磁振造影技術於診斷及藥物遞送之應用:以氧化鐵奈米粒子應用於非小細胞肺癌為例

Abstract

本研究建立於生醫分子影像之基礎上，發展具磁振造影顯影功能並攜帶治療藥物之雙功能顯影劑，以磁振造影之影像達成同時診斷及評估療效之目的。非小細胞肺癌為本研究之疾病模式，將粒徑小之氧化鐵奈米粒子表面鍵結非小細胞肺癌之標靶性藥物─gefitinib，利用腫瘤新生血管豐富及內皮細胞排列鬆散之特性使氧化鐵奈米粒子與藥物累積於腫瘤，對腫瘤進行標定及藥物治療。 Gefitinib作用機制乃上皮生長因子受體(EGFR)之酪胺酸酶(tyrosine kinase)抑制劑，阻斷EGFR活化後的訊息傳遞，進而控制腫瘤生長。近年來研究顯示，Gefitinib對於帶有EGFR突變之非小細胞肺癌細胞有特異性療效，東方人對其之治療反應更優於西方人，故選擇其做為研究之藥物。 在實驗設計方面，包括氧化鐵奈米粒子、修飾之藥物及兩者鍵結後三大部分。氧化鐵奈米粒子以活體外細胞株測試細胞毒性及顯影效果，以小鼠驗證活體顯影效果並持續追蹤氧化鐵奈米粒子被排出體外之時程；藥物部分，以核磁共振(NMR)確認經過修飾可與奈米粒子接合之藥物結構，並以活體外細胞株測試藥物抑制細胞生長之特異性；將氧化鐵奈米粒子與修飾藥物接合後，於活體外細胞株測試藥效、顯影效果、細胞內吞情形及細胞死亡機制，於活體異種移植非小細胞肺癌小鼠模型進行磁振造影分子影像觀測，再輔以組織切片之普魯士藍染色驗證標靶效果。 本研究中驗證合成修飾之gefitinib與結合至氧化鐵奈米粒子表面之gefitinib均可有效抑制具EGFR突變之非小細胞肺癌細胞株PC9的生長，亦藉由Annexin V螢光染色確認結合modified gefitinib之氧化鐵奈米粒子引發PC9細胞株大量邁向自我凋亡。相較於不具突變之wild-type EGFR非小細胞肺癌細胞株A549則無此效果，證明藥物之特異性及治療效果。在磁振造影活體腫瘤小鼠影像部分，注射結合modified gefitinib之氧化鐵奈米粒子，T2權重影像於腫瘤區域在4~8小時後達15%訊號改變，而體內其他組織如肌肉僅有5%的訊號變化，於腫瘤部位組織切片之普魯士藍染色也可以發現大量氧化鐵奈米粒子，從影像及組織切片均證實氧化鐵奈米粒子與藥物可經血流循環標靶運送並累積於腫瘤。 本研究合成出具顯影及攜帶藥物之雙功能顯影劑，藉由氧化鐵奈米粒子將藥物攜帶至腫瘤區域。未來將繼續朝向活體動物之治療效果評估、影像定量藥物局部濃度等方向努力，亦期望可結合不同疾病之藥物，移植於其他疾病模式之診斷及治療評估。

Molecular imaging is the technology that combined molecular biology and clinical medicine in the biomedical field. The goal of this study is to develop a bi-functional contrast agent used in magnetic resonance image (MRI) to achieve the simultaneous imaging and therapy on xenograft non-small cell lung cancer (NSCLC) murine model. The gefitinib is the targeting drug which is specifically efficient to NSCLC patient with EGFR mutation, it functionalized to inhibit the tyrosine kinase of EGFR for blocking the signal transduction pathway. The small molecules of modified gefitinib were conjugated on the surface of aqueous Fe3O4 nanoparticles, and it delivered through the blood system to the tumor site since the abundant blood vessels and leaky epithelium of tumors. To incorporate the gefitinib and Fe3O4 nanoparticles, we substituted the weak portion of inhibitors with carbon chain, here we called it as “modified gefitinib”. Further, the modified gefitinib and Fe3O4 nanoparticles were incorporated by N-(3-Dimethylamino propyl) -N’-ethylcarbodiimide hydrochloride (EDC). The in vitro assays showed both of the modified gefitinib and Fe3O4@gefitinib efficiently inhibited the cell viability, and cell apoptosis were dramatically induced by Fe3O4@gefitinib in EGFR mutant PC9 cells, whereas the wild-type EGFR A549 cells were not. In vitro assays demonstrated the Fe3O4@gefitinib with specificity of cell inhibitory to PC9 cells. In vivo assay showed the tumor was negative enhanced and signal intensity dropped 15% in T2-weighted imaging after Fe3O4@gefitinib administration 4~8 hours, while the muscle tissue presented less than 5% change. Furthermore, we provided the Prussian’s iron staining of tumor histology to verify the Fe3O4@gefitinib accumulated at tumor site substantially than other organs. As the result, it demonstrated that the Fe3O4@gefitinib could target to tumor through the blood circulation system. We also performed some preliminary study to verify the feasibility of MR technique applied in treatment response evaluation for PC9 animal model. Herein, we produced the bi-functional nanocontrast agent Fe3O4@gefitinib to achieve imaging and therapy simultaneous. In the future, except the improvement of chemical synthesis as well as we would work towards the treatment efficiency evaluation, quantification and prognosis for NSCLC.