Nanoscale presentation of antimicrobial peptides found at the ocular surface

描述（由申请人提供）：细菌性角膜炎是眼表疾病的主要原因，经常导致视力永久性受损，有时需要角膜移植来恢复足够的视力。通常导致这些感染的细菌菌株对传统抗生素治疗具有耐药性。由于其在眼表先天免疫中的作用，抗菌肽（AMPs）有望成为新一代抗耐药菌株的抗生素。然而，单个AMPs在达到与传统抗生素相同的杀微生物效果所需的浓度时可能对宿主细胞产生毒性，这一事实阻碍了AMPs作为眼表治疗剂的发展。目前的知识表明，由于AMPs具有较高的局部浓度、多重性和协同作用，它在体内具有有效的杀微生物活性，而不会对宿主细胞产生明显的毒性。我们的目的是了解眼表AMPs的空间排列、多聚态和协同作用如何增强杀微生物活性并防止对角膜上皮细胞的毒性。我们期望1)在高度定位的微结构域中单个AMP的多聚体将克服赋予强杀微生物特性所需的阈值浓度，同时保持对角膜上皮无毒；2)微结构域中AMP的协同作用将进一步降低角膜上皮细胞的细胞毒性和赋予杀微生物活性所需的每个AMP的有效浓度。我们将以不同的密度、比例和模式将amp固定在生物相容性表面上，并评估其对细菌和角膜上皮细胞的毒性，以此来验证我们的假设。我们使用导电AFM和薄膜沉积来制造amp的方法是创新的，因为它基于最先进的纳米制造技术来解决眼表面先天免疫的问题。在完成我们提出的研究后，我们希望了解多聚态和协同作用与杀微生物活性的关系。建立这种关系将增加我们对眼表先天免疫的理解，并进一步发展抗菌肽作为有效的抗菌药物。随着临床相关病原体对现有抗生素产生耐药性，必须开发对抗感染的新方法。先天免疫系统的抗菌肽是另一种选择，因为它们来源于宿主组织，不太可能导致耐药性。我们建议增加对这些肽如何发挥作用的理解，以便将它们开发为一类新的抗菌化合物。

DESCRIPTION (provided by applicant): Bacterial keratitis is a major cause of ocular surface morbidity that often leads to permanently compromised visual function, sometimes to an extent that necessitates corneal transplantation to restore adequate vision. Often the bacterial strains that cause these infections are resistant to treatment with traditional antibiotics. Due to their role in innate immunity at the ocular surface, antimicrobial peptides (AMPs) show promise as a new generation of antibiotics against resistant strains. However, single AMPs can be toxic to host cells at the concentrations necessary to have the same microbicidal efficacy as traditional antibiotics, a fact which has impeded progress in the development of AMPs as therapeutic agents for the ocular surface. The current knowledge base suggests that effective microbicidal activity of AMPs is achieved in vivo without significant host cell toxicity due to a combination of high local concentration, multimericity and synergism. Our objectives are to understand how the spatial arrangement, multimeric state, and synergism of AMPs present at the ocular surface enhance microbicidal activity and prevent toxicity to corneal epithelial cells. We expect that 1) multimers of a single AMP in highly localized micro-domains will overcome the threshold concentration necessary to confer strong microbicidal properties while remaining non-toxic to the corneal epithelium and 2) synergy of AMPs in micro-domains will further reduce both corneal epithelial cell cytotoxicity and the effective concentration of each AMP necessary to confer microbicidal activity. We will test our hypotheses by immobilizing AMPs in varying densities, ratios and patterns onto biocompatible surfaces and assessing the toxicity to bacteria and corneal epithelial cells. Our approach using conducting AFM and film deposition to pattern AMPs is innovative because it is based on state-of-the-art nanofabrication techniques to address questions about innate immunity of the ocular surface. Upon completion of our proposed studies, we expect to understand how multimeric state and synergism relate to microbicidal activity. Establishing this relationship will increase our understanding of innate immunity at the ocular surface and further the development of AMPs as effective antimicrobial agents. As clinically relevant pathogens become resistant to available antibiotics, new methods to combat infection must be developed. Antimicrobial peptides of the innate immune system present an alternative because they are derived from host tissue and are not likely to lead to resistance. We propose to increase the understanding of how these peptides function so that they can be developed as a new class of antimicrobial compounds.