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• W1967660067 abstract "The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) ≫ galactose) and the effects of ligand valency (tetraantennary > triantennary ≫ diantennary ≫ monoantennary) and sugar spacing (20 Å ≫ 10 Å ≫ 4 Å) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Hereto, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc3-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [3H]cholesteryl oleate-labeled long circulating liposomes (1–50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 ± 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes. The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) ≫ galactose) and the effects of ligand valency (tetraantennary > triantennary ≫ diantennary ≫ monoantennary) and sugar spacing (20 Å ≫ 10 Å ≫ 4 Å) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Hereto, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc3-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [3H]cholesteryl oleate-labeled long circulating liposomes (1–50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 ± 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes. asialoglycoprotein receptor asialoorosomucoid bovine serum albumin cholesteryl oleate 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanide perchlorate 3,3′-dioctadecyloxacarbocyanine perchlorate Dulbecco's modified Eagle medium galactose N-acetylgalactosamine galactose particle receptor lithocholic oleate γ-aminobutyric acid (3α(oleoyloxy)-5β-cholanoyl)-GABA-Gly-Tris(Gal)3 (3β(oleoylamido)-5β-cholanoyl)-GABA-Tyr-Gly-Tris(GalNAc)3 phosphate-buffered saline cholesterol monoantennary The hepatic asialoglycoprotein receptor (ASGPr)1 is a C-type (Ca2+-dependent) lectin that is expressed on the surface of hepatocytes (1Ashwell G. Harford J. Annu. Rev. Biochem. 1982; 51: 531-554Crossref PubMed Scopus (1524) Google Scholar) and plays a role in the clearance (endocytosis and lysosomal degradation) of desialylated proteins from the serum (2Spiess M. Biochemistry. 1990; 29: 10009-10018Crossref PubMed Scopus (374) Google Scholar, 3Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Scopus (714) Google Scholar) as has been shown for cellular fibronectin (4Rotundo R.F. Rebres R.A. Mckeown-Longo P.J. Blumenstock F.A. Saba T.M. Hepatology. 1998; 28: 475-485Crossref PubMed Scopus (32) Google Scholar) and all IgA2 allotypes (5Rifai A. Fadden K. Morrison S.L. Chintalacharuvu R. J. Exp. Med. 2000; 191: 2171-2181Crossref PubMed Scopus (129) Google Scholar). The human functional receptor is a noncovalent heterotetramer composed of two homologous type II membrane polypeptides with 55% sequence identity, generally called HL-1 (hepaticlectin 1) and HL-2, at a 2:2 stoichiometry (6Bider M.D. Wahlberg J.M. Kammerer R.A. Spiess M. J. Biol. Chem. 1996; 271: 31996-32001Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The ASGPr binds glycoproteins with either nonreducing terminal β-d-galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, at which the affinity for GalNAc is approximately 50-fold higher than for Gal (7Baenziger J.U. Maynard Y. J. Biol. Chem. 1980; 255: 4607-4613Abstract Full Text PDF PubMed Google Scholar, 8Connolly D.T. Townsend R.R. Kawaguchi K. Bell W.R. Lee Y.C. J. Biol. Chem. 1982; 257: 939-945Abstract Full Text PDF PubMed Google Scholar, 9Iobst S.T. Drickamer K. J. Biol. Chem. 1996; 271: 6686-6693Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). From studies using mice that are deficient in either the subunit HL-1 (10Ishibashi S. Hammer R.E. