開發載介孔二氧化矽粒子聚乳酸/褐藻酸之 複合纖維敷料

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姓名

林佑俞(Yu-Yu Lin) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

開發載介孔二氧化矽粒子聚乳酸/褐藻酸之複合纖維敷料
(The Development of Mesoporous Silica Nanoparticle-Laden Polylactic Acid/Alginate Composite Wound Dressings)

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摘要(中)

慢性傷口因癒合緩慢，造成感染風險增加，因此需開發多功能的傷口敷料以促進組織再生並達到抗菌的效果。本研究將褐藻酸鈉(Alg)與載入增加親水性的介孔二氧化矽奈米粒子(MSN)以及抗生素去氧羥四環素(DCH)的聚乳酸(PLA-DM)進行混合靜電紡絲以得到複合纖維，透過疏水PLA-DM纖維的加入，能解決Alg太過親水所致的細胞貼附不佳，並且改善Alg硬而易斷的機械性質，使其具適當的柔韌及抗張性。且由於DCH的抗菌效果，複合纖維均能達到高於99.9 %的抗菌率。另一方面，血小板衍生生長因子（PDGFB）基因能與聚乙烯亞胺(PEI)結合形成帶正電奈米顆粒，帶負電的褐藻酸鈉纖維能將其固定以藉此原位轉染貼附的細胞。體外實驗證實轉染細胞所表達的PDGFB能夠促進細胞的增生。將這些複合纖維應用於糖尿病傷口的感染模型，發現在有敷料的應用可以避免生物膜的生成，PLA-DM中的DCH具有良好的抗菌效果，可以避免炎症細胞的浸潤，而混紡纖維因為有固定PDGFB基因，能促進新的表皮生成，並提高傷口修復效果。因此本研究的所開發的傷口敷料確實可降低感染風險並減緩發炎反應，有效促進慢性傷口的修復。

摘要(英)

Chronic wounds are at risk of infection due to their slow healing, so multifunctional wound dressings with antibacterial effects and healing promotion are highly demanded. In this study, sodium alginate (Alg) acid and polylactic acid loaded with mesoporous silica nanoparticles (MSN) and antibiotic doxycycline hyclate (DCH), i.e. PLA-DM, were coelectrospun to fabricate composite fibers. Because Alg fibers are too hydrophilic, cells cannot well adhere to their surfaces. The incorporation of PLA-DM fibers reduced poor cell adhesion caused by hydrophilic Alg fibers. Furthermore, PLA-DM also improved the mechanical performance, so that the composite fiber demonstrated appropriate flexibility and tensile strength. Due to DCH loading, composite fibers all demonstrated antibacterial efficacies higher than 99.9%. On the other hand, the platelet-derived growth factor-B (PDGFB) gene complexed with polyethyleneimine (PEI) to form positively charged nanoparticles, which could be immobilized onto negatively charged sodium alginate fibers to in situ transfect adhered cells. The in vitro experiments showed that PDGFB expressed from transfected cells could promote cell proliferation. These composite fibers were applied to treat infected wounds on diabetic mice. Biofilm formation can be avoided by the coverage of wound dressing. Because DCH loaded in PLA-DM can inhibit bacterial infection, the infiltration of inflammatory cells was eliminated. Immobilizing PDGFB genes facilitated the formation of the newly born epidermis and enhance wound repair. Overall, wound dressings developed in this study indeed reduced the risk of infection, relieved inflammation, and effectively promoted the healing of chronic wounds.

關鍵字(中)

★ 電紡絲
★ 傷口敷料
★ 介孔二氧化矽粒子
★ 聚乳酸
★ 褐藻酸鈉

關鍵字(英)

