McKee T D, DeRome M E, Wu G Y, Findeis M A
TargeTech, Inc., Meriden, Connecticut 06450.
Bioconjug Chem. 1994 Jul-Aug;5(4):306-11. doi: 10.1021/bc00028a004.
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.
亚洲血型糖蛋白 - 聚赖氨酸（ASOR - PL）偶联物最近被开发出来，作为静电结合DNA的载体，用于靶向递送至肝脏去唾液酸糖蛋白受体（ASGPr）进行基因治疗。通过酸性尿素凝胶电泳，我们发现先前报道的ASOR - PL偶联物分级分离方法不能有效地从ASOR - PL中去除非共价结合的聚赖氨酸（PL）。用这些偶联物制备的DNA复合物溶解度低，这限制了它们在后续实验中的用途，特别是在体内实验。对于通过碳二亚胺介导与5 kDa PL交联制备的ASOR - PL，用1 M盐酸胍透析可有效去除低分子量未结合的PL。当使用更高分子量的PL时，透析不可行，但制备性洗脱酸性尿素凝胶电泳用于分离不含未结合PL的粗制ASOR - PL级分。通过透析或电泳去除PL的ASOR - PL通过阳离子交换HPLC在羧甲基官能化柱上进一步分级分离，用混合pH - 盐梯度洗脱。发现通过制备性洗脱酸性尿素凝胶电泳和阳离子交换HPLC相结合分离的早期洗脱ASOR - PL级分最适合形成可溶性DNA复合物。
