Construction Of Endoplasmic Reticulum Targeting Gene Deliv Ery System And Research On Transfection Performance

Gene therapy is a unique technology that uses genes to prevent or treat diseases.Gene therapy technology treats diseases by inserting genes into patient cells instead of using drugs or surgery.Gene delivery systems are essential for gene therapy of diseases.At present,T viral vectors and non-viral vectors were the wildly used for gene delivery.Among them,viral vectors have high gene transfection efficiency,but viral vectors were limited for using by their high toxicity and low gene loading capacity.The most widely used non-viral vector is polyethyleneimine(PEI).PEI has good proton buffering capacity and can escape from the endosome through the "proton sponge" effect.Large molecular weight PEI has strong cytotoxicity,and small molecular weight PEI is very toxic,but its transfection efficiency is also poor.Therefore,the development of high-efficiency and low-toxicity vectors is an important research hotspot.In addition,the gene delivery mechanism has very important guiding significance for the development of non-viral gene vectors.The endoplasmic reticulum is a continuous membrane-like structure,which is connected with a variety of organelles(nuclear membrane,Golgi apparatus,etc.),especially directly connected with the nuclear membrane.Therefore,the development of targeted gene vectors based on the endoplasmic reticulum may have very broad application prospects.Inspired by the structure of endoplasmic reticulum fluorescent dyes,this paper combined the endoplasmic reticulum targeting molecule4-[2-(4-sulfamoyl-phenyl)-ethylcarbamoyl]-butyric acid(SPEBA)with small molecular weight PEI in combination,through optimization and screening,the endoplasmic reticulum targeted gene delivery system PEI-ER was obtained.Research results:(1)The characterization of a series of physical and chemical properties proved the successful synthesis of the material,obtained the binding ratio,and verified that it has good DNA compounding ability and proton buffering ability.(2)In vitro experiments show that PEI-ER had good hemolysis and cytotoxicity,and the material prepared with SPEBA and PEI at a molar ratio of 0.5:1 had the best transfection efficiency.After different molecular weights of PEI modificated by SPEBA,transfection experiments proved that the modification of SPEBA can increase the transfection efficiency.(3)The results of cell uptake experiments indicated that most of the PEI-ER/DNA complexes enter cells through caveolin-mediated endocytosis,and then target the endoplasmic reticulum and release DNA.In summary,this study prepared an endoplasmic reticulum targeted gene delivery system(PEI-ER).The PEI-ER/DNA complex entered the cell through caveolin-mediated endocytosis,and then targeted to the endoplasmic reticulum.After that,the DNA was released,and finally realize the transfection process by means of the close contact between the endoplasmic reticulum and the nuclear membrane.The vector has simple structure,low toxicity,high gene transfection efficiency,and has great application prospects.