Nanoparticle-based Antimicrobial Photochemotherapy in Biofilms

描述（由申请人提供）：生长在牙齿表面的生物膜和排列在牙周袋/牙龈沟（龈下牙菌斑）上的上皮细胞是自然界中存在的最复杂的生物膜之一，可导致牙周疾病。我们的长期目标是开发一种临床合适的方法来提高光活性化合物（或光敏剂）在人类牙菌斑中的渗透和有效性，以预防、控制和/或治疗牙周炎。需要验证的假设是：a)负载亚甲基蓝（MB）的聚合物纳米颗粒可以在体外完全穿透由天然人类牙菌斑进化而来的口腔多物种生物膜；b)负载MB的纳米颗粒敏化后，再暴露在665 nm的红光下，可能会在体外完全光破坏这些微生物生物膜。该R21提案的具体目标是：1。制备和表征mb负载的生物相容性和可生物降解聚合物纳米颗粒。我们将(i)制备具有阴离子和正电荷表面的聚（乳酸-共乙醇酸）MB包封纳米颗粒，（ii）通过ZetaPALS仪器和扫描电子显微镜评估其大小、表面电荷和形态，（iii）通过光谱验证其MB包封能力和效率，（iv）研究MB在模拟环境中的体外释放，以确保有足够的药物可用于有效的光动力治疗。2. 研究负载mb纳米颗粒在生物膜中的分布及其对生物膜微生物的光毒性。我们(i)利用唾液和人类牙菌斑在体外培养多物种微观实验室生物膜，（ii）利用透射电子显微镜研究细菌对mb负载纳米颗粒的内化，（iii）利用共聚焦扫描激光显微镜评估它们对生物膜的渗透，以及（iv）通过总活菌计数评估它们对生物膜物种的光动力效应，利用DNA探针和PCR鉴定物种。

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.