A new peptide ligand that targets particles and heterologous proteins to hepatocytes in vivo (2003)

Hepatocytes are an important target in the treatment of various metabolic and infectious disorders [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar]. Most approaches for targeting hepatocytes have relied upon ligands reacting with the asialoglycoprotein receptor (ASGPr) [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 2Groman E.V. Enriquez P.M. Josephson L. Arabinogalactan for hepatic drug delivery.Bioconj. Chem. 1994; 5: 547-556Crossref PubMed Scopus (101) Google Scholar, 3Lee Y.C. et al.Binding of synthetic oligosaccharides to the hepatic gal/GalNAc lectin.J. Biol. Chem. 1983; 258: 199-202PubMed Google Scholar]. Such ligands have allowed hepatocyte targeting with small molecules and proteins [4Rogers J.C. Kornfeld S. Hepatic uptake of proteins coupled to fetuin glycoprotein.Biochem. Biophys. Res. Commun. 1971; 45: 622-629Crossref PubMed Scopus (121) Google Scholar, 5Fiume L. et al.Inhibition of hepatitis viral replication by vidarabine monophosphate conjugated with lactasaminated serum albumin.Lancet. 1988; 2: 13-15Abstract PubMed Scopus (68) Google Scholar]. However, the utility of ASGPr ligands for targeting particles is less clear, as judged from a number of often conflicting reports concerning the size limitation of ASGPr-reactive particles and liposomes. A wide variety of size limits for ASGPr-reactive particles, ranging from 10 to 70 nm, has been reported [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 6Rensen P.C. et al.Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.J. Biol. Chem. 2001; 276: 37577-37584Crossref PubMed Scopus (172) Google Scholar, 7Schlepper-Schafer J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar, 8Furumoto K. et al.Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.Biochim. Biophys. Acta. 2001; 1526: 221-226Crossref Scopus (40) Google Scholar]. Overall, the complexity of effects observed with particles and liposomes in hepatocyte delivery experiments suggests that the finding of new targeting ligands remains an important goal for creating robust delivery systems. New hepatocyte ligands of proteinaceous origin would also be beneficial in that they could be incorporated genetically into proteins and viral vectors. In our previous studies, we attempted to select organ-targeting peptides by using peptide libraries displayed on icosahedral T7 phage (∼60 nm in diameter) that mimics the size and shape of liposomes and DNA polyplexes (Fig. 1A). However, a T7 library displaying random peptides at the carboxy-terminus of the coat protein 10B (p10; 415 copies per phage particle) was rapidly inactivated by complement when injected into rats at a ratio of 109–1010 pfu per animal [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The activation of complement was mediated by a large population of natural IgM antibodies reacting with phage-displayed peptides in a sequence-specific manner [10Sokoloff A.V. et al.Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.Mol. Ther. 2001; 3: 821-830Abstract Full Text Full Text PDF Scopus (15) Google Scholar]. A portion of the injected phage library was taken up by liver [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar], presumably by nonparenchymal liver cells (NPCs) expressing complement receptors [11Ahearn J.M. Rosengard A.M. Complement receptors.in: Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Dekker, New York1998: 167-202Crossref Google Scholar]. In agreement with this model, the phage selected for long-term circulation in rats (clone 20-6) lost its p10 peptide display region (truncated after Q343) and, along with it, the ability to bind natural IgM, activate complement, and accumulate in liver [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The truncation after Q343 was the only structural change in the p10 of selected phage. Later on, we found that mice were a better model than rats for studying the interaction of IgM-reactive T7 phage with liver. The inactivation of phage by complement in mice was slower than in rats, and a majority of liver sequestered phage could be detected in an infectious state (data not shown). When we introduced the p10 truncation mutation into nonselected phage, the resultant phage, when injected into mice, unexpectedly disappeared from the circulation and rapidly accumulated in liver. Apparently, there was a complement-independent mechanism of phage uptake that was mediated by a phage determinant absent in the selected phage. Two-dimensional electrophoresis and DNA sequencing revealed that phage 20-6 also expressed a mutated tail fiber protein, p17, with residue K211 substituted with an E (Table 1). We confirmed the suspected role of the p17 mutated region in liver targeting by reverting the E211 in phage 20-6 back to a K present in this position in the wild-type phage. While phage 20-6 did not target liver to a noticeable degree, the reverted phage 20-6F (F stands for “fixed”) accumulated in liver with an average efficiency of ∼63% 5 min after injection and ∼75% (up to 90% in individual animals) 60 min after injection (Table 1). We evaluated accumulation of phage 20-6F in other organs (lung, heart, brain, muscle, spleen, kidney, pancreas, testes or