Preparation And Research Of A Gene And Drug Co-delivery System

In this study, a pH-responsive charge-reversal random copolymer polyethylenimine-poly(L-lysine)-poly(L-glutamic acid)(PELG) was synthesized by ring-opening polymerization of N-carboxyanhydride of ε-benzyl-oxycarbonyl-L-lysine (Lys(Z)-NCA) and N-carboxyanhydride of γ-benzyl-L-glutamate (BLG-NCA) monomers by using polyethylenimine (PEI) as initiator. This copolymer can be used as the shielding of nanocarriers. Doxorubicin (DOX) was modified with cis-aconitic anhydride (CA) to obtain an acid-sensitive small molecule drug cis-aconityl-doxorubicin (CAD). The structures and molecular weights of the products were characterized by1H NMR, GPC, FT-IR and ESI-MS. The results confirmed that the products were successfully synthesized.PELG/PEI/CAD drug delivery system was prepared by electrostatic binding. Zeta potential analysis confirmed the pH-responsive charge reversal function of the complexes. The acid-sensitive drug release of PELG/PEI/CAD was confirmed by drug release study. Cytotoxicity assay showed the carriers had good biological safety, and the PELG/PEI/CAD system had a high killing effect on HeLa cells. Cell uptake assay and CLSM results showed that the system could be endocytosed efficiently in pH6.8(similar to the acidic tumor environment), and a large amount of drug could be delivered into cells and distributed in cytoplasm and nucleus.Since the early work has verified the performance of the system as a gene carrier, so we further investigate the gene and drug co-delivery system PELG/PEI/(DNA+CAD). Zeta potential analysis confirmed the pH-responsive charge reversal function of the complexes. Gel retardation assay showed the co-delivery system had efficient DNA binding ability, and the transfection experiment showed the system had good transfection efficiency on HepG2cells. In the PELG/PEI/(DNA+CAD) co-delivery system, the loaded different concentrations of CAD did not affect the DNA transfection much, the mass ratio of DNA and CAD was also confirmed. The acid-sensitive drug release of PELG/PEI/(DNA+CAD) was confirmed by drug release study. We further used p53as the therapeutic gene. PELG/PEI/(p53+CAD) co-delivery system was prepared and characterized by in vitro evaluations. Cytotoxicity assay showed the half maximal inhibitory concentration (IC50) of the co-delivery system was lower than the gene or drug delivery system, the co-delivery system had the greatest killing effect on tumor cells. Confocal laser scanning microscopy (CLSM) showed the gene and drug could be simultaneously delivered into the cells, and the cell uptake of the complexes was higher in pH6.8, more drug and gene could be located in tumor cells. RT-PCR showed the expression of the tumor suppressor gene p53was significantly increased after the tumor cells were treated with co-delivery system. The co-delivery system could induce severe tumor apoptosis with80%apoptosis in24h and96%in48h. This co-delivery system will have great potential in anti-cancer therapy.