Polypeptide cationic micelles mediated co-delivery of docetaxel and siRNA for synergistic tumor therapy

Biomaterials 34 (2013) 3431e3438

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Polypeptide cationic micelles mediated co-delivery of docetaxel and siRNA for synergistic tumor therapy Cuifang Zheng a, b, c, Mingbin Zheng a, b, c, Ping Gong a, b, c, Jizhe Deng a, b, c, Huqiang Yi a, b, c, Pengfei Zhang a, b, c, Yijuan Zhang a, b, c, Peng Liu a, b, c, Yifan Ma a, b, c, Lintao Cai a, b, c, * a

CAS Key Lab of Health Informatics, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China c Shenzhen Key Laboratory of Cancer Nanotechnology, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 December 2012 Accepted 10 January 2013 Available online 31 January 2013

Combination of two or more therapeutic strategies with different mechanisms can cooperatively impede tumor growth. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a single nanoparticle (NP) provides a rational strategy for combined cancer therapy. Here, we prepared polypeptide micelle nanoparticles (NPs) of a triblock copolymer poly(ethylene glycol)-b-poly(L-lysine)-bpoly(L-leucine) (PEGePLLePLLeu) to systemically codeliver docetaxel (DTX) and siRNA-Bcl-2 for an effective drug/gene vector. The hydrophobic PLLeu core entrapped with anticancer drugs, while the PLL polypeptide cationic backbone allowed for electrostatic interaction with the negatively charged siRNA. The resulting micelle NP exhibited very stable, good biocompatible and excellent passive targeted properties. The micelle complexes with siRNA-Bcl-2 effectively knocked down the expression of Bcl-2 mRNA and protein. Moreover, the co-delivery system of DTX and siRNA-Bcl-2 (DTXesiRNAeNPs) obviously down-regulation of the anti-apoptotic Bcl-2 gene and enhanced antitumor activity with a smaller dose of DTX, resulting the significantly inhibited tumor growth of MCF-7 xenograft murine model as compared to the individual siRNA and only DTX treatments. Our results demonstrated well-defined PEG ePLLePLLeu polypeptide cationic micelles with the excellent synergistic effect of DTX and siRNA-Bcl-2 in combined cancer therapy. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

Keywords: Polypeptide micelle DTX siRNA Co-delivery Synergistic tumor therapy

1. Introduction The combination of two or more therapeutic approaches with different mechanisms is a promising strategy for effective treatments of cancers with synergistic or combined effects [1,2]. Small RNA interference (RNAi) was a powerful technique which has been regarded as a potential therapeutic option for silencing target genes in various diseases [3,4]. A variety of siRNA-based therapeutics have been developed, showing great promise in disease treatments [5,6]. However, siRNA cannot easily cross cell membranes because of negative charge and their downstream effects are delayed, compared to those of conventional small-molecule/protein-based therapeutics [7]. Additionally, owing to their short serum half-life

* Corresponding author. CAS Key Lab of Health Informatics, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Tel.: þ86 755 86392210. E-mail address: [email protected] (L. Cai).

and poor cellular uptake, successful clinical application of siRNA requires effective delivery vehicles to overcome the numerous cellular barriers [8]. On the other hand, small molecular anticancer drugs effect can be much faster than siRNA during their intracellular uptake [9], at the same time nanocarriers can make the drugs targeted and lower side-effect. Therefore, the combination of newly emerging siRNA-based therapy with traditional chemotherapy would be beneficial. So far, several promising systems for such co-delivery purpose have been developed based on polymeric [10,11], liposomal [12,13] and silica-based [14,15] cationic NPs. In comparison, polymer-based non-viral vectors have great advantages over cationic lipids with respect to safety, convenient large-scale production, and physiological stability. Up to now, a variety of synthetic and natural cationic polymers have been investigated as gene or siRNA carriers, including poly(ethylene imine) (PEI) [16], poly(L-lysine) [17], and chitosan [18]. On the other hand, although a large amount of small molecular drugs such as paclitaxel and doxorubicin have demonstrated their anticancer potency, their clinical applications are facing tremendous

