iRGD肽修饰的卡巴他赛白蛋白纳米粒的制备与评价

Preparation and Evaluation of iRGD-Modified Cabazitaxel Loaded Albumin Nanoparticles

目的制备一种白蛋白纳米递药体系,以人血白蛋白(human serum albumin,HSA)作为抗肿瘤药物载体,通过处方优化制备纳米级粒径且大小均一的环状多肽iRGD修饰的卡巴他赛(cabazitaxel,CTX)白蛋白纳米粒(iRGD-modified cabazitaxel-loaded albumin nanoparticles,iRGD-HSA-CTX NPs),并对制剂进行质量评价,初步考察其体外抗肿瘤活性。方法采用超声-均质法制备HSA-CTX NPs,响应面Box-Behnken Design筛选最优处方,然后利用sulfo-SMCC的交联作用,将环状多肽iRGD修饰到HSA-CTX NPs上,制得iRGD-HSA-CTX NPs。采用激光粒度仪、透射电镜对该制剂的形貌进行考察。为提高稳定性将其制成冻干粉末,并考察冻干前后制剂的粒径及包封率变化。采用透析法考察其体外释放规律。最后在细胞层面验证其体外抗肿瘤活性。结果按优化条件制备的iRGD-HSA-CTX NPs呈球形形貌,分布均匀,平均粒径为(84.37±3.44)nm,多分散系数(polydispersity index,PDI)为(0.237±0.02),Zeta电位为(-4.87±2.09)mV,包封率为(93.67±2.56)%,载药量为(5.13±0.78)%。iRGD-HSA-CTX NPs冻干后制剂稳定性较好,粒径和包封率与冻干前相比未发生明显变化。体外释放结果显示iRGD-HSA-CTX NPs具有一定的缓释效果。溶血实验表明,制备的iRGD-HSA-CTX NPs安全无毒,适宜静脉注射给药。细胞毒性实验表明,与游离的化疗药物卡巴他赛相比,iRGD-HSA-CTX NPs对小鼠乳腺癌4T1细胞及人非小细胞肺癌A549细胞有更强的细胞毒性。细胞摄取实验和细胞内吞实验表明纳米粒表面修饰的iRGD可以通过与肿瘤细胞高表达的ανβ3、ανβ5和NRP-1结合,提高iRGD-HSA-CTX NPs的肿瘤靶向性。结论采用超声-均质法和sulfo-SMCC交联作用制备的iRGD-HSA-CTX NPs粒径较小,且粒径分布均匀,药物包封率高,在降低了CTX的毒副作用的同时提高肿瘤靶向性,增强抗肿瘤活性。

OBJECTIVE To prepare and optimize cabazitaxel(CTX)-loaded iRGD-modified human serum albumin(HSA)nanoparticles(abbreviated as iRGD-HSA-CTXNPs)and evaluate the drug properties and anti-tumor activity.METHODS HSA-CTX NPs were prepared by ultrasonic homogenization.Box-Behnken design response surface method was used to optimize the prescription.The prepared HSA-CTX NPs were then modified with the cyclic peptide iRGD.The particle size and morphology of the preparation were examined using a laser particle sizer and transmission electron microscope respectively.It was made into a lyophilized powder to improve stability,and the changes in particle size and encapsulation efficiency were examined before and after lyophilization.The in vitro release pattern was investigated using a dialysis method.The in vivo anti-tumor activity was initially investigated by cytotoxicity assay.RESULTS The iRGD-HSA-CTX NPs prepared under the optimized conditions were spherical in shape and uniformly distributed.The particle size was(84.37±3.44)nm,the polydispersity coefficient(PDI)was(0.237±0.02),the Zeta potential was(-4.87±2.09)mV,the encapsulation efficiency was(93.67±2.56)%and the drug loading was 5.13±0.78%.The stability of the preparation was better after lyophilization and no significant change in particle size or encapsulation efficiency occurred.In vitro drug release results showed that iRGD-HSA-CTX NPs had a moderate release effect.The hemolysis test showed that iRGD-HSA-CTX NPs were safe,non-toxic and suitable for intravenous injection.Cytotoxicity assay showed that iRGD-HSA-CTX NPs were more cytotoxic than free CTX to 4T1 cells and A549 cells.Cellular uptake assay and cellular endocytosis assay showed that iRGD modification on the surface of nanoparticles could enhance the tumor targeting of iRGD-HSA-CTX NPs by binding to αvβ3,αvβ5 and NRP-1,which are highly expressed by tumor cells.CONCLUSION The iRGD-HSA-CTX NPs prepared by the ultrasonic-homogenization method and sulfo-SMCC crosslinking have exhibited uniform particle size distribution and high encapsulation efficiency,,which can reduce the side effects of CTX,and improve the tumor targeting of CTX,resulting in the enhanced antitumor activity.