) A490(untreated)

× 100%

Where A490 is the absorbance at 490 nm wavelength. 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, MMSN(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 preincubated 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 of 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 serumfree 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 precoated 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 passed through the membrane on the lower surface of the insets 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 precoat 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.

drug loading = [W(total DOX) − WDOX in supernatant)]/[W(nanoparticles)] × 100% Where W is the weight. The obtained M-MSN(DOX) were further modified with PEI-FA, a copolymer 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 (25 °C) before the PEI solution was added. The 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)/ PEI-FA 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 of 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 of MTS. Absorbance at 490 nm was measured using a microplate reader (ELx808, BioTek Instruments). The relative cell viability was calculated using the following equation: 13750


Research Article


■

RESULTS AND DISCUSION Synthesis and Characterization of M-MSN(DOX)/PEIFA/VEGF shRNA Nanocomplexes. M-MSN was synthesized by the classic sol−gel method with slight modifications. The SEM image in Figure 2A showed that the M-MSN had regular

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) ζPotential of M-MSN and above-mentioned three nanoparticles in water (pH 7.4) at room temperature (25 °C).

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. Because of 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 PEIFA layer on the M-MSN(DOX) surfaces. Then, DOX released from the nanocarriers. Because of 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

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) N 2 adsorption−desorption isotherms (inset: pore diameter distribution). (D) Particle size distribution of M-MSN as determined by dynamic light scattering (DLS).

morphology. As shown in the high resolution TEM image (Figure 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 confirm further 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 MMSN were uniform with an average size of 195 nm. 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 MMSN (Figures 3Aa′−c′), which may have been due to the increased surface charge of the nanocomplexes and VEGF shRNA electrostatic interaction. The average diameter of MMSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes slightly increased to 217 nm (Figure S1). Surface modification by PEIFA 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. 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, MMSN(DOX) displayed characteristic DOX absorption peaks at 13751


Research Article


Figure 4. In vitro payloads of DOX and VEGF shRNA and the serum stability of nanocomplexes. (A) UV−vis absorption spectra of free DOX, MMSN 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 shRNA; Lane 4, M-MSN(DOX)/PEIFA/VEGF shRNA incubated with heparin; 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 dissociation by heparin. (E) Serum stability of MMSN/PEI-FA/VEGF shRNA.

Figure 5. In vitro cytotoxic effects of various nanoparticles. (A) Cytotoxicity against HeLa cells or HUVECs after treatment with M-MSN or MMSN/PEI-FA nanoparticles for 72 h. (B) 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 < 0.05, **p < 0.01.

degradation. Moreover, when naked VEGF shRNA (Figure S3a′) and M-MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes (Figure S3b′) were incubated with 10% serum at 37 °C from 12 to 72 h, naked VEGF shRNA was completely degraded and could not be seen on an agarose gel, whereas MMSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could still be seen even after 72 h of incubation. This suggests that the MSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes could

been degraded (similar to the sample of naked shRNA treated with DNase I in Lane 2). The sample in lane 6 was MMSN(DOX)/PEI-FA/VEGF shRNA treated with DNase I, and no band appeared after electrophoresis. However, when the sample was mixed with heparin to rerelease the shRNA, an obvious band appeared. It is well documented that our MMSN(DOX)/PEI-FA/VEGF shRNA nanocomplexes effectively protect packaged VEGF shRNA from enzymatic 13752


Research Article


Figure 6. Intracellular uptake of DOX. (A) Confocal laser scanning microscope (CLSM) images showing cellular internalization of different formulations at 4 h. (B) Intracellular DOX was quantitatively detected by fluorescence spectrophotometry. *p < 0.05 vs M-MSN(DOX), and Δp < 0.05 vs M-MSN(DOX)/PEI-FA. (C) Investigation of cellular internalization mechanisms by pretreatment with various chemical inhibitors. HeLa cells were incubated with the indicated inhibitors (sodium azide [NaN3], chlorpromazine [Chl], genistein [Gen], nystatin [Nys], and cytochalasin D [Cyt D]). Intracellular DOX accumulation was detected by fluorescence spectrophotometry. *p < 0.05 and **p < 0.01 vs control.

