TSPO靶向分子探针CB86-DTPA-Gd的制备及类风湿关节炎模型MRI研究

目的制备相对分子质量1.8×104转位蛋白(TSPO)靶向分子探针2-(8-氨基-2-(4-氯苯基)咪唑1,2-a]吡啶-3-基)-N,N-二丙基乙酰胺(CB86)-二乙撑三胺五乙酸(DTPA)-Gd,探究其在类风湿关节炎(RA)模型的MRI效果。方法通过偶联双功能螯合剂制备CB86-DTPA,再与Gd螯合获得MRI靶向造影剂CB86-DTPA-Gd,测定其细胞毒性、MR弛豫率和体外稳定性。采用弗氏佐剂建立小鼠右踝RA模型,进行生物分布研究及MRI,以右踝关节炎性反应部位及其周围正常组织作为感兴趣区(ROI)计算信噪比(SNR)。采用两独立样本t检验分析数据。结果CB86-DTPA-Gd的MR纵向弛豫率r1为11.05 mmol·L-1·s-1。在Gd3+最大浓度(20μmol/L)时,小鼠巨噬细胞RAW264.7和小鼠乳腺癌4T1细胞的存活率均大于90%。室温下,CB86-DTPA-Gd在人血清和磷酸盐缓冲溶液(PBS;pH=7.4)中的稳定性良好。体内生物分布研究表明,CB86-DTPA-Gd具有较好的炎性反应靶向性,注射后120 min踝关节炎性反应部位摄取仍达(2.33±0.29)每克组织百分注射剂量率(%ID/g)。MicroMRI结果显示,右踝关节炎性反应部位在注射CB86-DTPA-Gd和Gd-DTPA后均表现为明显强化,CB86-DTPA-Gd组SNR最高可达23.21±1.44,最大强化时间为注射后90 min,且各时间点均能被CB86-DTPA所抑制(t值:6.083~12.451,均P<0.05),而Gd-DTPA组强化时间为注射后30 min,SNR为16.12±1.24。结论CB86-DTPA-Gd显示了良好的巨噬细胞靶向性,在关节炎性反应部位有良好的摄取,有望成为一种用于外周炎性反应显像的新型MRI分子探针。

Preparation and MRI of CB86-DTPA-Gd targeting TSPO in rheumatoid arthritis model

Objective To synthesize Gd labeled probe targeting transporter protein(TSPO)2-(8-amino-2-(4-chlorophenyl)imidazo1,2-a]pyridine-3-yl)-N,N-dipropylacetamide(CB86)-diethylene triamine pentaacetic acid(DTPA),and investigate its MRI effect in rheumatoid arthritis(RA)model.Methods CB86-DTPA was prepared by coupling a bifunctional chelating agent,and then chelated with Gd to obtain MRI targeted contrast agent CB86-DTPA-Gd.The cytotoxicity,MR relaxation rate and in vitro stability of CB86-DTPA-Gd were determined.RA model was established with Freund′s adjuvant and the biodistribution study and MRI was performed.The RA lesion and its surrounding normal tissue were used as regions of interest(ROI)to calculate the signal to noise ratio(SNR).Independent-sample t test was used to analyze the data.Results CB86-DTPA-Gd had excellent biosafety and a good MR relaxation rate(r1=11.05 mmol·L-1·s-1).The survival rate of RAW264.7 cells and 4T1 cells was still more than 90%at the maximum concentration(20μmol/L)of Gd3+.CB86-DTPA-Gd also exhibited good stability in human serum and phosphate buffer saline solution(PBS;pH=7.4).The in vivo biodistribution showed that CB86-DTPA-Gd had better inflammatory targeting efficiency,and the uptake of Gd in the inflamed site of the ankle joint was still(2.33±0.29)percent dose rate per gram of tissue(%ID/g)at 120 min after injection.MicroMRI showed that the inflammation of the right ankle joint displayed significant enhancement after the injection of CB86-DTPA-Gd and Gd-DTPA.The SNR of CB86-DTPA-Gd group was up to 23.21±1.44,and the maximum intensification time was 90 min after injection,and can be significantly inhibited by CB86-DTPA at all time points(t values:6.083-12.451,all P<0.05),while the Gd-DTPA group had a strengthening time of 30 min after injection with the SNR of 16.12±1.24.Conclusion CB86-DTPA-Gd shows good macrophage targeting and good uptake in arthritic reaction sites,and is expected to be a novel MRI molecular probe for peripheral inflammation imaging.