Ovarian cancer therapy by VSVMP gene mediated by a paclitaxel

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Ovarian cancer therapy by VSVMP gene mediated by a paclitaxel-enhanced nanoparticle Jianlin Long, Yuping Yang, Tianyi Kang, Wei Zhao, Hao Cheng, Yujiao Wu, Ting Du, Beibei Liu, Yang Li, Feng Luo, and Maling Gou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10796 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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Ovarian cancer therapy by VSVMP gene mediated by a paclitaxel-enhanced nanoparticle Jianlin Long†‡§#, Yuping Yang†#, Tianyi Kang†#, Wei Zhao†, Hao Cheng†, Yujiao Wu†, Ting Du†, Beibei Liu†, Yang Li†, Feng Luo†‡, Maling Gou†* †

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, SichuanUniversity, and Collaborative Innovation Center for Biotherapy. Chengdu Sichuan, 610041, P. R. China.

‡

Department of medical oncology, Lung Cancer Center, Cancer Center, West China Hospital, Medical School, Sichuan University. Chengdu Sichuan, 610041, P. R. China.

§

Department of oncology, West China Guang’an Hospital, Sichuan University. Guang’an, Sichuan, 638000, P. R. China.

ABSTRACT: Nanoparticles have great promise for gene delivery. However, the transfection efficiency of nanoparticle-based gene delivery systems is always unsatisfied to meet the requirement of effective gene therapy. Herein, we used low dosage paclitaxel to enhance a nanoscaled gene delivery system that was self-assembled

from

DOTAP

and

MPEG-PLA

(DPP),

creating

a

paclitaxel-encapsulated DPP nanoparticle (P-DPP). The encapsulated low-dosage paclitaxel significantly improved the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells, in some of which the transfection efficiency is as high as 92%. By the P-DPP nanoparticle, VSVMP that could induce cell apoptosis was delivered to treat ovarian cancer. The encapsulation of paclitaxel in DPP nanoparticles increased the expression of VSVMP, enhancing VSVMP to induce anti-proliferation and apoptosis in SKOV3 ovarian cancer cells. Intraperitoneal administration of P-DPP delivered VSVMP effectively inhibited the intraperitoneal metastasis of SKOV3 ovarian cancer, which was more efficient than DPP delivered VSVMP. Moreover, it was found that the tumor cell apoptosis induction, tumor cell

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proliferation inhibition and tumor angiogenesis suppression were involved in the anticancer mechanism of this nanocomplex. Our data suggest that the encapsulation of low-dosage paclitaxel can enhance the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells and exert a synergistic anticancer effect with VSVMP gene in ovarian cancer treatment. The VSVMP gene therapy delivered by the paclitaxel-enhanced nanoparticle has potential application in ovarian cancer therapy. Keywords: Nanoparticles; Gene delivery; VSVMP; Paclitaxel; Ovarian cancer INTRODUCTION Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer-related deaths worldwide.1 Surgical tumor debulking following paclitaxel and/or platinum-based chemotherapy are current standard therapeutic strategies for ovarian cancer treatment.2-3 Even quite a lot of progress has been made with respect to chemotherapy for ovarian cancer treatment, ovarian cancer is still the most common type among gynecologic malignancies. The 5-year survival rate of advanced ovarian cancer is less than 25% because of the development of multidrug resistance after repeated chemotherapeutic administration.4 Therefore, novel therapeutic strategies for ovarian cancer are urgently needed. Gene therapy is a potential treatment strategy that introduces exogenous genetic materials to modulate gene expression in specific cells to treat diabetes, cardia-cerebrovascular disease, rheumatism and cancer.5-7 Currently, two thirds of all gene therapy has been targeted to the treatment of cancer, with clinical trials being performed worldwide.8 However, lack of a safe and highly efficient gene delivery system remains the most significant challenge for human gene therapy.9 Nanotechnology has been used for delivery of various therapeutic substances, including small molecular drugs, genes, and biopharmaceuticals.10-11 Nanoparticles offer a platform to co-deliver cargos to reach the same cell for better pharmacokinetics and to ensure maximum intracellular cooperation between drugs.12 We have developed a novel nanoparticle that utilizes low-content N-[1-(2, 3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate (DOTAP) to modify



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Monomethoxy poly (ethylene glycol)-poly(d,l-lactide) (MPEG-PLA), forming a cationic nanoparticle named DPP, for gene delivery. Our previous results showed that the DPP exerted high transfection effects in SKOV3 ovarian cancer cells, almost as high as some viral vectors, but with very low toxicity. Recently, some microtubule-disrupting drugs were found to have the ability to increase nuclear gene entry efficiency and to further improve gene transfection efficiency.13 Paclitaxel, a commonly used microtubule-arresting drug, was found to improve gene transfection efficiency at a non-cytotoxic dosage.12,