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar) or HL-2 (11Tozawa R. Ishibashi S. Osuga J. Yamamoto K. Yagyu H. Ohashi K. Tamura Y. Yahagi N. Iizuka Y. Okazaki H. Harada K. Gotoda T. Shimano H. Kimura S. Nagai R. Yamada N. J. Biol. Chem. 2001; 276: 12624-12628Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), it is evident that both polypeptides are necessary for efficient clearance of asialoglycopeptides. In addition to the ASGPr on hepatocytes, a homologous Ca2+-dependent Gal-recognizing receptor that also recognizes GalNAc and fucose is present in the liver on Kupffer cells (galactose particle receptor (GPr), Gal/fucose receptor) (12Lehrman M.A. Hill R.L. J. Biol. Chem. 1986; 261: 7419-7425Abstract Full Text PDF PubMed Google Scholar, 13Lehrman M.A. Haltiwanger R.S. Hill R.L. J. Biol. Chem. 1986; 261: 7426-7432Abstract Full Text PDF PubMed Google Scholar) and is absent from all other types of macrophages (14Haltiwanger R.S. Lehrman M.A. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1986; 261: 7433-7439Abstract Full Text PDF PubMed Google Scholar, 15Hoyle G.W. Hill R.L. J. Biol. Chem. 1988; 263: 7487-7492Abstract Full Text PDF PubMed Google Scholar). Each polypeptide subunit of the ASGPr can bind at least a single terminal Gal or GalNAc residue (16Lee R.T. Lee Y.C. Biochemistry. 1986; 25: 6835-6841Crossref PubMed Scopus (26) Google Scholar), and the affinity of ligands for the ASGPr appears to be governed by the valency of sugar residues and their appropriate spacing. Studies using asialoglycopeptides from naturally occurring glycopeptides (7Baenziger J.U. Maynard Y. J. Biol. Chem. 1980; 255: 4607-4613Abstract Full Text PDF PubMed Google Scholar, 17Baenziger J.U. Fiete D. Cell. 1980; 22: 611-620Abstract Full Text PDF PubMed Scopus (155) Google Scholar) and synthetic cluster glycosides (8Connolly D.T. Townsend R.R. Kawaguchi K. Bell W.R. Lee Y.C. J. Biol. Chem. 1982; 257: 939-945Abstract Full Text PDF PubMed Google Scholar, 18Lee R.T. Lin P. Lee Y.C. Biochemistry. 1984; 23: 4255-4261Crossref PubMed Scopus (177) Google Scholar) have demonstrated that clustering of glycosides greatly enhances the affinity for the receptor through simultaneous occupation of the receptor sites of the polypeptide subunits, at the following binding hierarchy: tetraantennary > triantennary ≫ biantennary ≫ monoantennary galactosides. This effect is dependent on the structural organization of the receptor on the cell membrane, because it is not observed on the isolated receptor (8Connolly D.T. Townsend R.R. Kawaguchi K. Bell W.R. Lee Y.C. J. Biol. Chem. 1982; 257: 939-945Abstract Full Text PDF PubMed Google Scholar, 18Lee R.T. Lin P. Lee Y.C. Biochemistry. 1984; 23: 4255-4261Crossref PubMed Scopus (177) Google Scholar). In addition to this so-called “cluster effect,” Lee et al.(19Lee Y.C. Townsend R.R. Hardy M.R. Lönngren J. Arnarp J. Haraldsson M. Lönn H. J. Biol. Chem. 1983; 258: 199-202Abstract Full Text PDF PubMed Google Scholar) and Biessen et al. (20Biessen E.A.L. Beuting D.M. Roelen H.C.P.F. Van de Marel G.A. Van Boom J.H. Van Berkel T.J.C. J. Med. Chem. 1995; 38: 1538-1546Crossref PubMed Scopus (133) Google Scholar) have shown that optimal receptor recognition of synthetic cluster glycosides is also determined by appropriate spacing (at least 15 Å) of the sugar residues. Although the effects of sugar type and valency on the affinity of ligands for the ASGPr are now well established, the effects of ligand size on the binding characteristics to the receptor have still not been fully mapped. Early in vivo studies suggested that the ASGPr is mainly responsible for the uptake of small (≤15 nm) particles exposing galactose at relatively low density, such as high density lipoproteins that are lactosylated (21Bijsterbosch M.K. Van Berkel T.J.C. Mol. Pharmacol. 