Transcriptomically Revealed Oligo-Fucoidan Enhances the Immune System and Protects Hepatocytes via the ASGPR/STAT3/HNF4A Axis.

Oligo-fucoidan, a sulfated polysaccharide extracted from brown seaweed, exhibits anti-inflammatory and anti-tumor effects. However, the knowledge concerning the detailed mechanism of oligo-fucoidan on liver cells is obscure. In this study, we investigate the effect of oligo-fucoidan in normal hepatocytes by transcriptomic analysis. Using an oligo-fucoidan oral gavage in wild-type adult zebrafish, we find that oligo-fucoidan pretreatment enhances the immune system and anti-viral genes in hepatocytes. Oligo-fucoidan pretreatment also decreases the expression of lipogenic enzymes and liver fibrosis genes. Using pathway analysis, we identify hepatocyte nuclear factor 4 alpha (HNF4A) to be the potential driver gene. We further investigate whether hepatocyte nuclear factor 4 alpha (HNF4A) could be induced by oligo-fucoidan and the underlying mechanism. Therefore, a normal hepatocyte clone 9 cell as an in vitro model was used. We demonstrate that oligo-fucoidan increases cell viability, Cyp3a4 activity, and Hnf4a expression in clone 9 cells. We further demonstrate that oligo-fucoidan might bind to asialoglycoprotein receptors (ASGPR) in normal hepatocytes through both in vitro and in vivo competition assays. This binding, consequently activating the signal transducer and activator of transcription 3 (STAT3), increases the expression of the P1 isoform of HNF4A. According to our data, we suggest that oligo-fucoidan not only enhances the gene expression associated with anti-viral ability and immunity, but also increases P1-HNF4A levels through ASGPR/STAT3 axis, resulting in protecting hepatocytes.

Synthesis of pectin-deoxycholic acid conjugate for targeted delivery of anticancer drugs in hepatocellular carcinoma.

Sorafenib (SF) a chemotherapeutic drug is used in hepatocellular carcinoma (HCC) with vast side effects. The aim of the project ahead was synthesis of SF loaded co-polymeric micelles of pectin-deoxycholic acid (P-DOCA) to target the overexpressed asialoglycoprotein receptors of hepatocytes by pectin. DOCA was modified with ethylenediamine and conjugated to pectin. FT-IR and 1HNMR confirmed the bio-conjugation. Pyrene was used to measure critical micelle concentration (CMC) by fluorimetry technique. P-DOCA micelles were loaded with SF and their particle size, zeta potential, drug loading and release efficiency were measured. MTT assay was used for determining cytotoxicity. The cell cycle arrest was studied by flow cytometry analysis and the cellular uptake was studied using cumarin-6 as the fluorophore agent. The micelles capability in preventing the cells migration was tested by Transwell plates. The CMC of P-DOCA micelles was 10.747 µg/mL. The best formulation obtained from SF to polymer ratio of 1:2. SF loaded micelles showed 30% increased cytotoxicity. The micelles cellular uptake was more than the free drug. Relative migration of HepG2 cells treated with SF loaded micelles was reduced to 6.67% compared to free SF which was 26.67%. The designed micelles are promising for antitumor drug targeting to HCC.

Angelica sinensis polysaccharide nanoparticles as a targeted drug delivery system for enhanced therapy of liver cancer.

In recent years, the utilization of polysaccharides as targeted drug carriers has attracted considerable attention. Herein, Angelica sinensis polysaccharide (ASP), a plant polysaccharide with good biocompatibility, excellent aqueous solubility and intrinsic liver-targeted capability, was modified with hydrophobic group (deoxycholic acid) to fabricate amphiphilic conjugate (ASP-DOCA). Self-assembled nanoparticles were successfully developed for hepatoma-targeted delivery of therapeutic drug doxorubicin (DOX). The DOX loaded nanoparticles (DOX/ASP-DOCA NPs) were spherical in shape with a particle size of 228 nm and negatively charged around -17 mV. DOX was released from nanoparticles in a sustainable and pH-dependent manner. In vitro cellular uptake revealed that DOX/ASP-DOCA NPs were internalized into HepG2 cells through asialoglycoprotein receptor (ASGPR)-mediated endocytosis, resulting in a higher anti-proliferation effect than DOX-loaded dextran derivative DOX/DEX-DOCA NPs. Additionally, DOX/ASP-DOCA NPs showed higher inhibition on the growth of HepG2 multicellular spheroids (MCs) than DOX/DEX-DOCA NPs. In vivo imaging demonstrated that ASP-DOCA NPs specifically targeted HepG2 tumors via ASGPR, improving the accumulation of DOX/ASP-DOCA NPs in tumors and generating superior antitumor activity compared with free DOX and DOX/DEX-DOCA NPs. Taken together, ASP-DOCA NPs possess potential applications in drug delivery systems targeting liver cancer.

