利用對位疊氮基苯丙胺酸嵌入之攜鐵籠狀蛋白作為藥物載體開發之研究

Abstract

迄今，女性乳癌發生率仍是所有癌症之首，乳癌的高發病率對於世界各地的女性族群是潛在的威脅，因此乳癌治療是極為重要的研究課題。目前在乳癌治療的策略上，具有多重作用目標以及良好抗癌效率的聯合療法被廣泛運用於惡性乳癌的治療上。然而傳統聯合療法的主要問題是多種化療藥物引發的多重副作用和同時服用多種藥物的不便性，也大幅地降低了患者的生活品質。為了提高抗癌效率，降低聯合治療的脫靶效應，在此論文研究中我們致力於設計一種新穎的藥物輸送平台，該藥物載體可以攜帶多種化療藥物並且具有標靶乳癌細胞的選擇性。 攜鐵蛋白是一種中空蛋白籠，廣泛地存在於各種生物且具有一定的生物相容性和良好的溫度及酸鹼耐受性，是作為輸送蛋白質藥物和小分子藥物的理想載體。在此篇研究中，利用生物正交的pyrrolysyl-tRNA synthetase•tRNA配對將對疊氮基苯丙氨酸(p-azido-L-phenylalanine)嵌入攜鐵蛋白的F81和K143位點，且攜帶非典型胺基酸的攜鐵蛋白仍維持著籠狀結構。透過蛋白質譜分析及共振能量轉移分析(fluorescence resonance energy transfer)證明藉由鍵擊化學(click chemistry)能有效地將多重化療藥物或螢光分子裝載至攜鐵蛋白上。此外，攜鐵蛋白之末端與抗人類表皮生長因子受體2 (HER2)胜肽結合，透過共軛焦顯微成像分析，攜鐵蛋白能靶向HER2過表現之乳癌細胞並且藉由胞吞作用進入癌細胞。總體而言，非典氨基酸嵌入之攜鐵蛋白為具有多重載藥和細胞靶向功能的藥物運送載體。

Breast cancer occurs most frequently in women around the world. The high incidence rate of breast cancer is a potential threat to women, so it is a study emphasis in improving breast cancer therapy. So far, the combination therapy, with multiple targeting ability and improved anti-cancer efficiency, is widely used for treating malignant breast cancer. However, the main problems for conventional combination therapy are the numerous side effects of multiple chemotherapy drugs and inconvenience of taking multiple medications at the same time, which dramatically lower the life quality of patients. To elevate the efficiency and lower the off-target of combination therapy, in our research, we focus on designing a novel carrier-mediated drug delivery platform which can carry multiple chemotherapy drugs and target breast cancer cell selectively. Human ferritin, a hollow protein cage with potential biocompatibility and thermal/pH stability, is a suitable vehicle for peptide/protein drug and small molecule drug delivery. In this study, p-Azido-L-phenylalanine (pAzF) was incorporated into ferritin by evolved pyrrolysyl-tRNA synthetase•tRNA_CUA^Pyl pair at F81 and K143 positions with self-assembled capsid structure. Methodology in multi-drug loading through click chemistry is established and verified by protein mass spectrometry and fluorescence resonance energy transfer analysis. In HER2+ breast cancer cell specific targeting study, the anti-Her2/neu peptide fused ferritin (AHNP-ferritin) has been proved to entry cell through Her2 recognition/endocytosis in fluorescence imaging study. Overall, pAzF encoded AHNP-ferritin was proved can be displayed as a multi-drugs delivering platform with precise drug-loading and cell targeting function.