Study On Gene Transfection Mechanism Of Nano Hydroxyapatite

Objective:1. Synthesis nanoparticles of hydroxyapatite (HA) in simulated body fluid were characterized and transfection efficiency of study.2. Study on dispersion stability and long-term preservation method of nanoHA/pDNA complexes.3. Synthesis and optimization of a nanoHA/pDNA and fibrin based gene delivery system for in vitro use.4. Study on the endocytic pathways involved in the internalization and intracellular trafficking of the nanoHA/DNA complexes.Methods:1.The appropriate amount(Ca/P ratio=1.67) of K2HPO4·3H2O solution was added to CaCl2solution in simulated body fluid. The reaction condition is set to pH7.4,37℃,24hours. Powder was obtained by precipitation dried and then grinded. The nanoparticles of HA were characterized by used to XRD, FTIR, TEM, etc. The minimum of dose of HA was observed by it combined with DNA.And examined capacity of protect DNA from DNase Ⅰ.To contrast hydrothermal synthesis of HA nanoparticles in transfection efficiency of HeLa.2. The nanoparticles were prepared and stored either as an aqueous dispersion with or without sucrose or in the freeze-dried state with or without sucrose, at two different temperatures (4℃,25℃) for either2weeks or3weeks. Using a standard luciferase assay system to analyzed for luciferase activity. The particles sizes of the various treatment groups were assessed using Dynamic Light Scattering.3. The fibrin gels, synthesized using the various fibrinogen/thrombin ratios were analyzed using scanning electron microscopy (SEM). The gene transfection abilities of these gels were also analyzed(at1st,3rd,5th,7th day,respectively) by alkaline phosphatase activity assay.4. The cells were pre-treated with various concentrations of either phenylarsine oxide (PAO) or filipin. These cells were then transfected with the NanoHA/pDNA complexes. Using a standard luciferase assay system to analyzed luciferase expression of these cells.Also, the cytotoxic effects of PAO and filipin on HeLa were determined via a MTT cell viability colorimetric assay. And then, flow cytometry was used to assess the levels of NanoHA/pDNA internalization in HeLa cells. Finally, using the confocal microscopy to visualize the internalization and intracellular trafficking of the nanoHA/DNA complexes.Results:1. Biomimetic synthesis of HA nanoparticles were a calcium-deficiency hydroxyapatite. Ca/P ratio is1.658, the crystallinity slightly worse, the average particle size of40.58nm. The positively charged surfaces and the Zeta potential is about1.1±0.6mv.DNA binding capacity was maximum when DNA and HA particles combined within the mass ratio range of1:30were used. And the particles can protection DNA was not DNase I degradation. The hydrothermal synthesis of HA nanoparticles lower transfection effective as compared to HA particles synthesized within simulated body fluid in Hela.2. It was no significant difference between transfection effective of nanoparticles lyophilized with size and content10%sucrose and control at2or3weeks. By contrast, All other groups both an increase in particle size (≥2um) as well as significantly lower levels of gene expression relative to freshly prepared nano-particles.3. Scanning electron microscopy was used to assess the effect of varying the fibrinogen/thrombin ratio on the microstructure of the gels. Major amount of particle aggregation was observed in the using the lowest fibrinogen/thrombin ratio (fib/tht=0.02). At the same time,there was the large fiber strand thickness observed in gels synthesized using the fibrinogen/thrombin ratio increased from0.02to10.00. In all these gels, gene expression peaked between days3and5.The levels of gene expression of gel which have fibrinogen/thrombin ratio of0.02were Significantly lower than other groups.4. The cellular uptake data showed that the internalization of the NanoHA/pDNA complexes was strongly inhibited by treatment with filipin and PAO. The Cell viability of the HeLa cells was approximately85%to100%for all of the tested concentrations of PAO and filipin. The approximately93%of the HeLa cells were positive for the internalization of the NanoHA/pDNA complexes. Using the confocal microscopy to visualized nanoHA/pDNA complexe was mediated by both clathrin-and caveolae-dependent endocytosis as observed in HeLa cells.Conclusions:1. Synthesis nanoparticles of HA in simulated body fluid have good capacity of DNA-binding and DNA protection against nuclease digestion.This HA particles could be improved effective of transfection in Hela. HA nanoparticle is a new non-viral gene delivery vectors for transfection of cells.2. The NanoHA/pDNA complexes could be preserved both particle size and transfection efficiency for up to3weeks in storage at40℃by freeze-drying the nano-particles with sucrose.3. The gene transfection ability of the NanoHA/pDNA/fibrin gene delivery system could be controlled by varying the fibrinogen/thrombin ratio.4. HA-based gene delivery systems is mediated by both clathrin dependent and caveolae-dependent endocytosis in cell lines, with the latter pathway impacting cellular uptake more strongly.There are23figures,8tables,89references.