Development of Nanoparticle Antibiotic Adjuvant for the Treatment of Chronic Suppurative Otitis Media

项目概要/摘要 我们请求NIH支持研究功能化金纳米团簇作为辅助治疗的用途， 慢性化脓性中耳炎（CSOM）。CSOM的特点是慢性排放的感染中间 最常由铜绿假单胞菌（PA）引起的耳部，是永久性耳聋的主要原因。 听力损失在CSOM中，PA存在于被称为生物膜的细菌群落中。氟喹诺酮滴耳液是 由于其无耳毒性，是主要的治疗方法。然而，这种治疗在完全 根除感染这是由于氟喹诺酮不能靶向 代谢不活跃的细菌，称为生物膜内的持久细胞。持续存在的细胞重新填充 治疗停止后的生物膜生态位，导致CSOM复发。最终的结果是多轮的 手术和与这种疾病的终身斗争。有大量未满足的医疗需求，需要开发新的 针对CSOM生物膜中的持续细胞的医学疗法。 我们的实验室最近创建并验证了一种新的PA CSOM动物模型， PA允许对疾病进行真实的时间跟踪。它模仿人类的情况，持续超过六个月， 局部氟喹诺酮类药物治疗无效，并随时间推移导致毛细胞死亡（即听力损失）。使用 我们独特的PA CSOM模型，我们现在能够测试CSOM的新疗法。因此我们 已经产生了与以下共价缀合的阴离子介电官能化金纳米团簇： 细胞穿透肽（AuNC@CPP）作为氟喹诺酮类的佐剂。我们已经在试管中证明， AuNC@CPP与氧氟沙星的共同给药完全消除了两种细胞中的持续细胞， 固定相和生物膜。AuNC@CPP加氧氟沙星的特殊杀伤能力打开了 有可能开发出第一种专门针对PA CSOM的治疗药物。 我们推测我们的AuNC@CPP与氧氟沙星联合治疗可以在体内根除PA CSOM。因为 我们的初步数据显示，AuNC@CPP不增加细胞内氧氟沙星浓度， 假设AuNC@CPP既作为活性氧（ROS）介导的增强剂， 氧氟沙星致死性和抑制细菌DNA修复。我们建议确定背后的机制， AuNC@CPP通过测量细胞内ROS的产生增强氧氟沙星对持留细胞的致死性， 评价RecA抑制，随后进行与氧氟沙星相比的疗效和耳毒性体内研究 一个人这项工作将开发出第一种专门针对难治性CSOM的辅助治疗方法 并为今后研究该治疗方法在其他慢性生物膜感染中的应用奠定了基础。

Project Summary / Abstract We request NIH support to investigate the use of functionalized gold nanoclusters as an adjuvant therapy for chronic suppurative otitis media (CSOM). CSOM is characterized by a chronically discharging infected middle ear that is most frequently caused by Pseudomonas aeruginosa (PA) and is a leading cause of permanent hearing loss. In CSOM, PA exists in bacterial communities known as biofilms. Fluoroquinolone eardrops are the primary method of treatment due to its non-ototoxicity. However, this treatment is ineffective in fully eradicating the infection. This is a result of the inability of fluoroquinolone to target a subpopulation of metabolically inactive bacteria known as persister cells within the biofilms. Persister cells repopulate the biofilm niche after therapy is discontinued, causing a relapse of CSOM. The end result is multiple rounds of surgery and a lifelong struggle with this disease. There is a large unmet medical need to develop new medical therapies aimed at persister cells in biofilms of CSOM. Our lab has recently created and validated a novel PA CSOM animal model with bioluminescent strains of PA allowing real time tracking of disease. It mimics the human condition by persisting beyond six months, is recalcitrant to topical fluoroquinolone therapy, and leads to hair cell death (i.e. hearing loss) over time. Using our unique model of PA CSOM, we are now able to test novel therapeutics for CSOM. Consequently, we have created an anionic hydrophilically functionalized gold nanocluster covalently conjugated with cell penetrating peptide (AuNC@CPP) as an adjuvant for fluoroquinolones. We have shown, in vitro, that co-administration of the AuNC@CPP with ofloxacin completely eliminates persister cells in both stationary phase and biofilms of PA. The exceptional killing ability of AuNC@CPP plus ofloxacin opens up the possibility to develop the first therapeutic that specifically targets PA CSOM. We theorize that our AuNC@CPP combined with ofloxacin therapy can eradicate PA CSOM in vivo. Because our preliminary data shows that AuNC@CPP does not increase intracellular ofloxacin concentration, we hypothesize that AuNC@CPP acts as both an enhancer of reactive oxygen species (ROS)-mediated ofloxacin lethality and inhibits bacterial DNA repair. We propose to determine the mechanism(s) behind how AuNC@CPP potentiates ofloxacin lethality of persister cells by measuring intracellular ROS production and evaluating RecA inhibition followed by in vivo studies for efficacy and ototoxicity in comparison to ofloxacin alone. The proposed work will develop the first adjuvant therapy that specifically targets recalcitrant CSOM and lays the foundation for future research of this treatment in other chronic biofilm infections.