0142-9612/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.01.053

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challenges, including low water solubility, lack of targeting, leading to acute toxicity to normal tissues, and multi-drug resistance. Over the past two decades, polymeric micelles based on amphiphilic block copolymers which may potentially overcome these challenges have been extensively studied, resulting in the successful development of polymer-based micelle systems entering clinical trials [19,20] or receiving clinical approval (i.e. Genexol in Korea). We have recently developed a micelle system based on triblock copolymers poly(ethylene glycol)-b-poly-L-lysine-b-poly-L-leucine (PEGePLLePLLeu) for a highly effective gene vector [21]. The triblock polypeptide copolymers were amphiphilic and could selfassemble into micelle NPs, with PLLeu as the hydrophobic core, PLL as the cationic shell and PEG as the hydrophilic corona. These cationic micelles were expected to encapsulate the negatively charged siRNA and the hydrophobic DTX in a single NP as a codelivery gene/drug system. We characterized the properties of the micelle NPs, examined the ability of the micelleplex to simultaneously deliver siRNA-Bcl-2 and DTX into the same tumor cells, and further investigated the synergistic tumor suppression effect on the tumor xenograft model.

2.5. Stability test of DTXeNPs and characterization of micelleplex To evaluate the stability, DTXeNPs were diluted in phosphate buffer saline (PBS), fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM), then test the NPs size every week in one month. Also, we test the size and zeta potential of DTXeNPs and DTXesiRNAeNPs by Zetasizer Nano ZS instrument. 2.6. Cell culture MCF-7 human breast cancer cells, which are of Bcl-2 protein over-expression, were used. The DMEM medium supplemented with 10% FBS and 1% penicillin/ streptomycinin were utilized as the cell culture medium. Cells were cultivated at 37  C with 5% CO2. Before experiment, the cells were pre-cultured until confluence was reached to 75%. 2.7. Animals and tumor model Female BALB/c nude mice (4e6 weeks old and weighted 15e20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and all animals received care in compliance with the Guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. To set up the tumor xenograft model, MCF-7 cells (2  106) were administered by subcutaneous injection into the armpit of the mice. Tumor-bearing mice were used 2 weeks post-tumor inoculation.

2. Materials and methods 2.8. In vitro analysis of co-delivery of drug and siRNA into tumor cells 2.1. Materials Docetaxel (DTX) was purchased from Advanced Technology & Industrial Co. Ltd. (HK). Taxotere was purchased from Aventis Pharma (UK). The Lipofectamine 2000 transfection kit (Invitrogen, Carlsbad, CA) was used according to the supplied protocol. Antibody against human anti-apoptotic protein (Bcl-2) was purchased from Cell Signaling Technology (USA), b-actin conjugated IgGeHRP antibody was purchased from SigmaeAldrich (St. Louis, MO). Targeting human Bcl-2 siRNA (sence: 50 CCGGGAGAUAGUGAUGAAGdTdT-30 , anti-sence: 50 -CUUCAUCACUAUCUCCCGGdTdT30 ) and negative control siRNA (NCsiRNA) (sence: 50 -UUCUCCGAACGUGUCACGUdTdT-30 , anti-sence: 50 -ACGUGACACGUUCGGAGAAdTdT-30 ) were supplied by Shanghai GenePharma Co. Ltd. (Shanghai, China). Fluorescein-tagged siRNA (FAMsiRNA) was synthesized by modification of the 30 -end of the sense strand of the scrambled siRNA with fluorescein and was obtained from Shanghai GenePharma Co. Ltd. (Shanghai, China). 2.2. Preparation and characterization of PEG1ePLL10ePLLeu40 Synthesized the triblock polypeptide copolymers designated as PEG1ePLL10e PLLeu40 (the subscript number represents degree of polymerization of each block) using a previously reported procedure [21]. In brief, firstly synthesized the PEGe PLLZ copolymers by ring-opening polymerization of N-carboxyanhydride (NCA) of ε-benzyloxycarbonyl-L-lysine (LLZeNCA) using PEGeNH2 as initiator, then synthesized PEG1ePLLZ10ePLLeu40 copolymers by further ring-opening polymerization of LLeueNCA initiated by PEGePLLZ. The product (PEGePLLZePLLeu) was precipitated by diethyl ether and purified by repeated precipitation in diethyl ether and dried in vacuum. PEGePLLePLLeu copolymers were obtained by the deprotection of PEGe PLLZePLLeu. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker 400 MHz nuclear magnetic resonance instrument using CF3COOD as the solvents.

