摘要
目的旨在开发用^(125)I-NaI标记外泌体的方法,并通过γ计数仪考察其在Pan02胰腺癌荷瘤小鼠体内的生物分布特征。方法通过非放射性NaI对用于治疗胰腺癌的工程化外泌体进行冷标记,采用透射电子显微镜、纳米粒子跟踪分析和Western blotting实验对标记前后外泌体进行表征。在此基础上采用Iodogen法进行外泌体的放射性^(125)I-NaI标记,分离纯化后测定^(125)I-NaI的标记率,Radio-HPLC法测定给药前后^(125)I-外泌体的放化纯度以考察其稳定性;将^(125)I-外泌体单次尾iv于Pan02胰腺癌荷瘤小鼠体内,分别于给药后2、6、24、72 h(每个时间点雌雄各3只)经CO_(2)麻醉后心脏放血处死小鼠,取血清、主要组织器官及肿瘤,γ计数仪测量其放射性计数,计算各组织/血清在不同时间点的蛋白沉淀率;并计算在不同时间点的每克组织(或每毫升血清)放射性计数占总注入放射性计数的百分比(%ID·g^(−1)或%ID·mL^(−1))。结果外泌体表征的结果显示,标记前后的外泌体形态一致,均成圆形或茶托样结构;标记前外泌体粒径峰值为113 nm,标记后外泌体粒径峰值为122 nm,粒径大小主要分布在50~200 nm;均表达其标志性蛋白CD63及TSG101,符合外泌体特征。^(125)I-NaI标记外泌体的标记率为27.82%,纯化后HPLC法测得即时放化纯度为100%,给药后放化纯度为(93.34±5.48)%。在小鼠尾iv给药后2 h,标记的外泌体主要分布在肝脏[(10.8992±1.5181)%ID·g^(−1)]和脾脏[(2.5664±0.7998)%ID·g^(−1)],肿瘤中为[(0.2910±0.0560)%ID·g^(−1)],脑、心脏、脂肪和肌肉组织摄取较少;给药后72 h,肝脏中仍有较高摄取,肿瘤中仍有放射性分布。给药后2~6 h各组织脏器的蛋白沉淀率较低,表明^(125)I-NaI标记外泌体稳定性有所降低。结论外泌体可以进行^(125)I标记,而且标记同位素前后对外泌体物理形态、生物学活性无明显影响;^(125)I标记外泌体的方法简便,标记率和放化纯度�
Objective This study was aimed to develop a ^(125)I-NaI labeling method for exosomes,and to investigate their biological distribution in Pan02 pancreatic cancer tumor bearing mice by gamma counter.Methods Exosomes were cold-labeled with nonradioactive NaI.Exosomes were characterized before and after labeling by transmission electron microscopy,nanoparticle tracking analysis and Western blotting.On this basis,Iodogen method was used for radioactive ^(125)I-NaI labeling of exosomes,and the labeling rate of ^(125)I-NaI was determined after isolation and purification.Radio-HPLC method was used to determine the radiochemical purity of ^(125)I-exosomes before and after drug administration to understand its stability.^(125)I-exosome was injected into Pan02 pancreatic cancer tumor bearing mice by a single caudal vein,and the mice were killed by cardiac bloodletting after CO_(2) anesthesia at 2,6,24,72 h(three males and three females at each time point).Serum,major tissues,organs and tumors were collected and their radioactivity counts were measured.The percentage(%ID·g^(−1) or%ID·mL^(−1))of the total injected radioactivity count per gram of each tissue and organ at different time points was calculated,so as to investigate the biological distribution of ^(125)I-NaI labeled exosomes in tumor-bearing mice.Results The morphology of exosomes before and after labeling was consistent with that of round or saucer-like exosomes.The peak particle size of exosomes was 113 nm before labeling and 122 nm after labeling,and the particle size was mainly distributed in the range of 50~200 nm.Both of them expressed their signature proteins CD63 and TSG101,which were consistent with exosome characteristics,indicating that isotope labeling did not affect their biological characteristics.The labeling rate of ^(125)I-NaI labeled exosomes was 27.82%,and the purity was 100%by HPLC after purification,and(93.34±5.48)%after administration.The labeled exosome was mainly distributed in liver[(10.8992±1.5181)%ID·g^(−1)]and spleen[2.5664±0
作者
陶巧玉
许波华
秦天
赵晶
陈晓庆
李嫚琪
宋鑫
刘楚乔
付莉莉
常艳
TAO Qiaoyu;XU Bohua;QIN Tian;ZHAO Jing;CHEN Xiaoqing;LI Manqi;SONG Xin;LIU Chuqiao;FU Lili;CHANG Yan(School of Pharmacy,Anhui University of Traditional Chinese Medicine,Hefei 230012,China;InnoStar Biotechnology Nantong Co.,Ltd.,Nantong 226000,China;Yangtze Delta Drug Advanced Research Institute,Nantong 226133,China;ImmVira Co.,Ltd.,Shenzhen 518000,China 5.China;State Institute of Pharmaceutical Industry,Shanghai InnorStar Biotech Co.,Ltd.,Shanghai 201203,China)
出处
《药物评价研究》
CAS
2023年第5期1032-1038,共7页
Drug Evaluation Research
基金
江苏省新药一站式高效非临床评价公共服务平台建设(BM2021002)。