14-17

Thus, our goal was to develop a

paclitaxel-loaded nanoparticle with the purpose of further improving gene delivery efficiency and enhancing the antitumor effects of gene therapy. Vesicular stomatitis virus (VSV), an oncolytic virus, preferentially replicates in a large number of tumor cells and has been used for cancer treatment, exhibiting potent antitumor activity via apoptosis induction.18-20 However, its clinical application for cancer treatment has been restricted owing to the risks involved in its use.21-23 Previously, some efforts have been made to reduce virus-associated safety issues.24 The matrix protein (MP) of vesicular stomatitis virus (VSV), a structural component of the virion, plays a critical role in VSV-induced antitumor effects even in the absence of other viral structural components. The VSVMP acts via multiple mechanisms, including cytoskeletal element destruction,25 host cell gene expression inhibition,26 and host systemic immune response enhancement.27 VSVMP has also shown antitumor activity in multiple tumor-bearing models.28-31 Here, we designed a low-dosage paclitaxel-loaded DPP nanoparticle (P-DPP) to deliver pVSVMP for the treatment of SKOV3 ovarian cancer. The prepared P-DPP nanoparticles exhibited a weakly positive surface charge, appropriate shape and size, good drug release profiles and high drug encapsulation efficiency. We further evaluated the gene delivery efficiency of the P-DPP nanoparticles against cancer cells and the anticancer activity of the P-DPP/pVSVMP nanocomplex against SKOV3 human ovarian cancer. The results suggest that low-dosage paclitaxel-loaded nanoparticles represent a novel gene delivery system. The VSVMP delivered by low-dosage paclitaxel-enhanced DPP nanoparticle have significant potential for


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ovarian cancer therapy. EXPERIMENTAL SECTION Materials. Paclitaxel (PTX, Mw 853.91kDa, 99.9%) was purchased from Aladdin industrial corporation (Shanghai, China). Branched polyethylenimine (PEI, Mw

25

kDa),

N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl

sulfate (DOTAP) and 4', 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich

(St

Louis,

USA).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) and fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), streptomycin and penicillin were provided by HyClone™ (GE Healthcare UK Ltd, UK). Pre-stained protein ladder and DNA ladder were purchased from Fermentas (Thermo Fisher Scientific Inc., U.S.). Monomethoxy poly (ethylene glycol)-poly(d,l-lactide) (MPEG-PLA, Mw 4000) was synthesized in our lab.32 The therapeutic gene (VSVMP) was previously constructed into pVAX expression vector (pVSVMP) in our lab for tumor treatment,31 while pVAX was used as the empty vector. Plasmid DNA encoding luciferase (pGL6) was purchased from Beyotime Biotechnology (China). YOYO-1, LysoTracker red were purchased from Invitrogen (USA). Cell Culture. SKOV3, A549, MDA-MB-231, MCF-7, CT-26 and B16 cell lines were obtained from ATCC (American Tissue Culture Collection, USA) and were cultured in DMEM or RPMI-1640 medium added with 10% FBS, 100 µg/mL streptomycin, 2 mM l-glutamine, and 100 U/mL penicillin at 37˚C in a moist atmosphere containing 5% CO2. Animals. Female BALB/c nude mice of 4-6 weeks age were obtained from Beijing HFK Bioscience (Beijing, China) and housed and maintained under specific pathogen-free conditions at the laboratory animal room. All animals received care in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved and supervised by the Institutional Animal Care and Treatment Committee of Sichuan University. Preparation and Characterization of paclitaxel loaded DOTAP-MPEG-PLA nanoparticle (P-DPP). Paclitaxel loaded DPP (P-DPP) nanoparticles were prepared



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by the self-assembly method. In brief, 9 mg of MPEG-PLA diblock copolymer, 1 mg DOTAP and predetermined amount of paclitaxel were co-dissolved in methylene dichloride (KeLong Chemicals, China), and rotarily evaporated to form thin film at 60 ℃. P-DPP nanoparticle was obtained by hydrating the film with glucose injection (GS, 5%). The solution was filtered by using a 0.45 µm Millipore filter membrane. Coumarin-6 loaded P-DPP nanoparticle was prepared by additional incorporating coumarin-6. The DPP nanoparticle was produced in a similar way free of encapsulating drugs. The resultant micelles were adjusted to the final concentration of 2 mg/mL and stored at 4 ℃ for further utilization. The size and zeta potential of P-DPP nanoparticle, DPP nanoparticle, P-DPP/DNA nanocomplex and DPP/DNA nanocomplex were characterized by dynamic light scattering (DLS) using a Malvern Nano ZetaSize (Malvern Instruments Ltd., UK). The surface morphologies were examined by a Transmission Electron Microscope (TEM, Hitachi Ltd., Japan). Gel Retardation Assay. The P-DPP/DNA nanocomplex and the DPP/DNA nanocomplex were obtained at different mass ratios (DPP : DNA = 0:1 to 45:1) and incubated for 30 min at room temperature. Then, the nanocomplex were electrophoresed on 1% (w/v) agarose gel at 100 V for 30 min. The DNA bands were visualized and photographed by a gel documentation system (Bio-RAD Laboratories Inc, USA). Erythrocytes aggregation in vitro. Fresh blood from Sprague Dawley rats was collected in heparinized tubes. The blood was washed with normal saline (NS) until the supernatant was colorless. 100 µL of erythrocytes (4%) was co-incubated with 5 µg plasmid in samples (NS, DPP nanoparticles, P-DPP nanoparticles, DPP/DNA nanocomplex and P-DPP/DNA nanocomplex) in 100 µL of normal saline. After 2 hours culturing at 37 ℃, the condition of erythrocytes aggregation was taken under an optical microscope. Hemolysis assay in vitro. Briefly, different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) of P-DPP nanoparticles in 0.5 mL solution was added into 0.5 mL rat


