Hepatocyte Asialoglycoprotein Receptor Research Articles

In vivo evaluation of liver function by multimodal imaging in an alcohol-induced liver injury model.

Visually evaluating liver function is a hot topic in hepatology research. There are few reliable and practical visualization methods for evaluating the liver function in vivo in experimental studies. In this study, we established a multimodal imaging approach for in vivo liver function evaluation and compared healthy mice with chronic alcoholic liver injury (cALI) model mice to explore its potential applicability in experimental research. In vivo fluorescence imaging (IVFI) technology was utilized to visually represent the clearance of indocyanine green from the liver of both healthy mice and mice with cALI. The reserve liver function was evaluated via IVFI using the Cy5.5-galactosylated polylysine probe, which targets the asialoglycoprotein receptor of hepatocytes. Hepatic microcirculation was assessed through laser speckle perfusion imaging of hepatic blood perfusion. The liver microstructure was then investigated by in vivo confocal laser endomicroscopy imaging. Finally, hepatic asialoglycoprotein receptor expression, histology, and the levels of serum alanine aminotransferase and aspartate aminotransferase were measured. In vivo multimodal imaging results intuitively and dynamically showed that indocyanine green clearance [mean ± standard deviation (SD): 30.83±14.71, 95% confidence interval (CI): 20.3 to 41.35], the fluorescence signal intensity (mean ± SD: 1,217.92±117.63; 95% CI: 1,148.38 to 1,290.84) and fluorescence aggregation area (mean ± SD: 5,855.80±1,271.81; 95% CI: 5,051.57 to 6,653.88) of Cy5.5-galactosylated polylysine targeting the asialoglycoprotein receptor, and hepatic blood perfusion (mean ± SD: 1,494.86±299.33; 95% CI: 1,316.98 to 1,690.16) in model mice were significantly lower than those in healthy mice (all P<0.001). Compared to healthy mice, the model mice exhibited a significant decline in liver asialoglycoprotein receptor expression (mean ± SD: 219.03±16.34; 95% CI: 208.97 to 230.69; P<0.001), increased serum alanine aminotransferase (mean ± SD: 149.70±47.89 U/L; 95% CI: 81.75 to 128.89; P=0.01) and aspartate aminotransferase levels (mean ± SD: 106.30±36.13 U/L; 95% CI: 122.01 to 180.17; P=0.021), hepatocyte swelling and deformation, disappearance of the hepatic cord structure, partial necrosis, and disintegration of hepatocytes. The imaging features of fluorescence signals in liver regions, hepatic blood perfusion and microstructure were biologically related to hepatic asialoglycoprotein receptor expression, serum indices of liver function, and histopathology in model mice. Utilizing in vivo multimodal imaging technology to assess liver function is a viable approach for experimental research, providing dynamic and intuitive visual evaluations in a rapid manner.

NCOA4 Acts in Hepatocytes to Regulate Ferritin Subunit Levels and Hepatic Iron Storage

