Ciani Laura, Ristori Sandra, Bonechi Claudia, Rossi Claudio, Martini Giacomo
Department of Chemistry & CSGI, University of Florence, Via della Lastruccia 3, 50019 Sesto F no, Firenze, Italy.
Biophys Chem. 2007 Dec;131(1-3):80-7. doi: 10.1016/j.bpc.2007.09.011. Epub 2007 Oct 2.
Lipoplexes with different surface charge were prepared from a short oligonucleotide (20 mer, dsAT) inserted into liposomes of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE). The starting liposomes were prepared by two different procedures, i.e. progressive dsAT addition starting from plain liposomes (titration) and direct mixing of dsAT with pure liposomes (point to point preparation). Lipoplexes were characterized from a molecular point of view by Electron Spin Resonance (ESR) of a cationic spin probe and by Nuclear Magnetic Resonance. Structural and surface features were analysed by Zeta potential (zeta) measurements and Cryo-TEM micrographs. The complete set of results allowed to demonstrate that: i) the interactions between dsAT and cationic lipids were strong and occurred at the liposome surface; ii) the overall shape and physicochemical properties of liposomes did not change when short nucleic acid fragments were added before surface charge neutralization; iii) the bilayer structure of the lipids in lipoplexes was substantially preserved at all charge ratios; iv) the physical status of lipoplexes with electrical charge far from neutrality did not depend on the preparation method.
通过将短寡核苷酸(20聚体,双链AT)插入1,2 - 二油酰基-3 - 三甲基铵丙烷(DOTAP)和1,2 - 二油酰基-sn -甘油-3 - 磷酸乙醇胺(DOPE)的脂质体中,制备了具有不同表面电荷的脂质复合物。起始脂质体通过两种不同的方法制备,即从普通脂质体开始逐步添加双链AT(滴定法)和将双链AT与纯脂质体直接混合(点对点制备法)。从分子角度通过阳离子自旋探针的电子自旋共振(ESR)和核磁共振对脂质复合物进行表征。通过zeta电位测量和冷冻透射电子显微镜图像分析结构和表面特征。完整的结果表明:i)双链AT与阳离子脂质之间的相互作用很强,且发生在脂质体表面;ii)在表面电荷中和之前添加短核酸片段时,脂质体的整体形状和物理化学性质没有改变;iii)在所有电荷比下,脂质复合物中脂质的双层结构基本保持不变;iv)电荷远离中性的脂质复合物的物理状态不取决于制备方法。