Preparation of Asialoorosomucoid-Polylysine Conjugates

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Abstract

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.

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