本研究目標開發用於治療慢性糖尿病傷口的蠶絲絲質蛋白薄膜敷料 (SF-film)，並評估該敷料作為藥物載體對遞送第一型類胰島素生長因子 (IGF-1) 的動力及對傷口的治療效果。蠶絲經新式的高溫脫膠製程技術 (HD)可萃取出高純度的絲質蛋白，並可製成具有疏水層的薄膜敷料，相較於傳統鹼脫膠製程 (AD)，可提高薄膜透明度達42%，並提高BALB/3T3成纖維細胞增生率達32%。經傅立葉轉換紅外線光譜分析顯示，不論AD或HD製程均可維持 SF-film的二級結構，惟HD製程可提高β-折疊的結晶度。藥物動力分析顯示，SF-film的釋放速率遵循零級動力學 (zero-order kinetics)，經Ritger &Peppas公式計算確認，負載0.65、6.5 和 65 pmol IGF-1的 SF-film，在37°C下的釋放速率常數分別為每小時0.11、0.23 和 0.09 %，值得注意地是IGF-1負載在SF-film載體上可維持藥物活性，並持續釋放IGF-長達30天以上。在劑量評估中，顯示IGF-1具有劑量依賴性，高糖度培養基下的BALB/3T3 成纖維細胞添加0.65 pmol IGF-1對細胞刻痕癒合速度最佳；動物試驗顯示，施用SF-film與IGF-1對於糖尿病傷口癒合具有協同作用，相較於施用65 pmol 游離型 IGF-1，糖尿病鼠 (db/db) 於施用SF-film負載3.25 pmol IGF-1處理，於13天後可顯著加速傷口閉合率達13%。分析傷口修復參數顯示，可提高傷口組織再上皮率、表皮組織面積、肉芽組織生成和血管生成等。進一步探討分子機制，施用SF-film負載IGF-1的敷料於糖尿病鼠上，可顯著提高傷口組織中IGF-1受體（IGF1R）磷酸化的比率，推論由於SF-film持續遞送IGF-1，活化了IGF1R下游路徑，進而達到促進傷口細胞生合成與組織修復的效果。綜合上述，使用HD製程生產之SF-film具有極大的發展潛能，除可作為緩釋IGF-1的藥物載體，亦可作為傷口敷料，有效治癒糖尿病傷口。

This study aimed to develop a high purity patented silk fibroin (SF)-film and test its suitability to be used as a slow-release delivery for insulin-like growth factor-1 (IGF-1) for chronic diabetic wounds. Silk fibroin was purified through a newly developed heating degumming (HD) process and casted on a hydrophobic surface to form SF-films. The process allowed the fabricated film to achieve a 42% increase in transparency and a 32% higher proliferation rate for BALB/3T3 fibroblasts compared to that obtained by conventional alkaline degumming treatment. Fourier transform infrared analysis demonstrated that secondary structure was retained in both HD- and alkaline degumming-derived SF preparations, although the crystallinity of beta-sheet in SF-film after the HD processing was slightly increased. The release rate of the SF film delivering IGF-1 followed zero-order kinetics as determined via the Ritger and Peppas equation. The release rate constant was identified as 0.11, 0.23, and 0.09% h−1 at 37 °C for SF films loaded with 0.65, 6.5, and 65 pmol IGF-1, respectively. More importantly, the IGF-1 activity was preserved for more than 30 days when complexed with the SF film. This study also investigated whether IGF-1-loaded SF films accelerated in vitro (BALB/3T3) and in vivo (diabetic mice) wound healing and what the optimal dosage is. This study demonstrated that the optimal IGF-1 dosage to promote the BALB/3T3 cell growth in a hyperglycemic medium was approximately 0.65 pmol. Further analysis indicated that SF-films loaded with 3.25 pmol of IGF-1 showed significantly superior wound closure, a 13% increase at the 13th day after treatment relative to treatment with 65 pmol of free IGF-1 in a diabetic mice. IGF-1 also increased the epithelial tissue area and micro-vessel formation in a dose-dependent manner at a low dosage range (3.25 pmol) when loaded to SF-films. Improvement of diabetic wound healing was exerted synergistically by SF-film and IGF-1, as reflected by parameters including levels of re-epithelialization, epithelial tissue area, granulation tissue, and angiogenesis. Western blotting analysis demonstrated that IGF-1 receptor (IGF1R) phosphorylation in diabetic wounds increased more significantly in the IGF-1-loaded SF films group than in other experimental groups. The findings suggest that IGF-1 released from SF-films promotes wound healing through continuously activating the IGF1R pathway thereby accelerating the downstream wound repair process. Collectively, these results indicate that SF films produced using HD have considerable potential to be used as an IGF-1 drug delivery wound dressing for diabetic wound therapy.

Chapter 1：General Introduction 1-1
1.1 Diabetes mellitus and chronic wound 1-2
1.2 Insulin-like growth factor-1 1-2
1.3 Sericulture and Bombyx mori L. silkworm 1-3
1.4 Bombyx mori L. silk fiber fibroin 1-3
1.5 Wound healing 1-5
1.6 Wound dressing 1-5
1.7 Discussion 1-6
Chapter 2：An Insulin-Like Growth Factor-1 Conjugated Bombyx mori L. Silk Fibroin Dressing for Diabetic Wound Healing: Physicochemical Property, Characterization, and Dosage Optimization. 2-1
2.1 Introduction 2-2
2.2 Materials and methods 2-3
2.3. Results 2-9
2.4. Discussion 2-29
Chapter 3： An Insulin-Like Growth Factor-1 Conjugated Bombyx mori L. Silk Fibroin Dressing: Drug Delivery and Sustained Release 3-1
3.1 Introduction 3-2
3.2 Materials and methods 3-5
3.3 Results 3-8
3.4 Discussion 3-21
Chapter 4：Conclusion and Future Perspective 4-1
References R-1
Publication List R-14

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