Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts

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Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts☆ Zhi-Yao He a,1, Xia-Wei Wei b,1, Min Luo a, Shun-Tao Luo a, Yang Yang b, Yi-Yi Yu b, Yan Chen a, Cui-Cui Ma a, Xiao Liang a, Fu-Chun Guo a, Ting-Hong Ye a, Hua-Shan Shi a, Guo-Bo Shen a, Wei Wang a, Feng-Ming Gong a, Gu He a, Li Yang a, Xia Zhao c, Xiang-Rong Song a,⁎, Yu-Quan Wei a,⁎ a b c

State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, PR China Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China

a r t i c l e

i n f o

Article history: Received 19 April 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: Folate receptor α Claudin3 Lipoplex Ovarian cancer Gene therapy

a b s t r a c t Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p b 0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy. Published by Elsevier B.V.

1. Introduction Ovarian cancer gene therapy has been an important research field of human gene therapy since traditional therapeutics have hardly improved the overall survival rate in the last 20 to 30 years [1,2]. Up to now, 27 clinical phase I/II trials in the ovarian cancer gene therapy field still have been in progress. Many gene types have been used for ovarian cancer gene therapy in clinical trials, including cytokines, tumor suppressors, suicides, antigens, receptors and receptor suicide genes [3]. Therefore, targeted gene therapy is one of the development trends of ovarian cancer gene therapy. The claudin family (CLDNs) of transmembrane proteins is associated with the tight junctions [4,5], and a member of CLDNs, claudin3 (CLDN3) is one of the most highly and consistently upregulated genes in N85% of ovarian tumors [6–9]. It is studied that overexpression of CLDN3 played a key role in the survival, proliferation, invasion, cellular motility and

☆ The authors declare no conflict of interest. ⁎ Corresponding authors at: State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No. 17, Section 3, Renmin South Road, Chengdu, Sichuan 610041, PR China. Tel./fax: +86 28 85502796. E-mail addresses: [email protected] (X.-R. Song), [email protected] (Y.-Q. Wei). 1 These authors contributed equally to this workquery. 0168-3659/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jconrel.2013.10.015

metastasis of ovarian tumors [7,10,11], and is associated with poor survival [8]. The overexpressing CLDN3 may disrupt tight junction barrier function and contribute to tumorigenesis. The downregulation of CLDN3 is generally concerned with potential tumor growth inhibition and good prognosis, therefore CLDN3 is considered as a promising target for ovarian cancer gene therapy [7,10,11]. In the previous reports, the CLDN3 gene has been suppressed or silenced in ovarian tumor tissues by two non-viral vectors [7,11], namely, siRNA–lipidoid formulation (siRNA silencing CLDN3 gene and LipofectAMINE 2000 complexes) and shRNA–nanoparticle formulation (shRNA targeting CLDN3 incorporated in PLGA nanoparticle), however, the efficiency of ovarian cancer inhibition in both studies needs to be improved. As novel therapeutic approaches, targeting formulations have the potential to enhance the therapeutic efficacy [12–15]. Folate receptor α (FRα), overexpressing in approximately 90% of ovarian tumor tissues [16,17], is associated with ovarian tumor progression, and promotes ovarian tumor cell proliferation, migration and invasion [18]. The effects are blocked after stable knockdown of FRα. So FRα is an important target for treatment in the therapy of ovarian cancer. Farletuzumab, a humanized monoclonal antibody against FRα, has been entered in the phase III clinical trial in epithelial ovarian cancer [19]. Also, MOv19-BBζ, a FRα-specific scFv MOv19 with signaling domains comprised of T cell receptor-CD3ζ and 4-1BB (CD137), has

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entered a phase I clinical trial for ovarian therapy [20]. Therefore, FRαmediated gene therapy for ovarian cancer has broad development prospects in the foreseeable future. However, there are only limited studies on FRα-mediated ovarian cancer gene therapy in vivo [21,22]. Therefore, we developed a new non-viral vector of FRα-targeted lipoplex with CLDN3 shRNA for ovarian cancer treatment. A folate modified liposome (F-P-LP) was prepared and then complexed with the shRNA targeting CLDN3 gene to formulate a folate modified lipoplex (F-P-LP/CLDN3). The pharmaceutical properties of FP-LP/CLDN3 were characterized by size, zeta-potential, morphology, encapsulation efficiency, stability, and transfection activity in vitro. Next, the antitumor effects and antitumor mechanisms of F-P-LP/CLDN3 were studied in an orthotopic model of advanced ovarian cancer in vivo. Finally, safety evaluation for F-P-LP/CLDN3 was carried out by blood test and histological examination. 2. Materials and methods 2.1. Materials 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol (Chol) was purchased from Shanghai Bio Life Science & Technology Co. Ltd. (Shanghai, China). MPEG-succinyl-Cholesterol conjugate (mPEG-suc-Chol) and Folate-PEG-succinyl-Cholesterol conjugate (F-PEG-suc-Chol) were synthesized and purified by our research group [23,24]. The shRNA targeting CLDN3 plasmid (pCLDN3) was constructed according to previous reports [7,11]. The HK sequence (pHK), which had no homology with any known mammalian gene sequences and did not interfere with any gene expression, was used as the negative control [11]. The plasmid green fluorescent protein (pGFP) was used for a transfection experiment in vitro. Plasmid DNA was extracted according to the EndoFree Plasmid Purification Handbook (QIAGEN, Hilden, Germany). DNase I, DNA ladder and pre-stained protein ladder were provided by Fermentas (Thermo Fisher Scientific Inc., Waltham, MA, USA). Triton X-100 was obtained from Sanland Chemical Co., Ltd. (Los Angeles, CA, USA). All reagents were of analytical grade and were used without further purification except chloroform used to prepare liposomes.

