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Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery Yanfang Yang, Xuejun Xia, Wujun Dong, Hongliang Wang, Lin Li, Panpan Ma, Wei Sheng, Xueqing Xu, Yuling Liu* An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo.

1. Introduction The highly specific nature and clear-cut design principle demonstrate the potential of small interfering RNA (siRNA) as anticancer drugs of the future.[1] Routine Dr. Y. Yang, Prof. X. Xia, Dr. W. Dong, Dr. H. Wang, L. Li, P. Ma, W. Sheng, X. Xu, Prof. Y. Liu State Key Laboratory of Bioactive Substance and Function of Natural Medicines Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050, P.R. China E-mail: [email protected] Dr. Y. Yang, Prof. X. Xia, Dr. W. Dong, Dr. H. Wang, L. Li, P. Ma, W. Sheng, X. Xu, Prof. Y. Liu Beijing Key Laboratory of Drug Delivery Technology and Novel Formulation Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050, P.R. China Macromol. Biosci. 2016, 16, 759−773 © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

clinical use of siRNA therapeutics will depend particularly on the development of appropriate delivery vehicles. But enzyme degradation in vivo and the efficient cell membrane transport remains major challenge for siRNA delivery. Polymer micelles, formed by amphiphilic block copolymers self-assembling into a nanoscopic (10–200 nm) core/shell structures in an aqueous environment, show potential in siRNA delivery.[2] Unfortunately, relatively limited internalization efficiency necessitates further improvement for in vivo application of polymer micelles.[3] Cell penetrating peptides (CPPs), with potent cell penetrating ability, facilitating the intracellular delivery of various cargos without causing any cellular injury, have been widely used to improve cell internalization of polymer micelles.[4] Currently, activable CPPs were usually immobilized on the surface of these siRNA complexed polymer micelles to improve their cell internalization and the CPPs could be exposed via internal (over-expressed enzymes and lower pH value) or external stimulus (light and thermal).[5] Despite the great improvement on siRNA transfection of these nanocarriers, there still remain

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DOI: 10.1002/mabi.201500389

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barriers for these systems to be addressed: first, the immobilized CPPs on the surface of these nanocarriers may not be well protected from enzymatic degradation in vivo before approaching the targeting sites; second, because of the stiff structure and low spatial charge density of siRNA, it is not easily condensed by the commonly used cationic polymers. And the unstable siRNA complexed micelles can be easily destabilized by anionic serum proteins abundant in blood and extracellular fluid.[6] Therefore, how to combine activable CPPs and polymer micelles skillfully to improve CPPs’ shielding/ deshielding efficiency and micelle stability in the body circulation is important for biomedical applications of polymer micelles in siRNA transfection. It is reported that glutathione (GSH) concentration in the cell interior is 100–1000 times higher than that in the cell exterior, which made reversible disulfide bond linked siRNA conjugates noteworthy.[7] Reversible stability is an important feature for nucleic acid delivery vehicles, because the release of siRNA into the cytoplasm is required to access the RNAi pathway.[8] Therefore, disulfide bond linked siRNA-CPPs conjugates may be potential in siRNA delivery: siRNA could not only be transported into cell membrane with the help of CPPs, but also be dissociated from the carrier in the cytoplasm in response to intracellular GSH, thus siRNA-CPPs will lessen the influence of carrier on RNAi of siRNA.[9] However, nonspecificity and enzymatic degradation of CPPs in vivo limited siRNA transfection efficiency in vivo. Encapsulating CPPs into polymer micelles with triggered release mechanism to conceal their function in normal body circulation and expose them via stimulus at the target site may be a superior way to improve the shielding/deshielding efficiency of CPPs. As the CPPs were caged in the stimulus-responding nanocarrier, they were absolutely isolated from the blood in the body circulation and were prevented from being degraded by protease in the blood, thus their function masking efficiency was greatly improved. How to expose or activate CPPs efficiently at the target tumor extracellular is also important for CPPs mediated siRNA transfection, so the construction of stimulus-responding polymer micelles is vital. Many stimulus-responding nanocarriers were reported, including pH, light, or thermal triggered drug release of nanocarrier and so on.[10] External stimulus such as light or thermal, which needs precise positioning of the tumor, may not be suitable for some tumors of deep location in vivo. Since the pH differences between the solid tumor (pH 6.5–7.2) and blood (pH 7.4) are relatively simple and intrinsic, pH-sensitive polymeric micelle look like the most attractive candidate.[11] Poly (L-histidine) (PHIS) is a pH-sensitive polymer as the imidazole ring has an electron lone pair on the unsaturated nitrogen endowing PHIS with an amphoteric nature by

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protonation–deprotonation and has been extensively developed to construct pH-sensitive drug delivery nanocarrier.[12] Block copolymers of polyethylene glycol (PEG) and PHIS have been reported to have a pKb of 7.0–6.5, which means the micelles formed by this copolymer will dissociate at pH value of 7.0–6.5. And this is benefit for constructing tumor extracellular pH-responding drug delivery system.[13] Thus, caging the above mentioned disulfide bond linked siRNA-CPPs by PHIS-PEG-based micelles may have great potential in siRNA delivery for tumor-killing platform under dual stimulus of acid and intracellular redox environment. Although the nanoscale polymeric micelles could passively accumulated in the tumor via enhanced permeation and retention (EPR) effect, active targeting ligands modification on the surface could elicit cell surface binding and receptor-mediated endocytosis. Thus obviously boost the targeting effect of the nanocarrier, especially for some specific tumors. Folate receptor is highly expressed on a number of malignant tumor tissues including breast and lung cancers. Hence, folate conjugated polymer nanocarrier are extensively being explored for site specific delivery of anticancer drugs to cancer cells to improve their biodistribution and tumor therapy.[14] In this work, for the first time, utilizing the dual stimulus of lower pH value of tumor extracellular environment and redox effect of GSH in the cytosol, an active targeting delivery system (siRNA-CPPs/F-ASPM) combining folate (F), acid-sensitive polymer micelles (ASPM), and disulfide bond linked siRNA-CPPs would be constructed for efficiently siRNA transfection to MCF-7 cells. As shown in Figure 1: (i) Amphiphilic block copolymers PEG-PHIS and F-PEG-PHIS would be synthesized and self-assembled into acid-sensitive nanocarriers with active targeting ability (F-ASPM); (ii) CPPs (CKRRMKWKK), derived from penetratin were conjugated with siRNA via disulfide bond to form siRNA-CPPs conjugate. Then siRNA-CPPs would be entrapped into F-ASPM to conceal CPPs function in the normal body circulation. (iii) After intravenous injection, siRNA-CPPs/F-ASPM would bind to folate receptor over-expressed MCF-7 cells preferentially with the targeting ability of F; (iv) At the acid tumorous zone, F-ASPM would be destabilized, resulting in the release of siRNACPPs. The activated siRNA-CPPs then penetrated into the cell membrane and escaped from the endosomal entrapment into the cytosol with the help of CPPs; (v) Disulfide bond of siRNA-CPPs were reduced by GSH in the cytosol and dissociated siRNA from the carrier easily, resulting in efficiently gene knockdown of the tumor. The MCF-7 cells bearing nude female mice would be constructed to systematically evaluate the mechanism and corresponding therapeutic efficiency of siRNA-CPPs/F-ASPM both in vitro and in vivo.

