Department of Microbiology Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Nanobiotechnology. 2010 Feb 24;8:6. doi: 10.1186/1477-3155-8-6.
Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3beta-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 105 cells over a range of 500 ng - 10 mug of NPs containing 20-30 mug DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo.
质粒 DNA 编码的荧光素酶报告基因与六种不同的杂交纳米颗粒（NPs）复合，这些 NPs 是由聚（D，L-丙交酯-共-乙交酯酸）（PLGA 50:50）和阳离子脂质 DOTAP（1,2-二油酰基-3-三甲基铵丙烷）或 DC-Chol {3β-[N-（N'，N'-二甲氨基乙烷）-氨基甲酰基]胆固醇} 组成的混合物合成的。粒子直径为 100-400nm，所得复合物的表面吸附有 DNA（外）、包裹有 DNA（内）或吸附和包裹有 DNA（两者）。用荧光素酶报告基因检测法检测了 293 细胞中六种不同的 NP/DNA 复合物摄取的 DNA 表达。在 500ng-10ug 的 NPs 范围内，每 1mg NPs 含有 20-30ug DNA 时，对 105 个细胞的最佳 DNA 传递发生。从 NP/DNA 复合物中摄取的 DNA 效率比未结合的 DNA 高 500-600 倍。进行了回归分析，并绘制了 DNA 在四周时间间隔内的摄取曲线。吸附有 NPs（外）的 NP/DNA 复合物表现出较大的初始摄取，随后 DNA 下降斜率陡峭，下降角度大；吸附和包裹有 NPs（两者）的摄取线也表现出较大的初始摄取，但随后 DNA 下降斜率逐渐减小，下降角度较小，表明 293 细胞中的荧光素酶表达时间较长。仅包裹有 DNA 的 NPs（内），活性居中。后两种效应在 DOTAP-NPs 中表现最佳，而前一种效应在 DC-Chol-NPs 中表现最佳。这些结果为在体外和未来使用不同的杂交 NP/DNA 复合物提供了最佳条件，未来将在体内进行测试。
