Ligand For Asialoglycoprotein Receptor Research Articles

Hepatocyte targeting via the asialoglycoprotein receptor.

This review highlights the potential of asialoglycoprotein receptor (ASGPR)-mediated targeting in advancing liver-specific treatments and underscores the ongoing progress in the field. First, we provide a comprehensive examination of the nature of ASGPR ligands, both natural and synthetic. Next, we explore various drug delivery strategies leveraging ASGPR, with a particular emphasis on the delivery of therapeutic nucleic acids such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). An in-depth analysis of the current status of RNA interference (RNAi) and ASO-based therapeutics is included, detailing approved therapies and those in various stages of clinical development (phases 1 to 3). Afterwards, we give an overview of other ASGPR-targeted conjugates, such as those with peptide nucleic acids or aptamers. Finally, targeted protein degradation of extracellular proteins through ASGPR is briefly discussed.

GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy

Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.

Synthesis and inhibitory activity of atorvastatin conjugate with a branched asialoglycoprotein receptor ligand with 3 <i>N</i>-acetyl-d-galactosamine residues

The novel atorvastatin conjugate was synthesized in a multi-step procedure using branched N -acetylgalactosamine ligand for asialoglycoprotein receptor targeting. The molar solubility of the obtained conjugate was 6.1±0.9 mM, which is almost 60 times higher than that of unmodified atorvastatin. The hydrolysis products of the conjugate demonstrated effective inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase.

Synthesis and Inhibitory Activity of Atorvastatin Conjugate with a Branched Asialoglycoprotein Receptor Ligand Containing Three N-Acetyl-d-galactosamine Residues

Synthesis and Inhibitory Activity of Atorvastatin Conjugate with a Branched Asialoglycoprotein Receptor Ligand Containing Three N-Acetyl-d-galactosamine Residues

Water-soluble meso-ester substituted BODIPY with aggregation-induced emission property for ratiometric detection of carboxylesterases in living hepatoma cell

The sensing of carboxylesterases (CESs) activity in living systems is of great importance for drug design and biomedical research. Herein, water-soluble AIE-active fluorescent organic nanoparticles (2B3OTs) containing hydrophilic lactose and hydrophobic BODIPY-conjugated tetraphenylethene moieties were rationally designed for ratiometrically fluorescent sensing of CESs with the meso-ester BODIPY as the recognition unit. 2B3OTs were cell membrane permeable and successfully applied to detect the real activities of endogenous CESs in living cells. More importantly, 2B3OTs could preferentially recognize hepatoma cells rather than normal liver cells, since the lactose is an efficient ligand for asialoglycoprotein receptors which are over expressed in tumor liver cells. It is first time that water-soluble AIE-active fluorescence organic nanoparticle were designed and utilized for real-time sensing of CESs activities in living cells.

Asialoglycoprotein Receptor-Targeted Superparamagnetic Perfluorooctylbromide Nanoparticles.

Background The decrease in asialoglycoprotein receptor (ASGPR) levels is observed in patients with chronic liver disease and liver tumor. The aim of our study was to develop ASGPR-targeted superparamagnetic perfluorooctylbromide nanoparticles (M-PFONP) and wonder whether this composite agent could target buffalo rat liver (BRL) cells in vitro and could improve R2∗ value of the rat liver parenchyma after its injection in vivo. Methods GalPLL, a ligand of ASGPR, was synthesized by reductive amination. ASGPR-targeted M-PFOBNP was prepared by a film hydration method coupled with sonication. Several analytical methods were used to investigate the characterization and safety of the contrast agent in vitro. The in vivo MR T2∗ mapping was performed to evaluate the enhancement effect in rat liver. Results The optimum concentration of Fe3O4 nanoparticles inclusion in GalPLL/M-PFOBNP was about 52.79 µg/mL, and the mean size was 285.6 ± 4.6 nm. The specificity of GalPLL/M-PFOBNP for ASGPR was confirmed by incubation experiment with fluorescence microscopy. The methyl thiazolyl tetrazolium (MTT) test showed that there was no significant difference in the optical density (OD) of cells incubated with all GalPLL/M-PFOBNP concentrations. Compared with M-PFOBNP, the increase in R2∗ value of the rat liver parenchyma after GalPLL/M-PFOBNP injection was higher. Conclusions GalPLL/M-PFOBNP may potentially serve as a liver-targeted contrast agent for MR receptor imaging.

