口服奈米載體之基因傳遞研究

Oral Gene Delivery with Nano-Carrier

謝緯賢(Wei-Hsien Hsieh)

指導教授 ： 廖嘉鴻

臺北醫學大學/藥學院/藥學系(博士班)/博士(2012年)

摘要

本研究，以口服投予方式，每3個小時投予一個劑量，每個劑量為40 μg的DNA混合胜肽奈米管(PNTs)，連續投藥兩天至裸鼠體內，評估胜肽奈米管應用於口服基因載體的可行性。分別利用（1）掃描式電子顯微鏡（2）原子力顯微鏡（3）螢光顯微鏡（4）螢光光譜分析評估DNA與胜肽奈米管結合；DNA結合至胜肽奈米管上後，（5）DNA於DNase、人工胃液、或膽酸存在環境下之安定性；以及（6）DNA於離體十二指腸之穿透。實驗結果顯示，胜肽的濃度高於0.01 mg/ml時，會自發性地聚集形成胜肽奈米管；由掃描式電子顯微鏡、原子力顯微鏡、螢光顯微鏡的觀察發現有DNA吸附於胜肽奈米管表面，螢光光譜的實驗結果得知，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3.2×108 M-1；於DNase、人工胃液、或膽酸存在的環境之下，DNA會分別被胜肽奈米管保護長達50、60以及180分鐘；於離體十二指腸穿透實驗中，據計算擬穿透係數，發現DNA的擬穿透係數為49.2±21.6×10-10 cm/sec，而當DNA混合胜肽奈米管（P/PNTs）後，DNA的擬穿透係數顯著性地提升至395.6±142.2×10-10 cm/sec，而此擬穿透係數亦會因溫度降低、加入sodium azide、或者反向穿透而降低，推測進入十二指腸是須要能量參與。此外我們以chlorophenol red-β-D-galactopyranoside（CPRG）受質來定量β-galatosidase（β-Gal）酵素活性，實驗結果發現，於口服投予pCMV-Lac Z混合胜肽奈米管之後的第48小時，僅腎臟的β-Gal酵素活性會顯著性地增加41%，而於口服投予之後的第72小時，β-Gal酵素活性分別於胃、十二指腸、及肝臟會顯著性地增加達49%、63%、以及46%。而這些有β-Gal酵素活性增加的器官，同時利用南方墨點法的實驗證明有完整的DNA存在、以及投予已標記TM-rhodamine的pCMV-Lac Z、用thioflavin T預先染色的胜肽奈米管的方式，追蹤確實分別有pCMV-Lac Z以及胜肽奈米管存在，而且另外以反轉錄及時定量聚合酶鏈鎖反應（RT-qPCR）證明在這些器官內有Lac Z的mRNA存在。經由切片及X-Gal染色觀察，發現β-Gal酵素分佈在胃的粘膜表皮、胃小凹、胃底腺、壁細胞、主細胞；十二指腸絨毛的絨毛表皮、固有層、陷窩細胞；肝臟小葉的肝細胞、竇狀內皮細胞；腎臟皮質層近曲腎小管的內皮細胞、腎絲球的內皮細胞。除此之外，也用另一種可以表現出水母冷光酵素（Renilla luciferase）的質體DNA（pCMV-hRluc），進一步確認pCMV-hRluc混合胜肽奈米管以口服投予至動物體內後，確實有hRluc的mRNA存在於胃、十二指腸、肝臟、以及腎臟，而且以活體外（ex vivo）生物冷光照影，證實有水母冷光酵素存在於這些器官。而冷光酵素活性定量的結果發現，於口服投予pCMV-hRluc混合胜肽奈米管之後的第48小時，水母冷光酵素活性於十二指腸會顯著性地增加59%、於腎臟增加40%，而於口服投予之後的第72小時，水母冷光酵素活性分別於胃、十二指腸、及肝臟會顯著性地增加達53%、68%、以及43%。免疫螢光染色的結果亦觀察到水母冷光酵素同樣地存在於胃的粘膜表皮、胃小凹、胃底腺、壁細胞、主細胞；十二指腸絨毛的絨毛表皮、固有層、陷窩細胞；肝臟小葉的肝細胞、竇狀內皮細胞；腎臟皮質層近曲腎小管的內皮細胞、腎絲球的內皮細胞。這些結果顯示此胜肽奈米管可以當成口服的基因遞送載體，攜帶DNA至胃、十二指腸、肝臟、以及腎臟。