Non-targeted PEGylation liposomes (P-LP), composed of DOTAP/ Chol/mPEG-suc-Chol, were prepared by the same method as F-P-LP. Non-targeted PEGylation lipoplexes were prepared by mixing P-LP with pCLDN3 or pHK for 30 min at room temperature, formulating PLP/CLDN3 or P-LP/HK. Since P-LP/CLDN3 potentially targeted for the CLDN3 gene, actually P-LP/CLDN3 was a lipoplex with CLDN3 gene targeting. P-LP/HK was a non-targeted lipoplex. All experiments were performed in triplicate. 2.2.2. Characterization of liposomes and lipoplexes The mean particle size and zeta potential of liposomes and lipoplexes were measured by using a Zetasizer NanoZS ZEN 3600 (Malvern Instruments, Ltd., Malvern, Worcestershire, UK). The mean particle size was determined by dynamic light scattering at a fixed angle of 173°. The zeta potential was automatically calculated from the electrophoretic mobility. The morphological characteristic of F-P-LP/CLDN3 was examined by an atomic force microscope (AFM, SPI4000, SII NanoTechnology Inc., Chiba, Japan) as described previously [23]. Electrophoresis was conducted on 1% (w/v) agarose gel (Invitrogen Corp., Carlsbad, CA, USA) in pH7.4 TAE buffer (40mM Tris/HCl, 1% acetic acid, 1 mM EDTA) containing Golden View as nucleic acid stain after lipoplexes were prepared. 8 μL of lipoplexes mixed with 2 μL of loading buffer were applied to agarose at a constant voltage of 120 V for 25 min at room temperature. The electrophoresis gels were visualized and digitally photographed by a gel documentation system (Gel Doc 1000, Bio-Rad Laboratories, Hercules, CA, USA). DNase degradation was carried out as previously described [24]. Briefly, F-P-LP/CLDN3 containing 20 μg of pCLDN3 was incubated with 1 U of DNase I in a total volume of 500 μL in 50 mM Tris buffer (pH 7.4) containing 10 mM MgCl2. This mixture was incubated at 37 °C and aliquots of 50 μL were taken at 2 min, 10 min, 1 h, 6 h, 24 h, 48 h and 72 h, to which 4 μL of 250 mM EDTA was immediately added to stop DNA degradation. All samples were immediately placed in an ice bath. To release pCLDN3 from F-P-LP/CLDN3, 2 μL of Triton X-100 was used to dissociate F-P-LP/CLDN3 complexation for 5 min at room temperature. And then after the addition of 4 μL of 1 mg/mL sodium heparin, samples were incubated at room temperature for 15 min and 10 μL of each sample was analyzed by agarose gel electrophoresis as mentioned above.

2.2. Preparation and characterization of F-targeted liposomes and lipoplexes

2.3. Cell culture and transfection experiments

2.2.1. Preparation of F-targeted liposomes and lipoplexes F-targeted liposomes (F-P-LP) were prepared by a film dispersion method as described previously [25]. In brief, DOTAP, Chol, mPEG-sucChol and F-PEG-suc-Chol were dissolved in chloroform. The lipids solution was evaporated on a rotary evaporator to remove the organic solvent and then the formed thin film was further dried under high vacuum for 6 h. The dry lipid film was hydrated in glucose solution. And the suspension of lipids was sonicated by a probe until a translucent lipid suspension (F-P-LP) was obtained. Thereafter, F-P-LP were sterilized through a Millipore 0.22 μm microporous membrane and stored at 4 °C until use. F-targeted lipoplexes were prepared by mixing F-P-LP with pCLDN3 or pHK for 30min at room temperature, formulating F-P-LP/CLDN3 or FP-LP/HK. Details were as follows: F-P-LP was diluted into 1 mg/mL with glucose solution, and pCLDN3 or pHK was diluted into 1 mg/mL with TE buffer. Then F-P-LP was added to pCLDN3 or pHK. The obtained mixture was mixed quickly for 3 times to avoid larger particles or precipitant formation and after incubation for 30 min at room temperature, F-PLP/CLDN3 or F-P-LP/HK was finally achieved. It should be noted that F-P-LP/CLDN3 was potentially targeted for the CLDN3 gene besides targeting for FRα, so F-P-LP/CLDN3 was a lipoplex with vector and gene targeting. However, F-P-LP/HK only targeted for FRα, hence F-PLP/HK was a lipoplex with vector targeting.

Human ovarian carcinoma SKOV-3 cell line was obtained from the American Type Culture Collection. The cells were cultured as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified atmosphere containing 5% CO2 at 37 °C. Transfection experiments were done as described previously [24]. FP-LP/GFP and P-LP/GFP, containing 1 μg pGFP, were used to transfect SKOV-3 cells and transfection efficiency was determined by a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). 2.4. Orthotopic in vivo model of advanced ovarian cancer and tissue processing Female athymic nude mice (BALB/c-nude, specific pathogen-free conditions, SPF) were purchased from Vital River (Beijing, China). The mice were housed and maintained under SPF conditions in facilities. All studies were approved and supervised by the State Key Laboratory of Biotherapy Animal Care and Use Committee (Sichuan University, Chengdu, Sichuan, China). The mice were used in these experiments when they were 6 to 8 weeks old. Tumor models were established by intraperitoneal (i.p.) injection of SKOV-3 cells (about 1 × 107 cells/0.2 mL serum free DMEM) and mice