A Modular Ionophore Platform for Liver-Directed Copper Supplementation in Cells and Animals.

Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely on generic copper supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. Here we present a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, we designed and synthesized an N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying "Trojan Horse" that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease.

Engineering of pectin-capped gold nanoparticles for delivery of doxorubicin to hepatocarcinoma cells: an insight into mechanism of cellular uptake.

In this study, we have reported the fabrication and evaluation of pectin-capped gold nanoparticles (PEC-AuNPs) for delivery of anticancer drug, doxorubicin (DOX) to cells overexpressing asialoglycoprotein receptor (ASGPR). Pectin was used as a reducing, stabilizing and targeting agent. The pectin-capped gold nanoparticles demonstrated surface plasmon resonance band at 519 nm. The PEC-AuNPs were spherical in shape with a particle size of 14 nm and zeta potential value of -33 mV and were biocompatible and non-cytotoxic. The PEC-AuNPs exhibited a high drug loading efficiency of 78%. The DOX-loaded gold nanoparticles (DOX-PEC-AuNPs) showed excellent stability under varying pH and electrolytic conditions. The cytotoxicity study of the DOX-PEC-AuNPs in human Caucasian hepatocyte cells demonstrated their greater potency in killing these cells as compared to free DOX. The uptake and targeting potential of DOX-PEC-AuNPs was thoroughly investigated. Further, it was found that the PEC-AuNPs were taken up by HepG2 cells via a clathrin-dependent receptor-mediated endocytosis by asialoglycoprotein receptor present of the surface of these cells. Thus, the PEC-capped AuNPs can prove a promising carrier for anticancer drug in the treatment of hepatocellular carcinoma.

Carrier-free nanodrug by co-assembly of chemotherapeutic agent and photosensitizer for cancer imaging and chemo-photo combination therapy.

Nanosized drug delivery systems (NDDS) with photothermal therapy (PTT) and photodynamic therapy (PDT) have been extensively exploited to improve the therapeutic performance and bio-safety of chemotherapeutic drugs in cancer. In this work, a carrier-free nanodrug was developed by co-assembly of the anti-cancer agent ursolic acid (UA), an asialoglycoprotein receptor (ASGPR), which can recognize the target molecule lactobionic acid (LA), and the near-infrared (NIR) probe dye indocyanine green (ICG) to form UA-LA-ICG NPs by a simple and green self-assembly approach. The UA-LA-ICG NPs had suitable stability, showed controlled release profile of UA drugs, and exhibited preferable temperature response (∼59.4 °C) under laser irradiation (808 nm, 1 W/cm2). Compared with free ICG, the UA-LA-ICG NPs significantly enhanced the intracellular ICG uptake. Upon irradiation of the NIR laser, co-assembled nanodrugs demonstrated great performance as a reactive oxygen species (ROS) producer and exhibited more anti-proliferative activities on ASGPR-overexpressing HepG2 cells than ASGPR low-expressing HeLa cells. Meanwhile, in vivo NIR fluorescence imaging exhibited that the co-assembled nanodrugs were specifically targeted to the tumor by the active targeting property of LA, and its circulation time was much longer than that of free ICG. In addition, UA-LA-ICG NPs + NIR irradiation treatment displayed enhanced inhibitory effect on tumor growth in H22 tumor-bearing mice. Overall, the co-assembly of chemotherapeutic agent and photosensitizer by the self-assembly approach might open an alternative avenue and give inspiration to fabricate new carrier-free nanodrugs for cancer imaging and chemo-photo combination therapy. STATEMENT OF SIGNIFICANCE: The present study for the first time reported carrier-free nanoparticles (NPs) by co-assembly of a natural product ursolic acid (UA), an asialoglycoprotein receptor (ASGPR)-recognized sugar molecule lactobionic acid (LA), and the near-infrared dye indocyanine green (ICG) through a simple and green approach. The preparation process of nanodrugs is simple, rapid, effective, and labor-saving. The co-assembled nanodrugs were capable of stabilizing the ICG molecules and specifically targeting to the tumor, which could increase the tumor accumulation in cancer imaging and also enhance the efficacy of chemo-phototherapy.

Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.

Co-delivery of sorafenib and siVEGF based on mesoporous silica nanoparticles for ASGPR mediated targeted HCC therapy.

Combination with chemotherapeutic drug and gene therapy has been proven highly effective in suppressing tumor progression. Hence, an asialoglycoprotein receptor (ASGPR)-targeting nanodrug delivery system based on mesoporous silica (MSN) nanocarrier for co-delivery of sorafenib (SO) and vascular endothelial growth factor (VEGF) targeted siRNA (siVEGF) to hepatocellular carcinoma (HCC) was successfully designed and synthesized. The structure of nanoparticles was characterized by IR, particle size, zeta potential and N2 adsorption-desorption. The nanoparticles were further evaluated for drug release, cellular uptake, transfection, cell cytotoxicity and cell cycle against HepG2 and Huh7 cells. In vitro testing demonstrated that MSN-LA delivery system could not only induce S cell cycle arrest, enhance the cytotoxicity and improve the tumor target of SO and siVEGF, but also enhance the siVEGF transfection efficiency in ASGPR-overexpressing Huh7 cells. Overall, the MSN-LA delivery system can be a promising drug carrier which could further enhance the anti-cancer efficacy of SO and siVEGF via the active targeting property of LA.

Design of intrahepatocyte copper(I) chelators as drug candidates for Wilson's disease.

Wilson's disease is an autosomal recessive disease caused by mutations on the ATP7B gene found on chromosome 13. Since the corresponding ATPase is in charge of copper (Cu) distribution and excretion in the liver, its malfunctioning leads to Cu overload. This short review deals with treatments of this rare disease, which aim at decreasing Cu toxicity and are, therefore, based on chelation therapy. The drugs used since the 1950s are described first, then a novel approach developed in our laboratory is presented. Since the liver is the main organ of Cu distribution in the body, we targeted the pool of intracellular Cu in hepatocytes. This Cu pool is in the +1 oxidation state, and therefore soft sulfur ligands inspired from binding sites found in metallothioneins were developed. Their targeting to the hepatocytes by functionalization with ligands of the asialoglycoprotein receptor led to their cellular incorporation and intracellular Cu chelation.

Comparative in silico-in vivo evaluation of ASGP-R ligands for hepatic targeting of curcumin Gantrez nanoparticles.

The present study aims to design hepatic targeted curcumin (CUR) nanoparticles using Gantrez (GZ) as a polymer. Three carbohydrate-based hepatocyte asialoglycoprotein receptor (ASGP-R) ligands were selected for the study, namely kappa carrageenan (KC), arabinogalactan (AG), and pullulan (P). AG and KC are galactose based while P is a glucose-based polymer. CUR-GZ nanoparticles were prepared by nanoprecipitation and anchored with the ligands by nonspecific adsorption onto preformed nanoparticles. The change in zeta potential values confirmed adsorption of the ligands. Docking simulation was evaluated as a tool to predict ligand ASGP-R interactions, using grid-based ligand docking with energies (Glide). Monomers and dimers were used as representative units of polymer for docking analysis. The binding of ASGP-R was validated using D-galactose as monomer. The interaction of the ligands with the receptor was evaluated based on Glide scores and E model values, both for monomers and dimers. The data of the docking study based on Glide scores and E model values suggested higher affinity of AG and P to the ASGP-R, compared to KC. At 1 h, following intravenous administration of the nanoparticles to rats, the in vivo hepatic accumulation in the order CUR-GZAG > CUR-GZKC > CUR-GZP correlated with the docking data based on Glide scores. However, at the end of 6 h, pullulan exhibited maximum hepatic accumulation and arabinogalactan minimum accumulation (p < 0.05). Nevertheless, as predicted by docking analysis, arabinogalactan and pullulan revealed maximum hepatic accumulation. Docking analysis using dimers as representative stereochemical units of polymers provides a good indication of ligand receptor affinity. Docking analysis provides a useful tool for the preliminary screening of ligands for hepatic targeting.

A single-species approach considering additional physiological information for prediction of hepatic clearance of glycoprotein derivate therapeutics.

