Pharmaceutical Research Centre, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
J Gene Med. 2013 Jun-Jul;15(6-7):261-9. doi: 10.1002/jgm.2718.
Several strategies have been shown to improve the transfection efficiency of polyethylenimine (PEI) as a nonviral gene delivery vector. In the present study, a nucleic acid aptamer specific for protein tyrosine kinase 7 (PTK7) surface marker, sgc-8c, was conjugated electrostatically to pre-formed 10-kDa PEI/plasmid DNA polyplexes, and the ability of the conjugate to transfer genetic material was evaluated in MOLT-4 human acute lymphoblastic leukemia T-cells, which express PTK7 on their surface.
Polyplexes (plasmid DNA-vector conjugates), prepared using PEI-sgc-8c conjugate and pCMVLuc as a reporter gene, were characterized in terms of particle size, surface charge and the extent of DNA condensation. Polyplexes were also evaluated for cytotoxicity using the MTS colorimetric assay, as well as for transfection efficiency in MOLT-4 cells, and compared with the results obtained in U266 cells, which lack cell surface PTK7.
Relative to pDNA/PEI, the size of pDNA/PEI/sgc-8c aptamer polyplexes increased with decreasing zeta potential. In MOLT-4 cells, pDNA/PEI/sgc-8c aptamer polyplexes exhibited an almost six- to eight-fold increase in transfection efficiency compared to that of pDNA/PEI polyplex, indicating that conjugation of sgc-8c aptamer to pre-formed 10-kDa PEI/plasmid DNA polyplexes achieved effective targeting without covalent attachment, whereas receptor-mediated conducted transfection was confirmed by performing a competitive transfection experiment and a cellular uptake study.
The results of the present study provide an example of the usefulness of a nucleic acid aptamer in the form of noncovalent, electrostatic conjugates as an approach for enhancing the transfection efficiency of a polycation vector such as PEI without significant induced cytotoxicity.
已有多种策略被证明可以提高聚乙烯亚胺（PEI）作为非病毒基因传递载体的转染效率。在本研究中，一种针对蛋白酪氨酸激酶 7（PTK7）表面标志物的核酸适体 sgc-8c 通过静电作用与预先形成的 10 kDa PEI/质粒 DNA 超分子复合物结合，评估了该复合物在表达表面 PTK7 的人急性淋巴细胞白血病 T 细胞 MOLT-4 中传递遗传物质的能力。
使用 PEI-sgc-8c 缀合物和 pCMVLuc 作为报告基因，制备超分子复合物（质粒 DNA-载体缀合物），并从粒径、表面电荷和 DNA 凝聚程度等方面对其进行表征。还通过 MTS 比色法评估超分子复合物的细胞毒性，以及在 MOLT-4 细胞中的转染效率，并与缺乏细胞表面 PTK7 的 U266 细胞的结果进行比较。
与 pDNA/PEI 相比，pDNA/PEI/sgc-8c 适体超分子复合物的粒径随 ζ 电位的降低而增大。在 MOLT-4 细胞中，与 pDNA/PEI 超分子复合物相比，pDNA/PEI/sgc-8c 适体超分子复合物的转染效率提高了近 6 至 8 倍，表明 sgc-8c 适体与预先形成的 10 kDa PEI/质粒 DNA 超分子复合物的非共价静电结合实现了有效的靶向作用，而无需共价连接，通过进行竞争转染实验和细胞摄取研究证实了受体介导的转导转染。
本研究结果提供了一个实例，证明了核酸适体作为一种方法的有效性，以非共价、静电结合的形式作为一种增强多阳离子载体（如 PEI）转染效率的方法，而不会引起显著的细胞毒性。