論文目次

目錄

摘要 i
Abstract ii
致謝 iii
目錄 v
圖目錄 x
表目錄 xv
第一章緒論 1
1-1研究背景 1
1-2研究目的 3
1-3實驗架構 5
第二章文獻回顧 6
2-1慢性傷口 6
2-1-1糖尿病傷口修復 6
2-1-2促進傷口癒合的生長因子 11
2-1-3血小板衍生生長因子(PDGF)於糖尿病傷口之應用 12
2-2去氧羥四環素 16
2-2-1去氧羥四環素之簡介 16
2-2-2去氧羥四環素之特性 17
2-2-3去氧羥四環素於皮膚之應用 18
2-3基因治療 20
2-3-1基因載體 20
2-3-2基因治療之傷口修復應用 21
2-4電紡絲 23
2-4-1電紡絲原理 23
2-4-2電紡絲於傷口敷料之應用 25
2-4-3複合電紡絲 28
2-5褐藻酸鈉 30
2-5-1褐藻酸鈉之簡介 30
2-5-2褐藻酸鈉之性質 31
2-5-3褐藻酸鈉於傷口敷料之應用 33
2-6聚乳酸 34
2-6-1聚乳酸之簡介 34
2-6-2聚乳酸之性質 36
2-6-3聚乳酸於傷口敷料之應用與改善疏水材料的釋放特性 37
第三章材料與方法 41
3-1實驗材料 41
3-2實驗儀器 46
3-3實驗方法 48
3-3-1介孔二氧化矽奈米粒子合成 48
3-3-2電紡絲溶液製備 49
3-3-3電紡絲纖維製備 53
3-3-4電紡絲不同角度收集量之量測 55
3-3-5電紡絲纖維收集量之量測 55
3-3-6 電紡絲纖維之螢光染色測定 55
3-3-7ATR-FTIR之樣本製備 56
3-3-8萬能拉伸試驗機樣本製備 56
3-3-9電紡絲纖維溶脹率(Swelling ratio) 57
3-3-10電紡絲纖維水蒸氣穿透率(Water Vapor Transmission rate, WVTR) 57
3-3-11 SEM之樣本製備 58
3-3-12抗菌實驗 61
3-3-13質體DNA(PDGFB)純化 64
3-3-14 NIH 3T3細胞培養 65
3-3-15細胞存活率實驗(MTT assay) 70
3-3-16原位轉染實驗 72
3-3-17傷口癒合動物實驗 74
第四章結果與討論 77
4-1褐藻酸鈉/聚乳酸之複合奈米電紡絲性質 77
4-1-1複合電紡絲之收集量與直徑分佈 77
4-1-2複合電紡絲之螢光染色測定 82
4-1-3複合電紡絲之FTIR分析 84
4-1-4複合電紡絲之機械性質分析 86
4-1-5複合電紡絲之接觸角分析 89
4-1-6複合纖維之溶脹率 91
4-1-7複合纖維之水蒸氣穿透率(WVTR) 93
4-2複合電紡絲之抗菌活性 95
4-2-1固態培養之抗菌活性 95
4-3複合電紡絲對細胞之影響 99
4-3-1細胞培養於複合纖維上之SEM圖 99
4-3-2細胞培養於複合纖維上之增生 102
4-4於複合電紡絲上進行原位轉染PDGFB 104
4-4-1原位轉染之螢光影像 104
4-4-2原位轉染後分析PDGFB細胞增生 108
4-5傷口癒合動物實驗 111
第五章結論 116
第六章參考資料 118

