PCL-PEG-PCL奈米微胞裝載月桂酸對痤瘡丙酸桿菌活性之抑制效應研究

痤瘡發炎主要由痤瘡丙酸桿菌所引起，痤瘡丙酸桿菌可以月桂酸殺滅，但月桂酸屬難溶於水之脂肪酸，需添加二甲基亞碸增加月桂酸溶解度。本研究以兩性高分子形成微胞並包覆月桂酸之劑型，可減少二甲基亞碸(DMSO)的毒性且提高月桂酸抗菌效果。本研究中設計三種不同分子量的PCL-PEG-PCL，並分別利用氫核磁共振光譜、紅外線光譜、熱示差掃瞄卡量計及凝膠透層析儀分析合成高分子之化學結構，並用1,6-二苯基-1,3,5-己三烯做為螢光探針觀察其臨界微胞濃度。將PCL-PEG-PCL 以薄膜水合法製備微胞，以粒徑分析儀、表面電荷分析及穿透式顯微鏡觀察，微胞未包覆月桂酸前的粒徑介於49.8-198.1nm，而當包覆月桂酸後，微胞粒徑範圍下降至24.7-89.3 nm，並且觀察到含有不規則型態之粒子，其表面電荷也因月桂酸的羧酸基去質子化由-3.0 mV 至-9.7 mV 下降為-4.2 mV 至-18.4 mV。在以2-萘基溴甲基酮與微胞中月桂酸反應後，利用高效液相層析儀測定月桂酸包覆量；以PC50E40C50 的聚己內酯-聚乙二醇-聚己內酯可搭載 323.75 μg/mL 月桂酸，隨PCL 鏈長加長，藥物包覆率隨之增加。在抑菌及殺菌測試中，比較微胞包覆月桂酸(微胞劑型)及月桂酸溶於5% DMSO 的劑型效力，以最小抑菌濃度(MIC)及最小殺菌濃度(MBC)做評估；在5% DMSO 中，月桂酸的MIC 與MBC 分別為20 ug/mL 和80 μg/mL，而微胞劑型MIC 與MBC 分別介於10-20 μg/mL 與40-80 μg/mL，以微胞劑型只需較少月桂酸量便可達同樣抑菌和殺菌功效。以不同劑型對小鼠纖維母細胞和人類角質細胞做生物相容性評估，結果可見微胞劑型的生物相容性明顯優於溶劑劑型。因此利用PCL-PEG-PCL 包覆月桂酸可避免添加有機溶劑助溶的麻煩，並且仍可達到更佳的效果，未來可進一步觀察在痤瘡性發炎的動物體內，預期能同樣有效殺菌且降低痤瘡性的發炎。

關鍵字

聚己內酯-聚乙二醇-聚己內酯；微胞；痤瘡丙酸桿菌；月桂酸

並列摘要

Acne vulgaris is known as infection of pathogenic bacteria, Propionibacterium acnes (P. acnes). Lauric acid (LA) was found to kill P. acnes effectively. However, LA is a sparingly soluble fatty acid in water in which dimethly sulfoxide (DMSO) is used to increase solubility. In this study, amphiphilic micelles encapsulating LA was developed to reduce DMSO-induced toxicity and improve bactericidal effect of LA. Poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) triblock copolymer was sythysized by ring-opening polymerization. Different molecular weights of PCL-PEG-PCL were synthesized and chararerized by 1H nuclear magnegtic resonance, Fourier transform infared spectroscopy, differential scanning calorimeter and gel permeation chromatography, respectively. The critical micelle concentration(CMC) of the micelle solution was determined by using fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene(DPH). The results of particle size analyzer and transmission electron microscopy show that the averge particles size of placebo micelles and LA-encapsulated micelles were 49.8-198.1 nm and 24.7-89.3 nm, respectively. Upon encapsulation of LA, zeta potential decreased from -3.0 ~ -9.7mV to -4.2 ~ -18.4 mV, because of depronated carboxyl group in LA. A High-performance liquid chromatography was used to quantitate LA encapsulated in the micelles, after encapsulated LA was derived with naphthacyl ester. As the results, the payload of PC50E40C50 for LA was 323.75 μg/mL which was the highest among various PCL-PEG-PCL. In addition, as the length of PCL increased, the drug loading contents decreased. The effect of LA-encapsulated micelles(micelle formulation) were compared with LA dissolved in 5% DMSO(free LA formulation). For free LA formulation, as free lauric acid dissolved in 5% DMSO, the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) were 20 μg/mL and 80μg/mL, respectively. In contrasty, MIC and MBC of micelle formulation were only 10-20 μg/mL and 40-80 μg/mL, respectively. Therefore, the micelle formulation requires slightly lower LA to reach the same antibactirials effecacy of free LA formulation. The cytotoxicities of free LA formulation and micelle formulation in fibroblast and keratinocyte were conducted. It is found that micelle formulation I I I posses better biocompatibility over free LA. In summary, this study demonstrated that PCL-PEG-PCL was a potential drug carrier to encapsulate LA. It worthshile to further examine the efficacy to against the infection of P.acnes in animal study.

並列關鍵字

Lauric ； poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)

參考文獻

[65] 黃郁琪，柱孢藻之相剋物質研究，臺灣大學生態學與演化生物學研究所，

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PCL-PEG-PCL奈米微胞裝載月桂酸對痤瘡丙酸桿菌活性之抑制效應研究

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