FAM-labeled siRNA NPs (siRNAeNPs) and rhodamine-labeled micelle NPs (DTXeNPs) were prepared as described above. MCF-7 cells (5  104 cells/well) were seeded in an 8-well coverglass plate and incubated for 24 h at 37  C in 5% CO2, followed by adding the DTXeNPs, siRNAeNPs and DTXesiRNAeNPs solutions. After 2 h incubation, removal of the medium, cells were washed twice with the preheated PBS and fixed with 4% paraformaldehyde solution for 20 min, then the nuclei were stained by 10 mg/mL Hoechest 33258 for 5 min and washed thrice with PBS, finally the fixed cells were observed by confocal laser scanning microscope (CLSM, Leica TCS SP5, Germany). 2.9. In vitro siRNA-Bcl-2 transfection and analysis of Bcl-2 expression MCF-7 cells (5  104) were seeded in 6-well plates and incubated at 37  C in 5% CO2 for 24 h to reach 70% confluence. Various formulations (PBS, Blank NPs, NC-NPs, siRNAeNPs 50, 100, 150 nM of siRNA), and siRNAeLipofectamine (100 nM of siRNA) were added and incubated with the cells for 24 h (for mRNA isolation) or 72 h (for protein extraction). The cellular levels of Bcl-2 mRNA and protein were assessed using quantitative real-time PCR (qRT-PCR) and Western blot, respectively. In qRT-PCR analysis, total RNA from transfected cells was isolated using the AxyPrep Multisource total RNA Miniprep Kit (Axygen, USA) according to the protocol of manufacturer. One micrograms of total RNA were transcribed into cDNA using the Prime Script first Strand cDNA Synthesis Kit (Takara, Japan). Thereafter, 1 mL of cDNA was subjected to qRT-PCR analysis targeting Bcl-2 and b-actin using the SYBR Green 1 qPCR Mix (Maygene Biotech, China). Analysis was performed using the Applied Biosystems StepOne Real-Time PCR Systems. Relative gene expression values were determined by the DDCT method using Step One Software v 2.1 (Applied Biosystems). Data are presented as the fold difference in Bcl-2 expression normalized to the house keeping gene b-actin as the endogenous reference, and relative to the untreated control cells. Primers used in qRT-PCR for Bcl2 and b-actin are:

2.3. Preparation and characterization of drug loaded micelle NPs Micelle NPs and DTX loaded micelle NPs (DTXeNPs) were prepared by directly dissolving PEG1ePLL10ePLLeu40 copolymers (2 mg) without or with DTX in DMSO at a concentration of 2 mg/mL sonication for 10 min, then added water makes the copolymers concentration of 1 mg/mL, and moved to the dialysis bag with magnetic stirring for 24 h. The sizes and zeta potentials of the self-assembled micelles were determined at 25  C by transmission electronic microscopic (TEM) and Zetasizer Nano ZS (Malvern Instrument). The final volume was adjusted to 4 mL for further experiments. 2.4. Preparation of micelleplex and gel retardation assay Micelle NPs or DTXeNPs were diluted with DEPC water at different concentrations. Desired amount of siRNA (siRNA-Bcl-2 or NCsiRNA) in DEPC water was then mixed with equal volume of NPs by gentle pipetting. The formed micelleplex was allowed to stand at room temperature for 20 min before use. The electrophoretic mobility of micelleplex was visualized on a UV illuminator with gelred staining after electrophoresis on a 2% (w/v) agarose gel for 20 min at 60 V in TAE buffer (40 mM TriseHCl, 1% v/v acetic acid, 1 mM EDTA).

Bcl-2-forward 50 -AACATCGCCCTGTGGATGAC-30 Bcl-2-reverse 50 -AGAGTCTTCAGAGACAGCCAGGAG-30 and b-actin-forward 50 -CGCGAGAAGATGACCCAGATC-30 b-actin-reverse 50 -CATGAGGTAGTCAGTCAGGTCCC-30 PCR parameters consisted of 30 s of Taq activation at 95  C, followed by 40 cycles of PCR at 95  C, 5 s, 60  C, 30 s, and 1 cycle of 95  C, 15 s, 60  C, 60 s, and 95  C, 15 s. Standard curves were generated and the relative amount of target gene mRNA was normalized to bactin mRNA. Specificity was verified by melt curve analysis. In Western blot analysis, transfected cells were washed twice with cold PBS, and then resuspended in 50 mL of lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA) freshly supplemented with Roche’s Complete Protease Inhibitor Cocktail Tablets. The cell lysates were incubated on ice for 30 min and vortexed every 5 min. The lysates were then clarified by centrifugation for 10 min at 12,000 g. The protein concentration was determined using the BCA Protein Assay Kit (Tiangen, China). Total protein (60 mg) was separated on 12% BiseTrisepoly-acrylamide gels and then transferred (at 150 mA for 120 min) to PVDF membranes (Millipore, Bedford, MA). After incubation in 5% non-fat milk powder (Merck, Germany) in phosphate buffered

