Receptor-mediated uptake of human alpha1-acid glycoprotein into liver parenchymal cells in mice.

Human alpha(1)-acid glycoprotein (AGP), a serum glycoprotein, is thought to have anti-inflammatory effects by a mechanism that is not well understood. In this study, we investigated the pharmacokinetics of AGP in mice using enzymatically modified AGP (AGP with the sialic acids removed, asialo-AGP, and with both sialic acids and galactose removed, agalacto-AGP). It was observed that AGP was eliminated from the circulation slowly, and was mainly taken up by the liver. The elimination of labeled AGP, asialo-AGP and agalacto-AGP from the circulation was suppressed in the presence of excess unlabeled AGP, asialo-AGP and agalacto-AGP, respectively, suggesting the receptor-mediated uptake of these AGPs. Interestingly, the uptake of AGP by the liver competed with agalacto-AGP, but not with asialo-AGP, while agalacto-AGP competed with asialo-AGP. These results suggest that agalacto-AGP binds to at least two types of receptors, namely asialoglycoprotein receptor (ASGPR) and an as yet unidentified receptor that is shared with AGP, and that AGP is directly taken up by the liver through such a receptor and not via ASGPR. These findings help improve our understanding of the clearance mechanism of AGP.

Preparation of asialoorosomucoid-polylysine conjugates.

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.

Covalent protein-oligonucleotide conjugates for efficient delivery of antisense molecules.

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.