厚朴酚/和厚朴酚-PAMAM-COOH靶向缓释系统的防龋作用及机制研究

Dental caries is a wide-spread infectious disease caused by bacterial colonization on tooth surfaces. Severe caries can progress to pulpitis, apical periodontitis, and even loss of teeth. Furthermore, systemic diseases, such as cardiovascular diseases, can be induced. Therefore, it is generally considered to be very important to detect and treat the caries. However, despite the effort of the whole world all these years, dental caries continues to be a major health problem. Several kinds of anti-caries agents derived from herbal material have been proved effective. Because of their property of insolubility and instability, none of them had been used in clinical caries prevention. Magnolol and honokiol are anti-caries agents extracted from the magnolia bark and had been proved an excellent antimicrobial property. In order to improve the solubility and the stability of magnolol and honokiol, in this study we will load the drugs in the G4.5-PAMAM-COOH and certify the loading rate and release profile of the magnolol and honokiol. Then the targeted binding capability and the remineralization effect of the magnolol/ Honokiol- loaded PAMAM-COOH on demineralized enamel will be tested. The anti-bacterial property of the drug loaded PAMAM will be tested on five cariogenic bacteria and the mechanisms will be investigated using a high-throughput active attachment biofilm model inoculated by saliva. The result of this study will help to make use of magnolol and honokiol from “bench to beside”. Besides, it will set a new direction for the clinical application of herbal material in caries prevention.

龋病是口腔常见、多发的感染性疾病，严重危害口腔及全身健康。多年来龋病防治受到广泛关注，但至今尚未得到解决。天然防龋药物是龋病防治的重要研究方向，目前已发现多种天然药物提取物具备优越抗龋性，但多数难溶于水或不稳定，临床应用受到限制，厚朴提取物厚朴酚及和厚朴酚即是这一类药物。为了增加厚朴酚及和厚朴酚的溶解度及稳定性，同时赋予药物靶向缓释及再矿化功能，本研究拟将厚朴酚及和厚朴酚载入具有靶向再矿化功能的羧基改性PAMAM分子内疏水空腔中，制备出具有优良载药率及缓释效率的厚朴酚/和厚朴酚-PAMAM-COOH缓释系统，进一步通过荧光示踪和体外再矿化实验研究其靶向再矿化功能及机制，通过研究缓释系统对主要致龋菌抗菌性及对唾液多菌种生物膜形成及清除的影响，探讨其抗菌性及抗菌机制。本研究可为厚朴酚及和厚朴酚从实验室研究到临床应用的转化提供理论基础，同时为天然药物在龋病防治中的应用开辟新的方向。

龋病防治受到广泛关注，但至今尚未得到解决。天然防龋药物是龋病防治的重要研究方向，目前已发现多种天然药物提取物具备优越抗龋性，但多数难溶于水或不稳定，临床应用受到限制，厚朴提取物厚朴酚及和厚朴酚即是这一类药物。为了增加厚朴酚及和厚朴酚的溶解度及稳定性，同时赋予药物靶向缓释及再矿化功能，本研究成功将厚朴酚及和厚朴酚载入具有再矿化功能的羧基改性PAMAM分子(P-COOH)内疏水空腔中，制备了具有优良载药率及缓释效率的厚朴酚/和厚朴酚-PAMAM-COOH缓释系统(PM/PH)。通过筛选，确立最佳制备条件，使用DMSO、DDW 以体积比1:7 混合的共溶法，P-COOH浓度为6 mg/mL，厚朴酚及和厚朴酚浓度为2mg/mL，载药72h，得到PM的载药率为28.24±0.77%，包封率为84.72±1.84%，PH载药率为30.72±0.47%，包封率为92.16±1.41%。其在液体环境中缓释时间达264小时，且在酸性环境中释放速率更慢，这一缓释机制通过分子模拟得以解释；通过早期龋体外再矿化实验验证了缓释系统具有使釉质表层下再矿化的功能，TMR结果提示实验组脱矿深度小于对照组，而矿物损失量低于对照组；通过抑菌实验发现缓释剂能抑制变异链球菌（Sm）浮游菌生长(2 mg/mL PH 及4 mg/mL PM)并抑制生物膜形成（(4 mg/mL PH 及6 mg/mL PM)），且该抑菌作用具有持续性，PH的最低有效浓度低于PM，同时抑菌效果更强；进一步使用大鼠龋病模型验证PH在体内的抗龋作用, 结果发现使用PH组的大鼠，不论是一周内每日使用，还是每间隔3天使用一次，其Keyes计分均小于对照组，通过micro-CT计算其残余矿物体积和平均密度均高于对照组。通过本研究发现PH具有开发为预防龋病的潜在应用价值，为龋病防治工作提供新思路，但尚需要进一步研究以达到临床转化。