C. Zheng et al. / Biomaterials 34 (2013) 3431e3438 saline with Tween-20 (PBST, pH 7.2) for 1 h. The membranes were incubated in 5% non-fat milk powder in PBST with Bcl-2 antibodies (1:500) over night. After incubation in 5% non-fat milk powder in PBST with goat anti rabbit IgGeHRP antibody (1:3500) for 60 min, bands were visualized using the ECL system (Pierce).

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2.10. Cell proliferation analysis To evaluate the cytotoxicity of micelle NPs and DTXesiRNAeNPs, MCF-7 cells were grown with the NPs over a wide range of concentrations. The cell viability was measured by MTT assay. Cells (5  103 cells/well) were seeded into 96-well plates

Fig. 1. (A) Synthesis of PEGePLLePLLeu copolymers. (B) 1H NMR spectra of PEGePLLZePLL copolymers. (C) 1H NMR spectra of PEGePLLePLLeu triblock copolymers.

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and incubated at 37  C, 5% CO2 before experiment, and total volume was 100 mL. After 12 h, the old medium was replaced with the medium containing the NPs over a wide range of concentrations (from 0.5 to 80 mg/mL NPs) and different NPs samples and other controls (contain 0.05 mg/mL DTX). Untreated cells in growth media were used as the blank control. After 24 h, added 20 mL/well MTT solution (5 mg/mL) incubated for another 4 h, then the solution was carefully removed, and 150 mL DMSO was added to dissolve the MTT formazan crystal. The absorbance at a wavelength of 490 nm of each well was measured by a microplate reader (Synergy 4, Bio Tec, USA). The cell viability (%) was defined as the percentage of the absorbance of the cells containing the cells incubated with the NPs suspension over the blank control. All data were presented as the mean of four measurements (SD). The experiment was repeated three times.

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2.11. Micelle NPs distribution in nude mice The distributions of rhodamine-labeled micelle NPs (DTXeNPs) in nude mice were analyzed using a Maestro in vivo imaging system (CRi, Inc., USA). DTXeNPs and PBS control were intravenously injected into nude mice bearing MCF-7 tumors. 24 h later, the mice were sacrificed and the organs including heart, liver, spleen, lung, kidneys and tumor were collected and analyzed by the Maestro in vivo imaging system.

Fig. 3. Binding ability of Blank NPs and DTXeNPs to siRNA at different ratios of nitrogen in carrier to phosphate in siRNA (N/P ratio) demonstrated by the agarose gel retardation assay N/P ratios in the nanoparticles and siRNA.

2.14. Statistical analysis All of the data represent mean values  standard deviation of independent measurements. Statistical analysis was performed with a Student’s t-test (twotailed). Statistical significance was assigned at p < 0.05 (95% confidence level).

2.12. Tumor suppression study When the tumor volume was approximately 100 mm3, the mice were randomly divided into different groups (n ¼ 5), and treated with various formulations by i.v. injection. Details are listed as follows: Group 1: PBS, Group 2: Blank NPs, Group 3: NC-NPs, Group 4: siRNAeNPs, Group 5: Taxotere, Group 6: DTXeNPs, Group 7: siRNAeNPs þ DTXeNPs, Group 8: DTXesiRNAeNPs. The doses of siRNA-Bcl-2 (or NCsiRNA) and DTX of each injection were fixed at 0.2 mg/kg and 0.5 mg/kg, respectively. The injection was performed every 3 days within 24 days. The antitumor activity was evaluated in terms of the tumor size at different times postadministration, which was estimated by the following equation: V ¼ 6  larger diameter  (smaller diameter)2/p, as reported previously [22]. The tumor volumes and mice weights were measured after injection every time.