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erythrocyte suspension (4%) at 37 ℃. Distilled water and normal saline were employed as positive and negative control separately. After three hours incubation, the erythrocyte suspension was centrifuged at 1,800 rpm for 3 minutes, and the supernatant was collected to detect the light absorbance value. Red supernatant solution implies hemolysis while absolute achromatic supernatant solution indicates no hemolysis. Encapsulation Efficiency (EE) of P-DPP nanoparticle. The concentrations of paclitaxel were determined by high-performance liquid chromatography (1100 HPLC, Agilent Technologies, Santa Clara, CA). The mobile phase consisted of a mixture of acetonitrile and double-distilled water (60/40, v/v) pumped at a flow rate of 1.0 mL/min with determination wavelength of 227 nm at 35 ℃. The following equation was applied to calculate the encapsulation efficiency. Encapsulation Efficiency (%)= Wt of the drug in nanoparticles/ Wt of the feeding drugs×100% In Vitro Drug Release Assay. The amount of paclitaxel released from P-DPP nanoparticle and P-DPP/DNA nanocomplex were measured using the dialysis method. The P-DPP nanoparticle and P-DPP/DNA nanocomplex were dissolved in phosphate buffer saline (PBS) at pH 5.0 and 7.4. The solution was then sealed in a dialysis bag with a 3.5 kDa molecular weight cut-off (Sigma) and immersed into 10 mL of 0.1 mol/L PBS, pH 5.0 and 7.4 respectively at 37 ℃, 100 rpm. At designated time intervals, one milliliter samples were collected from the incubation medium and measured for paclitaxel concentration as described above. After sampling, an equal volume of fresh PBS was immediately added into the incubation medium. The concentration of paclitaxel released from P-DPP nanoparticle and P-DPP/DNA nanocomplex were shown as a percentage of the total paclitaxel in P-DPP nanoparticle and P-DPP/DNA nanocomplex and plotted as a function of time respectively. In Vitro Gene Transfection. Cancer cells were seeded in the 12 wells plate (Corning, NY, USA) at a density of 1×105 cells per well. After incubation for 24 h, the medium was replaced with fresh serum-free medium. PEI25K/pGFP, DPP/pGFP



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and P-DPP/pGFP (containing 2 µg of pGFP, at the mass ratio of 1:1, 25:1 and 25:1) were added. The equivalent concentration of PTX varied from 2 to 100 ng/mL in culture medium. After incubation for 6-8 h, the medium was replaced with complete medium. 48 h later, cells were visualized using an inverted fluorescence microscope (Olympus, Japan). Then cells were harvested and the transfection efficiency was quantitatively analyzed by a flow cytometer (BD Biosciences, USA). The relative luciferase activity was detected by using a dual luciferase reporter gene assay kit (Beyotime, China) on luminometer (BioTek, USA) according to the protocol of manufacturer. In Vitro Uptake of P-DPP/DNA Nanocomplex by SKOV3 Ovarian Cancer Cells. Plasmid was labeled by YOYO-1 to investigate the cellular uptake. SKOV3 cells were seeded at a density of 1.5×105 cells per well with DMEM supplemented with 10% fetal bovine serum. After 24 h, serum-free medium was applied to replace the complete medium, and different nanocomplex was used to treat cancer cells for different times. Then the cells were washed with cold PBS after incubation and immobilized with paraformaldehyde (4%) for 30 min. Finally, the cells were visualized using a confocal scanning laser microscope (Olympus, Japan). Cytotoxicity Assay. Cancer cells were seeded in 96 wells plate at a density of 5×103 cells per well and cultured for 24h. DPP, PEI25K and P-DPP nanoparticles (containing various concentration of paclitaxel in culture medium) were added to each well and incubated for another 48 h before MTT assay. Absorbance was measured with a multi-well spectrophotometer (BioTek, USA) at 570 nm. Cell viability was expressed as percentage of the untreated controls, which were not exposed to drugs. Real-time PCR assay. Total RNA was extracted from the transfected cells and tumor tissues using an RNAsimple Total RNA Kit (TIANGEN, China). Then the sample was reverse-transcribed by using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, China). The amount of template cDNA was normalized to GAPDH. The sets of primers were as following: VSVMP: 5′ -CGC GGA TCC ATC ATG AGT TCC TTA AAG AAG-3′ (forward) and 5′ -CGG AAT TCT CAT TTG AAG TGG CTG ATA GAA TCC-3′ (reverse). GAPDH: 5′ -CAGAACATCATCCCTGCATC -3′


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(forward) and 5′ -ACTCCTCAGCAACTGAGGG -3′ (reverse). The real time RT-PCR process contained a step at 95 ℃ for 30 seconds, followed by a 5-second step at 95 ℃ and 20-second step at 56 ℃ for 39 cycles with melting curve analysis. GAPDH was used as the internal reference. Western Blot Analysis. The total protein concentrations of transfected cells and tumor lysates were quantified by using the BCA Protein Assay Kit (Beyotime, China). Proteins from each sample were loaded into 12% sodium dodecyl sulphate– polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). The membranes were sealed by using 5% skimmed milk, then incubated with anti-β -actin (Santa Cruz Biotechnology Inc., USA) or anti-VSVMP (Chengdu Zen Bioscience Co., Ltd., China) primary antibody separately overnight at 4 ℃. The membranes were further incubated with relevant secondary antibody which is labeled with

horseradish

peroxidase

(HRP)

