Enzyme-Powered Self-Propelled DNA Nanoparticles for Disruption and Antibiotic Delivery in Topical Biofilms

项目摘要/摘要 细菌生物膜是大多数人类感染的罪魁祸首，导致数万人死亡和数十亿人死亡 每年的医疗费用。仅局部生物膜就会在开放的伤口上生长，从而对患者造成重大伤害， 皮肤损伤、烧伤或糖尿病溃疡等。众所周知，生物膜很难根除，在 很大程度上是由于胞外聚合物(EPS)，一种自我产生的细胞外基质，其中 生物膜细菌栖息在。EPS在许多方面对细菌有利，包括调节群体感应，提供 营养物质，并阻断抗生素的运输和宿主的免疫反应。能够积极地渗透到 EPS并在最需要的地方运送抗菌货物将绕过许多这些保护措施，并可能 从而对生物膜的修复产生变革性的影响。2004年首次引入人工自行车式 颗粒(SPP)可以在复杂的生物介质中自行推进，并将货物运送到特定位置。 因此，SPPs在生物膜修复等生物医学应用中具有巨大的潜力。然而，SPP 必须克服生物兼容性、跟踪和控制方面的重大挑战，才能在临床上可行 使用。在这里，我们建议利用新兴的DNA纳米技术领域来开发以尿素酶为动力的 基于DNA折纸的自行式颗粒(DNA-SPP)用于生物膜修复。作为一种模式生物，我们专注于 对研究较多的铜绿假单胞菌的研究。这项研究的目标1将量化对 DNA-SPPs对局部尿素浓度和pH的迁移及其作用程度 尿素梯度中的趋化性。目标2将检验假设，如果DNA-SPP被糖基修饰 水解酶(广泛用于破坏生物膜基质，特别是在铜绿假单胞菌的情况下)， 当它们穿过生物膜基质时，它们会降解生物膜基质，削弱EPS通常提供的保护 对细菌来说。AIM 2的成功将标志着一种模型抗生素(头孢他啶)的更高疗效，它已经 在治疗铜绿假单胞菌生物被膜方面证明有效)。在目标3中，我们将加载头孢他啶 使用对pH敏感的基序(例如，I基序)直接连接到DNA-SPP上，该基序经历结构变化作为响应 PH值降低，因此只在酸性区域释放货物。通过将传送的有效载荷与pH相关联 分布，我们将确认DNA-SPP优先在酸性地区运送货物的能力，在那里硬- 可触及的细菌往往聚集在一起。最后，我们将评估AIMS 2中各种方法的组合效益。 3通过使用DNA-SPP来增加生物膜的渗透性，并将抗生素深入到 生物膜。这项研究的主要成果将是基于DNA的酶动力SPP的设计标准来破坏 并在细胞外基质(ECM)环境中运送货物，这可能对治疗产生重大影响 并将为适用于各种ECM的可定制平台技术奠定基础。 媒介疾病。

PROJECT SUMMARY/ABSTRACT Bacterial biofilms are responsible for most human infections, causing tens of thousands of deaths and billions in medical costs per year. Topical biofilms alone cause significant harm to patients by growing on open wounds, skin lesions, burn injuries, or diabetic ulcers, and elsewhere. Biofilms are notoriously difficult to eradicate, in large part because of the extracellular polymeric substance (EPS), a self-produced extracellular matrix in which biofilm bacteria reside. The EPS benefits bacteria in many ways, including mediating quorum sensing, providing nutrients, and blocking transport of antibiotics and host immune response. The ability to actively penetrate the EPS and deliver anti-bacterial cargo where it is most needed would bypass many of these protections and could thus have a transformative impact on the remediation of biofilms. First introduced in 2004, artificial self-propelled particles (SPPs) can propel themselves through complex biological media and deliver cargo to specific locations. Thus, SPPs hold significant potential for biomedical applications such as biofilm remediation. However, SPPs must overcome significant challenges in the form of biocompatibility, tracking, and control to be viable for clinical use. Here, we propose to leverage the burgeoning field of DNA nanotechnology to develop urease-powered DNA-origami-based self-propelled particles (DNA-SPPs) for biofilm remediation. As a model organism, we focus on the well-studied pathogen Pseudomonas aeruginosa. Aim 1 of this study will quantify the dependence of DNA-SPPs’ locomotion on local urea concentration and pH and elucidate the extent to which they perform chemotaxis in urea gradients. Aim 2 will test the hypothesis that if DNA-SPPs are decorated with glycosyl hydrolase enzymes (which are widely used to disrupt the biofilm matrix, specifically in the case of P. aeruginosa), they will degrade the biofilm matrix as they move through it, weakening the protection the EPS normally provides to bacteria. The success of Aim 2 will be marked by greater efficacy of a model antibiotic (ceftazidime, which has demonstrated efficacy at treating P. aeruginosa biofilms) administered topically. In Aim 3, we will load ceftazidime directly onto DNA-SPPs using a pH-sensitive motif (e.g., I-motif) that undergoes structural changes in response to pH decrease, thus releasing cargo only in acidic regions. By correlating the delivered payload to the pH distribution, we will confirm the ability of DNA-SPPs to deliver cargo preferentially in acidic regions, where hard- to-reach bacteria tend to cluster. Finally, we will assess the combinatorial benefits of the approaches in Aims 2 and 3 by using DNA-SPPs to both increase the biofilm’s permeability and to deliver antibiotics deep inside the biofilm. The major output of this study will be design criteria for DNA-based enzyme-powered SPPs to disrupt and deliver cargo in extracellular matrix (ECM) environments, which could have a major impact on the treatment of biofilms, and will lay the foundation for a customizable platform technology applicable to a wide range of ECM- mediated diseases.