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

项目摘要/摘要 我们请求美国国立卫生研究院提供支持，以研究功能化的金纳米簇作为辅助治疗的应用 慢性化脓性中耳炎。CSOM的特点是慢性排出感染的中间体 由铜绿假单胞菌(PA)引起的最常见的耳部疾病，是导致永久性耳炎的主要原因 听力损失。在CSOM中，PA存在于称为生物膜的细菌群落中。氟喹诺酮类滴耳剂 由于其无耳毒性，因此是主要的治疗方法。然而，这种治疗完全无效。 根除感染。这是由于氟喹诺酮类药物不能针对 新陈代谢不活跃的细菌，称为生物膜内的持久细胞。永存细胞重新填充 治疗后生物被膜龛停止，导致CSOM复发。最终的结果是多轮的 手术和与这种疾病的终生斗争。有大量未得到满足的医疗需求需要开发新的 针对CSOM生物膜中的宿存细胞的药物治疗。 我们的实验室最近建立并验证了一种新的PA CSOM动物模型，该模型具有生物发光菌株 PA允许实时跟踪疾病。它通过持续六个月以上来模拟人类的情况，是 对局部氟喹诺酮类药物治疗顽固，随着时间的推移会导致毛细胞死亡(即听力损失)。vbl.使用 通过我们独特的PA CSOM模型，我们现在能够测试CSOM的新疗法。因此，我们 已经形成了共价连接的阴离子亲水功能化的金纳米簇 细胞穿透肽(AuNC@cpp)作为氟喹诺酮类药物的佐剂。我们已经证明，在体外， AUNC@CPP与氧氟沙星联合给药可完全消除 PA的固定相和生物膜。AuNC@CPP加氧氟沙星的非凡杀伤力打开了 有可能开发出第一种专门针对PA CSOM的治疗方法。 我们推测，我们的AuNC@CPP联合氧氟沙星治疗可以在体内根除PA CSOM。因为 我们的初步数据显示，AuNC@CPP不会增加氧氟沙星的细胞内浓度 假设AuNC@CPP既是ROS介导的增强剂 氧氟沙星致死和抑制细菌DNA修复。我们建议确定(S)背后的机制是如何 AuNC@CPP通过测量细胞内ROS的产生和增强氧氟沙星对宿存细胞的杀伤力 与氧氟沙星进行体内疗效和耳毒性研究后评价RecA抑制作用 独自一人。这项拟议的工作将开发第一种专门针对顽固性CSOM的辅助疗法。 为今后该疗法治疗其他慢性生物被膜感染的研究奠定了基础。

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.