Reducing wound bioburden and biofilm formation using a nanoscale wound surface en

描述(由申请人提供)：慢性伤口的管理是一项重大的医疗挑战，每年花费超过150亿美元。创面败血症，尤其是生物膜的形成，是导致创面无法愈合的重要因素。在慢性创面中，观察到60%的创面有明显的生物膜证据。此外，美国国立卫生研究院估计，人类65%-80%的微生物感染是由生物膜介导的。生物膜的特点是对宿主防御和治疗药物具有抵抗力，否则这些药物将对浮游状态的生物具有疗效。因此，生物膜面临着两个截然不同的挑战：1)必须去除或分散生物膜，以减少细菌的防御机制；2)应用有效的抗菌剂来抑制残留的细菌。在这一应用中，我们试图通过研究一种新的干预措施来解决这个问题，该干预措施有可能防止伤口的慢性定植和分散先前存在的生物膜。我们方法的创新围绕着对伤口床表面进行工程设计，以促进生物被膜细菌的溶解回到浮游状态，在这种状态下，它们更容易受到抗菌剂的影响，并在发生生物膜形成的伤口表面固定抗菌剂。我们的方法包括将装载有抗生物膜剂的可吸收纳米棒掺入纳米薄膜中，该薄膜由含有银纳米粒子的聚电解质多层制造，用于重新设计伤口表面，以增加其对微生物定植和生物膜形成的抵抗。这种结构化的工程方法允许以非常低的无毒活性物质浓度进行有效的抗菌作用。这些方法在单一纳米结构薄膜中的创新组合具有显著提高体内抗生物被膜和抗菌效率的潜力。尤其是纳米珠子的使用 允许精确控制抗生物膜剂的浓度和释放速率。它们还穿透并在生物膜内创建微区，使表面接触增加80%，以同时增加抗生物膜和抗菌剂的吸附，并在纳米级产生从微珠发出的渐进分散杀伤区。这项研究的中心假设得到了令人兴奋的初步数据的支持，即在伤口床中加入精选的D-和L-氨基酸将减少细菌生物膜的形成，并增加生物膜的溶解。第二个假设是，这种方法将增强固定在伤口表面的银纳米颗粒的抗菌活性。为了实现这些目标，我们提出了三个目标。在目标1中，我们将评价选定的D-和L-氨基酸固定在聚电解质薄膜中并负载到可吸收的聚乙交酯微球中防止生物被膜形成的能力，并在体外刺激现有的铜绿假单胞菌和金黄色葡萄球菌生物被膜的溶解。在目标2中，我们将使用聚电解质薄膜固定化方法，优化精选氨基酸和纳米微珠在模型伤口(全层和部分厚度皮肤创面)上的整合，并评估其在体内防止生物被膜形成和生物膜溶解方面的有效性；在目标3中，我们将验证这样一种假设，即联合应用银纳米颗粒和精选氨基酸可以进一步减少体内创面的生物被膜形成，并最大限度地减少微生物的生物堵塞。在这项研究的结论中，我们预计将提供概念证据，即在集成的纳米级伤口床工程平台中使用双臂方法来处理伤口生物膜将提高对生物膜的疗效，同时降低伤口床中的细胞毒性。该方法不稳定，可推广到其他抗生物膜和抗菌剂的固定化。因此，在成功完成R21应用后，我们将寻求对研究(通过R01机制)的支持，这些研究将扩大这些研究的范围，以优化这些策略，并评估一系列抗菌剂和抗菌膜剂的有效性，以期最大限度地抑制伤口床脓毒症和改善慢性开放性伤口的愈合。

DESCRIPTION (provided by applicant): Management of chronic wounds represent a major healthcare challenge responsible for over $15 billion in expenses annually. Wound surface sepsis, in particular biofilm formation, is a significant factor in nonhealing wounds. In chronic wounds, >60% have been observed to have clear evidence of a biofilm. Further, NIH has estimated that 65-80% of microbial infections in humans are biofilm-mediated. Biofilms are characterized by resistance to host defenses and to therapeutics that would otherwise have efficacy against the organisms planktonic state. Thus, biofilms present two distinct challenges: 1) the biofilm must be removed or dispersed to reduce bacterial defense mechanisms; and 2) effective antimicrobial agents applied to suppress the resident bacteria. In this application, we seek to address this problem by investigating a novel intervention with potential to prevent chronic colonization of wounds and disperse pre-existing biofilms. The innovation underlying our approach revolves around engineering the surfaces of wound beds to both promote the dissolution of biofilm bacteria back to a planktonic state, where they are more susceptible to antimicrobial agents, and immobilize antimicrobial agents at the wound surface where biofilm formation occurs. Our approach involves incorporation of absorbable nanobeads loaded with antibiofilm agents in a nanoscopic thin film, manufactured of polyelectrolyte multilayers containing silver nanoparticles, that is used to re-engineer the wound surface to increase its resistance to microbial colonization and biofilm formation. This structured engineered approach allows effective antimicrobial action with very low non-toxic concentrations of active agents. The innovative combination of these approaches in a single nanostructured film has the potential to markedly increase anti-biofilm and antimicrobial efficacy in vivo. In particular, use of nano-beads permits precise control over concentrations and release rates of the antibiofilm agent. They also penetrate and create microdomains within the biofilm, increasing surface contact by >80%, to increase adsorption of antibiofilm and antimicrobial agents concurrently and generating progressive dispersion-kill zones emanating from the beads at a nanoscale level. The central hypothesis of this study, supported by exciting preliminary data, is that incorporating select D- and L- amino acids into the wound bed will reduce bacterial biofilm formation and increase biofilm dissolution. A secondary hypothesis is that this approach will enhance the antimicrobial activity of silver nanoparticles immobilized at the wound surface. To address these goals we propose 3 Aims. In Aim 1, we will evaluate the ability of select D- and L- amino acids immobilized in polyelectrolyte thin films and loaded into absorbable PLGA beads to prevent a biofilm from forming and to stimulate dissolution of existing biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in vitro . In Aim 2, we will optimize the integration of select amino acids and nano-beads onto model wound surfaces (full and partial thickness skin wounds), using polyelectrolyte thin film immobilization methods, and evaluate their efficacy in preventing biofilm formation and biofilm dissolution in vivo; and in Aim 3, we will test the hypothesis that combined application of silver nanoparticles and select amino acids further reduces biofilm formation and minimizes microbial bioburden in wounds in vivo. At the conclusion of this study, we expect to provide proof of concept that a two-armed approach to wound biofilms contained in an integrated nanoscale wound bed engineering platform will have increased efficacy against biofilms with reduced cytotoxicity in the wound bed. This approach is labile and generalizable to immobilization of other antibiofilm and antimicrobial agents. Thus, upon successful completion of this R21 application, we will seek support for research (via the R01 mechanism) that will broaden the scope of these investigations in optimizing these strategies and in evaluating the efficacy of an array of antimicrobial and antibiofilm agents with the goal of maximizing the ability to suppress wound bed sepsis and improve healing of chronic open wounds.