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  • br We have searched the literature published and found


    We have searched the literature published and found many findings have illustrated that resveratrol-induced apoptosis was closely con-nected with p53-dependent pathway. However, to our knowledge, there is no paper reporting co-delivery of resveratrol and p53 for cancer therapy. So we chose the peptide cationic lipid prepared by our group as delivery vector for co-delivering resveratrol and p53. The chemical structure of the cationic lipid is shown in Fig. 1, it has double 14 carbon chains as tails, and a tripeptide as headgroup [27]. We hope to take full advantage of the synergistic effect of resveratrol and p53 via peptide cationic lipid to induce more apoptosis.
    2. Materials and methods
    The plasmid pcDNA3.1(+)-pEGFP-p53 encoding wide-type p53 and GFP Nutlin-3 and Lipofectimine 2000 (Lipo2000) were purchased from Invitrogen (USA). The plasmid pGFP-N1 was provided by Clontech (USA). Peptide-based cationic lipid CDO14 was prepared in our la-boratory. Human cervical carcinoma (Hela) cell line and human breast cancer (MCF-7) cell line were obtained from Cell Bank of Chinese Academy of Sciences. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were provided by Roche (Switzerland). MTT cell proliferation and cy-totoxicity assay kit was purchased from Sigma-Aldrich (Germany). Hoechst 33342 was purchased from Beyotime Institute of Biotechnology (China). Dulbecco's modified Eagle's medium (DMEM) was provided by Thermo (USA). Fetal bovine serum (FBS) was provided by Gibco (USA). All other chemicals were of the highest purity available and used as received.
    2.2. Preparation of resveratrol liposomes
    Resveratrol liposomes were prepared by thin-film and ultrasonic dispersing method [28], the cationic lipid CDO14 was formulated in combination with resveratrol at the CDO14/resveratrol weight ratios of
    5/1, 10/1, 20/1, 100/1, and 200/1, respectively, the molar ratio of CDO14 to the neutral lipid DOPE was 1/1. To prepare resveratrol li-posomes, 1 mg CDO14 was dissolved in 1 ml chloroform, then suitable amounts of resveratrol methanol (1 mg/ml) and DOPE methanol (10 mg/ml) were added to CDO14 solution dropwise. The resulting mixture was evaporated under a stream of nitrogen and the residual solvent was removed under vacuum overnight. The dried lipid film was resuspended in 1 ml distilled water to give liposomes in a concentration of approximately 1 mg/ml, followed by several times of sonication and vortex mixing until the solution was clear. Different weight ratios li-posomes were formulated (CDOR5, CDOR10, CDOR20, CDOR100, CDOR200). The blank liposome (CDOB) with no drug encapsulated was also prepared according to the above method.
    2.3. Particle size and zeta potential measurements
    For the measurement of particle size and zeta potential, 20 μl of resveratrol liposomes (1 mg/ml) were diluted to 1 ml with distilled water, then particle size and zeta potential were detected three times by a Nano-Partikelanalysator (HORIBA, SZ-100, Japan) at room tempera-ture.
    2.4. Encapsulation efficiency
    Encapsulation efficiency of resveratrol in the liposomes was mea-sured by indirect method through measuring the concentration of the free resveratrol in the aqueous suspension medium. 1 ml of resveratrol liposomes were centrifuged at 10,000 rpm for 15 min at 25 °C, and the supernatant was collected and subjected to high-performance liquid chromatography (HPLC) system (SHIMADZU, LC-20A, Japan) on a Kromasil® C18 column (4.6 mm × 250 mm) at room temperature. Elution was performed at a flow rate of 1 ml/min using a 80:20 mixture of methanol and ultrapure water, data were recorded at 306 nm. Encapsulation efficiencies (EE%) of CDOR5∼CDOR200 were calcu-lated by the following equation [29]:
    EE% = Initial amount of resveratrol in liposomes −Amount of free resveratrol × 100 Initial amount of resveratrol in liposomes
    To characterize the electrostatic binding interactions between the plasmid DNA and resveratrol liposomes, resveratrol liposomes were mixed with DNA at the N/P weight ratios from 0.5/1 to 8/1 and the lipoplexes were incubated for 20 min 20 μl of lipoplexes were mixed with loading buffer (1.6 μl) and then loaded onto 1.2% (w/v) agarose gel containing GelRed and 1 × TAE buffer. Electrophoresis was per-formed at 90 V for 50 min. DNA bands were visualized and photo-graphed by a gel imaging system (Syngene, LG3000, Britain).
    Hela and MCF-7 cells were grown in high-glucose DMEM medium supplemented with 10% (v/v) FBS and antibiotics (penicillin 100 U/ml and streptomycin 100 U/ml) at 37 °C in a humidified atmosphere containing 5% (v/v) CO2.
    2.7. In vitro pDNA transfection
    In vitro pDNA transfection of liposomes/pGFP-N1 lipoplexes was measured against Hela and MCF-7 cells. Hela and MCF-7 cells were seeded in 24-well plates (5 × 104 cells/well) in high-glucose DMEM until the required cell number (80% confluence) was obtained. The medium was removed and then 400 μl of serum-free DMEM was added to each well. To prepare liposomes/DNA (pGFP-N1) lipoplexes, they were diluted in 50 μl serum-free DMEM, respectively, the N/P weight