Huang Liyi, Xuan Yi, Koide Yuichiro, Zhiyentayev Timur, Tanaka Masamitsu, Hamblin Michael R
Department of Infectious Diseases, First Affiliated College & Hospital, Guangxi Medical University, Nanning 530021, China.
Lasers Surg Med. 2012 Aug;44(6):490-9. doi: 10.1002/lsm.22045. Epub 2012 Jul 3.
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).
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.
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.
抗菌光动力疗法（APDT）使用无毒的光敏剂（PS）和可见光，在氧气存在的情况下产生活性氧（ROS），如单线态氧（（1）O（2），通过II型机制产生）和羟基自由基（HO（.），通过I型机制产生）。本研究考察了（1）O（2）和HO（.）对APDT杀灭革兰氏阳性菌和革兰氏阴性菌的相对贡献。
研究设计/材料与方法：使用荧光探针3'-（对羟基苯基）-荧光素（HPF）和单线态氧传感器绿色试剂（SOSG）来测定两种光敏剂光照产生的HO（.）和（1）O（2）：三阳离子巴基球（BB6）以及聚乙烯亚胺与二氢卟吩（e6）的共轭物（PEI-ce6）。二甲基硫脲是HO（.）清除剂，而叠氮化钠（NaN（3））是（1）O（2）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（化学产生HO（.））杀菌实验。
共轭物PEI-ce6主要产生（1）O（2）（被NaN（3）猝灭），而当NaN（3）增强探针激活时，BB6除产生（1）O（2）外还产生HO（.）。NaN（3）也增强了芬顿试剂对HPF的激活。所有细菌都能被芬顿试剂杀灭，但革兰氏阳性菌所需浓度高于革兰氏阴性菌。NaN（3）增强了芬顿介导的对所有细菌的杀灭作用。对于BB6，革兰氏阳性菌与革兰氏阴性菌的APDT杀灭率之比为2或4:1，对于共轭物PEI-ce6为25:1。使用PEI-ce6和BB6对革兰氏阴性菌进行APDT杀菌时，NaN（3）的抑制作用呈剂量依赖性，而NaN（3）几乎未能抑制革兰氏阳性菌的杀灭。
叠氮基自由基可能由NaN（3）和HO（.）形成。可能革兰氏阴性菌对HO（.）更敏感，而革兰氏阳性菌对（1）O（2）更敏感。NaN（3）抑制作用的差异可能反映了光敏剂与细菌结合程度（微环境）的差异或NaN（3）进入细菌细胞壁的渗透差异。
