目的 旨在開發(fā)用 ¹²⁵I-NaI標記外泌體的方法，并通過 γ 計數(shù)儀考察其在 Pan02 胰腺癌荷瘤小鼠體內(nèi)的生物分布特征。方法 通過非放射性 NaI對用于治療胰腺癌的工程化外泌體進行冷標記，采用透射電子顯微鏡、納米粒子跟蹤分析和Western blotting實驗對標記前后外泌體進行表征。在此基礎(chǔ)上采用Iodogen法進行外泌體的放射性 ¹²⁵I-NaI標記，分離純化后測定 ¹²⁵I-NaI的標記率，Radio-HPLC法測定給藥前后 ¹²⁵I-外泌體的放化純度以考察其穩(wěn)定性；將 ¹²⁵I-外泌體單次尾iv于Pan02胰腺癌荷瘤小鼠體內(nèi)，分別于給藥后2、6、24、72 h（每個時間點雌雄各3只）經(jīng)CO₂麻醉后心臟放血處死小鼠，取血清、主要組織器官及腫瘤，γ計數(shù)儀測量其放射性計數(shù)，計算各組織/血清在不同時間點的蛋白沉淀率；并計算在不同時間點的每克組織（或每毫升血清）放射性計數(shù)占總注入放射性計數(shù)的百分比（%ID·g^-1或%ID·mL^-1）。結(jié)果 外泌體表征的結(jié)果顯示，標記前后的外泌體形態(tài)一致，均成圓形或茶托樣結(jié)構(gòu)；標記前外泌體粒徑峰值為 113 nm，標記后外泌體粒徑峰值為 122 nm，粒徑大小主要分布在 50～200 nm；均表達其標志性蛋白 CD63及 TSG101，符合外泌體特征。¹²⁵I-NaI標記外泌體的標記率為 27.82%，純化后 HPLC法測得即時放化純度為 100%，給藥后放化純度為（93.34±5.48）%。在小鼠尾 iv給藥后 2 h，標記的外泌體主要分布在肝臟［（10.899 2±1.518 1）%ID·g^-1］和脾臟［（2.566 4±0.799 8）%ID·g^-1］，腫瘤中為［（0.291 0±0.056 0）%ID·g^-1］，腦、心臟、脂肪和肌肉組織攝取較少；給藥后72 h，肝臟中仍有較高攝取，腫瘤中仍有放射性分布。給藥后 2～6 h各組織臟器的蛋白沉淀率較低，表明 ¹²⁵I-NaI標記外泌體穩(wěn)定性有所降低。結(jié)論 外泌體可以進行 ¹²⁵I標記，而且標記同位素前后對外泌體物理形態(tài)、生物學(xué)活性無明顯影響；¹²⁵I標記外泌體的方法簡便，標記率和放化純度均較高；該外泌體產(chǎn)品在荷瘤小鼠體內(nèi)大部分血流豐富的組織器官均有分布，且具有一定的腫瘤靶向定位能力。

Objective This study was aimed to develop a ¹²⁵I-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 non-radioactive 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 ¹²⁵I-NaI labeling of exosomes, and the labeling rate of ¹²⁵I-NaI was determined after isolation and purification. Radio-HPLC method was used to determine the radiochemical purity of ¹²⁵I-exosomes before and after drug administration to understand its stability. ¹²⁵I-exosome was injected into Pan02 pancreatic cancer tumor bearing mice by a single caudal vein, and the mice were killed by cardiac bloodletting after CO₂ 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 ¹²⁵I-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 ¹²⁵I-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 [(10.899 2±1.518 1) %ID·g^-1] and spleen [2.566 4±0.799 8)% ID·g^-1] at 2 h after administration in the tail vein of mice. Meanwhile, (0.291 0±0.056 0) %ID·g^-1 was found in tumor. After 72 h of administration, there was still high uptake in liver and radioactive distribution in tumor. The protein precipitation emissivity of various tissues and organs was low 2 ~ 6 h after administration, indicating a decrease in the stability of ¹²⁵I-NaI labeled exosomes.Conclusion Extracellular vesicles can be labeled with ¹²⁵I, and there is no significant effect on their physical morphology and biological activity before and after isotope labeling. The method of labeling exosomes with ¹²⁵I is simple, with high labeling rate and radiochemical purity. This exosomes product is distributed in most blood rich tissues and organs of tumor bearing mice, and has certain tumor targeting and localization capabilities.

R965.2

江蘇省新藥一站式高效非臨床評價公共服務(wù)平臺建設(shè)（BM2021002）