非RGD肽—超微超顺磁氧化铁分子探针的制备及鉴定

Preparation of molecular probe modified by non RGD peptide and its targeting effect on human umbilical vein endothelial cells

目的制备及鉴定非RGD肽(命名为P1c)—超微超顺磁氧化铁(USPIO)分子探针。方法采用化学交联法将P1c偶联于二巯基丁二酸(DMSA)修饰的USPIO,形成具有免疫活性的P1c-USPIO分子探针。采用CCK-8试剂盒检测P1c-USPIO分子探针对人脐静脉内皮细胞(HUVEC)增殖的影响,观察P1c-USPIO的细胞毒性。用固相结合实验、普鲁士蓝染色和体外MRI法观察P1c-USPIO的体外免疫学特性。结果成功构建USPIO标记的P1c肽分子探针P1c-USPIO。其对HUVEC增殖无影响。固相结合试验及普鲁士蓝染色证实该探针能特异性与整合素αvβ3结合。体外MRI显示在同一USPIO浓度时,用P1c-USPIO孵育的HUVEC与用USPIO孵育者相比T2加权相信号强度明显降低(P均<0.01)。结论化学交联法可成功制备P1c肽超微超顺磁氧化铁分子探针,该分子探针具有良好免疫学活性,可特异性结合整合素αvβ3分子。

Objective To develop an ultrasmall superparamagnetic iron oxide （USPIO） based molecular probe contai- ning non RGD peptide （named P1 e） and investigate its targeting effect on human umbilical vein endothelial cells （HU- VECs）. Methods Immunocompetent probe was prepared, and during this process P1 c was conjugated with USPIO coated with meso-2 and 3-dimercaptosuceinie acid （DMSA） , and the cytotoxicity of PI c-USPIO was observed . Cell counting kit-8 assay was conducted to ascertain the probe＇s effect on the HUVECs＇growth. The USPIO labeled Pie peptide was the experimental group, and the USPIO was the control group. The solid phase binding assay and Prussian blue staining were employed to evaluate the immunocompetence of Plc-USPIO. The probe＇s cell MR imaging in vitro was performed using HUVECs with high integrin αv33 expression. Results The P1 c-USPIO was constructed. The probe had no effect on the growth of HUVECs. The solid phase binding assay and Prussian blue staining showed that the probe could bind with integrin αvl33 on HUVECs with high specificity. MR cell imaging in vitro demonstrated that at the same concentration the T2 ＊ value in Plc-USPIO group was much shorter than that in USPIO group （when the concentration of USPIO was 0, 10, 20, 40 and 80 μg/mL respectively, the T2 ＊ value in Plc-USPIO group was 658.33 ±3.39, 618.67 ±4.55, 364.67 ±4.55, 246.33 ±_ 6.09 and 198.83 ± 5.23 ,while the T2 ＊ value in USPIO group was 660.33 ± 5.54,630.33 ± 5.13, 585.17 ± 4.71,495.50 ± 3.78 and 482.67 ± 8.02）. Conclusions The molecular probe P1 c-USPIO could be prepared successfully using chemical cross linking method. The Plc-USPIOs remain their immunocompetence. The probe could specifically bind with HUVECs.