Bio-Responsive and Immune Protein-Based Therapies for Inhibition of Proteolytic Enzymes in Dental Tissues

摘要 这项多学科的提案将一群技术高超的研究人员的努力转化为史无前例的 促进牙齿软硬组织保存的途径。在使用的方法中受到启发的尖端战略 医学领域被用来选择性地抑制内源性金属蛋白酶，这些酶负责介导 胶原蛋白的降解。在牙科，细胞外基质的这种基本成分的破坏是强烈的 与牙周病的进展和牙齿基质/修复材料的分解有关 界面导致细菌再繁殖和复发的龋齿，这是牙齿修复失败的主要原因。这些 在美国，近80%的拔牙都是由病理因素造成的。为了 针对这一严重的公共卫生问题，提出了两个创新平台。第一种策略是基于 两种有效的基质金属蛋白酶抑制剂--多酚类化合物--栎素和表没食子儿茶素没食子酸酯在纳米胶束中的包埋 可被基质金属蛋白切割。颗粒的外壳由可识别特定氨基酸序列的多肽组成。 并被靶向MMPs切割，导致胶束破裂和抑制剂分子的释放。在……里面 本质上，这个系统使基质金属蛋白酶能够抑制自身。第二个策略是一个免疫调节平台，基于 引入抗基质金属蛋白酶纳米抗体，即源自骆驼重链抗体的单域抗体，以 抑制/停止基质金属蛋白酶的蛋白分解活性。作为纳米颗粒或纳米体输送载体，牙科底漆配方和 一种新设计的多功能水凝胶分别用于粘接面和牙周袋。至 验证设计的平台、芯片上的牙齿和动物模型是否正在添加，以便快速跟踪它们的转换 变成了临床护理。这一建议的创新之处在于：(1)采用开创性的方法来停用 牙齿组织中的MMPs基于酶反应纳米颗粒来传递抑制剂，以及(2)引入一种 基于纳米体技术对基质金属蛋白酶活性进行调控的新范式。在完成这个项目后，我们 将对这两个平台进行量身定制和验证，以便在与当前临床 用于治疗牙周炎和在修复过程中杂交牙本质的技术，这将使 弥合尖端研究和改进的病人护理之间的差距。此外，这些平台可以是 结合或定制成更复杂和多功能的实体，并被编程为对各种酶做出反应 和细胞因子，这为发展创新的牙齿组织再生/保存提供了新的机会 减少牙齿缺失的治疗方法，特别是在高度脆弱的人群中。

SUMMARY This multidisciplinary proposal translates the efforts of a highly skilled group of researchers forging unprecedented pathways to promote hard and soft dental tissues preservation. Cutting-edge strategies inspired in approaches used in the medical field are employed to inhibit selectively endogenous metalloproteinases responsible for mediating the degradation of the collagen. In Dentistry, the destruction of this essential component of the extracellular matrix is strongly associated with the progression of periodontal disease and the breakdown of the dental substrate/restorative material interface leading to bacteria recolonization and recurrent caries, the primary cause of failure of dental restorations. These pathological conditions together are responsible for almost 80% of the tooth extractions in the United States. In order to address this serious public health issue, two innovative platforms are proposed. The first strategy is based on the encapsulation of two potent MMP inhibitors, the polyphenols quercetin and epigallocatechin gallate, in nano micelles cleavable by MMPs. The shell of the particles is composed of peptides with specific sequences of amino acids recognized and cleaved by the targeted MMPs, leading to rupture of the micelles and the release of the inhibitor molecules. In essence, this system enables the MMP to inhibit itself. The second strategy is an immunomodulatory platform based on the introduction of anti-MMP nanobodies, single domain antibodies derived from llama heavy-chain only antibodies, to inhibit/cease MMP proteolytic activity. As nanoparticles or nanobodies delivery vehicles, a dental primer formulation and a newly-designed multifunctional hydrogel are used for the adhesive interface and periodontal pockets, respectively. To validate the designed platforms, tooth-on-a-chip and animal models are being added in order to fast-track their translation into clinical care. The innovation of this proposal stems from: (1) the employment of a pioneering approach to inactivate MMPs in dental tissues based on enzymatic-responsive nanoparticles to deliver inhibitors, and (2) the introduction of a new paradigm based on the modulation of the MMPs activity by nanobody technology. After completing this project, we will have these two platforms tailored and validated to be delivered in vehicles compatible with the current clinical techniques employed to treat periodontitis and to hybridize the dentin during the restorative procedures, which will allow to bridge the gap between cutting-edge research and improved patient care. In addition, these platforms can be either combined or tailored in more complex and multi-functional entities and programmed to respond to a variety of enzymes and cytokines, which provides new opportunities to develop innovative dental tissue regeneration/preservation therapeutics to minimize tooth loss especially in highly vulnerable population.