Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria
Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria
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DOI：
10.1093/jac/dkf053
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发表时间：
2002-06-01
影响因子：
5.2
通讯作者：
Hasan, T
中科院分区：
文献类型：
--
作者：
Hamblin, MR;O'Donnell, DA;Hasan, T
目的：我们先前已经表明，聚-L-赖氨酸和光敏剂二氢卟酚（e6）之间的聚阳离子缀合物在革兰氏阳性和革兰氏阴性细菌的光动力灭活（PDI）中是有效的。在这份报告中，我们探讨了多聚赖氨酸链的大小与其介导革兰氏阴性菌和革兰氏阳性菌杀灭效果之间的关系。通过将一个二氢卟酚（e6）分子精确地连接到聚二氢卟酚的α-氨基上来制备缀合物。平均长度为8个和37个赖氨酸残基的（ε-苄氧基羰基）赖氨酸，然后脱保护ε-氨基，并通过等电聚焦表征。洗涤后，测量革兰氏阳性金黄色葡萄球菌（ATCC 27659）和革兰氏阴性大肠杆菌（ATCC 29181）对这些缀合物和游离二氢卟酚（e6）的吸收，作为光敏剂浓度（0-4 μ M二氢卟酚（e6）当量）和孵育时间的函数。培养后，将细菌暴露于低能量密度（10-40 J/cm（2））的660 nm半导体激光器发出的光，并在连续稀释后通过菌落形成试验评估存活率。aureus和E.大肠杆菌对这两种结合物的吸收量相当，但游离二氢卟酚（e6）仅被S.金黄色。光照后S.当加载8-赖氨酸缀合物和游离二氢卟酚（E6）时，金黄色葡萄球菌以通量依赖性方式被杀死，但加载37-赖氨酸缀合物时则稍弱。而E.大肠杆菌仅在浓度高达4 μ M的37-赖氨酸缀合物下有效。PDI使用8-赖氨酸缀合物和游离二氢卟酚（e6）在E.在100 μ M的浓度下观察到大肠杆菌。透射电子显微镜下可见与染色体损伤相一致的内部电子透明区。结论：这些结果可以用大聚阳离子穿透革兰氏阴性杆菌不渗透性外膜的必要性来解释。coli，而革兰氏阳性S.金黄色葡萄球菌更容易被小分子穿透。然而，由于S.金黄色葡萄球菌总体上比大肠杆菌敏感。大肠杆菌的37-赖氨酸缀合物可以有效地杀死这两种细菌。
Objectives: We have shown previously that a polycationic conjugate between poly-l-lysine and the photosensitizer chlorin(e6) was effective in photodynamic inactivation (PDI) of both Gram-positive and Gram-negative bacteria. In this report we explore the relationship between the size of the polylysine chain and its effectiveness for mediating the killing of Gram-negative and Gram-positive bacteria.Methods: Conjugates were prepared by attaching precisely one chlorin(e6) molecule to the alpha-amino group of poly-(epsilon-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the epsilon-amino groups, and were characterized by iso-electric focusing. The uptake of these conjugates and free chlorin(e6) by Gram-positive Staphylococcus aureus (ATCC 27659) and Gram-negative Escherichia coli (ATCC 29181) after washing was measured as a function of photosensitizer concentration (0-4 muM chlorin(e6) equivalent) and incubation time. After incubation the bacteria were exposed to low fluences (10-40 J/cm(2)) of 660 nm light delivered from a diode laser, and viability was assessed after serial dilutions by a colony-forming assay.Results: S. aureus and E. coli took up comparable amounts of the two conjugates, but free chlorin(e6) was only taken up by S. aureus. After illumination S. aureus was killed in a fluence-dependent fashion when loaded with the 8-lysine conjugate and free chlorin(e6) but somewhat less so with the 37-lysine conjugate. In contrast, PDI of E. coli was only effective with the 37-lysine conjugate at concentrations up to 4 muM. PDI using the 8-lysine conjugate and free chlorin(e6) on E. coli was observed at a concentration of 100 muM. Transmission electron micrographs showed internal electron-lucent areas consistent with chromosomal damage.Conclusion: These results can be explained by the necessity of a large polycation to penetrate the impermeable outer membrane of Gram-negative E. coli, while Gram-positive S. aureus is more easily penetrated by small molecules. However, because S. aureus is more sensitive overall than E. coli the 37-lysine conjugate can effectively kill both bacteria.