were randomly allocated into five groups based on their body weights. This model reflected the intraperitoneal growth pattern of advanced ovarian cancer, as intra-abdominal spread was the main mechanism of ovarian cancer metastasis [26–28]. To assess tumor growth, treatment began three days after inoculation. Mice were i.p. administered once every three days with liposomal plasmid DNA (5 μg) in 200 μL volume. Mice were monitored daily for adverse effects of therapy and were sacrificed when any of the mice appeared moribund. Treatment continued until the mice of the Control group became moribund (typically 4 to 6 weeks). At the time of sacrifice, tumor tissues, whole blood, ascitic fluid, and vital organs of mice were harvested, and mice weight, ascitic fluid volume, tumor weight and number of nodules were recorded. Blood (n = 3–4), ascitic fluid and tumor and normal tissues were used for further study. The tumor tissue specimen was divided into three parts: One was immediately lysed by RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), centrifuged, and supernatant was stored at −80 °C for western blot analysis; one was cut into pieces with surgical scissors and digested by collagenase I (Gibco, Invitrogen Corp., Carlsbad, CA, USA) for flow cytometry analysis; and another was fixed with paraformaldehyde in PBS (pH 7.4) and then embedded in paraffin for tissue sectioning. Vital organs (spleen, liver, kidney, heart and lung) were also harvested for evidence of tissue toxicity. Disposal of animal carcasses was done by a professional company (Dashuo Biotechnology Company, Ltd., Chengdu, Sichuan, China). 2.5. Flow cytometry analysis Tumor tissue specimens from Control, P-LP/HK, F-P-LP/HK, P-LP/ CLDN3 and F-P-LP/CLDN3 were digested into single-cell suspensions. The single-cell suspensions were centrifugated to remove collagenase I and then red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) was added to lyse red blood cells in the singlecell suspensions. And tumor cells were washed three times with PBS, counted, and resuspended in 99 μL PBS (pH 7.4). 1 μL mouse antihuman FOLR1 (FRα) antibody (R & D Systems, Minneapolis, MN, USA) was then added, cells were stained for 1 h at 4 °C and washed three times with 1 mL PBS to remove excess primary antibody and incubated at 4 °C for 30 min with 1 μL anti-mouse IgG-FITC secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) in 99 μL PBS. Cells were washed three times with PBS to remove excess secondary antibody and analyzed by the FACS Calibur flow cytometer. 2.6. Western blot analysis Total protein concentrations of tumor tissue lysates were measured using the Bradford protein assay reagent kit (Bio-Rad Laboratories, Hercules, CA, USA). The FRα protein was separated by 12% SDS-PAGE under non-reducing conditions and the CLDN3 protein was separated by 10% SDS-PAGE under reducing conditions, and then transferred to Millipore PVDF membranes. Membranes were blocked with 5% skimmed milk and incubated with anti-FRα antibody (R & D Systems, Minneapolis, MN, USA) or anti-CLDN3 antibody (Invitrogen Corp., Carlsbad, CA, USA) at 4 °C overnight. Antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody and developed with an enhanced chemiluminescence detection kit (Luminata Crescendo Western HRP Substrate, or Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA). Membranes were tested for β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) to confirm equal loading. 2.7. Immunohistochemistry and HE staining Immunohistochemistry analyses of FRα expression, CLDN3 expression, Ki67 antigen, and microvessel density (MVD, CD31) were done

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with mouse anti-human FRα, rabbit anti-human CLDN3, rabbit antihuman Ki67 (Novus Biologicals, Littleton, CO, USA), and rabbit antimouse CD31 antibodies (Abcam PLC, Boston, MA, USA) using the labeled streptavidin–biotin method. Briefly, sections (3–4 μm) were made from paraffin-embedded tumor tissue specimens of each group and were deparaffinized by sequential washing with xylene (I, II), 100% ethanol (I, II), 95% ethanol, 85% ethanol, 75% ethanol, and water. Endogenous peroxide was blocked with 3% H2O2 for 10 min at room temperature in the dark. Antigen retrieval was done by heating in a steam cooker in 10 mM sodium citrate buffer (pH 6.0). After being PBS washed, slides were blocked with serum according to the secondary antibody for 15 min at 37 °C followed by incubation with primary antibody overnight at 4 °C. After being PBS washed three times, the appropriate secondary antibody conjugated to HRP was added, and Histostain-Plus Kits (ZSGB-BIO, Beijing, China) were used in this step. HRP was detected with 3,3′-diaminobenzidine substrate (DAB Kit, Maixin Bio, Fuzhou, Fujian, China) for 30–45 s, washed, and counterstained with hematoxylin (Beyotime Institute of Biotechnology, Shanghai, China) for 20– 30 s. Control sections exposed to secondary antibody alone did not show nonspecific staining. The quantification of MVD was assessed according to the method of previous reports [29,30]. 10 random fields at ×200 magnification were examined for each section, and the microvessels within those fields were counted. A single microvessel was defined as a discrete cluster or single cell stained positive for CD31. To quantify Ki67 expression, the number of positive cells was counted in 10 random fields at ×200 magnification. After the sections were hydrated as above, the sections were stained with hematoxylin and eosin (HE) for histomorphometric analysis. All sections were observed or counted by two investigator pathologists in a blinded fashion. 2.8. TUNEL assay for tumor samples Apoptotic cells in tumor tissue specimens were detected on paraffin sections using the terminal deoxy-nucleotidyl transferasemediated dUTP nick end labeling DeadEnd Fluorometric (TUNEL) system assay kit (Promega, Madison, WI, USA) following the manufacturer's instructions. The sections were observed under a DM 2500 fluorescence microscope and digitally photographed by the Leica Application Suite (Leica Microsystems CMS GmbH, Wetzlar, Germany). Cells with pyknotic nucleus of dark green fluorescent staining were defined as TUNEL-positive cells. Percent apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in 10 random fields at × 200 magnification. 2.9. Safety evaluation of F-P-LP/CLDN3 in female mice 2.9.1. Body weight, general observation and HE staining To evaluate the potential side effects in the F-P-LP/CLDN3-treated mice, they were continuously observed for relevant indexes such as appearance, weight, independent activity, or toxic deaths. The body weights were measured and recorded. Vital organs (heart, liver, spleen, lung and kidney) were sectioned, stained with HE, and observed by two pathologists in a blinded manner. Those of Control, P-LP/HK, F-P-LP/HK and P-LP/CLDN3-treated mice were recorded and processed by the same procedure as F-P-LP/CLDN3-treated mice. 2.9.2. Blood test and serological biochemical analysis Whole blood, obtained from mice, was divided into two parts. One was directly used to conduct a blood test using a Celltac alpha MEK6318K fully automatic hematology analyzer (Nihon Kohden Corp., Shinjuku-ku, Tokyo, Japan). The other was processed with EDTA-2K for 2–3 h at room temperature and serum was obtained by centrifugation. And then the serum was used for serological biochemical

