透明质酸修饰阳离子脂质体的制备及初步评价

Preparation and Evaluation of Hyaluronic Acid-Modified Cationic Liposome

制备透明质酸(Hyaluronic acid,HA)修饰的载基因阳离子脂质体三元复合物(HA/liposome/DNA ternary complexes,TC),评价其物理化学和生物学性质。采用薄膜分散法制备空白阳离子脂质体(Liposome),与DNA孵育得liposome/DNA脂质复合物(Lipo/DNA),将其与HA孵育即得TC。考察其物理化学性质、体外抗核酸酶能力、血浆稳定性、细胞毒性及体外转染效率。制备的liposome、Lipo/DNA、TC均呈类球形,粒径分别为(93.1±3.0),(156.6±5.4),(234.8±2.7)nm;Zeta电位分别为(43.71±1.33),(29.00±0.71),(-22.13±2.94)mV;TC与核酸酶孵育4 h,其中的DNA几乎无降解;TC与血浆蛋白孵育24 h内吸光度变化不大;细胞毒性与Lipo/DNA相比,显著降低;体外转染实验显示TC在有、无血清条件下均有较好的转染效率。TC是一种制备工艺简单、细胞毒性低、体外转染效率高、极具应用潜力的非病毒纳米基因载体。

To Prepare hyaluronic acid-modified cationic liposome/DNA ternary complexes （HA/liposome/DNA ternary complexes, TC） ,the physicochemical and biological properties were investigated to evaluate the potential of TC as novel gene delivery systems. The cationic liposomes were prepared by film dispersion method. The formulation was optimized by single factor study and orthogonal design. Then, the plasrnid DNA was added dropwise to cationic liposome under gentle vortexing. The sample was incubated at room tem- perature for 30 minutes to form liposome/DNA lipoplex （Lipo/DNA）. Finally, TC was obtained after the lipoplex was incubated with HA aqueous solution at room temperature for 30 minutes. The physieoehemical properties, the protection effect on DNA from nuclease degradation,stability in plasma, in vitro cytotoxicity as well as transfection activity in B16 cells were investigated, respectively. The morphology of obtained liposome, Lipo/DNA and TC were approximately spherical with the average particle size of （ 93.1 _＋ 3.0 ）, （ 156.6 ~ 5.4 ）, （ 234.8 _＋ 2.7 ） nm and Zeta potential of （43.71 ＋ 1.33 ）, （ 29.00 ：t： 0.71 ）, （ - 22. l 3 ＋ 2.94 ） mV, respectively. The obtained TC could protect DNA from nuclease degradation after 4 h incubation at 37 ~C. Stability studies in plasma confirmed that TC remained relative!y ~taLlc in the presence of plasma after 24 h. In vitro cytotoxicity studies exhibited that the cytotoxicity of TC was lower than that of Lipo/DNA on B16 ceils. The results of gene transfection experiment suggested that TC could transfer the loaded gene on B16 ceils both in serum free medium and serum medium. TC could be prepared easily with low cytotoxicity and high transfection activities. It may be a good non-viral vector for application in gene delivery and has the potential to make in vivo gene therapy achievable.