Preparation And Evaluation Of Hyaluronic Acid Modified / Co - Loaded Hypericin And Hypoxia - Inducible Factor 1α - SiRNA Double - Targeting Nanoparticles

In this article, we synthesized Hyaluronic acid(HA) grafted PLGA-PEI nanoparticle that loaded Hypericin,small molecule drug,and gene. Eventually it formed a echelon-target nanoscale drug, we got the research of the nanoparticle’s physical and chemical properties at the same time of its tumor targeting in vivo and in vitro so that it can solve the problem of radio-resistance.In the first section,we used western blot method to detect the cell model which lack of oxygen. Western blot result showed that150μmol/L cobalt oxide could inhibit HIF-1alpha’s expression in nasopharyngeal carcinoma CNE2cells. After that,we studied whether the technology of RNA interference could be away of anti-cancergene therapy, siRNA containing the sequence of human HIF-1α mRNA coding region was obtained. We also detected the effect of inhibition by RT-PCR and Western blot after transfecting human gastric cancer cell line SGC-7901with liposomes. The results indicated that the expression of human HIF-1α gene was suppressed specific and effectively by S1RNA3724and siRNA3168compared to others.For the second part, at first we prepared PLGA-PEI nanoparticles that loaded Hypericin by emulsion solvent evaporation method and then took different proportions of hyaluronic acid on their surface modification in order to make it form spontaneously micelles structure in the water. Finally, we prepared and evaluated the stable hyaluronic acid coating nanoparticles that have hydrophobic cationic modification. After that, We used transmission electron microscopy(SEM) and atomic force microscope(AFM) to investigate the appearance of the nanoparticles. Those results showed that nanoparticle is conform to the relevant requirements, around200nm size, zeta potential is plus or minus20mv.Thirdly, The results showed that when the N/COOH is greater than or equal to4, nanoparticles size and potential could have some changes.These may due to the HA is a negative role. Moreover, determined by MTT experiment result showed the HA-Hypericin-PLGA-PEI nanoparticles than PLGA-Hypericin-PEI has low cytotoxicity.we can believe that hyaluronic acid affected the physical and chemical properties of nanoparticles as well as reduce the cytotoxicity of cationic liposomes.Fourthly, agarose gel electrophoresis method proved either through the HA modified or without HA modified sample can be combined strongly with the anionic siRNA. Heparin solution from the experiment and nuclease resistance experiment proved that the preparation of nanoparticles by carrier has better ability to protect RNA, makes the degradation of RNA from RNAase, stable to siRNA in the body,which acts on the target genes, achieve the goal of gene therapy. Finally, we examined the four kinds of material in the transfection efficiency of SGC7901cells by cell transfection technique.The results of the HA coating of PLGA-PEI nanoparticle liposome transfection rate is better than others. This is mainly due to the high expression of CD44on cancer cells’surface that increased the uptake rate of cells.In the last section,we studied the siRNA intracellular transfection effect by laser confocal microscopy. We took the PLGA-PEI NPs/Cy3-siRNA compounds into SGC7901after6h. The results showed the compounds can effectively deliver siRNA to cells. Its mechanism remains to be further research. Mainly research of the cellular uptake of Hypericin-PLGA-PEI in terms of qualitative and quantitative showed that the more Hypericin the more cellular uptake. In addition, the HA coating/PLGA-Hypericin-PEI have the best cellular uptake capacity.We consider that HA have the affinity of CD44that increase the combi-capability of the nanoparticles and the carrier. Flow cytometry instrument results consistent with the results. Secondly, receptor inhibition experiment proveded that HA can effectively improve the PLGA-PEI carrier targeted to cells. The targeted in vivo experiments proveded that the mice survival rate is reduced with the hypericin nanoparticles increased. In addition HE staining results showed that the cell edema and necrosis area have more obvious Fluorescence intensity. All these lay the foundation for the future experiments.