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Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery

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Figure 1. Proposed schematic diagram of our designed folate-targeted acid-sensitive polymeric micelles (F-ASPM) containing siRNA-CPPs conjugate for specific siRNA delivery to cancer cells under dual stimulus of acid tumor extracellular and intracellular redox environment. (i) Amphiphilic block copolymers PEG-PHIS and F-PEG-PHIS would be synthesized and self-assembled into acid-sensitive nanocarriers with active targeting ability (F-ASPM); (ii) CPPs (CKRRMKWKK), derived from penetratin were conjugated with siRNA via disulfide bond to form siRNACPPs conjugate. Then siRNA-CPPs would be entrapped into F-ASPM to conceal CPPs function in the normal body circulation. (iii) After intravenous injection, siRNA-CPPs/F-ASPM would bind to folate receptor over-expressed MCF-7 cells preferentially with the targeting ability of F; (iv) At the acid tumorous zone, F-ASPM would be destabilized, resulting in the release of siRNACPPs. The activated siRNA-CPPs then penetrated into the cell membrane and escaped from the endosomal entrapment into the cytosol with the help of CPPs; (v) Disulfide bond of siRNA-CPPs were reduced by GSH in the cytosol and dissociated siRNA from the carrier easily, resulting in efficiently gene knockdown of the tumor.

2. Experimental Section 2.1. Materials Hydroxyl-polyethyleneglycol-Maleimide (HO-PEG2000-Mal, HO-PEG3400-Mal) and Folic acid were supplied by Suzhou Bank valley Co., Ltd, (Jiangsu, China). Lyophilized c-myc siRNA against c-myc mRNA (5′-AACGUUAGCUUCACCAACAUU-3′) and Negative control siRNA (antisense strand, 5′-ACGUGACACGUUCGGAGAATT-3′), both of which were chemically modified with a thiol group at the 5′ end of one RNA strand and a 5′ FAM or Cy3 modification on the complementary strand, were obtained from GenePharma (Shanghai, China). All primers were synthesized by AuGCT Biotechnology (Beijing, China). Poly-L-histidine (PHIS) (MW 5000) and CPPs (CKRRMKWKK) were custom synthesized by Shanghai GL Biochem Co, Ltd, (Shanghai, China). Trypsin was obtained from Solarbio. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO, Invitrogen Corp. (Carlsbad, USA). Carbodicyclohexylimide (DCC), Diamide, 3-(4,5-Dimethylthia-zol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and Hoechst 33258 were purchased from

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Sigma-Aldrich (St Louis, MO). Penicillin and streptomycin were provided by North China Pharmaceutical Co., Ltd, (Hebei, China). LysoTracker Red and Annexin V-FITC apoptosis detection kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). TRNzol A+ reagent and Quantscript RT Kit were purchased from Tiangen Biotec Co., Ltd (Beijing, China). Anti-c-myc monoclonal antibody and rabbit anti-β-actin were supplied by Applygen Technologies Inc, (Beijing, China). All other chemicals were analytical or high performance liquid chromatography (HPLC) grade. Female BALB/c nude mice (18–22 g) were purchased from Vital River Laboratories (Beijing, China). All animals were handled according to the code of ethics in research, training, and testing of drugs as laid down by the Animal Care and Use Ethics Committee of Chinese Academy of Medical Sciences and Peking Union Medical College.

2.2. Synthesis of Folate-PEG-PHIS As shown in Figure 2a, folic acid was added in DMF solution of HO-PEG3400-Mal in the presence of DCC as catalyst in ice water bath and stirred for 48 h. The obtained mixture was

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Figure 2. a) Synthetic procedure for Folate-PEG-PHIS. b) Schemes on the synthesis of PEG-PHIS. c) MALDI-TOF mass spectra of the synthesized Folate-PEG-PHIS. d) MALDI-TOF mass spectra of the synthesized PEG-PHIS. concentrated and precipitated in excess diethyl ether. At last, the Folate-PEG-Mal was obtained. Then Folate-PEG-PHIS was synthesized via the thiol-ene “click” reaction of the maleimide group with the sulfhydryl group of PHIS. Briefly, Folate-PEG-Mal and PHIS (1.2:1, mol/mol) were mixed in DMSO and stirred at room temperature for 24 h under the nitrogen gas to produce Folate-PEG-PHIS. The obtained Folate-PEG-PHIS was dialyzed with cellulose ester membranes (MWCO 5 kDa) against DMSO firstly for 8 h and then against deionized water for another 48 h to remove the unreacted ones. Finally, the solution was lyophilized, confirmed using MALDI-TOF MS and stored at −20 °C for further utilization.

2.4. Synthesis of siRNA-CPPs Conjugates siRNA-CPPs conjugate was synthesized by coupling CPPs with siRNA via disulfide bond as reported by Muratovska et al.[15] As depicted in Figure 3a, c-myc siRNA, CPPs, and thiol oxidant diamide were dissolved in HEPES buffer (10 × 10−3 M HEPES, 1 × 10−3 M ethylenediamine tetraacetic acid (EDTA), pH 8.0) to obtain a final concentration of 17.5, 20, and 20 × 10−6 M, respectively. Then equimolar amounts of siRNA, CPPs and diamide were mixed and incubated for 1 h at 40 °C. The conjugates prepared were purified by minicolumn centrifugation method using dextran gel (Sephadex G-50) column. At last, the product with a yield of 82.7% was obtained and verified by MALDI-TOF MS.

2.3. Synthesis of PEG-PHIS An excess amount of HO-PEG2000-Mal (1.2:1, mol/mol) was added to DMSO solution containing 10 mg poly (L-histidine) and stirred at room temperature for 24 h under the nitrogen gas. The obtained products were dialyzed with cellulose ester membranes (MWCO 5 kDa) against DMSO first for 8 h and then against deionized water for another 48 h to remove the unreacted ones. Finally, the solution was lyophilized, confirmed using MALDITOF MS and stored at −20 °C for further utilization. The synthetic scheme was depicted in Figure 2b.

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2.5. Acid-Sensitive Micelles Containing siRNA-CPPs Folate-targeted or nontargeted acid-sensitive micelles carrying siRNA-CPPs were prepared as follows: Folate-PEG-PHIS (2 mg) and PEG-PHIS (3 mg) or PEG-PHIS (5 mg) was dissolved in DMSO and mixed with physiological saline (PBS, 10 × 10−3 M, pH 7.6) containing 10 × 10−6 M siRNA-CPPs (1:1, v/v) with vigorous agitation for 30 min at 4 °C. Then the solution was dialyzed with a preswollen dialysis membrane (MWCO 1500) against 10 × 10−3 M PBS (pH 7.6) at 4 °C for 24 h. Finally, the obtained micelles (Folate-acid

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Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery

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mean value ± standard deviation. The prepared siRNA-CPPs/F-ASPM was diluted with PBS (pH 7.4) and then droplets were deposited on the surface of a Formvar-coated copper grid, followed by a negative staining method using uranyl acetate solution (1%, w/v), and then air-dried overnight at room temperature. Then the particles were morphologically characterized by a transmission electron microscope (TEM, HITACHI, H-7650, Japan). Atomic force microscopy (AFM, NanoWizarc, JPK Ltd., Germany) was also used for morphologically characterization of siRNA-CPPs/F-ASPM diluted with PBS (pH 7.4). And serum stability of siRNA in its aqueous solution and siRNA-CPPs/FASPM was evaluated using agarose gel electrophoresis.

