Nanoparticle-based Antimicrobial Photochemotherapy in Biofilms

描述(申请人提供)：附着在牙齿表面和牙周袋/牙龈沟(牙周下牙菌斑)内的上皮细胞的生物膜是自然界中存在的最复杂的生物膜之一，会导致牙周疾病。我们的长期目标是开发一种临床上合适的方法来增强光活性化合物(或光敏剂)在人类牙菌斑中的渗透性和有效性，以预防、控制和/或治疗牙周炎。要测试的假设是：a)载亚甲基蓝(MB)的聚合物纳米颗粒在体外可以完全穿透天然人类牙菌斑形成的多物种口腔生物膜，以及b)这些微生物生物膜可能在体外被MB纳米颗粒敏化并暴露在665 nm的红光下完全被破坏。本R21提案的具体目的是：1.制备和表征MB负载的生物相容和可生物降解的聚合物纳米颗粒。我们将(I)制备表面带阴离子和正电荷的MB包裹的聚乳酸-乙醇酸共聚物纳米颗粒，(Ii)通过ZetaPALS仪器和扫描电子显微镜评估其尺寸、表面电荷和形貌，(Iii)通过光谱分析验证其包裹MB的能力和效率，以及(Iv)考察MB在模拟环境中的体外释放，以确保有足够的试剂可用于有效的光动力学治疗。2.研究亚甲基蓝纳米粒在生物膜中的分布及其对生物膜微生物的体外光毒性。我们(I)利用唾液和人类牙菌斑在体外建立多物种微宇宙实验室生物膜，(Ii)使用透射电子显微镜研究细菌对MB负载纳米颗粒的内化，(Iii)通过共聚焦扫描激光显微镜评估它们对生物膜的渗透，以及(Iv)通过总活菌计数来评估它们对生物膜物种的光动力学效应，这是一种使用DNA探针和聚合酶链式反应进行物种鉴定的方法。 与公众健康相关：该项目试图利用可见光和装载有光活性药物的微小颗粒来靶向牙菌斑细菌。我们的研究可能有助于将光动力疗法发展为一种预防、控制和/或治疗牙周病的非侵入性技术。

DESCRIPTION (provided by applicant): Biofilms that colonize tooth surfaces and epithelial cells lining the periodontal pocket/gingival sulcus (subgingival dental plaques) are among the most complex biofilms that exist in nature and lead to periodontal diseases. Our long-term goal is to develop a clinically appropriate way to enhance the penetration and effectiveness of photoactive compounds (or photosensitizers) in human dental plaque for prevention, control and/or treatment of periodontitis. The hypotheses to be tested are: a) Methylene blue (MB)-loaded polymeric nanoparticles can fully penetrate oral multispecies biofilms evolved from natural human dental plaque in vitro, and b) Complete photodestruction of these microbial biofilms in vitro may be possible after their sensitization with MB-loaded nanoparticles followed by exposure to red light of 665 nm. The specific aims of this R21 proposal are: 1. To prepare and characterize MB-loaded biocompatible and biodegradable polymeric nanoparticles. We will (i) prepare MB-encapsulated nanoparticles of poly(lactic-co-glycolic acid) with anionic and positive surface charge, (ii) evaluate their size, surface charge, and morphology by ZetaPALS instrument and scanning electron microscopy, (iii) verify their capacity and efficiency of MB encapsulation by spectroscopy, and (iv) investigate the in vitro release of MB in simulated environment to insure that sufficient agent is available for effective photodynamic therapy. 2. To investigate the distribution of MB-loaded nanoparticles in biofilms and their phototoxicity on biofilm microorganisms in vitro. We (i) develop multi-species microcosm laboratory biofilms in vitro using saliva and human dental plaque, (ii) investigate the internalization of MB-loaded nanoparticles by bacteria using transmission electron microscopy, (iii) assess their penetration into the biofilms by confocal scanning laser microscopy, and (iv) evaluate their photodynamic effects on biofilm species by total viable counts, an species identification using DNA probes and PCR. PUBLIC HEALTH RELEVANCE: This project is an attempt to target dental plaque bacteria using visible light and microscopic particles loaded with a photoactive drug. Our research may help develop photodynamic therapy as a non-invasive technique for prevention, control and/or treatment of periodontal disease.