1991; 41: 404-411Google Scholar) or provided with galactose-terminated monoantennary (mono-Gal-Chol) (22Roelen H.C.P.F. Bijsterbosch M.K. Bakkeren H.F. Van Berkel T.J.C. Kempen H.J.M. Buytenhek M. Van de Marel G.A. Van Boom J.H. J. Med. Chem. 1991; 34: 1036-1042Crossref PubMed Scopus (14) Google Scholar,23Bijsterbosch M.K. Bakkeren H.F. Kempen H.J.M. Roelen H.C.P.F. Van Boom J.H. Van Berkel T.J.C. Arterioscler. Thromb. 1992; 12: 1153-1160Crossref PubMed Google Scholar) and triantennary glycolipids (Tris-Gal-Chol) (24Van Berkel T.J.C. Kruijt J.K. Kempen H.-J.M. J. Biol. Chem. 1985; 260: 12203-12207Abstract Full Text PDF PubMed Google Scholar) and galactose-exposing gold particles (25Schlepper-Schäfer J. Hülsmann D. Djovkar A. Meyer H.E. Herbertz L. Kolb H. Kolb-Bachofen V. Exp. Cell Res. 1986; 165: 494-506Crossref PubMed Scopus (71) Google Scholar). In contrast, the GPr predominantly recognizes larger galactose-exposing particles (>15 nm) (26Bijsterbosch M.K. Van Berkel T.J.C. Biochem. J. 1990; 270: 233-239Crossref PubMed Scopus (36) Google Scholar, 27Biessen E.A.L. Bakkeren H.F. Beuting D.M. Kuiper J. Van Berkel T.J.C. Biochem. J. 1994; 299: 291-296Crossref PubMed Scopus (50) Google Scholar, 28Biessen E.A.L. Vietsch H. Van Berkel T.J.C. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1552-1558Crossref PubMed Scopus (5) Google Scholar), such as desialylated rat erythrocytes (29Kolb-Bachofen V. Schlepper-Schäfer J. Vogell W. Kolb H. Cell. 1982; 29: 859-866Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 30Kuiper J. Bakkeren H.F. Biessen E.A.L. Van Berkel T.J.C. Biochem. J. 1994; 299: 285-290Crossref PubMed Scopus (36) Google Scholar), low density lipoproteins that are lactosylated (26Bijsterbosch M.K. Van Berkel T.J.C. Biochem. J. 1990; 270: 233-239Crossref PubMed Scopus (36) Google Scholar) or provided with mono-Gal-Chol (22Roelen H.C.P.F. Bijsterbosch M.K. Bakkeren H.F. Van Berkel T.J.C. Kempen H.J.M. Buytenhek M. Van de Marel G.A. Van Boom J.H. J. Med. Chem. 1991; 34: 1036-1042Crossref PubMed Scopus (14) Google Scholar, 23Bijsterbosch M.K. Bakkeren H.F. Kempen H.J.M. Roelen H.C.P.F. Van Boom J.H. Van Berkel T.J.C. Arterioscler. Thromb. 1992; 12: 1153-1160Crossref PubMed Google Scholar) and Tris-Gal-Chol (31Van Berkel T.J.C. Kruijt J.K. Spanjer H.H. Nagelkerke J.F. Harkes L. Kempen H.-J.M. J. Biol. Chem. 1985; 260: 2694-2699Abstract Full Text PDF PubMed Google Scholar), and Tris-Gal-Chol-exposing liposomes (31Van Berkel T.J.C. Kruijt J.K. Spanjer H.H. Nagelkerke J.F. Harkes L. Kempen H.-J.M. J. Biol. Chem. 1985; 260: 2694-2699Abstract Full Text PDF PubMed Google Scholar). The affinity of glycosides for the GPr was shown to increase with particle size to reach a maximum at 15 nm (27Biessen E.A.L. Bakkeren H.F. Beuting D.M. Kuiper J. Van Berkel T.J.C. Biochem. J. 1994; 299: 291-296Crossref PubMed Scopus (50) Google Scholar). Furthermore, it has been shown that the GPr preferentially recognizes a high density of either fucose or galactose on either proteins (13Lehrman M.A. Haltiwanger R.S. Hill R.L. J. Biol. Chem. 1986; 261: 7426-7432Abstract Full Text PDF PubMed Google Scholar, 15Hoyle G.W. Hill R.L. J. Biol. Chem. 1988; 263: 7487-7492Abstract Full Text PDF PubMed Google Scholar) or particles (26Bijsterbosch M.K. Van Berkel T.J.C. Biochem. J. 1990; 270: 233-239Crossref PubMed Scopus (36) Google Scholar, 32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar). In contrast to these findings, providing low density lipoproteins with lactosaminated Fab fragments of anti-apoB100 antibodies induces a high uptake of low density lipoproteins by the ASGPr in vivo(33Bijsterbosch M.K. Bernini F. Bakkeren H.F. Gotto Jr., A.M. Smith L.C. Van Berkel T.J.C. Arterioscler. Thromb. 