三氧化二砷三元复合物纳米递送系统的构建及评价  

Construction and evaluation of a ternary complex nano-delivery system based on arsenic trioxide

作　　者：王笑红 张宇婷 吴婕 李俊松[1,2] 李文 王若宁[1,2] WANG Xiao-hong;ZHANG Yu-ting;WU Jie;LI Jun-song;LI Wen;WANG Ruo-ning(College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Engineering Research Center for Efficient Delivery System of TCM,Nanjing 210023,China;The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine,Nanjing 210001,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]江苏省中药高效给药系统工程技术研究中心,江苏南京210023 [3]南京中医药大学附属南京中医院,江苏南京210001

出　　处：《中草药》2022年第5期1382-1391,共10页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81903557);江苏省自然科学基金资助项目(BK20190802);江苏省高等学校大学生创新创业训练计划项目(省级重点)(202110315021);康缘中药学院创新创业项目(kyxysc12)。

摘　　要：目的以牛血清白蛋白(bovine serum albumin,BSA)为天然药物载体,通过形成砷硫键负载三氧化二砷(arsenic trioxide,ATO),酰胺化反应连接光敏剂二氢卟吩e6(chlorin e6,Ce6),构建基于BSA的载ATO三元复合物纳米递送系统,旨在解决ATO在治疗脑胶质瘤时体内生物分布不良、难以跨越血脑屏障(blood brain barrier,BBB)等问题,使其更有效地用于脑胶质瘤治疗。方法通过形成砷硫键和酰胺化反应分别将ATO和Ce6与BSA结合制备三元复合物纳米粒ATO-BSA@Ce6,采用纳米粒度分析仪考察ATO-BSA@Ce6的粒径分布、多分散系数(polydispersity index,PDI)、ζ电位;透射电子显微镜(TEM)观察其形态;紫外可见分光光度计(UV-vis spectrophotometer)考察UV-vis吸收光谱;荧光分光光度计考察荧光光谱和制剂活性氧(reactive oxygen species,ROS)产生;傅里叶变换红外(Fourier transform infrared,FTIR)光谱验证ATO的载入;电感耦合等离子体质谱(inductively coupled plasma mass spectrometry,ICP-MS)和UV-vis分光光度计分别测定ATO和Ce6的包封率、载药量;透析袋法考察不同pH值下的体外释放行为。对ATO-BSA@Ce6进行细胞摄取及机制、细胞毒性、LIVE/DEAD染色、ROS水平和体外跨越BBB等体外细胞学研究,并通过小鼠尾iv对其脑靶向性进行考察。结果成功制备了三元复合物纳米粒ATO-BSA@Ce6,在TEM下可观察到其形态规整,呈圆整球状分布,大小均匀,测得其粒径为(112.73±4.91)nm,ζ电位(−10.86±1.19)mV。通过ICP-MS测定ATO的包封率为(66.72±1.43)%、载药量为(10.83±0.21)%;Ce6的包封率为(91.50±0.51)%、载药量为(3.45±0.32)%。UV-vis吸收光谱、荧光光谱及FTIR光谱结果证明Ce6、ATO与BSA的成功连接。体外ROS生成实验结果表明Ce6与BSA的结合不影响其ROS产生。体外释放实验结果表明ATO及Ce6具有pH响应性释放。细胞摄取和细胞毒性实验表明其具有较强的入胞能力和体外抗肿瘤活性。体内活体成像实验表明其具有�Objective To solve the problem of poor biological distribution and difficulty in crossing the blood brain barrier(BBB)of arsenic trioxide(ATO)in glioma treatment,a pH-responsive ternary complex drug delivery system of ATO-BSA@Ce6 was constructed based on bovine serum albumin(BSA),where arsenic trioxide(ATO)was loaded through the formation of arsenic sulfur bond,and the photosensitizer chlorin e6(Ce6)was connected through amidation reaction.Methods The ternary complex nanoparticle,ATOBSA@Ce6,was prepared by combining ATO and Ce6 with BSA through arsenic-sulfur bonds and the amidation reaction,respectively.The particle size,polydispersity index(PDI),andζpotential of ATO-BSA@Ce6 were measured by Malvern particle size analyzer.The morphology was investigated by transmission electron microscopy(TEM).UV-vis absorption spectra were measured by UV-Vis spectrophotometer.The fluorescence spectrum and the production of reactive oxygen species(ROS)were investigated by fluorescence spectrophotometer.The loading of ATO was verified by Fourier transform infrared(FTIR)spectra.The encapsulation efficiency and drug loading of ATO and Ce6 were measured by inductively coupled plasma mass spectrometry(ICP-MS)and UV-vis spectrophotometer,respectively.The in vitro release behaviors at different pH conditions were investigated by dialysis bag method.The cellular uptake,uptake mechanism and intracellular ROS level were observed by inverted fluorescence microscope.The toxicity of free ATO and ATO-BSA@Ce6 on GL261 cells was investigated by MTT assay and LIVE/DEAD cell viability detection kit.The brain targeting was investigated by tail vein injection in mice.Results The drug delivery system(ATO-BSA@Ce6)was constructed successfully,and its morphology was regular,with a round spherical distribution and uniform size.The particle size andζpotential were(112.73±4.91)nm and(−10.86±1.19)mV,respectively.The encapsulation efficiency of ATO was(66.72±1.43)%,and drug loading efficiency was(10.83±0.21)%;The encapsulation efficiency of Ce6 was(

关 键 词：三氧化二砷 脑胶质瘤 纳米递送系统 二氢卟吩e6 牛血清白蛋白 电感耦合等离子体质谱 

分 类 号：R283.6[医药卫生—中药学]