and

colorated

by

the

enhanced

chemiluminescence detection reagents (EMD Millipore, USA). In Vitro Anticancer Activity. The synergistic antitumor activity of paclitaxel with VSVMP was evaluated by MTT assay and apoptosis detection. SKOV3 ovarian cancer cells were seeded in 96 wells plate at a density of 5×103 cells per well. After incubated for 24 h, the medium was then replaced by serum-free medium containing P-DPP/pVSVMP, DPP/pVSVMP, P-DPP/pVAX, P-DPP, DPP and GS (5 µg/DPP, 0.2 µg/pVSVMP, 2 ng/mL of paclitaxel in culture medium). After 6 h, the medium was replaced with fresh complete medium and cells were incubated for another 42 h. The in vitro cytotoxicity was determined by MTT assay. In apoptosis detection, SKOV3 cells were seeded in 6 wells plate at a density of 1.5×105 cells per well and the transfection procedure was the same as the above mentioned. Forty-eight hours after transfection, the rate of apoptosis was measured on the flow cytometry (Beckman-Coulter, USA) using the Annexin V-FITC Apoptosis Detection Kit I (BD biosciences, San Jose, CA) in accordance with the manufacture’s protocol. Biodistribution of P-DPP/pVSVMP Nanocomplex In vivo. The real-time



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distribution of P-DPP/pVSVMP nanocomplex in tumor tissue and other critical organs (heart, liver, spleen, lung and kidney) in BALB/c nude mice (four groups, four mice per group) bearing SKOV3 ovarian cancer peritoneal xenograft were monitored by a BioReal vivo imaging system (Bio-Real Quick View 3000, Austria). For tissue imaging, coumarin-6 was used to label P-DPP/pVSVMP nanocomplex. The mice of experimental group were administrated with 200 µL coumarin-6/P-DPP/pVSVMP nanocomplex via intraperitoneal injection 1 h, 3 h or 24 h before image taking. In Vivo Antitumor Efficacy. Female BALB/c nude mice were inoculated by intraperitoneal injection with 200 µL of SKOV3 ovarian cancer cell (1×107 cells) suspension. All the mice bearing tumor were allocated to six groups (n=5) randomly and treated with GS, DPP (5 mg/Kg), P-DPP (PTX 1µg/Kg, DPP 5 mg/Kg), P-DPP/pVAX (PTX 1µg/Kg, DPP 5 mg/Kg, pVAX 0.2 mg/Kg), DPP/pVSVMP (DPP 5 mg/Kg, pVSVMP 0.2 mg/Kg), P-DPP/pVSVMP (PTX 1µg/Kg, DPP 5 mg/Kg, pVSVMP 0.2 mg/Kg) via intraperitoneal injection 7 days after tumor inoculation. The treatment was performed every other day for 14 days. All mice were sacrificed by cervical vertebra dislocation on 30 days after first dosage. Vital organs and tumors were gathered and immobilized with 4% neutral paraformaldehyde, and frozen under the condition of liquid nitrogen immediately. Tumor weight, number of nodules and ascites volume were recorded. Histological Analysis. Tissues were fixed in neutral paraformaldehyde (4%) for at least 24 hours and then were embedded in paraffin. Immunohistochemical analysis of Ki67 antigen was conducted using rabbit anti-human Ki67 (Millipore Corporation, U.S.) according to that previously described. A commercially available TUNEL kit (Promega, Madison, WI, U.S.) was used to detect the apoptotic cells of tumor tissues in paraffin sections according to the manufacture. Microvessel density (MVD) was stained using anti-CD31 rabbit polyclonal antibody (Ebioscience, US) and Rho-labeling goat anti-rabbit secondary antibody (ZS-BIO, China) for immunofluorescence detection. Hematoxylin and eosin (H&E) staining of the sections was used to analyse the histomorphometric of tumor tissues. Safety Evaluation. Vital organs (liver, heart, lung, spleen and kidney) were


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harvested and stained with H&E using standard methods to evaluate the potential of P-DPP-pVSVMP. Statistical Analysis. All of the data were presented as mean values ± standard deviation. Statistical significance was evaluated by using unpaired Student’s t-test when only two groups were compared. In all the tests, differences were considered to be statistically significant at P0.05). The zeta potentials of DPP nanoparticles and P-DPP nanoparticles were almost the same at about 55 ± 2.1 mV. However, when DNA was absorbed, the zeta potentials of DPP/DNA and P-DPP/DNA nanocomplex decreased to about 29 ± 1.9 mV, as shown in Figure 2B and C. A gel retardation assay was performed to evaluate the DNA-binding ability of P-DPP and DPP nanoparticles. Figure 2D showed that complete retardation of DNA was achieved for both the DPP and P-DPP nanoparticles when the mass ratio of DPP to DNA reached 15:1. The results indicate that both DPP and P-DPP nanoparticles have similar DNA-binding ability and can compact genetic material efficiently. Furthermore, we carried out the erythrocyte aggregation and hemolysis analysis to evaluate the blood compatibility of P-DPP nanoparticles. Shown from Figure 2E, P-DPP nanoparticles caused no obvious hemolysis. However, DPP nanoparticles,



