以Integrin為目標之標靶鐵蛋白應用於腫瘤分子影像與藥物載體之可行性研究

Abstract

在傳統治療癌症方面，藥物都是屬於傾瀉系統。因此近年來人們致力於發展藥物載體來應用在癌症治療。最廣為人知的藥物載體就是微脂體，但它也存在某些缺點。 在近幾年陸續有許多研究尋找新的藥物載體，而鐵蛋白就是其中之一。 鐵蛋白質是由24個單元所組成外直徑為12奈米而內直徑為8奈米的球狀蛋白。而鐵蛋白質表面能夠經由化學或是基因學的方式進行修飾，使得鐵蛋白質能夠帶有發光物質、抗體、小片段RNA以及短胜肽等等，另一方面在球狀的中空部分也能夠將藥物放置於內，因此作為一個新興的藥物載體是非常有潛力的。 近來有研究建構出帶有RGD胜肽的鐵蛋白質應用在分子影像以及癌症治療方面。RGD胜肽有高度專一性與癌細胞表面上大量表現的integrin αVβ3結合的能力，但RGD胜肽在臨床上也會與正常內皮細胞結合導致促進癌細胞生長及促進血管新生作用。 所以本實驗中我們將利用另一種胜肽DGEA，能夠與癌細胞表面上另一種高度表現的Integrin α2β1結合，進而製作出一種新的、不同的藥物載體，使得在分子影像檢測或是癌症治療上可以有更進一步的發展。除了帶有DGEA胜肽，也嘗試使得鐵蛋白質能夠帶有兩段重複的DGEA胜肽，提升結合Integrin α2β1的能力。透過螢光標定蛋白質來確定經過胜肽修飾的鐵蛋白質能夠與癌細胞表面上的Integrin α2β1結合後，進一步將化療藥物阿黴素包裹在內並且對癌細胞進行測試，發現透過帶有胜肽的鐵蛋白質包裹藥物能夠提升藥物的治療能力，且能夠使用更少的濃度來達到與化療藥物直接作用的相同效果。未來我們會更進一步進行動物實驗來驗證其發展的可能性。甚至透過鐵蛋白質的特性做出帶有多種標靶的藥物載體使得在應用分子影像上能夠更廣泛更全面，或者是包裹其他不同的藥物來達到治療不同癌症的目的。

In tradition, drugs for cancer treatment always belong to a dump release system. Furthermore, people are devoted to developing the new drug delivery for cancer therapy. Recently, liposomes, primarily composed of phospholipids, are the major drug delivery system. However, liposomes still have some disadvantages as follows. In recent years, there are many research for find new drug delivery, and the ferritin is the one of them. Ferritin is composed of 24 subunits and able to self-assemble to form a nanocage structure with external and internal diameters of 12 and 8 nm, respectively. Ferritin can be easily modified and conjugated with various molecules onto the surface, including dye, peptides, siRNA or antibodies etc. the cavity of ferritin can be loaded drugs. Recent studies demonstrated RGD-modified ferritin had potential in cancer diagnosis and therapy. The RGD peptide has high affinity to integrin αVβ3, a tumor angiogenesis biomarker, and is currently applied to clinical cancer imaging. However, the RGD peptide has disadvantages that the peptide also targets to endothelial cells, leading to stimulation of tumor growth and angiogenesis. Hence, in this study, we wanted to construct a new drug delivery and applied on molecular imaging and cancer therapy. We intended to set up the integrin-targeted ferritin with the DGEA (Asp-Gly-Glu-Ala) peptide which has high affinity to integrin α2β1. In the thesis, we not only made the construct of DGEA-ferritin but also tried the 2xDGEA-ferritin to assess the effect of increasing the copy number of DGEA on the surface of ferritins. Next, we estimated the tumor targeting ability of DGEA-ferritin and 2xDGEA-ferritin on U-87 MG, glioblastoma cell line, or PC-3, human prostate cancer cell line, by incubating cells with FITC-labeled protein. After confirming that proteins could recognize cancer cells through the DGEA peptide, we loaded the doxorubicin into 2xDGEA-ferritin. Through the MTT assay, we demonstrated that the 2xDGEA-ferritin could kill cancer cells and improved drug efficiency, which could use less amount of doxorubicin than free doxorubicin to achieve the same cellular toxicity effect. In the future, we will do in vivo experiments to confirm the potential of developing this drug delivery. If the system succeeds, it will be a new, safe and efficient technology and has great potential in clinical application. Furthermore, multiple peptides could be constructed on the ferritin or other drugs be loaded into the ferritin, and apply these modified-ferritins on molecular imaging and cancer therapy.