Photodynamic Therapy of Localized Infections

描述(申请人提供)：本提案的总体目标是继续探索一种在局部感染模型中杀灭抗生素耐药病原菌或真菌的新的光化学方法。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，它会产生细胞毒性物质，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可以针对其目的细胞类型或组织，此外，照明可以在空间上定向到感染区域。在之前的资助期间，我们建立了聚阳离子载体，如聚L赖氨酸，可以连接到PS，如氯离子(E6)，这些PS的分子载体增加了对细菌的选择性结合，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灭作用。我们使用发光细菌和微光成像相机在创伤、烧伤和深层次感染的动物模型中证明了PDT可以在体内杀死革兰氏(-)菌(如铜绿假单胞菌)和革兰氏(+)菌(如金黄色葡萄球菌)。局部PDT可能还有一个额外的优势，因为它还可以灭活病原菌用来建立感染和入侵组织的分泌的细胞外毒力因子。这一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。四个具体目标将集中在(1)研究微生物(可能与癌细胞有很大不同)的光化学灭活机理，目的是设计简单的组合治疗；(2)调查新发现的低无毒浓度的过氧化氢显著增强抗菌PDT的数量级；(3)合成和测试第三代多阳离子PS与在所有条件下保持阳离子特性的季胺化氨基；(4)在感染致病菌(铜绿假单胞菌或金黄色葡萄球菌)的急慢性创面和烧伤小鼠模型上测试上述治疗方法，以及在不同品系的小鼠创面皮损中生长的绿色荧光蛋白白色念珠菌或烟曲霉菌的全新光谱分辨荧光成像模型。这些研究途径有望为优化抗菌光动力疗法提供简单的程序，并加速其广泛应用于临床实践。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引发了人们的担忧，即以前可以治疗的感染可能很快就会无法治愈。创伤性或外科创伤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)涉及无毒染料和无害可见光的组合，它们结合在一起会产生剧毒的活性氧物种。如果染料针对细菌细胞，光动力疗法可以成为一种高效的局部抗菌疗法，对宿主组织几乎没有损害。这项申请寻求确定抗菌光动力疗法的最佳参数，并将着眼于新的协同联合疗法。

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.