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analysis with an automatic analyzer (Hitachi High-Technologies Corp., Q18Minato-ku, Tokyo, Japan). Blood test and serological biochemical analysis were executed immediately once the samples were prepared. The interferon response of F-P-LP/CLDN3 was examined by a mouse IFN-γ instant ELISA kit (eBioscience, Inc., San Diego, CA, USA). 2.10. Statistical analysis Statistic analysis was performed using One-Way ANOVA in Statistical Product and Service Solutions (SPSS V 19.0, IBM Corp., New York, USA). When equal variances were assumed after homogeneity of variance test, Q19the Tukey multiple comparisons test was used. When equal variances were not assumed after homogeneity of variance test, Tamhane's T2 multiple comparisons test was used. Differences were considered statistically significant at p b 0.05. 3. Results and discussion 3.1. Preparation and characterization of F-P-LP and F-P-LP/CLDN3 F-P-LP and P-LP were produced by the film dispersion method [25]. As shown in Fig. 1a, the zeta-potential values of both liposomes (F-P-LP and P-LP) were higher than that of all lipoplexes (F-P-LP/CLDN3, F-P-LP/HK, P-LP/CLDN3 and P-LP/HK with the lipid/DNA charge ratio of 1.82:1), and the zeta-potential values were significantly decreased from 3842 mV of liposomes to 25-27 mV of lipoplexes (p b 0.001). When plasmid DNA with a negative charge bound with a liposome, the positive charge of a liposome was neutralized, which led to a decrease in the positive charge of the formed lipoplex. As shown in Fig. 1b, the size of F-P-LP and P-LP was about 80 nm. However, the size of F-P-LP/CLDN3 and F-PLP/HK, which were prepared by mixing F-P-LP with plasmid DNA (pCLDN3 or pHK, respectively) through electrostatic interactions, was significantly increased and reached about 180 nm-200 nm (compared with F-P-LP, p b 0.001). Similarly, the size of P-LP/CLDN3 and P-LP/HK appeared to have the same trend, significantly larger than that of P-LP (p b 0.001). The polydispersion indexes (PDIs) of liposomes (F-P-LP and P-LP) and lipoplexes (F-P-LP/CLDN3, F-P-LP/HK, P-LP/CLDN3 and P-LP/ HK) were about 0.2-0.3. As seen in Fig. 1c, the AFM observation showed that the morphological characteristic of F-P-LP/CLDN3 was spheroidal. Agarose gel electrophoresis with nanogram sensitivity was used for characterization of the encapsulation efficiency of liposome entrapped DNA. And a very small amount of the free DNA unentrapped into liposome was observed and performed as a bright band in the gel imaging system (Fig. 1d, lane 2 or lane 3). No band of free DNA appeared as shown in Fig. 1d (lanes 4 to 7). Thus we suggested that DNA (pCLDN3 or pHK) was completely incorporated into the liposomes and that lipoplexes were successfully prepared without free DNA (F-P-LP/CLDN3, F-P-LP/HK, P-LP/CLDN3 and P-LP/HK). We also studied the stability of pCLDN3 in F-P-LP/CLDN3 in the presence of DNase I as shown in Fig. 1e. Naked plasmid DNA was degraded within 2 min [24]. However, pCLDN3 encapsulated in F-PLP/CLDN3 remained stable for at least 72 h in the presence of DNase I. Therefore, F-P-LP/CLDN3 could preserve pCLDN3 from degradation of DNase I in vitro. 3.2. In vitro transfection activity of F-P-LP/GFP and in vivo antitumor effect of F-P-LP/CLDN3 As shown in Fig. 2a, F-P-LP/GFP more effectively transfected SKOV-3 cells than P-LP/GFP (⁎p = 0.041). It suggested that F-P-LP could condense plasmid DNA and transfect more SKOV-3 cells through the interaction between folate and FRα [24,31]. As shown in Fig. 2b, c and d, F-P-LP/CLDN3 and P-LP/CLDN3 more effectively and significantly inhibited tumor growth than Control, F-PLP/HK and P-LP/HK by i.p. administration, which was the most preferred administration route used in the clinical trials of ovarian

