Photodynamic Therapy of Localized Infections

描述（由申请人提供）：本提案的总体目标是继续探索一种新的光化学方法来杀死局部感染模型中的抗生素耐药病原菌或真菌。光动力疗法（PDT）使用一种称为光敏剂（PS）的无毒染料和低强度可见光，在氧气存在的情况下产生细胞毒性物质，破坏蛋白质、脂质和核酸并杀死细胞。PDT具有双重选择性的优点，即PS可以靶向其目标细胞类型或组织，并且光照可以在空间上定向到感染区域。在之前的资助期内，我们确定了聚阳离子传递载体（如聚l -赖氨酸）可以与PS（如氯）缀合（e6），并且这些分子传递载体增加了PS与细菌的选择性结合，并使PS能够穿透革兰氏（-）细菌的细胞壁，从而显著增强光介导的杀伤作用。我们使用发光细菌和低光成像相机来证明PDT可以在体内杀死革兰氏（-）种（如铜绿假单胞菌）和革兰氏（+）种（如金黄色葡萄球菌）在动物模型中的伤口，烧伤和深度感染。局部PDT可能还有一个额外的优势，因为它也有可能灭活病原细菌用来建立感染和侵入组织的分泌的细胞外毒力因子。这种竞争性的更新将寻求探索增加抗菌PDT的效力和适用性的新方法。具体目标将集中在四个方面：(1)研究微生物（可能与癌细胞非常不同）光动力失活的光化学机制，旨在设计简单的联合治疗；(2)研究低无毒浓度过氧化氢显著增强抗菌PDT的新发现；(3)合成并测试了具有季铵化氨基的第三代多阳离子PS偶联物，该偶联物在任何条件下都保持阳离子特性；(4)在感染致病菌（铜绿假单胞菌或金黄色葡萄球菌）的急性或慢性伤口和烧伤小鼠模型中测试上述治疗方法，并在不同品系小鼠创伤病灶中生长的GFP白色念珠菌或烟曲霉的全新模型中进行光谱分辨荧光成像。这些研究途径有望提出优化抗菌PDT的简单程序，并加速其广泛引入临床实践。致病菌中抗生素耐药性流行率的惊人上升使人们担心，以前可治疗的感染可能很快就无法治愈。创伤性或外科伤口和烧伤是常见的感染部位，如果不能通过抗生素控制，可能会发展为败血症和死亡。光动力疗法（PDT）包括无毒染料和无害可见光的结合，两者结合产生剧毒的活性氧。如果染料靶向细菌细胞，PDT可以是一种非常有效的局部抗菌治疗，对宿主组织的损害很小。本申请旨在确定抗菌PDT的最佳参数，并将研究新的协同联合疗法。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to continue to explore a novel photochemical method for killing antibiotic resistant pathogenic bacteria or fungi in models of localized infection. Photodynamic therapy (PDT) employs a non-toxic dye termed a photosensitizer (PS) and low intensity visible light, which in the presence of oxygen produce cytotoxic species that damage proteins, lipids and nucleic acids and kill cells. PDT has the advantage of dual selectivity in that the PS can be targeted to its destination cell type or tissue, and in addition the illumination can be spatially directed to the area of infection. In the previous funding period we established that polycationic delivery vehicles such as poly-L-lysine could be conjugated to PS such as chlorin(e6), and these molecular delivery vehicles for PS increased the selective binding to bacteria and enabled the PS to penetrate the cell walls of Gram (-) bacteria to dramatically potentiate light-mediated killing.. We used luminescent bacteria and a low-light imaging camera to demonstrate that PDT will kill both Gram (-) species (eg Pseudomonas aeruginosa) and Gram (+) species (eg Staphylococcus aureus) in vivo in animal models of wounds, burns and deep established infections. Localized PDT may have an additional advantage in that it is also possible to inactivate secreted extracellular virulence factors that pathogenic bacteria use to establish infections and invade tissue. This competing renewal will seek to explore new ways of increasing the potency and applicability of antimicrobial PDT. Four specific aims will focus on (1) studying the photochemical mechanisms of photodynamic inactivation of microbes (that may be very different from cancer cells) with the aim of devising simple combination treatments; (2) investigating the new discovery that low non-toxic concentrations of hydrogen peroxide dramatically potentiate antimicrobial PDT by orders of magnitude; (3) synthesizing and testing a third generation polycationic PS conjugates with quaternized amino groups that retain cationic character under all conditions; (4) testing the above treatments in mouse models of acute or chronic wounds and burns infected with pathogenic bacteria (P. aeruginosa or S. aureus), together with an entirely new model of spectrally resolved fluorescence imaging of GFP Candida albicans or Aspergillus fumigatus growing in traumatic lesions in various strains of mice. These avenues of research are expected to suggest simple procedures to optimize antimicrobial PDT and hasten its wide introduction into clinical practice. PUBLIC HEALTH RELEVANCE The alarming rise in prevalence of antibiotic resistance amongst pathogenic bacteria has led to worries that previously treatable infections could soon be incurable. Traumatic or surgical wounds and burns are common sites of infection that can progress to sepsis and death if they fail to be controlled by antibiotics. Photodynamic therapy (PDT) involves a combination of non- toxic dyes and harmless visible light that in combination produce highly toxic reactive oxygen species. If the dye is targeted to the bacterial cell PDT can be a highly effective local antimicrobial therapy with little damage to host tissue. This application seeks to determine the optimum parameters for antimicrobial PDT and will look at new synergistic combination therapies.