Effects Of VEGFR-3Sirna Delivered With PEI-alginate Nanoparticles On Inhibiting Lymphangiogenesis Of Endothelial Progenitor Cells

[Objective] To study the interfering effect of VEGFR-3siRNA delivered with PEI-alginate nanoparticles on lymphangiogenesis of endothelial progenitor cells, and explore the feasibility of acting lymphatic endothelial progenitor cell as a target to inhibit tumor lymphangiogenesis and lymphatic metastases.[Methods] The mononuclear cells of umbilical cord blood were isolated using density centrifugation with PercollTM. Then the VEGFR-3+cells were sorted from the mononuclear cells with a flow cytometer. Expression of CD34and CD133on VEGFR-3+cells were viewed using a confocal laser scanning microscope. The cells were induced with VEGF-C to proliferate. Prepare nanoparticle vector by PEI and alginate using aqueous method. The shape, size and surface charge of the nanoparticle was detected using a scanning electron microscope and dynamic light scatter. Chemical synthesis of three different VEGFR-3siRNAs in accordance with the sequence of target gene, the siRNA solution was droped into the PEI-alginate solution according to the different weigh ratios between PEI and alginate. Loading efficiency of PEI-alginate nanoparticles on the siRNA was detected by gel retardation analysis and chose the most effective siRNA. The fluorescence microscope assay detected the thansfection efficiency after PEI-alginate/siRNA nanocomplexes were transfected into the cells. MTT assay detected the toxicity of nanoparticles vector to cells. Distribution and degradation of nanocomplexes after transfecting for2h,4h,6h repectively was detected by a transmission electron microscope. Cell viability, number of proliferated cells and transmigrated cells, and length and area of capillary lymphatic tube-like structure was detected by MTT method, proliferating cell nuclear antigen immune cells staining, transmigration assay and capillary lymphatic tube-like structure formation assay in Matrigel respectively.[Results] Cytokine CD34and CD133express in the membrane of the VEGFR-3+cells. The amount of VEGFR-3+CD34+and VEGFR-3+CD133+cells was0.7%and was0.49%in the mononuclear cells respectively. The separated mononuclear cells appeared circle or elliptic, scattered in the dish. After3days, most of the cells began to stretch out, cells appeared fusiform or polygon. The VEGF-C induced cells distributed in a cluster, amount of cells increased and the size increased. Size and sueface charge of PEI-alginate nanoparticle carrier came up to basic condition of nanoparticle vector. The fluorescence microscope showed transfection efficiency of nanocomplexes was about80%. With the amount of PEI in nanoparticles carrier increased, the amount of carried siRNA increases in nanopartiels. When the ratio of nitrogen and phosphorus is above8, siRNA was almost completely sealed package in the nanoparticles. Nanoparticles with increased ratios of nitrogen and phosphorus were thansfered to LEPCs, cell activity gradually reduced. When the ratio of nitrogen and phosphorus is over16, cells viability reduced obviously. Comprehensive considerating, the best proportion is16and conduct the following experiments. The nanoparticles were thansfered to LEPCs, transmission electron microscope showed that the many nanocomplexes was distributed in the surface of the cells and few just got into cytoplasm after2h; after4h, many nanocomplexes had entered into in the cytoplasm, nanoparticles began to degrad; after6h, many nanoparticles in the cytoplasm had degradated to smaller particles. Different nanoparticles were transfectd to the cells, RT-PCR assay found the first siRNA showed the highest inhibition efficiency, so chose the first fragments for the following experiments. MTT assay showed cell viability was higher than the unmodified PEI. Transmigration assay results showed that number of transmigrated cells after transfection for4h decreased significantly. Proliferation cell nuclear antigen dying results found cell fluorescence brightness decreased after transfection with nanocomplexes. Tube formation assay showed that number and area of capillary lymphatic tube-like structure was less than that in VEGF-C group.[Conclusion] PEI-alginate nanoparticles delivered VEGFR-3siRNA into LEPCs, cell viability and capability of proliferation of LEPCs descreased, number of transmigrated cells and length and area of capillary lymphatic tube-like structure reduced. The study will provide a powerful experiment basis for inhibiting tumor lymphangiogenesis and lymphatic metastasis by interfering LEPCs with VEGFR-3siRNA.