The Construction And Evaluation Of Novel Chitosan Linked PEI Non-viral Gene Delivery Carrier

Objective: Gene therapy has received significant attention. The success of gene transfection is largely dependent on the development of vectors with high transfection efficiency and low toxicities. Non-viral vectors have been increasingly proposed due to its safety and ease of synthesis. Considering the character of PEI and chitosan, we linked them together and then grafted them with the targeted peptide to investigate their physico-chemical properties as well as the biochemical propertyies.Methods: 1. Plasmid were firstly incubated with chitosan. Subsequently, the complex was formed when the nanoparticles were incubated with PEI. The particle size and appearance were investigated. Using chitosan-PEI as the vector, PGL3 and GFP plasmid were transfected into HeLa cells and the expression level of the transfected genes was detected. MTT assay was also used to evaluate their cytotoxicity.2. Chitosan was linked with polyethylenimine (PEI, Mw = 1.8kDa) and the composition, particle size, as well as the zeta potential of this chitosan-linked-PEI (CP) complex was measured. The cell viability and long term transfection efficiency in HeLa, HepG2 and A549 cells were investigated by MTT and luciferase activity. The mechanism of intracellular transportation of CP was observed by transfer experiment under different temperature and by confocal laser scanning microscopy. 3. The targeted peptide was grafted to CP complex. The specific targeting properties were evaluated by transfering different tumor cells and by the competitive inhibition experiment. 4. The anti-tumor activity of gene-carried CP and CPT was evaluated by ex vivo and in vivo experiment using CCL22 and IL-12 plasmid on H22 cells .Results: When the N/P of PEI/Chitosan/DNA was 10:4:1, the incubated complex shows a high transfect efficiency and low totoxicity. PEI was successfully combined with chitosan to form a complex. In HepG2, A549 and HeLa cells, CP complex exhibited lower cytotoxicity and higher transfection efficiency in 96 h transfection as compared with PEI25KDa .The time for CP to enter the nucleus was about 4 h, and it was demonstrated that energy was required for the intracellμlar transportation of CP. The transfect efficiency of CPT on HepG2 cells (the peptide target) was higher than CP. And when peptide was added, the transfection efficiency was decreased. In ex vivo and in vivo experiment, both CP and CPT carried plasmid have shown some therapeutical potential.Conclusion: The incubation of chitosan and PEI could produce a new non-viral gene delivery vector which shows low cytotoxicity, high transfect efficiency and clinical therapy potentials.