Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery.

Asialoglycoprotein receptors (ASGPR) are hepatocyte bound receptors, which exhibit receptor mediated endocytosis (RME) for galactose specific moieties. Arabinogalactan (AG), a liver specific high galactose containing branched polysaccharide was hydrophobized using cholesterol (CHOL) as a lipid anchor via a two step reaction process to yield the novel polysaccharide lipid conjugated ligand (CHOL-AL-AG). CHOL-AL-AG was characterized by Fourier transform infra red (FTIR) spectroscopy, (1)H and (13)C nuclear magnetic spectroscopy (NMR), size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). Conventional liposomes (CL) and surface modified liposomes (SML) containing CHOL-AL-AG were prepared using reverse phase evaporation technique. Effect of CHOL-AL-AG concentration on particle size and zeta potential of SML was evaluated. Surface morphology of CL and SML was studied using cryo-transmission electron microscopy (cryo-TEM). In vitro binding affinity of SML and CL was evaluated using Ricinus communis agglutinin (RCA) assay. Cellular uptake of SML and CL was determined on ASGPR expressing HepG2 cell lines by confocal laser scanning microscopy technique (CLSM). FTIR spectra revealed bands at 1736 cm(-1) and 1664 cm(-1) corresponding to ester and carbamate functional groups, respectively. Signals at δ 0.5-2.5 corresponding to the cholestene ring and δ 3-5.5 corresponding to the carbohydrate backbone were observed in (1)H NMR spectrum of the product. CHOL-AL-AG possessed a mean average molecular weight of 27 KDa as determined by size exclusion chromatography. An endothermic peak at 207 °C was observed in the DSC thermogram of CHOL-AL-AG, which was not observed in thermograms of reactants and intermediate product. Synthesized CHOL-AL-AG was successfully incorporated in liposomes to yield SML. Both CL and SML possessed a mean particle size of ∼ 200 nm with polydispersity index of ∼ 0.25. The zeta potential of CLs was observed to be -17 mV whereas zeta potential of SMLs varied from -18 to -22 mV. RCA assay revealed enhanced binding of SML compared to CL confirming presence of galactose on surface of SML. CLSM studies demonstrated enhanced cellular uptake of SMLs compared to CL by HepG2 cells post 3 h administration indicating enhanced uptake by the ASGPR. Thus surface modified liposomes specific to target heptocytes demonstrate a promising approach for targeted drug delivery in liver cancer therapeutics.