2.8. Stability of FAM-siRNA and Acid Activation of siRNA-CPPs The molecular weight change of FAM-siRNA at different pH value that simulated the physiological environment (pH 7.4) and acid tumor environment (pH 5.0), respectively, was evaluated utilizing Q-TOF (QSTAR Elite, Applied Biosystem/MDS Sciex) equipped with ESI to study the stability of FAM-siRNA Figure 3. a) Schemes on the synthesis of siRNA-CPPs conjugate. b) MALDI-TOF mass at different pH value. spectra of siRNA. c) MALDI-TOF mass spectra of the synthesized siRNA-CPPs conjugate. FAM-siRNA-CPPs/F-ASPM were dispersed in PBS (pH = 5.0, 6.5, 7.0, and 7.4) and incubated at 37 °C with gentle shaking. Release of FAM-siRNA was sensitive polymer micelles named as F-ASPM or Acid sensitive monitored at several time intervals over 24 h. At each sampling polymer micelles named as ASPM) were filtrated by 0.22 μm time, the sample was thoroughly ultrafiltrated. And the released filter, freeze dried, and stored at −20 °C for further utilization. FAM-siRNA-CPPs was collected and determined using the spectrofluorometer previously described (see Section 2.6).

2.6. Encapsulation Efficiency (EE) Determination The EE of siRNA-CPPs was determined by dissolving the micelles containing FAM-siRNA-CPPs in DMSO for 1 h. The encapsulated FAM-siRNA-CPPs was quantified using a calibration line obtained with standard lead FAM-siRNA-CPPs solutions. The fluorescence of FAM-siRNA-CPPs was determined with the spectrofluorometer (Synergy 4, Biotek, WI, USA) using excitation and emission wavelengths of 495 and 525 nm, respectively. The EE was expressed as [(Mi-MU)/Mi]×100%. MU and Mi were defined as the mass of unencapsulated FAM-siRNA-CPPs and initially added FAM-siRNA-CPPs, respectively. Data are expressed as mean ± standard deviation (mean ± SD) of at least three replicates.

2.9. Cell Culture In this work, Human breast adenocarcinoma cells (MCF-7 cells) were used to evaluate the targeting effect of the constructed polymer micelles, as the MCF-7 cell lines were reported to use as a folate receptor positive cell model.[16] MCF-7 cells purchased from the Cell Resource Centre (IBMS, CAMS/PUMC) were maintained in the culture medium DMEM supplemented with 10% FBS, 100 IU mL−1 penicillin, and 100 mg mL−1 streptomycin. The cells were maintained in a 37 °C humidified incubator with a 5% CO2 atmosphere.

2.10. Cellular Uptake and Flow Cytometric Analysis 2.7. Physicochemical Characterization All size, zeta potential and polydispersity index measurements were made using Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., UK) at different pH values (pH 5.0, 6.5, 7.0, 7.4) of phosphate buffered saline (PBS). The results were determined in three serial measurements and reported as the

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The enhancement in cellular uptake offered by folate and acidactivable CPPs mediated drug delivery system was confirmed using flow cytometry with MCF-7 cells. MCF-7 cells were seeded into six-well plates (Corning, NY, USA) with a density of 3 × 105 cells/well, respectively. After incubating for 24 h, the medium in each well was replaced with fresh cell medium without FBS

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containing free FAM-siRNA, free FAM-siRNA-CPPs or an equivalent concentration of FAM-siRNA-CPPs/ASPM (incubated in pH 7.4), FAM-siRNA-CPPs/F-ASPM (incubated in pH 7.4), FAM-siRNACPPs/F-ASPM (incubated in pH 6.5 medium, which modeled tumor extracellular pH and adjusted with 0.1N NaOH or 0.1N HCL). As for the competitive binding assay of FAM-siRNA-CPPs/FASPM (incubated in pH 7.4) and FAM-siRNA-CPPs/ASPM (incubated in pH 7.4) to MCF-7 cells, excess free folate (1 mg mL−1) was added to the medium prior to the introduction of the two particles. The concentration of FAM-siRNA was 100 × 10−9 M. Following the treatment for 6 h, the cells were washed twice with cold PBS (0.1 M, pH 7.4), detached with 0.25% trypsin and washed another three times with cold PBS. Finally, the cells were resuspended in 0.2 mL PBS and subjected to flow cytometry (BD FACS Calibur). The autofluorescence of the cells was used as a control. All experiments were performed in triplicate.

2.11. In Vitro Confocal Image Following the culture of MCF-7 cells for 24 h on a petri dish (3 × 105 cells/well), free FAM-siRNA, free FAM-siRNA-CPPs, or an equivalent concentration of FAM-siRNA-CPPs/ASPM (pH 7.4), FAM-siRNA-CPPs/F-ASPM (incubated in pH 7.4 and pH 6.5, respectively) were added to each dish and incubated at 37 °C for 6 h. The medium was removed and discarded, and then the cells were washed with cold PBS for three times, followed by fixing with 4% Paraformaldehyde for 20 min. Nuclear staining was performed by Hoechst 33258 for 10 min at ambient temperature. The fluorescent images of cells were analyzed using a laser scanning confocal microscope (UltraVIEW Vox, PerkvnElmer, USA). FAM-siRNA and Hoechst 33258 were excited using 488 and 405 nm lasers, respectively. The endosomal release of FAM-siRNA-CPPs/F-ASPM (pH 6.5) was tracked by confocal image. MCF-7 cells were cocultured with FAM-siRNA-CPPs/F-ASPM (pH 6.5) for 6 h. Subsequently, the cells were washed three times with cold PBS and cultured in complete medium for an additional 1 h and 4 h, respectively. After incubating for 0.5 h, the endosome/lysosome was stained with 2 mL of Lyso-Tracker Red (75 × 10−9 M). Subsequently, the cells were rinsed with cold PBS and the nuclear staining was treated as described above. The cells were analyzed using confocal image and the LysoTracker Red was excited by a 561 nm laser.

2.12. In Vitro Transfection and Analysis of Gene Expression MCF-7 cells were seeded into 25 cm2 tissue culture flasks at a population of 2.0 × 106 cells/flask and incubated in a humidified atmosphere of 5% CO2 at 37 °C. Twenty-four hours later, the cells were cultured with free siRNA, free siRNA-CPPs, siRNA/ CPPs complex (siRNA and CPPs complexed via electrostatic interaction, zeta potential: 26.32 ± 2.01 mv), siRNA-CPPs/ASPM (pH 7.4), siRNA-CPPs/F-ASPM (incubated in pH 7.4 and pH 6.5, respectively), siN.C.-CPPs/F-ASPM (incubated in pH 6.5) at the concentration of 250 × 10−9 M in serum-free medium. After 6 h incubation, the cells were washed with PBS for three times and further incubated in complete medium for an additional 48 h (for mRNA assay) or 72 h (for protein quantification). Subsequently,

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qRT-PCR (quantitative real-time polymerase chain reaction) and western blot analysis were utilized to evaluate c-myc mRNA and protein, respectively. For the qRT-PCR assessment, cells were collected, and the total RNA was extracted by using TRNzol A+ reagent. 2 mg of total RNA was subjected to the synthesis of first strand cDNA using a Quantscript RT Kit (first strand cDNA synthesis kit). Then the synthesized cDNA was subjected to qRT-PCR analysis targeting c-myc and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the endogenous reference. The primers used for PCR amplification were: GAPDH forward: 5′-GGGTGTGAACCATGAGAAGT-3′; GAPDH reverse: 5′-GACTGTGGTCATGAGTCCT-3′; c-myc forward: 5′-GGCTATTCTGCCCATTTGGGGAC-3′; c-myc reverse: 5′-GGCAGCAGCTCGAATTTCTTC-3′. For western blot analysis, transfected cells were lysed in lysis buffer for 30 min on ice and then the lysates were collected by centrifugation for 10 min (14000 rpm, 4 °C). Subsequently, the protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to immobilon-P membranes (Millipore, Bedford, MA). The blots were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) for 1 h at ambient temperature and incubated overnight at 4 °C in 5% BSA in TBST with antic-myc monoclonal antibody (1:1000), and rabbit anti-β-actin (1:1500) was used as the internal control. The bands were visualized by the Molecular ImageChemiDoc TM XRS + imaging system (Bio-Rad).

