阳离子脂质/DNA复合物气雾剂的制备及其体外细胞转染研究

目的:制备阳离子脂质DOTAP和DNA的复合物(DOTAP/DNA)气雾剂并研究其细胞转染效率。方法:采用改良的Bligh and Dyer萃取法,制备能够在乙醚中均匀分散的不同氮-磷比的DOTAP/DNA复合物,采用定磷法测定载药率、激光粒度分布仪测定粒径分布等参数;进一步灌装DOTAP/DNA复合物气雾剂,以人肺腺癌细胞H1299为模型,检测气雾剂的细胞毒性(细胞相对存活率)和DNA转染效率(荧光强度)。结果:最佳氮-磷比(2∶1)的DOTAP/DNA复合物的载药率可达到(72±1.580)%,粒径为(188.700±42.770)nm;气雾剂的细胞相对存活率可达(91.643±6.970)%,荧光强度可达1.080×105RLU·mg-1。结论:研制的新型DOTAP/DNA复合物气雾剂,其毒性较低,细胞转染效率较高,具有被进一步开发研究成为呼吸道基因输送制剂的潜力。

Preparation of Aerosols Containing Cationic Lipid/DNA Complexes and in Vitro Cell Transfection Study

OBJECTIVE： To prepare Aerosols containing DOTAP/DNA complexes, and to study their cell transfection efficiencies in vitro. METHODS： DOTAP/DNA complexes were prepared by modified Bligh and Dyer extraction method, which could be scattered in ether evenly with different N/P ratios. Drug-loading rate and particle size distributions were examined by inorganic phosphate assay and laser granularity distributing apparatus. Aerosols containing DOTAP/DNA complexes were further filled. The cell transfection efficiencies and cytotoxicity of aerosols were determined using human lung adenocarcinoma cell line H1299 as a model. RESULTS ： The drugqoading rate of DOTAP/DNA complexes with optimal N/P ratio （2 ： 1 ） reached （72 ± 1.580） %, and the particle size was about （188.700 ± 42.770） nm. The cell survival rate after the aerosol application was （91.643 ± 6.970）%, and fluorescence intensity was 1.080×10^5RLU. mg-1. CONCLUSIONS： Developed novel Aerosols containing DOTAP/DNA complexes have low toxicity and high cell transfection efficiencies, which may have the potential to be an effective gene delivery formulation to the respiratory tract.