cancer gene therapy (as seen in the Supplementary content) [3]. After i.p. administration, positively charged lipoplexes (zeta-potential was 25-27 mV as mentioned above) could provide a high level of antitumor drug into the peritoneal cavity and remain constant at approximately 25% of the injected dose from 2 to 48 h [32]. This might contribute to the antitumor effects of F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and PLP/HK. F-P-LP/HK and P-LP/HK decreased tumor growth in tumor nodules (Fig. 2c, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001) and weight compared with Control (Fig. 2d, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001). Especially, with active targeting for FRα, F-P-LP/HK more effectively reduced tumor weight (Fig. 2d, ‡p = 0.032) than P-LP/HK. It was well known that liposomes or lipoplexes containing DOTAP had nonspecific cytotoxicity to tumor cells in vitro [24,25,33] and in vivo [34,35]. What is more, the tumor growth could be inhibited to a certain extent when the concentration of lipoplexes stayed at a high level in the peritoneal cavity after i.p. administration. Due to the notion that F-P-LP/HK was a folatetargeted gene delivery vector; it would be more favorable for tumor cells than P-LP/HK. Thus, F-P-LP/HK could more effectively inhibit the tumor growth than P-LP/HK. On the other hand, the antitumor activity of P-LP/CLDN3 was statistically superior to F-P-LP/HK (Fig. 2b and d, §§ p b 0.01; Fig. 2c, §§§p b 0.001). The reason for this was that pCLDN3 encapsulated in P-LP/CLDN3 actively targeted and silenced the CLDN3 gene, which specifically inhibited the tumor growth. This result was consistent with previously reported results [7,11]. Furthermore, F-P-LP/CLDN3 showed additional tumor growth inhibition compared with P-LP/CLDN3. As seen in Fig. 2b, F-P-LP/CLDN3, as well as P-LP/CLDN3, completely inhibited malignant ascites production and promoted advanced ovarian cancer benign differentiation [36]. F-PLP/CLDN3 significantly reduced the tumor weight (about 90% tumor growth inhibition, Fig. 2d, †††p b 0.001) and tumor nodule number (Fig. 2c, †††p b 0.001). It was demonstrated that additional folate moiety, F-P-LP/CLDN3 versus P-LP/CLDN3, brought significantly additional antitumor activity. And this difference of antitumor effect could be attributed to effective uptake and specific interaction between folate moiety in F-P-LP/CLDN3 and FRα expressed by tumor cells. Consequently, F-P-LP/CLDN3 with vector and gene targeting, which was specifically taken by tumor cells and actively targeted for silencing CLDN3 gene, was the most effective formulation than the other three lipoplexes groups (P-LP/HK, F-P-LP/HK and P-LP/CLDN3). 3.3. Downregulation of FRα in F-P-LP/CLDN3-treated tumor tissues As seen in Fig. 3, immunohistochemistry (IHC) staining (Fig. 3a), flow cytometry (Fig. 3b and d) and western blot (Fig. 3c) of tumor tissue specimens showed FRα was downregulated after treatment by F-P-LP/ CLDN3. As seen in Fig. 3a, IHC staining indicated FRα was expressed on the tumor tissues and few FRα positive cells were observed on the tissue sections treated by F-P-LP/CLDN3. As shown in Fig. 3c, western blot analysis demonstrated the FRα was significantly downregulated in the F-P-LP/CLDN3-treated sample. As shown in Fig. 3b, flow cytometry analysis suggested that FRα had changed in the tumor cell suspensions treated by F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and PLP/HK. It could be deduced that F-P-LP/CLDN3 decreased the amount of FRα indirectly. In F-P-LP/CLDN3-treated tumor tissues, F-P-LP/ CLDN3 was prone to be taken up by ovarian cancer cell expressing FRα, and then these cells expressing FRα would be apoptotic due to CLDN3 knockdown medicated by shRNA expression. Namely, the FRαexpressing cells would be killed after taking up F-P-LP/CLDN3, thereby decreasing the amount of FRα in the whole tumor tissues. The higher the shRNA expression was due to higher uptake of F-P-LP/CLDN3, the less the detected FRα was. Quantitative data from flow cytometry analysis were showed in Fig. 3d. The tumor cell suspensions treated by F-P-LP/CLDN3 significantly reduced tumor cells expressing FRα, shown as 9% shown in Fig. 3d (⁎⁎⁎p b 0.001, ‡‡‡p b 0.001, §§§p b 0.001, †††p b 0.001 compared with Control, P-LP/HK, F-P-LP/HK and P-LP/CLDN3,

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Fig. 1. Pharmaceutical properties of F-P-LP/CLDN3. (a) Zeta-potential of liposomes and lipoplexes (⁎⁎⁎p b 0.001 and †††p b 0.001 versus that of P-LP and F-P-LP, respectively, Mean ± SD, n = 3). (b) Particle size of liposomes and lipoplexes (⁎⁎⁎p b 0.001 and †††p b 0.001 versus that of P-LP and F-P-LP, respectively, Mean ± SD, n = 3). (c) Morphological characteristic of F-P-LP/ CLDN3 by AFM observation. F-P-LP/CLDN3 was spheroidal. (d) Agarose gel electrophoresis of DNA and lipoplexes. Lane 1, DNA marker; lanes 2 and 3, respectively naked pHK and pCLDN3; lane 4, P-LP/HK; lane 5, F-P-LP/HK; lane 6, P-LP/CLDN3; lane 7, F-P-LP/CLDN3. PCLDN3 or pHK was completely incorporated into P-LP or F-P-LP and lipoplexes were prepared without free DNA. (e) The stability of pCLDN3 in F-P-LP/CLDN3 in the presence of DNase I. F-P-LP/CLDN3 could preserve pCLDN3 from degradation of DNase I.

respectively). F-P-LP/CLDN3 with vector and gene targeting, which was specifically taken up by tumor cells bearing FRα and actively targeted for silencing the CLDN3 gene, minimized the FRα amount in the tumor tissues. It was reported that serous ovarian carcinoma patients with high FRα had reduce survival and cytotoxic chemoresponse [37]. FRα overexpression was associated with poor prognosis [38]. Also, downregulation of FRα in ovarian cancer was associated with benign differentiation of malignant tumor [39,40]. F-P-LP/CLDN3 significantly inhibited tumor growth and promoted benign differentiation of tumor. With non-targeted delivery of pCLDN3 to tumor cells, P-LP/CLDN3 reduced the tumor cells expressing FRα from 84% to 57% (⁎⁎⁎p b 0.001), and was superior to F-P-LP/HK (69%, §§p = 0.005) and P-LP/HK (74%, ‡‡‡p b 0.001). Therefore, P-LP/CLDN3 had a much weaker effect on inhibiting tumor growth compared with F-P-LP/CLDN3.