2.13. In Vitro Cytotoxicity of the Synthesized Materials Cytotoxicity of ASPM (nontargeted micelles without siRNACPPs, incubated in pH 7.4), F-ASPM (Folate-targeted micelles without siRNA-CPPs, incubated in pH 7.4), ASPM (nontargeted micelles without siRNA-CPPs, incubated in pH 6.5 medium, which modeled tumor extracellular pH and adjusted with 0.1 N NaOH or 0.1 N HCL) and F-ASPM (Folate-targeted micelles without siRNA-CPPs, incubated in pH 6.5 medium, which modeled tumor extracellular pH and adjusted with 0.1 N NaOH or 0.1 N HCL) was evaluated by MTT assay with MCF-7 cells. The cells were seeded into 96-well plates 24 h before the assay at a density of 2.0 × 104 cells/well and then incubated at 37 °C with 5% CO2. Then, the plates were washed twice with fresh media, and placed in the incubator with 20 μL of ASPM (pH 7.4 and pH 6.5) and F-ASPM (pH 7.4 and pH 6.5) for an additional 24 h. Next, 20 μL of MTT solution (5.0 mg mL−1) was added to each well, and the plates were incubated for 4 h at 37 °C. Next, the MTT-containing medium in each well was replaced with 200 μL of dimethyl sulfoxide (DMSO), and the mixture was shaken at room temperature to dissolve the reacted dye. The absorbance of each well was measured using a microplate reader (Model 680, BIO-RAD, USA) at a wavelength of 570 nm. All samples were evaluated in sextuplicate.

2.14. Cell Apoptosis Assay Induced by siRNA-CPPs/F-ASPM MCF-7 cells were seeded on 25 cm2 tissue culture flasks at a density of 2.0 × 106 cells per flask in 6 mL of complete DMEM

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medium. After 24 h, the cells were washed with PBS and incubated with free siRNA, free siRNA-CPPs, siRNA/CPPs complex, siRNA-CPPs/ASPM (pH 7.4), siRNA-CPPs/F-ASPM (incubated in pH 7.4 and pH 6.5, respectively), and siN.C.-CPPs/F-ASPM (incubated in pH 6.5) in serum-free medium at a concentration of 250 × 10−9 M. After incubating for 6 h, the cells were washed by PBS for three times and incubated in complete medium for an additional 72 h. Finally, the collected cells were stained with Annexin V-FITC apoptosis detection kit and analyzed by flow cytometer immediately.

2.15. In Vivo Imaging The antitumor efficacy in vivo was evaluated in MCF-7 tumorbearing female nude mice. Briefly, the mice were subcutaneously injected in right axilla with 0.2 mL of cell suspension containing 2 × 107 MCF-7 cells. When the tumor volumes reached approximately 200 mm3, the mice were administered with 0.2 mL of PBS, free Cy3-siRNA, free Cy3-siRNA-CPPs, Cy3-siRNA/F-ASPM, Cy3-siRNA-CPPs/ASPM, and Cy3-siRNA-CPPs/F-ASPM at a dose of 1.2 mg kg−1 via tail vein. At different time points (4 h and 24 h) after injection, the fluorescence signals from the whole body was monitored using an IVIS Lumina II in vivo imaging system (Caliper Life Sciences, USA). After 24 h, the mice were euthanized, and the excised tumors and major organs including heart, liver, spleen, lung, and kidney were also imaged.

2.16. In Vivo Tumor Growth Inhibition Study The antitumor efficacy was evaluated in MCF-7 tumor-bearing female nude mice as described above. Once tumors reached approximately 90 mm3, the mice were randomly divided into seven groups (n = 6) and received free c-myc siRNA, free c-myc siRNA-CPPs, c-myc siRNA/F-ASPM, c-myc siRNA-CPPs/ASPM, c-myc siRNA-CPPs/F-ASPM or si N.C.-CPPs/ F-ASPM at a dose of 1.2 mg kg−1 (0.2 mL), and PBS used as the control. Different samples were intravenously injected once every other day for a total of 14 d. During the whole experiment, the body weight and tumor size were measured every day. The tumor volume was calculated using the formula V = 1/2 × (larger diameter) × (smaller diameter)2. At the end of the experimental point, the mice were sacrificed and the tumors were separated for further in vivo gene silencing.

2.17. In Vivo Gene Silencing For the in vivo c-myc expression, tumor tissues were excised 48 h after the last administration. The tumor fragments were weighed and processed for total mRNA or protein extraction followed by qRT-PCR and western blot assays respectively as described in section 2.12.

2.18. Statistical Analysis All data are shown as means ± SD unless particularly outlined. Student’s t test or one-way analyses of variance (ANOVA) were performed in statistical evaluation. A p-value less than 0.05 was considered to be significant, and a p-value less than 0.01 was considered as highly significant.

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3. Results and Discussion Human breast adenocarcinoma is a malignant tumor and the leading cause of cancer death among women worldwide. Overexpression and activation of the c-myc gene induces various forms of cancers, including human breast adenocarcinoma.[17] However, expression of c-myc is also essential for proliferation and regulation in normal mammalian cells. Therefore, a targeted delivery system is required for specific delivery of c-myc siRNA. Utilizing the physiological characteristics of tumors such as receptor over-expressing on the surface of tumor cells, acid tumor extracellular environment and intracellular redox environment of cytoplasm, a delivery system combining folate targeting, pH sensitive polymer micelles, and reducible disulfide linked siRNA-CPPs conjugate was designed for transporting c-myc siRNA to human breast adenocarcinoma specifically. 3.1. Synthesis of Folate-PEG-PHIS, PEG-PHIS, and siRNA-CPPs Folate-PEG-PHIS and PEG-PHIS were synthesized according to the scheme depicted on Figure 2a,b. Folate-PEG-Mal was produced firstly, then conjugated PHIS via the thiol-ene “click” reaction of the maleimide group with the sulfhydryl group to produce the functional material of FolatePEG-PHIS. Successful synthesis of Folate-PEG-PHIS was confirmed by the molecular shifts in the MALDI-TOF MS (m/z, [M – H]−) analysis: 9140 (calculated), 9146 (observed) as depicted in Figure 2c. PEG-PHIS was also obtained via the thiol-ene “click” reaction of the maleimide group of HO-PEG-Mal with the sulfhydryl group of PHIS. The major peak showed at 7046 (Figure 2d, marked by red circle) mass-charge ratios was in agreement with the calculated mean MW (7038) of the corresponding product. c-myc siRNA was chemically modified with a free thiol group at the 5′ end of one strand so that it could react with a free thiol group from a cysteine amino acid on the CPPs (Figure 3a). Successful synthesis of siRNA-CPPs was confirmed by the molecular shifts in the MALDI-TOF MS (m/z, [M-H]−) analysis from 7104 to 8197. The increased value is the molecular weight of CPPs, as depicted in Figure 3b,c. In addition, siRNA is a double stranded structure. The linked bonds between the two stranded structures are very weak and they are easily dissociated from each other during the molecular weight detection using the MALDITOF mass. Therefore, the second peak in Figure 3b (6928) and c (6896) is the molecular weight of the other unmodified strand of siRNA, which remained unchanged before and after reaction with CPPs. The ionization of PHIS switches it from hydrophobic to hydrophilic and PHIS-based copolymers are usually used to devise pH sensitive nanocarrier. PHIS homopolymer has a pKb value of around 6.5. However, due to the

