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Sample records for asialoglycoproteins

  1. Glycomimetic ligands for the human asialoglycoprotein receptor.

    Science.gov (United States)

    Mamidyala, Sreeman K; Dutta, Sanjay; Chrunyk, Boris A; Préville, Cathy; Wang, Hong; Withka, Jane M; McColl, Alexander; Subashi, Timothy A; Hawrylik, Steven J; Griffor, Matthew C; Kim, Sung; Pfefferkorn, Jeffrey A; Price, David A; Menhaji-Klotz, Elnaz; Mascitti, Vincent; Finn, M G

    2012-02-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

  2. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  3. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R)

    OpenAIRE

    Anna Maria Bianucci; Ilaria Massarelli; Federica Chiellini; Emo Chiellini

    2010-01-01

    The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal...

  4. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity

    OpenAIRE

    Roggenbuck, Dirk; Mytilinaiou, Maria G.; Lapin, Sergey V.; Reinhold, Dirk; Conrad, Karsten

    2012-01-01

    Asialoglycoprotein receptor (ASGPR) autoantibodies have been considered specific markers of autoimmune hepatitis (AIH). The exact mechanisms responsible for the development of these autoantibodies and leading to autoimmunity to this peculiar liver receptor remain elusive. Furthermore, loss of T cell tolerance to ASGPR has been demonstrated in patients with AIH, but it is poorly understood whether such liver-specific T cell responses bear a pathogenic potential and/or participate in the precip...

  5. (18)F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model.

    Science.gov (United States)

    Kao, Hao-Wen; Chen, Chuan-Lin; Chang, Wen-Yi; Chen, Jenn-Tzong; Lin, Wuu-Jyh; Liu, Ren-Shyan; Wang, Hsin-Ell

    2013-02-15

    Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-(18)F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide ((18)F-FBHGal), a new (18)F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P ASGPR blocking agent. Animal studies showed the accumulation of (18)F-FBHGal in fibrosis liver (14.84±1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50±1.51 %ID/g, P ASGPR-related liver dysfunction.

  6. Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

    OpenAIRE

    McPhaul, M; Berg, P.

    1986-01-01

    The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to ...

  7. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R

    Directory of Open Access Journals (Sweden)

    Anna Maria Bianucci

    2010-10-01

    Full Text Available The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R, by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2, which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal oligomeric form, necessary for it to bind multi-antennary carbohydrate ligands. The ultimate target of the study is to contribute to increasing the knowledge of interactions between the human ASGP-R and carbohydrate ligands, at the molecular level, pertinent to applications in the field of hepatic tissue engineering.

  8. Asialoglycoprotein receptor (ASGPR) as target autoantigen in liver autoimmunity: lost and found.

    Science.gov (United States)

    Rigopoulou, Eirini I; Roggenbuck, Dirk; Smyk, Daniel S; Liaskos, Christos; Mytilinaiou, Maria G; Feist, Eugen; Conrad, Karsten; Bogdanos, Dimitrios P

    2012-12-01

    Asialoglycoprotein receptor (ASGPR) has attracted the attention of liver immunologists for many years. This liver-specific lectin was found to be a major B and T cell autoantigenic target in patients with autoimmune liver diseases, and in particular in autoimmune hepatitis (AIH). This review discusses the biological significance of ASGPR and its relevance to the pathogenesis of autoimmune and virus-triggered liver diseases. We also discuss emerging data on the diagnostic and clinical relevance of anti-ASGPR antibodies in light of recent reports based on commercially available anti-ASGPR enzyme-linked immunosorbent assays. Finally, we critically revisit the data reporting on disease-specific cellular immune responses against ASGPR and their relevance in relation to the pathogenesis of AIH.

  9. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity.

    Science.gov (United States)

    Roggenbuck, Dirk; Mytilinaiou, Maria G; Lapin, Sergey V; Reinhold, Dirk; Conrad, Karsten

    2012-12-01

    Asialoglycoprotein receptor (ASGPR) autoantibodies have been considered specific markers of autoimmune hepatitis (AIH). The exact mechanisms responsible for the development of these autoantibodies and leading to autoimmunity to this peculiar liver receptor remain elusive. Furthermore, loss of T cell tolerance to ASGPR has been demonstrated in patients with AIH, but it is poorly understood whether such liver-specific T cell responses bear a pathogenic potential and/or participate in the precipitation of AIH. Newly developed enzyme-linked immunosorbent assays have led to the investigation of the sensitivity and specificity of anti-ASGPR antibodies for AIH. The present review provides an overview of the diagnostic and clinical relevance of anti-ASGPR antibodies. A thorough investigation of the autoreactivity against ASGPR may assist efforts to understand liver autoimmunity in susceptible individuals.

  10. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  11. Clinical significance of anti-asialoglycoprotein receptor autoantibodies, detected by a capture-immunoassay, in autoimmune liver diseases

    OpenAIRE

    森末, 佳子

    2002-01-01

    In the development of autoimmune chronic acive hepatitis (AIH), the pathogenic relevance of antimmune responses against asialoglycoprotein recepter (ASGPR) has been implicated. We have previously developed a capture enzyme-linked immunsorbent assay (ELISA) for detection of anti-ASGPR antibodies and found a high prevalence of anti-ASGPR antibodies in AIH and primary biliary cirrhosis (PBC). IN this study, to clarify the clinical singnifi-cance of the measurement of anti-ASGPR antibodies in aut...

  12. Has asialoglycoprotein receptor (ASGP-R) a role to play in binding and processing of different parasites?

    OpenAIRE

    2002-01-01

    Liver asialoglycoprotein receptor (ASGP-R), which specifically recognizes and binds galactose and N-acetyl galactosamine, has been implicated in binding and endocytosis of glycoproteins. Therefore, the possibility that it may have a role in contacting and processing pathogenic organisms was investigated. The interaction in vitro between ASGP-R and surface oligosaccharide structures of Echinococcus granulosus and Trichinella spiralis was studied by immunohistochemical methods. Specific binding...

  13. Constant serum levels of secreted asialoglycoprotein receptor sH2a and decrease with cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Ron Benyair; Maria Kondratyev; Elena Veselki; Sandra Tolchinsky; Marina Shenkman; Yoav Lurie; Gerardo Z Lederkremer

    2011-01-01

    AIM: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum.METHODS: Production of recombinant sH2a and development of a monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA).This assay was used to determine the presence and concentration of sH2a in human sera of individuals of both sexes and a wide range of ages.RESULTS: The recombinant protein was produced successfully and a specific ELISA assay was developed. The levels of sH2a in sera from 62 healthy individuals varied minimally (147 ± 19 ng/mL).In contrast, 5 hepatitis C patients with cirrhosis showed much decreased sH2a levels (50 ± 9 ng/mL).CONCLUSION: Constant sH2a levels suggest constitutive secretion from hepatocytes in healthy individuals.This constant level and the decrease with cirrhosis suggest a diagnostic potential.

  14. Impact of asialoglycoprotein receptor deficiency on the development of liver injury

    Institute of Scientific and Technical Information of China (English)

    Serene ML Lee; Carol A Casey; Benita L McVicker

    2009-01-01

    The asialoglycoprotein (ASGP) receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis (RME). The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the missorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously,we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease (ALD). The studies revealed profound ethanolmediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations,studies were performed utilizing knockout mice (lacking a functional ASGP receptor) to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti-Fas (CD95) antibody, carbon tetrachloride (CCl4) and lipopolysaccharide (LPS)/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels,histopathological scores, as well as hepatocellular death.Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxinmediated liver diseases.

  15. Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

    Directory of Open Access Journals (Sweden)

    Hirai M

    2002-06-01

    Full Text Available Anti-idiotype antibodies (Ab2 play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH. We previously developed a mouse monoclonal antibody (clone 8D7 which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1. One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

  16. Tailored Presentation of Carbohydrates on a Coiled Coil-Based Scaffold for Asialoglycoprotein Receptor Targeting.

    Science.gov (United States)

    Zacco, Elsa; Hütter, Julia; Heier, Jason L; Mortier, Jérémie; Seeberger, Peter H; Lepenies, Bernd; Koksch, Beate

    2015-09-18

    The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate. We determined the binding of the glycopeptides to the hepatocytes, and we established the optimal distance and orientation of the galactose moieties for interaction with the ASGPR using flow cytometry. We confirmed that the binding occurs through endocytosis mediated by ASGPR via inhibition studies with cytochalasin D; fluorescence microscopy studies display the uptake of the carrier peptides inside the cell. Thus, this study demonstrates that the coiled-coil motif can be used as reliable scaffold for the rational presentation of ligands.

  17. Galactosylated manganese ferrite nanoparticles for targeted MR imaging of asialoglycoprotein receptor.

    Science.gov (United States)

    Yang, Seung-Hyun; Heo, Dan; Lee, Eugene; Kim, Eunjung; Lim, Eun-Kyung; Lee, Young Han; Haam, Seungjoo; Suh, Jin-Suck; Huh, Yong-Min; Yang, Jaemoon; Park, Sahng Wook

    2013-11-29

    Cancer cells can express specific biomarkers, such as cell membrane proteins and signaling factors. Thus, finding biomarkers and delivering diagnostic agents are important in the diagnosis of cancer. In this study, we investigated a biomarker imaging agent for the diagnosis of hepatic cancers. The asialoglycoprotein receptor (ASGPr) was selected as a biomarker for hepatoma cells and the ASGPr-targetable imaging agent bearing a galactosyl group was prepared using manganese ferrite nanoparticles (MFNP) and galactosylgluconic acid. The utility of the ASGPr-targetable imaging agent, galactosylated MFNP (G-MFNP) was assessed by several methods in ASGPr-expressing HepG2 cells as target cells and ASGPr-deficient MCF7 cells. Physical and chemical properties of G-MFNP were examined using Fourier-transform infrared spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. No significant cytotoxicity was observed in either cell line. Targeting ability was assessed using flow cytometry, magnetic resonance imaging, inductively coupled plasma atomic emission spectroscopy, absorbance analysis, dark-field microscopy, Prussian blue staining, and transmission electron microscopy. We demonstrated that G-MFNP target successfully and bind to ASGPr-expressing HepG2 cells specifically. We suggest that these results will be useful in strategies for cancer diagnoses based on magnetic resonance imaging.

  18. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    Science.gov (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  19. Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

    Institute of Scientific and Technical Information of China (English)

    YANG Yan; HUANG Huang; ZHANG Zhenghua; WANG Baoju; TIAN Yongjun; LU Mengji; YANG Dongliang

    2007-01-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  20. A New Splice Variant of the Major Subunit of Human Asialoglycoprotein Receptor Encodes a Secreted Form in Hepatocytes

    OpenAIRE

    Jia Liu; Bin Hu; Yan Yang; Zhiyong Ma; Yuan Yu; Shenpei Liu; Baoju Wang; Xiping Zhao; Mengji Lu; Dongliang Yang

    2010-01-01

    BACKGROUND: The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR geno...

  1. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice.

    Science.gov (United States)

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J

    2013-05-01

    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

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

    Science.gov (United States)

    Pathak, Pankaj Omprakash; Nagarsenker, Mangal Shailesh; Barhate, Chandrashekhar Rishikant; Padhye, Sameer Govind; Dhawan, Vivek Vijay; Bhattacharyya, Dibyendu; Viswanathan, C L; Steiniger, Frank; Fahr, Alfred

    2015-05-18

    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 m

  3. Cellular distribution of {sup 111}In-LDTPA galactose BSA in normal and asialoglycoprotein receptor-deficient mouse liver

    Energy Technology Data Exchange (ETDEWEB)

    Deal, Kim A.; Cristel, Michael E.; Welch, Michael J

    1998-05-01

    {sup 111}In-LDTPA galactose BSA (bovine serum albumin) was used to evaluate the asialoglycoprotein receptor (ASGPR) system in both normal and ASGPR-deficient mice. The radiolabeled glycoprotein had complete liver uptake in both normal and ASGPR-deficient mice. Metabolism and hepatic cell-type distribution studies were performed. The normal mouse excreted greater than 60% of the hepatic activity, while the ASGPR-deficient mouse excreted less than 40% of the hepatic activity. {sup 111}In-LDTPA galactose BSA was metabolized to {sup 111}In-LDTPA-L-lysine in both mouse types. Normal mice showed 70% of the radioactivity in the hepatocyte, whereas the homozygous ASGPR-deficient mouse had equal activity in the hepatocyte and the hepatic endothelial cell.

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

    Energy Technology Data Exchange (ETDEWEB)

    Vasanthan, Kirthanashri S.; Sethuraman, Swaminathan; Parthasarathy, Meera, E-mail: meera_p@scbt.sastra.edu

    2015-08-26

    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. - Highlights: • A methodology for electrochemical imaging of polymer scaffolds is proposed. • The new methodology allows

  5. Anti-asialoglycoprotein receptor autoantibodies, detected by a capture-immunoassay, are associated with autoimmune liver diseases.

    Directory of Open Access Journals (Sweden)

    Yoshioka M

    2002-04-01

    Full Text Available In autoimmune chronic active hepatitis (AIH and primary biliary cirrhosis (PBC, various autoantibodies including anti-asialoglycoprotein receptor (ASGPR antibodies have been found in patients' sera. We have previously developed a mouse monoclonal antibody against rat and human ASGPR. In this study, we developed a capture enzyme-linked immunosorbent assay (ELISA for detection of anti-ASGPR antibodies using this monoclonal antibody and investigated the occurrence of anti-ASGPR antibodies in the sera of patients with various liver diseases. Serum samples were obtained from 123 patients with various liver diseases, including 21 patients with AIH and 40 patients with PBC. In this capture ELISA, the target antigen in the crude rat liver membrane extracts was captured on the ELISA wells by the ASGPR-specific mouse monoclonal antibody. Thus, the cumbersome process of antigen purification was rendered unnecessary. Using this capture ELISA, we detected the anti-ASGPR antibody in 67% of the patients with AIH, in 100% of the patients with PBC, and in 57% of the patients with acute hepatitis type A. However, the anti-ASGPR antibody was rarely detected in patients with other liver diseases such as primary sclerosing cholangitis and obstructive jaundice. Our findings suggest that this capture ELISA would be useful for the detection of anti-ASGPR antibodies in autoimmune liver diseases.

  6. Physiological roles of asialoglycoprotein receptors (ASGPRs) variants and recent advances in hepatic-targeted delivery of therapeutic molecules via ASGPRs.

    Science.gov (United States)

    Hu, Jing; Liu, Jia; Yang, Dongliang; Lu, Mengji; Yin, Jian

    2014-01-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity C-type lectin receptor mainly expressed on mammalian hepatic cells. The physiological function of ASGPR has not been completely clarified and is thought to be specific binding and internalization of galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminating glycoproteins by hepatocytes. The human ASGPR is comprised of two homologous polypeptides, H1 and H2. ASGPR H1 has two splice variants (H1a and H1b) and ASGPR H2 has three splice variants (H2a, H2b, and H2c). These variants have been discovered to exist both in human liver tissues and in human hepatoma cells. Variant H1b, which has an in-frame deletion of exon 2 resulting in the loss of the transmembrane domain and is secreted as a soluble protein, encodes functional soluble ASGPR (s- ASGPR). Based on our previous results, we proposed the possible physiological function of s-ASGPR, which is well interpreted in the Galactosyl Homeostasis Hypothesis proposed by Weigel. ASGPR is one of the most promising targets for hepatic delivery. In this review, the recent progresses of cationic polysomes and liposomes as effective non-viral delivery system via ASGPR are also presented.

  7. Radiolabelled {sup 153}Sm-chelates of glycoconjugates: multivalence and topology effects on the targeting of the asialoglycoprotein receptor

    Energy Technology Data Exchange (ETDEWEB)

    Torres, S. [Centro de Quimica, Campus de Gualtar, Univ. do Minho, Braga (Portugal); Martins, J.A.; Andre, J.P.; Neves, M. [Inst. Tecnologico e Nuclear, Sacavem (Portugal); Santos, A.C.; Prata, M.I.M. [Servico de Biofisica, IBILI, Univ. de Coimbra (Portugal); Geraldes, C.F.G.C. [Dept. de Bioquimica, Centro de Espectroscopia RMN e Centro de Neurociencias e Biologia Celular, Univ. de Coimbra (Portugal)

    2007-07-01

    In this paper we report and discuss the biodistribution studies with Wistar rats of a series of {sup 153}Sm(III)-glycoconjugates, based on DO3A and DO2A(cis) scaffolds (DO3A = 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane; DO2A(cis) = 1,4-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane). The effects of changing the sugar type (galactose, lactose and glucose), valency (mono and divalent) and topology on the targeting ability of the liver asialoglycoprotein receptor (ASGPR) are evaluated. Divalent glycoconjugates with different topologies were generated by a pendant glycodendrimeric (generation 1) architecture on a DO3A scaffold and by a linear DO2A(cis)-bis derivative. The results show that the galactose conjugates are more target efficient than the lactose analogues, while the glucose conjugates have no liver targeting ability. Divalent galactose conjugates are more efficiently targeted to the liver than the monovalent ones, while the dendrimeric topology of DO3A-Gal{sub 2} has higher targeting efficiency than that of the DO2A(cis)-Gal{sub 2}. (orig.)

  8. Estimation of hepatic functional reserve by asialoglycoprotein receptor-binding, radiolabeled synthetic ligand 'Tc-99m-galactosyl-neoglycoalbumin'. Preclinical and clinical studies

    Energy Technology Data Exchange (ETDEWEB)

    Kudo, Masatoshi; Todo, Akio; Ikekubo, Katsuji

    1987-11-01

    Tc-99m-Galactosyl-Neoglycoalbumin (Tc-99m-NGA) is a receptor binding radiolabeled ligand, which binds to the asialoglycoprotein receptor of the plasma membrane of hepatocytes. NGA was prepared by covalently coupling D-galactose to normal human albumin. NGA was labeled with technetium-99m according to the electrolytic method by Benjamin, which produced Tc-99m-NGA with labeling yield in excess of 95%. Tc-99m-NGA was proved to be stable for at least 4 hours as determined by high performance liquid chromatography. Biodistribution studies of Tc-99m-NGA in normal rats demonstrated that liver is a sole target organ of Tc-99m-NGA. The lack of hepatic uptake of Tc-99m-NGA in healthy chicken corresponded to the lack of the asialoglycoprotein receptors in avian species. Tc-99m-NGA study in a normal volunteer showed apparently different images from those of Tc-99m-pyridoxyl-5-methyltryptophan (PMT) or Tc-99m phytate, suggesting that Tc-99m-NGA is a novel liver imaging radiopharmaceutical. The estimation of the time activity curves in the liver corresponded to the hepatic functional reserves which were evaluated by existing method in 9 clinical cases (2: Normal volunteers, 1: Chronic Active Hepatitis, 1: Liver Cirrhosis (Child A), 2: Liver Cirrhosis (Child B), 3: Liver Cirrhosis (Child C)). The NGA is useful as an imaging agent as well as in the estimation of the hepatic functional reserve, as the dynamic curves of the liver correlates to the asialoglycoprotein receptor concentration.

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

    Science.gov (United States)

    Vasanthan, Kirthanashri S; Sethuraman, Swaminathan; Parthasarathy, Meera

    2015-08-26

    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.

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

    Science.gov (United States)

    Li, Yan; Huang, Guifang; Diakur, James; Wiebe, Leonard I

    2008-10-01

    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.

  11. The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes.

    Science.gov (United States)

    Seested, Torben; Nielsen, Hanne M; Christensen, Erik I; Appa, Rupa S

    2010-12-01

    Recombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ perfused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.

