Gene Delivery Efficiency Of Dextran-peptides Vector

At present,cancer has become one of the most important diseases that threaten human health,and the incidence of cancer is increasing year by year.In recent years,gene therapy has attracted widespread attention as a promising method to treat cancer and genetic diseases.The success of gene therapy depends on a safe and efficient gene delivery system.Research indicates that significant progress has been made in the field of siRNA nanoformulations,but the important issue is to obtain non-toxic and effective vector that can successfully deliver siRNA.Dextran is widely used as the vector in drug delivery and the scaffold for 3D cell culture.The presence of a large number of hydroxyl groups in dextran can effectively connect other functional molecules.At present,the dextran derivative system is still at a research stage.This article mainly studies the transfection efficiency of Dextran-peptides vectors for two-dimensional and three-dimensional growth cells.Firstly,we studied the ability of DEX-Peptides vector to complex DNA and whether the vectors can transfect DNA.DEX-MA/VS-Peptides(the peptides are R3H7 C,R3H8C,R2H7C)were synthesized respectively,and the complexes Dex-Peptides/GFP-March5 were prepared at different N/P(0.5,1,2,3,4,5,10)conditions.The agarose gel electrophoresis experiment was used to determine the N/P of the complexes when it was completely wrapped.The particle size and potential of the complexes at different N/P was detected,which found that with the increase of N/P,the particle size of the complexes became smaller and the surface charge gradually increased,and the vector can transfect the plasmid into cells.Then,the ability of DEX-Peptides vector to complex SiRNA and whether the vectors can transfect SiRNA were studied.The complexes Dex-Peptides/SiRNA NC were prepared at different N/P(0.5,1,2,3,4,5,10)conditions,agarose gel electrophoresis experiment and detection of the particle size,potential of the complexes at different N/P(same as above)proved that the DEX-Peptides vector can complex SiRNA,and can transfect SiRNA into cells.Subsequently,the transfection efficiency and toxicity of the DEX-Peptides vectors were studied.Six kinds of vectors were used to overexpress March5 in Hela cells,real-time quantitative PCR experiments and Western Blot experiments proved that the vectors DEX-MA-R3H7,DEX-MA-R3H8,DEX-VS-R3H7,DEX-VS-R3H8 had higher transfection efficiency;Vectors were used to transfect SiRNApcsk9 into Hela cells,and the downexpression level of pcsk9 gene analysis showed that the vectors DEX-MA-R2H7 and DEX-VS-R2H7 had higher transfection efficiency for SiRNA;MTT experiments showed that the DEX-peptides vectors had lower toxicity to cells and cell survival rate was more than 80%,but PEI is more toxic to cells,cell survival rate was only about 65%.Finally,Hela cells were cultured with DEX-MA-HA hydrogels,we studied whether the vectors could transfect plasmids into cells.The hoechst staining nucleus and mouse model expriments showed that the hydrogel had good performance and can realistically simulate the three-dimensional tumor cells growth environment in vivo,cells were growed in the shape of a sphere.Fluorescence microscopy imaging showed that DEX-VS-R3H7 carrier can transfect plasmids into cells.Our experimental study will promote the progress of polymeric cationic carriers research and will provide an effective gene delivery system for gene therapy.