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). Microscopy 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 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 codelivery 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)/PEIFA/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 did not 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. We further determined the cell viability after HeLa cells were incubated with free DOX, M-MSN(DOX) or M-MSN(DOX)/ PEI-FA for 24 and 48 h. DOX exhibited dose-dependent cytotoxicity at a series of DOX concentrations from 0.1 to 15 μg/mL (Figure S5). As HeLa cells have high level expression of the FA receptor, M-MSN(DOX)/PEI-FA exhibited increased cytotoxicity compared with that of nontargeted M-MSN(DOX) at both 24 h (Figure S5A) and 48 h (Figure S5B), indicating that internalization of DOX via FA receptor-mediated endocytosis increased the antitumor effect of DOX. Therefore, M-MSN(DOX)/PEI-FA nanoparticles could increase the anticancer effects of DOX in FA receptor-positive tumor cells. Cellular Uptake and Uptake Pathways Studies. The efficiency of DOX delivered by M-MSN(DOX)/PEI-FA to HeLa cells was also determined. HeLa cells incubated with free DOX demonstrated notable fluorescence in the nucleus, which shows the excellent permeability and uptake of DOX into cells (Figure 6A). When treated with M-MSN(DOX), the uptake of DOX was markedly reduced. However, M-MSN(DOX)/PEIFA significantly enhanced the delivery of DOX to the cells compared with nontargeted M-MSN(DOX). In the presence of 1.25 mM free FA, the uptake efficiency decreased significantly, indicating that inhibition of cellular uptake is partly due to free FA occupying the folate receptor on cell surfaces. Quantitative analysis by fluorospectrophotometry also showed that MMSN(DOX)/PEI-FA increased DOX uptake approximately 1.7-fold more than M-MSN(DOX) or (Figure 6B). Cellular uptake of M-MSN/PEI-FA was also confirmed by Prussian blue 13753


Research Article


Figure 7. Dual-targeted images of HeLa cells treated with M-MSN(DOX)/PEI-FA nanoparticles (A) or pretreated with 1.25 mM free FA for 2 h before incubation with M-MSN(DOX)/PEI-FA nanoparticles (B) under an external magnetic field for 4 h. The left most panel shows where the fluorescence images 1−4 were taken, at different distances away from the magnet.

Figure 8. VEGF gene expression. (A) Suppression of VEGF mRNA as determined by quantitative real-time PCR. (B) Secretion of VEGF protein in culture media as detected by human VEGF ELISA kit. Both experiments were conducted at 72 h after transfection with M-MSN/PEI-FA/scrambled shRNA (M-MSN/PEI-FA/SC shRNA), M-MSN/PEI-FA/VEGF shRNA. The weight ratio of nanocomplexes to SC shRNA or VEGF shRNA was 30:1. Cells were transfected with VEGF shRNA using commercial LipoFiter or treated with suramin as the positive control. *p < 0.05 vs control.

internalization of these nanoparticles is an energy-dependent process. Intracellular Distribution and Endosome Escape. To clarify further the intracellular location of M-MSN(DOX)/PEIFA, we observed the intracellular localization of DOX at various time points using DAPI and LysoTracker staining, to label the nucleus and lysosomes, respectively (Figure S7). The images showed that the DOX reached the endosomes/lysosome after 0.5 h of incubation. As the incubation time increased, the amount of DOX also increased and maximum levels of colocalization could be observed at 1 h. After incubation for 4 h, few colocalization signals appeared in the merged images, indicating that most of the DOX had been released from the lysosomes. This result can be attributed to the successful escape of M-MSN(DOX)/PEI-FA from endosomes/lysosomes. Magnetic and FA Dual Targeting at the Cellular Level. Next, we further determined the magnetic and FA dual targeting effects of M-MSN(DOX)/PEI-FA in cell culture experiments. As shown in Figure 7A, HeLa cells incubated with M-MSN(DOX)/PEI-FA in a cell culture dish were exposed to a commercially available 0.42 T Nd−Fe−B magnet for 4 h. The cells were then washed three times to remove excess MMSN(DOX)/PEI-FA and fluorescence images were captured at

staining (Figure S6). The results showed that increasing concentrations of nanoparticles in the cell incubation medium enhanced the uptake of M-MSN/PEI-FA. Pretreatment with free FA, also decreased the uptake efficiency. Together, these data suggested the M-MSN(DOX)/PEI-FA can efficiently deliver DOX and this effect is highly dependent on the folate receptor. It has been reported that cells can internalize targetednanocarriers via various endocytic pathways.48−50 In this regard, several chemical inhibitors were used to explore the endocytic uptake pathways used by M-MSN(DOX)/PEI-FA. Chlorpromazine, an inhibitor for the clathrin-dependent pathway, markedly decreased the uptake of M-MSN(DOX)/PEI-FA (Figure 6C), suggesting the involvement of clathrin-mediated endocytosis. In contrast, caveolin-dependent pathway inhibitors, genistein and nystatin, and the micropinocytosis inhibitor cytochalasin D did not exhibit a significant difference compared to the control, suggesting the uptake of M-MSN(DOX)/PEIFA was not directly associated with these two pathways. NaN3 and 4 °C treatments were employed to investigate whether the uptake consumed energy, and the results showed that these treatments considerably decreased the uptake of M-MSN(DOX)/PEI-FA to 43% and 30%, respectively, indicating that 13754