  12. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  13. ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor, Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

    Directory of Open Access Journals (Sweden)

    Rebecca Louise Harris

    2012-01-01

    Full Text Available Background. The asialoglycoprotein receptor (ASGPR is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2, encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR, expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver.

  14. 肝脏去唾液酸糖蛋白受体的研究进展%Research progress of the asialoglycoprotein receptor in liver

    Institute of Scientific and Technical Information of China (English)

    石红; 刘健; 余进洪

    2014-01-01

    肝脏去唾液酸糖蛋白受体(ASGPR)与肝脏疾病的发生发展密切相关,去唾液酸糖蛋白受体用于临床,对于肝脏疾病的早期诊断以及指导治疗和评价预后有重要的临床意义。本文介绍了去唾液酸糖蛋白受体的发生发展过程及国内外的研究进展,并对其未来的发展方向做了展望。%The asialoglycoprotein receptor (ASGPR), which is closely related to the development of liver diseases, has important clinical significance in the early diagnosis of liver diseases and guiding treatment and evaluating prognosis. This paper introduced the progress of ASGPR both at home and abroad, and made the outlook for its future development direction.

  15. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

    Science.gov (United States)

    Chen, Cheng; Li, Kesang; Jiang, Hua; Song, Fei; Gao, Huiping; Pan, Xiaorong; Shi, Bizhi; Bi, Yanyu; Wang, Huamao; Wang, Hongyang; Li, Zonghai

    2017-04-01

    Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1(+)GPC3(-) tumor cells, but they exhibited a similar cytotoxicity against GPC3(+)ASGR1(-) and GPC3(+)ASGR1(+) HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3(+)ASGR1(+) HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3(+)ASGR1(+) HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3(+)ASGR1(+) HCC.

  16. Detection of circulating tumor cells in hepatocellular carcinoma using antibodies against asialoglycoprotein receptor, carbamoyl phosphate synthetase 1 and pan-cytokeratin.

    Directory of Open Access Journals (Sweden)

    Jun Li

    Full Text Available BACKGROUND: Asialoglycoprotein receptor (ASGPR-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK antibody have been demonstrated to detect circulating tumor cells (CTCs in hepatocellular carcinoma (HCC. The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. METHODS: The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1 antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. RESULTS: ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. CONCLUSIONS: Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.

  17. Understanding the Selectivity Mechanism of the Human Asialoglycoprotein Receptor (ASGP-R toward Gal- and Man- type Ligands for Predicting Interactions with Exogenous Sugars

    Directory of Open Access Journals (Sweden)

    Emo Chiellini

    2007-01-01

    Full Text Available A practical approach for addressing the computer simulation of protein-carbohydrate interactions is described here. An articulated computational protocol was setup and validated by checking its ability to predict experimental data, available in theliterature, and concerning the selectivity shown by the Carbohydrate Recognition Domain(CRD of the human asialoglycoprotein receptor (ASGP-R toward Gal-type ligands. Somerequired features responsible for the interactions were identified. Subsequently the sameprotocol was applied to monomer sugar molecules that constitute the building blocks foralginates and ulvans. Such sugar polymers may supply a low-cost source of rare sugars witha potential impact on several industrial applications, from pharmaceutical to fine chemicalindustry. An example of their applicative exploitation could be given by their use indeveloping biomaterial with adhesion properties toward hepatocytes, through interactionwith the ASGP-R. Such a receptor has been already proposed as a target for exogenousmolecules, specifically in the case of hepatocytes, for diagnostic and therapeutic purposes.The DOCK5.2 program was used to search optimal locations of the above ligands of interestinto CRD binding site and to roughly estimate interaction energies. Finally, the binding ∆G oftheoretical protein-ligand complexes was estimated by using the DelPhi program in which thesolvation free energy is accounted for with a continuum solvent model, by solving the Poisson-Boltzmann equation. The structure analysis of the obtained complexes and their ∆G values suggest that one of the sugar monomers of interest shows the desired characteristics.

  18. 去唾液酸糖蛋白受体介导的肝脏靶向性研究进展%Study on asialoglycoprotein receptor-mediated liver targeting:current progress

    Institute of Scientific and Technical Information of China (English)

    徐文; 殷正丰

    2006-01-01

    去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)又称半乳糖受体,主要表达于哺乳动物肝窦状隙的肝实质细胞表面,参与多种生理功能.多年来ASGPR一直被用于介导药物和基因的肝靶向递送以及肝脏成像等方面的研究,目前已取得许多进展.ASGPR介导的药物肝靶向递送研究主要集中在抗肿瘤药物、降胆固醇药物等.基因肝靶向递送多见于反义药物.肝脏成像研究包括评价肝脏功能、鉴别肝细胞肝癌和肿瘤肝转移灶等.近年来ASGPR的靶向性应用研究还进一步扩展到肝细胞立体培养、肝细胞筛选及肝细胞移植等领域.本文就这些内容作一综述.

  19. Selection and Identification of RNA Aptamer Specific for Hepatic Asialoglycoprotein Receptor by SELEX%肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    刘嘉; 杨东亮; 杨燕; 胡斌; 马智勇; 余源; 黄红平; 陆蒙吉; 冯新华; 郭培宣

    2009-01-01

    目的 获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础.方法 合成一个长度为115 nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX(systematic evolution of ligands by exponential enrichment)技术筛选具有高亲和力的ASGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力.结果 经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子.结论 成功地筛选出了具有高亲和力的肝脏ASGPR特异性RNA适配子库.

  20. Study on the preparation of ligand of hepatic asialoglycoprotein receptor%肝脏去唾液酸糖蛋白受体的配体制备研究

    Institute of Scientific and Technical Information of China (English)

    石红; 余进洪; 刘健

    2015-01-01

    目的:优化制备出具有高效性的肝脏去唾液酸糖蛋白受体(ASGPR)的配体半乳糖化多聚赖氨酸(Gal-PLL),为后期肝靶向性纳米液态氟碳微球超声造影剂的制备以及肝脏靶向分子成像提供依据。方法采用还原胺化法,根据各反应组分的比例不同总体分为实验组(A、B组)和对照组(C组),A、B组又分别细分成3组。 A组将3种不同摩尔比的D-半乳糖和多聚赖氨酸分别与等量的足量还原剂硼氢化钠(NaBH4)进行反应,B组将相同摩尔比的D-半乳糖和多聚赖氨酸分别与3种不同量的还原剂进行反应。各组产物均经葡聚糖凝胶柱分离纯化,得到不同化合物分子质量的达峰曲线并进行分析。结果当还原剂等量时,适量减少D-半乳糖的量,分离纯化的化合物分子质量的达峰曲线提前;当D-半乳糖与多聚赖氨酸摩尔比相等时,适当减少还原剂的量,分离纯化的化合物分子质量的达峰曲线亦出现提前;当D-半乳糖与还原剂的摩尔比为1∶1时,反应合成的半乳糖化多聚赖氨酸化合物与游离组分的曲线分离最为明显,且化合物的量达到最大值。结论当还原剂等量时,适量减少D-半乳糖可增强D-半乳糖与多聚赖氨酸的偶联效果;而当D-半乳糖与多聚赖氨酸摩尔比相等时,适量减少还原剂,D-半乳糖与多聚赖氨酸的偶联效果更佳;当D-半乳糖与还原剂的摩尔比为1∶1时,D-半乳糖与多聚赖氨酸的偶联效果最好。D-半乳糖与多聚赖氨酸的偶联效果不仅与2者的配比相关,也与D-半乳糖与还原剂的配比相关。%Objective To optimize the preparation of high-efficiency galactocylated poly-L-lysine (Gal-PLL) ligand of the asialoglycoprotein receptor in liver, providing premise and foundation for upper preparation of ultrasound contrast agent of liver targeted nanoscale perfluorocarbon microballoon and the liver

  1. The Experimental Research on vp3 Gene Targeting Therapy for Hepatoma by Asialoglycoprotein Receptor In Vivo%脱唾液酸糖蛋白受体介导的vp3基因靶向性治疗肝癌的实验研究

    Institute of Scientific and Technical Information of China (English)

    孙军; 王宇哲; 屈伸; 彭冬君; 宗义强

    2004-01-01

    目的利用肝细胞表面存在特异的脱唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR),探索ASGPR介导的vp3基因肝细胞靶向性治疗肝癌的方法.方法将携带vp3基因的质粒通过多聚左旋赖氨酸(poly-L-lysine,PLL)与该受体的天然配体脱唾液酸粘蛋白(asialoorosomucoid,Asor)结合,获得Asor-PLL-vp3复合物;通过体外转染、放射性同位素标记检测和动物实验鉴定该复合物的肝细胞靶向性.结果成功制备了较纯的可溶性的蛋白-核酸复合物;体内外实验结果表明Asor-PLL-vp3复合物具有良好的肝细胞靶向性.结论通过制备Asor-PLL-vp3复合物,利用ASGPR介导实现了vp3的肝细胞靶向性基因转移,证实了该复合物具有体内靶向性治疗肝癌的可行性.

  2. 去唾液酸糖蛋白受体抗体阳性的Ⅰ型自身免疫性肝炎生物学性状及其意义%Biological significance of autoantibodies to asialoglycoprotein receptor in patients with type Ⅰautoimmune hepatitis

    Institute of Scientific and Technical Information of China (English)

    吴虹杰; 徐芸

    2011-01-01

    AIM: To investigate the differences in clinical, biochemical, immunoserologic, genetic, histolog-ical features and response to treatment between patients with type 1 autoimmune hepatitis (AIH-Ⅰ) who were positive for autoantibodies to asialoglycoprotein receptor (ASGPR) and those negative for anti-ASGPR.METHODS: A total of 79 patients with AIH-Ⅰ were screened for the presence of anti-ASGPR by ELISA and were divided into anti-ASGPR-positive group and anti-ASGPR-negative group.RESULTS: There were 59 patients in the anti-ASGPR-positive group and 20 patients in the anti-ASGPR-positive group. No significant differences were found between anti-ASGPR-positive and-negative patients in age, gender,alanine transaminase (ALT) activity, aspar-tate aminotransferase (AST) activity, alkaline phosphatase (ALP) activity, gamma glutanmic transpeptidase (y-GT) activity, antinuclear autoantibodies (ANA), smooth muscle auto-antibodies (SMA), DR3, and DR4. The levels of immunoglobulin G, C3, histological inflammatory activity and fibrosis, and response to treatment differed significantly between the two groups.CONCLUSION: The presence of anti-ASGPR is correlated, to a certain extent, with inflammatory activity, pathological changes, and response to treatment in patients with AIH- Ⅰ.%目的:探讨去唾液酸糖蛋白受体抗体(Autoantibodies to asialoglycoprotein receptor,antiASGPR)阳性和阴性Ⅰ型自身免疫性肝炎(type Iauto-immune hepatitis AIH-I)在临床、生化、免疫学、遗传学、组织学特点及其治疗应答反应的差异.方法:应用酶联免疫分析法(ELISA)检测79例确诊的AIH-Ⅰ患者血清中anti-ASGPR,将其分为anti-ASGPR阳性组及阴性组.结果:患者中anti-ASGPR阳性的百分比为75%,阳性患者(n=59)中,男性(n=7),女性(n =52),平均年龄(49.1±8.6)岁;阴性患者(n=20)中,男性(n=2),女性(n=18),平均年龄(47.1±7.9)岁,阳性患者与阴性患者在年龄、性别、ALT、AST、ALP、GGT、ANA、SMA、DR3、DR4

  3. Cloning and expression of the human asialoglycoprotein receptor H1 subunit and preliminary application%去唾液酸糖蛋白受体H1亚单位基因片段的克隆表达及其检测自身免疫性肝炎的价值

    Institute of Scientific and Technical Information of China (English)

    张妍; 李永哲; 吴琳; 刘国振; 张蜀澜; 胡朝军; 佟大伟

    2009-01-01

    Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.%目的 克隆去唾液酸糖蛋白受体(asialoglycoprotein reeeptor,ASGPR)H1亚单位显性表位435 bp基因片段,构建表达重组质粒,进行表达、纯化,以

  4. Prokaryotic expression of asialoglycoprotein receptor H1 subunit and its application in the diagnosis of autoimmune hepatitis%去唾液酸糖蛋白受体H1亚单位表达及用于自身免疫性肝炎诊断的研究

    Institute of Scientific and Technical Information of China (English)

    乔健; 姜东林; 薛育政; 龚芳; 李成万

    2014-01-01

    去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是肝细胞膜表面特有的一种内吞性受体,H1是参与内吞功能的主要亚基.自身免疫性肝炎(autoimmune-hepatitis,AIH)患者存在抗-ASGPR的自身抗体.本研究以去唾液酸糖蛋白受体H1亚基在大肠杆菌中诱导表达,用纯化的重组去唾液酸糖蛋白受体H1亚单位rH1作为检测抗原,建立间接法rH1-IgG-ELISA,检测抗去唾液酸糖蛋白受体IgG抗体,观察其阳性和阴性符合率,并对rH1-IgG-ELISA检测的精确度、灵敏度和特异性进行评价.结果显示制备的rH1重组蛋白纯度在90%以上;以该重组抗原建立的rH1-IgG-ELISA的最佳检测条件为:重组抗原rH1的包被浓度为6 μg/ml,血清稀释度1 ∶ 100,酶标记的羊抗人IgG 1∶3000稀释;用rH1-IgG-ELISA对混合ASGPR阳性和阴性血清的重复检测表明:IgG阳性血清的检测值的变异系数(CV值)为9.9%,IgG阴性血清的CV值为9.7%;灵敏度检测表明血清稀释度在1∶50~1∶200均可检出阳性;特异性试验的抑制率为63.5%; rH1-IgG-ELISA与总符合率为87.36%,其中阳性和阴性符合率分别为82%(41/50)和93.33%(42/45).故建立的rH1-IgG-ELISA具有较好的灵敏性和特异性,与提供的ASGPR阳性血清有较高的符合率,表明该法具有较好的AIH诊断价值,可进一步推广应用.

  5. Study on the role of asialoglycoprotein receptor for human bone marrow mesenchymal stem cells against hepatitis B virus infection%骨髓间充质干细胞对乙型肝炎病毒的易感性及与无涎糖蛋白受体的关系

    Institute of Scientific and Technical Information of China (English)

    谢婵; 谢仕斌; 张绍全; 谢俊强; 林炳亮; 高志良

    2010-01-01

    Objective To investigate the susceptibility of bone marrow mesenchymal stem cell (BMSC) to hepatitis B virus (HBV) infection during induction toward hepatocyte and the role of asialoglycoprotein receptor (ASGPR) in BMSC HBV infection. Methods BMSC obtained from hepatitis B patients were tested for HBV infection and then cultured with HBV infectious serum in vitro and induced to differentiate into hepatocyte through exposure to hepatocyte growth factor (HGF), fibroblast growth factor-4(FGF-4), and epidermal growth factor(EGF). Subsequently these cells were determined for the presence of hepatitis B virus e antigen( HBeAg), hepatitis B virus surface antigen(HBsAg) and ASGPR. All experiments were repeated for 3 times in 5 different samples. The results were analyzed by non-parametric test. Results After 6 days of exposure, BMSC-derived hepatocyte-like cells expressed hepatic special genes and proteins, including alpha fetoprotein(AFP),cytokeratin18 (CK18), albumin (Alb), and manifested hepatocyte functions, including glycogen synthesis, urea secretion and albumin synthesis. Expressions of CK18 and Alb were increased, and AFP was decreased with time of induction. The BMSC were resistant to HBV infection both in vitro and in vivo or after induction toward hepatocyte. ASGPR expression level was low in BMSC, which was increased in the induced BMSC but still lower than that of the control HepG2 cells. Conclusions BMSC are resistant to HBV infection both in vitro and in vivo. The low level expression of ASGPR may be a reason for this.%目的 评价骨髓间充质干细胞(BMSC)向肝细胞诱导过程中对HBV的易感性及无涎糖蛋白受体(ASGPR)对BMSC感染HBV的作用.方法 体外使用肝细胞生长因子、成纤维细胞生长因子-4和表皮生长因子,将BMSC诱导分化为肝细胞.检测乙型肝炎患者BMSC的HBV感染情况,并对原代及诱导培养后的BMSC进行体外HBV感染实验,检测BMSC感染后的HBsAg、HBcAg表达情况,并检测BMSC

  6. Assessment of hepatic functional reserve by converted asialoglycoprotein receptor values calculated from parameters of indocyanine green test and thromboelastograph%应用吲哚青绿试验与血栓弹力图替代去唾液酸糖蛋白受体分析定量评估肝储备功能

    Institute of Scientific and Technical Information of China (English)

    张珂; 郭立民; 穆毅; 蒋力; 贾哲; 李宝亮; 赫嵘; 黄容海; 鲁岩; 李勤涛; 丁振昊

    2014-01-01

    目的:应用吲哚青绿实验与血栓弹力图检测指标,替代肝细胞表面去唾液酸糖蛋白受体分析,建立肝储备功能定量评估系统,并与Child-Pugh评分进行比较,了解其在肝切除术患者肝储备功能评估中的临床应用价值。方法对2012年1月1日至12月31日于本科室行肝部分切除术肝占位病变的患者共55例,测量PHCASGPR+、ICGR15、EHBF、R值与K值,建立以PHCASGPR+为因变量(Y), ICGR15、EHBF、R值与K值为自变量(Xn)的肝储备功能定量评估系统,与Child-Pugh评分进行比较,了解两种方法预测术后肝功能代偿情况的准确率。结果 Child-Pugh预测术后肝功能代偿良好准确率为56.67%,Y值预测术后肝功能代偿良好准确率为84.62%(χ2=5.374,P =0.020);Child-Pugh预测术后肝功能代偿不全准确率为76.00%,Y值预测术后肝功能代偿不全准确率为96.55%(χ2=5.400,P =0.020)。结论建立的肝储备功能定量评估系统能够更全面评价肝切除患者围手术期肝储备功能。%Objective To verify the value of asialoglycoprotein receptors (ASGPR) analyzed by lfow cytometry (FCM) in assessing hepatic functional reserve, and establish a conversion formula for the ASGPR value with the parameters of indocyanine green (ICG) test and thromboelastography (TEG). This assessment method was compared with the Child-Pugh score to evaluate its predictive value for hepatic functional reserve in patients undergoing liver resection.Methods Total of 55 patients with liver tumors had partial hepatectomy in our department from January 1st to December 31st, 2012. The percentages of ASGPR+hepatocyte (PHCASGPR+), ICGR15, effective hepatic blood lfow (EHBF), R value and K value were examined to establish the quantitative assessment method for liver functional reserve. The PHCASGPR+ was defined as a dependent variable (Y) while the ICGR15, EHBF, R value and K value were defined as independent variables

  7. Quantitative evaluation of liver functional reserve by asialoglycoprotein receptor and clinical test indexs%应用去唾液酸糖蛋白受体及临床指标建立肝储备功能定量评估系统

    Institute of Scientific and Technical Information of China (English)

