Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India.
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India.
Colloids Surf B Biointerfaces. 2016 Jun 1;142:130-140. doi: 10.1016/j.colsurfb.2016.02.044. Epub 2016 Feb 23.
Selective gene transfection could be strategy of interest for reducing off-target gene expression and toxicity. In this respect, sigma receptors are found to be over-expressed in many human tumors and liposomal formulations with ability to target these sigma receptors may improve the transfection efficiency to a significant level. To this direction, six novel lipids have been synthesized with different hydrophobic segments such as a long hydrophobic chain or a cholesteryl group and L-tryptophan as the head group. Three of them, Lipid 1, 3 and 5 possessed cationic Me3N(+) moiety at the distal end. In contrast each of the other three Lipid 2, 4 and 6 possessed sigma receptor targeting anisamide group with no cationic charge. Mixing of cationic and anisamide counterparts of the same lipid in a molar ratio of 1:1 produced co-liposomes L-M-1 (Lipid 1+2), L-M-2 (Lipid 3+4) and L-M-3 (Lipid 5+6). These co-liposomes, while keeping the sigma targeting anisamide tag intact, showed good DNA binding and release which were optimized from EB intercalation and gel electrophoresis assays. Inclusion of a zwitterionic, fusogenic natural lipid, DOPE, into the co-liposomes further improved the binding efficiencies of the lipid mixtures with DNA. These co-liposomes having cationic and anisamide lipids and DOPE were highly selective toward sigma positive HEK293 and HEK293T cells compared to the sigma negative HeLa cells. As evidenced from both FACS and luciferase assay, a lipid mixture comprising Lipid 3, 4 and DOPE in a molar ratio of 1:1:1 (L-M-2D1) was the best for transfection of reporter pEGFP-C3 and functional pCEP4-p53 gene plasmids. Anisamide mediated sigma receptor selectivity was further probed by pre-incubating the transfecting cells with lipids possessing anisamide and by quantification of the un-transfected plasmid DNA. Also each formulation was highly non-toxic in the cell lines examined.
选择性基因转染可能是减少脱靶基因表达和毒性的策略。在这方面，sigma 受体在许多人类肿瘤中过度表达，具有靶向这些 sigma 受体能力的脂质体制剂可能会显著提高转染效率。为此，已经合成了六种新型脂质，它们具有不同的疏水部分，如长疏水链或胆固醇基团以及 L-色氨酸作为头基。其中三种，脂质 1、3 和 5，在远端带有阳离子 Me3N(+)部分。相比之下，其他三种脂质 2、4 和 6 中的每一种都具有 sigma 受体靶向的anisamide 基团，没有阳离子电荷。将相同脂质的阳离子和 anisamide 对应物以 1:1 的摩尔比混合，产生 co-liposomes L-M-1（脂质 1+2）、L-M-2（脂质 3+4）和 L-M-3（脂质 5+6）。这些 co-liposomes 保持了 sigma 靶向 anisamide 标记的完整性，显示出良好的 DNA 结合和释放能力，这是通过 EB 插入和凝胶电泳实验进行优化的。将两性离子、融合天然脂质 DOPE 包含在 co-liposomes 中进一步提高了脂质混合物与 DNA 的结合效率。与 sigma 阴性 HeLa 细胞相比，这些具有阳离子和 anisamide 脂质和 DOPE 的 co-liposomes 对 sigma 阳性 HEK293 和 HEK293T 细胞具有高度选择性。正如 FAC 和荧光素酶测定所证明的那样，由脂质 3、4 和 DOPE 以摩尔比 1:1:1 组成的脂质混合物（L-M-2D1）是转染报告基因 pEGFP-C3 和功能性 pCEP4-p53 基因质粒的最佳选择。通过用具有 anisamide 的脂质预先孵育转染细胞并定量未转染的质粒 DNA，进一步研究了 anisamide 介导的 sigma 受体选择性。此外，每种制剂在研究的细胞系中均具有高度的非细胞毒性。