1991; 11: 1806-1813Crossref PubMed Scopus (7) Google Scholar). We have also recently shown that even larger (30 nm) liposomes may also be specifically taken up by the ASGPr in vivo, when provided with a relatively low amount (<10% w/w) of a nonexchangeable Gal-terminated triantennary glycolipid, with an intrinsic affinity for the ASGPr of 100 nm (32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar). In addition, in vitrostudies have suggested that the ASGPr may represent a potential pathway of entry for 28-nm hepatitis A virions (34Dotzauer A. Gebhardt U. Bieback K. Göttke U. Kracke A. Mages J. Lemon S.M. Vallbracht A. J. Virol. 2000; 74: 10950-10957Crossref PubMed Scopus (83) Google Scholar) and 42-nm hepatitis B virions (35Treichel U. Meyer zum Buschenfelde K.H. Stockert R.J. Poralla T. Gerken G. J. Gen. Virol. 1994; 75: 3021-3029Crossref PubMed Scopus (112) Google Scholar) into hepatocytes. These data indicate that particles larger than 15 nm with their sugars presented at a high local surface density (33Bijsterbosch M.K. Bernini F. Bakkeren H.F. Gotto Jr., A.M. Smith L.C. Van Berkel T.J.C. Arterioscler. Thromb. 1991; 11: 1806-1813Crossref PubMed Scopus (7) Google Scholar), at a low overall surface density (26Bijsterbosch M.K. Van Berkel T.J.C. Biochem. J. 1990; 270: 233-239Crossref PubMed Scopus (36) Google Scholar), or at an appropriate spatial orientation (32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar) can also be taken up by the ASGPrin vivo. The aim of the present study was to assess the intrinsic upper size limit for binding, uptake, and processing of ligands by the ASGPr. For this purpose, we synthesized a novel triantennary glycolipid that shows stable association with lipidic particles because of a highly lipophilic lithocholic oleate (LCO) structure (32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar, 36Rump E.T. De Vrueh R.L.A. Sliedregt L.A.J.M. Biessen E.A.L. Van Berkel T.J.C. Bijsterbosch M.K. Bioconjugate Chem. 1998; 9: 341-349Crossref PubMed Scopus (29) Google Scholar) and a predicted high affinity for the ASGPr by virtue of a triantennary GalNAc-terminated glycoside with 20Å spacing of the GalNAc residues (37Lee R.T. Lee Y.C. Glycoconj. J. 1987; 4: 317-328Crossref Scopus (101) Google Scholar, 38Lee R.T. Lee Y.C. Bioconjugate Chem. 1997; 8: 762-765Crossref PubMed Scopus (41) Google Scholar). Subsequently, we determined the effect of this glycolipid (LCO-Tyr-GalNAc3) on the ASGPr-mediated uptake of differently sized stable unilamellar liposomes (32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar, 39Rensen P.C.N. Schiffelers R.M. Versluis A.J. Bijsterbosch M.K. Van Kuijk-Meuwissen M.E.M.J. Van Berkel T.J.C. Mol. Pharmacol. 1997; 52: 445-455Crossref PubMed Scopus (49) Google Scholar) in vivo and in vitro. The data indicate that the novel glycoside displays a high intrinsic affinity for the ASGPr (2 nm). Moreover, we show that the glycolipid can induce effective recognition and uptake of liposomes with a diameter as large as 70 nm by the ASGPr on hepatocytes in vitro and in vivo, whereas larger particles do not bind to the ASGPr. These findings not only add to the further characterization of the structural requirements of ligands for proper recognition by the ASGPr but also have important implications for the design of particulate systems that are widely exploited for ASGPr-mediated targeting of drugs and genes to hepatocytes (40Wadhwa M.S. Rice K.G. J. Drug Targeting. 1995; 3: 111-127Crossref PubMed Scopus (113) Google Scholar, 41Wu G.Y. Wu C.H. Adv. Drug Deliv. Rev. 1998; 29: 243-248Crossref PubMed Scopus (73) Google Scholar, 42Yamazaki N. Kojima S. Bovin N.V. André S. Gabius S. Gabius H.-J. Adv. Drug Deliv. Rev. 2000; 43: 225-244Crossref PubMed Scopus (206) Google Scholar). 