3. Results and discussion 3.1. Preparation and characterization of drug loaded micelle NPs The synthesis and 1H NMR spectra of PEG1ePLL10ePLLeu40 were given in Fig. 1. As shown in Fig. 1A, the PEGePLLePLLeu copolymers were synthesized through three steps. The first step was to prepare the diblock copolymer PEGePLLZ by ring-opening polymerization of LLZeNCA using mPEGeNH2 as initiator. Then, the triblock copolymer PEGePLLZePLLeu was synthesized by further ringopening polymerization of LLeueNCA using amino-terminated PEGePLLZ as a macromolecular initiator. The amphiphilic PEGe PLLePLLeu triblock copolymers were obtained after the deprotection of PEGePLLZePLLeu by HBr/HAc in TFA solution. The 1H NMR spectra of PEGePLLZePLLeu and PEGePLLePLLeu are shown

2.13. Detection of Bcl-2 expression in tumor tissues Tumor tissues were collected 24 h after the last treatment, and lysed in 800 mL RIPA tissue lysis buffer (include 1% 100 mM PMSF) freshly supplemented with the lysates were incubated on ice and vortexed by the Tissue-Tearor for 10 min. The lysates were centrifuged for 10 min at 12,000 g and the protein concentration was determined using the BCA Protein Assay Kit. Total protein (60 mg) was then analyzed by the Western blot as described in Section 2.9.

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Fig. 2. (A) Schematic illustration of self-assembled cationic micelle and loading of siRNA and drug. (B) Transmission electronic microscopic image. (C) Zeta potential analysis of docetaxel micelle NPs.

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in Fig. 1B, C respectively. As shown in Fig. 1B, the peaks around 3.8 ppm are attributable to the protons (eCH2CH2O) in the PEG chain, the peaks around 7.3 ppm are attributable to the protons (e C6H5) in PLLZ, and the peaks present between 0.8 and 0.9 ppm are attributable to the protons (eCH3) in PLLeu. The polypeptide self-assembled a micelle structure in aqueous solution and exhibited the ability of simultaneous loading of siRNA and DTX (Fig. 2A). The DTXeNPs micelles showed compact and spherical morphology with a mean diameter of 30e50 nm (Fig. 2B), demonstrated by the transmission electronic microscopic (TEM) image. The zeta potential of the DTXeNPs was about þ38.8 mV (Fig. 2C). siRNA-Bcl-2 was subsequently absorbed to the assembly through a charge interaction with the PLL block to form the micelleplexes, denoted as DTXesiRNAeNPs. Efficient siRNA binding occurred at a 10/1 molar ratio of nitrogen/phosphate (N/P ratio) in the carrier and siRNA as demonstrated by a gel retardation assay (Fig. 3). After loaded DTX, the polypeptide micelles DTXeNPs were quite stable near the size of 100 nm in PBS, FBS and DMEM solution as shown in Fig. 4A. It was important for in vivo experiments

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3.2. In vitro siRNA transfection To demonstrate the simultaneous delivery, we analyzed the cellular uptake and intracellular distribution of DTXesiRNAeNPs in MCF-7 cells, where rhodamine and fluorescein (FAM) were labeled at DTXeNPs and siRNAeNPs respectively. Cells were incubated with DTXeNPs, siRNAeNPs and DTXesiRNAeNPs for 4 h. The confocal microscopy images (Fig. 5) showed a high degree of colocalization of the red and green fluorescence distributed in the cytoplasm, from which the simultaneous delivery of DTX and siRNA was confirmed. We incubated MCF-7 cells with siRNA-Bcl-2 packaged micelles (siRNAeNPs) for 24 h and then detected Bcl-2 mRNA expression using real-time PCR. After sequence-specific Bcl-2 gene silencing by siRNAeNPs, the Bcl-2 mRNA expression level was reduced in a siRNA-Bcl-2 dose-dependent manner (Fig. 6A). A higher siRNA-

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thereafter. As shown in Fig. 4B, the size of siRNA loaded DTXeNPs increased from 90.58  1.2 nm to 121.3  1.9 nm, and the zeta potential decreased from þ38.8  2.1 mV to þ20.48  1.8 mV.

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Fig. 5. Confocal laser scanning microscope (CLSM) images of intracellular distribution of DTXeNPs, siRNAeNPs and DTXesiRNAeNPs in MCF-7 cell after incubation for 4 h. DTXe NPs and siRNAeNPs were labeled with rhodamine (Rho, red) and fluorescein (FAM, green), respectively. Cell nuclei were stained with Hoechest 33258 (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. (A) Biocompatibility test of micelle NPs. (B) Co-delivery effect of DTX and siRNABcl-2 by micelleplex on the proliferation of MCF-7 cells, **p < 0.001. The concentration of DTX was 0.05 mg/mL, while the concentration of both siRNA-Bcl-2 and NCsiRNA was 100 nM. Free DTX was dissolved in DMSO for cell culture.