Research Article


Figure 9. HUVECs migration and invasion in a coculture model of HeLa and HUVECs. Representative images of migration (A), invasion (C), and quantitative analysis (B, D, insets are the model charts). The HeLa cells in the lower chamber were treated by the indicated nanoparticles, suramin (VEGF specific inhibitor, positive control) or VEGF (negative control). (a, control, equal medium addition; b, M-MSN/PEI-FA/SC shRNA; c, MMSN/PEI-FA/VEGF shRNA; d, FA+ M-MSN/PEI-FA/VEGF shRNA; e, suramin; f: VEGF). *p < 0.05 vs control.

In Vitro Antiangiogenic Effects. Angiogenesis is a process where new blood vessels are formed from pre-existing vessels and plays an important role in tumor growth and metastasis.53,54 During the early stages of tumorigenesis, a host of pro-angiogenic factors are produced by tumor cells to form a nascent vascular network.55 This kind of vasculature has the ability to transport oxygen and nutrients into the tumor and remove tumor metabolic waste products, making these vessels of considerable importance in tumor growth and progression.56 VEGF is an important factor regulating tumor angiogenesis.57 Here, we investigated whether VEGF secretion from HeLa cells transfected with M-MSN/PEI-FA/VEGF shRNA nanocomplexes could affect endothelial cell migration, invasion and microtubule formation. As shown in Figure 9A, cells treated with M-MSN/PEI-FA/SC shRNA were similar to control cells, which showed strong migration ability, and the addition of exogenous VEGF significantly enhanced cell migration. For cells treated with M-MSN/PEI-FA/VEGF shRNA and suramin, migration rates were 36% and 59% compared to the control, respectively (Figure 9B). However, after pretreatment with free FA the migration rate increased. It attributes to the free FA blocking the FA receptor, leading to reduce endocytosis of the M-MSN/PEI-FA/VEGF shRNA nanocomplexes into HeLa cells. To estimate further longitudinal motility of HUVECs, an invasion assay was performed. As shown in Figure 9C, consistent with the migration assay results, cells from the control group showed the highest invasion ability with cells passing through the membrane covering almost the entire lower surface. HUVECs treated with M-MSN/PEI-FA/SC shRNA showed no inhibition of longitudinal motility and the addition of exogenous VEGF markedly enhanced the invasion phenotype. Both M-MSN/PEI-FA/VEGF shRNA and suramin treated HUVECs displayed some degree of inhibition on invasion with coverage rates at 39% and 34% on the lower surface, respectively (Figure 9D). The antiangiogenic effects of the M-MSN/PEI-FA/VEGF shRNA were further investigated in vitro. HUVECs could organize into capillary-like structures in the Matrigel basement

different locations of the culture dish, showing an obvious decline in intracellular DOX fluorescence signals at an increasing distance from the magnet. When the cells were pretreated with free FA, the result was similar; however, the fluorescence intensity was significantly reduced compared to that without free FA pretreatment (Figure 7B). Our results showed that M-MSN(DOX)/PEI-FA nanoparticles could serve as an effective agent with both FA targeting and magnetic targeting capabilities. Analysis of VEGF Gene Silencing Effect in Vitro. To eliminate interference from DOX-induced apoptosis and evaluate the silencing efficiency of VEGF expression, MMSN/PEI-FA/VEGF shRNA nanocomplexes without DOX were used in an in vitro gene silencing assay. The inhibition of VEGF expression in HeLa cells was evaluated by qPCR and ELISA at the mRNA and protein levels, respectively. Here, suramin, a specific VEGF inhibitor, was chosen as a positive control.45,51,52 As shown in Figure 8A, cells incubated with LipoFiter/VEGF shRNA and suramin showed a decrease in VEGF mRNA levels when compared to either control cells or those treated with M-MSN/PEI-FA/SC shRNA. Importantly, the extent of the decrease afforded by M-MSN/PEI-FA/VEGF shRNA was notably outperformed by the LipoFiter/VEGF shRNA and suramin, and showed a dose-dependent effect (data not shown). M-MSN/PEI-FA/VEGF shRNA exhibited a significantly greater silencing efficiency of 90.4%. The addition of free FA resulted in a gene silencing efficiency by M-MSN/ PEI-FA/VEGF shRNA of 52.2%, which was remarkably decreased compared to cells that were not pretreated with free FA. Furthermore, VEGF expression at the protein level was also detected by ELISA as shown in Figure 8B, with the results similar to those obtained by qPCR. M-MSN/PEI-FA/VEGF shRNA dramatically downregulated VEGF expression (578 pg/ mL) in comparison to the control group (1640 pg/mL) and MMSN/PEI-FA/SC shRNA group (1474 pg/mL). Taken together, these findings show that M-MSN/PEI-FA/VEGF shRNA nanocomplexes efficiently target gene silencing by FAmediating cellular uptake. 13755


