人工合成胜肽ANGINEX 是一段由33個胺基酸組成β-sheet 構型短片段胜肽，在細胞實驗中抑制血管內皮細胞(vascular endothelial cell)生長並且藉由辨識腫瘤周邊血管細胞上的galectin-1 達到腫瘤生長的抑制。Galectin-1 大量表現在口腔癌的侵犯前緣癌細胞，以及口腔癌周邊的血管內皮細胞。在本次實驗中，我們希望合成連結ANGINEX 的奈米粒子，目的標靶癌細胞同時具有腫瘤顯影以及癌症治療效果。首先建構ANGINEX DNA序列，送入pET28a 載體表達ANGIEX 重組蛋白。我們觀察到了ANGINEX 如同少數一些其他抗血管新生的蛋白，帶有著抗細菌生長的活性，使得即便可以觀察到ANGINEX 在菌液當中的存在，而其濃度卻無法支持接下來的實驗分析，因此我們進一步透過胜肽合成儀來取得ANGINEX。我們將合成的ANGINEX 上的六組胺酸(histidine)經由自組裝方式結合至奈米鐵上的Ni-NTA，以產生連結ANGINEX 的奈米粒子(ANG-Fe@)，我們分析ANGINEX 和ANG-Fe@ 在血管內皮細胞生物活性上的影響，測定奈米氧化鐵吸收ANGINEX 的鍵結比例，同時將ANGINEX 接上奈米氧化鐵後，測試對於HUVEC 細胞在細胞實驗當中是否具有其標靶效果。
另一方面，經由致癌藥物4-NQO餵食B6小鼠， 經由初代培養得到的鼠類舌癌細胞，命名為W1ms(Wild type 1 mouse squamous cell) ，使用皮下注射到背部以及舌內注射打入到SCID mice 跟B6 mice ，希望能得到小鼠口腔癌動物模式，同時測定了這株細胞的基因型態以及是否具有癌細胞的特質，像是具有侵襲能力以及快速的生長速度。雖然最後無法由這樣的動物模式產生腫瘤，我們改用了人類口腔癌細胞OC2產生的腫瘤，在小鼠上驗證ANG-Fe@ 在腫瘤的標靶顯影以及抑制生長的效果。
小鼠的動物實驗發現，ANG-Fe@ 在帶有著OC2腫瘤的SCID 小鼠身上，在肝臟以及腎臟可以觀察到負顯影的產生，而在腫瘤的地方沒有能夠觀察到負顯影，可能是由於在肝臟以及腎臟的部分殘留了過多的ANG-Fe@ ，導致腫瘤部位的ANG-Fe@ 濃度降低，而ANGINEX 本身的標靶物為血管內皮細胞，在一些非腫瘤位置的血管內皮細胞表達Galectin-1 的同時，很可能也會帶著ANG-Fe@ 前往該位置而非腫瘤。如果能夠提升標靶蛋白對於腫瘤的辨識力，或是同時結合多種蛋白連結於奈米粒子上提高對於腫瘤細胞外圍帶有的受器的辨識能力，減低奈米粒子被代謝器官以及免疫系統吸收的比例。這樣一來奈米粒子結合標把蛋白對於腫瘤位置的顯影效果，未來可能成為一個高準確度的新穎顯影方式。

ANGINEX is an artificial beta-sheet peptide composed of 33 amino acids. It inhibits vascular endothelial cell growth in vitro and tumor growth in vivo by targeting galectin-1 on the tumor associate vascular endothelial cells. Galectin-1 is overexpressed at the invasive front of oral carcinoma and oral cancer-associated vascular endothelial cells. In this study, we wanted to synthesize ANGINEX-conjugated nanoparticles to develop tumor-targeted nanoparticles for oral cancer imaging and therapy. First, we constructed ANGINEX DNA sequence into a pET-28a plasmid for recombinant ANGINEX protein production. We observed that like other anti-angiogenesis peptides, ANGINEX also carries the activity of bacteria proliferation inhibition. Even though we can see the ANGINEX appeared at the bacteria lysate, the ANGINEX concentration is not sufficient to support the following experiments. Therefore, we further used the synthetic ANGINEX for the following experiments. Next, ANGINEX-conjugated nanoparticles (ANG-Fe@) have been synthesized through self-assembly of hexahistidine tag on recombinant ANGINEX to nickel nitrilotriacetate (Ni-NTA) coated Fe3O4 nanoparticles. On the other hand, we have isolated a murine oral cancer cell line, named W1ms, from 4-NQO carcinogen feeding B6 mice. We have subcutaneously injected this cell line into the posterior flank and the tongue of SCID and B6 mice trying to induce experimental mice oral cancer models. After two months of implantation, we found W1ms cells failed to form tumor in both murine systems. Hence, we evaluated the tumor targeting and inhibiting abilities of ANG-Fe@ by using human oral cancer OC2 tumor mice model.
On the tumor bearing SCID mice, the ANG-Fe@ could induce a strong negative signal on liver and kidney on the MRI scanning. We do not found a strong negative signal in the tumor position. It might be due to the over absorption from the liver and kidney, resulting a low concentration of ANG-Fe@ in the tumor site. ANGINEX targets the endothelial cell, some of the endothelial cell over expressing Galectin-1 might also being targeted by the ANG-Fe@ thus reduce the negative signal of the tumor. To increase the tumor targeting ability of the nano-particle conjugated protein or try conjugate multiple peptide to increase the combination of the tumor receptors through the different peptides, also keep lower the absorption of the liver, kidney and the immune system. The peptide conjugated nanoparticle in the MRI imaging developing system could be a high precision way in the future medical bio-imaging technique.

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