RNAi targeting heparin cofactor II promotes hemostasis in hemophilia A

Hemophilia A is a hemorrhagic disease due to congenital deficiencies of coagulation factor VIII (FVIII). Studies show that hemophilia patients with anticoagulant deficiency present less severe hemorrhagic phenotypes. We aimed to find a new therapeutic option for hemophilia patients by RNA interference (RNAi) targeting heparin cofactor II (HCII), a critical anticoagulant protein inactivating the thrombin. The optimal small interfering RNA (siRNA) was conjugated to an asialoglycoprotein receptor ligand (N-acetylgalactosamine [GalNAc]-HCII), promoting targeted delivery to the liver. After administration, GalNAc-HCII demonstrated effective, dose-dependent, and persistent HCII inhibition. After 7 days, in normal mice, GalNAc-HCII reduced HCII levels to 25.04% ± 2.56%, 11.65% ± 2.41%, and 6.50% ± 1.73% with 2, 5, and 10 mg/kg GalNAc-HCII, respectively. The hemostatic ability of hemophilia mice in the GalNAc-HCII-treated group significantly improved, with low thrombus formation time in the carotid artery thrombosis models and short bleeding time in the tail-clipping assays. After repeated administration, the prolonged activated partial thromboplastin time (APTT) was reduced. A 30 mg/kg dose did not cause pathological thrombosis. Our study confirmed that GalNAc-HCII therapy is effective for treating hemophilia mice and can be considered a new option for treating hemophilia patients.

Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins.

Targeted protein degradation (TPD) technology has drawn significant attention from researchers in both academia and industry. It is rapidly evolved as a new therapeutic modality and also a useful chemical tool in selectively depleting various protein targets. As most efforts focus on cytosolic proteins using PROteolysis TArgeting Chimera (PROTAC), LYsosome TArgeting Chimera (LYTAC) recently emerged as a promising technology to deliver extracellular protein targets to lysosome for degradation through the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we exploited the potential of the asialoglycoprotein receptor (ASGPR), a lysosomal targeting receptor specifically expressed on liver cells, for the degradation of extracellular proteins including membrane proteins. The ligand of ASGPR, triantennary N-acetylgalactosamine (tri-GalNAc), was conjugated to biotin, antibodies, or fragments of antibodies to generate a new class of degraders. We demonstrated that the extracellular protein targets could be successfully internalized and delivered into lysosome for degradation in liver cell lines specifically by these degraders. This work will add a new dimension to TPD with cell type specificity.

New Small-Molecule Glycoconjugates of Docetaxel and GalNAc for Targeted Delivery to Hepatocellular Carcinoma.

In this work, we have developed covalent and low molecular weight docetaxel delivery systems based on conjugation with N-acetyl-d-galactosamine and studied their properties related to hepatocellular carcinoma cells. The resulting glycoconjugates have an excellent affinity to the asialoglycoprotein receptor (ASGPR) in the nanomolar range of concentrations and a high cytotoxicity level comparable to docetaxel. Likewise, we observed the 21-75-fold increase in water solubility in comparison with parent docetaxel and prodrug lability to intracellular conditions with half-life values from 25.5 to 42 h. We also found that the trivalent conjugate possessed selective toxicity against hepatoma cells vs control cell lines (20-35 times). The absence of such selectivity in the case of monovalent conjugates indicates the effect of ligand valency. Specific ASGPR-mediated cellular uptake of conjugates was proved in vitro using fluorescent-labeled analogues. In addition, we showed an enhanced generation of reactive oxygen species in the HepG2 cells, which could be inhibited by the natural ligand of ASGPR. Overall, the obtained results highlight the potential of ASGPR-directed cytostatic taxane drugs for selective therapy of hepatocellular carcinoma.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Asialoglycoprotein receptor targeted micelles containing carborane clusters for effective boron neutron capture therapy of hepatocellular carcinoma

