非小細胞肺癌之磁振造影標定探針設計:以氧化鐵粒子修飾EGFR抗體為例

Abstract

本研究為整合生醫分子及活體細胞追蹤之磁振造影技術，針對非小細胞肺癌之疾病模式作為主要之疾病研究標的。於此研究中利用對細胞表面特異性抗原上皮生長因子受體(EGFR)有單株抗體導向性之氧化鐵奈米磁振顯影劑為探針，偵測異種移植非小細胞肺癌小鼠模式之腫瘤。作為磁振造影之氧化鐵奈米粒子是使用化學共沉澱法製備之水溶性粒子，此一製備模式之磁奈米粒子可利於合成具標靶性之氧化鐵生醫探針。 本研究包括活體外及活體內實驗兩大部分。活體外細胞實驗以西方浸漬法、免疫螢光染色、流式細胞儀分析確定非小細胞肺癌細胞株CL1-0、CL1-5、A549上EGFR之表現量，並以普魯士藍染色及磁振造影試管分析鑑定具標靶性之氧化鐵奈米磁振顯影劑於活體外細胞株之標定效率。於生物體疾病模式之偵測實驗則以免疫組織染色確定活體腫瘤細胞之EGFR表現，並以磁振造影分子影像觀測具標靶性之氧化鐵奈米磁振顯影劑於活體中之生物分佈與標定。其後再以組織切片之普魯士藍染色驗證標靶效果。 於本研究中，體外實驗結果顯示非小細胞肺癌細胞株中A549及CL1-5細胞具有EGFR高表現量，而CL1-0的EGFR表現量則較低，期間的差異約為40倍。同時也證實具EGFR單株抗體之氧化鐵奈米磁振顯影劑之專一性與導向性。於磁振造影之T2WI影像中，腫瘤區域訊號強度的變動於注射標靶性氧化鐵顯影劑後六小時達到最大下降點，其下降的程度為10 % ；T2*WI影像中，腫瘤區域訊號強度的變動於注射標靶性氧化鐵顯影劑後三到六小時間達到最大下降點，較施打顯影劑前下降17 %。於組織切片之普魯士藍染色驗證也發現於腫瘤部位切片染色中，施打具有標靶性氧化鐵之小鼠腫瘤切片染色結果，可以發現為數較多之氧化鐵奈米粒子，相對於控制組中施打無標靶氧化鐵奈米顯影劑則無明顯的標定結果，因此更突顯標靶性氧化鐵磁振顯影劑之導向性存在與實際效應。 經由本研究，建立起一套完整的活體外到活體內的實驗流程，並成功應用於非小細胞肺癌偵測上。未來也將建立轉移及原發型之非小細胞肺癌動物模式，以及發展診斷與治療雙功能之標靶性奈米氧化鐵顯影劑。

Molecular imaging is the technology that successfully combined molecular biology and clinical medicine, and it will be one of potential fields of medicine in the future. In this study, we successfully combined the biomolecular imaging and cellular specific imaging techniques to detect xenograft non-small cell lung cancer (NSCLC) murine model. And we used the aqueous Fe3O4-NH3+ nanoparticles conjugated with anti-epidermal growth factor receptor (EGFR) antibody as the biomedical probe to detect the NSCLC and animal disease model of lung cancer. Herein, the experiments of this study included in vitro and in vivo assay. In in vitro studies, we used western blotting, immunofluorescence, and flowcytometry to quantify EGFR expression of NSCLC cell lines, such as CL1-0, CL1-5, and A549. Then, the Prussian blue staining and MR in vitro assay would be utilized to confirm the targeting efficiency of iron oxide biomedical probe. As the results, we found that the cell lines of CL1-5 and A549 exhibited more expression level of EGFR than CL1-0 about forty times. Based on the over expression level of EGFR of cell, the Fe3O4-NH3+ nanoparticles conjugated with EGFR antibody could play as specific biomedical probe in NSCLC detection. Furthermore, we used immunohistochemistry and Prussian’s iron staining to ensure EGFR expression and the targeting efficiency of nanocontrast agent in tumor region and tissue section of animal model, respectively. And the molecular imaging of the biodistribution and targeting efficiency of iron oxide probe were observed under the 3T MR system. In vivo assay showed when mice were administrated with Fe3O4- anti-EGFR, the signal intensity of tumor region would be decreased by the time, and reached to 10 % at 6h in MR T2- weighted imaging. On the other hand, the MR T2*-weighted imaging also exhibited the variety of signal intensity of tumor region and it would be decreased about 17% as the mouse was administrated with the same dosage and detection time. Furthermore, we provide the Prussian’s iron staining of tumor histology to show the targeting of Fe3O4- anti-EGFR nanocontrast agent. Herein, the mice were injected with 10 mg/Kg of Fe3O4- anti-EGFR biomedical probe and Fe3O4 nanoparticles, respectively. And the Prussian’s iron staining of tumor histology showed that more iron oxide targeting on tumor region when the Fe3O4 nanoparticles conjugated with anti-EGFR antibody. As the results of Prussian’s iron staining, the Fe3O4-EGFR nanocontrast agent could be as the biomedical probe to detect NSCLC. Herein, we provided the platform and procedures of Fe3O4-anti-EGFR nanocontrast agen for in vitro and in vivo targeting, and we also used this strategy to detect NSCLC successfully. In the future, we will built up metastatic and primary murine animal model of NSCLC and develop novel Fe3O4-EGFR nanoparticles for dual-purposes for molecular diagnosis and therapy evaluation.