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P-DPP nanoparticles, DPP/DNA and P-DPP/DNA nanocomplex could resulted in erythrocyte aggregation, especially in DPP and P-DPP nanoparticles (Figure 2F). For the P-DPP nanoparticles, the encapsulation efficiency of paclitaxel was over 99.5% (Figure 2G). The in vitro drug release behaviors of P-DPP nanoparticles and P-DPP/DNA nanocomplex in aqueous solution at pH 5.0 and 7.4 at 37 ℃ were studied and illustrated in Figure 2H. The release profiles suggested that paclitaxel can be slowly released from P-DPP nanoparticles or P-DPP/DNA nanocomplex under both pH conditions. The cumulative release of paclitaxel only reached about 70% and 40% at pH of 5.0 and 7.4, respectively, even though it was monitored over 168 h. DNA absorption did not affect the release profiles of P-DPP nanoparticles. Comparing the release profiles at the two pH values, the release of paclitaxel was much faster at lower pH value. Transfection in vitro. We explored whether paclitaxel encapsulated into DPP nanoparticles could affect the transfection efficiency of DPP nanoparticles in multiple cancer cells by using luciferase and GFP as reporter genes. Flow cytometry analysis was performed to determine the percentage of fluorescent cells and a dual luciferase assay was carried out to measure the relative luciferase activity, both of which correspond to the gene expression efficiency. Fluorescence microscopy and flow cytometry analysis revealed that much more GFP-derived green fluorescence could be observed in SKOV3, A549, MDA-MB-231, MCF-7, CT-26 and B16 cells treated with P-DPP/pGFP nanocomplex than those treated with DPP/pGFP nanocomplex (Figure 3A). The GFP-derived green fluorescence increased when the concentration of paclitaxel increased from 2 to 10 ng/mL and then decreased when the concentration of paclitaxel was raised over 10 ng/mL due to cytotoxicity and cell death. The maximum GFP-derived green fluorescence was observed at paclitaxel concentration of 2 ng/mL in SKOV3 cells, 10 ng/mL in A549 cells, 10 ng/mL in MDA-MB-231 cells, 2 ng/mL in MCF-7 cells, 10 ng/mL in CT-26 cells and 5 ng/mL in B16 cells. The highest transfection efficiencies of P-DPP versus DPP were 81.5 ± 2.07 % versus 74.1 ± 3.8 % in SKOV3 cells, 92.35 ± 2.6 % versus 40.95 ± 0.7 % in A549 cells, 58.5 ± 0.4 % versus 40.95 ± 0.4 % in MDA-MB-231 cells, 18 ± 0.7 % versus 8.65 ± 0.2 % in


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MCF-7 cells, 66.85 ± 6.1 % versus 51 ± 3.2 % in CT-26 cells, and 79.6 ± 0.2 % versus 69.15 ± 0.6 % in B16 cells (Figure 3B and C). The dual luciferase assay result revealed that the highest relative luciferase activity of P-DPP had increased 2 fold in SKOV3 cells, 3.3 fold in A549, 2.1 fold in MDA-MB-231 cells, 1.3 fold in MCF-7, 8.8 fold in CT-26 cells, and 1.2 fold in B16 cells compare with that in the cells treated with DPP nanoparticles (Figure 3D). We evaluated the cytotoxicity of P-DPP (contain various transfection concentrations of paclitaxel), which demonstrated that the cell viabilities were all over 85% at the concentration of highest transfection efficiency (Figure 3E). These results suggest that low-dose paclitaxel-loaded DPP nanoparticles increased gene transfection efficiency more than the DPP nanoparticles. On the contrary, high-dose paclitaxel (over 10 ng/mL) decreased gene transfection efficiency sharply owing to cytotoxicity and cell death. Therefore, we chose a paclitaxel concentration of 2 ng/mL to perform the in vitro experiments in SKOV3 cells. We further compared the cytotoxicity of DPP and PEI25K in SKOV3 cells. MTT assay revealed that PEI25K nanoparticles presented about 90% growth inhibition of SKOV3 cells at a concentration of 25 µg/mL, while DPP nanoparticles only showed about 35% growth inhibition of SKOV3 cells even the concentration reached 200 µg/mL (Figure 3F). Cellular uptake analysis To investigate the mechanism of paclitaxel enhancing gene transfection efficiency of DPP, we further conducted the cellular uptake and distribution of P-DPP/DNA nanocomplex via confocal microscopy. pDNA was labeled with YOYO-1 (green) and the nuclei were stain with DAPI (blue). As shown in Figure 3G, the YOYO-1 labeled nanocomplex was distributed in cells time-dependently in both DPP and P-DPP groups. After 0.5 h incubation, the pDNA could be entrapped in the endosomes/lysosomes which is indicated by overlap of red and green fluorescence as orange/yellow dots. After 4 incubation, the green fluorescence of pDNA showed continuous accumulation in the nuclei, revealing the effective endosomal/lysosomal escape ability of DPP/DNA nanocomplex and P-DPP/DNA nanocomplex. However, compared with DPP/DNA nanocomplex group, more plasmid accumulated in the



