人参皂苷Rg_(3)和大黄酸纳米乳的制备及其联合aPD-L1治疗三阴性乳腺癌的药效学研究

Preparation of ginsenoside Rg_(3) and rhein nanoemulsion and pharmacodynamics study of its combination with aPD-L1 in treatment of triple negative breast cancer

目的根据中医治疗肿瘤时提倡的“扶正祛邪”治则,选用中药人参和大黄的主要成分人参皂苷Rg_(3)(ginsenoside Rg_(3),G-Rg_(3))和大黄酸(rhein,Rhe)进行组合,制备G-Rg_(3)/Rhe纳米乳(G-Rg_(3)/Rhe NE),同时提高G-Rg_(3)和Rhe溶解度以利于注射给药,并利用该纳米乳联合程序性死亡受体配体1单抗(anti-programmed cell death ligand 1,aPD-L1)治疗三阴性乳腺癌(triple negative breast cancer,TNBC)。方法采用紫外分光光度法分别测定G-Rg_(3)和Rhe在不同辅料中的溶解能力,筛选出合适的乳化剂、助乳化剂和油相。利用伪三元相图考察乳化剂与助乳化剂质量比(Km值)对纳米乳体系的影响,根据形成纳米乳区域面积大小选择适宜的Km值,优选G-Rg_(3)/Rhe NE的处方,并对其进行表征和稳定性评价。选用4T1Fluc原位乳腺肿瘤小鼠模型,评价G-Rg_(3)/Rhe NE联合aPD-L1抗TNBC的效果。结果G-Rg_(3)/Rhe NE的优化组分比为聚氧乙烯氢化蓖麻油EL35-聚乙二醇400-辛癸酸甘油酯-水,其质量比为1.33∶2.67∶2.80∶12.50,Km值为1∶2,G-Rg_(3)/Rhe NE的平均粒径为(84.8±1.1)nm,多分散性系数(polydispersity index,PDI)为0.21±0.02,ζ电位为(−11.30±0.43)mV。载药纳米乳中G-Rg_(3)、Rhe的包封率分别为96.51%、97.47%,两药共载的载药量为0.75%。G-Rg_(3)/Rhe NE联合aPD-L1,能显著抑制荷4T1Fluc小鼠的肿瘤生长。结论G-Rg_(3)/Rhe NE粒径均一、稳定性好,可联合aPD-L1增强其抗TNBC的效果。

Objective According to the“supporting healthy energy to eliminate evils”treatment principle advocated by traditional Chinese medicine in the treatment of tumors,the main components ginsenoside Rg_(3)(G-Rg_(3))and rhein(Rhe)in ginseng and rhubarb,respectively,were selected to prepare G-Rg_(3)/Rhe nanoemulsion.At the same time,the solubility of G-Rg_(3) and Rhe was improved to facilitate injection administration,and the nanoemulsion combined with anti-programmed cell death ligand 1(aPD-L1)was used to treat triple negative breast cancer(TNBC).Methods The solubility of G-Rg_(3) and Rhe in different excipients was determined by UV spectrophotometry,and the appropriate emulsifier,co-emulsifier and oil phase were screened out.The pseudo-ternary phase diagram was used to investigate the effect of Km value on the nanoemulsion system,and the appropriate Km value was selected according to the size of the nanoemulsion area.The prescription of G-Rg_(3)/Rhe NE was optimized,and its characterization and stability were evaluated.The 4T1^(Fluc) in situ mammary tumor mouse model was selected to evaluate the effect of G-Rg_(3)/Rhe NE combined with aPD-L1 on TNBC.Results The optimized component ratio of G-Rg_(3)/Rhe NE was as follows:polyoxyethylene hydrogenated castor oil EL35-polyethylene glycol 400-caprylic acid glyceride-water,and the mass ratio was 1.33:2.67:2.80:12.50,the Km value was 1:2.The average particle size of G-Rg_(3)/Rhe NE was(84.8±1.1)nm,the polydispersity index(PDI)was 0.21±0.02,and theζpotential was(−11.30±0.43)mV.The encapsulation rates of G-Rg_(3) and Rhe in drug-carrying nanoemulsions were 96.51%and 97.47%,respectively,and the drug loading capacity of the two drugs was 0.75%.G-Rg_(3)/Rhe NE combined with aPD-L1 could significantly inhibit the tumor growth in 4T1^(Fluc)-bearing mice.Conclusion The G-Rg_(3)/Rhe NE was of uniform particle size and good stability,it could synergistically enhance the antitumor effect of aPD-L1 on TNBC.