Investigation On Gene Transfection Mediated By Receptor By Use Of Polyethylenimine Grafted Chitosan With Modification Of Folate Acid

Objective:Chitosan(CS)is a typical cationic linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by β-(1,4)glycosidic bonds.Chitosan can form a complex with DNA through ionic bond and protect DNA from DNase degradation.It is considered to be a good gene delivery vector.However,in the process of gene delivery,chitosan has problems such as low transfection efficiency and low specificity,and poor proton buffering ability.In order to improve its gene transfection efficiency and proton buffering capacity,we modified it with polyethyleneimine(PEI).PEI has high charge density and good DNA binding ability,and its proton sponge effect can promote intracellular escape.Folate(FA)was conjugated on the carrier to enhance the targeted therapeutic effect of the vector.Related studies have showed that one of the most important reasons why cancer cells can escape the recognition of the body’s immune system is the high expression of PD-L1 on its surface.Based on this,we constructed an RNAi expression plasmid shPD-L1 that can target PD-L1.In summary,this paper designed and constructed CS-PEI-FA nanocomposite loaded shPD-L1 for the treatment of breast cancer.Research content and results:(1)Synthesis and characterization: The amino group of chitosan was activated by maleic anhydride(MAH),and the PEI reacted with the activated amino group of CS to synthesize CS-PEI.The FA is connected to the PEI.The carrier structure was determined using a nuclear magnetic resonance and infrared spectrometer for detection and characterization.The particle size and morphology of the carrier were characterized by dynamic light scattering and transmission electron microscopy,respectively.The particle size distribution was uniform with a diameter of 200 nm.The experimental results showed that CS-PEI-FA is successfully prepared.DNA gel retardation experiments indicate that CS-PEI-FA can carry DNA.By characterizing the pH buffering effect of the carrier,the experimental results show that the nanocarrier CS-PEI-FA has good proton buffering capacity.(2)Cell level in vitro: The cell cytotoxicity of CS-PEI-FA and CS-PEI-FA/DNA was detected by MTT assay using MCF-7 as a cell model,and detected by cell cycle detection kit and apoptosis detection kit.The cell cycle results showed that there was no significant difference between each group and NC group in cell number and each cycle(P>0.05).The apoptosis results showed no difference between each group that the cell number of each group and the NC group in each cycle.There was no significant difference(P >0.05).It indicates that the carrier and its composite object have good biocompatibility.Cell cycle and apoptosis showed that the in vitro biocompatibility of the vector before and after DNA loading was good.Subsequently,we conducted a study on the mechanism of cellular uptake.The results showed that CS-PEI-FA and CS-PEI-FA/DNA could have good uptake efficiency when the cells were ingested for 4 h.The addition FA significantly inhibited the cell uptake at low temperature.The uptake of CS-PEI-FA/DNA,the cellular uptake of CS-PEI-FA/DNA is energy-dependent and is taken up into cells by FA-specific receptor-mediated pathways.In order to investigate whether the vector can carry the plasmid into the cell and express it,we performed an in vitro cell transfection experiment,fluorescence was observed by fluorescence microscopy,and the transfection rate was 72.1% by flow cytometry.The CS-PEI-FA-encapsulated gene shPD-L1 was subjected to real-time quantitative PCR and western blot analysis.The PD-L1 mRNA expression of CS-PEI-FA/shPD-L1 was decreased to 17.5%.The PD-L1 protein expression level of CS-PEI-FA/shPD-L1 was reduced to 23.04%.(3)In vivo animal levels: CS-PEI-FA and CS-PEI-FA/shPD-L1 showed good in vivo biocompatibility in body weight and pathological sections of various organs.Relative tumor volume results indicate that CS-PEI-FA/shPD-L1 can significantly inhibit tumor volume growth.The relative tumor proliferation rate of CS-PEI-FA/shPD-L1 was calculated to be 54.76%,and the tumor inhibition rate was 77.96%.The results showed that CS-PEI-FA/shPD-L1 significantly inhibited tumor proliferation.The Q-PCR and Western blot results of PD-L1 of CS-PEI-FA/shPD-L1 indicated that CS-PEI-FA successfully carried the anti-tumor effect of shPD-L1 in mice.Conclusion: CS-PEI-FA nano-carrier was successfully constructed and its nucleic acid delivery ability was investigated in vitro and in vivo.The results showed that CS-PEI-FA nano-carrier had good RNAi ability and could effectively inhibit tumor proliferation.