The intracellular protein nuclear receptor coactivator 4 (NCOA4) has been shown to mediate the degradation of the iron storage protein ferritin (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014). Accordingly, mice with global disruption of Ncoa4 show ferritin accumulation in multiple organs (Bellelli R et al., Cell Rep 2016). How NCOA4 activity in different tissues contributes to local and systemic iron homeostasis, however, has not yet been clearly defined. Here we selectively dissect the role of NCOA4 in the liver, the body's major iron depot.To facilitate targeted Ncoa4 knockdown in hepatocytes, we utilized Ncoa4 -targeting siRNA that was conjugated to triantennary N-acetylgalactosamine (GalNAc- Ncoa4 siRNA) to promote uptake by the hepatocyte asialoglycoprotein receptor. We first validated the ability of GalNAc- Ncoa4 siRNA treatment to target endogenous murine Ncoa4 mRNA for degradation in murine Hepa1-6 hepatoma cells. We next assessed the ability of GalNAc- Ncoa4 siRNA to acutely lower Ncoa4 expression in mouse liver. Eight-week-old C57BL/6N female mice were injected subcutaneously with 1, 3, or 5 mg/kg body weight of a sterile, endotoxin-tested solution of GalNAc- Ncoa4 siRNA in saline or with vehicle (saline) alone, and tissues were collected for analysis 24 hrs after injection. Compared to vehicle alone, GalNAc- Ncoa4 siRNA effectively lowered Ncoa4 expression in total liver RNA; because the degree of Ncoa4 knockdown that was achieved by the 3 mg/kg and 5 mg/kg doses was similar, the 3mg/kg dose was selected for our further experiments to reduce the possibility of off-target effects.We next examined the durability of hepatocyte Ncoa4 knockdown achieved by a single injection of GalNAc -Ncoa4 siRNA as well as the resulting effects on hepatic and systemic iron parameters. Eight-week-old C57BL/6N female mice were injected once with GalNAc- Ncoa4 siRNA or saline vehicle, and blood and tissues were collected for analysis at 1, 2, 3, or 4 weeks after injection. Compared to saline treatment, a single injection of GalNAc -Ncoa4 siRNA produced a significant, sustained reduction in hepatic Ncoa4 mRNA; Ncoa4 mRNA remained maximally suppressed at the 1, 2, and 3-week timepoints and began to rise slightly 4 weeks after injection. Saline-treated mice showed a slight, progressive increase in mean liver non-heme iron concentration (LIC) over the 4 weeks following injection, consistent with the known effect of age on murine hepatic iron stores. However, in mice injected with a single dose of GalNAc- Ncoa4 siRNA, the rise in LIC over the 4-week time period was significantly greater, consistent with increased hepatic iron storage. Immunoblotting of liver lysates revealed that levels of the ferritin heavy (FTH) and light (FTL) subunits increased in mice treated with GalNAc- Ncoa4 siRNA compared to saline-treated controls. These increases in FTH and FTL were evident 1 week after GalNAc- Ncoa4 siRNA injection, and FTH and FTL levels continued to rise with additional weeks of Ncoa4 knockdown. In mice treated with saline, spleen non-heme iron concentration (SIC) increased over the 4 weeks following injection, consistent with the known effect of age on murine splenic iron stores. However, at the 3 and 4-week time points, mice treated with GalNAc- Ncoa4 siRNA showed a significant reduction in SIC compared to saline-treated controls, which correlated histologically with reduced iron retention in splenic macrophages. Compared to saline treatment, GalNAc -Ncoa4 siRNAtreatment did not produce significant changes in spleen weight, serum iron, complete blood count, or non-heme iron concentration of heart, pancreas, or muscle.Collectively, our findings indicate that NCOA4 acts in hepatocytes to limit LIC as well as hepatic levels of FTH and FTL. Furthermore, local NCOA4 activity in hepatocytes impacts iron distribution to the spleen. Our results are compatible with a model in which degradation of the ferritin protein complex in hepatocytes occurs as a constitutive process that is mediated by NCOA4. Importantly, our findings demonstrate that in the setting of hepatocyte Ncoa4 knockdown, other cellular pathways cannot compensate to maintain normal LIC and normal hepatic levels of FTH and FTL. Additionally, our findings raise the possibility that enhancement of NCOA4 activity in hepatocytes might serve as a therapeutic approach to promote hepatic iron mobilization in iron overload disorders. DisclosuresNo relevant conflicts of interest to declare.

Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

BackgroundThe SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified.MethodsThe specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1).ResultsE12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC.ConclusionThe absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.

Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Diverse Virus and Host-Dependent Mechanisms Influence the Systemic and Intrahepatic Immune Responses in the Woodchuck Model of Hepatitis B.

