Silva J P Neves, Oliveira I M S C, Oliveira A C N, Lúcio M, Gomes A C, Coutinho P J G, Oliveira M E C D Real
CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
CFUM (Centre of Physics of the University of Minho), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
Biochim Biophys Acta. 2014 Oct;1838(10):2555-67. doi: 10.1016/j.bbamem.2014.06.014. Epub 2014 Jun 27.
Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the β-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-)=6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.
二辛基二甲基溴化铵（DODAB）：单油酸甘油酯（MO）脂质复合物主要是在DODAB高摩尔比范围内进行研究的，在人胚肾（HEK，即293T）细胞系中具有显著的转染效率。在本研究中，我们旨在研究高MO含量对pDNA/DODAB:MO脂质复合物的结构和物理化学性质的影响，以使其与转染效率建立一定的关联。利用静态/动态光散射和冷冻透射电子显微镜成像来表征不同DODAB:MO含量（2:1、1:1、1:2）和电荷比（CRs）（+/ -）下DNA/DODAB:MO脂质复合物的尺寸/形态。通过尼罗红荧光发射检测DNA/DODAB:MO体系的微粘度、水合作用和极性变化。利用Förster共振能量转移（FRET）评估脂质复合物在生理pH值和存在阴离子脂质情况下的稳定性。利用β - 半乳糖苷酶报告基因活性测定法在HEK细胞中进行转染研究，对物理化学/结构数据进行补充。本研究报告了在富含MO的脂质复合物（DODAB:MO 1:2和1:4）中多层层状相和非层状反相共存的情况，这导致其转染效率与多层层状（富含DODAB）脂质复合物相当，但电荷比更高[CR（+/ -）=6.0]且无剂量效应响应。这些结果可能与高MO含量促进的脂质复合物结构变化有关。
