Characterization And Transfection Efficiency Of PEG-PEI/Fe₃O₄ Nanomagnetic Fluid As Gene Delivery Systems

Objective:To construct an ideal gene delivery system with high efficacy of gene transfer,targeting ability,effect of protection to DNA from DNase degration and good biocompatibility,especially high stability and the ability to prolong gene transfer.Methods:In this research,based on ours initial work,for the first time innovative choice PEI(polyethylenimines)and PEG(polyethylene glycol)prepares a PEG-PEI/Fe3O4 nanometer magnetic fluid through the co-precipitation law to take the gene transportation carrier,with concentration gradient design method arrangement experiment,optimal preparation technological conditions.We unify through the static electricity adsorption a PEG-PEI/Fe3O4 nanometer magnetic fluid and belt positive charge's DNA are forming together a PEG-PEI/Fe304 nanometer magnetic fluid,examines in a PEG-PEI/Fe3O4 nanometer magnetic fluid and the DNA compound through the spectrophotometer law the DNA density,thus calculates DNA the union efficiency.Hinders the experiment and the DNasd digestion experiment through the gelatin examines this gene carrier transportation system to DNA protection ability.Uses the MTT law research and the normal liver cell(L-02) accepts the grain of rice to person liver cancer cell(HepG2)(green fluorescence protein material particle)influence.The use carries pEGFP-C1(enhanced green fluorescent protein green fluorescence protein)to appraise a PEG-PEI/Fe3O4 nanometer magnetic fluid to dye the Hela cell in vitro extension as the report gene extension to dye the efficiency,commercialization lipin body(Lipofectamine2000,Invltrogen) as masculine comparison but bare DNA takes the negative comparison.Finally:Through the orthogonal experiment,we obtained have prepared a PEG-PEI/Fe3O4 nanometer magnetic fluid the most superior condition,its superficial electric potential for positive,obtained the PEG-PEI/Fe3O4 nanometer magnetic fluid particle size for 79.99±7.3nm, the superficial electric potential for 34.13±2.03m V,with DNA the union rate was 94.13±1.8.%;When H/79.528Am-1=6000 achieves when the magnetic saturation Ms =44.65A.m2.kg-1.A PEG-PEI/Fe3O4 nanometer magnetic fluid obtains union belt negative electricity DNA as a result of the superficial belt positive charge the ability.At the same time,the experiment also confirmed that a PEG-PEI/Fe3O4 nanometer magnetic fluid and the DNA compound can protect DNA which carries to be exempt from the nuclease the degeneration.This research discovery,a PEG-PEI/Fe3O4 nanometer magnetic fluid to the normal liver cell(7702) in certain dose range the non-cytotoxicity,displays certain cytotoxicity function under the high potency to the HepG2 cell.In vitro gene transportation,this nano particles may transport pEGFP-C1(enhanced green fluorescent protein green fluorescence protein)the reporter gene to express the material particle effectively to enter the Hela cell,under the magnetic field function,its transportation efficiency reaches 43%,strong the lipin body's extension dyes the efficiency under the same condition.Conclusion:Uses the co-precipitation law,the optimization technological conditions,the success preparation particle size is small,a distributed even PEG-PEI/Fe3O4 nanometer magnetic fluid.Causes this nano particles surface belt positive charge,thus union material particle DNA,assembles the PEG-PEI/Fe3O4 nanometer magnetic fluid gene transportation system.The gene extension DNA through in vivo outside experiments and so on dyes and protects,confirmed that this transportation system may protect carries DNA to be exempt from the nuclease the degeneration,and is one kind of low poisonous highly effective nanometer gene carrier,our research accepted the grain of rice for the application to realize the tumor gene therapy to lay the good scientific foundation.