Research Article


Figure 10. (A) Capillary-like structures of HUVECs in Matrigel basement membrane matrix. HeLa cells were treated for 72 h with a, culture medium (control); b, M-MSN/PEI-FA/SC shRNA; c, M-MSN/PEI-FA/VEGF shRNA; d, FA+ M-MSN/PEI-FA/VEGF shRNA; e, suramin; f, VEGF, and then the culture supernatant was collected. HUVECs were further incubated with the obtained culture supernatant. After incubation for 4 h at 37 °C, the cells were stained with calcein-AM solution and then observed under microscopy. HUVECs were treated with exogenous VEGF as negative control. (B) Quantitative analysis of tubule branch points. (a, control; b, M-MSN/PEI-FA/SC shRNA; c, M-MSN/PEI-FA/VEGF shRNA; d, FA+ M-MSN/PEI-FA/VEGF shRNA; f, suramin; e: VEGF). *p < 0.05 vs control.

membrane matrix. Because the tubule formation process is considered an essential step in angiogenesis, the number of tubule branch points (TBPs) is regarded as an important index of angiogenic activity.58 To determine further the effect of MMSN/PEI-FA/VEGF shRNA on angiogenesis, we executed the tube formation assay. As shown in Figure 10A, conditioned medium from HeLa transfected with M-MSN/PEI-FA/VEGF shRNA obviously decreased HUVECs capillary-like structure formation compared to transfected with M-MSN/PEI-FA/SC shRNA and control innocent HeLa conditioned medium, moreover, suramin treated HUVECs showed significantly lower TBPs due to the inhibition of microtubule formation, and the M-MSN/PEI-FA/VEGF shRNA group showed the same results, approximately 58% of the TBPs of the control group (Figure 10B), whereas the area and nets of the tubes in cells treated with MSN/PEI-FA/VEGF shRNA group were significantly less (Table S1). These data suggested that the VEGF secretion in HeLa cells was significantly reduced and might result in the inhibition of migration, invasion and microtubule formation in HUVECs.

■

AUTHOR INFORMATION

Corresponding Author

*Prof. Yiyao Liu, Ph.D. Tel: +86-28-8320-3353. Fax: +86-288320-8238. E-mail: [email protected] or liuyiyao@hotmail. com. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Nos. 81201192, 81471785, 81101147, 31470959, 11272083, 31470906, and 11502049), the Sichuan Youth Science and Technology Foundation of China (No. 2014JQ0008) for financial support.

■

CONCLUSION In summary, we have successfully designed and constructed DOX and VEGF shRNA codelivery nanocomplexes based on magnetic-mesoporous silica nanoparticles (M-MSN) and PEIFA modification. The nanocomplexes provided high DOX loading capacity and possessed an efficient siRNA protective effect. Drug release from the nanocomplexes was accelerated in acidic pH, and endo/lysosomal escape was also enhanced. Compared with M-MSN(DOX), the M-MSN(DOX)/PEI-FA nanoparticles could enter cells via the FA receptor-mediated pathway. VEGF shRNA loaded nanocomplexes demonstrated strong gene silencing activity and significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by transfected HeLa cells. The present work highlighted the potential of engineered nanocomplexes in the targeted codelivery of drugs/genes for cancer treatment.

■

targeting assay and identification of uptake pathways, in vitro synergistic effect assay of M-MSN(DOX)/PEI-FA/ VEGF shRNA, particle size distribution by dynamic light scattering(DLS), Prussian blue staining of HeLa cells, and various parameters of capillary-like structures formed by the HUVECs (PDF).

