目的 制备小豆蔻明纳米混悬剂（cardamonin nanosuspensions，Car-NPs），研究其体外抗肿瘤活性。方法 采用反溶剂沉淀联合高压均质法制备Car-NPs，以平均粒径及多分散指数（polydispersity index，PDI）为评价指标，对药载比、均质压力、均质次数进行单因素考察，利用Box-Behnken效应面设计法优化处方工艺；对最优处方工艺制备的Car-NPs进行表征，以动态光散射法测定平均粒径、粒度分布和ζ电位，在不同生理介质中监测粒径变化考察介质稳定性，透析法考察载药纳米粒的体外药物释放；通过细胞计数试剂盒-8（CCK-8）法测定并评估小豆蔻明游离药物及Car-NPs对人肝癌HepG2细胞、小鼠乳腺癌4T1细胞的体外细胞毒性。结果 选用P188为稳定剂制备Car-NPs，最佳工艺处方为药载比4∶1，均质压力为65.37 MPa，均质次数11次；所制备的Car-NPs平均粒径为（198.5±5.4）nm，PDI为0.191±0.020，载药量为（62.54±0.13）%，包封率为（95.65±0.26）%，透射电子显微镜（TEM）下呈均一的圆球状；Car-NPs在PBS、5%葡萄糖、生理盐水、人工胃肠液中都能够稳定存在，满足口服和iv给药要求；以0.1%聚乙烯吡咯烷酮（PVP）为保护剂，Car-NPs冻干复溶后粒径基本不变，便于长期保存；Car-NPs在磷酸盐缓冲液中，呈现出先速释后缓释的两相释放曲线，释药过程符合一级释放方程，Q＝1－e^{-0.104 8 t＋73.586}，R²＝0.990 3。在168 h内累积释放率达到76.49%，而相同条件下小豆蔻明物理混悬液的累积释放率仅有22.65%；Car-NPs显著提高了小豆蔻明对4T1细胞［半数抑制浓度（IC₅₀），113.2μg/mL vs 33.89 μg/mL，P＜0.001］和HepG2细胞的杀伤作用（IC₅₀，126.4 μg/mL vs 52.94 μg/mL，P＜0.001）。结论 采用Box-Behnken效应面法优化了Car-NPs的处方和工艺，所制备的Car-NPs有效解决了小豆蔻明难溶、难给药问题，冻干后便于长期保存。Car-NPs提高了小豆蔻明的体外释放度和对肿瘤细胞的杀伤作用，口服有望提高生物利用度；基于滞留效应（enhanced permeability and retention effect，EPR），iv给药有望提高药物在肿瘤组织中的分布。

Objective To prepare cardamonin nanosuspensions (Car-NPs) and study their antitumor activity in vitro. Methods The Car-NPs was prepared by anti-solvent precipitation combined with high-pressure homogenization, and the average particle size and polydispersion index (PDI) were used as evaluation indexes to investigate the drug loading ratio, homogenization pressure and homogenization times and the Box-Behnken response surface design method was used to optimize the formulation and process. The particle size, PDI and the surface potential of the obtain Car-NPs were measured by dynamic light scattering technology. The obtain Car-NPs were co-incubated with a series of physiological media for particle size change so as to evaluate their stability in these media. Dialysis in PBS was employed to assess the in vitro drug release of Car-NPs, and in vitro cytotoxicity of free cardamonin and Car-NPs on human hepatocellular carcinoma HepG2 cells and mouse mammary carcinoma 4T1 cells were determined and evaluated by the cell counting kit-8 (CCK-8) analysis.Results Poloxamer 188 (P188) was screened to be the best stabilizer, the optimal formulation and process parameters was as follows: the drug loading ratio of 4:1, the homogenization pressure being 65.37 MPa, and the homogenization times being 11 times. The prepared Car-NPs showed an average particle size of (198.5 ±5.4) nm, a PDI value of 0.191 ±0.020, a high drug loading content of (62.54 ±0.13)%, a high encapsulation effenciency of (95.65 ±0.26)%, and a uniform spherical shape under transmission electron microscopy. Car-NPs were quite stable in PBS, 5% glucose, normal saline, and artificial gastroenteric fluid, and thus meeting the requirement for both oral administration and intravenous injection. With 0.1% polyvinylpyrrolidone (PVP) as the cryoprotective agent, Car-NPs could be easily lyophilized and reconstituted into nanoparticle suspensions with nearly unchanged particle size, thus convenient for long-term storage; Car-NPs showed a bi-phase drug release in PBS, and the release process was in accordance with the first-order release equation, the cumulative release rate reached 76.49% within 168 h, while under the same conditions, the cumulative release rate of cardamonin physical suspension was only 22.65%. The nanosuspensions significantly improved the killing effect of free cardamonin against 4T1 cells (IC₅₀, 113.2 μg/mL vs 33.89 μg/mL, P < 0.001) and HepG2 cells (IC₅₀, 126.4 μg/mL vs 52.94 μg/mL, P < 0.001). Conclusion The Box-Behnken effect surface design method was used to optimize the formulation and process parameters of Car-NPs, and the nanosuspensions technology effectively solved the problem of poor solubility and difficulty for the in vivo drug delivery confronted by free cardamonin, the obtained Car-NPs were convenient for long-term storage after lyophilization. The nanosuspensions improved the in vitro release of cardamonin and the inhibition on tumor cells. The oral administration of Car-NPs was expected to improve the bioavailability, and intravenous administration was hopeful to enhance the drug distribution in tumors on basis on the well-known EPR effect.

R283.6

中国医学科学院医学与健康创新工程（2021-I2M-1-071）；黑龙江省应用技术研究与开发计划项目（GA19C107）