Woodchuck infected with woodchuck hepatitis virus (WHV) represents the pathogenically nearest model of hepatitis B and associated hepatocellular carcinoma (HCC). This naturally occurring animal model also is highly valuable for development and preclinical evaluation of new anti-HBV agents and immunotherapies against chronic hepatitis (CH) B and HCC. Studies in this system uncovered a number of molecular and immunological processes which contribute or likely contribute to the immunopathogenesis of liver disease and modulation of the systemic and intrahepatic innate and adaptive immune responses during hepadnaviral infection. Among them, inhibition of presentation of the class I major histocompatibility complex on chronically infected hepatocytes and a role of WHV envelope proteins in this process, as well as augmented hepatocyte cytotoxicity mediated by constitutively expressed components of CD95 (Fas) ligand- and perforin-dependent pathways, capable of eliminating cells brought to contact with hepatocyte surface, including activated T lymphocytes, were uncovered. Other findings pointed to a role of autoimmune response against hepatocyte asialoglycoprotein receptor in augmenting severity of liver damage in hepadnaviral CH. It was also documented that WHV in the first few hours activates intrahepatic innate immunity that transiently decreases hepatic virus load. However, this activation is not translated in a timely manner to induction of virus-specific T cell response which appears to be hindered by defective activation of antigen presenting cells and presentation of viral epitopes to T cells. The early WHV infection also induces generalized polyclonal activation of T cells that precedes emergence of virus-specific T lymphocyte reactivity. The combination of these mechanisms hinder recognition of virus allowing its dissemination in the initial, asymptomatic stages of infection before adaptive cellular response became apparent. This review will highlight a range of diverse mechanisms uncovered in the woodchuck model which affect effectiveness of the anti-viral systemic and intrahepatic immune responses, and modify liver disease outcomes. Further exploration of these and other mechanisms, either already discovered or yet unknown, and their interactions should bring more comprehensive understanding of HBV pathogenesis and help to identify novel targets for therapeutic and preventive interventions. The woodchuck model is uniquely positioned to further contribute to these advances.

Synthesis of a new betulinic acid glycoconjugate with N-acetyl-d-galactosamine for the targeted delivery to hepatocellular carcinoma cells

A new promising conjugate of betulinic acid with N-acetyl-d-galactosamine was synthesized by the simple reaction sequence: esterification and copper-catalyzed azide-alkyne cycloaddition. The obtained glycoderivative exhibited high activity against hepatocarcinoma cell lines in vitro, selectivity of cytotoxic action, and excellent binding to the asialoglycoprotein receptor (ASGPR) of hepatocytes. Its affinity to the ASGPR was established by surface plasmon resonance spectroscopy and confirmed by molecular docking in silico. An original approach was proposed to enhance the cytotoxic properties of C-28 betulinic esters by introducing a hemioxalate fragment bearing free carboxyl group into the C(3) position of ring A.

New ASGPR-targeted ligands based on glycoconjugated natural triterpenoids

New ligands for targeted delivery to the liver based on glycoconjugated natural triterpenoids are proposed. Simple chemical transformations of starting lupane triterpenes led to betulin or betulinic acid conjugates with N-acetyl-d-galactosamine linked through an isopropenyl fragment of the terpene skeleton. The obtained compounds were non-toxic in vitro to a series of cancer cell lines and normal cells. Molecular docking in silico and surface plasmon resonance spectroscopy revealed a high specificity of the synthesized glycotriterpenoids to the asialogly-coprotein receptor of hepatocytes.

Synthesis of allobetulin-based asialoglycoprotein receptor-targeted glycoconjugates

New allobetulin conjugates were obtained through its O-esterification with hex-5-ynoic acid followed by [3+2]-cycloaddition with three azido derivatives of N-acetyl- d -galactosamine. The conjugates are non-toxic in micromolar range against hepatocellular carcinoma cell lines and have a high affinity towards the HO asialoglycoprotein receptor of hepatocytes based on molecular docking and surface plasmon resonance data.

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.

Hydroxyl-Rich PGMA-Based Cationic Glycopolymers for Intracellular siRNA Delivery: Biocompatibility and Effect of Sugar Decoration Degree.

