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  • br A br Particle size

    2022-05-04


    A
    Particle size (nm) 
    Zeta potential (mV) 
    efficiency
    Encapsulation 60
    Fig. 3. Encapsulation efficiency of resveratrol liposomes.
    DNA-binding ability of liposomes enhanced with the increase of N/P weight ratios. CDOR5, CDOR10, CDOR20, CDOR100 and CDOR200 retarded plasmid DNA completely at the N/P weight ratios of 3/1, 1/1, 3/1, 4/1 and 2/1, respectively. The low N/P weight ratios of resveratrol liposomes for retardation may reduce its dose required to obtain great transfection efficiency, and improve the safety of the vector itself to normal tissues and organs. So they were promising for being ideal carriers to deliver gene therapy agents into cells, the liposomes/DNA lipoplexes ensured the entry of pDNA into Concanamycin A and avoid degradation of DNA by DNase.
    3.4. In vitro pDNA transfection
    The transfection efficiency of CDOB/pGFP-N1 lipoplexes and CDOR/pGFP-N1 lipoplexes was investigated through the detection of GFP expression (Fig. 4 and Fig. 5). The transfection efficiency increased with the increase of the N/P ratios, as the net positive charge on the lipoplexes also increased, leading to lipoplexes internalization more easily by fusion with the negatively charged cell membrane [34]. The highest transfection efficiency of all the lipoplexes was achieved at the  Journal of Drug Delivery Science and Technology 51 (2019) 746–753
    N/P ratio of 3/1 in Hela cells and MCF-7 cells, whereas higher N/P ratios led to lower trasnfection efficiency due to strong DNA-binding capacity of liposomes. It indicated that optimal positive charge and appropriate DNA-binding capacity helped the lipoplexes enter into cells, and then released DNA. The expression of GFP mediated by CDOR exhibited comparable trasnfection efficiency with CDOB and commer-cial reagents Lipofectamine 2000 and DOTAP. The results demonstrated that the addition of resveratrol had no effect on transfection efficiency of the blank liposome. Otherwise, the lipoplexes exhibited greater transfection efficiency against Hela cells than against MCF-7 cells as they may have selectivity for certain cells in terms of transfection.
    Cationic liposomes are mostly utilized as gene carriers for plasmid DNA (pDNA) [35,36], antisense oligonucleotides [37] or small inter-fering RNA (siRNA) [38,39]. Through the above experiments, the li-posomes with the tri-peptide headgroups could combine with the plasmid DNA, and then the lipoplexes destabilized the endosomal membrane, resulting in a flip-flop reorganization of phospholipids. These phospholipids then diffused into the lipoplex and interacted with the cationic lipids causing the DNA to dissociate into the cytoplasm for the subsequent transcription [40,41]. In brief, resveratrol liposomes showed great ability for delivering genes and exhibited good perspec-tive in the field of anti-cancer drug delivery.
    3.5. In vitro cell proliferation inhibition
    MTT assay is widely used to assess the growth inhibitory effects of drugs on primary patient cells or cell lines, so the cytotoxicity of re-sveratrol and p53 gene against MCF-7 and Hela cells were determined by this assay. Fig. 6 showed that the cells treated with the blank lipo-some showed comparable cell viability (> 90%) with Lipofectamine 2000 and DOTAP, and the cells treated with resveratrol liposomes showed slightly lower cell viability compared with that of blank lipo-some. The cells treated with CDOR20/p53 lipoplexes exhibited stronger tumor growth inhibition effect than that of resveratrol with the relative cell viability 56.5%–70.5% (Fig. 6B). It illustrated that resveratrol and p53 gene showed synergistic effect on cells growth inhibition. The anti-tumor effect of resveratrol liposmes and lipoplexes against MCF-7 cells were better than against Hela cells. Studies have shown that Hela cells expressed lower level of wide-type p53 proteins compared with MCF-7 cells [42,43], thus resveratrol liposomes exhibited better cell pro-liferation inhibition against MCF-7 cells due to additionally synergistic effect of reveratrol and wide-type p53 proteins. Otherwise, higher cy-totoxicity of resveratrol liposomes against MCF-7 cells induce more cell
    apoptosis, so MCF-7 cells exhibited inferior transfection compared with Hela cells (Fig. 5).
    3.6. In vitro cell apoptosis studies
    Hoechst 33342 is a blue fluorescent dye, which can penetrate the cell membrane and staining the cell nucleus, it has low cytotoxicity. Hoechst 33342 assay is often used for cell apoptosis detection, and the apoptosis can be detected by the fluorescence of apoptotic cells using
    fluorescence microscope and flow cytometry. The apoptosis of lipo-plexes against Hela and MCF-7 cells was presented in Fig. 7 and Fig. 8. The cells treated with resveratrol liposomes displayed greater blue fluorescence than those treated with the blank liposome, therefore, resveratrol could enhance apoptosis against Hela and MCF-7 cells. Si-milarly, the cells treated with Lipofectamine 2000/p53 and DOTAP/ p53 showed stronger fluorescence than those treated with Lipofecta-mine 2000/pGFP-N1 and DOTAP/pGFP-N1. It illustrated that p53 gene could induce cell apoptosis. The cells containing CDOR20/p53