正十二醇修饰的多西紫杉醇前药纳米结构脂质载体的设计及抗肿瘤活性评价

Design and Antitumor Activity Evaluation of n-Dodecanol Modified Docetaxel Prodrug Nanostructured Lipid Carrier

目的设计合成正十二醇修饰的多西紫杉醇(docetaxel,DTX)前药,并制备纳米结构脂质载体(nanostructured lipid carrier,NLC),对其制剂学性质,体外抗肿瘤活性及体内药效进行研究。方法以正十二醇、硫代二乙酸及多西紫杉醇为原料合成DTX前药,采用超声法制备纳米结构脂质载体(DNLC),单因素法和响应面法筛选出较优的处方和工艺条件;采用透射电镜观察纳米制剂的形态;采用高效液相色谱测定DNLC在不同介质中的降解特征;采用马尔文粒径仪测定纳米制剂的粒径及多分散指数(PDI),并考察其长期稳定性;采用MTT法对DNLC的体外细胞毒性进行评价;采用鼠源乳腺癌细胞(4T1)荷瘤balb/c小鼠模型,评价DNLC在荷瘤小鼠体内的抗肿瘤活性。结果合成并制备正十二醇修饰的DTX前药纳米结构脂质载体,较优处方和工艺条件为:乳化剂与助乳化剂质量比(Km) 1∶3,固液脂质比1.43∶1,药脂比1∶10,乳化剂质量浓度60 mg·mL^-1,温度70℃,搅拌速度800 r·min^-1。制得的纳米制剂外观圆整,呈均一球形,在30 d(4℃)的保存条件下较为稳定。在空白PBS及含有10 mmol·L^-1DTT和H2O2的介质中,24 h时DTX前药累积降解率分别为(9.07±0.01)%、(21.52±0.35)%和(96.72±4.12)%。DTX-Sol组及DNLC组与4T1孵育72 h后,IC50分别是(1.2±0.2)和(13.2±4.3) nmol·L^-1,DTX-Sol组的细胞毒性强于DNLC组。第18天药效学实验结束时,生理盐水、DTX-Sol和DNLC组的小鼠肿瘤体积分别是(1 930.39±215.20)、(1 013.64±138.65)和(765.16±177.43) mm3,小鼠体重变化率分别是(-19.69±4.44)%、(-14.85±3.61)%和(-2.61±1.70)%。DNLC与生理盐水组和DTX-Sol组相比,肿瘤体积和小鼠体重有显著性统计学差异(P<0.05)。结论制备的DNLC稳定性较好,具有氧化还原双敏感性和明显的抗乳腺癌作用,同时毒性较低,为DTX前药纳米给药系统的开发提供新的实验基础。

OBJECTIVE To design Ndodecanol modified docetaxel(DTX) prodrug,prepare nanostructured lipid carrier(NLC)and investigate in vitro antitumor activity and in vivo pharmacodynamic.METHODS Nanostructured lipid carrier(DNLC) encapsulating n-dodecanol-modified DTX prodrug was prepared by ultrasonic method.The formulation was optimized by single-factor experiment and response surface optimization.The accumulated rates of DTX degraded from DNLC in different media was evaluated by high performance liquid chromatography(HPLC).The morphology of DNLC was observed by transmission electron microscopy(TEM).The particle size and PDI of DNLC were determined by Malvern particle size analyzer.The long-term stability of the preparations was investigated.In vitro cytotoxicity of DNLC was measured by MTT method.In vivo pharmacodynamics of DNLC were performed in 4 T1 tumor xenograft balb/c mice using saline and DTX-Sol as control.RESULTS n-Dodecanol-modified DTX prodrug was synthesized and used to prepare DNLC.The optimal formulation was as following: mass ratio of emulsifier to co-emulsifier(Km) of 1∶3,solid-liquid lipid ratio of 1.43∶1,drug-lipid ratio of 1∶10,the emulsifier concentration of 60 mg·mL^-1,the temperature of 70 ℃ and the stirring speed of 800 r·min^-1.DNLC had a round appearance and a uniform spherical shape.And the particle size and PDI remained substantially stable within 30 d.The accumulated rates of DTX degraded from DNLC in PBS(pH 7.4),PBS(pH 7.4) containing 10 mmol·L^-1 DTT and 10 mmol·L^-1 H2 O2 was(9.07±0.01) %,(21.52±0.35) % and(96.72±4.12) % at 24 h,respectively.After incubation of DTX-Sol and DNLC with 4 T1 cells for 72 h,IC50 of DTX-Sol and DNLC were(1.2±0.2) and(13.2±4.3)nmol·L^-1,respectively.The cytotoxicity of DTX-Sol group was stronger than that of DNLC group.At the end of the pharmacodynamics,the tumor volumes of the mice in saline,DTX-Sol and DNLC groups were(1 930.39±215.20),(1 013.64±138.65),and(765.16±177.43) mm3,respectively.And the change percentage of body weight in saline,DTX-Sol and DNLC groups were(-19.69±4.44) %,(-14.85±3.61) % and(-2.61±1.70) %.There were significant differences in tumor volume and body weight between the DNLC and DTX-Sol group(P<0.05).CONCLUSION The prepared DNLC shows good stability,redox sensitivity,obvious anti-tumor effect and lower toxicity.These results could provide a new experimental basis for the development of DTX prodrug loaded nano-drug delivery system.