    李勤涛; 丁振昊; 郭立民; 穆毅; 蒋力; 张珂; 黄容海; 李宝亮; 贾哲; 鲁岩; 赫嵘

    2014-01-01

    目的:探讨应用肝细胞表面ASGPR流式细胞分析及临床指标建立肝储备功能定量评估系统,并与Child-Pugh评分进行比较,了解其在患者术前肝储备功能评估中的临床应用价值。方法选择32例肝占位病变行肝部分切除术患者,术前检测ICGR15与EHBF、R值与K值以及临床生化指标、Child-Pugh评分,以此作为自变量(Xn),以肝组织标本PHCASGPR+为因变量(Y),通过多元线性回归分析,建立肝储备功能定量评估系统,并与Child-Pugh评分进行比较,了解两种方法预测术后肝功能代偿情况的准确率。按照回归方程计算46例肝占位病变接受肝部分切除术及肝硬化并门脉高压症接受断流术患者肝储备功能定量值,评估肝储备功能情况,预测术后肝功能恢复情况,比较术前不同Child-Pugh分级与Y值患者术后肝功能恢复情况。结果在预测术后肝功能代偿良好准确率方面,Y值优于Child-Pugh分级(P =0.030);而在预测术后肝功能代偿不良准确率方面,Y值与Child-Pugh分级无统计学差异(P =1.000)。结论新肝储备功能定量评估系统能一定程度提高肝切除或肝硬化患者术前肝储备功能评价准确度,具有临床应用价值。%Objective To investigate a quantitative assessment of hepatic functional reserve using flow cytometric analysis of asialoglycoprotein receptors (ASGPR) on the liver cell surface and regular clinical indexes. This assessment method was compared with the Child-Pugh score to evaluate its predictive value for hepatic functional reserve in patients undergoing liver resection or devascularization. Methods Total of 32 patients with liver tumors had partial hepatectomy. The percentage of the ASGPR+hepatocyte (PHCASGPR+), ICGR15, effective hepatic blood flow (EHBF), the R value, the K value and regular clinical parameters were examined to evaluate a quantitative assessment method for liver functional

  8. 去唾液酸糖蛋白受体介导的肝靶向纳米脂质超声造影剂的制备及体外实验%Preparation of asialoglycoprotein receptor-mediated hepatic targeting nano- lipid ultrasound contrast agent and its ultrasound imaging in vitro

    Institute of Scientific and Technical Information of China (English)

    余进洪; 王志刚; 郑元义; 项莹霞; 李奥; 李巧

    2011-01-01

    Objective To prepare the liver targeting nano-liquid perfluorocarbon ultrasound contrast agent and observe its general characteristics;to observe the targeting combined effects of the human liver cells L02 and the targeted ultrasound contrast agent ;to evaluate the gathering imaging effects of the targeted ultrasound contrast agent. Methods Amine method was used to prepared asialoglycoprotein Gal specific ligand polylysine (Gal-PLL), rotary evaporator and sonicated liquid fluorocarbon were used to prepare nano lipid ultrasound contrast agent. Human liver cell L02 were cultured, the combined effects were observed according to the reacting time of the cells and the targeted nano-lipid ultrasound contrast agent. The nanolipid ultrasound contrast agent and the degassed_ water_ were loaded into cysts and their ultrasound imaging effects were observed by ultrasound diagnostic apparatus Philips iU22. Results The particle size of the liquid fluorocarbon nano-targeted lipid ultrasound contrast agent was extremely small, uniform, cylindrical and spherical. The cysts in vitro showed that the side of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent showed high echo. Conclusions The targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent can be effectively targeted to the human liver cells L02 due to carrying home-made Gal-PLL. The targeted ultrasound contrast agents can be imaging by ultrasound and be confirmed in vitro.The size of the contrast agent was small, therefore, it can be considered an ideal ultrasonic molecular probe and achieve the ultrasound molecular imaging in cell level.%目的 以肝细胞膜特异性受体去唾液酸糖蛋白受体为靶向受体,利用共价结合的原理,制备出肝靶向性液态氟碳纳米超声造影剂,观察该造影剂的一般特性、与人肝细胞L02的靶向结合及体外聚集显像效果.方法 利用还原胺法制备去唾液酸糖蛋白特异性配体半乳糖化多聚赖氨酸(Gal

  9. Relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kindberg, G.M.; Refsnes, M.; Christoffersen, T.; Norum, K.R.; Berg, T.

    1987-05-25

    The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with /sup 125/I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

  10. Asialoglycoprotein receptor-magnetic dual targeting nanoparticles for delivery of RASSF1A to hepatocellular carcinoma

    Science.gov (United States)

    Xue, Wan-Jiang; Feng, Ying; Wang, Fei; Guo, Yi-Bing; Li, Peng; Wang, Lei; Liu, Yi-Fei; Wang, Zhi-Wei; Yang, Yu-Min; Mao, Qin-Sheng

    2016-02-01

    We developed a nanovector with double targeting properties for efficiently delivering the tumor suppressor gene RASSF1A specifically into hepatocellular carcinoma (HCC) cells by preparing galactosylated-carboxymethyl chitosan-magnetic iron oxide nanoparticles (Gal-CMCS-Fe3O4-NPs). After conjugating galactose and CMCS to the surface of Fe3O4-NPs, we observed that Gal-CMCS-Fe3O4-NPs were round with a relatively stable zeta potential of +6.5 mV and an mean hydrodynamic size of 40.1 ± 5.3 nm. Gal-CMCS-Fe3O4-NPs had strong DNA condensing power in pH 7 solution and were largely nontoxic. In vitro experiments demonstrated that Gal-CMCS-Fe3O4-NPs were highly selective for HCC cells and liver cells. In vivo experiments showed the specific accumulation of Gal-CMCS-Fe3O4-NPs in HCC tissue, especially with the aid of an external magnetic field. Nude mice with orthotopically transplanted HCC received an intravenous injection of the Gal-CMCS-Fe3O4-NPs/pcDNA3.1(+)RASSF1A compound and intraperitoneal injection of mitomycin and had an external magnetic field applied to the tumor area. These mice had the smallest tumors, largest percentage of TUNEL-positive cells, and highest caspase-3 expression levels in tumor tissue compared to other groups of treated mice. These results suggest the potential application of Gal-CMCS-Fe3O4-NPs for RASSF1A gene delivery for the treatment of HCC.

  11. Generation and characterization of a pseudotyped gene transfer system with selectivity for the hepatocyte-specific asialoglycoprotein receptor

    OpenAIRE

    Spiegel, Martin

    2000-01-01

    Ansätze zum therapeutischen in vivo Lebergentransfer erfordern leberrestringierte Gentransfersysteme. Die derzeit verwendeten amphotropen Moloney Murine Leukemia Virus (MoMLV)- Vektoren sind aufgrund ihres breiten Transduktionsspektrums für in vivo Applikationen nicht geeignet. In diesem Zusammenhang wurde die Möglichkeit der Pseudotypbildung zwischen ecotropem MoMLV und Sendai Virus (SeV), einem Parainfluenzavirus, untersucht. Der Einbau beider SeV- Hüllproteine (HN und F) ...

  12. Binding site of hepatitis B virus preS1 region to the asialoglycoprotein receptor of human liver

    Institute of Scientific and Technical Information of China (English)

    JIN Zhe-zhu; LI Mei-hua; WANG Yu-shu; CAI Ying-ji; JIN He-kui; ZHU Wei

    2002-01-01

    @@ The hepatitis B virus (HBV) is a major pathogen of chronic inflammatory liver disease and liver cirrhosis and is known to be infected to the hepatocytes via HBV specific receptors[1]. However, the specific receptor for HBV has not yet been identified. The HBV envelops consist of three related proteins, called major-(S region), middle-(S + preS2) and large protein (S + preS2 + preS1).

  13. Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation.

    Science.gov (United States)

    Pathak, Pankaj; Dhawan, Vivek; Magarkar, Aniket; Danne, Reinis; Govindarajan, Srinath; Ghosh, Sandipto; Steiniger, Frank; Chaudhari, Pradip; Gopal, Vijaya; Bunker, Alex; Róg, Tomasz; Fahr, Alfred; Nagarsenker, Mangal

    2016-07-25

    We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

  14. 人去唾液酸糖蛋白受体单克隆抗体制备及其初步应用%Preparation of mouse anti-human asialoglycoprotein receptor monoclonal antibody and its preliminary application

    Institute of Scientific and Technical Information of China (English)

    李军; 陈磊; 孙斌; 张妤; 钱海华; 殷正丰

    2013-01-01

    目的 制备人去唾液酸糖蛋白受体(ASGPR)的小鼠单克隆抗体,并应用该抗体检测ASGPR在细胞系和组织中的表达.方法 对ASGPR H1大亚基进行序列分析,选取ASGPR1全长序列制备免疫原.通过RT PCR合成ASGPR1cDNA,构建ASGPR1表达重组载体,在E.coli BL21中表达后纯化,获得目的抗原.应用传统融合杂交瘤技术制备小鼠单克隆抗体,常规检测单抗亚类和效价,竞争抑制实验鉴定单抗特异性.用流式细胞术和免疫组织化学法分别检测ASGPR在多种肝源性与非肝源性细胞系以及多种肝组织中的表达.结果 制备的单克隆抗体亚型为IgG1,效价达1∶12 800.竞争抑制实验结果显示该单抗具有很好的ASGPR结合特异性,而且识别的肽段位于ASGPR胞外段.流式细胞术检测结果显示,ASGPR不同程度地表达于肝源性细胞系,但不表达于非肝源性细胞系.免疫组织化学检测结果显示,ASGPR专一性表达于正常肝组织和肝癌组织,在肝癌组织的表达与分化程度有关,高分化肝癌中的阳性率高于低分化肝癌(75.0% vs 28.6%,P<0.05).结论 成功制备高特异性人ASGPR小鼠单克隆抗体,适用于用流式细胞术和免疫组织化学法检测ASGPR表达,可用于临床病理鉴定原发性肝癌与转移性肝癌.

  15. 去唾液酸糖蛋白受体及其在药物肝靶向递送中的应用%Asialoglycoprotein Receptor and Its Application in Liver-targeted Drug Delivery

    Institute of Scientific and Technical Information of China (English)

    黄渊余; 梁子才

    2015-01-01

    去唾液酸糖蛋白受体(ASGPR)是肝细胞特异性表达的受体,且是一种高效的内吞型受体,去唾液酸糖蛋白、半乳糖、半乳糖胺、N-乙酰半乳糖胺等糖分子对其有高亲和性.该综述回顾了ASGPR的发现历程、结构特征、生物学功能、表达模式、胞吞特点.总结了影响ASGPR与其配体亲和、介导胞吞的影响因素(包括配体类型、触角数量、空间距离与颗粒粒径).概述了早期ASGPR与其特异性配体在药物递送中的应用.最后介绍了最近利用N-乙酰半乳糖胺的缀合或修饰来实现肝靶向核酸药物递送的研究进展.

  16. 非唾液酸糖蛋白受体显像在评估肝储备功能方面的应用价值%The clinical value of asialoglycoprotein receptor imaging for determining liver functional reserve.

    Institute of Scientific and Technical Information of China (English)

    张海山; 房学东; 何尔斯泰; 徐文贵; 郑泽霖

    2001-01-01

    目的探讨肝非唾液酸糖蛋自受体显像在评估肝储备功能方面的临床应用价值.方法静注T-新半乳糖白蛋白行肝非唾液酸糖蛋白受体显像,利用标准核医学设备和计算机软件获得肝非唾液酸糖蛋白受体浓度ASGPR,测定常用肝储备功能指标和吲哚绿15min滞留率.结果对照组和肝硬化肝功能Ⅰ级、Ⅱ级、Ⅲ级组[ASGPR]分别为0.780±0.163、0.576±0.150、0.512±0.124和0.265±0.158,除肝功能Ⅰ级和肝功能Ⅱ级组无显著性差异外(P>0.05),其余各组间均存在显著性差异(P<0.001);对照组和肝硬化组[ASGPR]与凝血酶原时间(%)、血清白蛋白血胆碱脂酶、吲哚青绿15min滞留率呈显著正相关(均P<0.001),与血总胆红素呈负相关(P=0.154).结论肝非唾液酸糖蛋白受体显像是一种有推广价值的评估肝储备功能的新方法,且可能较经典肝储备功能指标更敏感.

  17. 肝细胞表面去唾液酸糖蛋白受体的流式细胞分析%Flow Cytometric Evaluation of Asialoglycoprotein Receptor on the Surface of Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李文新; 张荣军; 谭成; 陶永辉; 金坚

    2000-01-01

    建立肝细胞表面去唾液酸糖蛋白受体(ASGPR)的流式细胞分析方法(FCM),对正常及损伤鼠肝细胞、肝癌细胞(BEL-7402)表面的ASGPR作同步比较分析.以异硫氰酸荧光素标记的新半乳糖白蛋白(FITC-NGA)为ASGPR的特异性配体,以培养的正常肝细胞(L-02)为靶细胞,建立肝细胞表面ASGPR的FCM.测定并计算正常及损伤鼠肝细胞,BEL-7402细胞与同一浓度的FITC-NGA同步反应后的平均荧光强度(MIF)值.FITC-NGA与L-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/L的EDTA完全抑制.FCM能够良好地揭示FITC-NGA同ASGPR之间的受配体结合特性.该方法证实BEL-7402细胞表面几乎没有ASGPR,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少.

  18. 去唾液酸糖蛋白受体H1亚单位的表达、纯化及其单链抗体的筛选与鉴定%Expression and purification of the human asialoglycoprotein receptor H1 subunit and selection of anti-CRDH1 scFv

    Institute of Scientific and Technical Information of China (English)

    曹利民; 司进; 朱荫昌; 王炜煜; 赵晓蓉; 叶庆; 李文涵; 朱慧芬; 沈关心

    2005-01-01

    目的去唾液酸糖蛋白受体(ASGPR)H1亚单位的重组、表达、纯化及其单链抗体的筛选与鉴定.方法将ASGPR H1亚单位糖识别区基因(CRDH1)定向克隆至原核表达载体pET-32c经IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫印迹技术(WB)检测;用纯化的CRDH1对人源噬菌体抗体库进行4轮固相筛选,阳性克隆用表达标记(Trx-His-s tag)进行差异筛选.取阳性噬菌体C3感染宿主菌HB2151,37℃振荡培养过夜,终浓度为1 mmol/L的IPTG诱导表达,12%SDS-PAGE与Western blot鉴定以及免疫组化鉴定纯化的单链抗体C3与肝细胞结合的特性.结果CRDH1/pET-32c在原核系统经诱导表达出相对分子质量(Mr)约35×103大小的融合蛋白,以包涵体的形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白;经4轮筛选和差异筛选后,有2株可分泌性表达与CRDH1特异性结合的单链抗体,DNA序列分析表明单链抗体重链基因和轻链基因分别属于人免疫球蛋白VH3家族和VKI家族.噬菌体C3经诱导表达出Mr约28×103大小的融合蛋白,可溶性分泌表达于培养基中;通过Ni2+亲和柱纯化获得单链抗体,ELISA、Western blot表明单链抗体C3能较好结合rCRDH1,免疫组化显示该单链抗体可与肝癌细胞、肝细胞特异结合.结论成功表达ASGPR并筛选出其人源单链抗体.该单链抗体可与表达的rCRDH1以及肝细胞和肝癌细胞特异性结合,提示对肝脏疾病的靶向治疗有潜在的应用价值.

  19. 肝去唾液酸糖蛋白受体显像:三维分段肝功能评估的前景%Liver asialoglycoprotein receptor scintigraphy:a new prospect of segmentational three-dimensional evaluation of liver function

    Institute of Scientific and Technical Information of China (English)

    张涛; 毛一雷

    2006-01-01

    去唾液酸糖蛋白受体(ASGPR)是哺乳动物肝细胞表面的特异性受体,肝硬化和肝癌时ASGPR水平下降.利用99mTcGSA进行该受体的显像,能够得到HH15、LHL15、[R]0、R0等指标用于肝功能的评估;将这种功能性显像与单光子发射型计算机断层显像(SPECT)技术相结合,可以模拟肝脏切除的范围,并预测术后剩余肝脏的功能.此技术在国际上是一个新的课题,在国内尚无开展,用它来数字化的评估手术风险对患者手术方式的选择及预后具有重要影响.笔者结合正在进行的这方面部分研究,向读者作一介绍,以期在临床上发挥更好的作用.

  20. 中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域的原核表达及复性%Prokaryotic Expression, Purification and Renaturation of CRDH1 and CRDH2 of Chinese Marmata Asialoglycoprotein Receptor

    Institute of Scientific and Technical Information of China (English)

    黄凰; 杨燕; 刘嘉; 张振华; 田拥军; 王宝菊; 郝友华; 杨东亮

    2006-01-01

    目的 对中国旱獭去唾液酸糖蛋白受体(ASGPR)H1和H2亚基糖基识别域(CRD)的克隆、表达、纯化及复性.方法 用RT-PCR从中国旱獭肝组织中扩增ASGPR CRDH1和CRDH2 cDNA,分别将其克隆到原核表达载体pRSET-B上,在埃希菌BL21(DE3)pLysS内诱导表达含6个组氨酸标签的融合蛋白.融合蛋白经Ni2+螯合柱亲和纯化后,在体外行透析复性.结果 ASGPR CRDH1和CRDH2经原核表达后得到分子量约为22 ku和15 ku的目的蛋白,以包涵体形式存在.经Ni2+螯合柱亲和纯化后获得纯度大于95%的融合蛋白.利用仅识别天然构像的单克隆抗体对复性后产物进行检测,证明复性成功.结论 成功地表达了具有活性的ASGPR CRDH1和CRDH2的融合蛋白,在肝脏疾病的靶向治疗研究中具有潜在的应用价值.

  1. 去唾液酸糖蛋白受体特异性单链抗体的优化表达及亲和常数的测定%Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 易继林; 邓云华; 司进; 王从俊; 李翔; 曹利民

    2006-01-01

    目的: 表达及纯化抗去唾液酸糖蛋白受体(ASGPR)的单链抗体的可溶性,并测定其亲和常数.方法: 用噬菌体C1克隆感染E.coli HB2151,挑取单个菌落接种于2×TY培养基中,于37℃震荡培养过夜.将培养物作1∶ 100稀释并转种后,用终浓度为0.25、0.5、1.0 mmol/L的IPTG,分别在37℃、25℃和20℃下诱导表达过夜.取其培养上清,用饱和硫酸铵沉淀后,以120 g/L SDS-PAGE分析.另外,将饱和硫酸铵沉淀物用30 mL PBS重新溶解、透析除盐后,用Ni2+螯合柱进行纯化,再以120 g/L SDS-PAGE鉴定纯化scFv C1的纯度.用非竞争酶免疫法测定scFv的亲和常数.结果: 用0.5 mmol/L IPTG在25℃诱导过夜,表达的scFv C1的量较多,其相对分子质量(Mr)约为28 000,以可溶性的形式存在于培养基中.通过Ni2+亲和柱纯化后scFv C1的纯度在95%以上,产量约为0.8 mg/L.scFv的亲和常数为(2.31±0.36)×10-7 mol/L.结论: 以筛选的C1噬菌体感染E.coli HB2151后可表达低亲和力的可溶性scFv,对肝癌的基因治疗具有潜在的应用价值.

  2. 去唾液酸糖蛋白受体H1亚基CRD的原核表达、纯化及特性分析%Cloning, Expression, Purification and Immunoresponse Evaluation of the Carbohydrate Recognition Domain of H1 Subunit of the Asialoglycoprotein Receptor

    Institute of Scientific and Technical Information of China (English)

    乔健; 黄建萍; 曹利民; 司进

    2006-01-01

    目的研究去唾液酸糖蛋白受体H1亚基糖识别区(CRD)在大肠杆菌中的诱导表达,为从噬菌体抗体库中筛选其特异性抗体提供靶分子.方法以pET3-CRDH1为模板设计引物,通过PCR扩增CRDH1基因,采用分子克隆技术将其定向克隆至原核表达载体pET-32c中.将含CRDH1/pET-32c的BL21单菌落接种至LB肉汤培养基中培养,并通过IPTG诱导表达.表达产物经Ni2+螯合柱亲和纯化,采用免疫印迹技术(WB)分析融合蛋白的免疫反应性.结果表达产物经SDS-PAGE在35 kd左右显示条带,符合CRDH1与表达标签融合蛋白的理论值, 并证明以包涵体的形式表达; 通过Ni2+亲和柱纯化获得的CRDH1重组融合蛋白经WB证实,重组CRDH1融合蛋白能被羊抗人ASGPR血清特异性地识别.结论 rCRDH1在原核系统获得高效表达,亲和纯化的rCRDH1具有较强的免疫反应性,为进一步从噬菌体抗体库中筛选特异性单链抗体奠定了基础.

