[关键词]
[摘要]
目的 采用分支状聚乙二醇化聚合物G2制备不同药载比的和厚朴酚(HK)纳米粒,考察其稳定性、体外释放及细胞毒等体外表征,以期丰富现有的纳米载药系统并筛选合适的药载比。方法 采用超声-蒸发法将有机相(HK和G2的丙酮溶液的混合相)缓慢注入到去离子水中,减压旋蒸除去有机溶剂即得HK-G2纳米粒(HK-G2-Nps),分别制备药载比为1∶1、2∶1、4∶1、8∶1的纳米粒。利用马尔文粒度分析仪测定HK-G2-Nps粒径、Zeta电位及聚合物分散性指数(PDI)值;高效液相色谱(HPLC)法测定各药载比载药量;用扫描电镜观察其形态;动态膜透析法进行体外释药试验;以乳腺癌4T1细胞为模型用MTT法评价质量浓度为50.0、10.0、5.0、2.5、1.0、0.5、0.1 μg/mL的各药载比HK-G2-Nps细胞毒作用。结果 各药载比HK-G2-Nps的粒径分布较为集中,分散较为均匀;载体G2的用量影响HK-G2-Nps的粒径大小,随着药载比的增加,粒径逐渐减小,载药量提高。稳定性结果显示,纳米粒室温下放置稳定;纳米粒在0.9% NaCl中粒径明显增大,在5%葡萄糖溶液、PBS缓冲液和血浆中可稳定存在。电镜结果显示,药载比为1∶1时,纳米粒没有均一的形态;药载比为2∶1时,纳米粒呈现出不规则的类球状;药载比为4∶1时,呈现出规则均一的球形;药载比达到8∶1时,纳米粒呈现出明显的立方柱状形态同时存在不规则球状。体外释放试验结果显示,在前24 h,纳米粒能够迅速释放,药载比为4:1时累计释放最高,可达70%左右;24 h之后,4个药载比均缓慢释放至120 h,药载比1∶1和2∶1的累计释放率均在60%左右,药载比4∶1和8∶1的累计释放率可达80%、89%。HK-G2-Nps各药载比4T1细胞增殖抑制率均大于游离组HK,药载比为4∶1时作用最显著,其中质量浓度50.0、10.0、2.5、1.0、0.5、0.1 μg/mL显著高于HK组(P<0.05),半数抑制浓度(IC50)值为3.83 μg/mL。结论 成功筛选并制备药载比为4∶1的HK-G2-Nps,其对乳腺癌4T1细胞增殖抑制率大于HK。
[Key word]
[Abstract]
Objective To prepare honokiol (HK) nanoparticles with different drug loading ratios, using the branching oligoethylene glycols (OEG) dendron (G2) the in vitro characterizations of nanoparticles such as stability, in vitro release properties, and cytotoxicity were investigated in order to filter the appropriate drug loading ratio for further in vitro studies.Method The ultrasonicevaporation method was used to inject the organic phase (mixture phase of HK and G2 acetone solution) into deionized water slowly and spin down to remove the organic solvent under reduced pressure, HK-G2 nanoparticles (HK-G2-Nps) with drug loading ratios of 1:1, 2:1, 4:1 and 8:1 were prepared respectively. The particle size Zeta potential and polymer dispersion index (PDI) of HK nanoparticles was determined using a Malvern particle size analyzer, the specific drug loading was determined by HPLC, the morphology of HK nanoparticles was observed by scanning electron microscopy, the drug release test in vitro was performed by dynamic membrane dialysis, and the cytotoxicity of HK nanoparticles of 50.0, 10.0, 5.0, 2.5, 1.0, 0.5 and 0.1 μg/mL was evaluated by MTT assay using 4T1 cell line. Results The particle size distribution of each drug-loading ratio HK-G2-Nps was concentrated and uniform; The amount of carrier G2 affects the particle size of HK-G2-Nps; With the increase of drug-loading ratio, the particle size decreases gradually, and the drug loading increases. The stability results showed that the nanoparticles were stable at room temperature, and the particle size increased significantly in 0.9% Nacl. The nanoparticles were stable in 5% glucose solution, PBS buffer and plasma. The results of electron microscopy showed that when the drug loading ratio was 1:1, the nanoparticles had no uniform morphology; When the drug loading ratio was 2:1, the nanoparticles showed irregular spherical shape; When the drug loading ratio was 4:1, the nanoparticles showed regular spherical shape; When the drug loading ratio was 8:1, the nanoparticles showed obvious cubic columnar shape and irregular spherical shape. The results of in vitro release test showed that the nanoparticles could be released rapidly in the first 24 hours, and the cumulative release rate was about 70% when the drug-loading ratio was 4:1. After 24 h, the four drug-loading ratios were released slowly to 120 h, the cumulative release rates of drug-loading ratio 1:1 and 2:1 were about 60%, and the cumulative release rates of drug-loading ratio 4:1 and 8:1 were 80% and 89%. The inhibitory rate of proliferation of HK-G2-Nps 4T1 cells was higher than that of free HK cells. The inhibitory rate of drug-loading ratio 4:1 at concentration of 50.0, 10.0, 2.5, 1.0, 0.5 and 0.1 μg/mL was significantly higher than that of HK (P<0.01), and the half inhibitory concentration (IC50) was 3.83 μg/mL. Conclusion The preparation and screening of HK nanoparticles with a drug-loading ratio of 4:1 is needed for further study.
[中图分类号]
[基金项目]
中国医学科学院医学与健康科技创新工程经费资助(2017-I2M-1-013)