摘要
目的 利用荧光染料Hoechst 332 5 8与DNA结合后 ,荧光强度显著增强的特点 ,建立了基因治疗非病毒载体中DNA的含量测定方法。方法 对Hoechst 332 5 8-DNA溶液进行荧光扫描 ,并建立了标准曲线。分别测定了纳米粒胶体溶液超速冷冻离心后的上清液和用酶消化后的脂质体提取液中DNA的含量。结果 Hoechst332 5 8-DNA溶液的最佳激发波长为 35 3.6nm ,最佳发射波长为 4 5 4 .4nm ,标准曲线的线性范围为 0 .2～ 1.0 μg·ml-1。测得包载DNA纳米粒的平均包封率为 75 .1%± 8.6 %(n =5 ) ,DNA阳离子脂质体的平均抗核酸酶能力为 84 .9%± 7.8% (n =5 )。结论 荧光染料Hoechst332 5 8可用于测定基因治疗非病毒载体中DNA的含量 ,所建立的荧光分光光度法为载基因纳米粒、脂质体制备工艺和质量标准的建立提供了依据。
OBJECTIVE To develop a simple and sensitive assay method for quantitative determination of DNA in nanoparticles and liposomes.METHODS The fluorescence excitation spectrum and the fluorescence emission spectrum were scanned in a RF-5000 spectrofluoromrter.A standard curve with liner range of 0.2-1.0 μg·ml -1 was built up by using 0.15 mg·ml -1 Hoechst 33258.DNA loaded nanoparticles and DNA-liposome complexes were prepared by using PLGA and phospholipids respectively.The embedding ratio of DNA loaded nanoparticles and the stability in nuclease of DNA-liposome complexes were determined to optimize the formulations.RESULTS The maximum excitation wavelength and the maximum emission wavelength were 353.6 nm and 454.4 nm respectively.The average embedding ratio of DNA loaded nanoparticles and the average stability in nuclease of DNA-liposome complexes in optimized formulation were75.1%±8.6%(n=5) and 84.9%±7.8%(n=5),respectively.CONCLUSION The assay can be used in the formulation optimization and quality control for non-viral vectors of gene therapy.
出处
《华西药学杂志》
CAS
CSCD
2003年第6期412-415,共4页
West China Journal of Pharmaceutical Sciences
基金
国家高技术研究发展计划 ( 863计划 )资助项目 (批准号 :2 0 0 1AA2 180 2 1)