  3. 基于去唾液酸糖蛋白受体的循环肝癌细胞磁性分选检测法及其改良%Detection of circulating tumor cells in patients with hepatocellular carcinoma using an improved asialoglycoprotein receptor-based separation method

    Institute of Scientific and Technical Information of China (English)

    余锋; 陈磊; 徐文; 曹璐; 张妤; 施乐华; 殷正丰

    2013-01-01

    目的:介绍一种肝细胞癌(hepatocellularcarcinoma,HCC)循环肿瘤细胞(circulatingtumor cells,CTCs)富集和检测系统,并对该系统进行改良,以提高循环肿瘤细胞的检出率.方法:去唾液酸糖蛋白受体(asialoglycoproteinreceptor,ASGPR)是一种特异表达于肝实质细胞膜的跨膜蛋白.我们建立了一种基于ASGPR的磁性分选和Hep par 1免疫鉴定的循环肝癌细胞富集和计数系统.首先以生物素化去唾液酸胎球蛋白作为配体与肝癌细胞结合,然后用抗生物素抗体磁珠进行间接磁性标记,从而磁性捕获循环肝癌细胞,接着用Hep par 1进行免疫荧光染色鉴定,并对阳性细胞进行计数.文献报道和实际操作中发现,检测过程中采用肝素抗凝会引起细胞悬液中出现凝胶,影响细胞通过分离柱,降低分离效率.我们将部分步骤改用乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)替代肝素抗凝,采用Hep3B肝癌细胞掺入试验对两种方法回收情况进行比较.结果:部分步骤改用EDTA抗凝后,细胞悬液中未出现凝胶现象.细胞掺入试验显示,5 mL健康志愿者外周血中分别掺入10、30、90、270和810个Hep3B细胞,在每个掺入水平,改良法Hep3B细胞的平均回收率均≥70%,回收效率明显增加(P<0.05).结论:基于ASGPR的循环肝癌细胞富集和计数系统是一种具有临床应用潜能的循环肝癌细胞检测方法.经改良后能够有效消除细胞悬液中凝胶形成的现象,肝癌细胞回收效率较原检测法明显提高.

  4. 中国旱獭去唾液酸糖蛋白受体糖基结合域的原核表达与多克隆抗体的制备%Cloning, prokaryotic expression and polyclonal antibody preparation of the asialoglycoprotein receptor of marmota himalayan

    Institute of Scientific and Technical Information of China (English)

    杨燕; 黄凰; 刘慎沛; 张振华; 王宝菊; 田拥军; 杨东亮

    2007-01-01

    目的:构建中国旱獭去唾液酸糖蛋白受体(ASGPR)H1和H2亚基糖基识别域(CRD)的原核表达质粒,体外表达纯化后制备多克隆抗体.方法:RT-PCR扩增出中国旱獭肝组织中ASGPR CRDH1和CRDH2 cDNA,将其克隆至原核表达载体pRSET-B中,在大肠杆菌BL21(DE3)pLysS内诱导表达.用纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,并采用酶联免疫吸附试验、Western blot及免疫组织化学检测抗体的灵敏度和特异性.结果:成功构建了中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域原核表达质粒pRSET-B.CRDH1和pRSET-B.CRDH2,目的蛋白可以高效表达,用其免疫BALB/c小鼠获得了高效价的特异性多克隆抗体.结论:首次成功表达了中国旱獭去唾液酸糖蛋白受体H1和H2亚基糖基识别域多肽,且纯度高,免疫原性强,用其免疫小鼠获得的多克隆抗体特异性好、效价高,为在HBV感染模型-中国旱獭体内进行肝脏疾病的靶向治疗奠定了实验基础.

  5. Optimization of enzymatic synthesis of asialoglycoprotein receptor ligand cholesterol-vinyl sebacate-lactitol%去唾液酸糖蛋白受体配体胆固醇-半乳糖苷的酶促合成优化研究

    Institute of Scientific and Technical Information of China (English)

    陈静; 程怡; 郑品劲; 聂华; 陈宇潮; 仝一丹; 罗利华; 李朝

    2015-01-01

    目的 在有机相中利用酶促反应合成能被去唾液酸糖蛋白受体(ASGPR)特异性识别的配体胆固醇-半乳糖苷分子,对其合成工艺进行优化.方法 采用质谱和核磁碳谱鉴别产物结构;单因素和正交设计法优化酶促合成条件.结果 酶促最佳条件为底物胆固醇癸二酸乙烯酯(CH-VS)与乳糖醇(LA)物质的量之比4∶1,脂肪酶Novozym 435 25 mg,反应32 h,产率高达92%.结论 该方法高效、反应条件可行、产物专一.

  6. Establishment of enzyme linked immunosorbent assay of autoantibodies to asialoglycoprotein receptor and its clinical application%抗去唾液酸糖蛋白受体酶联免疫检测法的建立及临床初评

    Institute of Scientific and Technical Information of China (English)

    刘海英; 范列英; 沈芳; 邓安梅; 屠小卿; 周晔; 陈燕; 仲人前

    2004-01-01

    目的建立肝脏特异性自身抗体的ELISA检测方法,辅助自身免疫性肝炎的诊断.方法利用亲和层析法从人肝脏组织中直接纯化去唾液酸糖蛋白受体(ASGPR),经免疫学鉴定后以此纯化抗原包被微孔板,酶联免疫固相吸附试验检测去ASGPR的自身抗体(抗-ASGPR).结果本研究从10 g肝组织纯化ASGPR,总量为1.6 mg.经SDS-PAGE和Dot-ELISA验证,纯化ASGPR纯度高、具有抗原活性.ASGPR预吸附可阻断血清抗体与ASGPR间的免疫反应,类风湿因子、梅毒阳性血清不干扰本ELISA法的抗原抗体反应.33例自身免疫性肝炎(AIH)患者中检出抗-ASGPR 24例,阳性率为72.7%.在临床上高度怀疑AIH的10例患者中,抗-ASGPR阳性者有7例.原发性胆汁性肝硬化、病毒性肝炎、肝炎后肝硬化、肝癌、胆囊炎患者中阳性检出率分别为21.4%(9/42)、16.8%(16/95)、16.1%(10/62)、10.7%(3/28)、14.3%(1/7).药物性肝炎、酒精性肝炎、系统性红斑狼疮和类风湿性关节炎患者中未检测到抗-ASGPR.200名健康体检者阳性率为4.6%.抗-ASGPR检测对AIH诊断的特异性为89.6%.结论本研究所建立的抗-ASGPR ELISA检测方法可靠、特异性好.抗-ASGPR检测有助于AIH的诊断,尤其是对那些抗核抗体、平滑肌抗体、肝肾微粒体-1型抗体等自身抗体均阴性、临床高度怀疑AIH病例的诊断可能更为有用.

  7. 去唾液酸糖蛋白受体H1亚基的原核表达及多克隆抗体的制备%Prokaryotic Expression of the H1 Subunit of the Asialoglycoprotein Receptor and the Preparation of Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 易继林; 王健; 司进; 朱荫昌; 曹利民

    2007-01-01

    目的 在原核系统中表达并纯化去唾液酸糖蛋白受体(ASGPR)H1亚基,制备兔抗人ASGPR1多克隆抗体.方法 以质粒pEA1为模板,设计引物,将聚合酶链反应(PCR)扩增产物H1基因克隆到原核表达载体pET-32c中.接种含H1/pET-32c的菌株BL21单菌落至LB肉汤中,1∶100稀释转种后用1 mmol/L 终浓度的异丙基硫代-β-半乳糖苷(IPTG)诱导表达,表达产物用Ni2+螯合柱亲和纯化,用纯化的ASGPR1免疫新西兰兔制备多克隆抗体.结果 H1/pET-32c在原核系统中成功表达和纯化出约50.3 kD大小的融合蛋白,用纯化的H1成功制备了兔抗人H1多克隆抗体,并用6His-H1和GST-H1重组蛋白进行免疫印迹技术(Western blot)分析,证实了抗体的正确性.结论 应用多克隆抗体可以检测体内外ASGPR H1亚基基因的表达,为临床上测定血清可溶性ASGPR奠定基础.

  8. Generating the human asialoglycoprotein receptor specific scFv by capture phage ELISA%捕获噬菌体酶联免疫吸附法筛选抗去唾液酸糖蛋白受体单链抗体的价值

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 易继林; 邓云华; 曹利民

    2006-01-01

    目的评价捕获噬菌体酶免疫吸附试验法(ELISA)在筛选抗去唾液酸糖蛋白受体(ASGPR)单链抗体(scFv)中的价值.方法以含ASGPR基因的质粒(CRDH1/pET3b)为模板,通过聚合酶链反应扩增获得ASGPR H1亚单位糖识别区基因(CRDH1),定向克隆至原核表达载体pET-32c,并经IPTG诱导表达,其表达产物用Ni2+螯合柱亲和纯化;然后,采用捕获噬菌体ELISA对人源噬菌体抗体库进行筛选和鉴定,同时对筛选的抗CRDH1单链抗体进行DNA测序、表达、纯化和免疫印记鉴定.然后,与间接噬菌体ELISA比较.结果 CRDH1/pET-32c在原核系统中经诱导表达出分子量约35 000的融合蛋白,且以包涵体形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白后,采用捕获噬菌体ELISA进行四轮筛选,在60个噬菌体克隆中有45株与CRDH1特异性结合,其中仅1株与重组蛋白标签有交叉结合反应.DNA序列分析表明,有9株DNA序列各异,9株可分泌性表达与CRDH1特异性结合的scFv,纯化的scFv也可特异性识别rCRDH1.计算该筛选方法的假阳性率为2%,低于间接噬菌体ELISA筛选的假阳性率(58%).结论捕获噬菌体ELISA筛选CRDH1特异性scFv可有效降低硫氧还蛋白标签(Trx·Tag)引起的假阳性,提高筛选阳性率,并成功筛选出9株具有rCRDH1特异性的scFv.

  9. Evaluation of hepatic functional reserve using Child-Pugh score, ICGR15, asialoglycoprotein receptor assay and transient elastography in partial hepatectomy patients%应用Child-Pugh评分、吲哚青绿15 min潴留率、去唾液酸糖蛋白受体与肝瞬时弹性值建立肝切除患者肝储备功能定量评估系统的初步研究

    Institute of Scientific and Technical Information of China (English)

    贾哲; 张珂; 蒋力; 黄容海; 李宝亮; 鲁岩; 赫嵘; 丁振昊; 李勤涛

    2013-01-01

    目的 联合运用Child-Pugh评分、吲哚青绿(ICG)试验、去唾液酸糖蛋白受体(ASGPR)及肝瞬时弹性值(TE),建立肝切除患者肝储备功能定量评估系统.方法 对2011年1月至2012年12月在我科行肝切除的104例患者,术前测定Child-Pugh评分、吲哚青绿15 min潴留率(ICGR15)、肝细胞ASGPR荧光强度及TE,并使用术后2周Child-Pugh评分评估肝功能恢复情况.利用多元线性逐步回归分析法筛选影响术后肝功能恢复的指标,以术后肝功能Child-Push评分为因变量(Y值),以筛选指标为自变量(Xn),建立肝储备功能评估方程.结果 利用多元线性逐步回归分析法筛选得出ASGPR、Child-Pugh评分、ICGR15与TE是影响术后肝功能恢复的敏感指标,并可建立多元线性回归方程为Y=6.3050-0.0543X1+0.4980X2+0.1150X3-0.0637X4,该模型能够解释术后肝功能85.8%变差,而单独使用Child-Pugh评分仅能解释术后肝功能66.0%变差.结论 术前联合应用Child-Pugh评分、ICGR15、ASGPR与TE能够提高Child-Pugh评分评估肝切除患者肝储备功能准确率,具有一定的临床应用价值.%Objective To establish an innovative quantitative assessment of hepatic functional reserve using Child-Pugh score,ICGR15,ASGPR and transient elastography (TE).Methods A total of 104 patients with partial hepatectomy in our department from January 2011 to December 2012 were involved in this study.The Child-Pugh score,ICGR15,ASGPR and TE were examined to establish the quantitative assessment method for liver functional reserve.Child-Pugh score was examined after two weeks of operation and defined as a dependent variable (Y) while the pre-operative Child-Pugh score,ICGR15,ASGPR and TE were defined as independent variables (Xn).Results ASGPR,Child-Pugh score,ICGR15 and TE were sensitive values to predict hver function after surgery through stepwise multiple regression analysis,as well as the multiple regression equation was established with Y =6.3050-0.0543X1 + 0.4980X2 + 0.1150X3-0.0637X4.Compared with Child-Pugh score alone,the new method was more accurate to predict the variation of postoperative hepatic function.Conclusion The combined application of Child-Pugh score,ICGR15,ASGPR and TE could improve the accuracy of evaluating hepatic functional reserve in partial hepatectomy patients.

  10. ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

    Science.gov (United States)

    Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

    2016-04-01

    Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

  11. Superfluorinated PEI Derivative Coupled with (99m) Tc for ASGPR Targeted (19) F MRI/SPECT/PA Tri-Modality Imaging.

    Science.gov (United States)

    Guo, Zhide; Gao, Mengna; Song, Manli; Li, Yesen; Zhang, Deliang; Xu, Duo; You, Linyi; Wang, Liangliang; Zhuang, Rongqiang; Su, Xinhui; Liu, Ting; Du, Jin; Zhang, Xianzhong

    2016-07-01

    Fluorinated polyethylenimine derivative labeled with radionuclide (99m) Tc is developed as a (19) F MRI/SPECT/PA multifunctional imaging agent with good asialoglycoprotein receptors (ASGPR)-targeting ability. This multifunctional agent is safe and suitable for (19) F MRI/SPECT/PA imaging and has the potential to detect hepatic diseases and to assess liver function, which provide powerful support for the development of personalized and precision medicine.

  12. DNA Nanocarriers for Systemic Administration: Characterization and In Vivo Bioimaging in Healthy Mice

    OpenAIRE

    David, Stéphanie; Passirani,Catherine; Carmoy, Nathalie; Morille, Marie; Mevel, Mathieu; Chatin, Benoit; Benoit, Jean-Pierre; Montier, Tristan; Pitard, Bruno

    2013-01-01

    International audience; We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should imp...

  13. CLINICAL DIAGNOSTIC VALUE OF AUTOANTIBODIES IN THE DIAGNOSIS OF AUTOIMMUNE LIVER DISEASES

    Directory of Open Access Journals (Sweden)

    V. V. Bazarnyi

    2015-01-01

    Full Text Available We are studied the 15 patients with autoimmune liver diseases and 36 patients without autoimmune pathology found the diagnostic value of antinuclear and antimitochondrial autoantibodies (AMA-M2 tests, and antibodies to asialoglycoprotein receptor (anti-ASGPR. Based on the ROC analysis showed that the diagnostic sensitivity and diagnostic specificity of AMA-M2 was 73% and 100% and for anti-ASGPR – 60% and 77%, respectively. Therefore, the test for anti-ASGPR in autoimmune diseases of the liver showed no advantages over standart tests, and its using in clinical practice requires clarification. 

  14. ASGPR单链抗体-蜂毒肽融合蛋白的构建及其溶红细胞效应

    Institute of Scientific and Technical Information of China (English)

    曹利民; 朱慧芬; 赵晓蓉; 叶庆; 吴砂; 李文涵; 赵晓萍; 朱荫昌; 司进; 沈关心

    2007-01-01

    去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是一种异源低聚物的内吞受体,定位于肝脏实质细胞朝向窦状隙一侧的细胞膜表面,在介导肝脏疾病靶向治疗方面具有潜在应用价值。我们在获得抗ASGPR单链抗体CI的基础上,

  15. Role of sialylation in determining the pharmacokinetics of neutrophil inhibitory factor (NIF) in the Fischer 344 rat.

    Science.gov (United States)

    Webster, R; Taberner, J; Edgington, A; Guhan, S; Varghese, J; Feeney, H; Blocker, L; Jezequel, S G

    1999-11-01

    1. Recombinant neutrophil inhibitory factor (NIF) is a glycoprotein. Its amino acid sequence remains constant and has a molecular weight of 28.9 kD. However, approximately 40% of the total molecular weight consists of glycans with variable structure. 2. The pharmacokinetics of 11 different NIF batches with varying extents and patterns of sialylation have been investigated in the Fischer 344 rat following intravenous administration. These data indicate that reducing the extent of NIF sialylation reduces the half-life of the molecule due to an increase in the systemic clearance. Also, an increase in the number of unsialylated or neutral glycans may increase the volume of distribution of NIF, although this effect is marginal. 3. Isolated perfused rat liver (IPRL) investigations have shown that sialylated NIF has a low hepatic extraction (NIF has an extraction that is > 20-fold higher. Co-administration of asialo NIF with asialo fetuin (a protein cleared by hepatic asialoglycoprotein receptor (possibly galactose)-mediated uptake reduced the hepatic extraction of asialo NIF. 4. These data suggest that NIF molecules that have free sugar moieties (possibly galactose) interact with an asialoglycoprotein receptor (possibly galactose-mediated) in the liver (parenchymal cells/hepatocytes). Interaction with this receptor leads to cellular internalization and degradation.

  16. Evaluation of usefulness of [sup 99m]Tc-GSA liver scintigraphy on fatty liver in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Kimoto, Mitsunori; Akaki, Shiro; Kohno, Yoshihiro; Gohbara, Hideo; Sakae, Katsuyoshi; Nagaya, Isao; Takeda, Yoshihiro; Hiraki, Yoshio (Okayama Univ. (Japan). School of Medicine)

    1994-10-01

    [sup 99m]Tc-GSA is a new liver-imaging radiopharmaceutical which binds to the asialoglycoprotein receptors on the hepatocytes. We evaluated liver injury induced by fatty infiltration in the rats. Studies were performed in the Wistar rats under control conditions (6 cases), and with choline deficiency diet for 2, 4, 6, 10 and 12 weeks (6 cases respectively). [sup 99m]Tc-GSA was administered via the inferior vena cava. Immediately after injection, a dynamic imaging study was performed for 30 min. t[sub 90] (the time at which the liver time-activity curve reached 90% of its peak), K[sub u] and K[sub d] (calculated by 2 compartment model) were used as parameters which reflect on asialoglycoprotein receptors on the hepatocytes. t[sub 90] prolonged, and K[sub u] and K[sub d] decreased according to the severity of fatty infiltration. These results suggest that [sup 99m]Tc-GSA is useful for evaluating liver injury induced by fatty infiltration. (author).

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

    Science.gov (United States)

    Heo, Dan; Lee, Chanjoo; Ku, Minhee; Haam, Seungjoo; Suh, Jin-Suck; Huh, Yong-Min; Park, Sahng Wook; Yang, Jaemoon

    2015-08-01

    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.

  18. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome

    Institute of Scientific and Technical Information of China (English)

    Si-Yuan Wen; Xiao-Hong Wang; Li Lin; Wei Guan; Sheng-Qi Wang

    2004-01-01

    AIM: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors.METHODS: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors,red blood cell hemolysis,in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc.RESULTS: Liposome L8-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency.CONCLUSION: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

  19. [Enzyme-catalyzed synthesis of ASGPR ligand-targeted modifier in non-aqueous medium].

