Effect of hydrophile-lipophile balance of the linker in Gal/GalNAc ligands on high-affinity binding of galactosylated liposomes by the asialoglycoprotein receptor.

In this study, we explored the effect of the hydrophile-lipophile balance (HLB) in the linker unit of Galactose (Gal)/N-acetylgalactosamine (GalNAc) ligands on their affinity toward asialoglycoprotein receptors (ASGPRs). Two Gal/GalNAc ligands with lipophilic linkers-{(5-cholesten-3b-ol)[(2-acetamido-2-deoxy-d-galactopyranose-6-o)sebacate]} (CHS-6-GalNAc) and {(5-cholesten-3b-ol)[(d-galactopyranose-6-o)sebacate]} (CHS-6-Gal)-and two with hydrophilic linkers-{(5-cholesten-yl)[(4-O-b-D-galactopyranosyl)-D-glucitol-6-yl]sebacate} (CHS-1-Gal) and {(5-cholesten-3a-ol)[(2-acetamido-2-deoxy-d-galactopyranose-6-o)3,6-dioxa-octanedioate]} (CHS-PEG2-6-GalNAc)-were synthesized by enzymatic catalysis. Compared with unmodified liposomes, all Gal/GalNAc ligand-modified liposomes showed higher efficiency toward the hepatocyte target as evaluated by weighted-average overall drug-targeting efficiency (Te*) in vivo and HepG2 cell uptake efficiency in vitro. The ligands containing linkers with high HLB values (i.e., CHS-PEG2-6-GalNAc and CHS-1-Gal) exhibited higher ASGPR affinity than those containing linkers with low HLB values (i.e., CHS-6-GalNAc and CHS-6-Gal). We used molecular-dynamics (MD) simulations to investigate the structure-activity relationship between the HLB value of the linker in a ligand and ASGPR affinity. MD simulation results indicated that a Gal/GalNAc ligand with a more hydrophilic linker (i.e., higher HLB value) unit tended to have a higher solvent-accessible surface area (SASA), leading to lower steric hindrance for effective ASGPR recognition. The results of this study will provide an improved design for Gal/GalNAc ligand-based surface-modified liposomes with high ASGPR affinity.

Effect of gal/GalNAc regioisomerism in galactosylated liposomes on asialoglycoprotein receptor-mediated hepatocyte-selective targeting in vivo.

In this study, we describe a novel synthesis of galactosylated lipids by lipase catalysis. Lactitol (Lac), galactose (Gal), or N-acetyl galactosamine (GalNAc) was coupled with cholesterol (CHS) as target head groups by enzyme-catalyzed regioselective esterification to produce three kinds of lipids: CHS-1-Gal, CHS-6-Gal, or CHS-6-GalNAc1. The biological effects of galactosylated lipids carrying different constitutional isomers of the pendent sugar species were investigated. LP-1-Gal (liposomes containing 5.0 molar% of CHS-1-Gal) showed strong binding to tetrameric lectins of Ricinus communis agglutinin (RCA120) in vitro, while LP-6-Gal (liposomes containing 5.0 molar% of CHS-6-Gal) and LP-6-GalNAc (liposomes containing 5.0 molar% of CHS-6-GalNAc) did not. After intravenous injection, LP-6-GalNAc, LP-1-Gal and LP-6-Gal rapidly disappeared from the blood and accumulated rapidly in liver (up to 74.88 ± 4.11%, 58.67 ± 5.75%, and 47.66 ± 4.56% of injected dose/g organ within 4 h, respectively). This is significantly higher than the uptake of unmodified liposomes (Unmod-LP) (18.67 ± 6.07%). Pre-injection of asialofetuin significantly inhibits liver uptake of Gal-liposomes (P < 0.01), with the degree of inhibition appearing in the following order: LP-6-GalNAc (73.29%) > LP-1-Gal (67.06%) > LP-6-Gal (53.61%). More importantly, LP-6-GalNAc was preferentially taken up by hepatocytes and the uptake ratio by parenchymal cells (PC) and nonparenchymal cells (NPC) (PC/NPC ratio) was 11.03 higher than LP-1-Gal (7.32), LP-6-Gal (5.83) and Unmod-LP (2.39). We suggest that liposomes containing the novel galactosylated lipid CHS-6-GalNAc have potential as drug delivery carriers for hepatocyte-selective targeting.