AIMTo study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

METHODSDipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

RESULTSThe co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

CONCLUSIONCo-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.