The asialoglycoprotein receptor (ASGP-R) is viewed as an ideal target for hepatocyte-specific delivery. And the galactose residue is a promising ASGP-R ligand because of its high receptor affinity. Herein, a novel polymer based on PEGylated galactose was developed to achieve boron neutron capture therapy (BNCT) for active targeting hepatocellular carcinoma (HCC) by loading carborane clusters. Notably, the polymer could self-assemble into micelles with an average diameter of 135 nm under physiological conditions. The micelle had the high selectivity and low cytotoxicity to HepG2 cells (IC50 >1000 μM). Kinetically, the micelle had the higher uptake in HepG2 cells than the positive control group sodium borocaptate (BSH) in vitro. After the HepG2 cells were treated with the micelle, the cytoskeleton was changed and the migration ability was weakened during BNCT. Apoptosis was remarkably induced by breaking of DNA double strands of cancer cells. In addition, the concentration of 10B in the tumor was 4.5 times higher than that of the BSH group at 4 h after the micelle administration in the tumor-bearing mice. The tumor/blood ratio of 10B concentration reached over 25 at 24 h after micelle injection. In the normal mice, the micelles were mainly distributed among the liver and kidney tissues and could be effectively eliminated from the body within 24 h. No systemic toxicity was observed after administration. Thus, the carborane-containing PEGylated galactose micelles with ASGP-R targeting can be used as a promising therapeutic vector for effective boron neutron capture therapy of hepatocellular carcinoma.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Synthesis and biological evaluation of novel doxorubicin-containing ASGP-R-targeted drug-conjugates

Asialoglycoprotein receptor (ASGP-R) belongs to a wide family of C-type lectins and it is currently regarded as an attractive protein in the field of targeted drug delivery (TDD). It is abundantly expressed in hepatocytes and can be found predominantly on the sinusoidal surface especially of HepG2 cells. Therefore, ASGP-R can be used for the TDD of anticancer therapeutics against HCC and molecular diagnostic tools. To date, a variety of mono- and multivalent selective ASGP-R ligands have been discovered. Although many of these compounds have demonstrated a relatively high binding affinity towards the target, the reported synthetic schemes are not handled, complicated and include many non-trivial steps. In the current study, we describe a convenient and versatile synthetic approach to novel monovalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose fragment as an ASGP-R-recognition “core-head” and well-known nonselective cytostatic – Doxorubicin (Dox). This is the first example of the direct conjugation of a drug molecule to the ASGP-targeted warhead by a really convenient manner via a simple linker sequence. The performed MTS-based biological evaluation in HepG2 cells revealed the novel conjugates as having anticancer activity. Confocal microscopy showed that the molecules readily penetrated HepG2 membrane and were mainly localized within the cytoplasm instead of the nucleus. Per contra, Dox under the same conditions demonstrated good anticancer activity and was predominantly concentrated in the nucleus. Therefore, we speculate that the amide “trigger” that we have used in this study for linker attachment is a sufficiently stable inside the cells to be enzymatically or spontaneously degraded. As a consequence, we did not observe the release of the drug. Ligands containing triggers that are more liable towards endogenous hydrolysis within the tissue of targeting are strongly required.

Asialoglycoprotein receptor targeted delivery of doxorubicin nanoparticles for hepatocellular carcinoma

We report asialoglycoprotein receptor (ASGPR)-targeted doxorubicin hydrochloride (Dox) nanoparticles (NPs) for hepatocellular carcinoma (HCC). Polyethylene sebacate (PES)-Gantrez® AN 119 Dox NPs of average size 220 nm with PDI < 0.62 and ∼20% Dox loading were prepared by modified nanoprecipitation. ASGPR ligands, pullulan (Pul), arabinogalactan (AGn), and the combination (Pul-AGn), were anchored by adsorption. Ligand anchoring enabled high liver uptake with a remarkable hepatocyte:nonparenchymal cell ratio of 85:15. Furthermore, Pul-AGn NPs exhibited an additive effect implying incredibly high hepatocyte accumulation. Galactose-mediated competitive inhibition confirmed ASGPR-mediated uptake of ligand-anchored NPs in HepG2 cell lines. Subacute toxicity in rats confirmed the safety of the NP groups. However, histopathological evaluation suggested mild renal toxicity of AGn. Pul NPs revealed sustained reduction in tumor volume in PLC/PRF/5 liver tumor-bearing Nod/Scid mice up to 46 days. Extensive tumor necrosis, reduced collagen content, reduction in the HCC biomarker serum α-fetoprotein (p < 0.05), a mitotic index of 1.135 (day 46), and tumor treated/tumor control (T/C) values of <0.42 signified superior efficacy of Pul NPs. Furthermore, weight gain in the NP groups, and no histopathological alterations indicated that they were well tolerated by the mice. The high efficacy coupled with greater safety portrayed Pul Dox NPs as a promising nanocarrier for improved therapy of HCC.

Well-Defined Multivalent Ligands for Hepatocytes Targeting via Asialoglycoprotein Receptor.