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nuclei along with the time increasing of incubation in P-DDP/DNA nanocomplex group. In addition, the nuclei were nearly entirely overlapped with luminous green fluorescence after 6 h of incubation, which indicated that paclitaxel could promote escape of nanocomplex from endosomal/lysosomal and increased nuclear entry efficiency of pDNA. Expression of VSVMP and antitumor activity in vitro. We detected the expression of VSVMP gene in SKOV3 cells by real-time PCR and western blot. The expression of VSVMP was observed in cells treated with DPP/pVSVMP nanocomplex and P-DPP/pVSVMP nanocomplex and it was hardly observed in cells treated with GS, DPP nanoparticles, P-DPP nanoparticles, and P-DPP/pVAX (Figure 4A and B). Furthermore, the expression of VSVMP in SKOV3 cells treated with P-DPP/ pVSVMP nanocomplex was significantly higher than that in cells with DPP/ pVSVMP nanocomplex. These results indicate that DPP and P-DPP can efficiently deliver the target gene into cancer cells, inducing expression of the therapeutic gene in SKOV3 cells in vitro. Paclitaxel-loaded DPP was found to enhance the expression of the VSVMP gene more than DPP nanoparticles. To investigate whether paclitaxel has a synergistic anticancer activity of DPP/pVSVMP nanocomplex in vitro, MTT assays and cell apoptosis were performed. MTT assays revealed that both DPP/pVSVMP nanocomplex and P-DPP/pVSVMP nanocomplex sharply inhibited the growth of SKOV3 cells, resulting to about 64.1 ± 4.87 % and 37.4 ± 2.22 % cell viability, respectively. Meanwhile DPP and P-DPP nanoparticles as well as P-DPP/pVAX nanocomplex only had a small effect on cell viability: 97.5 ± 5.01 % for DPP nanoparticles, 94.2 ± 2.60 % for P-DPP nanoparticles, and 90.8 ± 3.79 % for P-DPP/pVAX nanocomplex (Figure 4C). As shown in Figure 4D and E, P-DPP/pVSVMP nanocomplex clearly induced cell apoptosis to 56.9 ± 4.63% of cells, which was significantly higher than that induced by the DPP/pVSVMP nanocomplex (32.4 ± 6.93%). In addition, neither DPP nor P-DPP nanoparticles induced signiﬁcant cell apoptosis. Notably, P-DPP/pVAX also induced a certain amount of cell apoptosis (25.9 ± 1.56%). Biodistribution. The real-time distribution images of P-DPP/pVSVMP


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nanocomplex in vivo demonstrated that coumarin-6-derived fluorescence was mainly observed in tumor nodes, reaching the top at 3 h, after that, increasingly dying over time

and

almost

disappeared

24

h

post-administration.

In

addition,

coumarin-6-derived fluorescence could also be detected in liver and kidney, where the fluorescence was much weaker than that in tumor nodes and also vanished in 24 h (Figure 5). It suggests that P-DPP/pVSVMP nanocomplex is biodegradable. In vivo antitumor activity. To study the antitumor effects of VSVMP delivered by the paclitaxel-enhanced nanoparticles for the intraperitoneal metastatic tumor model of SKOV3 ovarian cancer cells, the treatment regimen was presented in Figure 6A. As shown in Figure 6B, the representative images showed that although both DPP/pVSVMP and P-DPP/pVSVMP nanocomplex displayed significant tumor growth inhibition effects compared to GS, the inhibition extent of P-DPP/pVSVMP nanocomplex was dramatically higher than that of DPP/pVSVMP nanocomplex. In contrast, P-DPP and DPP vesicles hardly influenced tumor growth (Figure 6C). We also found that tumors treated with P-DPP/pVAX were slightly inhibited compared with those treated with GS. The average tumor weight in the P-DPP/pVSVMP nanocomplex group was 0.162 ± 0.065 g, 1.558 ± 0.233 g in the GS group, 1.578 ± 0.108 g in the DPP group, 1.476 ± 0.111 g in the P-DPP group, 0.922 ± 0.322 g in the P-DPP/pVax group, and 0.314 ± 0.109 g in the DPP/pVSVMP nanocomplex group (Figure 6D). In addition, compared with that in the DPP/pVSVMP nanocomplex group, the number of tumor nodules and the ascites volume markedly decreased in the P-DPP/pVSVMP nanocomplex group (Figure 6E and F). These results suggest that P-DPP/pVSVMP nanocomplex has higher antitumor efficiency against the intraparietal metastasis of SKOV3 ovarian cancer than DPP/pVSVMP nanocomplex. Furthermore, we verified the expression of the VSVMP mRNA and protein in tumor tissues via real-time PCR and western blot. As shown in Figure 6G and H, compared with that in the DPP/pVSVMP nanocomplex group, the mRNA and protein expression levels of VSVMP was markedly increased in tumors treated with P-DPP/pVSVMP nanocomplex, whereas in the other groups, no expression of VSVMP was observed.



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Antitumor mechanism study. To clarify the underlying mechanisms of the antitumor effects of P-DPP/pVSVMP nanocomplex in vivo, TUNEL, Ki67, CD31, and H&E staining were carried out (Figure 7A, B, C and D). As shown in Figure 7A, C, E and G, tumor tissues treated with either DPP/pVSVMP nanocomplex or P-DPP/pVSVMP

nanocomplex

both

remarkably

increased the

number

of

TUNEL-positive tumor cells and decreased the percentage of proliferating ki67-positive tumor cells in comparison with the GS, DPP or P-DPP treatments. However, when compared to DPP/pVSVMP nanocomplex, P-DPP/pVSVMP nanocomplex significantly induced cell apoptosis and inhibited cell proliferation. In addition, CD31 staining indicated that DPP/pVSVMP and P-DPP/pVSVMP nanocomplex also demonstrated anti-angiogenic effects in tumors compared with GS, DPP and P-DPP, while P-DPP/pVSVMP nanocomplex inhibited angiogenesis more effectively than DPP/pVSVMP nanocomplex (Figure 7B and F). We found that P-DPP/pVAX indicated a weak apoptotic induction, anti-proliferative effect, and angiogenesis inhibition compared with GS, DPP and p-DPP (P > 0.05). H&E staining of tumor sections indicated that tumor tissues treated with DPP/pVSVMP nanocomplex and P-DPP/pVSVMP nanocomplexes showed fewer blood vessels and increased number of dead cancer cells. On the contrary, there were more cancer cells， red blood cells and vessels in the tumors treated with GS, DPP, P-DPP or P-DPP/pVAX (Figure 7D). Thus, the results suggest that tumor cell apoptosis induction, tumor cell inhibition, and tumor angiogenesis suppression were all involved in the anticancer mechanism of the nanocomplex. Furthermore, we also evaluated the side effects of P-DPP/pVSVMP nanocomplex in mice by H&E staining. Figure 8 showed that there were no significant pathological changes in the liver, kidney, lung, spleen, or heart. These results indicated that P-DPP/pVSVMP nanocomplex had no obvious side effects. DISCUSSION In this study, a paclitaxel-enhanced nanoparticle, called P-DPP, was designed as a novel gene delivery system for ovarian cancer gene treatment. The low dosage paclitaxel can significantly improve gene delivery efficiency of the nanoparticle to