ovaries, thyroid, adrenal, skin, bone marrow, intestine, stomach, uterus, and eye) in a similar fashion, by injecting 1011 pfu of phage into the tail vein and, 5 min later, perfusing the whole animal with PBS/heparin. In contrast to the liver, these organs accumulated individually and together <0.5% and <4.5% of injected phage, respectively. The results from immunohistochemical experiments with different tissues also indicated that a predominant majority of injected 20-6F phage was targeted to the liver (Figs. 2A2 and 2A3 ; data for nonliver tissues are not shown). Thus, a determinant that apparently included the K211 residue of p17 was necessary for selective phage targeting to liver.TABLE 1T7 phage uptake by liverPhage typeP17 sequenceaThe critical amino acid residues in p17 position 211 are shown in bold. Both clones had a truncated p10 coat protein ending in sequence VVFQ343.Live phage in liver,b109 pfu of T7 phage was injected into the tail vein of mice, and the amount of infectious phage sequestered by liver was evaluated at 60 min after injection. The results are presented as mean ± standard deviation for 6 mice.%Phage per hepatocytec1011 pfu of T7 phage was injected into the tail vein of mice, and the percentage of phage-containing hepatocytes was determined at 5 or 30 min after phage injection. The amount of intracellular material reactive with anti-T7 antibody was semi-quantitated by confocal fluorescence microscopy (described in the figrue legend for Fig. 2). Five randomly chosen cells in each group were examined in 4-fold enlarged reconstructed images (3 μm thick) acquired at a 630-fold magnification. At this magnification, the smallest “phage particle” appeared as a red pixel. The average amount of phage in an individual hepatocyte was estimated as a number of stained pixels normalized to the intensity of staining. The number of bright red pixels was multiplied by 4, the number of pixels with an intermediate brightness was multiplied by 2, and the number of weakly stained pixels was multiplied by 1.5 min30 min20-6R207DEAE211RF3.7 ± 1.661–5 in 20% cells3–7 in 50% cells20-6FdT7-phage 20-6F was prepared by amplifying the wild-type gene 17 region of T7 DNA with 5′-gagtacttggagcGTGCACtggacccaac-3′ and 5′-ccatcagtGGCCacaacGGCCtgacctac-3′ primers containing unique ApaL I and Sfi I sites, respectively. Amplified DNA was digested with Sfi I and ApaL I and ligated to the genomic DNA isolated from T7 phage 20-6 and digested with Sfi I and ApaL I. The ligation product was packaged (Novagen [21]) and the resultant infectious phage was amplified.R207DEAK211RF74.8 ± 5.7050–250 in 100% cells500–2000 in 100% cellsNote. T7 phage (109 pfu) was injected into the tail vein of anesthetized male ICR mice (18–22 g) in 1 ml of PBS under low pressure (over 60 sec). Animals were preinjected with 10 U of heparin and the liver was perfused with PBS containing 2 U/ml heparin through the portal vein, with the right atrium cut. Livers were homogenized in PBS containing 2% Triton X-100 and 1 M NaCl and the amount of liver-associated phage was evaluated by a plaque assay 9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar.a The critical amino acid residues in p17 position 211 are shown in bold. Both clones had a truncated p10 coat protein ending in sequence VVFQ343.b 109 pfu of T7 phage was injected into the tail vein of mice, and the amount of infectious phage sequestered by liver was evaluated at 60 min after injection. The results are presented as mean ± standard deviation for 6 mice.c 1011 pfu of T7 phage was injected into the tail vein of mice, and the percentage of phage-containing hepatocytes was determined at 5 or 30 min after phage injection. The amount of intracellular material reactive with anti-T7 antibody was semi-quantitated by confocal fluorescence microscopy (described in the figrue legend for Fig. 2). Five randomly chosen cells in each group were examined in 4-fold enlarged reconstructed images (3 μm thick) acquired at a 630-fold magnification. At this magnification, the smallest “phage particle” appeared as a red pixel. The average amount of phage in an individual hepatocyte was estimated as a number of stained pixels normalized to the intensity of staining. The number of bright red pixels was multiplied by 4, the number of pixels with an intermediate brightness was multiplied by 2, and the number of weakly stained pixels was multiplied by 1.d T7-phage 20-6F was prepared by amplifying the wild-type gene 17 region of T7 DNA with 5′-gagtacttggagcGTGCACtggacccaac-3′ and 5′-ccatcagtGGCCacaacGGCCtgacctac-3′ primers containing unique ApaL I and Sfi I sites, respectively. Amplified DNA was digested with Sfi I and ApaL I and ligated to the genomic DNA isolated from T7 phage 20-6 and digested with Sfi I and ApaL I. The ligation product was packaged (Novagen 21Novagen (1999). T7Select System ManualGoogle Scholar) and the resultant infectious phage was amplified. Open table in a new tab Note. T7 phage (109 pfu) was injected into the tail vein of anesthetized male ICR mice (18–22 g) in 1 ml of PBS under low pressure (over 60 sec). Animals were preinjected with 10 U of heparin and the liver was perfused with PBS containing 2 U/ml heparin through the portal vein, with the right atrium cut. Livers were