A novel anti-caries approach to modulate virulence of cariogenic biofilms

项目摘要 针对致龋性生物膜的新的化疗方法的开发是具有挑战性的。细菌 在体内，生物膜被包裹在富含胞外多糖(EPS)的基质中。此外，嵌入式EPS 细菌还能创造高度保护和酸性的微环境，促进致龋性生物膜的形成。 牙釉质的酸溶作用。为了克服这些显著的挑战，我们之前的NIH支持 (DE018023)研究开发了一种有效的防龋剂，将食品衍生的抗生物膜制剂结合在一起 (杨梅素和法尼醇)和氟化物。我们证明了这些毒剂的组合严重妥协了 EPS-基质组装和致龋性生物膜的发展，导致高效的防龋治疗 活着。尽管前景看好，但该技术的进一步开发和临床翻译仍存在局限性。 接近。金合欢醇和杨梅素都不溶于水。此外，这些制剂的保留率 在牙齿-生物膜界面可以增强，以最大限度地发挥其体内效果。为了解决这些障碍，我们 已经开发出能够共包裹杨梅素(Myr)和 法尼醇(FAR)是完全水溶性的，对人类使用的实际制剂很重要。 此外，局部应用的NPC可强烈结合到膜和EPS上，并在生物膜中积累。令人兴奋的是， NPC对酸性pH的反应在酸性(病理)比中性(生理)更快地释放药物 PH，大大提高了体外抗生物膜的活性(约为游离剂的20倍)。我们假设 NPC将通过增加溶解度、保留率而大大增强我们联合疗法(CT)的疗效 PH激活的活化剂与氟化物的释放。为了支持我们的假设，目标1将优化 鼻咽癌的物理化学性质，以提高我们的药物的靶向性，从而增强其 抗生物膜功效。我们将重点提高NPC pH响应性药物的释放动力学，以确保 当pH与酸性生物膜环境一致时，药剂的最大释放量。然后，目标2将评估 优化的含MyR和FAR的鼻咽癌细胞(CT-NPC)的体外致龋性生物膜 模特。我们以前已经确定了主要的生物学作用(EPS合成和产酸)和 我们治疗的分子靶点(GTFB，atpD)。因此，我们将研究CT-NPC是如何破坏这些毒力的 使用新方法比CT更有效地评估EPS矩阵的时空发展， 在完整的3D生物膜中，酸性pH生态位和基因原位表达。目标3将评估 CT-NPC在体内破坏致龋性生物膜和减少龋齿的研究 临床相关局部治疗方案下的龋齿。CT-NPC也将被比作黄金 龋病预防(氟化物)和抗菌药物治疗(洗必泰)标准。成功完成 这些目标将导致一种高效的、临床可转化的疗法，这种疗法可能优于目前的疗法。 抗菌斑/防龋齿的方法，并将推动临床研究的配方开发。

Project Summary The development of novel chemotherapeutic approaches against cariogenic biofilms is challenging. Bacteria within biofilms are enmeshed in an exopolysaccharides (EPS)-rich matrix. Furthermore, EPS-embedded bacteria also create highly protected and acidic microenvironments that promote cariogenic biofilm build-up and acid-dissolution of tooth enamel. To overcome these remarkable challenges, our previous NIH supported (DE018023) studies developed a potent anti-caries approach by combining food-derived antibiofilm agents (myricetin and farnesol) with fluoride. We demonstrated that these agents in combination severely compromise EPS-matrix assembly and cariogenic biofilm development, resulting in a highly effective anti-caries therapy in vivo. Despite promising activity, there are limitations for further development and clinical translation of this approach. Both farnesol and myricetin are insoluble in aqueous solutions. In addition, retention of these agents at tooth-biofilm interface could be enhanced to maximize their efficacy in vivo. To address these hurdles, we have developed pH-responsive nanoparticle carriers (NPC) capable of co-encapsulating myricetin (Myr) and farnesol (Far) which were completely water-soluble, important towards practical formulations for human use. Furthermore, topically applied NPC bind avidly to pellicle and EPS, and accumulate within biofilms. Excitingly, NPC respond to acidic pH to release agents more rapidly at acidic (pathological) versus neutral (physiological) pH, greatly improving (~20-fold more effective than free agents) antibiofilm activity in vitro. We hypothesize that NPC will substantially amplify the efficacy of our combination therapy (CT) via increased solubility, retention and pH-activated release of active agents with fluoride. To support our hypothesis, Aim 1 will optimize physicochemical properties of NPC to improve targeted delivery of our agents, and thereby potentiate their antibiofilm efficacy. We will focus on increasing the kinetics of NPC pH-responsive drug release to ensure maximal release of the agents at pH consistent with the acidic biofilm milieu. Then, Aim 2 will evaluate the efficacy of optimized NPCs containing Myr and Far with fluoride (CT-NPC) using our in vitro cariogenic biofilm model. We have previously identified the major biological actions (EPS synthesis and acidogenicity) and molecular targets (gtfB, atpD) of our therapy. Thus, we will investigate how CT-NPC disrupts these virulence properties more effectively than CT using novel methods to assess spatiotemporal development of EPS matrix, acidic pH niches and gene expression in situ within intact 3D biofilms. Aim 3 will evaluate the efficacy of the developed CT-NPC in disrupting cariogenic biofilms and reducing dental caries in vivo using a rodent model of dental caries under clinically-relevant topical treatment regimen. CT-NPC will be also compared to `gold standards' of caries prevention (fluoride) and antimicrobial therapy (chlorhexidine). Successful completion of these aims will lead to a highly efficacious and clinically-translatable therapy that may be superior to current anti-plaque/anti-caries modalities and will motivate formulation development for clinical studies.