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.
McKee
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TargeTech, Inc., Meriden, Connecticut 06450.
DeRome
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Wu
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Findeis
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D000818:Animals; D001212:Asialoglycoproteins / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004337:Drug Carriers; D004586:Electrophoresis; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D011107:Polylysine / Q000138:chemical synthesis*
D001212:Asialoglycoproteins; D004337:Drug Carriers; D009961:Orosomucoid; C017030:asialoorosomucoid; D011107:Polylysine; D004247:DNA