叶酸修饰蟾毒灵阳离子脂质体的制备及体外评价

Preparation and evaluation in vitro of folic acid-modified bufalin cationic liposomes

目的制备叶酸修饰蟾毒灵阳离子脂质体(FA-BF-CLs),并对其进行质量评价及抗肝癌研究。方法采用薄膜-超声分散法制备FA-BF-CLs,以粒径、Zeta电位及包封率等为指标,采用单因素和Box-Behnken响应面分析法对处方及工艺进行优化。透射电镜观察脂质体显微形态,并考察其体外释放过程、溶血性及空白阳离子脂质体体外细胞安全性。采用CCK-8法比较FA-BF-CLs、BF-CLs(未加叶酸修饰的蟾毒灵阳离子脂质体)及BF-solution(蟾毒灵溶液)的体外细胞毒性。结果最佳条件为磷脂与药物质量比14.17∶1、磷脂与胆固醇质量比3.8∶1、超声时间13 min,制备得到脂质体粒径(135.5±1.40)nm,多分散系数0.192±0.025,Zeta电位(16.23±0.46)mV,包封率69.42%±2.18%。体外释放研究表明,FA-BF-CLs具有明显的缓释效果,溶血试验结果显示该载体材料无溶血现象。体外细胞安全性结果显示阳离子脂质体浓度在0~20μmol/L范围内安全无毒。体外细胞毒性表明FA-BF-CLs对HepG2肝癌细胞的抑制作用大于BF-CLs和BF-solution(P<0.0.1)。结论制备得到的FA-BF-CLs外观均一稳定、具有缓释性能,空白阳离子脂质体安全无毒,FA-BF-CLs具有更强的体外抗肝癌研究,可用于进一步的研究。

Objective To prepare folic acid-modified bufalin cationic liposomes(FA-BF-CLs)and to evaluate their quality and anti-liver cancer.Methods FA-BF-CLs were prepared by thin-film ultrasonic dispersion method.Moreover,the formulation and process of FA-BF-CLs were optimized by single factor and Box-Behnken response surface analysis with particle size,Zeta potential and encapsulation rate as indexes.The micromorphology of liposomes was observed by transmission electron microscopy,and the in vitro release process,hemolysis and in vitro cell safety of blank cationic liposomes were investigated.The in vitro cytotoxicity of FA-BF-CLs,BF-CLs(bufalin cationic liposomes without folic-acid modification)and BF-solution(bufalin solution)was compared by the CCK-8 method.Results The optimum conditions were as follows:the mass ratio of phospholipid to drug was 14.17∶1,the mass ratio of phospholipid to cholesterol was 3.8∶1,and the ultrasonic time was 13 min.The particle size of liposomes was(135.5±1.40)nm,the polydispersity index was 0.192±0.025,the Zeta potential was(16.23±0.46)mV,and the encapsulation rate was 69.42%±2.18%.The in vitro release study showed that FA-BF-CLs had an obvious sustained release effect,and hemolysis test showed that the carrier material had no hemolysis.The in vitro cell safety result showed that the cationic liposomes were safe and non-toxic in the concentration range from 0 to 20μmol/L,and the in vitro cytotoxicity tests demonstrated that the inhibitory effects of FA-BF-CLson HepG2 hepatoma cells were greater than those of BF-CLs and BF-solution(P<0.01).Conclusion The prepared FA-BF-CLs appears uniform and stable,and possesses sustained release properties,of which the blank cationic liposomes are safe and non-toxic.Moreover,FA-BF-CLs have stronger anti-liver cancer in vitro and can be used for further research.