| 摘要: | 本實驗室先前於裸鼠以口服方式投予質體DNA混合胜肽奈米管(peptide nanotube, PNTs)，結果發現質體DNA能於裸鼠體內進行表現。然而，因先前製程在強酸進行，本實驗室希望能夠找到一個簡易且安全的製程，對胜肽奈米管進行優化。本實驗使用乙醇水溶液，利用各種顯微鏡及動態光散射法，評估胜肽奈米管的大小，以及質體DNA與胜肽奈米管的結合狀態；再利用DNase I評估質體DNA結合至胜肽奈米管上後之安定性。實驗結果顯示，胜肽在濃度高於0.06 mg/mL時，會自發性地聚集形成胜肽奈米管，由螢光顯微鏡、掃描式顯微鏡、原子力顯微鏡確認奈米管形成；再由原子力顯微鏡的觀察發現有質體DNA吸附於胜肽奈米管表面，而螢光光譜的實驗結果得知，質體DNA會與胜肽奈米管上的酪胺酸(Tyr)產生交互作用，質體DNA與胜肽奈米管的結合常數經計算為5.42×1010 M-1；於DNase存在的環境下，DNase會被胜肽奈米管保護長達40分鐘。此外我們以coelenterazine受質來定量luciferase酵素活性，實驗結果發現，於口服投予pCMV-hRluc混合胜肽奈米管之後的第48小時，luciferase的酵素分別於胃、十二指腸、肝臟、腎臟、肺臟、腦顯著性增加達623%、173%、306%、556%、778%、217%，投予用Thioflavin T預先染色地胜肽奈米管的方式，追蹤確實有胜肽奈米管的存在，免疫螢光染色的結果觀察到水母冷光酵素會存在胃的胃底腺、胃小凹、壁細胞、主細胞；十二指腸絨毛的絨毛表皮、固有層、腺窩；肝臟小葉的肝細胞、竇狀內皮細胞；腎臟皮質層的近曲小管；肺臟的第二型肺泡上皮細胞；腦部灰質區。此結果顯示改變胜肽奈米管的製程會改變口服基因載體的全身性分佈。
In the previous study, the feasibility of peptide nanotubes (PNTs) as oral gene delivery carrier has been confirmed. However, because such a process of making peptide nanotube contained a strong acidic solvent, we want to find a process which could optimize peptide nanotube. In this experiments, we used ethanol-water co-solvent to make peptide nanotube, we performed fluorescence microscope, scanning electron microscope, atomic force microscope, and dynamic light scattering to evaluate the size of PNT. And we performed atomic force microscope and fluorescence spectrum assay to evaluate whether DNA associated with PNTs. In addition, we estimated the stability of PNT-associated DNA with DNase I. The results showed that the PNTs self-associated at concentration above 0.06 mg/mL. The small PNTs was found by fluorescence microscope, scanning electron microscope, atomic force microscope. Plasmid DNA was found associated with PNTs by atomic force microscope. Plasmid DNA associated with tyrosine of PNTs with a binding constant of 5.42×1010 M-1 calculated by fluorescence quenching assay. PNTs were able to protect DNA from DNase I for 40 min. The Renilla luciferase activity in tissues was quantitatively assessed using coelenterazine, and was significantly increased by 623% in stomach, 173% in duodenum, 306% in liver, 556% in kidney, 778% in lung, 217% in brain, respectively, at 48 h after the first dose of oral delivery of pCMV-hRluc/PNTs formulation. The organs with luciferase activity were confirmed the presence of PNTs with intracellular tracing the thioflavin T pre-stained PNTs. The immunostaining of tissue revealed the Renilla luciferase was also exsited in gastric pits, fundus glands, parietal cells and chief cells of stomach, in villous epithelium, lamina propria and crypt of villi of duodenum, in hepatocytes, sinusoidal endothelial cells of liver lobules, in proximal tubular of kidney, in type II pneumocytes of lung, and in grey matter of brain. These results implicate that using a different process to make peptide nanotubes would change the biodistribution of oral gene delivery. |