10–12-Week-old male C57Bl/6KH mice weighing 24–28 g and Wistar rats weighing 250–300 g (from Broekman Instituut BV, Someren, The Netherlands) fed ad libitum with regular chow were used for the in vivo experiments. [1α,2α-3H]Cholesteryl oleate ([3H]CO) and 125I (carrier-free) in NaOH were purchased from Amersham Pharmacia Biotech. Egg yolk phosphatidylcholine (lipoid E PC; 98%) was from Lipoid (Ludwigshafen, Germany). Galactose oxidase (EC 1.1.3.9) from Dactylium dendroides (crude) and collagenase (EC 3.4.24.3) from Clostridium histolyticum(type IV) were from Sigma. Cholesteryl oleate (CO; 97%) was from Janssen (Beersse, Belgium), and Percoll® was from Fluka (Buchs, Switzerland). 2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate (6Bider M.D. Wahlberg J.M. Kammerer R.A. Spiess M. J. Biol. Chem. 1996; 271: 31996-32001Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar)] diammonium salt, horseradish peroxidase type II (200 units/mg), Precipath® L, EDTA, and collagen S (type I) from calf skin were from Roche Molecular Biochemicals. Ketamine (HCl salt, 100 mg/ml) was from Eurovet (Bladel, The Netherlands). Hypnorm (0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone) and thalamonal (0.05 mg/ml of fentanyl and 2.5 mg/ml of droperidol) were from Janssen-Cilag Ltd. (Saunderton, UK). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanide perchlorate (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) were from Molecular Probes (Leiden, The Netherlands). Asialoorosomucoid (ASOR) was prepared by enzymatic desialylation (approximately 70%, as judged by the extent of sialic acid release) of human α1-acid glycoprotein (orosomucoid) from Cohn Fraction VI (99%) from Sigma as described (43Whitehead P.H. Sammons H.G. Biochim. Biophys. Acta. 1966; 124: 209-211Crossref PubMed Scopus (40) Google Scholar). Multiwell cell culture dishes were from Costar (Cambridge, MA). Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were obtained from Flow Laboratories (Irvine, UK). All other chemicals were of analytical grade. The synthesis of the ether-linked triantennary galactoside Z-Gly-Tris(Gal)3 (Gal3;Mr =1484) and its γ-aminobutyric acid (GABA)-mediated coupling product with the steroid structure 3α-oleoyloxy cholenic acid, leading to the bifunctional glycolipid (3α(oleoyloxy)-5β-cholanoyl)-GABA-Gly-Tris(Gal)3(LCO-Gal3; Mw 2058) (see Fig. 1A) has been recently reported in full detail (32Sliedregt L.A.J.M. Rensen P.C.N. Rump E.T. Van Santbrink P.J. Bijsterbosch M.K. Valentijn A.R.P.M. Van der Marel G.A. Van Boom J.H. Van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (138) Google Scholar). A novel triantennaryN-acetylgalactosamine-terminated cluster (Z-Tris(GalNAc)3; Mw 1532) has been synthesized and conjugated with a nearly identical steroid structure via a tyrosine residue to allow for trace labeling with 125I, yielding (3β(oleoylamido)-5β-cholanoyl)-Tyr-Gly-Tris(GalNAc)3(LCO-Tyr-GalNAc3; Mw 2182) (see Fig.1B). The synthesis of this glycolipid will be described in full detail elsewhere. The homogeneity and identity of both glycolipids has been fully established by high pressure liquid chromatography, NMR spectroscopy, and mass spectroscopy. The freeze-dried glycolipids were dissolved in PBS at a final concentration of 25–50 μg/μl and stored at −80 °C under argon before use. Their stability (which exceeded 12 months) was routinely checked by thin layer chromatography (n-butanol, n-propanol, 25% NH4OH, and H2O 15:40:30:15 v/v/v/v, or isopropanol and 25% NH4OH 1:1 v/v) and subsequent visualization of carbohydrate and cholesterol moieties by charring with H2SO4and ethanol (1:4 v/v) and MnCl2 (44Goswami S.K. Frey C.F. J. Chromatogr. 