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 acid-modified 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，BBD-RSM）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-oxi-Ms/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）mV，活性氧响应后ζ电位为（−4.7±0.29）mV；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＋H₂O₂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（half maximal inhibitory concentration，IC₅₀）值为18.94 μmol/L；体外细胞摄取实验和Transwell实验结果表明，与非靶向Pod-Ms相比，透明质酸的修饰明显增加了卵巢癌细胞对胶束的摄取，HA-oxi-Ms/Pod具有更显著抑制卵巢癌细胞迁移的能力。结论 成功制备的HA-oxi-Ms/Pod展现出良好的药物包封和释放特性，透明质酸的修饰显著增强了其对卵巢癌细胞的靶向能力，表明其在肿瘤治疗中具有潜在应用价值。

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 and ζ potential 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 + H₂O₂ was (80.73 ±1.82)%, indicating that the podophyllotoxin loaded in HA-oxi-Ms/Pod could be responsively released in an oxidative environment. Cytotoxicity assays showed that the IC₅₀ value of HA-oxi-Ms/Pod was 18.94 μmol/L. In vitro cellular uptake and Transwell assays demonstrated that hyaluronic acid modification significantly increased the uptake of micelles by ovarian cancer cells and that HA-oxi-Ms/Pod had a more pronounced ability to inhibit the migration of ovarian cancer cells compared to non-targeted Pod-Ms. Conclusion HA-oxi-Ms/Pod have been successfully prepared and can be further applied in tumor prevention and treatment research.

R283.6

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）