Silva J P Neves, Oliveira A C N, Casal M P P A, Gomes A C, Coutinho P J G, Coutinho O P, Oliveira M E C D Real
Centre of Physics, University of Minho, CP, Campus of Gualtar, Braga, Portugal.
Biochim Biophys Acta. 2011 Oct;1808(10):2440-9. doi: 10.1016/j.bbamem.2011.07.002. Epub 2011 Jul 20.
DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.
DNA/阳离子脂质体复合物（脂质复合物）已被广泛用作非病毒转染载体。脂质体制剂中的中性脂质是决定这些载体转染效率的关键因素。在本研究中，我们探讨了单油酸甘油酯（MO）作为辅助脂质用于细胞转染的潜力。研究了由质粒DNA（pDNA）与不同摩尔比（4:1、2:1和1:1）的溴化二辛基二甲基铵（DODAB）/1-单油酰-rac-甘油（MO）以及不同阳离子脂质/DNA比例组成的脂质复合物。通过动态光散射（DLS）、zeta电位（ζ）和冷冻透射电子显微镜（cryo-TEM）研究了脂质复合物的物理化学性质（大小、电荷和结构）。还通过溴化乙锭（EtBr）排除试验和电泳研究了MO对pDNA凝聚的影响以及肝素和硫酸乙酰肝素对脂质复合物中pDNA释放百分比的影响。使用293T细胞评估了这些脂质复合物的细胞毒性和转染效率，并与黄金标准辅助脂质1,2-二油酰-sn-甘油-3-磷酸乙醇胺（DOPE）和胆固醇（Chol）以及一种商业转染试剂（Lipofectamine™ LTX）进行了比较。还使用同一细胞系观察了转染的荧光标记pDNA的内化情况。结果表明，MO的存在不仅提高了pDNA的凝聚效率，还影响了脂质复合物的物理化学性质，这可能会干扰脂质复合物与细胞的相互作用。所测试的DODAB:MO制剂显示出低毒性，并成功介导了体外细胞转染。荧光显微镜研究支持了这些结果，表明脂质复合物能够进入细胞质并将pDNA递送至细胞核。因此，基于DODAB:MO的脂质复合物被验证为用于哺乳动物细胞基因修饰的无毒、高效脂质转染载体。了解基于MO的脂质复合物的结构与活性之间的关系将进一步推动这些新型递送系统的发展。