■

REFERENCES

(1) Yu, M. K.; Park, J.; Jon, S. Targeting Strategies for Multifunctional Nanoparticles in Cancer Imaging and Therapy. Theranostics 2012, 2, 3−44. (2) Yin, F.; Yang, C.; Wang, Q.; Zeng, S.; Hu, R.; Lin, G.; Tian, J.; Hu, S.; Lan, R. F.; Yoon, H. S.; Lu, F.; Wang, K.; Yong, K. T. A LightDriven Therapy of Pancreatic Adenocarcinoma Using Gold NanorodsBased Nanocarriers for Co-Delivery of Doxorubicin and siRNA. Theranostics 2015, 5, 818−833. (3) Dong, D. W.; Xiang, B.; Gao, W.; Yang, Z. Z.; Li, J. Q.; Qi, X. R. pH-Responsive Complexes Using Prefunctionalized Polymers for Synchronous Delivery of Doxorubicin and siRNA to Cancer Cells. Biomaterials 2013, 34, 4849−4859. (4) Zhang, W.; Guo, Z.; Huang, D.; Liu, Z.; Guo, X.; Zhong, H. Synergistic Effect of Chemo-Photothermal Therapy Using Pegylated Graphene Oxide. Biomaterials 2011, 32, 8555−8561. (5) Shi, S.; Shi, K.; Tan, L.; Qu, Y.; Shen, G.; Chu, B.; Zhang, S.; Su, X.; Li, X.; Wei, Y.; Qian, Z. The Use of Cationic mPEG-PCL-g-PEI Micelles for Co-delivery of Msurvivin T34A Gene and Doxorubicin. Biomaterials 2014, 35, 4536−4547. (6) Drozd, E.; Krzyszton-Russjan, J.; Gruber, B. Doxorubicin Treatment of Cancer Cells Impairs Reverse Transcription and Affects the Interpretation of RT-qPCR Results. Cancer Genomics Proteomics. 2016, 13, 161−170.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02963. Drug payload and release assay, agarose gel electrophoresis assay of VEGF shRNA protection, hemolysis and stability assay, HeLa cell viability assay, magnetic 13756


