根據衛生署2007年統計資料顯示，口腔癌為台灣男性十大癌症死因排名第四位，且其盛行率有逐年攀升的趨勢，流行病學研究發現台灣地區口腔癌的成因與嚼食檳榔有顯著之關連性。經治療後之口腔癌的五年預後期一般而言不甚良好，存活率由29.8%到54.5%，尤其是晚期就醫的病患其預後更不理想。從過往研究中已知癌症之早期診斷有助於良好的臨床治療以及預後，現行的影像診斷技術，施行率雖然已經十分普及並且可以提供大部分的癌症診斷，然而對於非常初期的疾病情報資訊以及疾病影像仍有許多進步空間。分子影像診斷技術能夠在疾病的早期提供相關病灶的影像、顯示病灶的形成機制，對於臨床醫師或是基礎科學研究人員都是一個新興的影像領域。人類上皮細胞生長因子接受器(EGFR)以及其突變型(EGFR variant III)已知在許多腫瘤細胞過度表現，尤其是口腔癌細胞，並且嚴重影響到預後的效果，這些特殊的腫瘤細胞表面標記常被用來作為標定物以利疾病影像研究。經由M13嗜菌體表面展示技術篩選生產的擬人化單域抗體(single domain antibody, SdAb)可有效地獲得具有高表面標定性的癌症細胞，並且單域抗體具有良好的環境耐受性、組織滲透性以及低致免疫反應等等優點。尤其單域抗體最重要的優點就是其尺寸僅有一般IgG抗體的十分之一，而小尺寸的特性較不會顯著增加奈米粒子的流體動力學直徑。因此在本研究中，我們利用標定EGFR以及EGFR variant III的EG-2單域抗體接合磁性奈米粒子期許能夠作為癌症診斷的工具。首先，利用EG-2單域抗體篩選口腔癌細胞株以了解其專一性標定能力，此研究結果與利用市售anti-EGFR以及anti-EGFRvIII單株抗體所進行之西方墨點法結果一致，進而挑選出人類上皮細胞生長因子接受器表現量最高的KOSC3細胞株以及最低的人類正常口腔黏膜角化細胞進行後續的磁性奈米粒子接合實驗。由於EG-2單域抗體在羧端(C-terminus) 標記6-Histidine序列，為了使與磁性奈米粒子接合後的抗體仍具有方向性以及專一標定性，因此將與EG-2單域抗體接合之磁性奈米粒子在其表面修飾Ni-NTA結構，以利形成專一性Ni-NTA-¬¬Hexa Histidine自組裝。在此研究中，被選用的磁性奈米粒子有二:其一為負載釓(Gadolinium, Gd)的樹狀奈米粒子(dendrimer)，為可以提供磁振造影T1影像之對比劑; 另一為四氧化三鐵奈米粒子(Fe3O4)，為可以提供T2影像之對比劑。此兩種磁性奈米材料皆具有小尺寸的特性，直徑約為6到10奈米。測試兩者的細胞毒殺特性發現Gd-dendrimer的IC50為5x10-4M而Fe3O4為1x10-2M。此濃度亦能夠提供良好的磁振造影效果。進一步的研究顯示當EG-2 單域抗體接合上Gd-dendrimer，其鬆弛係數relaxivity1以及relaxivity2均低於尚未接合上EG-2單域抗體的Gd-dendrimer，但是兩者之鬆弛係數仍高於市售顯影劑Magnevist。此結果顯示不論是Gd-dendrimer或是EG-2單域抗體接合後的Gd-dendrimer，其顯影效果均較Magnevist優異。反觀之，研究顯示當EG-2單域抗體接合上Fe3O4，其鬆弛係數relaxivity1以及 relaxivity2較高於尚未接合上EG-2單域抗體的Fe3O4，顯示接合上單域抗體會提升Fe3O4的顯影效果。將接合上磁性奈米粒子Gd-dendrimer或是Fe3O4的EG-2單域抗體作用在KOSC3以及HNOK，且進行磁振造影分析。結果顯示此接合後的複合物仍然具有高度專一性標定且不論是在T1或是T2皆具有良好顯影的能力。在後續的研究中，我們將會針對奈米粒子與單域抗體之複合體在生物體內的標定造影進行深入的研究以利提升其作為臨床醫學應用上專一性標定對比劑的能力。

Oral squamous cell carcinoma (OSCC) is now ranked the forth-leading cause of cancer deaths in the Taiwanese male population owing to the prevalent betel quid chewing habit. Oral cancer has a general poor prognosis with a five-year survival rate ranging from 29.8%~54.5%, especially in late disease stages. Early diagnosis contributes to effective clinical treatment and better prognosis. Current imaging technology, however, usually failed to provide informative disease profile at the very early stage. Molecular imaging technology is an emerging field of research that has enabled insights of pathogenesis and has revealed true disease progress at their earliest possible stage. EGFR and EGFRvIII are selectively over expressed in many tumor cells including oral cancer with prognostic significance. Thus these unique surface markers are often selected as the targets for functional imaging of cancers. M13 phage display system engineered with humanized single domain antibody (SdAb) could effectively target unique cancer cell surface markers with high environmental tolerance and low immunogenic activity in vivo. The single domain antibody has a size only about one tenth of a normal IgG antibody and bioconjugation normally will not significantly increase the overall hydrodynamic size. Thus better tissue penetration could be achieved. The EG-2 SdAb targeting EGFR and EGFRvIII is selected to conjugate with nanoparticles in this study for cancer diagnostic imaging. We first screened EG-2 SdAb in our oral cancer cell line library for their endogenous expression of EGFR. The results were consistent with those from Western Blot using anti-EGFR and EGFRvIII antibodies. The positive and the negative clones were selected for in vitro experiments. For improved orientation control of EG-2, we proposed the idea of self-assembly on the particle surface. Ni-NTA was modified on the surface of magnetite particles through cross-linking between NTA and the nanoparticle surface. The poly-histidine at the C-terminal of the SdAb was known to interact with Ni-NTA specifically; thus, forming a self-assembled monolayer with high orientation control of the targeting moiety. Two types of nanoparticles with T1 (Gd3+ loaded dendrimer) and T2 (Fe3O4 nanoparticle) imaging contrast, respectively, were chosen for the following molecular imaging studies. Both have a small diameter of about 6-10 nm. The cytotoxicity of these materials revealed satisfactory results at concentrations below 5x10-4M and 1x10-2M for the dendrimer and the Fe3O4 nanoparticles, respectively. These concentration ranges were able to generate significant MRI contrasts. Conjugation of SdAb significantly increased r1 and r2 of the Fe3O4 nanoparticles, while decreased those of the dendrimers. EG-2 conjugated nanoparticles target EGFR over-expression cancer cells and presented a significant MRI contrast effect in vitro with positive contrast for the dendrimers and inversed contrast for the magnetite particles. Future in vivo molecular imaging is warranted to evaluate their potentials as nano-molecular imaging contrast agents for clinical applications.

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