基于D-构型生物活性肽的牙体靶向抗菌涂层的构建及其龋病防治效能评价

Dental caries is one of the most prevalent chronic diseases of human being. Without proper dental plaque control, the development of caries can be aggressive. Antimicrobial peptides (AMPs) with anti-biofilm and immunomodulatory properties are promising novel agents for combating biofilm-mediated infections. Since ecological preventive strategies are raising concern, we suggest that the establishment of tooth targeting bioactive peptides based antibacterial coatings should bring new insight into caries prevention and control. Based on our previous pilot investigations, hydroxyapatite binding peptides (HABPs) are proved to be suitable carriers due to their programmability and tooth-binding affinity. By linking a multifunctional innate defense regulator peptide-1018 (IDR-1018) to a stronger binder (s-HABP), enhanced antimicrobial activity of the chimeric peptide (s-HABP@IDR-1018) on HA surface was confirmed. In the present project, a protease-resistant D-enantiomeric peptide DJK-5 will be incorporated with the HABPs-based delivery system. DJK-5 and IDR-1018 share the same anti-biofilm mechanism, while the former is considerably more active and stable for endodontic applications. Replica exchange molecular dynamics (REMD) simulation in combination with experimental data will provide detailed information about the organic-inorganic interface. The adsorption and release kinetics of fluorescein isothiocyanate (FITC) labeled chimeric peptides on HA surface will be investigated in vitro. The activities of chimeric peptides can be optimized by carefully choosing the synergistic functional groups and designing linkers to avoid undesirable interferences. The biological characteristics of D-enantiomeric peptide based antibacterial coatings will be investigated in vitro and in a rodent caries model for the first time. This project aims to provide valuable information on adsorption and desorption behaviors of the bioactive peptides on HA surface, propose general rules for rational design of tooth-targeting chimeric peptides, and pave novel alternative pathways for caries prevention and control in the clinical practice.

龋病以牙体组织进行性破坏为主要特征，是最常见的口腔慢性感染性疾病。新发现的生物活性肽（兼具抗生物膜和免疫调节活性）有较好的龋病防治潜能。项目提出构建亲牙体的嵌合肽抗菌涂层，利用羟磷灰石结合肽（HABPs）特殊的“吸附-解吸附”界面行为可实现其负载药物在牙面的靶向涂布和释控。项目组已证实HABPs可增强免疫调节肽IDR-1018的牙面抗菌活性。本项目拟选用与IDR-1018作用机制相似但兼具抗菌性和稳定性的D-构型生物活性肽（DJK-5）进行嵌合肽抗菌涂层的构建。分子动力学模拟结合实验数据反馈优化HABPs@DJK-5活性，体外模型中实现HABPs@DJK-5的牙面涂布和释控，进而在龋病动物模型中验证利用HABPs@DJK-5进行龋病干预的效能。项目的实施将提供实验室证据以丰富“有机-无机”界面分子识别机制假说并为生物活性肽的口腔靶向应用提供理论依据。

龋病和根尖周炎的发生均与细菌的持续发展有关，抗菌肽DJK5具有较好的抗菌性能，但应用受限于难以控制和维持局部有效药物浓度，而羟基磷灰石结合肽（HABPs）对牙齿主要矿物成分羟基磷灰石（HA）具有标靶亲和力。项目组对前期噬菌体展示工作中获取的87个非重复HABPs序列的标靶亲和力进行评价，通过免疫荧光技术成功筛选出4条高亲和力多肽，并使用滴度测定、质量耗散法、荧光图像法等验证其对羟基磷灰石的标靶亲和力。REMD 模拟肽序列稳态结构并结合圆二色谱、HA标靶亲和数据等反馈优化分子模拟参数，进而选择能与 AMPs发挥协同作用的HABPs基团，最后使用微量肉汤稀释法和HA体外生物膜模型结合激光共聚焦显微镜（CLSM）评价复合肽的生物性能。结果显示，M330和M423对HA的亲和力最高，受低pH影响最小，且体外合成的HABPs@AMPs对HA有一定的亲和行为。厌氧下，DJK5、M423@DJK5和DJK5@M423对变异链球菌和粪肠球菌的最小抑菌浓度（MIC100）为31.25μg/mL，最小抑制生物膜浓度（MBIC100）为31.25μg/mL。体外HA生物膜模型中，5μg/mL DJK5@M423处理三天厌氧变异链球菌生物膜和粪肠球菌生物膜3min，灭菌效果略优于DJK5，但没有统计学差异；10μg/mL M423@DJK5和DJK5@M423与HA过夜孵育后在4h内可明显抑制细菌生物膜形成，优于DJK5（P＜0.05）。结合HABPs的改良抗菌肽复合物HABPs@AMPs对浮游状态下的口腔常见致病菌及生物膜具备一定的抗菌能力，为口腔感染的控制提供了一种新的潜在措施。

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