Investigation On Liver-Targeted Immune Gene Delivery

In this study, a novel non-viral gene delivery system was developed. We used the cationic polymer PEI to compress plasmid DNA, Then,added PEG-liposomes to coated PEI/pDNA compression.the liposome-PEI-pDNA complexes was called lipoplexes(LPD).Through the hydrosulfide group conjugating with biotin, The anti-human insulin receptor Monoclonal antibody(8314-SA) connect with DSPE-PEG2000-biotin. Monoclonal antibodies are active targeting group of LPD and improve the targeting capacity of this carrier. This is a long cycling and active targeting of liver gene vector.The research contents and results are as follows:In the first step,Escherichia coli was used to amplify the reporter gene and Plasmid DNA was isolated and purified from the escherichia coli cells using the Qiagen Endo-Free Maxi Plasmid Purification kit. DNA concentration and Purity were quantified by ultraviolet absorption at 260nm and 280nm. Further analysis of purified DNA was by agarose gel electrophoresis. The second step, Preparated liposomes and DNA/PFI respectively.Using the film-vibration to prepare liposomes. The Lipid composition is 94% of POPC,2% of DDAB,3% of DSPE-PEG2000 and 1% of DSPE-PEG2000-biotin. The third step, added liposomes to PEI/pDNA, vortexed 30 second, the LPD was completed. Single-factor experiment results are:Plasmid concentration below 40μg/ml, the particle size changed little, as the DNA concentration increasing, complex particle size increases; N/P ratio exceeds 1.5, the plasmid is encapsulated entirely, with the N/P increasing, the PEI/pDNA particle size decreases,zeta potential increase,at the N/P equal to 15, the particle size of PEI/pDNA was smallest, and then increasing N/P ratio, particle size did not change significantly;Lipid/pDNA molar ratio of 50:1, liposome can encapsulate PEI/DNA compression completely. with the lipid/pDNA ratio of increasing, LPD particle size decreases, potential reduce at 170:1, the particle size was smallest, as the lipid/ pDNA ratio further increasing,the complexes particle size began to get bigger. LPD uniform design optimization of experimental results are:At pH 7.0 of HEPES buffer, the plasmid concentration of 40μg/ml, N/P=15, lipid/pDNA=170:1,LPD mean diameter was about 130 nm, the average zeta potential is around 1.5mV. further modificated LPD structure, the outer lipid membrane of PEG chain conjugated with anti-human insulin receptor monoclonal antibody, the formation of liver active targeting monoclonal antibody-modified Lipo/PEI/pDNA complexes was completed.which is called 8314-LPD. By TEM observation.8314-LPD complexes approximate spherical shape, neat and not adhesions; analysis of their particle size and zeta potential, the particle has an average diameter about 150nm, and has an average zeta potential 4.5mv. We use green fluorescent protein gene as a reporter gene to study of the this complexes protective effect of the plasmid, gel electrophoresis result show that the gene vector could effectively protect the plasmid DNA do not degraded by DNase I; Serum stability experiment results show that 8314-LPD complexes are stable in the serum; long-term stability experiment results show, both 4℃and 25℃nitrogen sealed storage condition, complexes size would increase, but the degree of change is not the same, which indicated that the temperature has an obvious effect on the stability of the 8314-LPD complexes, at 25℃storage condition,8314-LPD particle size will occur a obviously increasing within one month.In vitro cell transfection and cell toxicity experiments, LPD vector transfection efficiency is lower than PEI/DNA compression, but after modified by the monoclonal antibody,8314-LPD gene vector can be good at human hepatocellular carcinoma cell SMMC-7721 intaking, compared with the PEI/pDNA and LPD vectors,8314-LPD transfection rate was increased significantly. MTT experiments show toxicity of LPD and 8314-LPD complexes are lower than the PEI/DNA compression.In order to improve the long-term stability of the 8314-LPD,we used freeze-drying method. firstly, Select a suitable freeze-dried agent, freeze-dried powder. at their appearance, color, surface refinement level, and dispersing ability, we found freeze-dried powder prepared with trehalose, the appearance does not collapse, non-foaming, color uniform, fine texture, re-dissolved within a short time after they dispersed.after re-dissolved,the complex particle size and zeta potential have no significant change. Trehalose is the optimal freeze-dried agent. Secondly quality evaluation of freeze-dried powder, through the re-dissolved anti-enzyme experiments, the freeze-dried formulations can still maintain the ability of anti-nuclease, this shows that the carrier structure in freeze-drying process has not been destroied, transfection experiments show that the re-dissolved complexes transfection activity was unchanged.The results of this study show,8314-LPD complexes preparation method is simple, can protect plasmid DNA from being nuclease degradation effectively, have a high stability in serum and high cells transfection, low cell toxicity, last one,this carrerier can be prepared as freeze-dried powder by freeze-drying for long-term preservation. This liver active targeting of long-circulating non-viral gene vector, utilize the advantages of cationic polymers and liposomes, overcome the shortcomings of both, in the future of gene therapy of liver cancer, will have a good development prospects.