Photodynamic Therapy of Localized Infections

描述（由申请人提供）：本次修订的第二次竞争性更新的总体目标是继续我们对光动力治疗（PDT）在治疗局部感染中的作用的研究。PDT使用无毒光敏剂（PS）和无害的可见光（通常是红光，以增加组织穿透），它们在氧气存在下结合产生活性氧，活性氧会破坏生物分子，如蛋白质、脂质和核酸，并随后导致细胞死亡。在该基金的两个资助期内，我们合成并鉴定了几种新型高活性抗菌PS，并证明了它们在治疗感染伤口、烧伤和脓肿的小鼠模型中的有效性。在某些情况下，PDT可以使小鼠免于因未经治疗的局部感染而导致的败血症而死亡。适当的PS分子设计，加上局部或局部应用于感染区域，以及较短的药物光照间隔，使微生物细胞比周围的宿主细胞具有更高的选择性。这条研究线的广泛动机是世界范围内病原微生物对抗生素耐药性的无情增加，并且已经发现多重抗生素耐药菌株通常和光动力失活（PDI）一样敏感naïve菌株，而且微生物细胞不能对PDI产生耐药性。Aim 1将探索临床批准的PS、亚甲基蓝和相关的戊噻嗪盐可以通过添加碘离子等简单离子增强其抗菌PDT作用的发现。虽然我们最初将其解释为涉及羟基自由基的电子转移（氧化），但我们已经

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.