3.4. Downregulation of CLDN3 in tumor tissues after treatment by F-P-LP/CLDN3 After ovarian tumors treated with F-P-LP/CLDN3, the effectiveness of gene silencing and therapeutic efficacy were determined by IHC staining and western blot analysis. As seen in Fig. 4, CLDN3 expression was almost completely inhibited in F-P-LP/CLDN3-treated tumors compared with those in other lipoplexes groups and Control (Fig. 4, F-P-LP/CLDN3 (1) and (2) completely inhibited CLDN3 expression as shown in western blot; F-P-LP/CLDN3 (3) and (4) almost completely inhibited CLDN3 expression; IHC staining shown CLDN3 expression was almost completely inhibited in F-P-LP/CLDN3-treated tumors), and F-P-LP/CLDN3 was the most efficacious agent of CLDN3 gene silencing. CLDN3 expression was reduced by 70% in P-LP/CLDN3treated tumors compared with Control and by 55-60% in P-LP/CLDN3-

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Fig. 2. In vitro transfection activity of F-P-LP/GFP and in vivo antitumor effect of F-P-LP/CLDN3. (a) In vitro transfection activity of F-P-LP/GFP (Mean ± SD, n = 3, ⁎p b 0.05). F-P-LP/GFP more effectively transfected SKOV-3 cells than P-LP/GFP. (b), (c) and (d): in vivo antitumor effect of F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK (⁎⁎p b 0.01, ⁎⁎⁎p b 0.001, F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK versus Control; ‡p b 0.05, ‡‡p b 0.01, ‡‡‡p b 0.001, F-P-LP/CLDN3, P-LP/CLDN3 and F-P-LP/HK versus P-LP/HK; §§p b 0.01, §§§p b 0.001, F-P-LP/CLDN3 and P-LP/CLDN3 versus F-P-LP/HK; †††p b 0.001, F-P-LP/CLDN3 versus P-LP/CLDN3. Mean ± SD, n = 8). (b) Ascites; (c) Tumor nodules; (d) Tumor weight. F-P-LP/CLDN3 was specifically taken by tumor cells, actively targeted for silencing CLDN3 gene and was the most effective formulation than the other three lipoplex groups.

treated tumors compared with P-LP/HK and F-P-LP/HK. So, it was indicated that pCLDN3 in P-LP/CLDN3 was able to inhibit CLDN3 expression but it still needs improvement to achieve better inhibition ability. F-P-LP/CLDN3 had the efficacious ability to silence the CLDN3 gene compared with P-LP/CLDN3. And F (folate), as the ligand targeting for FRα, was clearly an important moiety in the F-P-LP/CLDN3. And F in the F-P-LP/CLDN3 improved the inhibition ability to silence the CLDN3 gene compared with P-LP/CLDN3. Therefore, by i.p. administration, FP-LP/CLDN3 almost completely downregulated CLDN3 expression in tumor tissues by dual targeting: (i) F-P-LP/CLDN3 liposomal particles were extensively taken by the ligand F in F-P-LP/CLDN3 targeting for FRα on tumor cells; (ii) pCLDN3 in F-P-LP/CLDN3 was an extremely efficient gene targeting for silencing CLDN3. However, some nonspecific CLDN3 gene inhibitions were also observed in tumor tissues treated by P-LP/HK (15% reduction) and F-P-LP/HK (10% reduction) as seen in Fig. 4. 3.5. Effect of F-P-LP/CLDN3 on tumor cell proliferation, angiogenesis and apoptosis F-P-LP/CLDN3 has showed a direct antitumor effect by vector and gene targeting, but the potential mechanisms underlying the efficacy need to be further studied. It was reported [7,11] that the CLDN3 gene knockdown inhibited tumor cell proliferation and angiogenesis, and promoted tumor cell apoptosis. We investigated the change of cell proliferation (Ki67), angiogenesis (CD31) and cell apoptosis (TUNEL) in tumors of each group to determine the antitumor mechanisms of F-PLP/CLDN3 as shown in Fig. 5. First, the effects of F-P-LP/CLDN3 on ovarian tumor cell proliferation were determined by Ki67 staining. F-P-LP/CLDN3, P-LP/CLDN3 and F-P-LP/HK were able to reduce the Ki67 expression

compared with Control and P-LP/HK (⁎⁎⁎p b 0.001 and †††p b 0.001). However, F-P-LP/CLDN3 significantly reduced Ki67 expression by 10% compared with 57% of P-LP/CLDN3 (†††p b 0.001) and 69% of F-P-LP/HK (§§§p b 0.001). F-P-LP/CLDN3 with the CLDN3 gene and FRα targeting most effectively inhibited Ki67 expression and cell proliferation. Although single targeted lipoplexes, P-LP/CLDN3 with CLDN3 and F-P-LP/HK with F, were superior to non-targeted P-LP/HK (‡‡‡pb0.001). P-LP/CLDN3 with a single CLDN3 gene targeting more effectively decreased Ki67 expression than F-P-LP/HK with a single F-targeting vector (§p = 0.036). Since CLDN3 expression in ovarian epithelial cells enhanced invasion and was associated with increased matrix metalloproteinase2 activity (MMP-2) [41], enhanced expression of MMP-2 was concerned with markedly increased vascularization [42]. Next, in order to study the effect of F-P-LP/CLDN3 on angiogenesis, CD31 expression was examined by IHC staining. F-P-LP/CLDN3 with CLDN3 gene targeting and FRα sharply reduced CD31 positive cells by b1% and significantly decreased microvessel density (MVD) compared with P-LP/CLDN3 (††p = 0.002), F-P-LP/HK (§§§p b 0.001), P-LP/HK (‡‡‡p b 0.001) and Control (⁎⁎⁎p b 0.001). And P-LP/CLDN3 with CLDN3 gene targeting significantly decreased MVD than P-LP/HK (‡‡p = 0.001) and Control (⁎⁎p = 0.001). However, F-P-LP/HK effectively reduced MVD in tumor tissue by targeting FRα compared with P-LP/HK (‡p = 0.027) and Control (⁎p = 0.013). Finally, the TUNEL method was used to evaluate tumor cell apoptosis. F-P-LP/CLDN3 significantly increased tumor cell apoptosis by 81% compared with 15% of P-LP/CLDN3 (††p = 0.002), 1% of F-P-LP/HK (§§p = 0.004), b 1% of P-LP/HK (‡‡p = 0.005) and Control (⁎⁎p = 0.005). P-LP/CLDN3 with CLDN3 gene targeting significantly elevated the percentage of apoptotic cells than F-P-LP/HK with F-targeting vector (§§p = 0.003), as indicated that CLDN3 gene targeting contributed to