Research Article

ACS Applied Materials & Interfaces (7) Dong, X.; Mumper, R. J. Nanomedicinal Strategies to Treat Multidrug-Resistant Tumors: Current Progress. Nanomedicine (London, U. K.) 2010, 5, 597−615. (8) Yin, Q.; Shen, J.; Zhang, Z.; Yu, H.; Li, Y. Reversal of Multidrug Resistance by Stimuli-Responsive Drug Delivery Systems for Therapy of Tumor. Adv. Drug Delivery Rev. 2013, 65, 1699−1715. (9) Al-Lazikani, B.; Banerji, U.; Workman, P. Combinatorial Drug Therapy for Cancer in the Post-Genomic Era. Nat. Biotechnol. 2012, 30, 679−692. (10) Kajimoto, K.; Sato, Y.; Nakamura, T.; Yamada, Y.; Harashima, H. Multifunctional Envelope-Type Nano Device for Controlled Intracellular Trafficking and Selective Targeting in Vivo. J. Controlled Release 2014, 190, 593−606. (11) Liu, J.; Huang, Y.; Kumar, A.; Tan, A.; Jin, S.; Mozhi, A.; Liang, X. J. pH-Sensitive Nano-Systems for Drug Delivery in Cancer Therapy. Biotechnol. Adv. 2014, 32, 693−710. (12) Chen, S.; Hao, X.; Liang, X.; Zhang, Q.; Zhang, C.; Zhou, G.; Shen, S.; Jia, G.; Zhang, J. Inorganic Nanomaterials as Carriers for Drug Delivery. J. Biomed. Nanotechnol. 2016, 12, 1−27. (13) Li, J.; Wang, Y.; Zhu, Y.; Oupicky, D. Recent Advances in Delivery of Drug-Nucleic Acid Combinations for Cancer Treatment. J. Controlled Release 2013, 172, 589−600. (14) de Fougerolles, A.; Vornlocher, H. P.; Maraganore, J.; Lieberman, J. Interfering with Disease: A Progress Report on siRNA-Based Therapeutics. Nat. Rev. Drug Discovery 2007, 6, 443− 453. (15) Zamore, P. D.; Tuschl, T.; Sharp, P. A.; Bartel, D. P. Rnai: Double-Stranded RNA Directs the Atp-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals. Cell 2000, 101, 25−33. (16) Dominska, M.; Dykxhoorn, D. M. Breaking Down the Barriers: siRNA Delivery and Endosome Escape. J. Cell Sci. 2010, 123, 1183− 1189. (17) Yang, Z. Z.; Li, J. Q.; Wang, Z. Z.; Dong, D. W.; Qi, X. R. Tumor-Targeting Dual Peptides-Modified Cationic Liposomes for Delivery of siRNA and Docetaxel to Gliomas. Biomaterials 2014, 35, 5226−5239. (18) Yang, H.; Deng, L.; Li, T.; Shen, X.; Yan, J.; Zuo, L.; Wu, C.; Liu, Y. Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery into Human Breast Cancer Cells and Ultrasound Cellular Imaging. J. Biomed. Nanotechnol. 2015, 11, 2124−2136. (19) Yin, T.; Wang, P.; Li, J.; Zheng, R.; Zheng, B.; Cheng, D.; Li, R.; Lai, J.; Shuai, X. Ultrasound-Sensitive siRNA-Loaded Nanobubbles Formed by Hetero-Assembly of Polymeric Micelles and Liposomes and Their Therapeutic Effect in Gliomas. Biomaterials 2013, 34, 4532−4543. (20) Hannon, G. J. RNA Interference. Nature 2002, 418, 244−251. (21) Lee, S. J.; Yook, S.; Yhee, J. Y.; Yoon, H. Y.; Kim, M. G.; Ku, S. H.; Kim, S. H.; Park, J. H.; Jeong, J. H.; Kwon, I. C.; Lee, S.; Lee, H.; Kim, K. Co-Delivery of VEGF and Bcl-2 Dual-Targeted siRNA Polymer Using a Single Nanoparticle for Synergistic Anti-Cancer Effects in Vivo. J. Controlled Release 2015, 220, 631−641. (22) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2590−2605. (23) Wang, Y.; Gao, S.; Ye, W. H.; Yoon, H. S.; Yang, Y. Y. Codelivery of Drugs and DNA from Cationic Core-Shell Nanoparticles Self-Assembled from a Biodegradable Copolymer. Nat. Mater. 2006, 5, 791−796. (24) Tang, S.; Yin, Q.; Su, J.; Sun, H.; Meng, Q.; Chen, Y.; Chen, L.; Huang, Y.; Gu, W.; Xu, M.; Yu, H.; Zhang, Z.; Li, Y. Inhibition of Metastasis and Growth of Breast Cancer by pH-Sensitive Poly (BetaAmino Ester) Nanoparticles Co-delivering Two siRNA and Paclitaxel. Biomaterials 2015, 48, 1−15. (25) Sun, T. M.; Du, J. Z.; Yao, Y. D.; Mao, C. Q.; Dou, S.; Huang, S. Y.; Zhang, P. Z.; Leong, K. W.; Song, E. W.; Wang, J. Simultaneous Delivery of siRNA and Paclitaxel via a ″Two-in-One″ Micelleplex Promotes Synergistic Tumor Suppression. ACS Nano 2011, 5, 1483− 1494.

