奈米粒子由於尺寸效應、量子效應及高表面/體積比，近年來被廣泛地應用於藥物攜帶載體及在活體分子影像等等。而隨著病毒相關的研究愈漸深入，科學家也利用病毒進行遺傳工程、基因治療以及快速篩選並量產具標定功能的專一性結合子等。在此研究中，我們欲結合奈米粒子及噬菌體，以製造具有超順磁特性的噬菌體系統並探討其臨床診斷及治療之應用。首先，我們利用具有超順磁性的氧化鐵奈米粒子，表面被覆幾丁聚醣並攜帶抗癌藥物epirubicin。在核磁共振造影系統中，不論是在口腔癌細胞株SCC15之體外或是動物活體都可偵測到氧化鐵奈米粒子所造成T2及T2*訊號加成影像擷取模式下之負顯影效果；而抗癌藥物對癌細胞的毒殺性也不受到奈米粒子連結影響。然而，如何在生物體內增加磁場區域性導引作用外，又使奈米粒子對局部疾病細胞組織具有專一性標的將是活體應用成功關鍵之一。為了使噬菌體呈現技術的快速篩選及專一性能與超順磁氧化鐵奈米粒子做分子方位正確之自組裝結合，利用葉晨聖教授團隊合成之表面修飾Ni-NTA的氧化鐵奈米粒子作為研究模型。因修飾Ni-NTA的氧化鐵奈米粒子對與含六個組氨酸的胜肽具有高度的親附能力，因此若將此胜肽表現在M13噬菌體第七個基因所轉譯的外鞘蛋白質上，將能與修飾Ni-NTA的奈米粒子結合以成為具磁性的噬菌體。為此，我們成功地利用寡核定位突變的技術在第七個基因之5’-端（朝菌體外端）插入XhoI的限制酶切點，此插入序列將有利於未來接合能與奈米粒子形成專一連結之胜肽的DNA序列。本研究利用pET-20b(+)載體上具有的hexahistidine及pelB 領導序列，並利用定點定位突變在插入片段的兩端設計XhoI限制酶切點，進行接合反應以產生重組M13噬菌體。而藉由重組的M13噬菌體結合磁性奈米粒子除可增加在細胞及動物體上標的專一性外，也可藉由磁性而簡短純化專一噬菌體的時間等應用。然而從實驗結果顯示，我們並沒有得到重組的M13噬菌體；推測可能與插入片段太長導致噬菌體無法直接自行組裝，或修飾的新插入胜肽干擾病毒之生命週期，而其真正原因仍有待探討。同時推論利用噬質體的系統可改善此問題。未來，我們將更進一步生產具有磁性的噬菌體並應用在生物醫學上。

Nanoparticles have recently been widely investigated for biomedical applications as drug carriers and molecular imaging purposes due to their size effect, quantum effect and surface chemistry. Viruses have been engineered for gene therapy and rapid selection and amplification of functional ligands. In this study, we aimed to combine the two platforms for a novel magnetic phage system and to explore their potential applications in clinical diagnosis and therapy. We first synthesized superparamagnetic nanoparticles with chitosan surface modification to carry anti-cancer drug epirubicin. In MRI, the nanoparticles showed significant inversed contrast effect in the oral cancer cell line SCC15 model and in rat animal model under T2 and T2* weighed imaging. The drug carrier with MRI constract effect also successfully demonstrated anti-cancer efficacy in vitro, which enabled tracking delivery in future application. However, for in vivo application, a local targeting moiety is required to improve the specificity of the therapy in addition to the magnetic force regional guidance. The phage display system offered rapid selection, amplification and optimization of targeting ligands with specificity. To assemble phages with correct orientation on the nanoparticles, we synthesized the iron oxide nanoparticles which are modified with Ni-NTA and conjugated this onto the nanoparticles with superparamagnetic properties. The Ni-NTA complex is known to exhibit high affinity and specificity to a consecutive hexahistidine peptide domain. To artificially create this peptide tag onto a phage particle, gVII of M13 was modified by insertion of a restriction site through oligonucleotide-directed mutagenesis. The insertion sequence which contains hexahistidine and pelB leader sequence came from a pET-20b(+) vector. The insertion fragment was further modified by site-directed mutagenesis to create the same XhoI restriction sites in the two terminal ends. T4 DNA ligase was used to ligate the modified M13 vector and insertion fragment to create recombinant M13 phages. The desired phages were expected to exhibit high affinity with Ni-NTA nanoparticles. However the desired recombined M13 phages were not achieved through this strategy. It is speculatated that size of the insertion sequence maybe too long for the viral packing or the modified pVII may significantly affect the life cycle of M13 phages. It is suggested that phagemid system may provide a solution for the insertion of larger external peptides. We will continue research on this and evaluate functional applications of desired magnetic phages in medicine.

張立德、牟季美 奈米材料和奈米結構。滄海出版
張揚狀 (2005)表面被覆幾丁聚醣之多功能磁性奈米載體的製備與應用。國立成功大學化學工程研究所博士論文。
楊文仁、黃玫琪、曹雯珊、宣大衛 (2003)噬菌體呈現技術及其在生物科技上之應用。生物科學 第四十六卷 第一期 12 - 26 頁
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