    Science.gov (United States)

    Cheng, Yi; Wu, Wei; Zhang, Dong-qing; Mai, Yan-zhen

    2010-09-01

    The asialoglycoprotein receptor (ASGPR) was used to mediate drug carrier for hepatic targeted drug delivery, this article showed the enzyme-catalyzed esterification of galactose and vinyl stearate and a kind of ASGPR ligand-targeted which was used to insert the surface of liposome has been synthesized. The structure of product has been confirmed by TLC, ESI-MS and 1H NMR. The factors of types and quantity of enzyme, organic solvents, molar ratio of substrate, temperature and time of reaction have been studied. Results showed when using acetone as reaction medium, the quantity of Novozym 435 immobilized lipase was 30 mg mL(-1), molar ratio of galactose to vinyl stearate was 1:5, and reacted at 60 degrees C for 12 h, the transformation of vinyl stearate reached more than 70%. This study provides a novel and efficient route to the synthesis of ligand-targeted modifier.

  20. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

    Science.gov (United States)

    Rajeev, Kallanthottathil G; Nair, Jayaprakash K; Jayaraman, Muthusamy; Charisse, Klaus; Taneja, Nate; O'Shea, Jonathan; Willoughby, Jennifer L S; Yucius, Kristina; Nguyen, Tuyen; Shulga-Morskaya, Svetlana; Milstein, Stuart; Liebow, Abigail; Querbes, William; Borodovsky, Anna; Fitzgerald, Kevin; Maier, Martin A; Manoharan, Muthiah

    2015-04-13

    We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

  1. 肝靶向性聚集超声分子显像的实验研究

    Institute of Scientific and Technical Information of China (English)

    余进洪; 王志刚; 李奥; 郑元义; 周洋

    2011-01-01

    @@ 去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是哺乳动物肝实质细胞特有的高效内吞受体,肝细胞表面ASGPR的量与肝功能状态明显相关,在肝细胞损害时,其表面的ASGPR会相应减少[1],该指标可以直接反映肝的储备功能.实验中针对ASGPR这一受体,将靶向纳米脂质微囊液态氟烷超声造影剂团注入正常大鼠体内,观察该靶向超声造影剂对正常大鼠的肝特异性显影,为超声造影在体评价肝功能提供实验依据.

  2. 半乳糖基多聚赖氨酸介导自杀基因对肝癌细胞的杀伤效应

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 曹利民; 司进; 李兴睿; 王健; 易继林

    2005-01-01

    去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASG—PR)是哺乳动物肝实质细胞特有的一种高效内吞受体,专一性识别、结合并内吞循环血液中一些带有末端半乳糖基的糖蛋白,并使其在肝细胞内进行代谢。可利用该受体介导的内吞机制进行基因的肝靶向运送,其中制备实用而高效的肝靶向配体是实现肝靶向运送的关键之一。ASGPR的内源性配体与其亲和力较高,但由于存在来源少、

  3. Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand.

    Science.gov (United States)

    Tep, Karona; Korb, Virginie; Richard, Cyrille; Escriou, Virginie; Largeau, Céline; Vincourt, Véronique; Bessodes, Michel; Guellier, Adeline; Scherman, Daniel; Cynober, Luc; Chaumeil, Jean-Claude; Dumortier, Gilles

    2009-01-01

    An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (+/- DOPE) (+/- Lac-10-Chol) that present reproducible absolute entrapment value (1.45 +/- 0.10%), with a size of 109 +/- 7 nm and a slight positive charge (3.77 +/- 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (P > 0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (P < 0.05).

  4. Targeting and molecular imaging of HepG2 cells using surface-functionalized gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Rathinaraj, Pierson [Auckland University of Technology, Institute of Biomedical Technologies (New Zealand); Lee, Kyubae; Choi, Yuri; Park, Soo-Young [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of); Kwon, Oh Hyeong [Kumoh National Institute of Technology, Department of Polymer Science and Engineering (Korea, Republic of); Kang, Inn-Kyu, E-mail: ikkang@knu.ac.kr [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of)

    2015-07-15

    Mercaptosuccinic acid (M)-conjugated gold nanoparticles (GM) were prepared and characterized by transmission electron microscope and dynamic light scattering. M was used to improve the monodispersity and non-specific intracellular uptake of nanoparticles. Lactobionic acid (L) was subsequently conjugated to the GM to target preferentially HepG2 cells (liver cancer cells) that express asialoglycoprotein receptors (ASGPR) on their membrane surfaces and facilitate the transit of nanoparticles across the cell membrane. The mean size of lactobionic acid-conjugated gold nanoparticle (GL) was approximately 10 ± 0.2 nm. Finally, the Atto 680 dye (A6) was coupled to the nanoparticles to visualize their internalization into HepG2 cells. The interaction of surface-modified gold nanoparticles with HepG2 cells was studied after culturing cells in media containing the GM or L-conjugated GM (GL)

  5. Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin.

    Science.gov (United States)

    Wang, Ying; Liu, Xinquan; Liu, Guangpu; Guo, Hejian; Li, Caiyun; Zhang, Yongchun; Zhang, Fang; Zhao, Zhongxi; Cheng, Huiling

    2016-04-11

    Nanoparticles based on the newly synthesized copolymers of linear PLGA blocked with two TPGS ends and galactosylated TPGS were successfully constructed as carriers of oridonin for liver-targeting. The novel copolymers were characterized by (1)H-NMR and TGA. The drug-loaded nanoparticles were prepared by a nanoprecipitation technique and characterized in terms of physicochemical properties, such as particle size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and physical state of the entrapped drug. The ORI-Gal-PT NPs were found to have the highest antitumor efficacy in comparison with the oridonin solution and non-galactosylated nanoparticles and induced a higher apoptotic rate of tumor cells. The targeting nanoparticles could enhance the therapeutic effect of oridonin by increasing uptake of the nanoparticles through asialoglycoprotein receptor-mediated endocytosis. The ORI-Gal-PT NPs system could be a highly promising drug delivery system to be used in liver cancer therapy.

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

    Science.gov (United States)

    Gateau, Christelle; Delangle, Pascale

    2014-05-01

    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.

  7. Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes.

    Science.gov (United States)

    Kinberger, Garth A; Prakash, Thazha P; Yu, Jinghua; Vasquez, Guillermo; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Murray, Heather M; Gaus, Hans; Swayze, Eric E; Seth, Punit P

    2016-08-01

    Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

  8. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.

    Science.gov (United States)

    Goh, John S Y; Chan, Kah Fai; Song, Zhiwei

    2015-01-01

    The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

  9. Protease-triggered siRNA delivery vehicles.

    Science.gov (United States)

    Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H; Benson, Jonathan D; Carlson, Jeffrey C; Klein, Jason J; Almeida, Lauren J; Nicholas, Anthony L; Hamilton, Holly L; Chu, Qili; Hegge, Julia O; Wong, So C; Trubetskoy, Vladimir S; Hagen, Collin M; Kitas, Eric; Wolff, Jon A; Lewis, David L

    2015-07-10

    The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

  10. Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR.

    Science.gov (United States)

    Hu, Bin; Yang, Yan; Liu, Jia; Ma, Zhiyong; Huang, Hongping; Liu, Shenpei; Yu, Yuan; Hao, Youhua; Wang, Baoju; Lu, Mengji; Yang, Dongliang

    2010-10-01

    To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

  11. Lactoferrin-modified PEGylated liposomes loaded with doxorubicin for targeting delivery to hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Wei MY

    2015-08-01

    Full Text Available Minyan Wei,1,2 Xiucai Guo,1,3 Liuxiao Tu,1 Qi Zou,1 Qi Li,1 Chenyi Tang,1 Bao Chen,1 Yuehong Xu,1 Chuanbin Wu1 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Sun Yat-sen University, 2Department of Pharmaceutics, School of Pharmaceutical Sciences, Guangzhou Medical University, 3Department of Pharmacy, 12th People’s Hospital of Guangzhou City, Guangzhou, People’s Republic of China Abstract: Lactoferrin (Lf is a potential-targeting ligand for hepatocellular carcinoma (HCC cells because of its specific binding with asialoglycoprotein receptor (ASGPR. In this present work, a doxorubicin (DOX-loaded, Lf-modified, polyethylene glycol (PEGylated liposome (Lf-PLS system was developed, and its targeting effect and antitumor efficacy to HCC was also explored. The DOX-loaded Lf-PLS system had spherical or oval vesicles, with mean particle size approximately 100 nm, and had an encapsulation efficiency of 97%. The confocal microscopy and flow cytometry indicated that the cellular uptake of Lf-PLS was significantly higher than that of PEGylated liposome (PLS in ASGPR-positive cells (P<0.05 but not in ASGPR-negative cells (P>0.05. Cytotoxicity assay by MTT demonstrated that DOX-loaded Lf-PLS showed significantly stronger antiproliferative effects on ASGPR-positive HCC cells than did PLS without the Lf modification (P<0.05. The in vivo antitumor studies on male BALB/c nude mice bearing HepG2 xenografts demonstrated that DOX-loaded Lf-PLS had significantly stronger antitumor efficacy compared with PLS (P<0.05 and free DOX (P<0.05. All these results demonstrated that a DOX-loaded Lf-PLS might have great potential application for HCC-targeting therapy. Keywords: asialoglycoprotein receptor, immunoliposome, PEGylated modification, post-insertion, hepatic cancer, active targeting

  12. Verification of the interaction between ASGPR and HBV preS1 protein%ASGPR 与 HBV preS1蛋白之间相互作用的验证

    Institute of Scientific and Technical Information of China (English)

    张曦; 刘小静; 陈云茹; 孔颖; 杨雪亮; 叶峰; 蔺淑梅

    2016-01-01

    目的:验证去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)与乙肝病毒前 S1蛋白(HBV preS1蛋白)之间的相互作用,确认 ASGPR 作为乙肝病毒肝细胞膜受体在介导乙肝病毒感染的分子机制中的作用。方法分别用哺乳动物双杂交及体外免疫共沉淀技术验证 ASGPR 与 HBV preS1蛋白之间的相互作用,操作方法参照试剂盒说明书进行。结果哺乳动物双杂交实验结果提示,ASGPR 与 HBV preS1蛋白在细胞环境中具有相互作用;免疫共沉淀实验结果提示,ASGPR 与 HBV preS1蛋白在非细胞环境中具有相互作用。结论ASGPR 可能是介导 HBV 入侵的肝细胞膜受体之一。%Objective To verify the interaction between asialoglycoprotein receptor (ASGPR)and hepatitis B virus (HBV)preS1 protein in vivo and in vitro ,and identify ASGPR as a cell-surface receptor for HBV,which could elucidate the molecular mechanism of HBV infection.Methods The preS1-ASGPR interaction was examined in mammalian two-hybrid and coimmunoprecipitation system by strictly following the manufacturer’s instructions.Results ASGPR interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro .Conclusion ASGPR may be a candidate receptor for HBV that mediates further step of HBV entry.

  13. Biodistribution of {sup 99m}Tc-lactosylated serum albumin in mice with diethylnitrosamine or thiacetamide induced liver injury

    Energy Technology Data Exchange (ETDEWEB)

    Whang, Jae Seok; Ahn, Byeong Cheol; Sung, Young Ok; Seo, Ji Hyoung; Bae, Jin Ho; Jeong, Shin Young; Yoo, Jung Soo; Lee, Jae Tae; Lee, Kyu Bo [Kyungpook National University Medical School, Daegu (Korea, Republic of); Jeong, Jae Min [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2005-02-15

    Tc-99m labeled diethylenetriaminepentaacetic acid (DTPA)-coupled galactosylated human serum albumin (GSA) is a currently used imaging agent for asialoglycoprotein receptor (ASGPR) of the liver, but it has several shortcomings. Recently a new ASGPR imaging agent, {sup 99m}Tc-lactosylated human serum albumin (LSA), with simple labeling procedure, high labeling efficiency, high stability was developed. In order to assess the feasibility of the {sup 99m}Tc-LSA as a ASGPR imaging radiopharmaceuticals, we performed biodistribution study of the tracer in liver injured mice model and the results were compared with histologic data. To induce hepatic damage in ICR mice, diethylnitrosamine (DEN) (60 mg/kg/week X 5 time, low dose or 180 mg/kg/week X 2 time, high dose) and thioacetamide (TAA) (50 mg/kg X 1 time) were administrated intraperitoneally. Degree of liver damage was evaluated by tissue hematoxilin-eosin stain, and expression of asialoglycoprotein receptor (ASGPR) was assessed by immunohistochemistry using ASGPR antibody. {sup 99m}Tc-LSA was intravenously administrated via tail vein in DEN or TAA treated mice, and biodistribution study of the tracer was also performed. DEN treated mice showed ballooning of hepatocyte and inflammatory cell infiltration in low dose group and severe hepatocyte necrosis in high dose group, and low dose group showed higher ASGPR staining than control mice in immunohistochemical staining. TAA treated mice showed severe hepatic necrosis. {sup 99m}Tc-LSA biodistribution study showed that mice with hepatic necrosis induced by high dose. DEN or TAA revealed higher blood activity and lower liver activity than control mice, due to slow clearance of the tracer by the liver. The degree of liver uptake was inversely correlated with the degree of histologic liver damage. But low dose DEN treated mice with mild hepatic injury showed normal blood clearance and hepatic activity, partly due to overexpression of ASGPR in mice with mild degree hepatic

  14. Amidine-bearing lipoplex targeting to hepatocyte cells

    Institute of Scientific and Technical Information of China (English)

    Yasuya Kudo; Kazunori Koiwai; Kazuhiro Shimizu; Shota Kusuki; Mina Sakuragi; Naohiko Shimada; Yoichi Takeda; Kazuo Sakurai

    2008-01-01

    A lipoplex (i.e., pDNA#1/lipid complex and transfection reagent for pDNA delivery) containing galactosylceramide (GalCer) and an amidine-bearing lipid (TRX) was examined whether the bound pDNA was specifically ingested by hepatocyte via asialoglycoprotein receptor (ASGPR) and then expressed protein. Gel electrophoresis and small-angle X-ray scattering (SAXS) confirmed that the TRX-GalCer liposome#2 complexed with pDNA and the resultant lipoplex took a hexagonally packed inverted cylinder structure when the GalCer composition was less than 20 wt.% of the total lipid. When the lipoplex carrying pGL3 (luciferase-cording pDNA) was administrated to HepG2, the luciferase activity was increased with increasing the GalCer composition until it reached 3 wt.% and then decreased upon further addition of GalCer. When we added galactose itself as a competitor, the luciferase activity was decreased, while glucose did not show such decrease, suggesting that HepG2 ingested the lipoplex via ASGPR-mediated endocytosis. This paper indicated that the hexagonally packed inverted cylinder structures of lipoplex may not always provide excellent transfection and presented a possibility that the TRX lipoplex#3 can obtain a cellular-targeting ability through the receptors for oligosaccharide.

  15. Cloning expeditions: risky but rewarding.

    Science.gov (United States)

    Lodish, Harvey

    2013-12-01

    In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine.

  16. New competition assay for the solubilized hepatic galactosyl receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ray, D.A.; Weigel, P.H.

    1985-02-15

    The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total /sup 125/I-asialo-orosomucoid (/sup 125/I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both /sup 125/I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the /sup 125/I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.

  17. Superparamagnetic Iron Oxide Nanoparticles Coated with Galactose-Carrying Polymer for Hepatocyte Targeting

    Directory of Open Access Journals (Sweden)

    Mi Kyong Yoo

    2007-01-01

    Full Text Available Our goal is to develop the functionalized superparamagnetic iron oxide nanoparticles (SPIONs demonstrating the capacities to be delivered in liver specifically and to be dispersed in physiological environment stably. For this purpose, SPIONs were coated with polyvinylbenzyl-O-β-D-galactopyranosyl-D-gluconamide (PVLA having galactose moieties to be recognized by asialoglycoprotein receptors (ASGP-R on hepatocytes. For use as a control, we also prepared SPIONs coordinated with 2-pyrrolidone. The sizes, size distribution, structure, and coating of the nanoparticles were characterized by transmission electron microscopy (TEM, electrophoretic light scattering spectrophotometer (ELS, X-ray diffractometer (XRD, and Fourier transform infrared (FT-IR, respectively. Intracellular uptake of the PVLA-coated SPIONs was visualized by confocal laser scanning microscopy, and their hepatocyte-specific delivery was also investigated through magnetic resonance (MR images of rat liver. MRI experimental results indicated that the PVLA-coated SPIONs possess the more specific accumulation property in liver compared with control, which suggests their potential utility as liver-targeting MRI contrast agent.

  18. Gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugate of arabinogalactan as a potential liver-targeting magnetic resonance imaging contrast agent.

    Science.gov (United States)

    Xiao, Yan; Xue, Rong; You, Tianyan; Li, Xiaojing; Pei, Fengkui; Wang, Xuxia; Lei, Hao

    2014-08-18

    A novel biocompatible macromolecule (AG-CM-EDA-DOTA-Gd) was synthesized as a liver magnetic resonance imaging (MRI) contrast agent. AG-CM-EDA-DOTA-Gd consisted of a carboxymethyl-arabinogalactan unit conjugated with gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd-DOTA) via ethylenediamine, and was specifically designed to bind to hepatocyte asialoglycoprotein in vivo, in an effort to develop a potential new tool for the diagnosis of liver diseases. The T1-relaxivity (8.87mmol(-1)Ls(-1)) of AG-CM-EDA-DOTA-Gd was 1.86 times than that of Gd-DOTA (4.76mmol(-1)Ls(-1)) in D2O at 9.4 T and 25°C. MRI experiments showed significant enhancement in rat liver following the intravenous administration of AG-CM-EDA-DOTA-Gd (0.094mmol Gd(3+)/kg body weight), which persisted for longer than Gd-DOTA (0.098mmol Gd(3+)/kg body weight). The mean percentage enhancements in the liver parenchyma were 85.2±6.5% and 19.3±3.3% for AG-CM-EDA-DOTA-Gd and Gd-DOTA, respectively. The results of this study therefore indicate that AG-CM-EDA-DOTA-Gd could be used as a potential liver-targeting contrast agent for MRI.

  19. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

    Science.gov (United States)

    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  20. Clinical evaluation of hepatic scintigraphy using sup 99m Tc-GSA

    Energy Technology Data Exchange (ETDEWEB)

    Ishii, Katsumi; Nishiyama, Shogo; Nishimaki, Hiroshi; Tadokoro, Katsumi; Ikeda, Toshiaki; Matsubayashi, Takashi; Ishii, Kodo (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)

    1992-06-01

    Functional hepatic imaging was performed using {sup 99m}Tc-DTPA-galactosyl human serum albumin ({sup 99m}Tc-GSA), a radiolabeled ligand that reacts specifically to the asialoglycoprotein receptor that resides at the plasma membrane of hepatocytes, in 21 patients: five with chronic active hepatitis (CAH), 14 with compensative liver cirrhosis (LC), one with chronic inactive hepatitis and one with acute hepatitis. The former two diseases were mainly investigated. Serial liver images were acquired at the rates of 10 sec/frame for 0-5 min and 2 min/frame for 6-30 min after the injection of {sup 99m}Tc-GSA, and the images were compared with {sup 99m}Tc-phytase images in 2 patients with CAH and 11 with LC, and those with portal scintigrams using {sup 123}I-iodoamphetamine (IMP) in 3 patients with LC. The images using {sup 99m}Tc-GSA were in better agreement with hepatic function than those using {sup 99m}Tc-phytase, and with the findings of portal scintigraphy using {sup 123}I-IMP. LHL15 (liver/liver and heart radioactivities at 15 min after injection of {sup 99m}Tc-GSA) correlated with the hepaplastin test (r=0.978 in CAH, and r=0.544 in LC), indicators of hepatic reserve. These results suggest that liver scintigraphy using {sup 99m}Tc-GSA might be a useful method for evaluating liver function. (author).