(26) Han, L.; Tang, C.; Yin, C. Dual-Targeting and pH/RedoxResponsive Multi-Layered Nanocomplexes for Smart Co-Delivery of Doxorubicin and siRNA. Biomaterials 2015, 60, 42−52. (27) Wang, M.; Zhang, J.; Yuan, Z.; Yang, W.; Wu, Q.; Gu, H. Targeted Thrombolysis by Using of Magnetic Mesoporous Silica Nanoparticles. J. Biomed. Nanotechnol. 2012, 8, 624−632. (28) Li, L.; Liu, T.; Fu, C.; Liu, H.; Tan, L.; Meng, X. Multifunctional Silica-Based Nanocomposites for Cancer Nanotheranostics. J. Biomed. Nanotechnol. 2014, 10, 1784−1809. (29) Shen, J.; Kim, H. C.; Su, H.; Wang, F.; Wolfram, J.; Kirui, D.; Mai, J.; Mu, C.; Ji, L. N.; Mao, Z. W.; Shen, H. Cyclodextrin and Polyethylenimine Functionalized Mesoporous Silica Nanoparticles for Delivery of siRNA Cancer Therapeutics. Theranostics 2014, 4, 487− 497. (30) Tang, F.; Li, L.; Chen, D. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. 2012, 24, 1504−1534. (31) Wang, L. S.; Wu, L. C.; Lu, S. Y.; Chang, L. L.; Teng, I. T.; Yang, C. M.; Ho, J. A. Biofunctionalized Phospholipid-Capped Mesoporous Silica Nanoshuttles for Targeted Drug Delivery: Improved Water Suspensibility and Decreased Nonspecific Protein Binding. ACS Nano 2010, 4, 4371−4379. (32) Cho, Y.; Borgens, R. B. The Preparation of Polypyrrole Surfaces in the Presence of Mesoporous Silica Nanoparticles and Their Biomedical Applications. Nanotechnology 2010, 21, 205102. (33) Zhao, X.; Zhu, D. M.; Gan, W. J.; Li, Z.; Zhang, J. L.; Zhao, H.; Zhou, J.; Li, D. C. Lentivirus-Mediated shRNA Interference Targeting Vascular Endothelial Growth Factor Inhibits Angiogenesis and Progression of Human Pancreatic Carcinoma. Oncol. Rep. 2013, 29, 1019−1026. (34) Bao, X.; Wang, W.; Wang, C.; Wang, Y.; Zhou, J.; Ding, Y.; Wang, X.; Jin, Y. A Chitosan-Graft-PEI-Candesartan Conjugate for Targeted Co-delivery of Drug and Gene in Anti-Angiogenesis Cancer Therapy. Biomaterials 2014, 35, 8450−8466. (35) Pan, Y.; Wu, Q.; Qin, L.; Cai, J.; Du, B. Gold Nanoparticles Inhibit VEGF165-Induced Migration and Tube Formation of Endothelial Cells via the Akt Pathway. BioMed Res. Int. 2014, 2014, 418624. (36) Ferrara, N. Vascular Endothelial Growth Factor. Arterioscler., Thromb., Vasc. Biol. 2009, 29, 789−791. (37) Kalishwaralal, K.; Banumathi, E.; Pandian, S.; Deepak, V.; Muniyandi, J.; Eom, S. H.; Gurunathan, S. Silver Nanoparticles Inhibit VEGF Induced Cell Proliferation and Migration in Bovine Retinal Endothelial Cells. Colloids Surf., B 2009, 73, 51−57. (38) Almubarak, M.; Newton, M.; Altaha, R. Reinduction of Bevacizumab in Combination with Pegylated Liposomal Doxorubicin in a Patient with Recurrent Glioblastoma Multiforme Who Progressed on Bevacizumab/Irinotecan. J. Oncol. 2008, 2008, 942618. (39) Chen, Y.; Bathula, S. R.; Li, J.; Huang, L. Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer. J. Biol. Chem. 2010, 285, 22639−22650. (40) Chang, B. S.; Chen, D.; Wang, Y.; Chen, Y. Z.; Jiao, Y. F.; Sha, X. Y.; Yang, W. L. Bioresponsive Controlled Drug Release Based on Mesoporous Silica Nanoparticles Coated with Reductively Sheddable Polymer Shell. Chem. Mater. 2013, 25, 574−585. (41) Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. I.; Nel, A. E. Engineered Design of Mesoporous Silica Nanoparticles to Deliver Doxorubicin and P-glycoprotein siRNA to Overcome Drug Resistance in a Cancer Cell Line. ACS Nano 2010, 4, 4539−4550. (42) Yang, H.; Li, Y.; Li, T. T.; Xu, M.; Chen, Y.; Wu, C. H.; Dang, X. T.; Liu, Y. Y. Multifunctional Core/Shell Nanoparticles Cross-Linked Polyetherimide-Folic Acid as Efficient Notch-1 siRNA Carrier for Targeted Killing of Breast Cancer. Sci. Rep. 2014, 4, 7072. (43) Feng, W.; Nie, W.; He, C.; Zhou, X.; Chen, L.; Qiu, K.; Wang, W.; Yin, Z. Effect of pH-Responsive Alginate/Chitosan Multilayers Coating on Delivery Efficiency, Cellular Uptake and Biodistribution of Mesoporous Silica Nanoparticles Based Nanocarriers. ACS Appl. Mater. Interfaces 2014, 6, 8447−8460. 13757


