Merdan Thomas, Callahan Jon, Petersen Holger, Kunath Klaus, Bakowsky Udo, Kopecková Pavla, Kissel Thomas, Kopecek Jindrich
Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 30 South, 2000 East, Salt Lake City, Utah, USA.
Bioconjug Chem. 2003 Sep-Oct;14(5):989-96. doi: 10.1021/bc0340767.
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'.
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，该抗体针对OA3表面抗原。大多数人卵巢癌细胞系都表达OA3。为了证明PEG-PEI-Fab'有效结合DNA的能力，进行了溴化乙锭排除试验。与PEG-PEI或25 kDa的PEI相比，在凝聚DNA的能力上仅显示出微小差异。由于Fab'与PEG-PEI的偶联可能影响复合物的稳定性，通过用增加量的肝素孵育复合物来解决这个问题。该试验揭示了与未修饰的PEG-PEI/DNA或PEI 25 kDa/DNA复合物相似的稳定性。复合物的大小约为150 nm，zeta电位接近中性。后一特性对于潜在的体内应用特别有意义，因为中性表面电荷可减少非特异性相互作用。使用流式细胞术和荧光标记DNA的结合研究表明，与未修饰的PEG-PEI/DNA复合物相比，PEG-PEI-Fab'/DNA复合物与表达表位的细胞系的结合程度高出6倍以上。在表达OA3的OVCAR-3细胞中，与PEG-PEI相比，荧光素酶报告基因表达提高了80倍，甚至高于25 kDa的PEI。该系统的优点是其特异性，这在转染实验期间通过在细胞培养基中与游离Fab'的竞争实验以及使用OA3阴性细胞得以证明。在后一种情况下，PEG-PEI-Fab'只能实现低水平的报告基因表达。