關鍵字

胜肽奈米管、基因傳遞、口服

並列摘要

The feasibility of peptide nanotubes (PNTs) as oral gene delivery carrier was investigated in nude mice with eight 40 μg doses of pCMV-Lac Z in 2 days at 3 h intervals. We performed scanning electron microscope, atomic force microscope, fluorescence microscope, and fluorescence spectrum assay to evaluate whether DNA associated with PNTs. In addition, we estimated the stability of PNTs-associated DNA with DNase I, simulated gastric acid, and bile treatment. Furthermore, in vitro permeability of DNA was also estimated. The results showed that the PNTs self-associated at concentration above 0.01 mg/ml. Plasmid DNA was found associated with PNTs by scanning electron microscope, atomic force microscope, and fluorescence microscope observation. Plasmid DNA associated with tyrosine of PNTs with a binding constant of 3.2×108 M-1 calculated by fluorescence quenching assay. PNTs were able to protect DNA from DNase I, acid, and bile digestion for 50, 60, and 180 min, respectively. The in vitro duodenal apparent permeability coefficient of pCMV-Lac Z was increased from 49.2±21.6×10-10 cm/sec of naked DNA to 395.6±142.2×10-10 cm/sec of pCMV-Lac Z/PNTs formulation. The permeation of pCMV-Lac Z formulated with PNTs was found decreased in 4℃, sodium azide condition, or reverse permeated direction indicated the involvement of energy-dependent process. Furthermore, β-galatosidase (β-Gal) activity in tissues was quantitatively assessed using chlorophenol red-β-D-galactopyranoside (CPRG), and was significantly increased by 41% in kidney at 48 h and by 49, 63, 46% in stomach, duodenum, and liver, respectively, at 72 h after the first dose of oral delivery of pCMV-Lac Z/PNTs formulation. The organs with β-Gal activity were confirmed for the presence of pCMV-Lac Z DNA with Southern blotting analysis and intracellular tracing the TM-rhodamine-labeled DNA, the presence of PNTs with intracellular tracing the thioflavin T pre-stained PNTs, and the presence of mRNA by reverse transcription-real time quantitative PCR (RT-qPCR). The observation of tissue section stained with X-gal solution found β–Gal was existed in mucosa surface epithelium, gastric pits, fundus glands, parietal cells, and chief cells of stomach, in villous epithelium, lamina propria, and crypt cells of duodenum, in hepatocytes and sinusoidal endothelial cells of liver lobules, and in proximal tubular and glomerular of kidney. Another plasmid (pCMV-hRluc) encoding Renilla reniformis luciferase was used to confirm the results. An increased hRluc mRNA and luciferase in stomach, duodenum, liver, and kidney were detected by RT-qPCR and ex vivo bioluminescence imaging, respectively. Luciferase activity in tissues was significantly increased by 59% in duodenum and 40% in kidney at 48 h and by 53, 68, 43% in stomach, duodenum, and liver, respectively, at 72 h after the first dose of oral delivery of pCMV-Lac Z/PNTs formulation. The immunostaining of tissue revealed the Renilla luciferase was also existed in mucosa surface epithelium, gastric pits, fundus glands, parietal cells, and chief cells of stomach, in villous epithelium, lamina propria, and crypt cells of duodenum, in hepatocytes and sinusoidal endothelial cells of liver lobules, and in proximal tubular and glomerular of kidney. These results implicate the potential application of PNTs being a nano-vector for oral gene delivery to duodenum, stomach, liver and kidney.

並列關鍵字

peptide nanotube, gene delivery, oral

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