甘露糖修饰雷公藤红素脂质体处方工艺优化及体外靶向性评价

Optimization of mannose-modified celastrol liposome formulation and targeting evaluationinvitro

目的制备甘露糖修饰的雷公藤红素脂质体(mannose-celastrol-liposomes,Man-Cel-Lips),对其处方进行优化,并初步评价其体外靶向性。方法采用薄膜分散法制备Man-Cel-Lips。以包封率为考察指标,采用Box-Behnken设计-响应面法优化处方中胆固醇加入量、Cel加入量和探头超声时间间隔;按最优处方制备3批脂质体,对其进行表征并考察其体外释放情况。以香豆素为荧光探针,采用流式细胞仪和荧光显微镜法考察甘露糖对脂多糖诱导的RAW264.7细胞的摄取和靶向作用。结果通过Box-Behnken响应面法,确定最优处方工艺为胆固醇4 mg、Cel 1 mg、磷脂22 mg,超声间隔时间5 s,总处方量为5 mL。所制得3批脂质体平均粒径为(93.50±1.04)nm,多分散指数为0.22±1.81,ζ电位为(-5.70±0.26)mV,平均包封率为(92.75±0.61)%,相比于游离Cel,Man-Cel-Lips具有一定缓释效果。体外细胞摄取实验结果表明,Man-Cel-Lips对细胞的摄取效率高于其他组,且呈剂量相关性(P<0.05)。结论成功制备并优化Man-Cel-Lips,经甘露糖修饰的Cel-Lips靶向性增强,为后续研究其抗炎活性奠定基础。

Objective To optimize the prepared prescription of Mannose celastrol liposomes(Man-Cel-Lips) and preliminarily evaluate its in vitro targeting. Methods Man-Cel-Lips was prepared by thin-film dispersion method. Box-Behnken design-response surface methodology was used to optimize the dosage of cholesterol, the dosage of Cel and the ultrasonic time interval using encapsulation efficiency as index. Three batches of liposomes were prepared according to the optimal formula, and the liposomes were characterized and their release in vitro was investigated. The uptake and targeting effects of MAN on lipopolysaccharide induced RAW264.7 cells were investigated by flow cytometry and fluorescence microscopy using coumarin as fluorescence probe.Results Box-Behnken design-response surface method was used to determine the optimal prescription process: 4 mg cholesterol, 1mg Cel, 22 mg phospholipid, 5 s ultrasonic interval and 5 mL total prescription. The average particle size, polydispersion index, ζpotential and encapsulation efficiency of the three batches of liposomes were(93.50 ± 1.04) nm, 0.22 ± 1.81,(-5.70 ± 0.26) mV and(92.75 ± 0.61)% respectively. Compared with free Cel, MAN-Cel-lips had a certain sustained release effect. In vitro cell uptake experiment results showed that the uptake efficiency of Man-Cel-Lips was higher than that of other groups(P < 0.05), and increased with the increase of dose(P < 0.05). Conclusion Man-Cel-Lips were successfully prepared and optimized, and the targeting activity of Man-Cel-Lips was enhanced, which laid a foundation for the follow-up study of its anti-inflammatory activity.