Biofilm Elimination and Caries Prevention using Multifunctional Nanocatalysts

尽管生物膜相关的口腔疾病如龋齿的患病率很高，但临床上没有 破坏有毒生物膜的有效疗法，在美国每年产生超过400亿美元的支出。 生龋生物膜的有效控制是众所周知的挑战，因为细菌陷入在一个 富含胞外多糖（EPS）的保护性细胞外基质。此外，EPS-缠结的细菌产生 高度酸性的微环境，促进牙釉质的酸溶解，导致牙本质损害的发生。 龋齿目前的抗微生物剂不能破坏EPS基质或影响其物理性质。 化学方面的龋齿，往往不能有效地杀死微生物内的生物膜，导致有限的 体内功效为了克服这些显著的障碍，我们开发了一种令人兴奋的治疗策略 使用具有催化活性和pH响应特性的生物相容性氧化铁纳米颗粒（IO-NP）， 显示抗生物膜和抗龋齿作用。IO-NP在酸性pH值下表现出过氧化物酶样活性， 原位快速活化过氧化氢（H2 O2），同时降解保护性生物膜EPS基质 并在5分钟内以优异的功效（细胞存活率的对数减少>5）杀死包埋的细菌。此外，委员会认为， IO-NP还在酸性条件下减少磷灰石脱矿。我们假设IO-NP与 H2 O2通过纳米催化作用增强抗生物膜效应并预防体内龋齿的发生， 在酸性pH下增强的抗菌剂、EPS降解剂和脱矿物质阻断剂的原位产生。 这项工作的意义在于开发一种可行且上级的抗生物膜和防龋方法 与目前的化学品模式相比。为了验证我们的假设，我们将优化IO-NP/H2 O2的功效， 进一步提高抗生物膜和脱矿物质阻断活性（目的1）。我们将提高催化活性 的IO-NP的纳米粒子，并探讨各种葡聚糖的影响， 基于涂层以增加IO-NP在生物膜结构内的定位。此外，我们还将 磷酸钙转化为IO-NP，以增强其对脱矿作用。然后，我们将评估 增强的IO-NP/H2 O2用于使用混合物种的致龋生物膜模型的体外生物膜控制（目的2）。我们 将使用时间推移共聚焦和生物物理方法进一步阐明IO-NP/H2 O2的生物学作用 以检查EPS-基质的时空降解、细菌杀灭和完整生物膜内的内聚性。 将使用显微硬度和显微CT评估对釉质脱矿的影响。在目标3中，我们 评价开发的IO-NP/H2 O2疗法在阻碍致龋生物膜方面的生物相容性和有效性 以及使用啮齿动物龋齿模型和临床相关的局部治疗在体内龋齿损伤的发生 方案.这些目标的成功实现将为进一步的制剂开发提供一个框架 临床疗效研究。重要的是，IO-NP可以以低成本大规模合成，而H2 O2是 容易获得，这可能导致用于局部使用的可行的新的抗生物膜/抗龋齿治疗平台。

Despite the high prevalence of biofilm-related oral diseases such as dental caries, there are no clinically effective therapies to disrupt virulent biofilms, resulting in >$40 billion expenditures annually in the US. Effective control of cariogenic biofilms is notoriously challenging because the bacteria are enmeshed in a protective extracellular matrix rich in exopolysaccharides (EPS). Furthermore, EPS-enmeshed bacteria create highly acidic microenvironments that promote acid-dissolution of tooth enamel, leading to the onset of dental caries. Current antimicrobial agents are incapable of disrupting the EPS matrix or affecting the physico- chemical aspects of caries and often fail to efficiently kill the microbes within biofilms, resulting in limited efficacy in vivo. To overcome these remarkable hurdles, we have developed an exciting therapeutic strategy using biocompatible iron oxide nanoparticles (IO-NP) with catalytic activity and pH-responsive properties that display both anti-biofilm and anti-caries actions. IO-NP exhibit peroxidase-like activity at acidic pH values that rapidly activates hydrogen peroxide (H2O2) in situ to simultaneously degrade the protective biofilm EPS-matrix and kill embedded bacteria with exceptional efficacy (>5-log reduction of cell viability) in 5 minutes. Moreover, IO-NP also reduce apatite demineralization in acidic conditions. We hypothesize that IO-NP synergizes with H2O2 to amplify anti-biofilm effects and prevent the onset of dental caries in vivo via nanocatalysis and enhanced in situ production of antibacterial, EPS-degrading and demineralization-blocking agents at acidic pH. The significance of this work is to develop a feasible and superior anti-biofilm and caries preventive approach compared to current chemical modalities. To test our hypothesis, we will optimize the efficacy of IO-NP/H2O2 to further improve anti-biofilm and demineralizing-blocking activities (Aim 1). We will enhance the catalytic activity of IO-NP by inclusion of specific metal salts into the nanoparticles, and explore the effects of various dextran- based coatings to increase IO-NP localization within biofilm structure. Furthermore, we will incorporate calcium-phosphate into IO-NP to enhance its effects on demineralization. Then, we will evaluate the efficacy of enhanced IO-NP/H2O2 for biofilm control in vitro using a mixed-species, cariogenic biofilm model (Aim 2). We will further elucidate the biological actions of IO-NP/H2O2 using time-lapsed confocal and biophysical methods to examine spatiotemporal degradation of EPS-matrix, bacterial killing and cohesiveness within intact biofilms. The effects on enamel demineralization will be assessed using micro-hardness and micro-CT. In Aim 3, we will evaluate the biocompatibility and efficacy of the developed IO-NP/H2O2 therapy in hindering cariogenic biofilms and the onset of carious lesions in vivo using a rodent caries model with a clinically-relevant topical treatment regimen. Successful completion of these aims will provide a framework for further formulation development and clinical efficacy studies. Importantly, IO-NP can be synthesized with low cost at large scale while H2O2 is readily available, which could lead to a feasible new anti-biofilm/anti-caries therapeutic platform for topical use.