  1. Lactosamination of liposomes and hepatotropic targeting research

    Institute of Scientific and Technical Information of China (English)

    Yong Peng Chen; Lian Zhang; Qiao Sheng Lu; Xiao Rong Feng; Kang Xian Luo

    2000-01-01

    Site-specific delivery of therapeutic drugs to their target cells is a major scientific challenge for the pharmaceutical sciences. It offers a number of advantages over conventional drug administration. With drug targeting, high local concentrations of the drug can be achieved, thus circumventing many unwanted side effects. Various carriers have been suggested for the delivery of drugs, including liposomes[1 - 5] and (neo ) glycoproteins[6-8]. The asialoglycoprotein receptor (ASGP-R) has frequently been utilized for targeting drugs to the parenchymal liver cell[6- 12]. Liposomes have several advantageous characteristics as drug carrier, and particularly, ligandtacked liposomes achieve a highly effective targeting[13]. Hara et al reported that asialofetuin (AF)-tacked liposomes distributed to rat hepatocytes selectively in vivo[14], and ASGP-R mediated the uptake of AF-liposomes encapsulating IFN-γ by isolated rat hepatocytes in vitro[15]. Lactosaminated human serum albumin (L-HSA) is a neoglycoprotein taking number of galactose residue as terminal sugar[6].

  2. Improved nanoparticles preparation and drug release for liver targeted delivery

    Directory of Open Access Journals (Sweden)

    Qiao Weili

    2009-05-01

    Full Text Available "nTargeted delivery of drugs and proteins to liver can be achieved via asialoglycoprotein receptor, which can recognize and combine the galactose- and N-acetygalatosamine-terminated glycoproteins. Glycosyl is usually conjugated with drugs directly to fabricate prodrugs or with nanoparticles encapsulated drugs via forming covalent bonds, while the covalent bonds may lead to some shortages for drug release. Therefore, we have a hypothesis that we can prepare nanoparticles for efficient targeting by glycosylation using galactosylated poly (L-glutamic acid (Gal-PLGA as a carrier to entrap the model drugs in nanoparticles core physically rather than forming covalent drug conjugation. The means of incorporation of drug in nanoparticles may improve drug release to maintain its activity, raise its therapeutic index and diminish the adverse effect. Based on previous researches, it is achievable to obtain nanoparticles that we hypothesize to prepare. Due to their nanometer-size and galactosyl, the nanoparticles may be a potential delivery system for passive and active targeting to liver parenchymal cells for therapy of hepatitis and liver cancer.

  3. Establishment of Stable Cell Line Expressing Human Hepatocyte Surface Molecular ASGPR%表达人去唾液酸糖蛋白受体分子细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    胡斌; 杨燕; 刘嘉; 马智勇; 郝友华; 黄红平; 杨东亮

    2009-01-01

    目的 构建稳定表达人肝细胞表面分子去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)的细胞系.方法 逆转录PCR扩增人肝组织ASGPR大亚基H1全编码序列,插入到真核表达载体pIRES2EGFP中,重组质粒pIRES2EGFP/ASGPRH1转染HeLa细胞,G418筛选,RT-PCR,Western印迹及免疫荧光检测ASGPRH1的表达.结果 成功构建了pIRES2EGFP/ASGPRH1重组质粒,该质粒转染HeLa细胞后,Western印迹及免疫荧光均检测到ASGPRH1蛋白的表达.结论 成功建立了稳定表达人ASGPRH1的细胞系,为进一步研究ASGPR分子奠定了基础.

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

    Science.gov (United States)

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    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. PMID:27321376

  5. Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

    Science.gov (United States)

    Sakashita, Mizuha; Mochizuki, Shinichi; Sakurai, Kazuo

    2014-10-01

    Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.

  6. Lactoferrin-modified PEGylated liposomes loaded with doxorubicin for targeting delivery to hepatocellular carcinoma.

    Science.gov (United States)

    Wei, Minyan; Guo, Xiucai; Tu, Liuxiao; Zou, Qi; Li, Qi; Tang, Chenyi; Chen, Bao; Xu, Yuehong; Wu, Chuanbin

    2015-01-01

    Lactoferrin (Lf) is a potential-targeting ligand for hepatocellular carcinoma (HCC) cells because of its specific binding with asialoglycoprotein receptor (ASGPR). In this present work, a doxorubicin (DOX)-loaded, Lf-modified, polyethylene glycol (PEG)ylated liposome (Lf-PLS) system was developed, and its targeting effect and antitumor efficacy to HCC was also explored. The DOX-loaded Lf-PLS system had spherical or oval vesicles, with mean particle size approximately 100 nm, and had an encapsulation efficiency of 97%. The confocal microscopy and flow cytometry indicated that the cellular uptake of Lf-PLS was significantly higher than that of PEGylated liposome (PLS) in ASGPR-positive cells (PASGPR-negative cells (P>0.05). Cytotoxicity assay by MTT demonstrated that DOX-loaded Lf-PLS showed significantly stronger antiproliferative effects on ASGPR-positive HCC cells than did PLS without the Lf modification (P<0.05). The in vivo antitumor studies on male BALB/c nude mice bearing HepG2 xenografts demonstrated that DOX-loaded Lf-PLS had significantly stronger antitumor efficacy compared with PLS (P<0.05) and free DOX (P<0.05). All these results demonstrated that a DOX-loaded Lf-PLS might have great potential application for HCC-targeting therapy.

  7. Intracellular delivery of cytochrome c by galactosylated albumin to hepatocarcinoma cells.

    Science.gov (United States)

    Yeh, Tzyy-Harn; Wu, Fe-Lin Lin; Shen, Li-Jiuan

    2014-07-01

    In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery.

  8. Liver-targeting of interferon-alpha with tissue-specific domain antibodies.

    Science.gov (United States)

    Coulstock, Edward; Sosabowski, Jane; Ovečka, Milan; Prince, Rob; Goodall, Laura; Mudd, Clare; Sepp, Armin; Davies, Marie; Foster, Julie; Burnet, Jerome; Dunlevy, Gráinne; Walker, Adam

    2013-01-01

    Interferon alpha (IFNα) is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb) specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR). Our results show that the murine IFNα2 homolog (mIFNα2) fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb VHD2 (which does not bind ASGPR) was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections.

  9. Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

    Science.gov (United States)

    Shah, Sanket M; Goel, Peeyush N; Jain, Ankitkumar S; Pathak, Pankaj O; Padhye, Sameer G; Govindarajan, Srinath; Ghosh, Sandipto S; Chaudhari, Pradip R; Gude, Rajiv P; Gopal, Vijaya; Nagarsenker, Mangal S

    2014-12-30

    Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.

  10. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

    Science.gov (United States)

    Matsuda, Shigeo; Keiser, Kristofer; Nair, Jayaprakash K; Charisse, Klaus; Manoharan, Rajar M; Kretschmer, Philip; Peng, Chang G; V Kel'in, Alexander; Kandasamy, Pachamuthu; Willoughby, Jennifer L S; Liebow, Abigail; Querbes, William; Yucius, Kristina; Nguyen, Tuyen; Milstein, Stuart; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2015-05-15

    Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

  11. The preparation and characterization of porous galactosylated chitosan scaffolds%半乳糖修饰大孔壳聚糖支架的制备及表征

    Institute of Scientific and Technical Information of China (English)

    包志明; 潘继伦; 吴晨; 俞耀庭

    2004-01-01

    生物人工肝反应器中肝细胞的数量和功能是能否有效替代已衰竭肝脏功能的关键,与此紧密相关的是细胞支架材料结构和性能的优化.采用冷冻干燥法,制备了大孔壳聚糖支架,孔隙率在90%以上,平均孔径在100~200μm之间.以肝细胞表面去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)的特异性配体-半乳糖,对材料表面进行糖基化修饰,制备了半乳糖基修饰的大孔壳聚糖支架,肝细胞在其上生长状况良好,细胞培养密度高,为高密度培养肝细胞提供了一种性能优良的支架材料.

  12. Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells.

    Science.gov (United States)

    Li, Dapeng; Romain, Gabrielle; Flamar, Anne-Laure; Duluc, Dorothée; Dullaers, Melissa; Li, Xiao-Hua; Zurawski, Sandra; Bosquet, Nathalie; Palucka, Anna Karolina; Le Grand, Roger; O'Garra, Anne; Zurawski, Gerard; Banchereau, Jacques; Oh, Sangkon

    2012-01-16

    Dendritic cells (DCs) can initiate and shape host immune responses toward either immunity or tolerance by their effects on antigen-specific CD4(+) T cells. DC-asialoglycoprotein receptor (DC-ASGPR), a lectinlike receptor, is a known scavenger receptor. Here, we report that targeting antigens to human DCs via DC-ASGPR, but not lectin-like oxidized-LDL receptor, Dectin-1, or DC-specific ICAM-3-grabbing nonintegrin favors the generation of antigen-specific suppressive CD4(+) T cells that produce interleukin 10 (IL-10). These findings apply to both self- and foreign antigens, as well as memory and naive CD4(+) T cells. The generation of such IL-10-producing CD4(+) T cells requires p38/extracellular signal-regulated kinase phosphorylation and IL-10 induction in DCs. We further demonstrate that immunization of nonhuman primates with antigens fused to anti-DC-ASGPR monoclonal antibody generates antigen-specific CD4(+) T cells that produce IL-10 in vivo. This study provides a new strategy for the establishment of antigen-specific IL-10-producing suppressive T cells in vivo by targeting whole protein antigens to DCs via DC-ASGPR.

  13. Evaluating the potential of a new isotope-labelled glyco-ligand for estimating the remnant liver function of schistosoma-infected mice.

    Science.gov (United States)

    Cheng, P-C; Chiang, P-F; Lee, K-M; Yeh, C-H; Hsu, K-L; Liu, S-W; Shen, L-H; Peng, C-L; Fan, C-K; Luo, T-Y

    2013-01-01

    A new glyco-derivative compound (OCTAM) was developed and labelled with isotope to form (188) Re-OCTAM as a candidate nuclear medicine imaging agent for testing the liver function. We evaluated the potential of isotope-labelled OCTAM for estimating the remnant liver function in vitro and in vivo schistosoma-infected mice. The affinity of OCTAM to liver asialoglycoprotein receptors (ASGPR) was assessed by competitive inhibition assay in vitro. In vivo assessments were performed to score the remnant liver function in mice at different schistosomal infection stages. OCTAM binds specifically to ASGPR and showed competitive inhibition of anti-ASGPR antibody binding to hepatocytes, and was higher than that of other galactosyl ligands. Micro-SPECT/CT images of uninfected mice revealed strong liver uptake. Quantified serial images of mice infected for 9, 12 and 18 weeks showed delayed liver uptake, and the retention of uptake was inversely correlated with stage and grade of schistosoma infection. Pathological and biochemical analysis demonstrated that gradually accumulating liver injury caused by infection significantly influenced uptake of (188) Re-OCTAM. Hepatic ASGPR expression diminished only in the chronic infection stage. This study demonstrated that the isotope-labelled OCTAM could accumulate in the liver, might have potential as an imaging agent for in vivo hepatic function evaluation of schistosomiasis.

  14. 去唾液酸糖蛋白受体介导的肝靶向研究进展%Progress on ASGPR-Mediated Hepatic Targeting

    Institute of Scientific and Technical Information of China (English)

    梁美好; 沈正荣; 马臻; 王尊元

    2010-01-01

    目的 介绍去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)介导的药物和基因肝靶向作用和机制,及其在治疗肝癌和乙型肝炎实验研究方面的最新进展.方法 查阅和选取针对性强、相关度高的文献,总结和归纳ASGPR介导的药物和基因肝靶向用于治疗肝癌和乙型肝炎的最新研究进展.结果 ASGPR是肝细胞的一种重要且高效的内吞受体,可用于介导药物和基因的肝靶向递送.结论 ASGPR介导的药物和基因肝靶向有望成为肝癌和乙型肝炎治疗的有效手段.

  15. 去唾液酸糖蛋白受体与慢性肝病自身免疫

    Institute of Scientific and Technical Information of China (English)

    刘海英; 仲人前; 孔宪涛

    2003-01-01

    @@ 去唾液酸糖蛋白受体(Asialoglycoprotein Receptor ,ASGPR)是肝细胞的一种重要且高效的内吞受体,主要分布于肝小叶门静脉周围肝细胞的窦面膜上,又名肝凝集素(liver lectin).近年来研究发现该受体除了在肝脏基因定向转移、靶向药物治疗当中具有较高的应用价值之外,还相继在自身免疫性肝炎(AIH)、原发性胆汁性肝硬化(PBC)、病毒性肝炎等慢性肝病患者血清中检测到其相应的自身抗体,从这些患者外周血、肝活检组织分离到ASGPR特异性的T淋巴细胞.由于ASGPR的肝脏特异性与其在肝脏的特殊分布位置和某些慢性肝病的组织病理学特征极为吻合,从而激起了人们探讨ASGPR与慢性肝病自身免疫关系的兴趣.

  16. Hepatocyte-mediated cytotoxicity and host defense mechanisms in the alcohol-injured liver.

    Science.gov (United States)

    McVicker, Benita L; Thiele, Geoffrey M; Tuma, Dean J; Casey, Carol A

    2014-09-01

    The consumption of alcohol is associated with many health issues including alcoholic liver disease (ALD). The natural history of ALD involves the development of steatosis, inflammation (steatohepatitis), fibrosis and cirrhosis. During the stage of steatohepatitis, the combination of inflammation and cellular damage can progress to a severe condition termed alcoholic hepatitis (AH). Unfortunately, the pathogenesis of AH remains uncharacterized. Some modulations have been identified in host defense and liver immunity mechanisms during AH that highlight the role of intrahepatic lymphocyte accumulation and associated inflammatory cytokine responses. Also, it is hypothesized that alcohol-induced injury to liver cells may significantly contribute to the aberrant lymphocytic distribution that is seen in AH. In particular, the regulation of lymphocytes by hepatocytes may be disrupted in the alcoholic liver resulting in altered immunologic homeostasis and perpetuation of disease. In recent studies, it was demonstrated that the direct killing of activated T lymphocytes by hepatocytes is facilitated by the asialoglycoprotein receptor (ASGPR). The ASGPR is a well-characterized glycoprotein receptor that is exclusively expressed by hepatocytes. This hepatic receptor is known for its role in the clearance of desialylated glycoproteins or cells, yet neither its physiological function nor its role in disease states has been determined. Interestingly, alcohol markedly impairs ASGPR function; however, the effect alcohol has on ASGPR-mediated cytotoxicity of lymphocytes remains to be elucidated. This review discusses the contribution of hepatocytes in immunological regulation and, importantly, how pathological effects of ethanol disrupt hepatocellular-mediated defense mechanisms.

  17. The liver-targeting study of the N-galactosylated chitosan in vivo and in vitro.

    Science.gov (United States)

    Liang, Meihao; Zheng, Xiaoliang; Tu, Linglan; Ma, Zhen; Wang, Zunyuan; Yan, Dongmei; Shen, Zhengrong

    2014-12-01

    In order to study the liver targeting of the N-galactosylated chitosan (GC) polymer in liver, we first conjugated the lactobionic acid with chitosan (CS) to obtain the carrier of GC with different degree of substitution of lactosyl group. Western blot was performed to detect the expression levels of the asialoglycoprotein receptors (ASGPR) in the cell lines of HepG2, SMMC-7721, and HL-7702. The protein level of ASGPR was lower in HepG2 compared to HL-7702 and SMMC-7721. Although all treated by CS, viabilities of HL-7702 and HepG2 did not experience any significant drop, while viability of SMMC-7721 decreased 15% on average from control. It was the first data about the inhibitory effect of GC on the liver cells. Fluorescein isothiocyanate (FITC) labeled GC (GC-FITC) was injected intravenously into mice at a dose of 0.02 μmol/mouse. GC-FITC showed maximum liver localization at 5 min and even detectable at 48 h after injection. Further, the accumulation of GC in liver was about 5.4-fold higher than that of CS. In conclusion, GC demonstrated its higher efficacy in drug liver targeting and thus could be a more promising drug or gene carrier in future therapies.

  18. Self-assembled drug delivery systems. Part 7: hepatocyte-targeted nanoassemblies of an adefovir lipid derivative with cytochrome P450-triggered drug release.

    Science.gov (United States)

    Du, Lina; Wu, Lailong; Jin, Yiguang; Jia, Junwei; Li, Miao; Wang, Yu

    2014-09-10

    A novel strategy was used in the design of self-assembled drug delivery systems (SADDSs) in this study. The nanoassemblies of an amphiphilic adefovir lipid derivative were prepared and demonstrated to have the functions of hepatocyte targeting, enzyme-triggered drug release and high anti-hepatitis effect. An amphiphilic adefovir lipid derivative, N-lauroyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (LCPA) was prepared and formed the nanoassemblies by injecting the mixture of LCPA and another amphiphilic polymer, d-galactide polyoxyethylene (20) cetyl ether (GPCE) (ca. 20:1, mol/mol) into water. The nanoassemblies were very stable and showed negative charge. LCPA was sensitive to the cytochrome P450 isozymes that were expressed predominantly in the hepatocytes to produce adefovir. GPCE contained a long hydrophilic chain and a galactose ligand targeting the asialoglycoprotein receptors overexpressed on the surface of hepatocytes. The nanoassemblies showed the long-circulating and liver targeting effects according to the results of pharmacokinetics, tissue distribution and fluorescence imagination after bolus intravenous administration of the nanoassemblies to the mice. The highly efficient hepatitis B treatment was achieved by 10 day continuous administration of the nanoassemblies to the HBV-infected mice. Many functions were combined in the nanoassemblies, including prodrug, molecular self-assembly, nanotechnology, long-circulating, hepatocyte targeting and hepatocyte over expressing enzyme-triggered drug release.

  19. Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

    Science.gov (United States)

    Wong, Siew Heng; Zhang, Tao; Xu, Yue; Subramaniam, V. Nathan; Griffiths, Gareth; Hong, Wanjin

    1998-01-01

    Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. PMID:9614193

  20. A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

    Energy Technology Data Exchange (ETDEWEB)

    Yannam, Govardhana Rao [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Han, Bing [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Setoyama, Kentaro [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamamoto, Toshiyuki [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Ito, Ryotaro; Brooks, Jenna M. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Guzman-Lepe, Jorge [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Galambos, Csaba [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Fong, Jason V. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Deutsch, Melvin; Quader, Mubina A. [Department of Radiation Oncology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamanouchi, Kosho [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York (United States); Kabarriti, Rafi; Mehta, Keyur [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Soto-Gutierrez, Alejandro [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); and others

    2014-02-01

    Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

  1. Simultaneous SPECT imaging of multi-targets to assist in identifying hepatic lesions

    Science.gov (United States)

    Guo, Zhide; Gao, Mengna; Zhang, Deliang; Li, Yesen; Song, Manli; Zhuang, Rongqiang; Su, Xinhui; Chen, Guibing; Liu, Ting; Liu, Pingguo; Wu, Hua; Du, Jin; Zhang, Xianzhong

    2016-01-01

    Molecular imaging technique is an attractive tool to detect liver disease at early stage. This study aims to develop a simultaneous dual-isotope single photon emission computed tomography (SPECT)/CT imaging method to assist diagnosis of hepatic tumor and liver fibrosis. Animal models of liver fibrosis and orthotopic human hepatocellular carcinoma (HCC) were established. The tracers of 131I-NGA and 99mTc-3P-RGD2 were selected to target asialoglycoprotein receptor (ASGPR) on the hepatocytes and integrin αvβ3 receptor in tumor or fibrotic liver, respectively. SPECT imaging and biodistribution study were carried out to verify the feasibility and superiority. As expected, 99mTc-3P-RGD2 had the ability to evaluate liver fibrosis and detect tumor lesions. 131I-NGA showed that it was effective in assessing the anatomy and function of the liver. In synchronized dual-isotope SPECT/CT imaging, clear fusion images can be got within 30 minutes for diagnosing liver fibrosis and liver cancer. This new developed imaging approach enables the acquisition of different physiological information for diagnosing liver fibrosis, liver cancer and evaluating residual functional liver volume simultaneously. So synchronized dual-isotope SPECT/CT imaging with 99mTc-3P-RGD2 and 131I-NGA is an effective approach to detect liver disease, especially liver fibrosis and liver cancer. PMID:27377130

  2. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  3. [Preparation of hepatic targeting antivirus agent NGA-ACV and its targeting property].