1970; 53: 389-390Crossref PubMed Scopus (25) Google Scholar), respectively. LCO-Tyr-GalNAc3 was radioiodinated with carrier-free 125I at pH 7.4 using a Iodogen-coated (10 μg) reaction tube, and ASOR at pH 10.0 according to the ICl method (45McFarlane A.S. Nature. 1958; 182: 53-54Crossref PubMed Scopus (1497) Google Scholar), respectively. Free 125I was removed by Sephadex G-50 medium gel filtration. The radioiodinated glycolipid migrated as a single band on TLC (n-butanol, n-propanol, 25% NH4OH, and H2O 15:40:30:15 v/v/v/v) as determined by imaging, and more than 98% of the radiolabel in ASOR was 10% trichloroacetic acid-precipitable. The specific activities of LCO-Tyr-GalNAc3 and ASOR were 1300–4300 dpm/ng of glycolipid and 260 dpm/ng of protein, respectively. Protein concentrations were determined according to Lowry et al. (46Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 93: 265-275Abstract Full Text PDF Google Scholar) using BSA as a standard. Hepatocytes were isolated from anesthetized rats or mice by perfusion of the liver with collagenase (type IV, 0.05%, w/v) for 10 min at 37 °C according to the method of Seglen (47Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar) as detailed earlier (27Biessen E.A.L. Bakkeren H.F. Beuting D.M. Kuiper J. Van Berkel T.J.C. Biochem. J. 1994; 299: 291-296Crossref PubMed Scopus (50) Google Scholar). The cells were ≥99% pure as judged by light microscopy, and their viabilities were ≥95% (rat) and ≥80% (mouse) as determined by 0.2% trypan blue exclusion. Hepatocytes were incubated (2 h at 4 °C) in DMEM containing 2% BSA (1 × 106 cells/ml) with 5 nm 125I-ASOR in the presence of increasing amounts of unlabeled galactose (0.2–200 mm), Z-Gly-Tris(Gal)3 (1–1000 nm), or Z-Tyr-Gly-Tris(GalNAc)3 (0.2–200 nm) under gentle shaking in a circulating lab shaker (Adolf Kühner AG, Basel, Switzerland) at 150 rpm. After incubation, the cells were pelleted by centrifugation (1 min at 50 g), and unbound125I-ASOR was removed by washing twice with ice-cold 50 mm Tris-HCl, 150 mm NaCl, 5 mmCaCl2 (Tris-buffered saline), pH 7.4, containing 0.2% BSA and once with Tris-buffered saline without BSA. The cell pellet was lysed in 0.1 n NaOH, the radioactivity and protein content was measured, and 125I-ASOR binding was calculated (dpm/mg of cell protein). Nonspecific binding was determined in the presence of 100 mm GalNAc. Displacement binding data were analyzed according to a single-site binding model. Inhibition curves were calculated by nonlinear regression analysis (GraphPad, ISI Software, Philadelphia, PA). Liposomes (mean diameters, 30, 50, and 70 nm) were prepared by sonication as described (39Rensen P.C.N. Schiffelers R.M. Versluis A.J. Bijsterbosch M.K. Van Kuijk-Meuwissen M.E.M.J. Van Berkel T.J.C. Mol. Pharmacol. 1997; 52: 445-455Crossref PubMed Scopus (49) Google Scholar). In short, egg yolk phosphatidylcholine (25 mg), CO (1 mg), and [3H]CO (50–100 μCi) were hydrated in 10 ml of 0.1 m KCl, 10 mm Tris-HCl, pH 8.0, and subsequently sonicated at 54 °C using a Soniprep 150 (MSE Scientific Instruments, Crawley, UK) at 18 μm output. Alternatively, liposomes (mean diameters, 50 and 90 nm) were prepared after hydration of the lipids in 2.0 ml of buffer and multiple extrusion (11 times) at 54 °C through 50- and 100-nm Whatman Nuclepore®(Pleasanton, CA) polycarbonate filters, respectively, using a Liposofast-Pneumatic (Avestin Inc., Ottawa, Canada) (48MacDonald R.C. MacDonald R.I. Menco B.P. Takeshita K. Subbarao N.K. Hu L.R. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1389) Google Scholar). All liposomes were purified and concentrated (1.014 g/ml) by density gradient ultracentrifugation according to Redgrave et al. (49Redgave T.G. Roberts D.C. West C.E. Anal. Biochem. 