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Fig. 6. (A) Expression of Bcl-2 mRNA determined by quantitative real-time PCR. (B) Representative Bcl-2 protein expression determined by Western blot analysis. (C) Analysis of light intensities of Bcl-2 protein expression as the ratio of Bcl-2 to b-actin from Western blot results. MCF-7 cells were transfected with micelleplex siRNA-Bcl-2 at N/P of 10:1 with different siRNA-Bcl-2 doses. The concentrations of siRNA-Bcl-2 with Lipofectamine 2000 (siRNAeLipofectamine) and NCsiRNA with micelle NPs micelleplex (NC-NPs) were 100 nM. Transfection experiments were performed independently three times. *p < 0.05, **p < 0.001 as compared with controls (n ¼ 3).

Bcl-2 concentration resulted in more significant knockdown efficacy. For example, 50 and 100 nM of siRNA-Bcl-2 led to approximately 32% and 78% knockdown of Bcl-2 mRNA, respectively, whereas there was no clear knockdown efficiency on PBS, blank micelle NPs (Blank NPs), and micelle carrying negative control siRNA (NC-NPs). The siRNAeNPs transfection is similar to that of Lipofectamine 2000 transfection reagent carrying 100 nM of siRNABcl-2 (siRNAeLipofectamine), indicated the highly effective gene vector of the polypeptide micelles. A reduction in Bcl-2 mRNA was subsequently accompanied by decreased Bcl-2 protein expression in a similar dose-dependent manner (Fig. 6B, C) following transfection with siRNAeNPs, as determined by Western blot analyses of Bcl-2 protein in the cell lysates 72 h after transfection.

when the NPs was 0.5 mg/mL the cell viability was 100.01%. As indicated in Fig. 7B, the micelle DTXeNPs treatment showed better inhibitory effect than that of the free DTX, indicating that DTXeNPs can enhance the cytotoxicity of DTX at certain dose. Moreover, DTXesiRNAeNPs significantly reduced cell proliferation to 8.9%, achieving a synergistic inhibitory effect through simultaneous delivery of siRNA-Bcl-2 with DTX at a low concentration (0.05 mg/mL), and at this concentration the NPs (0.5 mg/mL) were almost no cytotoxicity. It was worth noting that blank micelles (Blank NPs) and NC-NPs did not exhibit a significant inhibitory effect on cell proliferation. 3.4. Micelle distribution in nude mice A fluorescence imaging approach was used to investigate the DTXeNPs distribution in nude mice. Rhodamine-labeled DTXeNPs (DTXeNPs) and PBS control were delivered via tail vein injections. Fig. 8 showed the fluorescence signals from the DTXeNPs group nude mice were located in the liver and tumor, and the signal from the tumor was very strong, which could be contributed to the excellent passive targeting effective of the PEGePLLePLLeu micelle delivery system.

3.3. Cell proliferation analysis 3.5. Tumor suppression study in vivo Cell proliferation was determined by MTT assay. As shown in Fig. 7A, the blank micelle NPs did not significantly affect the viability of MCF-7 cells from 0.5 mg/mL to 80 mg/mL concentration, and

The tumor growth inhibitions with a series of DTX/siRNA/ micelle formulation in vivo were performed to evaluate the

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Fig. 8. Fluorescence images of organs and tumors in MCF-7 tumor-bearing mice 24 h after i.v. injection of DTXeNP and PBS. Fluorescence signal was mainly localized in tumor and liver.

synergistic effect of DTXesiRNAeNPs. Mice bearing MCF-7 xenografts were treated by DTXesiRNAeNPs or various other formulations through i.v. injection every three days from the 15th day after xenograft implantation. As shown in Fig. 9A, the delivery of DTX by DTXeNPs at a lower DTX dose (0.5 mg/kg per injection) hardly affected tumor growth compared with PBS treatment. Delivery of siRNA-Bcl-2 (0.2 mg/kg per injection) by siRNAeNPs only moderately inhibited tumor growth. However, simultaneous delivery of the same doses of DTX and siRNA-Bcl-2 by DTXesiRNAeNPs and DTXeNPs þ siRNAeNPs exhibited particularly significant inhibition