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cancer cells, and exert a synergistic anticancer effect with VSVMP gene. The P-DPP nanoparticle delivered VSVMP gene can efficiently inhibit the ovarian cancer in vitro and in vivo. Lack of safe and efficient gene delivery system remains the main obstacle for human gene therapy.9 Compared to viral-vectors, non-viral vectors exhibit higher gene-loading capacity, lower immune response, easier large-scale production, and safer application.9 DOTAP enabled cationic liposomes were widely used as transfection reagents. However, the high content of cationic DOTAP always causes cytotoxicity, restricting its clinical application. Here, we used DOTAP and MPEG-PLA, both of which are already used in some approved therapeutic agents,33-34,35-36 to composite a hybrid nanoparticles (DPP). In this nanoparticle, the content of DOTAP is as low as 10%, which promises a low cytotoxicity. Meanwhile, this DPP nanoparticle can efficiently deliver genes into cancer cells such as SKOV3 ovarian cancer cells and B16 melanoma cells. Moreover, a low dosage paclitaxel was encapsulated into the DPP nanoparticle, forming a paclitaxel-enhanced DPP nanoparticle (P-DPP) which has much higher transfection efficiency than DPP nanoparticles in many kinds of cancer cells including SKOV3, A549, MDA-MB-231, MCF-7, CT-26 and B16. Endosomal/lysosomal escape and subsequent efficient nuclear entry is pivotal to improve gene transfection efficiency. As the nuclear envelope disassembles and reassembles during mitosis, some microtubule-disrupting drugs such as paclitaxel, nocodazole could increase nuclear gene entry efficiency and further improve gene transfection efficiency through inducing cell synchronization at G2-M phase. Wang et al found that paclitaxel could enhance intracellular gene release from its carrier and/or endosome/lysosomes, thus improving the nuclear entry efficiency.15 Qiu et al found that low dose of paclitaxel could improve gene transfection efficiency while the high dose couldn’t for the induction of massive cell death.16 The underlying mechanism of low dose paclitaxel improving gene delivery efficiency might be that paclitaxel could promote endosome/lysosome escape and increase nuclear entry efficiency of DNA at a non-cytotoxicity dossage.15,

17

Our

results suggested that the low dosage paclitaxel-enhanced DPP nanoparticle is a novel



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gene delivery system with high transfection efficiency and low cytotoxicity, which could also inspire the design of future paclitaxel-enhanced nanoscaled gene delivery system. PEI is one of the most effective non-viral gene carrier for facilitating endosomal/lysosomal escape by “proton sponge effect”. However, the application of PEI is restricted for its nonbiodegradable nature and relatively high cytotoxicity. In this study, we developed a novel DPP nanoparticle as gene vector. Our results indicated that DPP nanoparticles exerted higher gene transfection efficiency in most cancer cells but much lower cytotoxicity in SKOV3 cells compared with PEI25K. The combination of gene and chemotherapeutic drugs presents a promising therapeutic strategy for effective cancer treatment.12,

15, 17, 28, 37-38

To exert their

maximal intracellular synergistic effect, gene and chemotherapeutic drug are expected to be simultaneously delivered to one tumor site.12, 39 Here, low dosage paclitaxel was encapsulated into DPP nanoparticle as a whole to deliver VSVMP for ovarian cancer treatment, which ensures all the cargos reach one tumor site. The encapsulation of low dosage paclitaxel in DPP nanoparticles did not exert obvious systemic toxicity or tumor growth inhibition while could significantly increase the expression of VSVMP, enhancing VSVMP to induce anti-proliferation and apoptosis in SKOV3 ovarian cancer cells, showing synergistic antitumor effects. Abdominal cavity is the most common metastatic site of ovarian cancer and intraperitoneal infusion is also a commonly used method for ovarian cancer treatment.40 Intraperitoneal administration displays advantage in pharmacokinetic for making the therapeutic agents reaching the cancerous cells directly and protecting normal tissues outside the peritoneal cavity free from unwanted harmness.41-42 Intraperitoneal administration of VSVMP which is delivered by the low dosage paclitaxel encapsulated DPP nanoparticles could efficiently inhibit the growth of the ovarian cancer intraperitoneal metastatic tumor, showing potential application in ovarian cancer therapy. Our results indicated that combing low dosage paclitaxel which is encapsulated into DPP nanoparticles with therapeutic VSVMP gene could be a new strategy for cancer therapy for its advantage of synergistic antitumor effect and non-significant cytotoxicity. This strategy also


