Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes for Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors
Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes for Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors
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DOI：
10.1021/bc200596a
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发表时间：
2012-06-01
影响因子：
4.7
通讯作者：
Cheng, Zhen
中科院分区：
文献类型：
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作者：
Qi, Shibo;Miao, Zheng;Cheng, Zhen
表皮生长因子受体1（EGFR）已成为肿瘤分子显像和治疗的一个有吸引力的靶点。已经报道了对EGFR具有强结合亲和力的亲和体蛋白Z（EGFR：1907）。我们有兴趣将Affibody分子转化为EGFR阳性癌症的潜在临床光学成像。在本研究中，因此制备了四种基于抗EGFR Affibody的近红外（NIR）荧光探针，并在携带EGFR阳性皮下A431肿瘤的小鼠中评价了它们的体内性能。方法：采用固相多肽合成法合成亲和体类似物Ac-Cys-Z（EGFR：1907）。然后将纯化的小蛋白与四种NIR荧光染料Cy 5. 5-单马来酰亚胺、Alex-Fluor-680-马来酰亚胺、SRfluor 680-马来酰亚胺或IRDye-800 CW-马来酰亚胺位点特异性缀合，以产生四种光学探针Cy 5.5-Z（EGFR：1907）、Alexa 680-Z（EGFR：1907）、SR 680-Z（EGFR：1907）和800 CW-Z（EGFR：1907）。使用高EGFR表达的A431细胞和低表达的MCF 7细胞通过荧光显微镜研究四种NIR荧光亲和体探针的EGFR结合特性和特异性。通过流式细胞术进一步测定探针（K-D）与EGFR的结合亲和力。在携带皮下A431肿瘤的小鼠中进行四种探针的体内光学成像。结果：制备出纯度较高的四种近红外光学探针。体外细胞成像研究表明，它们都可以特异性结合EGFR阳性的A431细胞，而在低表达EGFR的MCF 7细胞中显示最小摄取。流式细胞术显示Cy5.5-Z（EGFR：1907）和Alexa 680-Z（EGFR：1907）在低纳摩尔范围内具有高结合亲和力（分别为43.6 +/- 8.4和28.3 +/- 4.9）。四种探针的体内光学成像显示，它们都显示出快速的肿瘤靶向能力和良好的肿瘤与正常组织的对比度，早在注射后0.5小时（p.i.）。肿瘤与正常组织的比率在感染后2至4小时达到峰值。通过图像的感兴趣区域（ROI）分析。离体研究进一步证明，四种探针具有高肿瘤摄取。特别是Cy5.5-Z（EGFR：1907）和Alex 680-Z（EGFR：1907）显示出比其他两种探针更高的肿瘤与正常组织比率。结论：这项工作表明，亲和体蛋白可以用不同的NIR荧光染料修饰，并用于EGFR表达肿瘤的成像。不同的NIR荧光染料对所得的基于亲和体的探针的体外结合和体内性能具有可变的影响。因此，选择合适的NIRF标记物对于光学探针的开发是重要的。所开发的探针有希望用于进一步的肿瘤成像应用和临床翻译。特别是，Alex 680-Z（EGFR：1907）和Cy5.5-Z（EGFR：1907）是用于光学成像的EGFR靶向探针的优秀候选者。
The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, Z(EGFR:1907), has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. Methods: The Affibody analogue, Ac-Cys-Z(EGFR:1907), was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-Z(EGFR:1907), Alexa680-Z(EGFR:1907), SR680-Z(EGFR:1907), and 800CW-Z(EGFR:1907). The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (K-D) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. Results: The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-Z(EGFR:1907) and Alexa680-Z(EGFR:1907) Possessed high binding affinity in low nanomolar range (43.6 +/- 8.4 and 28.3 +/- 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-Z(EGFR:1907) and Alex680-Z(EGFR:1907) displayed higher tumor-to-normal tissue ratios than those of the other two probes. Conclusion: This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-Z(EGFR:1907) and Cy5.5-Z(EGFR:1907) are excellent candidates as EGFR-targeted probes for optical imaging.