評估含有抗真菌藥物氟康唑的幾丁聚醣水膠對抗念珠菌的效果

Abstract

念珠菌屬的真菌中約有二十種會造成感染，其中最常見的是白色念珠菌。當宿主免疫力低下時，在口腔、皮膚、生殖道等部位易產生念珠菌症。過去實驗室研究發現以幾丁聚醣(chitosan)和抗真菌藥物氟康唑(fluconazole)兩者結合的水膠，對於白色念珠菌有良好的協同抑菌效果。因此本研究著重於將此水膠以實用性為目的進行測試，包括進行水膠藥物釋放、細胞毒性測試及動物實驗等。延續實驗室先前的研究，為了了解以幾丁聚醣(chitosan)和抗真菌藥物氟康唑(fluconazole)兩者結合的水膠是否對於白色念珠菌、熱念念珠菌及其個別的抗藥性菌株具有顯著抑菌效果，本研究以水膠紙錠擴散實驗(hydrogel disk diffusion assay)對白色念珠菌、熱念念珠菌及其個別的抗藥性菌株進行測試，結果顯示加入fluconazole的水膠對白色念珠標準菌株具有顯著的抑菌效果。以人類成纖維母細胞(HDFa) 進行細胞毒性試驗，Calcein AM測試結果顯示，水膠100％浸泡液對細胞具有毒性，50% 水膠浸泡液處理後，則能降低細胞毒性。此外，考慮到細胞微環境和水膠用作傷口敷料實際使用情形遠比體外測試來得複雜許多，以及實際使用上水膠並僅有局部接觸到傷口，無法直接將體外細胞毒性測試結果直接連結至傷口癒合表現。將水膠利用高效液相層析（High Performance Liquid Chromatography; HPLC）進行藥物釋放率測量，針對水膠作用時間長短來預測此水膠中fluconazole的藥物釋放量。最後，利用大鼠背部開創性皮膚傷口感染白色念珠菌，結果顯示相較於紗布與不含fluconazole水膠，含有fluconazole的水膠在第十二天時具有顯著修復能力。綜合以上結果，本研究開發外用水膠敷料能夠有較佳的傷口感染修復效果，未來希望能夠進一步改良水膠功能與特性，期望能為皮膚真菌感染提供另一有效的治療方式。

Approximately twenty species of fungi in the Candida genus can cause infections, with Candida albicans being the most common. Candidiasis, caused by Candida fungi, commonly occurs in areas such as the mouth, skin, and genital tract when the host's immune system is weakened. Previous laboratory studies have discovered that a hydrogel combining chitosan and the antifungal drug fluconazole exhibits a synergistic antimicrobial effect against C. albicans. Therefore, this study focuses on testing the practicality of this hydrogel, including drug release, cytotoxicity testing, and animal experiments. Continuing from previous research in the laboratory, this study aims to determine whether the chitosan-fluconazole hydrogel demonstrates significant inhibitory effects on C. albicans, C. tropicalis, and their drug-resistant strains. Hydrogel disk diffusion assays were conducted to test the hydrogel's efficacy against C. albicans, C. tropicalis, and their respective drug-resistant strains. The results showed a significant inhibitory effect of the fluconazole-containing hydrogel on C. albicans SC5314. Cell toxicity tests were performed using human dermal fibroblasts (HDFa), and the results obtained from the Calcein AM test indicated that the 100% soaking solution of the hydrogel was toxic to the cells, while treatment with a 50% soaking solution of the hydrogel reduced cell toxicity. Furthermore, considering the complex cellular microenvironment and the practical use of hydrogel as a wound dressing, which involves localized contact with the wound, it is not directly feasible to correlate the results of in vitro cytotoxicity tests with wound healing outcomes. Additionally, High-Performance Liquid Chromatography (HPLC) was utilized to measure the drug release rate from the hydrogel and predict the amount of fluconazole released based on the duration of hydrogel action. Finally, an experimental model using rats with dorsal skin wounds infected with C. albicans showed that the fluconazole-containing hydrogel exhibited significant reparative capability on the twelfth day compared to gauze and fluconazole-free hydrogel. Considering the overall results, this study demonstrates the potential of developing a topical hydrogel dressing that improves efficacy in repairing wound infections caused by Candida fungi. Future efforts aim to further enhance the functionality and characteristics of the hydrogel, with the hope of providing an alternative promising treatment approach for cutaneous fungal infections.