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inspires the design of future combined therapy of chemotherapeutic drug and gene. CONCLUSION In this study, we demonstrate a paclitaxel-enhanced gene delivery system by encapsulation of low-dosage paclitaxel into a DPP nanoparticle which is consisted of DOTAP and PLA-PEG. The transfection efficiency of DPP nanoparticles is improved by the encapsulation of low-dosage paclitaxel. Meanwhile, low-dosage paclitaxel has synergistic anticancer effect with VSVMP gene. Delivery by this paclitaxel-enhanced gene delivery system, VSVMP could efficiently inhibit the growth of the intraperitoneal metastatic ovarian cancer through apoptosis induction, showing potential application in ovarian cancer therapy. AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

ORCID iD Maling Gou: 0000-0003-0431-0340 Jianlin Long: 0000-0002-2718-0860 Yuping Yang: 0000-0002-3836-5238 Tianyi Kang: 0000-0002-8444-9889 Wei Zhao: 0000-0001-9176-4871 Hao Cheng: 0000-0003-4731-7665 Yujiao Wu: 0000-0002-6787-8139 Ting Du: 0000-0001-6124-8438 Beibei Liu: 0000-0001-7522-4170 Yang Li: 0000-0002-6924-1297 Author list Jianlin Long

[email protected]



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Yuping Yang

[email protected]

Tianyi Kang

[email protected]

Wei Zhao

[email protected]

Hao Cheng

[email protected]

Yujiao Wu

[email protected]

Ting Du

[email protected]

Beibei Liu

[email protected]

Yang Li

[email protected]

Author Contributions #

These authors equally contributed to this work.

Conceived and designed the experiments: Maling Gou; Analyzed the data: Jianlin Long, Wei Zhao, Yuping Yang, Tianyi Kang; Performed the experiments: Jianlin Long, Wei Zhao, Yuping Yang, Tianyi Kang, Yujiao Wu, Hao Cheng, Ting Du, Beibei Liu, Yang Li, Feng Luo; Wrote the paper: Jianlin Long, Yuping Yang, Tianyi Kang, Maling Gou. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This study was financed by the Foundation for Distinguished Young Scientists of Sichuan Province(2016JQ0020), Sichuan province science and technology support plan (2015FZ0040, 2015SZ0049, 2016SZ0006) and the National Natural Science Foundation (81422025, 81572990). REFERENCES (1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet‐Tieulent, J.; Jemal, A. Global Cancer Statistics, 2012. Ca-Cancer J. Clin. 2015, 65 (2), 87-108. (2) Banerjee, S.; Kaye, S. B. New Strategies in the Treatment of Ovarian Cancer: Current Clinical Perspectives and Future Potential. Clin. Cancer Res. 2013, 19 (5), 961-968. (3) Guarneri, V.; Piacentini, F.; Barbieri, E.; Conte, P. F. Achievements and Unmet Needs in the Management of Advanced Ovarian Cancer. Gynecol. Oncol. 2010, 117 (2), 152-158.


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FIGURE LEGENDS Figure 1: (A) The chemical formula of DOTAP, MPEG-PLA and paclitaxel and plasmid map of pVSVMP. (B) Schematic the prepared procedure of P-DPP/DNA nanocomplex. Figure 2: Characterization of P-DPP/DNA nanocomplex. (A) Morphologic feature determined by transmission electron microscopic image. Scale bar, 200nm. (B) Size



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distribution spectrum and (C) zeta potential spectrum determined by Zetasizer Nano ZS. (D) The DNA binding ability determined by gel retardation assay. (E) Hemolytic test. 1) Distilled water (positive control); 2) Normal saline (negative control); 3) 1 mg/mL; 4) 0.8 mg/mL; 5) 0.6 mg/mL; 6) 0.4 mg/mL; 7) 0.2 mg/mL. (F) Erythrocytes aggregation assay. (G) Encapsulation efficiency and (H) Release profile of paclitaxel determined by HPLC. Figure 3: Transfection efficiency and cytotoxicity of P-DPP nanoparticles and Cellular uptake image. (A) Fluorescent image of multiple cancer cell lines transfected by P-DPP nanoparticles, DPP nanoparticles, PEI25K. green fluorescence image (right panels), Brilliant image (left panels) observed under fluorescent microscopy. Scale bar, 50 µm. (B) and (C) Transfection efficiency of P-DPP nanoparticles, DPP nanoparticles and PEI25K were determined by flow cytometry analysis. (D) Relative luciferase activity of P-DPP nanoparticles, DPP nanoparticles and PEI25K at different concentration of paclitaxel were measured by dual luciferase assay. (E) Cell viability of different transfection concentration of paclitaxel was determined by MTT. (F) Comparision of cell viability of DPP and PEI25K. (G) Cellular uptake image analyzed by confocal laser scanning microscope (CLSM). SKOV3 cells treated with DPP and P-DPP nanoparticles for 0.5 h, 2 h, 4 h and 6 h, respectively. pDNA was labeled with YOYO-1, the endosomes/lysosomes were stained with LysoTracker Red, while the nuclei were stained with DAPI. Scale bar, 10um. Figure 4: Antitumor activity of P-DPP/pVSVMP nanocomplex in SKOV3 cells in vitro. Expression of VSVMP was examined by real time PCR (A) and Western blot assay (B). (C) Cell viability were measured by MTT assay. (D) and (E) Apoptotic ratio induced by different formations was determined by flow cytometric analysis. All data were representative of three independent experiments. *p

Ovarian cancer therapy by VSVMP gene mediated by a paclitaxel

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