构建多功能外泌体用于治疗未分化甲状腺癌

Multifunctional exosome for the treatment of anaplastic thyroid carcinoma

目的观察131I标记、内化精氨酸-甘氨酸-天冬氨酸多肽(iRGD)靶向的多功能外泌体(iRGD-Exo-131I)对未分化甲状腺癌(ATC)细胞的杀伤作用。方法蛋白印迹检测Hth7、Cal-62(购自上海生命科学院细胞所)两种ATC细胞中整合素αvβ3的表达;设计包含酪氨酸、iRGD肽段及外泌体膜蛋白(Lamp2b)基因的质粒,将其转染HEK-293T(天津医科大学肿瘤医院)细胞后利用超高速离心法得到靶向外泌体(iRGD-Exo);共聚焦显微镜观察Hth7细胞对靶向及无靶向外泌体的摄取情况;氯胺T法制备iRGD-Exo-131I;细胞毒性实验验证iRGD-Exo的细胞安全性,并比较131I标记的靶向及无靶向外泌体对Hth7细胞的毒性;构建Hth 7荷瘤鼠模型,当瘤体直径约8 mm时按随机数字表法分为4组(每组5只),分别尾静脉注射磷酸盐缓冲液、无靶向外泌体(blank-Exos)、blank-Exos-131I或iRGD-Exos-131I溶液,在注射后不同时间点(0、3、6、9、12、15、18 d)记录瘤体体积及荷瘤鼠体重。采用两样本t检验、单因素方差分析统计数据。结果整合素αvβ3在Hth7、Cal-62细胞中明显高表达。共聚焦实验显示外泌体经iRGD修饰后可增强Hth7细胞对其的摄取能力。iRGD-Exo-131I的标记率为(50.16±4.21)%,放化纯>95%。共孵育48 h后,131I标记的靶向与无靶向外泌体组Hth7细胞存活率均显著低于游离Na131I组[靶向组:(40.97±6.55)%比(83.80±5.21)%,t=12.531,P<0.05;无靶向组:(67.32±8.92)%比(83.80±5.21)%,t=3.912,P<0.05];且无靶向组高于靶向组[(67.32±8.92)%比(40.97±6.55)%,t=5.833,P<0.05];动物实验亦证实iRGD-Exo-131I能更有效地抑制ATC细胞的增殖。结论成功制备iRGD-Exo-131I,且该标志物较稳定,靶向性良好;体外及体内实验证实其能有效杀伤ATC细胞。

Objective To observe the killing effects of 131I-labeled,internalizing Arg-Gly-Asp peptide-targeted multifunctional exosome(iRGD-exo-131I)on anaplastic thyroid carcinoma(ATC)cells.Methods The expression of integrinαvβ3 in Hth7 and Cal-62 cells(purchased from Institute of Cell Science,Shanghai Academy of Life Sciences)was detected by Western blotting.A plasmid containing tyrosine,iRGD peptide and lysosome-associated membrane glycoprotein 2b(Lamp2b)was designed and transfected into HEK-293T cells(donated by professor Zhang Haiyang,Tianjin Medical University Cancer Hospital)to produce iRGD-exo.Confocal microscopy was used to detect the uptake ability of Hth7 cells to absorb iRGD-exos or blank-exos.iRGD-exo-131I was prepared by chloramine-T method.Cell counting kit-8(CCK-8)assay was used to verify the safety of iRGD-exo on Nthy-ori 3-1 cells and observe the in vitro cytotoxicity of 131I-labeled exosomes on Hth7 cells.The Hth7xenograft mouse model was constructed,and the mice were randomly divided into 4 groups(5 mice every group).The phosphate buffer saline(PBS),blank-Exos,blank-Exos-131I and iRGD-Exos-131I were intravenously injected via tail vein,respectively when the tumor diameter was about 8 mm.Tumor volume and body weight of the mice were recorded at 0,3,6,9,12,15 and 18 days after injection.Two sample t test and one-way analysis of variance were used.Results Integrinαvβ3 was highly expressed in Hth7 and CAL-62 cells.Confocal microscopy confirmed that exosomes modified with iRGD could enhance the uptake ability of Hth7 cells.The labeling rate of iRGD-exo-131I was(50.16±4.21)%,and the radiochemical purity was more than 95%.After 48h co-incubation,the survival rate of Hth7 cells in the 131I-labeled targeted and non-targeted exosome groups was significantly lower than that in the Na 131I group[targeted group:(40.97±6.55)%vs.(83.80±5.21)%,t=12.531,P<0.05;non-targeted group:(67.32±8.92)%vs.(83.80±5.21)%,t=3.912,P<0.05].In addition,the survival rate in the non-targeted group was higher than that in the targeted group[(67.32±8.92)vs.(40.97±6.55)%,t=5.833,P<0.05].iRGD-exo-131I showed excellent in vivo cancer inhibition effect.Conclusion iRGD-exo-131I has been successfully prepared,which has been confirmed to possess good ability to target Hth7 cells in vitro,providing a beneficial exploration for the treatment of ATC.