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of tumor growth compared with PBS treatment (p < 0.001). Further more, a synergistic inhibitory effect of the two therapeutic agents on tumor growth was demonstrated. In contrast, combinatorial delivery of separate siRNA-Bcl-2 and DTX by siRNAeNPs þ DTXe NPs only showed moderate inhibition of tumor growth and no synergistic effect was observed, primarily due to the more separate internalization of the two micelle NPs by tumoral cells as demonstrated above. Taxotere is a clinically used formulation of DTX dissolved in Cremophor EL and 50% ethanol. The DTXeNPs group was not effective as in vitro experiment because the dose of the DTX was low. The doses of siRNA and DTX for injection in the therapeutic process were only 0.5 mg/kg and 0.2 mg/kg every injection. Such a dose of DTX was comparable to and much lower than the doses used only (10 mg/kg for DTX NPs and 30 mg/kg for free DTX [23]). We improved that the polypeptide micelle mediated drug and siRNA simultaneous delivery system was more effective than the physical mixture system. Fig. 9B showed that the weights of the nude mice from different groups have not significant change compared to the PBS control, which indicated that the micelle NPs were safety material. The time course survival rates of nude mice receiving different formulations are in line with the results of tumor growth inhibition (Fig. 9C). All animals in the PBS control group died in 21 days. Although the treatment groups (DTXesiRNAeNPs and DTXe NP þ siRNAeNPs) demonstrated similar therapeutic effect (Fig. 9A), the survival rate of the DTXeNPs þ siRNAeNPs group was 40% in 27 days, in comparison, 100% of the DTXesiRNAeNPs group.

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Fig. 9. Antitumor effect of DTXesiRNAeNPs and other controls (PBS, Blank NPs, NC-NPs, siRNAeNPs, Taxotere, DTXeNPs, DTXeNPs þ siRNAeNPs, DTXesiRNAeNPs) through i.v. injection in MCF-7 xenografts tumor-bearing nude mice. (A). The growth curve of tumor showed that the treatment groups significantly inhibited the growth of tumor as compared with the controls **p < 0.001 (n ¼ 5). (B). The body weights of the mice in all groups. (C). Cumulative survival rate in all groups. (D). A representative mouse from each group and Bcl-2 protein expression of tumor tissue from each group determined by Western blot analysis. Dose per injection: DTX 0.5 mg/kg and siRNA 0.2 mg/kg.

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Analysis of Bcl-2 protein of each tumor mass by Western blot showed consistent knockdown efficiency. Down-regulation of Bcl-2 protein occurred only when the micelle NPs containing siRNA-Bcl-2 (Fig. 9D). It should also be noted that such a dose of DTX and siRNABcl-2 delivered by the micelle NPs also make the drug and siRNA more sensitive. 4. Conclusions In summary, we designed the self-assembled polypeptide cationic micelle for systemic and simultaneous delivery of siRNA and a chemotherapeutic drug demonstrating synergistic tumor growth inhibition in vivo. The micelleplex system showed great superiority in simultaneously delivering the two pay-loads into the tumor cell, passive targeting to the tumor in vivo and could remarkably inhibit tumor growth in a synergistic manner. The synergistic tumor suppression effect was obviously correlative to the simultaneous delivery of siRNA and DTX into tumor cells. Additionally, the micelleplex system was highly stable and non-cytotoxic. Such welldefined polypeptide cationic micelle provides a promising codelivery system with high effective and synergistic tumor growth inhibition for combined cancer therapy. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81071249, 81171446, 20905050), Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies (GIRTF-LCHT), the Key Project of Science and Technology of Guangdong (2009A030301010), the Shenzhen Science and Technology Program (Grant No. JC201005260247A, CXB201005250029A, JCYJ20120615125829685) and the “Hundred Talents Program” of Chinese Academy of Sciences. References [1] Sun TM, Du JZ, Yao YD, Mao CQ, Dou S, Huang SY, et al. Simultaneous delivery of siRNA and paclitaxel via a “two-in-one” micelleplex promotes synergistic tumor suppression. ACS Nano 2011;5:1483e94. [2] Greco F, Vicent MJ. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev 2009;61:1203e13. [3] Li B, Tang Q, Cheng D, Qin C, Xie FY, Wei Q, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in rhesus macaque. Nat Med 2005;11(9):944e51.

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