BACKGROUND AND OBJECTIVES: Existing methods for the prediction of human clearance of therapeutic proteins involve the use of allometry approaches. In general, these approaches have concentrated on the role of body weight, with only occasional attention given to more specific physiological parameters. The objective of this study was to develop a mechanism-based model of hepatic clearance (CL(H)), which combines a single-species scaling approach with liver physiology, for predicting CL(H) of selected glycoprotein derivate therapeutics, and to compare the outcome of this novel method with those of two empirical methods obtained from the literature - namely, the single-exponent theory and multiple-species allometry. Thus, this study was designed as an explanatory study to verify if the addition of physiological information is of benefit for extrapolating clearance of selected therapeutic proteins from one species to another. METHODS: Five glycoprotein derivate therapeutics that are known to be principally eliminated by asialoglycoprotein receptors (ASGPRs) under in vivo conditions were selected. It was assumed that the interspecies differences in CL(H) reported for these compounds are reflected by the interspecies differences in the abundance of these receptors. Therefore, key scaling factors related to these differences were integrated into one model. Fourteen extrapolation (prediction) scenarios across species were used in this study while comparing the single-species model, based on physiology, with the single-exponent theory. In addition, the physiological model was compared with multiple-species allometry for three proteins. RESULTS: In general, the novel physiological model is superior to the derived allometric methods. Overall, the physiological model produced a predicted CL(H) value with levels of accuracy of 100% within 3-fold, 100% within 2-fold and about 82% within 1.5-fold, compared with the observed values, whereas the levels of accuracy decreased to 93%, 77% and 53%, respectively, for allometry. The proposed physiological model is also superior to allometry on the basis of the root mean square error and absolute average fold error values. CONCLUSIONS: It has been demonstrated that interspecies differences in the abundance of ASGPRs principally govern interspecies variations in CL(H) of compounds that are principally eliminated by ASGPRs. Overall, the proposed physiological model is an additional tool, which should facilitate investigation and prediction of human CL(H) of specific glycoproteins solely on the basis of clearance data determined in a single preclinical species.

Targeted delivery of macromolecular drugs: asialoglycoprotein receptor (ASGPR) expression by selected hepatoma cell lines used in antiviral drug development.

The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.

Radiolabeled constructs for evaluation of the asialoglycoprotein receptor status and hepatic functional reserves.

Transplantation of isolated hepatocytes may eventually replace a whole liver transplantation for the treatment of selected liver metabolic disorders and acute hepatic failure. To understand the behavior of transplanted hepatocytes, methods for longitudinal assessment of functional activity and survival of hepatocyte transplants must be developed. Targeting of asialoglycoprotein receptor (ASGPr) with various radiolabeled or Gd-labeled constructs of asialofetuin (AF) is expected to allow noninvasive and quantitative assessments of the ASGPr status in functioning hepatocytes before and after the transplant. Six new constructs of (125)I-, (99m)Tc-, (153)Gd-, and (111)In-radiolabeled AF with distinct stabilities and clearance rates were prepared and evaluated in vitro in mice, rat, porcine, and human hepatocytes, and in vivo in mice and rats. The blood and organ clearance rates, as well as liver and spleen uptake, were measured. Even extensive chemical modifications of AF with poly-l-lysine and various chelating agents do not appear to diminish AF's binding to ASGPr. Binding to isolated hepatocytes and the in vivo liver uptake studies indicate unimpaired functional activity of AF as evidenced by the rapid (<10 min) and nearly complete hepatic extraction of AF constructs from the systemic circulation. The catabolic processing and elimination of AF constructs from liver depend on the chemical modification used in the preparation of a given reagent. Radioiodinated AF has by far the shortest postabsorption (5.1 min +/- 0.05 min) and elimination half-lives (2.8 +/- 0.06 h) in liver. In comparison, the AF construct prepared by conjugation of DTPA- and 2-iminothiolane-substituted p-Lys with N-sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB)-modified AF (AF-SMPB-Traut-p-Lys-((111)In-DTPA)(20)(-)(30)) has a hepatic postabsorption time of 9.1 +/- 0.1 min and an elimination half-life of 44.3 +/- 3.08 h, whereas [(99m)Tc]technetium-labeled AF appears to be permanently retained in liver. These differences in rates of liver uptake and clearance of catabolized radiolabeled AF can be used to determine functional activity of liver and transplanted hepatocytes.

The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor.

The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation.