Construction And Evaluation Of Gene Delivery System Based On Tannic Acid Modified PEI

As a new type of disease treatment,gene therapy has broad development prospects.However,the lack of safe and efficient gene delivery vectors remains one of the major challenges facing the development of gene therapy.Viral vectors have high gene delivery efficiency,but the immunogenicity and preparation difficulties have limited their development.Therefore,non-viral gene vectors are considered to be the hope of promoting the development of gene therapy.Polyethyleneimine（PEI）is an outstanding representative of non-viral vectors due to its strong gene carrying capacity and excellent"proton sponge"effect.However,its excessive positive charge causes drastic cytotoxicity,which limits its application.To solve this problem,the existing strategies are mainly to reduce the surface charge,including two strategies of this study:（1）grafting strategy by the neutral molecular to reduce toxicity;（2）screening strategy to reduce toxicity.Tannic acid（TA）has dendritic structure owing to its many highly reactive phenolic hydroxyl group that can be modified facilely.In addition,TA has good biological adhesion and is cross-linked with various metal ions through complexation.Based on the above two ideas,this study attempts to construct and preliminary evaluate the gene delivery system based on TA and PEI,the specific contents are as follows:1.By using the grafting strategy to reduce the positive charge of PEI,scilicet three different molecular weight PEIs were grafted onto TA（PTA）by using chloroacetic acid as a bridging molecule.The characterization of PTAs proved the successful synthesis of PTAs.In vitro studies have shown that PTA2k and PTA30k could completely condense DNA at N/P of 2,and PTA600 could only completely condense DNA at N/P of 50.The PTAs polymers could protect DNA from degrading by nucleases and have high DNA encapsulation efficiency.Biocompatibility assay showed that PTAs had no significant cytotoxicity compared with PEIs,and PTA30k promotes cell proliferation significantly.PTAs could be efficiently uptaked by cells,and its transfection efficiency is higher than PEIs.Compared with PTA600 and PTA2k,PTA30k has the highest transfection efficiency.At N/P of 3,PTA30k showed the highest transfection efficiency of 24.13%,which is much higher than PEI30k.These results indicate that PTAs,especially PTA30k,is expected to develop into safe and effective non-viral gene delivery.2.By using the shielding strategy to reduce the positive charge of PEI.Firstly,TA/PEI/DNA complex was formed by the hydrogen bond interaction of TA hydroxyl group and PEI amino.And then,Mⁿ⁺（TA/PEI/DNA）nanoparticles were prepared by the complexation of TA with metal ions(Mⁿ⁺),which further compresses the complexes volume.Compared with PEI30k/DNA,when PEI70k/DNA was chaewn as the shielding object,the preparation of TA/PEI70k/DNA complex and Mⁿ⁺（TA/PEI70k/DNA）nanoparticles has a higher transfection efficiency,and the weight ratio of 0.0125/1/60/80,K⁺（TA/PEI70k/DNA）has the highest transfection efficiency of 67%.Furthermore,compared with PEI70k/DNA complex and TA/PEI70k/DNA complex,K⁺（TA/PEI70k/DNA）nanoparticles showed smaller particle size,stronger stability and lower cytotoxicity,and its ability to bind and release DNA was not affected.In addition,Mⁿ⁺（TA/PEI/DNA）can be effectively absorbed by cells,especially K⁺（TA/PEI70k/DNA）has higher cellular uptake efficiency.In summary,K⁺（TA/PEI70k/DNA）nanoparticles based on TA shielding and K⁺complexation is expected to be an ideal platform for gene delivery.