Hirsch-Lerner D, Barenholz Y
Laboratory of Membrane and Liposome Research, Department of Biochemistry, The Hebrew University - Hadassah Medical School, Jerusalem 91120, Israel.
Biochim Biophys Acta. 1999 Nov 9;1461(1):47-57. doi: 10.1016/s0005-2736(99)00145-5.
Lipoplexes, which are formed spontaneously between cationic liposomes and negatively charged nucleic acids, are commonly used for gene and oligonucleotide delivery in vitro and in vivo. Being assemblies, lipoplexes can be characterized by various physicochemical parameters, including size distribution, shape, physical state (lamellar, hexagonal type II and/or other phases), sign and magnitude of electrical surface potential, and level of hydration at the lipid-DNA interface. Only after all these variables will be characterized for lipoplexes with a broad spectrum of lipid compositions and DNA/cationic lipid (L(+)) mole (or charge) ratios can their relevance to transfection efficiency be understood. Of all these physicochemical parameters, hydration is the most neglected, and therefore the focus of this study. Cationic liposomes composed of DOTAP without and with helper lipids (DOPC, DOPE, or cholesterol) or of DC-Chol/DOPE were complexed with pDNA (S16 human growth hormone) at various DNA(-)/L(+) charge ratios (0.1-3.2). (DOTAP=N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DC-Chol=(3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholester ol; DOPC=1, 2-dioleoyl-sn-glycero-3-phosphocholine; DOPE=1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine). The hydration levels of the different cationic liposomes and the DNA separately are compared with the hydration levels of the lipoplexes. Two independent approaches were applied to study hydration. First, we used a semi-quantitative approach of determining changes in the 'generalized polarization' (GP) of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). This method was recently used extensively and successfully to characterize changes of hydration at lipid-water interfaces. Laurdan excitation GP at 340 nm (GP(340)DOTAP. The GP(340) of lipoplexes of all lipid compositions (except those based on DC-Chol/DOPE) was higher than the GP(340) of the cationic liposomes alone and increased with increasing DNA(-)/L(+) charge ratio, reaching a plateau at a charge ratio of 1. 0, suggesting an increase in dehydration at the lipid-water interface with increasing DNA(-)/L(+) charge ratio. Confirmation was obtained from the second method, differential scanning calorimetry (DSC). DOTAP/DOPE lipoplexes with charge ratio 0.44 had 16.5% dehydration and with charge ratio 1.5, 46.4% dehydration. For DOTAP/Chol lipoplexes with these charge ratios, there was 17.9% and 49% dehydration, respectively. These data are in good agreement with the laurdan data described above. They suggest that the dehydration occurs during lipoplex formation and that this is a prerequisite for the intimate contact between cationic lipids and DNA.
阳离子脂质体与带负电荷的核酸之间自发形成的脂质体复合物，常用于体外和体内的基因及寡核苷酸递送。作为聚集体，脂质体复合物可通过多种物理化学参数进行表征，包括尺寸分布、形状、物理状态（层状、六方II型和/或其他相）、表面电势的符号和大小，以及脂质 - DNA界面处的水合水平。只有在对具有广泛脂质组成和DNA/阳离子脂质（L(+)）摩尔（或电荷）比的脂质体复合物的所有这些变量进行表征之后，才能理解它们与转染效率的相关性。在所有这些物理化学参数中，水合作用是最被忽视的，因此也是本研究的重点。由不含和含有辅助脂质（DOPC、DOPE或胆固醇）的DOTAP组成的阳离子脂质体，或由DC - Chol/DOPE组成的阳离子脂质体，在各种DNA(-)/L(+)电荷比（0.1 - 3.2）下与pDNA（S16人生长激素）复合。（DOTAP = N-(1-(2,3 - 二油酰氧基)丙基)-N,N,N - 三甲基氯化铵；DC - Chol = (3β-[N-(N',N' - 二甲基氨基乙烷)-氨基甲酰基]-胆固醇；DOPC = 1,2 - 二油酰 - sn - 甘油 - 3 - 磷酸胆碱；DOPE = 1,2 - 二油酰 - sn - 甘油 - 3 - 磷酸乙醇胺）。将不同阳离子脂质体和DNA各自的水合水平与脂质体复合物的水合水平进行比较。采用了两种独立的方法来研究水合作用。首先，我们使用了一种半定量方法来测定劳丹（6 - 十二烷酰 - 2 - 二甲基氨基萘）的“广义极化”（GP）变化。该方法最近被广泛且成功地用于表征脂质 - 水界面处的水合变化。在340nm处的劳丹激发GP（GP(340)DOTAP）。所有脂质组成（基于DC - Chol/DOPE的脂质体复合物除外）的脂质体复合物的GP(340)高于单独阳离子脂质体的GP(340)，并随DNA(-)/L(+)电荷比的增加而增加，在电荷比为1.0时达到平台期，表明随着DNA(-)/L(+)电荷比的增加，脂质 - 水界面处的脱水增加。通过第二种方法差示扫描量热法（DSC）得到了证实。电荷比为0.44的DOTAP/DOPE脂质体复合物有16.5%的脱水，电荷比为1.5时，脱水率为46.4%。对于具有这些电荷比的DOTAP/胆固醇脂质体复合物，脱水率分别为17.9%和49%。这些数据与上述劳丹数据高度一致。它们表明脱水发生在脂质体复合物形成过程中，并且这是阳离子脂质与DNA紧密接触的先决条件。