Targeted delivery of therapeutic agents to hepatocytes is a particularly attractive strategy for the treatment of hepatocellular carcinoma and other liver diseases. The asialoglycoprotein receptor (ASGP-R) is abundantly expressed on hepatocytes and minimally found on extra-hepatic cells, making it an ideal entry gateway for hepatocyte-targeted therapy. Numerous multivalent ligands have been developed to target ASGP-R, among which well-defined multivalent ligands display especially high binding affinity to the receptor. Recently, several gene delivery systems based on such ligands for ASGP-R showed encouraging clinical results, drawing increasing interest in the scientific community and eventually promoting the improvement of current treatment for liver diseases. Here, we review ASGP-R targeting with a special emphasis on well-defined systems and properties such as the linker's length, hydrophilic-hydrophobic balance of the linker, and the spatial geometry of the scaffold. The present manuscript provides important guidelines for the design of multivalent ligands for ASGP-R.

Identification of Novel Small-Molecule ASGP-R Ligands.

During the past decade asialoglycoprotein receptor (ASGP-R) expressed predominantly by hepatocytes has attracted a considerable attention as a convenient biomolecular trap for targeted drug delivery. Currently, several selective galactose-containing ligands equipped by drug molecules, e.g. known anticancer therapeutics, as well as diagnostic tools are under active preclinical development. In this paper, we have carried out a rational in silico screening among the molecules available in ChemDiv collection and compounds provided by our colleagues to reveal potential ASGP-R binders. Thus, 3D molecular docking approach provided a set of 100 `high score` molecules that was subsequently evaluated in vitro using an advanced Surface Plasmon Resonance (SPR) technique. As a result, dozens of novel small-molecule ASGP-R ligands with high diversity in structure were identified. Several hits showed the binding affinity much more better than that determined for galactose and Nacetylgalactosamine which were used as reference compounds. The disclosed molecules can be reasonably regarded as promising molecular devices for targeted drug delivery to hepatocytes.

Asialoglycoprotein receptor facilitates infection of PLC/PRF/5 cells by HEV through interaction with ORF2.

Although the biological and epidemiological features of hepatitis E virus (HEV) have been studied extensively in recent years, the mechanism by which HEV infects cells is still poorly understood. In this study, coimmunoprecipitation, pull-down, and ELISA were used to show that the HEV ORF2 protein interacts directly with the ectodomain of both ASGR1 and ASGR2. Susceptibility to HEV correlated positively with the expression level of surface asialoglycoprotein receptor (ASGPR) in cell lines. ASGPR-directed small interfering RNA (siRNA) in HEV-infected PLC/PRF/5 cells had no significant effect on HEV release, suggesting that ASGPR mainly regulates the viral binding and entry steps. Both the purified ASGPR ectodomain and anti-ASGPR antibodies disturbed the binding of HEV to PLC/PRF/5 cells. The classic ASGPR ligands asialofetuin, asialoganglioside, and fibronectin competitively inhibited the binding of HEV to hepatocytes in the presence of calcium. HeLa cell lines stably expressing ASGPR displayed increased HEV-binding capacity, whereas ASGPR-knockout PLC/PRF/5 cell lines had lower HEV-binding capacity. Thus, our study demonstrates that ASGPR is involved in and facilitates HEV infection by binding to ORF2. J. Med. Virol. 88:2186-2195, 2016. © 2016 Wiley Periodicals, Inc.

Electrochemical evidence for asialoglycoprotein receptor – mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds

Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell–cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Stealth lipoplex decorated with triazole-tethered galactosyl moieties: a strong hepatotropic gene vector.

Mono-antennary galacto derivatives of cholesterol are being actively developed to direct lipoplexes to the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Here we report on a novel ASGP-R ligand cholest-5-en-3-yl [1-(β-D-galactopyranosyl)-1H-1,2,3-triazol-4-yl]methylcarbamate (4), assembled by a copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry), and compare it with cholest-5-en-3-yl-β-D-galactopyranoside (2) and cholest-5-en-3-yl [1-(β-D-galactopyranosyl-1'-oxy)phen-4-yl]carbamate (3), in liposome formulations with or without 5 mol% distearoylphosphatidylethanolamine poly(ethylene glycol)2000, intended for DNA delivery to ASGP-R-positive hepatocyte-derived HepG2 cells and the ASGP-R-negative embryo kidney cell line HEK293. Transfection levels attained with lipoplex 4 were 100 and 300% greater than those for lipoplexes 2 and 3 respectively in HepG2 cells, while competition assays reduced transfection levels by up to 98%. Transfection activities achieved in HEK293 cells were up to three orders of magnitude lower. Therefore, 4 is representative of a new class of promising hepatotropic ligands for gene delivery.