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Fig. 3. In vivo study of FRα in tumor tissues after treatment by F-P-LP/CLDN3. (a) IHC staining; (b) Flow cytometry analysis; (c) Western blot analysis; (d) Quantitative data from flow cytometry analysis (⁎p b 0.05, ⁎⁎⁎p b 0.001, F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK versus Control; ‡‡‡p b 0.01, F-P-LP/CLDN3 and P-LP/CLDN3 versus P-LP/HK; §§p b 0.01, §§§ p b 0.001, F-P-LP/CLDN3 and P-LP/CLDN3 versus F-P-LP/HK; †††p b 0.001, F-P-LP/CLDN3 versus P-LP/CLDN3. Mean ± SD, n = 3). According to the results of IHC staining (original magnification, ×400), and flow cytometry and western blot analyses, F-P-LP/CLDN3, with downregulating FRα in ovarian cancer cells bearing FRα by specific uptake and actively targeting for silencing CLDN3 gene, significantly inhibited tumor growth.

Fig. 4. In vivo downregulation of CLDN3 with F-P-LP/CLDN3 was determined by IHC staining and western blot analysis, quantitative data from western blot analysis. IHC staining suggested that F-P-LP/CLDN3 clearly reduced CLDN3 expression and that P-LP/CLDN3 was able to decrease CLDN3 expression (original magnification, ×400). Western blot analysis was done for CLDN3 expression: F-P-LP/CLDN3 remarkably reduced CLDN3 expression, and P-LP/CLDN3 was able to decrease CLDN3 expression (20 μg total protein was used in the band of Control, P-LP/HK, F-P-LP/HK, P-LP/CLDN3 and F-P-LP/CLDN3 (1), (2)-treated tumor tissues, and about 60 μg total protein was used in the band of F-P-LP/CLDN3 (3), (4)-treated tumor tissues).

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Fig. 5. Effect of F-P-LP/CLDN3 on cell proliferation, angiogenesis and apoptosis determined by Ki67, CD31 staining and TUNEL analysis. F-P-LP/CLDN3 with vector and gene targeting significantly inhibited cell proliferation (†††p b 0.001 versus P-LP/CLDN3, §§§p b 0.001 versus F-P-LP/HK, ‡‡‡p b 0.001 versus P-LP/HK, ⁎⁎⁎p b 0.001 versus Control) and MVD (††p b 0.01 versus P-LP/CLDN3, §§§p b 0.001 versus F-P-LP/HK, ‡‡‡p b 0.001 versus P-LP/HK, ⁎⁎⁎p b 0.001 versus Control), and increased apoptosis (††p b 0.01 versus P-LP/CLDN3, §§p b 0.01 versus F-P-LP/HK, ‡‡ p b 0.01 versus P-LP/HK, ⁎⁎p b 0.01 versus Control). P-LP/CLDN3 with CLDN3 gene targeting was superior to other controls in reducing cell proliferation (§p b 0.05 versus F-P-LP/HK, ‡‡‡ p b 0.001 versus P-LP/HK and ⁎⁎⁎p b 0.001 versus Control), inhibiting MVD (‡‡p b 0.01 versus P-LP/HK, ⁎⁎p b 0.01 versus Control) and increasing apoptosis (§§p b 0.01 versus F-P-LP/ HK, ‡p b 0.05 versus P-LP/HK and ⁎p b 0.05 versus Control). Cell morphology changes including cell shrinkage, chromatin condensation and nuclear fragmentation were observed by HE staining.

cell apoptosis but not to F-targeting. But, in combination with Ftargeting, F modified vector significantly enhanced the ability of the CLDN3 gene to induce cell apoptosis according to the results (†††p b 0.001, compared with P-LP/CLDN3). Cell morphology changed in F-P-LP/CLDN3-treated tumor tissues. Cell shrinkage, chromatin condensation and nuclear fragmentation were observed by HE staining, which further confirmed an increase in tumor cell apoptosis.

3.6. Safety and toxicity evaluations of F-P-LP/CLDN3 in female mice As the BALB/c nude breeding system was introduced to Vital River (Beijing, China) from Charles River Laboratories Inc. (Kohoku-ku, Yokohama, Japan), we referred to the related technical sheet of Charles River Laboratories Japan (CRLJ) [43] while doing the weight measurements and blood test. The appearance of mice remained hairless and albino in background until they were sacrificed. Although the skin of the control group became shrunken and not shiny, the skin of the mice treated by lipoplexes (F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK) maintained full albino background and glossy. Therefore, treatment by lipoplexes (F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and PLP/HK) kept the appearance of the mice. Normal urine and fecal appearances were observed during treatment by lipoplexes. As seen in Fig. 6a, there were no differences in total body weight among the mice treated by lipoplexes, and the growth curves were similar to the technical sheet of CRLJ. But the total body weight of the control group mice abnormally decreased compared with that of lipoplexes-treated mice (⁎p b 0.05 or ⁎⁎p b 0.01). In a word, mice bearing ovarian tumor did not grow normally unless the ovarian tumor got treated and inhibited by lipoplexes. Consequently, no obvious toxicities were observed in the mice as determined by appearance, body weight, and fecal and urinary excretions. F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK were all evaluated as safe formulations in i.p. administration. It had been explained that local delivery of lipoplexes could provide sustained, elevated concentration of lipoplexes and reduce local and systemic toxicities by i.p. administration [32].

