光動力殺菌幾丁聚醣水凝膠之開發及齒槽模型建置

Developments of Chitosan Hydrogel and Dental Gingival Model for Antimicrobial Photodynamic Inactivation

陳玨頻(Chueh-Pin Chen)

指導教授 ： 陳進庭

國立臺灣大學/生命科學院/生化科技學系/博士(2015年)

https://doi.org/10.6342/NTU.2015.10884

摘要

自然界中的微生物大部份以生物膜形式存在，生物膜對殺菌劑有極強之抗性。此外，抗生素的廣泛及不當使用導致抗藥性菌株的產生。而這些抗藥性菌株的出現，讓許多研究學者開始尋求其他治療的方式。光動力殺菌為一新興的微生物感染治療方式，研究結果顯示這一個殺菌方式並不會對微生物造成抗藥性。然而光動力殺菌目前於動物實驗及臨床使用上仍存在許多問題，像是對革蘭氏陰性菌及生物膜殺菌效果不如預期。先前研究發現，利用幾丁聚醣結合光動力治療，光動力能夠對菌體造成破壞、殺死菌體，再加入幾丁聚醣可增強殺菌之作用，降低藥物的使用量，進而提高殺菌之效果。於臨床應用上，如果藥物劑型流動性高，不易附著於感染部位，將導致治療效果不彰。為因應光動力殺菌實際需求，須找出一最佳治療之配方，希望可長時間附著於感染部位上，且不會影響藥物釋放。本研究中，首先將開發一應用於治療微生物感染之幾丁聚醣水凝膠搭配光動力殺菌技術平台，並利用牙周治療來確認其效益。實驗中，以HPMC為增稠劑來提高幾丁聚醣水凝膠黏度，隨著HPMC濃度增加，幾丁聚醣水凝膠黏度及黏附力皆會增加。將幾丁聚醣水凝膠與金黃色葡萄球菌及綠膿桿菌生物膜共同培養後，進行照光，於照光後持續培養，殺菌效果隨幾丁聚醣水凝膠黏度增加，有下降之趨勢，且由共軛焦顯微鏡觀察，TBO釋放受到HPMC濃度之影響，故培養時間長短影響光動力效益。動物實驗上，使用幾丁聚醣水凝膠可減少感染傷口之菌數。接著，建立一體外牙齦槽模型並於模型中測試幾丁聚醣水凝膠之光動力效益，發現增加照光劑量及培養時間，會增加光動力殺菌之效果；照光的數目對殺菌效果也有顯著的影響。以牙周病致病菌株作測試，亦有同樣的殺菌效果。其次，探討光動力對生物膜之作用機制，發現光動力除造成菌體死亡外，亦會破壞生物膜之胞外聚合物，使幾丁聚醣能進一步達到協同殺菌之效果。最後，基於光動力處理會破壞生物膜之機制，於光動力處理後加入抗生素，發現不需提高藥物濃度即可增加殺菌效果，且此效果不僅針對標準菌株生物膜，對抗藥性菌株之生物膜亦有相同效果。

關鍵字

光動力殺菌 ； 生物膜 ； 幾丁聚醣 ； 水凝膠 ； 抗生素

並列摘要

Antibiotics are the most common antimicrobial agents used for the treatment of bacteria and fungi infection. Drug-resistance is a growing problem largely due to the widespread use of antibiotics. Biofilms are the main growth forms of microbes in nature and have stronger resistance to antibiotics compared to planktonic cells. Photodynamic inactivation (PDI) is an emerging method to treat microbial infections. Presently, there is no report related microbial resistance to PDI. However, there are several problems to be resolved for PDI application. Previously, we showed that chitosan could increase the efficacy of PDI against both Gram-positive and Gram-negative bacteria in planktonic cells and biofilms. The purpose of this study was to develop a chitosan hydrogel containing hydroxypropyl methylcellulose (HPMC), chitosan and toluidine blue O (TBO) to improve the PDI efficacy for topical application in clinic. The PDI efficacy of hydrogel was examined in vitro against the biofilms of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Confocal scanning laser microscopy (CSLM) was performed to investigate the penetration level of TBO into viable S. aureus biofilms. Incorporation of HMPC could increase the physicochemical properties of chitosan hydrogel including the hardness, viscosity as well as bioadhesion; however, higher HMPC concentration also resulted in reduced antimicrobial effect. CSLM analysis further demonstrated that higher HPMC concentration constrained TBO diffusion into the biofilm. The bactericidal efficacy could be significantly augmented by prolonged retention of hydrogel in the biofilm as well as in the infected skin burn wounds of rat after light irradiation. We found that increasing the illumination dose and incubation time could enhance the PDI efficacy. The PDI efficacy of chitosan hydrogel was also verified in the biofilm of periodontal pathogenic strains in a 3D gingival model. Further studies indicate that PDI could damage the extracellular polymer substrate of biofilm. Finally, we showed that combination of PDI and antibiotics could significantly increase the bactericidal effect against biofilm of wild type strains as well as drug-resistant strains.

並列關鍵字

photodynamic inactivation ； biofilm ； chitosan ； hydrogel ； antibiotic

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