Study On The Liver Targeting Of Galactosylated Liposome-protamine-DNA Complexes(LPD)

Gene transfer to hepatocytes should be of great therapeutic potential since hepatocytes are related to many genetic and metabolic diseases and tumorigenesis. Also, hepatocytes are responsible for the synthesis of a wide variety of proteins, which play important physiological roles in or outside the hepatocytes after various post-translational modifications and translocation. However, most of the established viral and non-viral gene carriers lack cell specificity and, therefore, their in vivo application is limited. Receptor-mediated gene transfer is an important gene transfer strategy using the recognization and internalization to the specific ligands by the receptor existed on the cell surface delivering outside genes into cells for expressing.Hepatocytes exclusively express large numbers of high affinity cell-surface receptors—asialoglycoprotein receptor (ASGP-R), which can recognize the galactose or N-acetylgalactosamine residues of desialylated glycoproteins. This mechanism would be an effective way to achieve hepatocyte targeting utilizing the cationic liposomes modified by galactose ligands.In an effort to construct a stable, targetable vector for gene transfer, we focused on the specific recognization to galactose of ASGP-R and synthesized a series of novel galactosylated cholesterol derivatives, and prepared cationic liposomes with galactose residues. And then a novel non-viral gene delivery system, Gal-LPD (galactosylated liposomeprotamine-DNA complexes) was formed by mixing the galactosed liposomes with protamine-DNA complexes. Structurally, this formulation was virus-like particles. The highly condensed core composed of protamine and DNA was surrounded by a lipid coat.In the first step, E. Coli was employed to amplify the reporter genes for our experiments. Plasmid DNA was isolated and purified from the cells using the Qiagen Gifa Endo-free plasmid purification kit. DNA concentration and purity were quantified by UV absorbance at 260 nm and 280 nm. The structural integrity and topology of purified DNA was analyzed by agarose gel electrophoresis.And then, 18 cholesterylated thiogalactosides with different spacer lengths and galactose residues were synthesized.A simple and sensitive assay method for quantitative determination of DNA was developed by fluorescent spectrophometer with Hoechst 33258 as the fluorechrome. we prepared cationic liposomes using cholesterylated thiogalactosides with different spacer lengths and galactose residues, and formulated galactosylated LPDs (Gal-LPD) and non-galactosylated LPDO. The resulting LPDs were regularly spherical, with encapsulation efficiencies of more than 85%. The diameter and zeta potential of Gal-LPDs were changed with the number of galactose residues and the length of spacer between the anchor and galactose residues. LPDs can prevent plasmid DNA from degradation by nuclease.The in vitro transfection ability of LPDs was evaluated on hepatoma cells HepG2 and lung cancer cells A549, using pORF-LacZ reporter genes. Transfection efficiencies of LPDIIb, LPDIIIc and LPDIVe exhibited 1.6-, 1.8-and 2.1-fold higher in HepG2 cells than those of non-galactosed control LPDO, respectively. But their transfection activities were lower in A549 cells compared with LPD0. The presence of galactose can significantly inhibited the gene expression of Gal-LPDs, but not the LPDO. Further experiments indicated that the transfection efficiencies are dose-dependent, and affected by the ratio of DDAB/ DNA. The results of MTT assay showed that LPDs had no obvious toxicities.Our research demonstrated that galactosylated LPDs can recognized by the asialoglycoprotein receptor, and their affinities to ASGPR and transfecion efficiencies are enhanced with the increasing of galactose residues. This system may be a promising candidate to be further investigated in vivo for systemic gene delivery.