    Science.gov (United States)

    Fan, J Z; Li, T L; Pang, Q J; Guan, C T; He, Y; Su, K Y

    1996-01-01

    Neoglycoalbumin (NGA), a special ligend of asialoglycoprotein receptor on the hepatocyte, was linked via a butanediacyl bridge to acyclovir to form a conjugate NGA-ACV. By using DTA (Differential thermoanalysis) and HPLC analysis, ACV was shown to be connected with NGA by covalent bonds and stable in blood. The radio-biodistribution of 131I-NGA-ACV with high drug density in vivo was carried out in mice. The maximum absorption of 131I-NGA-ACV in liver was 81.7 +/- 10.4% at 5 min. The radioimage of 131I-NGA-ACV with high or low drug density in rabbit showed no significant difference in liver targeting property. The competitive connection tests indicated that 131I-NGA-ACV was concentrated in liver through receptor mediated mechanism. A tentative test of antihepatitis B of NGA-ACV and ACV in vitro showed that the effective dose of the former was significantly lower than that of the latter.

  4. Cellular uptake of fluorophore-labeled glyco-DNA-gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Witten, Katrin G.; Ruff, Julie [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany); Mohr, Anne; Goertz, Dieter; Recker, Tobias; Rinis, Natalie [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Rech, Claudia; Elling, Lothar [RWTH Aachen University, Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering (Germany); Mueller-Newen, Gerhard [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Simon, Ulrich, E-mail: ulrich.simon@ac.rwth-aachen.de [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany)

    2013-10-15

    DNA-functionalized gold nanoparticles (AuNP-DNA) were hybridized with complementary di-N-acetyllactosamine-(di-LacNAc, [3Gal({beta}1-4)GlcNAc({beta}1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP-DNA-di-LacNAc. While AuNP-DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.

  5. Cellular uptake of fluorophore-labeled glyco-DNA-gold nanoparticles

    Science.gov (United States)

    Witten, Katrin G.; Ruff, Julie; Mohr, Anne; Görtz, Dieter; Recker, Tobias; Rinis, Natalie; Rech, Claudia; Elling, Lothar; Müller-Newen, Gerhard; Simon, Ulrich

    2013-10-01

    DNA-functionalized gold nanoparticles (AuNP-DNA) were hybridized with complementary di- N-acetyllactosamine-( di-LacNAc, [3Gal(β1-4)GlcNAc(β1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP-DNA- di-LacNAc. While AuNP-DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.

  6. Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.T.; Lee, Y.C.

    1987-10-06

    The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which (43 kilodalton (kDa)) exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an /sup 125/I-labeled high-affinity reagent the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. The authors postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work.

  7. DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

    Science.gov (United States)

    David, Stephanie; Passirani, Catherine; Carmoy, Nathalie; Morille, Marie; Mevel, Mathieu; Chatin, Benoit; Benoit, Jean-Pierre; Montier, Tristan; Pitard, Bruno

    2013-01-08

    We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.Molecular Therapy - Nucleic Acids (2013) 2, e64; doi:10.1038/mtna.2012.56; published online 8 January 2013.

  8. Radiopharmaceuticals in China. Current status and prospects

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Hong-Mei; Liu, Bo-Li [Beijing Normal Univ. (China). Key Laboratory of Radiopharmaceuticals

    2014-04-01

    The review provides an overview of the current status of radiopharmaceuticals in China for in vivo clinical use and also describes some important advances in the past three decades. Development of the diagnostic and therapeutic radiopharmaceuticals as well as basic research on radiopharmaceutical chemistry are being introduced. The radiotracers developed in China include: (1) Brain perfusion imaging agents and CNS radiotracers for β-amyloid plaques, σ{sub 1} receptors, and dopamine D{sub 2} or D{sub 4} receptors; (2) {sup 99m}Tc- and {sup 18}F-labeled myocardial perfusion imaging agents; (3) tumor imaging agents including integrin-targeting radiotracer, novel sentinel lymph node imaging agents, hypoxia imaging agents, {sup 99m}Tc-labeled glucose derivatives, σ{sub 2} receptor imaging agents, folate receptor imaging agents, and potential radiotracers for imaging of human telomerase reverse transcriptase expression; (4) Potential infection imaging agents; (5) Potential asialoglycoprotein receptor imaging agents; (6) Other imaging agents. Moreover, some prospects of research and development of radiopharmaceuticals in the near future are discussed. (orig.)

  9. A galactose-functionalized dendritic siRNA-nanovector to potentiate hepatitis C inhibition in liver cells

    Science.gov (United States)

    Lakshminarayanan, Abirami; Reddy, B. Uma; Raghav, Nallani; Ravi, Vijay Kumar; Kumar, Anuj; Maiti, Prabal K.; Sood, A. K.; Jayaraman, N.; Das, Saumitra

    2015-10-01

    A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting ``out'' in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the `proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated

  10. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Fatima Khaja

    2016-01-01

    Full Text Available Since its discovery, small interfering RNA (siRNA has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA not easily accessed by conventional drugs. Hence, RNA interference (RNAi therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs. This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF, an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP, showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  11. Hemocompatible pullulan-polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency.

    Science.gov (United States)

    Rekha, M R; Sharma, Chandra P

    2011-01-01

    Polyethyleneimine (PEI; 25 kDa)-conjugated pullulans (PPE1, PPE2 and PPE3) were developed and investigated for possible use in gene delivery applications. The cytotoxicity, blood component interactions such as red blood cell/white blood cell aggregation, platelet and complement activation, and protein interaction of the pullulan-conjugated PEI was drastically reduced in comparison to PEI-based nanocomplexes. Based on the blood compatibility studies, PPE1 was selected for further study. The buffering capacity of this derivative was similar to that of PEI, which plays an important role in efficient gene transfection. The particle size, zeta potential, stability in the presence of plasma and resistance to nuclease degradation were evaluated. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of HepG2 cells. Endocytosis inhibitors, confocal laser scanning microscopy and fluorescent labeling techniques were used to visualize the nanoplex uptake mechanism, cellular distribution and nuclear localization. The results from inhibitor experiments in the presence of asialofetuin indicated that the asialoglycoprotein receptor is involved in transfection of hepatocytes with pullulan-PEI complexes. The conjugation of pullulan with PEI did not hinder the plasmid nuclear localization ability of PEI. The transfection efficiency of pullulan conjugate was similar to PEI, with the added advantage of hemocompatibility and non-cytotoxicity. The transfection efficiency of PEI and PPE1 was 1.6- and 2-fold more, respectively, in the presence of serum than in the absence of serum. Therefore, the pullulan-PEI conjugate seems to be a promising gene delivery vector with good hemocompatibility and low toxicity but without compromising the transfection efficacy of PEI.

  12. Recent Findings Concerning PAMAM Dendrimer Conjugates with Cyclodextrins as Carriers of DNA and RNA

    Directory of Open Access Journals (Sweden)

    Keiichi Motoyama

    2009-08-01

    Full Text Available We have evaluated the potential use of various polyamidoamine (PAMAM dendrimer [dendrimer, generation (G 2-4] conjugates with cyclodextrins (CyDs as novel DNA and RNA carriers. Among the various dendrimer conjugates with CyDs, the dendrimer (G3 conjugate with α-CyD having an average degree of substitution (DS of 2.4 [α-CDE (G3, DS2] displayed remarkable properties as DNA, shRNA and siRNA delivery carriers through the sensor function of α-CDEs toward nucleic acid drugs, cell surface and endosomal membranes. In an attempt to develop cell-specific gene transfer carriers, we prepared sugar-appended α-CDEs. Of the various sugar-appended α-CDEs prepared, galactose- or mannose-appended α-CDEs provided superior gene transfer activity to α-CDE in various cells, but not cell-specific gene delivery ability. However, lactose-appended α-CDE [Lac-α-CDE (G2] was found to possess asialoglycoprotein receptor (AgpR-mediated hepatocyte-selective gene transfer activity, both in vitro and in vivo. Most recently, we prepared folate-poly(ethylene glycol-appended α-CDE [Fol-PαC (G3] and revealed that Fol-PαC (G3 imparted folate receptor (FR-mediated cancer cell-selective gene transfer activity. Consequently, α-CDEs bearing integrated, multifunctional molecules may possess the potential to be novel carriers for DNA, shRNA and siRNA.

  13. Cholesterol lowering effects of mono-lactose-appended β-cyclodextrin in Niemann–Pick type C disease-like HepG2 cells

    Directory of Open Access Journals (Sweden)

    Keiichi Motoyama

    2015-11-01

    Full Text Available The Niemann–Pick type C disease (NPC is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD has been applied for the treatment of NPC. HP-β-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by actively targeted-β-CyD to hepatocytes is expected. In the present study, to deliver β-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended β-CyD (Lac-β-CyD and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-β-CyD (degree of substitution of lactose (DSL 1 significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-β-CyD was associated with NPC-like HepG2 cells higher than TRITC-β-CyD. In addition, TRITC-Lac-β-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-β-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-β-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.

  14. Cholesterol lowering effects of mono-lactose-appended β-cyclodextrin in Niemann–Pick type C disease-like HepG2 cells

    Science.gov (United States)

    Motoyama, Keiichi; Hirai, Yumi; Nishiyama, Rena; Maeda, Yuki; Higashi, Taishi; Ishitsuka, Yoichi; Kondo, Yuki; Irie, Tetsumi; Era, Takumi

    2015-01-01

    Summary The Niemann–Pick type C disease (NPC) is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has been applied for the treatment of NPC. HP-β-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by actively targeted-β-CyD to hepatocytes is expected. In the present study, to deliver β-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended β-CyD (Lac-β-CyD) and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-β-CyD (degree of substitution of lactose (DSL) 1) significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-β-CyD was associated with NPC-like HepG2 cells higher than TRITC-β-CyD. In addition, TRITC-Lac-β-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-β-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-β-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease. PMID:26664628

  15. Receptor-mediated delivery of all-trans-retinoic acid (ATRA) to hepatocytes from ATRA-loaded poly(N-p-vinylbenzyl-4-o-{beta}-D-galactopyranosyl-D-gluconamide) nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Seog-Jin [School of Agricultural Biotechnology, Seoul National University, Seoul 151-742 (Korea, Republic of) ; Moon, Hyun-Seuk [School of Agricultural Biotechnology, Seoul National University, Seoul 151-742 (Korea, Republic of) ; Guo, Ding-Ding [School of Agricultural Biotechnology, Seoul National University, Seoul 151-742 (Korea, Republic of) ; Kim, Sang-Heon [Korea Institute of Science and Technology, Seoul 136-791 (Korea, Republic of) ; Akaike, Toshihiro [Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama 226-8501 (Japan); Cho, Chong-Su [School of Agricultural Biotechnology, Seoul National University, Seoul 151-742 (Korea, Republic of) ]. E-mail: chocs@plaza.snu.ac.kr

    2006-01-15

    All-trans-retinoic acid (ATRA) plays a role in regulating CYP26 gene expression in hepatocytes. Poly(N-p-vinylbenzyl-4-o-{beta}-D-galactopyranosyl-D-gluconamide) (PVLA) nanoparticles have been used as hepatocyte-specific targeting candidates. The objective of this study was to investigate receptor-mediated delivery of ATRA using PVLA nanoparticles. ATRA-loaded PVLA nanoparticles were confirmed by {sup 1}H-nuclear magnetic resonance ({sup 1}H-NMR) and powder X-ray diffraction (XRD). In the {sup 1}H-NMR study, the proton signals of ATRA disappeared in the spectrum of ATRA-loaded PVLA nanoparticles in D{sub 2}O, whereas in dimethylsulfoxide-d{sub 6}, the spectrum seemed like an addition of the respective spectrum of each of the pure components. The crystalline peaks of ATRA disappeared in the XRD pattern of ATRA-loaded PVLA nanoparticles after ATRA was loaded into PVLA nanoparticles. In the measurement of size distribution, diameter of PVLA and ATRA-loaded PVLA nanoparticles in aqueous solution was 6.9 and 61.2 nm in number average, respectively. Flow cytometric analysis showed that the internalization of FITC-PVLA nanoparticles by hepatocytes in the absence of a competitive inhibitor was larger than preincubated with galactose. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, ATRA-loaded PVLA nanoparticles induced CYP26A1 gene in hepatocytes in the absence of a competitive inhibitor but not preincubated with galactose. The results indicate that the ATRA-loaded PVLA nanoparticle can induce CYP26A1 gene in aqueous phase by an asialoglycoprotein receptor (ASGPR)-mediated delivery system.

  16. [In vitro targeting effect of lactoferrin modified PEGylated liposomes for hepatoma cells].

    Science.gov (United States)

    Wei, Min-yan; Zou, Qi; Wu, Chuan-bin; Xu, Yue-hong

    2015-10-01

    A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma.

  17. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy.

    Science.gov (United States)

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing

    2015-01-01

    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy.

  18. In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes.

    Science.gov (United States)

    Ketkar-Atre, Ashwini; Struys, Tom; Dresselaers, Tom; Hodenius, Michael; Mannaerts, Inge; Ni, Yicheng; Lambrichts, Ivo; Van Grunsven, Leo A; De Cuyper, Marcel; Himmelreich, Uwe

    2014-01-01

    The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

  19. Diagnosis of abnormal biliary copper excretion by positron emission tomography with targeting of (64)Copper-asialofetuin complex in LEC rat model of Wilson's disease.

    Science.gov (United States)

    Bahde, Ralf; Kapoor, Sorabh; Bhargava, Kuldeep K; Palestro, Christopher J; Gupta, Sanjeev

    2014-01-01

    Identification by molecular imaging of key processes in handling of transition state metals, such as copper (Cu), will be of considerable clinical value. For instance, the ability to diagnose Wilson's disease with molecular imaging by identifying copper excretion in an ATP7B-dependent manner will be very significant. To develop highly effective diagnostic approaches, we hypothesized that targeting of radiocopper via the asialoglycoprotein receptor will be appropriate for positron emission tomography, and examined this approach in a rat model of Wilson's disease. After complexing (64)Cu to asialofetuin we studied handling of this complex compared with (64)Cu in healthy LEA rats and diseased homozygous LEC rats lacking ATP7B and exhibiting hepatic copper toxicosis. We analyzed radiotracer clearance from blood, organ uptake, and biliary excretion, including sixty minute dynamic positron emission tomography recordings. In LEA rats, (64)Cu-asialofetuin was better cleared from blood followed by liver uptake and greater biliary excretion than (64)Cu. In LEC rats, (64)Cu-asialofetuin activity cleared even more rapidly from blood followed by greater uptake in liver, but neither (64)Cu-asialofetuin nor (64)Cu appeared in bile. Image analysis demonstrated rapid visualization of liver after (64)Cu-asialofetuin administration followed by decreased liver activity in LEA rats while liver activity progressively increased in LEC rats. Image analysis resolved this difference in hepatic activity within one hour. We concluded that (64)Cu-asialofetuin complex was successfully targeted to the liver and radiocopper was then excreted into bile in an ATP7B-dependent manner. Therefore, hepatic targeting of radiocopper will be appropriate for improving molecular diagnosis and for developing drug/cell/gene therapies in Wilson's disease.

  20. Research Progress of Assessment of Hepatic Function before Hepatectomy%肝切除术术前肝功能评价的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨波

    2011-01-01

    评价肝切除手术患者的肝功能储备是术前的重点和难点.目前常用的评估方法有Child-Turcotte-Pugh评分、终末期肝病模型评分、肝脏体积测量、去唾液酸糖蛋白受体功能显像技术和吲哚青绿排泄试验等.但是,目前没有一种完善、单一的评估方法可以全面地反映肝脏耐受手术的能力,尽管在这方面全球都在努力地探索.现总结具有代表性的相关文献,介绍各种肝功能评价的方法和优缺点.%The preoperative evaluation of the hepatic functional reserve is a difficult and important for hepatectomy. Child-Turcotte-Pugh scoring, model for end-stage liver disease scoring, hepatic volumetry, an imaging technique of asialoglycoprotein receptor and retention of indocyanine green are common measures for assessing the liver function that can be used before hepatectomy. However,there is no impeccable and single method that is capable of assessing the liver function comprehensively, regardless of the great effort being made all over the world. Here is to introduce some typical and most correlated literatures to set forth the advantages and disadvantages of different hepatic function assessment measures.

  1. Liver-targeting of interferon-alpha with tissue-specific domain antibodies.

    Directory of Open Access Journals (Sweden)

    Edward Coulstock

    Full Text Available Interferon alpha (IFNα is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR. Our results show that the murine IFNα2 homolog (mIFNα2 fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb VHD2 (which does not bind ASGPR was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections.

  2. Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin.

    Science.gov (United States)

    Takeya, Akira; Hosomi, Osamu; Nishijima, Hironori; Ohe, Yoshihide; Sugahara, Kunio; Sagi, Morihisa; Yamazaki, Kentaro; Hayakawa, Hideyuki; Takeshita, Hiroshi; Sasaki, Chizuko; Kogure, Tadahisa; Mukai, Toshiji

    2007-01-16

    Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.

  3. Development of {sup 68}Ga-labelled DTPA galactosyl human serum albumin for liver function imaging

    Energy Technology Data Exchange (ETDEWEB)

    Haubner, Roland [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); Medizinische Universitaet Innsbruck, Universitaetsklinik fuer Nuklearmedizin, Innsbruck (Austria); Vera, David R.; Farshchi-Heydari, Salman [University of California, Department of Radiology, School of Medicine, and the UCSD Molecular Imaging Program, San Diego, CA (United States); Helbok, Anna; Rangger, Christine; Putzer, Daniel; Virgolini, Irene J. [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria)

    2013-08-15

    The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [{sup 99m}Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([{sup 99m}Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [{sup 68}Ga]GSA for the potential use with positron emission tomography (PET). Labelling of GSA with {sup 68}Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [{sup 68}Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [{sup 68}Ga]GSA distribution in Lewis rats was compared with [{sup 99m}Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [{sup 68}Ga]GSA and [{sup 99m}Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). [{sup 68}Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free {sup 68}Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98 %). Stability after 120 min incubation at 37 C was high in PBS (>95 % intact tracer) and low in human serum ({proportional_to}27 % intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [{sup 68}Ga]GSA compared to [{sup 99m}Tc]GSA. The [{sup 99m}Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [{sup 68}Ga]GSA curves. The mean

  4. Inhibition of hepatitis B virus and induction of hepatoma cell apoptosis by ASGPR-directed delivery of shRNAs.

    Science.gov (United States)

    Ma, Jingwei; Huang, Chunmei; Yao, Xinxin; Shi, Chuan; Sun, Lifang; Yuan, Lu; Lei, Ping; Zhu, Huifen; Liu, Hongbo; Wu, Xiongwen; Ning, Qin; Zhou, Chun; Shen, Guanxin

    2012-01-01

    Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.

