| 摘要: | 由於多數腫瘤細胞表面之胎盤型鹼性磷酸酶過量表達 (69±44 U/g)，因此被視為一個良好的引信訊號來促使藥物釋放。然而，市售的阿黴素微脂體並無任何引信釋放機制。因此我們設計一個智能響應的胜肽微脂體，以鹼性磷酸酶作為引信訊號，增加藥物微脂體在較高引信訊號環境 (例如腫瘤細胞) 釋放藥物。
我們以抗菌胜肽 Magainin 2 作為破膜多肽骨架，設計鹼性磷酸酶引信響應破膜胜肽，藉由將磷酸根修飾在酪胺酸上來遮蔽抗菌胜肽破膜活性，並可藉由去磷酸化恢復破膜活性，再將胜肽透過錨定脂質連接到微脂體上形成引信響應多肽微脂體。在實驗室先前數據中，一個磷酸根修飾之胜肽 (1pY19-Magainin 2 和 1pY8-Magainin 2) 無法有效遮蔽破膜活性，有顯著使藥物洩漏的現象，而兩個磷酸根多肽 (2pY8,19-Magainin 2) 則可較佳抑制抗菌胜肽破膜活性但仍有些許藥物背景洩漏現象。在此研究中，我們合成三個磷酸根修飾的胜肽 (3pY8,15,19-Magainin 2) 企圖進一步壓抑抗菌胜肽破膜活性阿黴素微脂體。
在胜肽微脂體控制藥物釋放結果中，我們證實鹼性磷酸酶能去除微脂體上胜肽的磷酸基團，使胜肽恢復破膜活性，進而破膜釋放藥物。在圓二色性光譜實驗中，沒有磷酸基團遮蔽破膜活性的胜肽 3Y8,15,19-Magainin 2 錨定到微脂體後，其二級結構為 β-sheet ，而 3pY8,15,19-Magainin 2 錨定到微脂體時，則以無序纏繞，這說明磷酸基團能有效遮蔽破膜胜肽與微脂體作用，而去磷酸化之後，多肽產生二級結構並進一步破壞微脂體的膜。另外，在冷凍電子顯微鏡實驗結果中，引信響應胜肽 3pY8,15,19-Magainin 2 在去磷酸化之前已經在微脂體膜表面上聚集，但此聚集無法造成藥物釋放；直到去磷酸化發生後，胜肽仍有聚集現象並使微脂體相互聚集，但微脂體內的阿黴素結晶已消失，表明藥物的釋放。
最後，我們在離子種類及濃度接近人體血漿的模擬體液 (沒有磷酸鹽) 中，以 HS-5 細胞 (鹼性磷酸酶表達極少) 和 KB 細胞 (鹼性磷酸酶過度表達) 進行細胞實驗的比較。由於 2pY8,19-Magainin 2 和 3pY8,15,19-Magainin 2 胜肽微脂體都可以透過細胞原生性的鹼性磷酸酶達到控制釋放。期許未來能作為具潛力的智能微脂體應用於臨床研究。
關鍵字 : 鹼性磷酸酶、微脂體、破膜活性胜肽、引信響應釋放
;Placental alkaline phosphatase was a suitable trigger signal for drug release since it was overexpressed on most tumor cells′ surfaces (69±44 U/g). However, commercially available DOX liposomes lack a triggered release mechanism. Hence, we designed alkaline phosphatase (ALP)-responsive peptidyl liposomes to increase drug release from liposomes in a higher triggering signal environment (such as tumor cells).
We design alkaline phosphatase trigger-responsive membrane lytic peptides based on antibacterial peptide Magainin 2. The membrane lytic activity of the antibacterial peptides was masked by modifying the phosphate group on tyrosine and peptide was conjugated on liposome surface. The phosphoryl group can be cleaved by ALP to restore the membrane lytic activity and rupture liposome membrane to cause content release. In previous results, peptides with one phosphoryl tyrosine group (1pY19-Magainin 2 and 1pY8-Magainin 2) could not effectively masked the membrane lytic activity, causing significant drug background leakage. In contrast, peptides modified with two phosphoryl groups (2pY8,19-Magainin 2) could have a better inhibition of the membrane lytic activity. Nevertheless, slight background releases of drugs still happened. Therefore, this study aimed to synthesize peptides with three phosphoryl tyrosine (3pY8,15,19-Magainin 2) to masked the membrane lytic activity further.
In the triggered release results, we confirmed that ALP can remove the phosphoryl group of peptides on the liposomes to rupture membrane and release drugs. In circular dichroism spectroscopy results, the secondary structure of unmasked 3Y8,15,19-Magainin 2 peptides was β-sheet, while the secondary structure of masked 3pY8,15,19-Magainin 2 peptides (with phosphoryl group) was random coil when peptides were conjugated with the liposome. It indicates that the phosphoryl group can effectively controlled the secondary structure to control the interaction between membrane lytic peptides and liposomes. In cryo-electron microscopy results, trigger-responsive peptides (3pY8,15,19-Magainin 2) were laterally preaggregated on the surface of the liposome membrane before dephosphorylation, but this aggregation won′t cause drug release. After dephosphorylation, the peptides remain laterally aggregated but liposomes contents were released. Moreover, there were inter-liposome aggregation connected at the peptide aggregating location.
In the cellular experiment, we tested liposomal content release caused by endogenous cellular ALP using HS-5 cells (less alkaline phosphatase expression) and KB cells (overexpressing alkaline phosphatase) in simulated body fluids. Both 2pY8,19-Magainin 2 and 3pY8,15,19-Magainin 2 peptidyl liposomes can be triggered by endogenous cellular ALP, showing the potentials for further possible medical applications in the future.
Keyword : Alkaline phosphatase, liposomes, membrane lytic peptides, trigger-responsive release |
