活性氧响应型透明质酸修饰的鬼臼毒素纳米胶束的处方优化与体外评价  

Optimization of formulation and in vitro evaluation of reactive oxygen species-responsive hyaluronic acid-modified podophyllotoxin nano-micelles

作　　者：谷丽艳[1] 孙朝渭 董禹何 李学涛[2,3] 于洋 GU Liyan;SUN Zhaowei;DONG Yuhe;LI Xuetao;YU Yang(Key Laboratory of Ministry of Education for TCM Viscera State Theory and Applications,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;School of Pharmacy,Liaoning University of Traditional Chinese Medicine,Dalian 116600,China;Shenyang Key Laboratory of Targeted Delivery of Traditional Chinese Medicine,Shenyang 110000,China)

机构地区：[1]辽宁中医药大学中医脏象理论及应用教育部重点实验室,辽宁沈阳110847 [2]辽宁中医药大学药学院,辽宁大连116600 [3]沈阳市中药靶向递送重点实验室,辽宁沈阳110000

出　　处：《中草药》2024年第22期7663-7673,共11页Chinese Traditional and Herbal Drugs

基　　金：辽宁省教育厅科学技术研究重点攻关项目(202064);辽宁省教育厅高校基本科研项目(JYTQN2023471);辽宁中医药大学中医脏象理论及应用教育部重点实验室开放基金资助(zyzx2301)。

摘　　要：目的 制备活性氧响应型透明质酸修饰的鬼臼毒素纳米胶束(reactive oxygen species-responsive hyaluronic acidmodified podophyllotoxin nano-micelles,HA-oxi-Ms/Pod),并对其进行处方优化、理化表征以及体外生物活性评价。方法 采用薄膜分散法制备HA-oxi-Ms/Pod;利用Box-Benhken设计-响应面法(Box-Benhken design-response surface method,BBDRSM)对影响其包封率的3个因素[聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇接枝共聚物(Soluplus)与维生素E琥珀酸酯聚乙二醇1000(TPGS1000)质量比、Soluplus与鬼臼毒素质量比、水化温度]进行优化;利用激光散射粒度仪测定胶束的粒径和ζ电位;利用透射电子显微镜(transmission electron microscope,TEM)观察胶束的形态;利用透析袋法考察胶束的体外释放行为;采用CCK-8法考察胶束对卵巢癌细胞的毒性;采用流式细胞仪和荧光显微镜考察细胞摄取情况;利用Transwell实验评价胶束对人卵巢癌SK-OV-3细胞迁移能力的影响。结果 HA-oxi-Ms/Pod的最佳制备工艺为Soluplus与TPGS1000质量比2∶1,Soluplus与鬼臼毒素质量比40∶1,水化温度30℃;按最佳处方以Soluplus、TPGS1000、DSPE-PEG2000-活性氧响应键-PEG5000及DSPE-PEG2000-HA为膜材,制备的HA-oxi-Ms/Pod包封率为(94.28±0.51)%;激光散射粒度仪测得HA-oxiMs/Pod的粒径为(104.85±1.03)nm,活性氧响应后粒径缩短为(86.94±0.62)nm;HA-oxi-Ms/Pod的ζ电位为(-6.40±0.43)m V,活性氧响应后ζ电位为(-4.7±0.29)m V;TEM观察结果显示,HA-oxi-Ms/Pod为类球形。体外释放结果显示,在第30小时,鬼臼毒素游离药体外释放率为(87.12±6.62)%,Pod-Ms体外释放率为(40.04±3.18)%,HA-oxi-Ms/Pod体外释放率为(35.42±3.91)%,HA-oxi-Ms/Pod+H_(2)O_(2)体外释放率为(80.73±1.82)%,表明HA-oxi-Ms/Pod中装载的鬼臼毒素能够在氧化环境中响应性的释放。细胞毒实验结果表明,HA-oxi-Ms/Pod的半抑制浓度(halfmaximalinhibitory concentration,IC_(50))值为18.94μmol/L;体外细胞摄取实验Objective To prepare reactive oxygen species(ROS)-responsive hyaluronic acid(HA)-modified podophyllotoxin(Pod)nano-micelles(HA-oxi-Ms/Pod),optimize formulation,characterize physicochemical properties,and evaluate in vitro bioactivity.Methods HA-oxi-Ms/Pod were prepared using thin film dispersion method.The Box-Behnken surface response method was employed to optimize the preparation process by examining the effects of three factors(Soluplus/TPGS1000 mass ratio,Soluplus/Podophyllotoxin mass ratio,and hydration temperature)on encapsulation efficiency.The particle size andζpotential of the micelles were measured using laser scattering particle size analysis.The morphology of micelles was observed using transmission electron microscopy(TEM).The in vitro release behavior of micelles was studied using the dialysis bag method.The cytotoxicity of the micelles against ovarian cancer cells was assessed using the CCK-8 assay.Cellular uptake was examined by flow cytometry and fluorescence microscopy,and the effect of the micelles on the migration ability of ovarian cancer cells was evaluated using the Transwell assay.Results The optimal preparation conditions for HA-oxi-Ms/Pod were Soluplus at 40 mg,Soluplus/TPGS1000 mass ratio of 2:1,Soluplus/Pod mass ratio of 40:1,and a hydration temperature of 30℃.Using the optimized formulation,the encapsulation efficiency of HA-oxi-Ms/Pod prepared with Soluplus,TPGS1000,DSPE-PEG2000-ROS-responsive linkage-PEG5000,and DSPE-PEG2000-HA as the membrane materials was(94.28±0.51)%.The particle size of HA-oxi-Ms/Pod measured by laser scattering was(104.85±1.03)nm,which decreased to(86.94±0.62)nm after ROS response.The ζ potential of HA-oxi-Ms/Pod was(−6.40±0.43)mV,which changed to(−4.70±0.29)mV after ROS response.TEM revealed that HA-oxi-Ms/Pod were spherical.In vitro release studies showed that at 30 h,the release rate of free podophyllotoxin was(87.12±6.62)%,Pod-Ms was(40.04±3.18)%,HA-oxi-Ms/Pod was(35.42±3.91)%,and HA-oxi-Ms/Pod+H_(2)O_(2) was(80.73±1.82)%,indicating that th

关 键 词：鬼臼毒素 活性氧响应型胶束 透明质酸 靶向药物递送 卵巢癌 

分 类 号：R283.6[医药卫生—中药学]