Photodynamic Therapy of Localized Infections

描述（由申请人提供）：本次修订后的第二次竞争性更新的总体目标是继续研究光动力疗法（PDT）在治疗局部感染中的作用。PDT采用无毒的光敏剂（PS）和无害的可见光（通常为红光以增加组织穿透），其在氧存在下联合收割机结合以产生活性氧物质，其破坏生物分子如蛋白质、脂质和核酸并随后引起细胞死亡。在该资助的两个资助期内，我们合成并表征了几种新型高活性抗菌PS，并证明了它们在治疗感染伤口、烧伤和烫伤的小鼠模型中的有效性。在某些情况下，PDT可以使小鼠免于因败血症而死亡，败血症是由未经治疗的局部感染引起的。PS的适当分子设计与PS局部或局部应用于感染区域以及短的药物-光照间隔的组合允许与周围宿主细胞相比对微生物细胞的高选择性。这一系列研究的广泛动机是病原微生物中抗生素耐药性在全球范围内的持续增加，并且已经发现，多抗生素耐药菌株通常与幼稚菌株一样对光动力学灭活（PDI）敏感，此外，微生物细胞不能对PDI产生抗性。目的1将探索一个发现，即临床批准的PS，亚甲蓝和相关的吩噻嗪鎓盐可以通过添加简单离子如碘化物来增强其抗微生物PDT效果。虽然我们最初将其解释为涉及来自羟基自由基的电子转移（氧化），但我们 现在发现我们在没有氧气的情况下仍然可以杀人。除了研究1型和2型光化学机制的贡献外，我们现在正在研究一种可能的机制，该机制涉及通过激发态MB直接氧化碘（和叠氮化物）阴离子以产生有效杀死微生物细胞的碘/叠氮化物自由基。目的2提出了一种解决体内抗菌PDT有效性的最大障碍的方法。e.将光敏剂（PS）递送到感染的组织中。由于所有高效抗菌PS都带有阳离子电荷，因此它们非常适合通过电力输送到组织中。我们将探索单独和组合使用离子电渗和电穿孔将抗菌PS（和碘化物）递送到离体猪皮和无毛小鼠皮肤中。目的3通过探索PDT与传统全身性抗生素的组合来防止PDT后细菌的再生长来回应评论者的批评。初步数据显示，妥布霉素的亚治疗方案可以协同地联合收割机与由富勒烯和白色光介导的PDT方案组合，以挽救假单胞菌伤口感染模型中的小鼠免于死亡。我们将使用PDT和/或抗生素的MIC测定来研究体外可能的协同作用。在目标4中，为了响应评论者的批评，我们将研究相对于哺乳动物皮肤细胞（小鼠和人）杀死微生物细胞（真菌和细菌）的选择性，并观察从小鼠移除的组织中可能的损伤。我们已经发现了一些非常有效的抗真菌PS，并将在体外和体内研究这些强调选择性。我们现在可以使用经过基因工程改造的白色念珠菌来表达Gaussia princeps荧光素酶，以及表达萤火虫荧光素酶的烟曲霉菌，它们可以形成局部感染，可以通过生物发光成像进行成像。

DESCRIPTION (provided by applicant): The overall objective of this revised second competitive renewal is to continue our investigations on the role of photodynamic therapy (PDT) in treating localized infections. PDT employs non-toxic photosensitizers (PS) and harmless visible light (frequently red light for increased tissue penetration) that combine in the presence f oxygen to produce reactive oxygen species that damage biological molecules such as proteins, lipids and nucleic acids and subsequently cause cell death. In the two funding periods of this grant we have synthesized and characterized several novel highly active antimicrobial PS and demonstrated their effectiveness in treating mouse models of infected wounds, burns and abscesses. In some cases PDT can save mice from a certain death due to sepsis that develops from an untreated localized infection. A combination of the appropriate molecular design of the PS, together with topical or local application of the PS to the infected area and a short drug-ligh interval allows high selectivity for microbial cells compared to the surrounding host cells. The broad motivation for this line of research is the relentless worldwide increase in antibiotic resistance amongst pathogenic microbes, and it has been found that multi-antibiotic resistant strains are in general as sensitive to photodynamic inactivation (PDI) as naïve strains, and moreover that microbial cells are unable to develop resistance to PDI. Aim 1 will explore a finding that the clinically approved PS, methylene blue and related penothiazinium salts could have their antimicrobial PDT effect potentiated by addition of simple ions like iodide. While we initially interpreted this to involve electron transfer (oxidation) from hydroxyl radicals, we have now discovered that we can still get killing in the absence of oxygen. In addition to studying the contribution of Type 1 and Type 2 photochemical mechanisms we are now investigating a possible mechanism involving direct oxidation of iodide (and azide) anions by excited state MB to produce iodide/azide radicals that efficiently kill microbial cells. Aim 2 proposes a solution t the biggest barrier to the effectiveness of antimicrobial PDT in vivo i. e. delivering the photosensitizer (PS) into the infected tissue. Since all highly effective antimicrobial PS have cationic charges they are ideally suited for delivery into tissue by electricity. We will explore te use of iontophoresis and electroporation singly and in combination to deliver antimicrobial PS (and iodide) into ex vivo pigskin and into hairless mouse skin. Aim 3 responds to the reviewers' criticisms by exploring the combination of PDT with traditional systemic antibiotics to prevent regrowth of bacteria after PDT. Preliminary data has shown that a sub-therapeutic regimen of tobramycin can synergistically combine with a PDT regimen mediated by a fullerene and white light to save mice from dying in a Pseudomonas wound infection model. We will study possible synergy in vitro using MIC determinations with PDT and/or antibiotics In aim 4 in response to the reviewers' criticisms we will study selectivity for killing microbial cells (both fungi and bacteria) over mammalian skin cells (both mouse and human) and look at possible damage in tissue removed from mice. We have found some highly effective antifungal PS and will study these in vitro and in vivo with emphasis on selectivity. We now have access to Candida albicans that has been genetically engineered to express Gaussia princeps luciferase and Aspergillus fumigates expressing firefly luciferase that form localized infections that can be imaged by bioluminescence imaging.