圖目錄
圖 1傷口癒合四階段[3] 2
圖 2糖尿病傷口示意圖[15] 10
圖 3 MMP在傷口癒合中的角色[5] 10
圖 4 PDGF在傷口癒合中的作用[19] 13
圖 5使用S4PL與PDGFB14天後的傷口癒合情況[17] 13
圖 6使用S4PL與PDGFB傷口邊緣的傷口上皮細胞覆蓋率的量化[17] 13
圖 7 (a)VEGF/PDGFB逐層結構敷料(b)VEGF與PDGFB釋放曲線[20] 15
圖 8糖尿病鼠術後第7天與第14天癒合情形[20] 15
圖 9 Doxycycline hyclate化學結構式[21] 16
圖 10 DCH/PLA奈米纖維(a)接觸角(b)藥物釋放曲線[30] 19
圖 11 DCH/PLA對糖尿病大鼠傷口處理7天後的傷口癒合情況(a)傷口外觀(b)傷口大小(DCH+PLA group DCH直接加到PLA奈米纖維上，DCH/PLA group：載有 DCH的PLA奈米纖維)[30] 19
圖 12 phEGF NF、LPEI NF、phEGF solution、未處理傷口在第7天與第14天的癒合率[33] 22
圖 13靜電紡絲裝置圖[34] 24
圖 14 AM/ST/PEO與AM/ST/PVA的藥物釋放[43] 26
圖 15 Chit/PEO/SiO2/Cip電紡絲傷口癒合程度[44] 27
圖 16複合電紡絲製備方法[37] 29
圖 17 PCL及PVA複合纖維的傷口癒合率[45] 29
圖 18褐藻酸鈉結構式[46] 30
圖 19褐藻酸鈉與鈣離子螯合示意圖[48] 31
圖 20市售敷料SorbAlgon與褐藻酸鈉纖維敷料在15天內傷口癒合率與外觀[53] 33
圖 21 PLA合成途徑[54] 35
圖 22左旋乳酸與右旋乳酸[55] 36
圖 23複合纖維表面接觸角變化[63] 40
圖 24載藥纖維的藥物累積釋放曲線[63] 40
圖 25超音波分散示意圖 50
圖 26電紡絲裝置圖 54
圖 27 Alg纖維與PLA-DM纖維收集速率關係 79
圖 28改變噴頭角度示意圖(θ=噴頭與收集面法線夾角) 79
圖 29以進料流速為0.2 mL/hr的PLA-DM進行不同角度電紡絲，並量測其PLA-DM纖維的收集速率 80
圖 30複合電紡絲纖維之SEM圖與其直徑分布圖(scale bar=1 μm ) 81
圖 31 電紡30秒所收集的Alg/PLA-DM複合纖維之螢光染色影像與明視野影像(scale bar=50 μm ) 83
圖 32複合纖維之FTIR紅外光譜儀分析圖 85
圖 33褐藻酸鈉與聚乳酸複合纖維之應力應變曲線圖 88
圖 34 (a)Alg(b)A3P1(c)A1P1(d) A1P3(e) PLA-DM之接觸角分析 90
圖 35複合電紡絲纖維的溶脹率 (**: p<0.01 compared to the Alg group) 92
圖 36複合電紡絲纖維之水蒸氣穿透率(**: p<0.01, *: p<0.05 compared to the Alg group) 94
圖 37 (a)Alg(b)A3P1(c)A1P1(d)A1P3(e)PLA-DM (f)DCH以紙錠擴散法檢測不同比例之複合纖維對金黃色葡萄球菌之抑菌力(scale bar = 1 cm) 96
圖 38以紙錠擴散法檢測不同比例之複合纖維對金黃色葡萄球菌之抑菌區環形面積(*: p<0.05,**: p<0.01) 96
圖 39 (a)control(b)Alg(c)A3P1(d)A1P1(e)A1P3(f) PLA-DM液態培養之塗盤法結果 98
圖 40 NIH 3T3細胞於(a) Alg、(b)A3P1、(c)A1P1、(d)A1P3、(e) PLA-DM纖維之生長型態SEM圖(scale bar=10 μm) 100
圖 41 NIH 3T3細胞培養於(a)Alg(b) A3P1(c)A1P1(d) A1P3(e) PLA-DM纖維上之光學顯微鏡圖(scale bar = 100 μm) 101
圖 42 NIH 3T3培養於不同比例之複合電紡絲纖維第1、3、5天後之存活率分析(**: p < 0.01 compared to the Alg group on the same day, #: p<0.05, ##: p < 0.01 compared to the PLA-DM group on the same day) 103
圖 43 (a)赫斯特螢光染劑33258標定複合纖維上的DNA奈米粒子與(scale bar = 100 μm) (b)螢光影像量化結果(**: p < 0.01 compared to the Alg) 106
圖 44 (a)複合纖維吸附PEI與pPDGFB/GFP奈米粒子後對NIH 3T3進行原位轉染與(scale bar = 100 μm) (b)螢光量化結果(**: p < 0.01 compared to the A3P1) 107
圖 45於(a) Alg (b) A3P1 (c) A1P1 (d) A1P3(e)PLA-DM對NIH 3T3細胞進行基因的原位轉染後1、3、5天的細胞存活率分析(*: p < 0.05, **p < 0.01 compared to the control group on the same day) 109
圖 46不同複合纖維對NIH 3T3細胞進行基因的原位轉染後第5天的細胞存活率分析(*: p < 0.05,* *: p < 0.01 compared to the PLA-DM group, #: p < 0.05, ##: p < 0.01 compared to the control groups) 110
圖 47使用不同敷料覆蓋一周後的傷口外觀，紅色虛線圓圈為6mm傷口原本的範圍，(scale bar = 1 mm)(n=3) 113
圖 48使用不同複合纖維敷料覆蓋傷口一周後之傷口癒合率 114
圖 49使用不同敷料覆蓋一周後的H&E染色的組織病理學分析(scale bar =100 μm)(黑色箭頭:炎症細胞浸潤, 紅色箭頭:巨噬細胞, 藍色箭頭:新生血管, 綠色箭頭:毛囊) 114
圖 50使用不同敷料覆蓋一周後的TRI染色的組織病理學分析(scale bar =100 μm) 115

表目錄
表 1生長因子在傷口癒合的角色[16] 11
表 3超音波分散參數 50
表 4褐藻酸鈉與聚乳酸複合纖維之拉伸性質 88
表 5複合纖維對金黃色葡萄球菌之抗菌率分析 98

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2022-9-16

推文