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increased hydration of the PEG, PEG-PHIS copolymers showed inflexion points at around 7.0.[13] Because the measured tumor extracellular pH values of most solid tumors range from 6.5 to 7.2,[2] micelles consisted of PEGPHIS will collapse or destabilize at the tumor extracellular environment. Therefore, PEG-PHIS and F-PEG-PHIS were used to construct acid-sensitive polymer micelles with active targeting to folate receptor over-expressed MCF-7 cells in this work. Meanwhile, as mentioned above, redox effect of GSH in the cell cytosol made the reducible disulfide bond linked siRNA-CPPs conjugate an ideal system to transport siRNA. Disulfide bond benefits the effective dissociation of naked siRNA from the carrier in the cancer cells cytosol, thereby minimizing the CPPs’ interference on the silence processing and maximizing the antisense effect of siRNA.[9] 3.2. Preparation and Characterization of siRNA-CPPs Encapsulated F-ASPM In this work, folate active targeting acid sensitive polymer micelles encapsulating siRNA-CPPs (named as siRNACPPs/F-ASPM) were devised to avoid the nonspecificity and enzyme degradation of CPPs in the normal circulation. The morphology of siRNA-CPPs/F-ASPM was determined by TEM and AFM. As shown in Figure 4a,b, the micelles showed well dispersed circular shape and had diameters ranging from 80 to 100 nm, which were close to the values

measured by the laser particle analyzer (99 ± 1 nm). This diameter facilitates the vesicles to be transported in the vasculature of the tumor tissue, permits greater accumulation of the micelles in the tumor tissue by the enhanced permeability retention (EPR) effect, and evades elimination by the kidney.[18,19] The encapsulation efficiency of siRNACPPs/ASPM and siRNA-CPPs/F-ASPM determined in this work is 79.89 ± 1.56% and 78.97 ± 1.62%, respectively. As shown in Figure 4d, pure siRNA was fully degraded after 8 h. In contrast, siRNA in siRNA-CPPs/F-ASPM did not fully degrade even after 24 h. The mean diameter, PDI and zeta potential of siRNACPPs/F-ASPM at different pH values (5.0, 6.5, 7.0, and 7.4) are shown in Table 1. The results indicate the micelle size increased as pH decreased. When pH drops from 7.4 to 6.5, as explained before, PHIS of the micelles becomes highly protonated and its hydrophilicity greatly increased, leading to the disruption of the micelles. Therefore, due to the disruption of the micelles at pH value of 6.5 and 5.0, the particle size of micelles cannot be measured in these two values. This result also suggests siRNA-CPPs/F-ASPM micelles, which will destabilize at tumor pH (pH 6.5), is benefit for our constructed acid-sensitive delivery system. The zeta potential of siRNA-CPPs/F-ASPM at different pH value from pH 7.4 to pH 5.0 was −5.30 ± 0.36 mV, −3.30 ± 0.23 mV, 15.98 ± 1.32 mV, and 16.01 ± 1.45 mV, which is increased with pH decreased, respectively. The results also suggest the micelles size and zeta potential did not show

Figure 4. Physicochemical characterization of siRNA-CPPs/F-ASPM: a) Transmission electron micrographs. b) Atomic force microscopic images (3D image). c) Particle size distribution. d) siRNA serum stability assay.

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Table 1. Particle size, PDI, and zeta potential of siRNA-CPPs/F-ASPM at different pH values. The data are expressed as the mean ± SD value for at least three different preparations.

Samples

Diameter [nm]

PDI

Zeta potential [mv]

siRNA-CPPs/F-ASPM (pH 7.4)

99 ± 1

0.156 ± 0.027

−5.30 ± 0.36

siRNA-CPPs/F-ASPM (pH 7.0)

154 ± 0

0.263 ± 0.035

−3.30 ± 0.23

siRNA-CPPs/F-ASPM (pH 6.5)





15.98 ± 1.32

siRNA-CPPs/F-ASPM (pH 5.0)





16.01 ± 1.45

significant difference at pH 6.5 and pH 5.0, indicating pH 6.5 is enough to trigger destabilization of siRNA-CPPs/ F-ASPM. Overall, these results confirmed our hypothesis that the functions of CPPs could be shielded by encapsulation into F-ASPM and activated at the acid tumor site. 3.3. Acid-Triggered Activation of siRNA-CPPs In this work, since the cumulative release of siRNA, cellular internalization as well as endosomal escape of siRNA

were all detected via fluorescence labeled FAM-siRNA, the stability of FAM-siRNA at the medium with different pH value is important. The molecular weight change of FAMsiRNA at PBS of pH 7.4 and pH 5.0 was investigated to study the stability of FAM-siRNA. The data shown in Figure 5a,b suggest that there is no change of the molecular weight of FAM-siRNA at pH 7.4 (the molecular weight is 7102.97 and 7104.98 calculated from 1016.2848 and 1185.4956 in Figure 5a) and pH 5.0 (the molecular weight is 7102.94 and 7104.96 calculated from 1016.1369 and 1185.4893 in

Figure 5. Stability of FAM-siRNA at a,b) different pH value and c) acid-triggered activation of siRNA-CPPs. a) Molecular weight of FAM-siRNA determined by Q-TOF equipped with ESI at pH 7.4; b) molecular weight of FAM-siRNA determined by Q-TOF equipped with ESI at pH 5.0; and c) acid-triggered release behaviors of siRNA-CPPs from the fabricated siRNA-CPPs/F-ASPM at different pH values (5.0, 6.5, 7.0, and 7.4) (n = 3).

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Figure 6. Cellular uptake of nanocarriers by MCF-7 cells. a) Competitive binding assay of nanocarriers. b) FACS analysis of pH-triggered cellular uptake after incubation with various formulations. The data are presented as the means ± SD (n = 3). * indicates p < 0.05; ** indicates p < 0.01.

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Figure 5b), indicating that FAM-siRNA is stable at acid tumor environment. To validate that siRNA-CPPs could be activated at the acidic tumor extracellular environment, FAM-siRNACPPs/F-ASPM was incubated in PBS with different pH value (5.0, 6.5, 7.0, and 7.4) at 37 °C. The results were depicted in Figure 5c. The data indicate the activation of siRNA-CPPs was pH-dependent. The release value was very low at pH 7.4 and the optimal activation pH value for siRNA-CPPs is pH 5.0 and pH 6.5. These results are mainly originated from PHIS, the major component of F-ASPM, which has an amphoteric nature and will transform from hydrophobic to hydrophilic at acid environment. This could cause the disruption of F-ASPM, and thus releasing the encapsulated siRNA-CPPs.[20]

were considerably different. As shown in Figure 6b, due to the dual effect of folate receptor-mediated endocytosis and the efficient internalization of acid activated CPPs, siRNA-CPPs/F-ASPM (pH 6.5) exhibited the highest mean intensity of 376.27 ± 2.98. The mean intensities of fluorescently labeled siRNA were higher in MCF-7 cells treated with siRNA-CPPs/F-ASPM (pH 7.4) than cells treated with siRNA-CPPs/ASPM (pH 7.4). This indicates the folate ligand increased the delivery efficiency of the micelles for folate receptor over-expressing MCF-7 cells. With the help of CPPs, free siRNA-CPPs display higher cellular uptake than siRNA-CPPs/F-ASPM (pH 7.4). However, the large MW (≈13 kDa), hydrophilic nature as well as negative zeta potential of siRNA made no translocation of naked siRNA into the MCF-7 cells detected.[21]

3.4. Competitive Binding Assay of Nanocarriers and pH-Triggered Cellular Uptake

3.5. Acid-Triggered Confocal Microscopy Analysis and Endosomal Escape of siRNA

First, the competitive binding of siRNA-CPPs/F-ASPM (pH 7.4) and siRNA-CPPs/ASPM (pH 7.4) to MCF-7 cells was performed by adding excess free folate (1 mg mL−1) to the media prior to the introduction of the two micelles. As shown in Figure 6a, the cellular uptake of siRNA-CPPs/ F-ASPM in the presence of excess free folate (1 mg mL−1) in MCF-7 cells was suppressed significantly, becoming almost equivalent to that of siRNA-CPPs/ASPM. However, cellular internalization of siRNA-CPPs/ASPM was not influenced by free folate. The in vitro internalization efficiency of acid-sensitive formulations was quantitatively evaluated. The flow cytometric analysis reveals that fluorescent intensities of MCF-7 cells incubated with different formulations

Confocal laser scanning microscopy (CLSM) was further used to track the distribution of gene delivery systems following cellular uptake. As shown in Figure 7, there is limited green florescence inside cells treated with free FAM-siRNA only, demonstrating that FAM-siRNA alone is difficult to enter and accumulate in cells. Otherwise, the strongest green fluorescence was found in the cytoplasm of MCF-7 cells treated with siRNA-CPPs/F-ASPM (pH 6.5). Being consistent with the results of flow cytometric, the intracellular distribution of various formulations was in the order of siRNA-CPPs/ F-ASPM (pH 6.5) > siRNA-CPPs > siRNA-CPPs/F-ASPM (pH 7.4) > siRNA-CPPs/ASPM (pH 7.4) > free siRNA. Escaping the lysosomal entrapment and deliver siRNA into the cytoplasm is crucial for siRNA transporting

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ability of CPPs used in this work, which is rich in lysine amino acid residues. Lysine-rich peptides have already been reported that could mimic the endosomal disruptive properties of viral particles, penetrate cells and facilitate the delivery of nucleic acids.[23] 3.6. c-myc Gene Silencing Efficiency In Vitro and Cell Apoptosis Assay

Figure 7. Confocal laser scanning microscopy (CLSM) analysis of the uptake of various formulations by MCF-7 cells.

system as siRNA produced its effects in the cytoplasm.[22] Thus, the endosomal/lysosomal escape of siRNA-CPPs/FASPM (pH 6.5) was evaluated in MCF-7 cells after 1 and 4 h incubation, respectively. As shown in Figure 8, there were a relatively high colocalization spots of the green FAM-siRNA and red endosomes/lysosomes for 1 h, suggesting that the major of released FAM-siRNA-CPPs were within endosomes/lysosomes. After transfecting for 4 h, the green FAM-siRNA fluorescence was almost totally separated from the endosomes/lysosomes, indicating the successful endosomal/lysosomal escape. The result may be attributed to the endosomal/lysosomal escape

To further assay the effect of the dual stimulus activated delivery system against c-myc gene in MCF-7 cells, we examined its inhibitory activity of c-myc expression through qRT-PCR and western blot analysis. As shown in Figure 9a,b, being consistent with the above results, siRNACPPs/F-ASPM (pH 6.5) showed the largest down-regulation efficiency among the various formulations. MCF-7 cells treated by siRNA-CPPs/F-ASPM (pH 7.4) showed smaller c-myc mRNA and protein expression than those treated with siRNA-CPPs/ASPM (pH 7.4), which may benefit from the targeting effect of folate. Free c-myc siRNA and siN.C.CPPs/F-ASPM (pH 6.5) did not show any gene silencing activity on c-myc. More importantly, the decrease in c-myc expression levels of cells treated with siRNA-CPPs conjugate was larger than those treated with siRNA/CPPs complex. The results suggest siRNA-CPPs conjugate enables an advantageous improvement in the endogenous genesilencing efficiency when compared to the simple electrostatic complex of siRNA/CPPs counterparts. As a delivery material, it must be less or no toxicity, thus MTT assay was used to evaluate the safety of our delivery system. Because CPPs were reported that have no cytotoxicity,[5c] the safety of this delivery system was mainly focused on the cytotoxicity of F-ASPM and ASPM. The result of MTT assay suggests that there is no apparent cytotoxicity observed for F-ASPM (pH 7.4 and pH 6.5) and ASPM (pH 7.4 and pH 6.5) within MCF-7 cells (shown in Figure 9c). Knockdown of c-myc has been shown to induce

Figure 8. Intracellular trafficking of FAM-siRNA in MCF-7 cells undergoing 1 h or 4 h of routine culture following 6 h of incubation with siRNA-CPPs/F-ASPM (pH 6.5). Hoechst33258 for nuclei staining (blue), FAM-siRNA fluorescence (green) and LysoTracker Red for endosomes/ lysosomes (red) were recorded.

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Figure 9. In vitro evaluation of various c-myc siRNA contained samples. a) The level of c-myc mRNA determined by qRT-PCR. b) c-myc protein expression determined by western blot analysis. c) In vitro toxicity of micelles without siRNA at different pH value. The data are presented as the means ± SD (n = 6). d) Cell apoptosis following exposure to different formulations. MCF-7 cells were individually treated with various formulations carrying c-myc siRNA or siN.C. (10 0 × 10−9 M) at 37 °C for 6 h, followed by a) 48 h or b,d) 72 h of regular culture. The data are presented as the means ± SD (n = 3). *p < 0.05; **p < 0.01.

apoptosis in tumor cells, so cell apoptosis induced by various formulations carrying c-myc siRNA and siN.C. was evaluated by flow cytometry. As shown in Figure 9d, cells exposed to c-myc siRNA-loaded formulations showed significant apoptosis, whereas only a slight effect was observed in the control (PBS), siN.C.-CPPs/F-ASPM (pH 6.5) and free c-myc siRNA groups. Cell apoptosis was in the order of siRNA-CPPs/F-ASPM(pH6.5, 60.28%)>siRNACPPs(56.34%)>siRNA/CPPs(42.78%)>siRNA-CPPs/F-ASPM (pH 7.4, 38.78%)>siRNA-CPPs/ASPM(pH 7.4, 29.78%)>free siRNA(3.61%)>the control (2.01%)>siN.C.-CPPs/F-ASPM (pH 6.5, 1.32%), which is consistent with the c-myc protein expression results described above. In this study, the strongest gene knockdown and cell apoptosis induced by siRNA-CPPs/F-ASPM (pH 6.5) is related with the folate targeting ability, cell penetrating and endosomal/lysosomal escape ability of acid-activiated

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CPPs, and the release of naked siRNA in the cytosol via disulfide bond broken. All of these improved sequencespecific mRNA degradation in MCF-7 cells. As PHIS had proton capacity in acidic endosomes, micelles consisted of PHIS also had endosomal escaping capability, thus both siRNA-CPPs/F-ASPM (pH 7.4) and siRNA-CPPs/ASPM (pH 7.4) could inhibit c-myc expression in the treated cells to some extent. Meanwhile, since the targeting effect of siRNA-CPPs/F-ASPM (pH 7.4), cells treated with this formulation showed higher level of c-myc gene silencing efficiency than siRNA-CPPs/ASPM (pH 7.4). In addition, it has been reported that peptides binding too strongly to siRNA will prevent successful RNAi.[24] Therefore, the superior efficacy of siRNA-CPPs to siRNA/CPPs in silencing c-myc in vitro was in virtue of its effective dissociation of siRNA from CPPs upon redox stimulus by GSH, which reduced the RNAi interference from the carrier. The negligible

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gene knockdown efficacy of siN.C.-CPPs/F-ASPM (pH6.5) in vitro suggested the c-myc siRNA used in this work is gene-specific. Taken together, these results verified siRNA-CPPs/F-ASPM is capable of inhibiting c-myc expression in MCF-7 cells under the dual stimulus of acid and intracellular redox environment.

no fluorescence signals were detected in the mice treated with PBS, suggesting no auto fluorescence interferes in vivo images. The fluorescent intensity of the group treated only with siRNA disappeared after 24 h. The reason for this phenomenon is related with the instant degradation by RNase (a type of nucleases) and rapid renal excretion of siRNA after intravenous injection. At the site of the tumor, it exhibited higher fluorescence intensity when siRNA-CPPs/ F-ASPM was administered in comparison with siRNA-CPPs/ ASPM. Importantly, the fluorescence intensity at the tumor site following siRNA-CPPs/F-ASPM injection did not significantly decay after 24 h. Although the folate targeting effect made siRNA/F-ASPM show accumulation in tumor at first 4 h, the released siRNA at the tumor extracellular was rapidly eliminated from the body after 24 h. The nonspecificity of CPPs made siRNA-CPPs treated mice show discrete fluorescence distribution in vivo. Moreover, the fluorescence intensity in the siRNA-CPPs group decayed after 24 h, suggesting the disulfide linked conjugates might be destroyed before their arrival at the tumor sites. To provide more direct evidence, major organs (e.g., heart, liver, spleen, lung, kidney, and tumor) were further observed by sacrificing the mice after 24 h of administration (Figure 10b). As observed from the images, strong fluorescence intensity in the tumor tissue was observed in the siRNA-CPPs/F-ASPM group, implying that siRNACPPs/F-ASPM could efficiently target to solid tumors and obviously decrease nonspecific accumulation in normal organs. Since siRNA-CPPs/ASPM may undergo nonspecific RES (reticular endothelial system) uptake in vivo, mice treated by this formulation exhibited fluorescence signals in many organs (liver, spleen, lung, and kidney) except the tumor tissue. Less or no fluorescence in isolated tumor but bright fluorescence intensity in the kidney in free siRNA treated mice groups revealed the in vivo fate of siRNA involved renal excretion. Although siRNA/F-ASPM could target to the tumor with the help of folate, less fluorescence in tumor and bright signals in kidney suggested siRNA of this formulation cannot penetrate into the tumor and has the same in vivo fate with free siRNA. The free siRNA-CPPs treated groups showed fluorescence in most isolated organs involved liver, spleen, lung, kidney, and tumor, resulting from the nonspecificity of CPPs in vivo.

3.7. In Vivo Fluorescence and Biodistribution

3.8. In Vivo Antitumor Efficacy

Delivering siRNA directly into the tumor tissues for tumorspecific therapy is vital for effective delivery system. In this work, we evaluated the time-dependent biodistribution of various Cy3-siRNA formulations in a MCF-7 cells xenograft female nude mouse model by in vivo imaging system. Through the whole-animal imaging (Figure 10a),

The constructed siRNA-CPPs/F-ASPM showed ideal antitumor activity in vitro and had strong tumor accumulation in vivo. To further detect the antitumor efficiency of the micelles in vivo, the antitumor growth effect in MCF-7 tumor-bearing female nude mice was performed with various formulations carrying c-myc siRNA via tail vein injection. As illustrated

Figure 10. Biodistribution of Cy3-siRNA contained in various samples in mice bearing MCF-7 tumor xenograft. a) Whole body imaging at different time points after systemic administration. b) Fluorescence detection of isolated main tissues and organs from mice at the end point of observation.

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Figure 11. Anticancer efficacy in the MCF-7 xenografts in female nude mice after treatments with PBS and various samples carrying c-myc siRNA or siN.C. a) Antitumor activity. *p < 0.05 indicates siRNA-CPPs/ASPM versus PBS, free siRNA, siRNA-CPPs, siRNA/F-ASPM, siRNA-CPPs/FASPM and siN.C.-CPPs/F-ASPM, whereas **p < 0.01 indicates siRNA-CPPs/F-ASPM versus PBS, free siRNA, siRNA-CPPs, siRNA/F-ASPM, and siN.C.-CPPs/F-ASPM (n = 6). b) Body weight changes. Arrows represent drug administration. Data are presented as means ± SD (n = 6). Expression of c) c-myc mRNA and d) c-myc protein in tumors was detected 24 h after the last administration. *p < 0.05, **p < 0.01 (n = 3). The exposure was performed at a 1.2 mg kg−1 siRNA equivalent dose once every other day for a total of 14 d.

in Figure 11a,b, treatment with free siRNA and siRNA/FASPM did not show significantly tumor growth inhibition (501.63 ± 79.23 mm3 and 493.97 ± 78.12 mm3, respectively) in comparison with the control groups (PBS 526.31 ± 79.56 mm3 and siN.C.-CPPs/F-ASPM 535.69 ± 78.98 mm3). Free siRNACPPs slightly inhibited tumor growth with the final tumor volume of 475.81 ± 80.12 mm3, which may result from the destruction of siRNA-CPPs in the normal body circulation. In comparison, formulations of siRNA-CPPs/F-ASPM showed a significant inhibition efficiency of tumor growth (142.49 ± 28.09 mm3) due to its excellent tumor accumulation and combined effects, which was also much higher than that of siRNA-CPPs/ASPM (240.61 ± 29.03 mm3). In addition, as shown in Figure 11b, except siRNA-CPPs, all tested micelles did not induce significant changes in the body weight of tumor-bearing mice. The significant weight loss of the siRNA-CPPs-treated mice groups may result from the nonspecific targeting of CPPs, which might lead to the damage of normal tissues.

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To further evaluate whether retarded tumor growth by siRNA-CPPs/F-ASPM was related to c-myc downregulation in tumors, the tumors were excised 48 h after the last injection. And c-myc mRNA and protein level in tumors were assayed by qRT-PCR and western blot, respectively. Figure 11c shows that c-myc mRNA levels were significantly downregulated to about 33% with siRNA-CPPs/F-ASPM administrations in comparison with those receiving PBS solution (p < 0.01). This value was significantly lower than that with the treatment of siRNA-CPPs/ASPM (approximately 57%, p < 0.01). Treatments with free siRNA, siN.C.-CPPs/F-ASPM, and siRNA/F-ASPM did not show obvious reductions in c-myc mRNA levels in tumors when compared to the PBS control. Whereas, due to the non-specific function of CPPs and destruction of siRNA-CPPs in body circulation, free siRNA-CPPs exhibited only slight downregulation of c-myc mRNA. On the other hand, c-myc protein expression in tumors assessed by western blot analyses showed

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consistent results (Figure 11d). The results demonstrated that the dual stimulus sensitive siRNA-CPPs/F-ASPM with higher tumor accumulation of payloads and synergistically inhibition effect exhibited excellent antitumor activity in a combination manner. The outstanding advantage of siRNA-CPPs/F-ASPM may result from the following combined effects: (i) active targeting of folate to MCF-7 cells; (ii) function masking of siRNA-CPPs by F-ASPM in body circulation and activation at the acid tumor extracellular environment; (iii) siRNACPPs had much stronger cell penetrating and endosomal escape ability than siRNA; and (iv) efficient dissociation of naked siRNA from CPPs in the cytosol with GSH stimulus, which improved knockdown efficiency of siRNA by reducing the interference from the carriers. Furthermore, the results in this study also confirmed that free siRNACPPs, which displayed efficient gene silencing effect in vitro but not in vivo, must be protected by vesicles in vivo to maximize their gene silencing effect.

4. Conclusions In summary, utilizing dual stimulus of acid tumor extracellular environment and redox effect of GSH in the cytosol, a functional delivery system (siRNA-CPPs/ F-ASPM) combining folate targeting, acid sensitive micelles, and CPPs was constructed to suppress c-myc gene expression of breast cancer both in vitro and in vivo. The data demonstrated the vesicle could not only overcome various physiological and biological barriers, but also be “switched on” in a smart fashion in response to the tumorous zone. Therefore, the vesicle could selectively and efficiently deliver siRNA into the cytosol of MCF-7 cells and showed excellent gene silencing efficiency. To the best of our knowledge, no others reported nanosystems so far are flexible enough to response to dual stimulus of the tumor environment and redox effect of GSH. Overall, good body safety and higher gene silencing efficiency made the constructed nanocarrier a tremendous potential siRNA transporting vector for oncotherapy.

Acknowledgments: This work was supported by PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (Grant No. 3332015139). The authors also acknowledge the financial support from the National Science Foundation of China (Grant No. 81402874) and National Megaproject for Innovative Drugs (Grant No. 2012ZX09301002-001).

Received: October 21, 2015; Revised: December 30, 2015; Published online: January 29, 2016; DOI: 10.1002/mabi.201500389

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Keywords: acid sensitive polymer micelle; cell penetrating peptides; dual stimulus; reducible disulfide linked siRNA-CPPs; siRNA delivery

[1] H. J. Mok, T. G. Park, Macromol. Biosci. 2012, 12, 40. [2] H. Wu, L. Zhu, V. P. Torchilin, Biomaterials 2013, 34, 1213. [3] A. K. Hyun, N. Kihoon, W. K. Sung, Biomaterials 2014, 35, 7543. [4] N. P. Gabrielson, H. Lu, L. C. Yin, K. H. Kim, J. J. Cheng, Mol. Ther. 2012, 20, 1599. [5] a) Y. Maitani, Y. Hattori, Expert Opin. Drug Delivery 2009, 6, 1065; b) A. Ouahab, N. Cheraga, V. Onoja, Y. Shen, J. S. Tu, Int. J. Pharm. 2014, 466, 233; c) Y. Z. Huang, Y. F. Jiang, H. Y. Wang, J. X. Wang, M. C. Shin, Y. R. Byund, H. N. He, Y. Q. Liang, V. C. Yang, Adv. Drug Delivery Rev. 2013, 65, 1299. [6] S. J. Lee, S. Son, J. Y. Yhee, K. Choi, I. C. Kwon, S. H. Kim, K. Kim, Biotechnol. Adv. 2013, 31, 491. [7] M. Wang, K. Alberti, A. Varone, D. Pouli, I. Georgakoudi, Q. B. Xu, Adv. Healthcare Mater. 2014, 3, 1398. [8] Y. Qe, R. J. Christie, M. Naito, S. A. Low, S. Fukushima, K. Toh, Y. Miura, Y. Matsumoto, N. Nishiyama, K. Miyata, Biomaterials 2014, 35, 7887. [9] P. Lundberg, S. WI-Andaloussi, T. Sütlü, H. Johansson, U. Langel, FASEB J. 2007, 21, 2664. [10] a) T. N. Hao, Acta Pharm. Sin. 2008 , 43 , 123 ; b) M. G. Von , Nat. Mater. 2011 , 10 , 545 ; c) A. Agarwal , M. A. Mackey, M. A. EI-sayed , R. V. Bellamkonda , ACS Nano 2011 , 5 , 4919 . [11] E. S. Lee, Z. G. Gao, D. G. Kim, K. S. Park, I. C. Kwon, Y. H. Bae, J. Controll. Release 2008, 129, 228. [12] B. X. Zhang, Y. Zhao, Y. Huang, L. M. Luo, P. Song, X. Wang, S. Chen, K. F. Yu, X. Zhang, Q. Zhang, Biomaterials 2012, 33, 2508. [13] E. S. Lee, H. J. Shin, K. Na, Y. H. Bae, J. Controll. Release 2003, 90, 363. [14] Y. Chen, W. B. Cao, J. L. Zhou, B. Pidhatika, B. Xiong, L. Huang, Q. Tian, Y. W. Shu, W. J. Wen, I. M. Hsing, H. K. Wu, ACS Appl. Mater. Interfaces 2015, 7, 2919. [15] A. Muratovska, M. R. Eccles, FEBS Lett. 2004, 558, 63. [16] K. Jain, U. Gupta, N. K. Jain, Eur. J. Pharm. Biopharm. 2014, 87, 500. [17] D. J. Hehir, G. Mcgreal, W. O. Kirwan, W. Kealy, M. P. Brady, J. Surg. Oncol. 1993, 54, 207. [18] Y. F. Yang, X. Y. Xie, Y. Yang, H. Zhang, X. G. Mei, Acta Pharm. Sin. 2013, 48, 1644. [19] Y. F. Yang , Y. Yang , X. Y. Xie , X. S. Cai , H. Zhang , W. Gong , Z. Y. Wang , X. G. Mei , Biomaterials 2014 , 35 , 4368 . [20] J. Liu, Y. R. Huang, A. Kumar, A. Tan, S. B. Jin, A. B. Mozhi, X. J. Liang, Biotechnol. Adv. 2014, 32, 693. [21] B. Y. Shi, H. Zhang, J. X. Bi, S. Dai, Colloid. Surf. B: Biointerfaces 2014, 119, 55. [22] R. J. Christie , Y. Matsumoto, K. Miyata , T. Nomoto , S. Fukushima , K. Osada , J. Halnaut , F. Pittella , H. J. Kim , N. Nishiyama , K. Kataoka , ACS Nano 2012 , 6 , 5174 . [23] M. C. Morris, L. Chaloin, J. Méry, F. Heitz, G. Divita, Nucl. Acids Res. 1999, 27, 3510. [24] J. C. Geoghegan, B. L. Gilmore, B. L. Davidson, Mol. Ther.: Nucl. Acids 2012, 1, e53.

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An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular en...
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