Rodríguez-Pulido Alberto, Ortega Francisco, Llorca Oscar, Aicart Emilio, Junquera Elena
Departamento de Química Física I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
J Phys Chem B. 2008 Oct 2;112(39):12555-65. doi: 10.1021/jp804066t. Epub 2008 Aug 27.
A 1:1 mixture of the cationic lipid 3beta-[ N-( N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl- sn-glycero-3-phosphoetanolamine (DOPE), has been used to compact calf-thymus DNA (CT-DNA) in aqueous buffered solution at 298.15 K. The formation process of this lipoplex has been analyzed by means of electrophoretic mobility, cryo-TEM, dynamic light scattering, and fluorescence spectroscopy techniques. The experimental results indicate that DC-Chol/DOPE liposomes are mostly spherical and unilamellar, with a mean diameter of around 99 +/- 10 nm and a bilayer with a thickness of 4.5 +/- 0.5 nm. In the presence of CT-DNA, DC-Chol/DOPE/CT-DNA lipoplexes are formed by means of a strong entropically driven surface electrostatic interaction, as confirmed by zeta potential and fluorescence results, as a consequence of which DNA is compacted and condensed at the surface of the cationic liposomes. The negative charges of DNA phosphate groups are neutralized by the positive charges of cationic liposomes at the isoneutrality L/ D ratio, ( L/ D) varphi around 4, obtained from electrophoretic, fluorescence, and DLS measurements. The decrease in the fluorescence emission intensity of ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the association between the biopolymer and the cationic liposomes takes place has permitted one to confirm its electrostatic character as well as to evaluate the different microenvironments of varying polarity of DNA-double helix, liposomes, and/or lipoplexes. Electronic microscopy reveals a rich scenario of possible nanostructures and morphologies for the lipoplexes, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures and several intermediate morphologies.
阳离子脂质盐酸3β-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇(DC-Chol)与两性离子脂质1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)按1:1混合，已用于在298.15 K的水性缓冲溶液中压缩小牛胸腺DNA(CT-DNA)。通过电泳迁移率、冷冻透射电子显微镜、动态光散射和荧光光谱技术分析了这种脂质体复合物的形成过程。实验结果表明，DC-Chol/DOPE脂质体大多为球形且单层，平均直径约为99±10 nm，双层厚度为4.5±0.5 nm。在CT-DNA存在的情况下，DC-Chol/DOPE/CT-DNA脂质体复合物通过强烈的熵驱动表面静电相互作用形成，zeta电位和荧光结果证实了这一点，其结果是DNA在阳离子脂质体表面被压缩和凝聚。从电泳、荧光和动态光散射测量中获得，在等电点L/D比((L/D)φ约为4)时，DNA磷酸基团的负电荷被阳离子脂质体的正电荷中和。最初插入DNA碱基对之间的溴化乙锭(EtBr)荧光发射强度的降低，只要生物聚合物与阳离子脂质体之间发生缔合，就可以证实其静电性质，并评估DNA双螺旋、脂质体和/或脂质体复合物不同极性的微环境。电子显微镜揭示了脂质体复合物可能的纳米结构和形态的丰富情况，从不带DNA的单层脂质体到多层脂质体复合物，经过簇状结构和几种中间形态。