The ErbB family of proteins, structurally related to the epidermal growth factor receptor (EGFR), is found to be overexpressed in many cancers such as gliomas,a lung and cervical carcinomas. Gene therapy allows to modify the expression of genes like ErbB and has been a promising strategy to target oncogenes and tumor suppressor genes. In the current work, novel hydroxyl-rich poly(glycidyl methacrylate) (PGMA)-based cationic glycopolymers were designed for intracellular small interfering RNA (siRNA) delivery to silence the EGFR gene. The cationic polymers with different sugar decoration degrees (0, 9, and 33%) were synthesized by ring-opening reaction of PGMA with ethanolamine and a lactobionic acid-derived aminosaccharide (Lac-NH2). Specific EGFR knockdown of the protein tyrosine kinase ErbB-overexpressing HeLa cells was achieved using these hydroxyl-rich polycation/siRNA complexes. Higher sugar content improved the biocompatibility of the polymers, but it also seems to decrease the EGFR knockdown capability, which should mainly be related to the surface charge of polyplexes. An optimum balance was observed with PGEL-1 (9% sugar content) formulation, achieving ∼52% knockdown efficiency as well as high cell viability. Considering the specific recognition between galactose residues and asialoglycoprotein receptor in hepatocytes, our novel PGMA-based cationic glycopolymers exhibited promising future to serve as a safe and targeting gene delivery vector to hepatoma cell line like HepG2.

Promising positive liver targeting delivery system based on arabinogalactan-anchored polymeric micelles of norcantharidin

Liver cancer is the third most common cause of global cancer-related deaths. This study focused on newly developed drug delivery systems with hepatocyte asialoglycoprotein receptor binding targeting the liver. Although norcantharidin (NCTD) is effective in primary liver cancer treatment, its toxicity in the urinary system remains. Positive liver-targeting effect could be achieved by preparing polymer micelles by arabinogalactan on the surface of N-(4-methylimidazole)-hydroxyethyl-chitosan (MHC). HepG2 cells were used to analyze the cytotoxicity, invasion, apoptosis and uptake of NCTD-loaded micelles. The in vivo antitumor efficacy of NCTD-M was evaluated using tumor-bearing nude mice. Successful preparation of NCTD-M was shown. In vivo imaging showed that micelles significantly increased positive liver drug targeting. Laser confocal microscopy showed increased cellular uptake of micelles. NCTD-M also enhanced cell invasion and the proportion of apoptotic cells. Compared with the other groups, the micelles showed better antitumor effects in vivo. Therefore, the positive liver-targeting NCTD-M, which can enhance antitumor efficacy and reduce toxicity, could be a promising and effective therapeutic agent for liver cancer treatment.

Augmented liver targeting of exosomes by surface modification with cationized pullulan.

Exosomes have shown potentials as therapeutics for various inflammatory disease models. This study is the first to show the specific accumulation of exosomes in the liver and enhanced anti-inflammatory effect via the surface modification of exosomes using pullulan, which is specifically recognized by the asialoglycoprotein receptor (AGPR) on HepG2 cells and hepatocytes. The pullulan was expressed on the surface of PKH-labeled exosomes, and it led increased accumulation of PKH into HepG2 cells, whereas the accumulation was canceled by AGPR inhibitor. In the mouse liver injury model, the modification of PKH-labeled exosomes with pullulan enabled increased accumulation of PKH specifically in the injured liver. Furthermore the greater therapeutic effects against the liver injury compared with unmodified original exosomes was observed.

Fabrication of 3D-culture platform with sandwich architecture for preserving liver-specific functions of hepatocytes using 3D bioprinter.

The development of new three-dimensional (3D) cell culture system that maintains the physiologically relevant signals of hepatocytes is essential in drug discovery and tissue engineering research. Conventional two-dimensional (2D) culture yields cell growth, proliferation, and differentiation. However, gene expression and signaling profiles can be different from in vivo environment. Here, we report the fabrication of a 3D culture system using an artificial scaffold and our custom-made inkjet 3D bioprinter as a new strategy for studying liver-specific functions of hepatocytes. We built a 3D culture platform for hepatocytes-attachment and formation of cell monolayer by interacting the galactose chain of galactosylated alginate gel (GA-gel) with asialoglycoprotein receptor (ASGPR) of hepatocytes. The 3D geometrical arrangement of cells was controlled by using 3D bioprinter, and cell polarity was controlled with the galactosylated hydrogels. The fabricated GA-gel was able to successfully promote adhesion of hepatocytes. To observe liver-specific functions and to mimic hepatic cord, an additional parallel layer of hepatocytes was generated using two gel sheets. These results indicated that GA-gel biomimetic matrices can be used as a 3D culture system that could be effective for the engineering of liver tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1583-1592, 2017.