In order to further study the effects of lipoplexes on the physiology of mice treated by lipoplexes, blood test and serological biochemical analysis were carried out as shown in Fig. 6b. WBC, RBC, HGB and PLT counts were all present in normal range after therapy by lipoplexes. Although there were individual differences, all biochemical indexes suggested that the functions of lipoplexes-treated mice's vital organs were present at the same levels according to the technical sheet of CRLJ, previous report [7] or the data of normal mice. The IFN-γ level in the serum was lower than the limit of quantification (15.6 pg/mL), thus inferring that the F-PLP/CLDN3 system would not induce the interferon response. The dose and charge ratios of lipoplexes were much lower than that reported by other researchers [44], so the interferon response was not presented in F-P-LP/CLDN3. HE staining indicated that no significantly toxic pathological changes in the heart, liver, spleen, lung and kidney were detected as shown in Fig. 7. So the mice were in good physical and health conditions, and F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK were all safe formulations by i.p. administration.

4. Conclusions A non-viral gene vector targeting FRα, with 80 nm in particle size and 42mV in zeta-potential, F-P-LP was produced by the film dispersion method. ShRNA targeting for silencing the CLDN3 gene (pCLDN3), was extracted and purified by using the QIAGEN Endofree Kit. A vector and gene-targeted lipoplex, F-P-LP/CLDN3, prepared by mixing F-P-LP with pCLDN3 through electrostatic interaction, had a larger size around 200 nm and a lower zeta-potential of 27 mV compared with F-P-LP, and the morphological characteristic of F-P-LP/CLDN3 was spheroidal as observed by AFM. It was proved by agarose gel electrophoresis that pCLDN3 was completely incorporated into F-P-LP and F-P-LP/CLDN3 was formed without free pCLDN3. Also, F-P-LP/CLDN3 could preserve pCLDN3 from degradation of DNase I over 72 h. F-P-LP/GFP transfected effectively SKOV-3 cells, which suggested that F-P-LP could condense plasmid DNA and transfect more SKOV-3 cells through the interaction between folate and FRα. These results indicated that F-P-LP/CLDN3,

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Fig. 6. Safety and toxicity evaluations of F-P-LP/CLDN3 in female mice. (a) Body weight change; (b) Blood test and serological biochemical analysis (WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; PLT, platelet; ASP, AST, aspartate aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; TBil, total bilirubin; CK, creatine kinase; LDH, lactate dehydrogenase; GLU, glucose; CREA, creatinine; UREA, urea; μTP, micro total protein; TG, triglycerides; TC, total cholesterol; HDL, high density lipoprotein-cholesterol; LDL, low density lipoprotein-cholesterol.⁎p b 0.05, ⁎⁎p b 0.01. Mean ± SD, n = 3). No obvious toxicities were observed in the mice as determined by body weight, blood test and serological biochemical analysis, and mice were in good physical and health conditions. Therefore, F-P-LP/CLDN3, P-LP/CLDN3, F-P-LP/HK and P-LP/HK were all safe preparations by i.p. administration.

with a small size, controlled structures of regular morphology and good stability [45], could be a potential candidate in ovarian cancer gene therapy. In vivo antitumor experiments showed that F-P-LP/CLDN3 significantly inhibited ovarian tumor growth. In addition to that, malignant ascites formation was completely inhibited, and that tumor nodule number and tumor weight significantly decreased in F-P-LP/CLDN3treated mice (p b 0.001). In the further study we found that CLDN3 expression was downregulated in the F-P-LP/CLDN3 group and F-P-LP/ CLDN3 was extensively taken by tumor cells which resulted in an inhibition of cell proliferation. These are the reasons why F-P-LP/CLDN3 with F had an improved antitumor activity while compared with P-LP/ CLDN3 (p b 0.001). Furthermore, downregulation of FRα expression

was observed in F-P-LP/CLDN3 while compared with F-P-LP/HK without pCLDN3 (p b 0.001). This might reduce the degree of malignant ovarian tumor growth and promote the benign transformation of malignant ovarian tumor. Therefore, CLDN3 gene and FRα targeting exhibited synergistic antitumor effect in the treatment using F-P-LP/CLDN3. In FP-LP/CLDN3, FRα targeting significantly enhanced the effect of pCLDN3 on silencing the CLDN3 gene, and CLDN3 gene targeting significantly increased the down-regulation of FRα. Consequently, F-P-LP/CLDN3 with vector and gene targeting had the optimal antitumor activity and could significantly inhibit ovarian tumor growth. Furthermore, the potential mechanisms underlying the efficacy of FP-LP/CLDN3-based therapy were studied by TUNEL assay, Ki67 staining and CD31 staining. F-P-LP/CLDN3 sharply promoted tumor cell apoptosis,

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Fig. 7. Histological examination of HE-stained vital organ sections. Compared with normal BALB/c nude mice, after ovarian tumor inoculation, inflammatory response in lung was observed. There was still an inflammatory response but it has been reduced after gene therapy, especially for the specimen treated by F-P-LP/CLDN3. Histological examination result of other vital organs was normal by HE-stained sections.

significantly inhibited tumor cell proliferation and statistically reduced MVD, which were proved by TUNEL assay, Ki67 staining and CD31 staining, respectively. These might have contributed to the efficacious antitumor effect of F-P-LP/CLDN3. Finally, safety and toxicity evaluations of F-P-LP/CLDN3 were performed in female mice. No obvious toxicities were observed in the mice as determined by appearance, body weight, and fecal and urinary excretions. The morphology and function of vital organs were all present at the normal levels. Therefore, we draw a conclusion that F-P-LP/CLDN3 was a safe and efficient formulation when used in i.p. administration. F-P-LP/CLDN3 was a novel and potential targeting formulation and a promising candidate for clinical ovarian cancer gene therapy.

Acknowledgments This work was supported by the National Basic Research Program of China (No. 2010CB529900); and the National Natural Science Foundation of China (No. 81123003); and the National High-tech R&D Program of China (No. SS2014AA020708).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2013.10.015.

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