電場對於複合奈米絲進行原位基因傳送之影響

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丁仁傑(Jen-Chieh Ting) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

電場對於複合奈米絲進行原位基因傳送之影響
(The effects of electric field on in situ gene delivery using composite electrospun nanofiber)

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摘要(中)

在本研究中，將褐藻酸鈉電紡絲製備於聚吡咯表面，用以吸附以聚乙烯亞胺包覆的DNA奈米粒子。藉由外加電場的輔助使帶正電奈米粒子往位於陰極的褐藻酸鈉絲泳動。結果證明有通電的組別粒子吸附量有顯著增加，但是在不同電壓下粒子吸附量則無明顯差異。測重實驗證實褐藻酸鈉絲中交聯的鈣離子會在電場作用下游離出並增加褐藻酸鈉絲的降解速率，因此推測電場處理造成褐藻酸鈉絲數量減少會影響到粒子的吸附。為了將此系統應用於原位轉染並彌補褐藻酸鈉生物適合性不佳的問題，我們將此系統在支架中加入聚己內酯纖維。複合絲在電場處理下其重量損耗會加速，且與所施加的電壓成正相關，以接觸角來測定複合纖維的親疏水性及FTIR測定複合纖維中官能基的特徵峰，證實所降解的成分為親水性的褐藻酸鈉。最後我們將電場處理過的複合絲植入HEK293T細胞進行原位轉染實驗，顯示相較於未通電的組別，電場可以促進原位轉染，且轉染的效率會隨著電壓增加而提升，同時由於電壓越大，褐藻酸鈉降解越多，聚己內酯絲的比例上升，進而促進細胞的貼附、活性與增生速率。因此本研究結果顯示利用電場輔助確實有利於基因在支架上的裝載量，並可以調控材料的降解速率以改變複合纖維的比例，進而達到兼顧促進基因傳送與改善生物適合性的目的。

摘要(英)

In this study, alginate was electrospun onto polypyrrole surface for polyethyleneimine (PEI) encapsulated DNA nanoparticle adsorption. By treating by an external electric field, positive charged nanoparticles were guided to contact cathodic alginate nanofibers. Although the electric field significantly increased the adsorption of nanoparticles, which did not increase with increasing voltages. The weight experiments demonstrated that the degradation of alginate was promoted under the electric field, which should be relative to the movement of calcium ions within the alginates and thus the structure was destabilized. It also explained that the increasing electric fields may also harmful to the nanoparticle adsorption due to the decrease of alginate fibers. To apply this system for in situ transfection, polycaprolactone (PCL) was coelectrospun with alginate to promote the biocompatibility. After electric field treatment, the loss of composite nanofiber increased with increasing voltages. The contact angle and Fourier-transform infrared spectrometry (FTIR) were applied to investigate the hydrophobicity and functional groups of composite nanofibers, respectively. These results indicated that the loss of composite nanofibers were mainly due to the degradation of alginate fibers. Finally, DNA/PEI nanoparticles were loaded composite nanofibers with electric field treatment and HEK293T cells were seeded onto the fibers for in situ transfection. Compared to the control group, electric fields greatly improve the transfection efficiency which increased with increasing voltages. In addition, higher voltages induced more degradation of alginate nanofibers, which rose the ratio of PCL within composite nanofibers that the cell adhesion, viability, and proliferation were thus improved. Therefore, the use of electric field indeed facilitated the load of gene onto scaffolds. Additionally, it can control the degradation rates to regulate the constitute of composite nanofibers. These properties suggested that our developed scaffold systems can not only provide suitable environment for cell ingrowth, but also be efficient carriers to regulate drug delivery.

關鍵字(中)

★ 電紡絲
★ 奈米粒子
★ 電場

關鍵字(英)

★ Electrospinning
★ Nanoparticles
★ Electric field

論文目次

摘要 I
Abstract II
致謝 IV
目錄 V
圖目錄 VIII
緒論1
1-1 背景 1
1-2 實驗目的 4
第二章文獻回顧 6
2-1 組織工程 6
2-2 生物可降解性材料用於組織工程 8
2-3 電紡絲應用於組織工程 11
2-3-1 電紡絲原理 11
2-3-2 褐藻酸鈉製備電紡絲介紹 14
2-3-3 聚己內酯製備電紡絲介紹 16
2-4 基因治療 18
2-4-1 基因治療介紹 18
2-4-2 基因載體—正電高分子聚乙烯亞胺 20
2-4-3 電紡絲應用於基因治療 24
2-5 導電高分子聚吡咯介紹 27
2-6 電吸附應用於組織工程 28
2-7 電刺激對於褐藻酸鈉之影響 35
第三章材料與方法 38
3-1 實驗藥品 38
3-2 實驗儀器 40
3-3 實驗方法 43
3-3-1 電紡材料製備 43
3-3-2 導電高分子Polypyrrole之合成 45
3-3-3 纖維表面分析實驗 45
3-3-4 粒子通電吸附定量實驗 49
3-3-5 生物實驗 50
第四章結果與討論 55
4-1 電壓對於奈米粒子吸附的影響 55
4-1-1 通電時間對於奈米粒子的吸附量 55
4-1-2 不同電壓對於奈米粒子吸附量 56
4-2 電壓對於纖維的影響 61
4-2-1 通電後褐藻酸鈉纖維之型態 61
4-2-2 通電後褐藻酸鈉纖維之測重分析 61
4-2-3 通電後混紡纖維之型態 65
4-2-4 通電後混紡纖維之測重分析 69
4-3 混紡纖維降解之表面測定 71
4-3-1 接觸角測定 71
4-3-2 FTIR-ATR測定 76
4-4 生物實驗 78
4-4-1 不同電壓與HEK293T細胞之轉染 78
4-4-2 複合纖維施加不同電壓後之生物適合性 81
第五章結論 84
第六章參考資料 86

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指導教授

胡威文

審核日期

2014-8-20

推文