Nanoscale presentation of antimicrobial peptides found at the ocular surface

描述(申请人提供)：细菌性角膜炎是眼表并发症的主要原因，常常导致永久性的视觉功能受损，有时需要进行角膜移植以恢复足够的视力。引起这些感染的细菌菌株通常对传统抗生素的治疗具有抗药性。由于抗菌肽在眼表天然免疫中的作用，抗菌肽有望成为对抗耐药菌株的新一代抗生素。然而，单一的AMPs对宿主细胞的毒性达到与传统抗生素相同的杀菌效果所需的浓度，这一事实阻碍了AMPS作为眼表治疗剂的发展。目前的知识基础表明，由于高局部浓度、多聚体和协同作用的结合，AMPS在体内实现了有效的杀菌活性，而没有明显的宿主细胞毒性。我们的目标是了解AMPs在眼表面的空间排列、多聚态和协同作用如何增强杀菌活性和防止对角膜上皮细胞的毒性。我们预计，1)单一AMP在高度局域化的微域中的多聚体将超过赋予强大杀菌性能所需的阈值浓度，同时保持对角膜上皮细胞的无毒性；2)AMP在微域中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菌活性所需的每种AMP的有效浓度。我们将通过将不同密度、比例和图案的AMP固定到生物兼容表面并评估其对细菌和角膜上皮细胞的毒性来验证我们的假设。我们使用导电原子力显微镜和薄膜沉积来构图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天然免疫的问题。在我们建议的研究完成后，我们希望了解多聚体状态和协同作用如何与杀微生物活性有关。建立这种关系将增加我们对眼表天然免疫的了解，并进一步发展AMPs作为有效的抗菌剂。随着临床相关病原体对现有抗生素产生抗药性，必须开发新的方法来对抗感染。先天免疫系统的抗菌肽提供了另一种选择，因为它们来自宿主组织，不太可能导致耐药性。我们建议增加对这些多肽如何发挥作用的了解，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.