抗菌光动力疗法的 I 型和 II 型机制：革兰氏阴性和革兰氏阳性菌的体外研究。,Lasers in Surgery and Medicine

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抗菌光动力疗法的 I 型和 II 型机制：革兰氏阴性和革兰氏阳性菌的体外研究。
Lasers in Surgery and Medicine ( IF 2.2 ) Pub Date : 2012-07-03 , DOI: 10.1002/lsm.22045
Liyi Huang ₁ , Yi Xuan , Yuichiro Koide , Timur Zhiyentayev , Masamitsu Tanaka , Michael R Hamblin

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背景和目的抗菌光动力疗法 (APDT) 采用无毒光敏剂 (PS) 和可见光，在有氧的情况下产生活性氧 (ROS)，例如单线态氧 ((1) O(2)，产生通过 II 型机制）和羟基自由基（HO(.)，通过 I 型机制产生）。这项研究审查了 (1) O(2) 和 HO(.) 对革兰氏阳性菌和革兰氏阴性菌的 APDT 杀灭的相对贡献。研究设计/材料和方法荧光探针、3'-(对羟基苯基)-荧光素 (HPF) 和单线态氧传感器绿色试剂 (SOSG) 用于测定由照明产生的 HO(.) 和 (1) O(2)两个 PS：三阳离子-巴克敏斯特富勒烯 (BB6) 和聚乙烯亚胺和二氢卟酚 (e6) 之间的共轭物 (PEI-ce6)。二甲基硫脲是一种 H2O(.) 清除剂，而叠氮化钠 (NaN(3)) 是 (1) O(2) 的猝灭剂。对革兰氏阳性菌（金黄色葡萄球菌和粪肠球菌）和革兰氏阴性菌（大肠杆菌、奇异变形杆菌和铜绿假单胞菌）进行了 APDT 和芬顿反应（H2O(.) 的化学生成）的杀灭。结果共轭 PEI-ce6 主要产生 (1) O(2)（由 NaN(3) 淬灭），而当 NaN(3) 增强探针激活时，BB6 除 (1) O(2) 外还产生 HO(.)。NaN(3) 还通过芬顿试剂加强了 HPF 的激活。所有细菌都被芬顿试剂杀死，但革兰氏阳性菌需要比革兰氏阴性菌更高的浓度。NaN(3) 增强了芬顿介导的所有细菌的杀灭作用。革兰氏阳性菌和革兰氏阴性菌之间的 APDT 杀灭比例对于 BB6 为 2 或 4:1，对于缀合物 PEI-ce6 为 25:1。有使用 PEI-ce6 和 BB6 对革兰氏阴性细菌的 APDT 杀伤的 NaN(3) 剂量依赖性抑制，而 NaN(3) 几乎无法抑制革兰氏阳性细菌的杀伤。结论叠氮基自由基可由 NaN(3) 和 HO(.) 形成。可能是革兰氏阴性菌对 HO(.) 更敏感，而革兰氏阳性菌对 (1) O(2) 更敏感。NaN(3) 抑制的差异可能反映了 PS 与细菌（微环境）结合程度的差异或 NaN(3) 进入细菌细胞壁的差异。可能是革兰氏阴性菌对 HO(.) 更敏感，而革兰氏阳性菌对 (1) O(2) 更敏感。NaN(3) 抑制的差异可能反映了 PS 与细菌（微环境）结合程度的差异或 NaN(3) 进入细菌细胞壁的差异。可能是革兰氏阴性菌对 HO(.) 更敏感，而革兰氏阳性菌对 (1) O(2) 更敏感。NaN(3) 抑制的差异可能反映了 PS 与细菌（微环境）结合程度的差异或 NaN(3) 进入细菌细胞壁的差异。

"点击查看英文标题和摘要"

Type I and Type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria.

BACKGROUND AND OBJECTIVES Antimicrobial photodynamic therapy (APDT) employs a non-toxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen ((1) O(2), produced via Type II mechanism) and hydroxyl radical (HO(.), produced via Type I mechanism). This study examined the relative contributions of (1) O(2) and HO(.) to APDT killing of Gram-positive and Gram-negative bacteria. STUDY DESIGN/MATERIALS AND METHODS Fluorescence probes, 3'-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO(.) and (1) O(2) produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI-ce6). Dimethylthiourea is a HO(.) scavenger, while sodium azide (NaN(3)) is a quencher of (1) O(2). Both APDT and killing by Fenton reaction (chemical generation of HO(.)) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). RESULTS Conjugate PEI-ce6 mainly produced (1) O(2) (quenched by NaN(3)), while BB6 produced HO(.) in addition to (1) O(2) when NaN(3) potentiated probe activation. NaN(3) also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN(3) potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN(3) dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN(3) almost failed to inhibit killing of Gram-positive bacteria. CONCLUSION Azidyl radicals may be formed from NaN(3) and HO(.). It may be that Gram-negative bacteria are more susceptible to HO(.) while Gram-positive bacteria are more susceptible to (1) O(2). The differences in NaN(3) inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN(3) into cell walls of bacteria.

更新日期：2019-11-01

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