1975; 65: 42-49Crossref PubMed Scopus (876) Google Scholar) using NaCl/KBr/EDTA density solutions in a Beckman SW 40 Ti rotor at 40,000 rpm for 18–22 h at 4 °C. Particle sizes were determined by photon correlation spectroscopy (Malvern 4700 C System, Malvern Instruments, Malvern, UK) at 27 °C and a 90° angle between laser and detector. Sonication for 60, 15, and 10 min resulted in liposomes with mean particle diameters of 29.4 ± 2.2, 55.7 ± 0.9, and 72.3 ± 3.6 nm (mean ± S.D.; n = 3, 2, and 3) that were homogeneous with respect to size (polydispersities of 0.14–0.17, 0.28–0.29, and 0.26–0.27). Extrusion led to liposomes of 48.3 nm (50 nm filter; n = 1) and 90.3 ± 6.1 nm (100 nm filter; mean ± S.D.; n = 4) with polydispersities of 0.15 and 0.11–0.15, respectively. When indicated, liposomes were labeled with 1% (w/w) DiO or DiI by adding 0.25 mg from 10 mg/ml stock solutions in CHCl3:CH3OH (1:1 v/v) before hydration of lipids. The phosphatidylcholine and cholesterol ester contents were determined with the Roche Molecular Biochemicals enzymatic kits for phospholipid and cholesterol, respectively. Precipath® L was used as an internal standard. The particles were stored at 4 °C under argon and used for characterization and metabolic studies within 7 days following preparation, in which period no physicochemical changes occurred. Liposomes (100 μg of phospholipid) were incubated (30 min at 37 °C) with (radioiodinated) glycolipid in PBS, pH 7.4. The mixtures were subjected to 0.75% (w/w) agarose gel electrophoresis at pH 8.8, and the resulting gels were stained for lipid using Sudan Black. Radioactivity was visualized by imaging using a Packard Instant Imager (Hewlett-Packard Co., Palo Alto, CA). The electrophoretic mobility (Rf) of the Coomassie Brilliant Blue-stained liposomes (0.18 ± 0.01) was determined relative to the front marker bromphenol blue. Alternatively, incubation mixtures (50 μl) were subjected to fast protein liquid chromatography (SMART System; Amersham Pharmacia Biotech) using a Superose® 6 (PC 3.2/30) column at a flow rate of 50 μl/min and with PBS, 1 mm EDTA, 0.02% NaN3, pH 7.4, as eluent. The galactose content of the collected fractions was determined using a galactose oxidase assay (recovery, 85–100%). In short, samples were incubated in the dark (30 min at room temperature) with 0.9 mm 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate (6Bider M.D. Wahlberg J.M. Kammerer R.A. Spiess M. J. Biol. Chem. 1996; 271: 31996-32001Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar)] diammonium salt, 66.5 milliunits/ml peroxidase, 2.2 units/ml galactose oxidase, 0.1 m KPi buffer, pH 7.0, and the absorbance was measured at 405 nm. LCO-Tyr-GalNAc3 was used as a standard. The number of associated glycolipid molecules/30-nm particle was calculated assuming 7.62 × 1013liposomes/mg of phospholipid (39Rensen P.C.N. Schiffelers R.M. Versluis A.J. Bijsterbosch M.K. Van Kuijk-Meuwissen M.E.M.J. Van Berkel T.J.C. Mol. Pharmacol. 1997; 52: 445-455Crossref PubMed Scopus (49) Google Scholar). Mice were anesthetized by subcutaneous injection of a mixture of ketamine (120 mg/kg body weight), thalamonal (0.03 mg/kg fentanyl and 1.7 mg/kg droperidol), and hypnorm (1.2 mg/kg fluanisone and 0.04 mg/kg fentanyl citrate), and the abdomens were opened. [3H]CO-labeled liposomes (100 μg of phospholipid) were injected via the inferior vena cava, after previous incubation (30 min at 37 °C) with PBS" @default.
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• W1967660067 title "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytesin Vitro and in Vivo" @default.
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