  5. 肝脏功能评估在临床中的应用与发展%Liver function assessment:Application and improvement in clinical practice

    Institute of Scientific and Technical Information of China (English)

    杜顺达; 车璐; 毛一雷

    2012-01-01

    Preoperative liver function assessment is very important for surgical planning and prognosis. A comprehensive scoring system incorporates various parameters which reflect a certain aspect of liver function such as synthesis and secretion, as well as impact of related organs. Currently, various metabolic liver function tests differ in their ability to reflect liver function. Combination of the scoring system with metabolic tests can better assess liver function and prognosis. However, these assessments only account for overall liver function. Liver volume assessment allows obtaining every segment volume without function, especially in case of cirrhosis. Imaging technique based on asialoglycoprotein receptor (ASGPR) reflects liver function in three dimensions and is now considered a promising liver function assessment. When combined with computer technology, it allows surgeons to perform resection simulation and evaluate surgical risk and prognosis preoperatively.%术前肝功能评估对制定手术方案、判断预后非常重要.由各指标组成的综合评分系统,考虑了肝脏合成、分泌等功能及相关脏器的影响.各种代谢性肝功能定量评估试验反映肝脏功能的能力不一,与综合评分系统共同应用,能更好的评估肝功能和预后,但这些评估只是针对肝脏整体,肝脏体积计算可以获得各部分的体积,但在肝硬化肝功能受损时体积并不能准确的反映功能,去唾液酸糖蛋白受体显像技术可以三维反映肝脏功能,结合计算机技术,进行术前模拟切除,可以获得手术风险信息,判断预后,目前被认为是一个有前途的肝功能评估方法.

  6. N-Acetylgalactosamine Block-co-Polycations Form Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery.

    Science.gov (United States)

    Dhande, Yogesh K; Wagh, Bharat S; Hall, Bryan C; Sprouse, Dustin; Hackett, Perry B; Reineke, Theresa M

    2016-03-14

    The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve

  7. 去唾液酸糖蛋白受体介导的肝靶向脂质体配体的酶促催化合成研究%Study on the Enzyme-Catalyzed Synthesis of Ligands for ASGPR-mediated Liver Targeting Liposomes

    Institute of Scientific and Technical Information of China (English)

    郭波红; 程怡; 林绿萍; 吴卫; 林德晖

    2012-01-01

    目的 用酶促催化乳糖酸与十八胺合成一种可用于镶嵌脂质体表面的去唾液酸糖蛋白靶向配体修饰物.方法 通过红外光谱(IR)、质谱(ESI-MS)和核磁共振(1H-NMR)对产物结构进行确证,并对酶种类、反应介质、酶的加入量、底物摩尔比、反应温度等影响因素进行考察.结果 二甲基亚砜作为反应介质;Novozym 435固定化脂肪酶作为催化剂、酶加入量为400U·mL-1、乳糖酸和十八胺的摩尔比为2:1、40℃下反应24 h,十八胺的转化率可达99%以上.结论 酶促催化法可用来合成肝靶向脂质体配体.%OBJECTIVE To synthesize asialoglycoprotein receptor ligand-targeted modifier which is used to insert the surface of liposome by enzyme-catalyzed amidation of lactobionic acid and stearamine. METHODS The structure of the product was confirmed-by IR, ESI-MS and ' H-NMR. The effects of types and quantity of enzyme, organic solvents, molar ratio of substrate and temperature of reaction were studied. RESULTS When using DMSO as reaction medium, Novozym 435 immobilized lipase at 400 U ? mL ~ , molar ratio of lactobionic acid to stearamine at 2- 1, and reacting at 40 °C for 24 h, the transformation of stearamine reached more than 99%. CONCLUSION The enzyme catalysis is useful for synthesizing liver targeting liposomes.

  8. Inhibition of hepatitis B virus and induction of hepatoma cell apoptosis by ASGPR-directed delivery of shRNAs.

    Directory of Open Access Journals (Sweden)

    Jingwei Ma

    Full Text Available Hepatitis B virus (HBV infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC. Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg and HBV e antigen (HBeAg, and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR, jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.

  9. IMMUNOGLOBULIN A (IgA AND ITS RECEPTORS

    Directory of Open Access Journals (Sweden)

    V. B. Klimovich

    2006-01-01

    Full Text Available Abstract. Daily IgA production in human organism comprises 3 to 5 g, thus exceeding total synthesis of other Ig classes. IgA in human body is presented by 9 structural variants. Its molecules belong to two subclasses, IgA1 and IgA2, the latter represented by two allotypes. In human serum, IgA1 monomers predominate, that are produced by the bone marrow cells. Mucosa-associated lymphoid tissues produce dimeric IgA1 and IgA2 molecules containing an accessory polypeptide J-chain. When transported across epithelial layer to the mucosal surface, an extracellular segment of polymeric IgA receptor (pIgAR is joining the dimeric IgA1, which becomes a ‘secretory’ component being a part cesretory IgA (sIgA molecule. The main function of sIgA is to bind bacteria and viruses at the mucosal surfaces, thus preventing pathogens to invade the internal spaces of the organism (immune exclusion. If transferred across epithelium, IgA may neutralize the viruses penetrating the cells, like as bind and deliver proteins and other antigens to the mucosal surface. The leukocyte IgA receptor (FcαRI, CD89 is expressed on the neutrophils, eosinophiles, monocytes/macrophages, as well as dendritic and Kupffer cells. The cytoplasmic domain FcαRI is devoid of an activation ITAM motif. To transduce signal, an FcαRI-associated chain of Fcγ receptor is used. Due to this mechanism, IgA binding leads to activation of phagocytosis, endocytosis, antigen presentation, synthesis of proinflammatory mediator and other immune functions. Fcα/μR receptor is a structural homologue of pIgR, and it is able to bind IgA and IgM, being, however, expressed only at the surface of mature B lymphocytes and macrophages. Interaction of IgA with asialoglycoprotein and transferrin (CD71 receptors, like as with some other molecules, that have yet undetermined role in immune defense and development of pathological events.

  10. Lactobionic acid enhances mPEG-PLGA-PLL nanoparticles targeting to hepatocellular carcinoma cell Huh7%乳糖酸修饰mPEG-PLGA-PLL纳米粒靶向肝癌细胞Huh7的研究

    Institute of Scientific and Technical Information of China (English)

    孙彦明; 朱明洁; 王炳武; 孙颖; 刘培峰; 段友容

    2012-01-01

    背景与目的 去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是一种肝细胞特异性表达的膜表面蛋白,能够特异性地识别带有半乳糖残基的糖蛋白.乳糖酸含有半乳糖基团,可以作为靶向肝癌的特异性配基.该研究旨在探讨乳糖酸修饰的聚乙二醇/聚丙交酯-乙交酯/聚赖氨酸[methoxypoly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide)-b-poly(L-lysine)(mPEG-PLGA-PLL)纳米粒,mPEG-PLGA-PLL-GAL NPs)]对肝癌Huh7靶向效果,为构建新型的靶向肝癌的纳米递送系统提供实验数据.方法 MTT法确定Huh7细胞摄取mPEG-PLGA-PLL NPs与mPEG-PLGA-PLL-GAL NPs适当的浓度;通过激光共聚焦和荧光显微镜定性观察Huh7对罗丹明B标记的mPEG-PLGA-PLL NPs和mPEG-PLGA-PLL-GAL NPs的摄取;并采用流式细胞计数仪定量研究Huh7细胞对两者的摄取差别;尾静脉注射荷Huh7瘤裸鼠研究两者体内分布情况.结果 mPEG-PLGA-PLL NPs与mPEG-PLGA-PLL-GAL NPs的浓度在0.2 mg/mL时细胞存活率较高且对Huh7细胞的毒性较小.激光共聚焦断层扫描显示Huh7细胞可以较好地摄取mPEG-PLGA-PLL-GAL NPs,同时流式细胞仪定量显示mPEGPLGA-PLL-GAL NPs在Huh7细胞的分布较mPEG-PLGA-PLL NPs高40%(P<0.05).mPEG-PLGA-PLL NPs与mPEG-PLGAPLL-GAL NPs在移植瘤中的分布明显多于其他脏器,并且随时间的延长mPEG-PLGA-PLL-GAL NPs体现了更好的靶向效果.结论 体外与体内实验证明乳糖酸修饰的mPEG-PLGA-PLL NPs对肝癌细胞Huh7有很好的靶向效果,可为肝癌的靶向治疗提供较好的药物载体.%Background and purpose: It is a wonderful approach to deliver drugs to hepatocellular carcinoma cell by receptor-mediated targeting. The asialoglycoprotein receptor (ASGPR) specifically recognized by galactose moiety residue is a mainly expressing membrane protein on the surface of hepatocellular carcinoma cell. Methoxy-poly (ethylene glycol)-b-poly(D, L-lactide-co-glycolide)-b-poly(L-lysine) (m

  11. 自身抗体在自身免疫性肝病中的预测价值%Value of autoantibodies in predicting autoimmune liver disease

    Institute of Scientific and Technical Information of China (English)

    王嘉森; 姚定康

    2011-01-01

    自身抗体作为自身免疫性肝病的诊断标志物已经在临床上得到了广泛应用,但其在病情严重程度及预后判断方面的价值报道并不多,事实上某些自身抗体除本身的诊断价值外,还具有预测病情的作用:如抗可溶性肝抗原抗体、抗肌动蛋白抗体、抗肝细胞溶质抗原Ⅰ型抗体、抗去唾液酸糖蛋白受体抗体、抗染色体抗体、抗环瓜氨酸多肽抗体和抗肝肾微粒体Ⅲ型抗体与自身免疫性肝炎的发生、发展及恶化有一定的相关性,其中抗可溶性肝抗原抗体与某些自身免疫性肝炎患者严重肝组织学改变、长期维持治疗、停药后再度恶化以及肝衰竭的发生相关;抗gp210抗体、抗Sp100抗体和抗着丝点抗体在原发性胆汁性肝硬化中有一定的预测意义,其中抗gp210抗体可提示原发性胆汁性肝硬化患者有较严重的界面性肝炎、小叶炎症以及易进展为肝衰竭等.%Autoantibodies have been widely used in autoimmune liver disease as diagnostic markers, but the value of estimating disease severity and prognosis has not been commonly comprehended.Actually, autoantibodies are valuable in predicting disease severity and treatment outcome as well as directing the diagnosis.Antibodies to soluble liver antigen, actin, liver cytosol type 1, asialoglycoprotein receptor, chromatin,cyclic citrullinated peptide, and uridine glucuronosyltransferases have been considered to be associated with the occurrence, severity and progression of autoimmune hepatitis.For example, antibody to soluble liver antigen is associated with severe histological changes, long durations of treatment, relapse after drug withdrawal and high frequency of liver failure.Besides, antibodies to Sp100, gp210 and centromere have prognostic value in primary biliary cirrhosis, for instance, antibody to gp210 is associated with severe interface hepatitis, lobular inflammation, and progression to liver failure.

  12. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan

    2006-01-01

    with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A,beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class Ⅱ lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86(LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).Conclusions Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation,carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S 1 protein.

  13. Study on hepatic ASGP receptors in normal and chronic liver disease model animals%肝受体研究

    Institute of Scientific and Technical Information of China (English)

    张荣军; 万卫星; 陶永辉; 王铁生; 肖志坚; 蔡刚明; 张莲芬; 金坚; 王博诚

    2001-01-01

    Objective To evaluate the effects of functional analysis of hepatic asialoglycoprotein receptors (ASGPR) for differentiating diseased liver from the normal ones in vitro and in vivo. Methods The rat model of chronic liver disease was established with inhaling CCl4.Galactosylneoglycoalbumin (NGA) was labeled with fluorescien isothiocyanate (FITC) by Marshal's method, or was labeled directly with Na99TcmO4 by SnCl2 method. The ASGPR on hepatocytes was analyzed by flow cytometry method (FCM) in vitro and by SPECT in vivo. Results It was showed that the amounts of ASGPR on normal and injured hepatocytes were very different in vitro. The worse the hepatocytes were injured, the lower the mean intensity of fluorescein (MIF) would be detected.Biodistribution analysis in animals showed that 99 Tcm-NGA could be uptaken fast by liver, no significant accumulation in other organs was obtained. Intestinal accumulation was also minimal and increased with time, that indicated the major excretory route of 99 Tcm-NGA was the biliary system and, eventually, the gastrointestinal tract. The blood clearance was fast in normal animals but retarded in liver injured model animals. The simple kinetics analysis indicated that radioactivity curves overtime of both hearts and livers in normal animals were obviously different from those in model animals. The receptor indexes (LHL15) were 0.980±0.010 and 0.949±0.025, and the clearance indexes (HH15) were 0.675±0.106 and 0.696±0.103 respectively. Conclusions The FCM is a good method for analyzing the amount of ASGPR on the surface of normal and injured heptocytes in vitro. Hepatic imaging using 99 Tcm-NGA can reflect specific hepatocyte function as the radioligand is metabolized only by the ASGPR on hepatocytes. As hepatic uptake of 99 Tcm-NGA and LHL15 analysis is sensitive for quantitation of ASGPR , fairly good estimation of liver function in liver disease is possible based on visual 99 Tcm-NGA imaging of the liver alone

  14. Preliminary experimental study on evaluation of liver injury by ultrasonic targeting imaging%靶向性超声显影评价不同程度肝损伤的初步实验研究

    Institute of Scientific and Technical Information of China (English)

    余进洪; 郑元义; 李奥; 王志刚; 项莹霞

    2011-01-01

    目的 使用靶向超声分子显像对比分析大鼠不同程度急性肝损伤后峰值回声强度的改变与去唾液酸糖蛋白受体(ASGPR)的含量及血清学检查的关系.方法 根据腹腔灌注四氯化碳分析纯的不同剂量(10%、20%、30%、40%、50%),将45只健康雄性SD大鼠随机分为相应5组(组Ⅰ~组Ⅴ),每组9只,每组中再随机抽取4只取肝组织,分别检测ASGPR的含量;另5只仅常规麻醉,胸腹部备皮,经尾静脉团注靶向液态氟碳纳米脂质超声造影剂.从注射开始至0.5 h内连续观察并记录,0.5~1 h内每10 min观察并记录,1 h至造影剂消退每30 min观察并记录肝实质超声图像.DFY型超声图像定量分析诊断仪对各时间点采集到的超声图像进行分析.另选10只健康雄性SD大鼠作为靶向超声造影对照组,方法同前.结果 随着肝损伤程度的增加,肝组织内的ASGPR含量逐渐减少,且各组样本均数两两比较差异均有统计学意义(P0.05).结论 自制靶向肝实质细胞膜受体ASGPR的液态氟碳纳米超声造影剂的肝靶向性显影效果佳;不同程度肝损伤后的靶向超声显影结果改变与ASGPR结果改变一致,有利于超声显影定量评价肝脏功能.%Objective To comparatively analyze the relationship of peak - echo intensity of targeted ultrasound molecular imaging and asialoglycoprotein receptor ( ASGPR) content in different degree of rat acute liver injury. Methods Acute liver injury model was established by intraperitoneal infusion of carbon tetrachloride ( CCl4 ) in 45 healthy male SD rats,the rats were divided int0 5 groups ( group Ⅰ - groupⅤ ) according to doses of CCl4 ( 10% ,20% ,30% ,40% ,50% ) with 9 rats in each group. The content of liver ASGPR in 4 rats of each group was detected respectively. The others were anesthetized generally , thoracic and abdominal skin was prepared, the liquid perfluorocarbon nanoparticles targeted ultrasound contrast agents were injected

  15. 酵母双杂交系统筛选HBV PreS1相互作用蛋白%Screening the hepatitis B virus PreS1 associated proteins in yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    叶峰; 张曦; 邸莹; 王小清; 刘小静; 孔颖; 赵英仁; 陈天艳; 刘敏

    2012-01-01

    Objective To screen the interacted protein hepatitis B virus (HBV) PreSl with human hepatocytes from normal human liver cDNA library by sos-recruitment system (SRS) and explore the mechanism of HBV endocylosis. Methods PCR was performed to amplify the gene of HBV PreSl from the plasmid PCP10/ HBV ayw subtype containing the whole fragment of HBV;the PCR product was cloned into yeast expression plasmid pSos. and reconstituted plasmid was tested by auto-sequencing assay and named pSos- PreSl. The pSos-PreSl fusion protein expressed in the yeast cells was confirmed by Western blot after pSos- PreSl was transfected into the yeast cell cdc25.Self-activation of the bait protein was determined by cotransformation of pSos- PreSl and pMyr-Lamin C, and the cytoplasmic localization of the bait protein was verified by cotransformation of pSos- PreSl and pMyr SB. Yeast cells co-transfected with pSos- PreSl and the normal human liver cDNA library grew in selective nutrition and temperature. The true positive clones were submitted for sequencing. The results were submitted to the BLAST notebook of NCBI to seek homologous sequence. Results The yeast expression vector of HBV PreSl gene was constructed successfully and its expression in yeast was verified. The recombinant bait plasmid did not have self-activation and toxicity to yeast cdc25H cells. Furthermore, the cytoplasmic localization of the bait protein was verified correctly. After yeast cells were co-transfected with pSos-PreSl and the normal human liver cDNA library, 5 clones were positive and showed high homology with KCMF1, cyt C, vitamin D binding protein, Homo sapiens albumin (ALB) and Homo sapiens asialoglycoprotein receptor (ASGPR). Conclusion We obtained 5 proteins which may interact with HBV PreSl protein by SRS. This is helpful for exploring the mechanism of HBV endocytosis.%目的 利用Sos招募系统(SRS),构建含HBV PreS1基因的酵母双杂交诱饵载体,筛选人肝细胞与HBV PreS1蛋白相互作用的

  16. Editorial: advances in therapeutic glycopeptides.

    Science.gov (United States)

    Zeng, Wenbin; Chen, Yue-Lei

    2014-01-01

    glycoproteins, with a focus on available therapeutic glycoproteins. Next three papers discuss the synthetic chemistry of glycopeptides. Dr. Zhu and coauthor describe a facile synthesis of differently protected cystathionines by the reaction of γ-bromohomoalanine with cysteine derivatives in an ethyl acetate/water biphasic system. Dr. Chen et al. report a neoglycopeptides synthesis by aqueous Suzuki-Miyaura reaction between glycosyl boronic acid and iodopeptides. Dr. Zeng et al. developed a novel strategy to prepare glycopeptide-based molecular imaging and therapy agents using fluorine-rich (fluorous) technology. The following review by Dr. Li et al. outlines a sample of mAbs currently approved for cancer treatment by the FDA, as well as antibody platforms in the research pipeline and clinic that have been engineered for greater tumor penetration, binding, and therapy efficacy. The asialoglycoprotein receptor (ASGPR) is a high-capacity C-type lectin receptor expressed on mammalian hepatocytes. Research in this field is summarized by Dr. Lu, Dr. Yin and coauthors. Recent progresses of cationic polysomes and liposomes as effective non-viral delivery system via ASGPR are also presented by these authors. Proteoglycans (PGs), a core protein and glycosaminoglycans (GAGs) chain, play important roles in amyloid-beta protein as well as tau processing, and have potential significance in Alzheimer's disease (AD) therapy. Next, Dr. Zeng and coworkers summarized recent advances of the chemistry and biology of glycopeptides with antibiotic activity. The last review of this issue by Dr. Ding and coworker provide the progress of PGs and GAGs in AD and their therapeutic implication. In summary, experts from different fields of therapeutic glycopeptides have showcased new results and expressed their opinions in this special thematic issue of Protein & Peptide Letters. As the guest editors, we wish that this diverse collection of valuable intellectual contributions will positively influence this