目的 采用分支狀聚乙二醇化聚合物G2制備不同藥載比的和厚樸酚（HK）納米粒，考察其穩(wěn)定性、體外釋放及細胞毒等體外表征，以期豐富現(xiàn)有的納米載藥系統(tǒng)并篩選合適的藥載比。方法 采用超聲-蒸發(fā)法將有機相（HK和G2的丙酮溶液的混合相）緩慢注入到去離子水中，減壓旋蒸除去有機溶劑即得HK-G2納米粒（HK-G2-Nps），分別制備藥載比為1∶1、2∶1、4∶1、8∶1的納米粒。利用馬爾文粒度分析儀測定HK-G2-Nps粒徑、Zeta電位及聚合物分散性指數(shù)（PDI）值；高效液相色譜（HPLC）法測定各藥載比載藥量；用掃描電鏡觀察其形態(tài)；動態(tài)膜透析法進行體外釋藥試驗；以乳腺癌4T1細胞為模型用MTT法評價質(zhì)量濃度為50.0、10.0、5.0、2.5、1.0、0.5、0.1 μg/mL的各藥載比HK-G2-Nps細胞毒作用。結(jié)果 各藥載比HK-G2-Nps的粒徑分布較為集中，分散較為均勻；載體G2的用量影響HK-G2-Nps的粒徑大小，隨著藥載比的增加，粒徑逐漸減小，載藥量提高。穩(wěn)定性結(jié)果顯示，納米粒室溫下放置穩(wěn)定；納米粒在0.9% NaCl中粒徑明顯增大，在5%葡萄糖溶液、PBS緩沖液和血漿中可穩(wěn)定存在。電鏡結(jié)果顯示，藥載比為1∶1時，納米粒沒有均一的形態(tài)；藥載比為2∶1時，納米粒呈現(xiàn)出不規(guī)則的類球狀；藥載比為4∶1時，呈現(xiàn)出規(guī)則均一的球形；藥載比達到8∶1時，納米粒呈現(xiàn)出明顯的立方柱狀形態(tài)同時存在不規(guī)則球狀。體外釋放試驗結(jié)果顯示，在前24 h，納米粒能夠迅速釋放，藥載比為4：1時累計釋放最高，可達70%左右；24 h之后，4個藥載比均緩慢釋放至120 h，藥載比1∶1和2∶1的累計釋放率均在60%左右，藥載比4∶1和8∶1的累計釋放率可達80%、89%。HK-G2-Nps各藥載比4T1細胞增殖抑制率均大于游離組HK，藥載比為4∶1時作用最顯著，其中質(zhì)量濃度50.0、10.0、2.5、1.0、0.5、0.1 μg/mL顯著高于HK組（P<0.05），半數(shù)抑制濃度（IC₅₀）值為3.83 μg/mL。結(jié)論 成功篩選并制備藥載比為4∶1的HK-G2-Nps，其對乳腺癌4T1細胞增殖抑制率大于HK。

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.

中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程經(jīng)費資助（2017-I2M-1-013）