Pegylated polyethylenimine-Fab′ antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells
Pegylated polyethylenimine-Fab′ antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells
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DOI：
10.1021/bc0340767
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发表时间：
2003-09-01
影响因子：
4.7
通讯作者：
Kopecek, J
中科院分区：
文献类型：
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作者：
Merdan, T;Callahan, J;Kopecek, J
本研究的目的是利用与OV-TL16抗体的抗原结合片段(Fab‘)偶联的聚乙二醇化聚乙烯亚胺(PEG-PEI)特异性靶向卵巢癌细胞。OA3在大多数人卵巢癌细胞系中都有表达。为了证明聚乙二醇PEI-Fab‘有效地络合DNA的能力，进行了溴化乙锭排斥实验。与聚乙二醇化PEI或聚乙二醇化聚乙二醇单乙醇胺相比，25 kDa聚乙二醇单核苷酸在凝聚DNA的能力上仅有微小的差异。由于Fab‘与PEG-PEI的结合可能会影响络合物的稳定性，因此可以通过增加肝素孵育络合物来解决这个问题。该方法的稳定性与未修饰的PEI-PEI/DNA或PEI 25 kDa/DNA复合物的稳定性相似。络合物的尺寸约为150 nm，Zeta电位接近中性。由于中性表面电荷减少了非特异性的相互作用，后一种性质在体内的潜在应用中特别有意义。用流式细胞仪和荧光标记DNA进行的结合研究表明，与未经修饰的聚乙二醇PEI/DNA复合体相比，聚乙二醇PEI-Fab‘/DNA复合体与表达表位的细胞株的结合度提高了6倍以上。在表达OA3的OVCAR-3细胞中，荧光素酶报告基因的表达比PEI高达80倍，甚至高于PEI的25 kDa。该系统的优势在于其特异性，这一点通过在细胞培养上清中与游离Fab‘的竞争实验以及在OA3阴性细胞中的竞争实验证明了这一点。在后一种情况下，使用PEG-PEI-Fab‘只能获得低水平的报告基因表达。
Specific targeting of ovarian carcinoma cells using pegylated polyethylenimine (PEG-PEI) conjugated to the antigen binding fragment (Fab') of the OV-TL16 antibody, which is directed to the OA3 surface antigen, was the objective of this study. OA3 is expressed by a majority of human ovarian carcinoma cell lines. To demonstrate the ability of the PEG-PEI-Fab' to efficiently complex DNA, an ethidium bromide exclusion assay was performed. Comparison with PEG-PEI or PEI 25 kDa showed only minor differences in the ability to condense DNA. Since conjugation of Fab' to PEG-PEI might influence complex stability, this issue was addressed by incubating the complexes with increasing amounts of heparin. This assay revealed stability similar to that of unmodified PEG-PEI/DNA or PEI 25 kDa/DNA complexes. Complexes displayed a size of approximately 150 nm with a zeta potential close to neutral. The latter property is of particular interest for potential in vivo use, since a neutral surface charge reduces nonspecific interactions. Binding studies using flow cytometry and fluorescently labeled DNA revealed a more than 6-fold higher degree of binding of PEG-PEI-Fab'/DNA complexes to epitope-expressing cell lines compared to unmodified PEG-PEI/DNA complexes. In OA3-expressing OVCAR-3 cells, luciferase reporter gene expression was elevated up to 80-fold compared to PEG-PEI and was even higher than that of PEI 25 kDa. The advantage of this system is its specificity, which was demonstrated by competition experiments with free Fab' in the cell culture media during transfection experiments and by using OA3-negative cells. In the latter case, only a low level of reporter gene expression could be achieved with PEG-PEI-Fab'.