Photodynamic Therapy of Localized Infections

描述（由申请方提供）：本提案的总体目标是继续探索一种新的光化学方法，用于杀灭局部感染模型中的抗生素耐药致病菌或真菌。光动力疗法（PDT）采用称为光敏剂（PS）的无毒染料和低强度可见光，其在氧的存在下产生细胞毒性物质，所述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，因为PS可以靶向其目的细胞类型或组织，并且另外照射可以在空间上指向感染区域。在之前的资助期间，我们确定了聚阳离子递送载体如聚-L-赖氨酸可以与PS如二氢卟酚（e6）缀合，并且PS的这些分子递送载体增加了与细菌的选择性结合，并使PS能够穿透革兰氏（-）细菌的细胞壁以显著增强光介导的杀伤。我们使用发光细菌和低光成像相机来证明PDT将在伤口、烧伤和深度建立的感染的动物模型中在体内杀死革兰氏（-）菌种（例如铜绿假单胞菌）和革兰氏（+）菌种（例如金黄色葡萄球菌）。局部PDT可能具有额外的优势，因为它也可以抑制分泌的细胞外毒力因子，致病菌使用这些因子来建立感染和侵入组织。这种竞争性的更新将寻求探索新的方法来增加抗菌PDT的效力和适用性。本论文的主要目标是：（1）研究光动力灭活微生物的光化学机理（这可能与癌细胞非常不同），目的是设计简单的组合治疗;（2）研究新发现，即低无毒浓度的过氧化氢以数量级显著增强抗微生物PDT;（3）合成和测试具有季铵化氨基的第三代聚阳离子PS缀合物，其在所有条件下保持阳离子特性;（4）在感染病原菌（铜绿假单胞菌或S. aureus），以及在各种品系小鼠的创伤性病变中生长的GFP白色念珠菌或烟曲霉的光谱分辨荧光成像的全新模型。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。公共卫生相关性病原菌中抗生素耐药性流行率的惊人上升导致人们担心以前可治疗的感染可能很快无法治愈。创伤或手术伤口和烧伤是感染的常见部位，如果不能通过抗生素控制，则可能进展为败血症和死亡。光动力疗法（PDT）涉及无毒染料和无害可见光的组合，所述无毒染料和无害可见光组合产生高毒性活性氧物质。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，对宿主组织的损伤很小。本申请旨在确定抗微生物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.