Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors
Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors
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DOI：
10.1021/bc0601119
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发表时间：
2006-09-20
影响因子：
4.7
通讯作者：
Kissel, Thomas
中科院分区：
文献类型：
--
作者：
Germershaus, Oliver;Merdan, Thomas;Kissel, Thomas
在这项研究中，我们描述了由聚（乙二醇2000 Da）（10）-接枝-聚（乙烯亚胺25 kDa）（PEG-PEI）通过N-琥珀酰亚胺基-3-（2-吡啶二硫代）丙酸酯（SPDP）共价偶联到曲妥珠单抗（赫赛汀）组成的缀合物的合成和表征，用于特异性基因递送到Her 2表达细胞系。使用溴化乙锭排阻测定研究DNA缩合的效率，并证明与PEG-PEI相比差异可忽略不计。通过动态光散射测定缀合物复合物的尺寸为130-180 nm。不同氮磷比下的xi电位接近中性。流式细胞术和共聚焦显微镜显示使用Her 2阳性SK-BR-3细胞的曲妥珠单抗-PEI-PEG复合物的有效结合和摄取。相反，Her 2阴性OVCAR-3细胞的结合和摄取可忽略不计。与这些发现良好相关的是，在SK-BR-3细胞中使用靶向复合物的报告基因表达比未修饰的PEG-PEI复合物高7倍。在使用OVCAR-3细胞的情况下，在缀合物和PEG-PEI复合物之间未观察到表达效率的显著差异。使用游离曲妥珠单抗的抑制实验显示，使用SK-BR-3细胞的报告基因表达显著降低，但使用OVCAR-3细胞的报告基因表达没有降低，这强烈支持特异性Her 2受体介导的摄取机制。我们的研究结果表明，曲妥珠单抗-PEI-PEG可能是一种有前途的新的生物缀合物，用于体内靶向Her 2阳性肿瘤细胞的基因转移。
In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2000 Da)(10)-graft-poly(ethylene imine 25 kDa) (PEG-PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG-PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130-180 nm. xi potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab-PEI-PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG-PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG-PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab-PEI-PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.