Fabrication of stable galactosylated alginate microcapsules via covalent coupling onto hydroxyl groups for hepatocytes applications

Galactose moieties are covalently coupled with sodium alginate to enhance liver-specific functions in microcapsules owing to the specific interaction between the galactose moieties and the asialoglycoprotein receptors (ASGPRs) of hepatocytes. In this study, galactosylated alginate (L-NH2-OH-alginate) based microcapsules with desirable stability and a suitable 3D microenvironment are designed and fabricated for primary hepatocyte applications. The designed L-NH2-OH-alginate is fabricated via the application of ethylenediamine grafted lactobionic acid (L-NH2) onto the hydroxyl groups of sodium alginate so that the negatively charged carboxyl groups intact in L-NH2-OH-alginate can effectively bond with Ca2+ to form a stable three-dimensional gel network; a subsequent reaction with polycations forms a stable membrane of microcapsules. As a result, L-NH2-OH-alginate based microcapsules exhibit an excellent mechanical stability. Moreover, with a higher degree of substitution in L-NH2-OH-alginate (DS 0.41), the hepatocytes entrapped in L-NH2-OH-alginate microcapsules exhibit better viability and well-maintained liver-specific functions.

CD8(+) T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia.

In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP.

Multistage Delivery Technologies: Multifunctional, Interdisciplinary Approaches to Nanomedicine

Nanomedicine frequently employs single particles ranging from very simple1 to highly multifunctional.2 However, the diversity of barriers to effective transport can limit the global optimization of delivery for such systems. Multistage particles present an effective means to overcome such issues, allowing scientists to better segment the delivery mechanism into discrete steps by combining multiple formulations for sequential utility in vivo. A recent article in Nature Biotechnology highlights such a system,3 delivering nanoformulated doxorubicin (Dox) within larger disc-shaped, mesoporous silica particles (MSPs).

Galactosylated magnetic nanovectors for regulation of lipid metabolism based on biomarker-specific RNAi and MR imaging

The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic–inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.

Novel bivalent spermine-based neutral neogalactolipids for modular gene delivery systems

New bivalent spermine-based neutral neogalactolipids have been synthesized to develop effective modular gene delivery systems targeting hepatocyte asialoglycoprotein receptors.

Hepatocyte targeting using pegylated asialofetuin-conjugated liposomes

Background and purpose: The hepatocyte asialoglycoprotein receptor mediates uptake of desiaylated glycoproteins by receptor-mediated endocytosis. This work explores a hepatocyte-specific targeting strategy using asialofetuin (AF) covalently coupled to pegylated liposomes.Methods: AF was conjugated to the distal end of polyethylene glycol–functionalized phospholipids. Chemical modification of AF did not interfere with its receptor interaction. AF-liposomes had a size of less than 130 nm, were judged to be monodisperse and were labelled with fluorescent organic dyes or loaded with quantum dots.Results: In vitro, binding and cellular uptake of fluorescent AF-liposomes by HepG2 hepatocellular carcinoma cells were reduced at low temperature and could be competitively inhibited by an excess of unbound AF. Hepatocyte-specific targeting and internalization of AF-liposomes in vivo was confirmed in the rat and could be competitively inhibited by co-injection of unbound AF. In contrast, non-pegylated liposomes accumulated in cells of the reticuloendothelial system such as hepatic Kupffer cells and spleen after intravenous administration.Conclusion: We conclude that the use of AF-conjugated, pegylated liposomes is a promising strategy to avoid the reticuloendothelial system and specifically target hepatocytes via the asialoglycoprotein receptor in vitro as well as in vivo.

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20–80min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA–primaquine conjugate (TriGalNAc-PGA–PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250ng per million cells of the conjugate bound to one million rat hepatocytes at 0°C, and approximately 2μg per million cells of the conjugate was taken up over 7h incubation at 37°C. Furthermore, our results suggested that TriGalNAc-PGA–PQ was almost completely degraded over 24h, and small degradation products were secreted into cell culture medium.The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.