Photodynamic Therapy of Localized Infections

描述(由申请人提供)：本次修订后的第二次竞争更新的总体目标是继续我们对光动力疗法(PDT)在治疗局部感染中的作用的研究。PDT使用无毒的光敏剂(PS)和无害的可见光(通常是红光，以增加组织的穿透性)，在氧气存在的情况下结合起来产生活性氧物种，从而破坏蛋白质、脂类和核酸等生物分子，从而导致细胞死亡。在这笔赠款的两个资助期，我们已经合成和表征了几种新型的高活性抗菌剂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发生的脓毒症而死亡。适当的PS分子设计，以及将PS局部或局部应用于感染区域，以及较短的药物光间隔相结合，使微生物细胞比周围的宿主细胞具有更高的选择性。这项研究的广泛动机是全球范围内病原微生物对抗生素耐药性的不断增加，人们已经发现，耐多种抗生素的菌株通常与幼稚菌株一样对光动力灭活(PDI)敏感，而且微生物细胞无法对PDI产生耐药性。目的1将探索一项发现，临床批准的PS、亚甲基蓝和相关的吩噻嗪盐可以通过添加简单的离子(如碘)来增强其抗菌PDT效应。虽然我们最初的解释是涉及羟基自由基的电子转移(氧化)，但我们已经 现在发现，在没有氧气的情况下，我们仍然可以杀人。除了研究类型1和类型2的光化学机制的贡献外，我们现在正在研究一种可能的机制，涉及激发态MB直接氧化碘(和叠氮)阴离子产生碘/叠氮自由基，从而有效地杀死微生物细胞。目的2提出了一种解决体内抗菌剂PDT有效性的最大障碍的方法，即将光敏剂(PS)输送到感染组织中。由于所有高效的抗菌剂PS都带有阳离子电荷，因此它们非常适合通过电传递到组织中。我们将探索单独和联合使用离子导入和电穿孔将抗菌剂PS(和碘)输送到体外猪皮和无毛小鼠皮肤中。目的3通过探索PDT与传统的全身抗生素的结合来预防PDT后细菌的再生长，回应了评价者的批评。初步数据表明，妥布霉素亚治疗方案可以与富勒烯和白光介导的PDT方案协同结合，使小鼠免于在假单胞菌伤口感染模型中死亡。我们将在目标4中使用PDT和/或抗生素的MIC测定在体外研究可能的协同作用针对评审者的批评，我们将研究对哺乳动物皮肤细胞(包括小鼠和人类)杀灭微生物细胞(真菌和细菌)的选择性，并观察从小鼠身上取出的组织可能造成的损害。我们已经发现了一些高效的抗真菌PS，并将在体外和体内进行研究，重点是选择性。我们现在可以接触到白色念珠菌，这种白色念珠菌已经经过基因工程，表达了表达萤火虫荧光素酶的高斯白念珠菌和烟曲霉，这些真菌形成了局部感染，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.