Research Article

ACS Applied Materials & Interfaces (44) Yang, H. W.; Huang, C. Y.; Lin, C. W.; Liu, H. L.; Huang, C. W.; Liao, S. S.; Chen, P. Y.; Lu, Y. J.; Wei, K. C.; Ma, C. C. Gadolinium-Functionalized Nanographene Oxide for Combined Drug and microRNA Delivery and Magnetic Resonance Imaging. Biomaterials 2014, 35, 6534−6542. (45) Yu, Q. D.; Liu, Y. N.; Cao, C. W.; Le, F. L.; Qin, X. Y.; Sun, D. D.; Liu, J. The Use of pH-Sensitive Functional Selenium Nanoparticles Shows Enhanced in vivo VEGF-siRNA Silencing and Fluorescence Imaging. Nanoscale 2014, 6, 9279−9292. (46) Dong, D.; Gao, W.; Liu, Y.; Qi, X. R. Therapeutic Potential of Targeted Multifunctional Nanocomplex Co-delivery of siRNA and Low-dose Doxorubicin in Breast Cancer. Cancer Lett. 2015, 359, 178− 186. (47) Raskopf, E.; Vogt, A.; Sauerbruch, T.; Schmitz, V. Sirna Targeting VEGF Inhibits Hepatocellular Carcinoma Growth and Tumor Angiogenesis in vivo. J. Hepatol. 2008, 49, 977−984. (48) Khalil, I. A.; Kogure, K.; Akita, H.; Harashima, H. Uptake Pathways and Subsequent Intracellular Trafficking in Nonviral Gene Delivery. Pharmacol. Rev. 2006, 58, 32−45. (49) Sahay, G.; Alakhova, D. Y.; Kabanov, A. V. Endocytosis of Nanomedicines. J. Controlled Release 2010, 145, 182−195. (50) Ran, R.; Liu, Y.; Gao, H.; Kuang, Q.; Zhang, Q.; Tang, J.; Huang, K.; Chen, X.; Zhang, Z.; He, Q. Enhanced Gene Delivery Efficiency of Cationic Liposomes Coated with Pegylated Hyaluronic Acid for Anti P-Glycoprotein siRNA: A Potential Candidate for Overcoming Multi-Drug Resistance. Int. J. Pharm. 2014, 477, 590− 600. (51) Gagliardi, A.; Hadd, H.; Collins, D. C. Inhibition of Angiogenesis by Suramin. Cancer Res. 1992, 52, 5073−5075. (52) Bhargava, S.; Hotz, B.; Hines, O. J.; Reber, H. A.; Buhr, H. J.; Hotz, H. G. Suramin Inhibits Not Only Tumor Growth and Metastasis But Also Angiogenesis in Experimental Pancreatic Cancer. J. Gastrointest. Surg. 2007, 11, 171−178. (53) Wang, H. Y.; Yi, W. J.; Qin, S. Y.; Li, C.; Zhuo, R. X.; Zhang, X. Z. Tyroserleutide-Based Gene Vector for Suppressing VEGF Expression in Cancer Therapy. Biomaterials 2012, 33, 8685−8694. (54) Gu, X.; Cun, Y.; Li, M.; Qing, Y.; Jin, F.; Zhong, Z.; Dai, N.; Qian, C.; Sui, J.; Wang, D. Human Apurinic/Apyrimidinic Endonuclease siRNA Inhibits the Angiogenesis Induced by X-Ray Irradiation in Lung Cancer Cells. Int. J. Med. Sci. 2013, 10, 870−882. (55) Carmeliet, P.; Jain, R. K. Molecular Mechanisms and Clinical Applications of Angiogenesis. Nature 2011, 473, 298−307. (56) Wang, J.; Li, G.; Wang, Y.; Tang, S.; Sun, X.; Feng, X.; Li, Y.; Bao, G.; Li, P.; Mao, X.; Wang, M.; Liu, P. Suppression of Tumor Angiogenesis by Metformin Treatment via a Mechanism Linked to Targeting of Her2/Hif-1alpha/VEGF Secretion Axis. Oncotarget. 2015, 6, 44579−44592. (57) Jo, D. H.; Kim, J. H.; Yu, Y. S.; Lee, T. G.; Kim, J. H. Antiangiogenic Effect of Silicate Nanoparticle on Retinal Neovascularization Induced by Vascular Endothelial Growth Factor. Nanomedicine 2012, 8, 784−791. (58) Arnaoutova, I.; George, J.; Kleinman, H. K.; Benton, G. The Endothelial Cell Tube Formation Assay on Basement Membrane Turns 20: State of the Science and the Art. Angiogenesis 2009, 12, 267−274.

13758
