開發以鹼性去磷酸酶為引信之多肽微脂體

Abstract

癌症又名惡性腫瘤(malignant tumor)，是指不正常的細胞增生，進而侵略其他細胞的現象，目前位居台灣十大死因第一名。早期臨床上治療癌症的其中一種方法為投放藥物治療，直接將藥物注入患者體內，由於抗癌藥物毒性較強，且生物分布選擇性差，導致患者全身細胞都會受到傷害。近期研發出來的微脂體藥物在體內運送中正常細胞不會直接接觸到藥物，因此大幅地降低了細胞毒性，但其釋放緩慢的特性也限制了藥效。所以，選擇性將治療藥物瞬間大量暴露在癌細胞周圍以提高藥物被利用的效果為治療的重要課題。 引信響應釋放在奈米藥物中是一項重要的技術，可以利用在癌細胞中大量表達的酵素作為引信訊號。本實驗為開發以鹼性去磷酸酶為訊號的引信響應多肽微脂體藥物。根據實驗室已經建立起的多肽微脂體系統加以改良。多肽微脂體在我們實驗室已經被廣泛用來做為微脂體引信釋放的產物。我們首先合成磷酸化的多肽鍵結到微脂體表面，產生穩定的微脂體等待訊號。透過鹼性去磷酸酶，將修飾於微脂體上的多肽去磷酸化，產生不穩定的多肽微脂體，並造成微脂體內藥物的釋放。 我們首先以滴定共價成鍵的方式找出最適當的多肽在微脂體上的鍵結比例，多肽數量足夠讓微脂體有顯著釋放，卻可以在等待訊號時又非常穩定。動態光散射分析儀可以用來分析去磷酸化前後的多肽微脂體之粒徑與界達電位之改變。試管實驗證明我們開發的引信響應多肽微脂體能順利於信號出現後釋放藥物。圓二色光譜可以看出來在微脂體表面的磷酸化多肽二級結構趨近於一級結構，但在去磷酸化之後二級結構改變成雙性螺旋。冷凍電子顯微鏡可看出多肽在去磷酸化之前就已經在膜表面上先行聚集，但此聚集無法破膜，直到去磷酸化發生後，微脂體內容物才會消失。除此之外，藉由高通量顯微鏡與流式細胞儀細胞實驗也可以觀察，與沒有多肽的微脂體以及沒有去磷酸化的多肽微脂體比較起來，細胞能從去磷酸化的多肽微脂體獲得較多的藥物，在酵素夠多的環境下也能造成細胞凋亡，展現出此系統能在活體應用及轉譯醫學的可能性。Cancer, also called malignant tumor, is a group of diseases involving abnormal cell growth with the potential to invade to other parts of the body and it is the first of the top ten causes of deaths in Taiwan. Traditional intravenous injection of free drug has systemic toxicity due to unfavored biodistribution. Liposomal drug can lowered the systemic toxicity and prolong the circulatory time. However, the slow release also limits its drug efficacy. Hence, design trigger release liposome that can rapidly unload liposomal content around tumor cell is an important topic. Triggered release is one of the key technologies for nanomedicine. Many enzymes are specifically over-expressed in diseased tissues and therefore can be used as a useful trigger. The purpose of this study is to develop a triggered release system that response to phosphatase activity. We first conducted the covalent titration experiment to find the best peptide-to-lipid ratio. Dynamic light scattering (DLS) was used to study the size and zeta potentials of the peptidyl liposomes before and after dephosphorylation. In vitro experiment shows the peptidyl liposome can release significant amount content after triggered. Circular dichroism (CD) spectrum shows that the primary structure of the peptide on liposome surface is random coil and turns into amphiphilic helix after dephosphorylation. We also visualized our peptidyl liposome by cryo-EM. Imaging evidences shows that peptide has aggregated upon peptide conjugating to the liposome surface (before dephosphorylation) but the liposomal content release only occurs after dephosphorylation. In the cell experiments, our peptidyl liposome released the drug when incubated with phosphatase-rich KB cells. We have successfully demonstrated that our peptidyl liposome is responsive to dephosphorylation and has the potential to be used as a novel therapeutic tool.