homogenized in PBS containing 2% Triton X-100 and 1 M NaCl and the amount of liver-associated phage was evaluated by a plaque assay 9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar. All clones found in the phage population selected for long-term circulation in rats and analyzed soon after the selection contained the p17-E211 [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. We found less common p17 mutations after a prolonged storage of selected phage at −80°C, apparently due to preferential inactivation of p17-E211 clones. These mutations included R207 substituted with an L, R200 substituted with a C, and R200 substituted with an S. The T7 phage tail fiber is composed of three parallel p17 chains that form proximal (residues 1 to ∼267) and distal (residues ∼268–553) half-fibers separated by a kink (Fig. 1C). Residues 151–267 of the proximal half-fiber form a triple-stranded coiled coil, known as the rod domain (Fig. 1B; [17]). All mutations disrupting the liver targeting were within the rod domain and occupied the exterior positions, b and e, of two adjacent coiled-coil heptad repeats. Thus, the mutations delineated a compact area on the coiled-coil surface that presumably served as a liver-targeting determinant or constituted a part of this determinant. We analyzed the association of phage sequestered by liver with hepatocytes and NPCs (Kupffer, endothelial, and stellate cells) by immunohistochemistry (Figs. 2A1–2A3 and 2B1). Hepatocytes were typically seen as large polygonal cells with round-shaped nuclei. Many hepatocytes contained double nuclei, which is a common morphological marker of these cells [12Severin E. Willers R. Bettecken T. Flow cytometric analysis of mouse hepatocyte ploidy. II. The development of polyploidy pattern in four mice strains with different life spans.Cell Tissue Res. 1984; 238: 649-652Crossref PubMed Scopus (30) Google Scholar]. Another distinctive marker of hepatocytes was the presence of highly counterstained F-actin along bile capillaries [13Benkoel L. et al.Effect of ischemia–reperfusion on bile canalicular F-actin microfilaments in hepatocytes of human liver allograft image analysis by confocal laser scanning microscopy.Dig. Dis. Sci. 2001; 46: 1663-1667Crossref PubMed Scopus Google Scholar]. NPCs typically had less and contained often small nuclei. The morphological NPCs and hepatocytes with selective of NPCs by antibodies reacting with or cells (data not shown). experiments that phage expressing was present in liver in occupied by NPCs and (Fig. We observed only particles of this phage in In phage expressing the wild-type was with hepatocytes (Figs. and 2B1). A large amount of phage 20-6F was with hepatocyte as as 5 min after injection (Fig. We observed a portion of this phage as small The accumulation of phage in hepatocytes presumably was due to the of hepatocytes and the by nm in diameter) that are large to phage J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar]. The amount of phage hepatocytes by 30 min after injection (Fig. as ∼75% of phage infectious at 60 min after that the phage observed in hepatocytes was by phage The of phage 20-6 and phage 20-6F present in at 30 min after injection were similar (Figs. and of mice with 1 of did not the amount of 20-6F phage accumulated in hepatocytes (data not that the ASGPr was not in phage uptake [4Rogers J.C. Kornfeld S. Hepatic uptake of proteins coupled to fetuin glycoprotein.Biochem. Biophys. Res. Commun. 1971; 45: 622-629Crossref PubMed Scopus (121) Google Scholar]. A portion of phage observed hepatocytes was in the as hepatocyte (Fig. 2B1). The analysis of through a of the hepatocyte indicated that the phage in the that did not the of the cells (data not shown). We the of phage into the hepatocyte by the liver by injecting highly phage 20-6 into a bile and that the phage in the liver in the bile (data not shown). Thus, the phage seen hepatocytes was the of infectious 20-6F phage in the mouse liver, we isolated mouse liver cells 60 min after injecting 109 pfu of phage into the tail vein and to as by et into mouse liver. of and of the of and J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar]. We cells with an of PBS containing 2% Triton X-100 and 1 M NaCl and evaluated the amount of phage by a plaque assay [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The of liver cells into NPCs and and containing hepatocytes [12Severin E. Willers R. Bettecken T. Flow cytometric analysis of mouse hepatocyte ploidy. II. The development of polyploidy pattern in four mice strains with different life spans.Cell Tissue Res. 1984; 238: 649-652Crossref PubMed Scopus (30) Google Scholar], that a contained on average 20-6F phage particles than a (data not shown). that cells were by NPCs and that hepatocytes constituted of liver cells G. J. The of In The and New in our the ratio the of phage sequestered by hepatocytes and NPCs in the whole liver was ratio to of phage in which is a on the that cells are by We also the presence in liver of two different of sequestered phage, apparently by hepatocytes and by by the for phage with hepatocytes or A phage expressing p17-E211 and a natural IgM-reactive peptide on p10 [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google complement and accumulated in NPCs (data not [11Ahearn J.M. Rosengard A.M. Complement receptors.in: Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Dekker, New York1998: 167-202Crossref Google Scholar]. of phage was present in an infectious state in the liver prepared at 60 min after phage injection. However, only of this phage was in an infectious state at after injection. In the percentage of infectious phage 20-6F in the liver only from ∼75% at 60 min (Table to at after phage injection. that phage 20-6F was than phage and was in a different liver mice of by treatment with liposomes et or of cells to and prolonged hepatic using Ther. 7: Full Text Full Text PDF Scopus Google in phage 20-6F liver phage uptake was (data not shown). as the of hepatocyte targeting by the p17 determinant be that the uptake of 20-6F phage by nonparenchymal cells be at with phage that has structural experiments have shown that prolonged storage of 20-6 phage at to the recognition of this phage by natural IgM antibodies (data not shown). Thus, the of 20-6F and 20-6 phage with natural IgM, to be by complement could for the phage uptake by liver p17 its ability to target hepatocytes when from its structural within phage, we expressed the p17 in The gene 17 region was by of gene 17 into by We and primers containing and sites, and respectively. We the resultant the in with the We the p17 gene from mutated phage 20-6 into using the We expressed proteins using cells and using proteins were by that isolated p17 that are to the phage tail fiber et of a viral protein the of Biol. 1988; PubMed Scopus Google Scholar], was that p17 would of its structural p17 injected into mice accumulated in liver in the manner as the phage from which they were a amount of was with p17-E211 was observed in (Figs. and We also observed a similar in hepatocyte targeting p17 expressing K211 or E211 for truncated p17, the proximal half-fiber (residues the rod domain with the distal half-fiber (residues to and the rod domain (residues to (data not shown). The hepatocyte targeting observed with the rod domain was with the results of the mutation that this domain contained the determinant. We evaluated the utility of the hepatocyte targeting p17 determinant for protein delivery by genetically the p17 to protein this we the region of from the using and primers with incorporated We the digested with and ligated into the the wild-type or the p17-E211 We expressed and proteins as protein injected into the mouse tail vein accumulated in the protein was observed in (Figs. and We also observed hepatocyte targeting with a protein composed of to the p17 rod We the human region by using the as a with incorporated into and and respectively. We digested the with and into the the wild-type or the p17 rod domain (residues We expressed proteins as and evaluated in liver by liver for phage protein (Figs. and or (Figs. and on We detected the protein in hepatocytes (Figs. and the protein, as a was seen in (Figs. and The observed for stained with antibody (Fig. was apparently we also observed similar in the of injected protein (Fig. The ability of the p17 determinant to target hepatocytes was not to We also observed targeting of hepatocytes by when this protein was to (Fig. In we detected only a small amount of p17-E211 in hepatocytes (Fig. We observed the majority of p17-E211 in association with nonparenchymal in this the of p17-E211 in mice (Fig. the of the p17 targeting determinant could a of viral particles and proteins into K. K. K. Hepatic by in the treatment of liver Scopus Google Scholar, E. and of drug Scopus Google Scholar, L. et of in rat and mouse liver cells by a of monophosphate with Full Text PDF PubMed Scopus Google Scholar]. The of the p17 targeting determinant within a coiled-coil domain suggests that this determinant be incorporated into viral displaying as in a et or of cells to and prolonged hepatic using Ther. 7: Full Text Full Text PDF Scopus Google Scholar]. the of the p17 mutations disrupting liver is that the determinant is composed of two or three heptad repeats. The by hepatocytes of phage particles suggests that the phage be also for delivery to hepatocytes of large as liposomes and acid is important the that are from through the mediated by the ASGPr [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 6Rensen P.C. et al.Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.J. Biol. Chem. 2001; 276: 37577-37584Crossref PubMed Scopus (172) Google Scholar, 7Schlepper-Schafer J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar, 8Furumoto K. et al.Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.Biochim. Biophys. Acta. 2001; 1526: 221-226Crossref Scopus (40) Google Scholar]. The p17 determinant also hepatocytes in (Fig. that be in as the of the determinant be the important of this determinant in to its this determinant is to be its by its and its size be to its We have found in experiments that the determinant be its targeting which of for (data not shown). is that the p17 is also with other as or reactive with the of ligands could the of delivery the are under to the receptor and its natural the utility of the determinant for also be determined by the of by the to hepatocytes through the phage The experiments at the of protein and DNA molecules through this are in