EGFR靶向性MRI探针的制备及其与NSCC细胞靶向结合的研究*

Preparation of EGFR-targeted MRI probe and its targeted binding to nasopharyngeal squamous carcinoma cells*

摘要:

目的 探讨表皮生长因子受体（EGFR）靶向性磁共振成像（MRI）探针的制备及其与鼻咽部鳞状细胞癌（NSCC）细胞靶向结合的研究。方法 利用NSCC表面EGFR作为靶受体，构建一种具有EGFR靶向性MRI探针，检测探针的性质，将探针与NSCC和正常鼻咽细胞孵育，进行普鲁士蓝染色、间接免疫荧光 及MRI，观察铁染色、荧光染色及T2WI信号强度变化，并计算信号强度变化率。结果 EGFR靶向性MRI探针的粒径为（77.0±1.5）nm，超顺磁性氧化铁（SPIO）负载率为（306.0±4.9）μg/ml，阿霉素（DOX）负载率为（103.3± 3.1）μg/ml，弛豫率为（110.4±2.9），提示EGFR靶向性MRI探针制备成功。EGFR靶向性MRI探针与NSCC细胞共孵育后，细胞内可见不同量的蓝色铁Fe颗粒，随着SPIO浓度的升高而增加；EGFR靶向性MRI探针与正常鼻咽细胞共孵育，细胞内未见明显铁颗粒存在。西妥昔单抗（阳性对照）对NSCC细胞膜有强烈的绿色荧光表达，阴性对照呈现很微弱的绿色荧光表达，EGFR靶向性MRI探针与DOX荧光强度相近。随浓度5、10、 20、40和80?mg/ml梯度增高，EGFR靶向性MRI探针T1信号基本无变化，EGFR靶向性MRI探针T2信号强度逐渐减弱，EGFR靶向性MRI探针在20、40和80 mg/ml 3个时间点比较，T2信号强度明显下降，其中80?mg/ml时T2信号强度下降最显著。EGFR靶向性MRI探针与NSCC细胞共孵育后MRI信号在T2WI上有不同程度的降低（P?<0.05），SPIO浓度越高MRI信号强度降低越明显（P?<0.05）。结论 EGFR靶向性MRI探针的粒径小，可降低MRI的T2信号强度，具有高度的靶向性。

Abstract:

Objective To investigate the preparation of epidermal growth factor receptor (EGFR) targeted magnetic resonance imaging (MRI) probe and its targeted binding to nasopharyngeal squamous cell carcinoma (NSCC) cells. Methods Using NSCC surface EGFR as a target receptor, a EGFR-targeted MRI probe was constructed to detect the properties of the probe. The probe was incubated with NSCC and normal nasopharyngeal cells for Prussian blue staining, indirect immunofluorescence and MRI, and to observed iron staining, fluorescent staining and T2WI signal intensity changes, and to calculate signal intensity change rate. Results The particle size of the EGFR-targeted MRI probe was (77.0?±?1.5) nm, the SPIO loading rate was (306.0?±?4.9) μg/ml, and the DOX loading rate was (103.3?±?3.1) μg/ml. The heave rate Rz was (110.4?±?2.9), suggesting that the EGFR-targeted MRI probe was successfully prepared. After EGFR-targeted MRI probe was co-incubated with NSCC cells, different amounts of blue iron particles were observed in the cells, which increased with the increase of SPIO concentration. EGFR-targeted MRI probes were incubated with normal nasopharyngeal cells, and no obvious iron particles were present inside. Cetuximab (positive control) showed strong green fluorescence expression on NSCC cell membranes, but the negative control showed a very weak green fluorescence expression, and the EGFR-targeted MRI probe was similar to cetuximab. The T1 signal of the EGFR-targeted MRI probe was almost unchanged with the gradient of 0, 5, 10, 20, 40 and 80?mg/ml, and the T2 signal intensity of the EGFR-targeted MRI probe was gradually weakened. Compared with the EGFR-targeted MRI probes at 20, 40 and 80?mg/ml, the T2 signal intensity decreased significantly, and the difference of T2 signal intensity was the most significant at 80?mg/ml. MRI signal was significantly decreased on T2WI after EGFR-targeted MRI probes co-incubated with NSCC cells (P?