Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad, India.
Bioconjug Chem. 2011 Nov 16;22(11):2244-54. doi: 10.1021/bc2002874. Epub 2011 Oct 26.
In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.
在设计用于基因转染的新型阳离子脂质体时，从降解的角度广泛研究了连接子的化学性质，而从其对生物物理性质的贡献角度研究得较少。我们合成了两种带有谷氨酸酰胺作为主链和两个赖氨酸以提供阳离子头基的十二烷基脂质。脂质 1 与脂质 2 的不同之处在于存在酰胺键而不是特征在于脂质 2 的酯键。当以胆固醇作为共脂质时，带有脂质 1 的脂质体对中国仓鼠卵巢 (CHO) 和 HepG2 细胞系的转染效率非常高，而脂质 2 对 HepG2 细胞仅显示部分转染效率。尽管活性较低，但在 HepG2 和 CHO 细胞中测试时，发现脂质 1 在存在血清时稳定。基于荧光染料结合和琼脂糖凝胶的测定表明，在电荷比> 1:1 时，脂质 1 比脂质 2 更有效地与 DNA 结合。通过共焦显微镜显示，带有脂质 1 的寡核苷酸摄取量高于脂质 2。透射电子显微镜 (TEM) 图像显示，带有脂质 1 的脂质体和脂质体形成明显的囊泡和脂质体，而带有脂质 2 的则是碎片和无序结构。与组成类似于质膜的阴离子囊泡融合表明，脂质 2 的融合非常迅速，与融合事件不同，而脂质 1 囊泡的融合动力学则更加明确。差示扫描量热法 (DSC) 显示脂质 1 的高 T(m)（65.4°C），而脂质 2 的 T(m)为 23.5°C。与脂质 2 相比，脂质 1 的表面积-压力等温线的压缩性较低。然而，使用 1,6-二苯基-1,3,5-己三烯 (DPH) 测量的微粘度显示，用任一种脂质制成的囊泡具有相同的值。酰胺键的存在显然导致了稳定的囊泡形成、更高的熔点和低压缩性，同时保持了膜的流体性质，这表明脂质 1 的分子间氢键产生了具有高转染效率的稳定的脂质体。
