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抑制多菌种生物膜的生物响应纳米疗法用于龋病预防|Bioactive Materials

BAM BioactMater生物活性材料 2022-09-30

近期,武汉大学口腔医院杜民权团队西南大学材料与能源学院许志刚团队在科爱出版创办的期刊Bioactive Materials上联合发表原创文章:抑制多菌种生物膜的生物响应纳米疗法用于龋病预防。该工作开发了一种简单有效的纳米光动力疗法,能有效穿透并杀伤深层生物膜中的致龋菌。该疗法有潜力用于儿童的日常口腔清洁,从而预防早期儿童龋。


01

研究内容简介


龋病是一种牙体硬组织渐行性破坏的慢性感染性疾病。早期儿童龋(Early childhood caries, ECC)是一种发展迅猛,龋坏程度严重的特殊类型龋,累及约 90%的 3-5岁儿童。如果患儿龋病早期未得到及时治疗,其发音、咀嚼、牙颌发育以及心理健康均会受到影响。细菌、宿主、食物和时间四联因素综合作用下将导致 ECC 的快速进展。微生物插入到其自身分泌的胞外多糖(Extracellular polysaccharides, EPS)等物质构成的细胞外基质中,形成动态复杂的菌斑生物膜结构。健康状态下,菌斑生物膜能抵御外部环境的变化,维持牙齿表面的相对稳定和平衡。当碳水化合物摄入过多或口腔卫生情况较差,产酸耐酸性细菌成为优势菌,从而破坏生物膜的菌群平衡,形成以产酸耐酸菌为主导菌群的致龋性生物膜致龋性生物膜的微环境呈酸性状态,pH 值约为 4.5-5.5长期持续的酸性微环境可引起牙体硬组织脱矿,最终导致龋病的形成。因此,有效抑制致龋性生物膜对 ECC 的预防和治疗具有重要意义。


光动力抗菌疗法(Antimicrobial photodynamic therapy, aPDT)通过光源激发光敏剂产生活性氧簇,在病变部位进行特异性抗菌治疗的新型技术。aPDT 具有无创无痛、抗菌效率强以及不易产生耐药性等优点,广泛应用于口腔感染性疾病的控制。然而,大多数光敏剂具有疏水性,不能有效深入到结构复杂的致龋性生物膜内部;同时,带负性电荷的光敏剂,难以进入同样呈负电性的细菌和生物膜中,严重影响了 aPDT 的临床应用。近些年,基于病变部位的特殊微环境,大量研究针对光敏剂进行修饰和改性,以增强其对细菌和生物膜的吸附渗透能力,使其在病变部位具有足够的滞留率。其中,pH 响应性的两亲性二嵌段共聚物在中性条件下可以形成粒径较小的纳米胶束,并能通过自组装包载疏水性药物,提高药物的分散性和吸附性;在病变部位酸性条件下,聚合物纳米胶束能够 pH 响应性破裂分解,局部释放出大量药物;而在正常口腔环境中,纳米颗粒维持稳定,不影响正常组织的功能和菌群平衡。


基于以上研究背景,作者开发了负载光敏剂二氢卟吩 e6 的 pH 响应性聚合物纳米颗粒(MPP-Ce6),以探究纳米技术对 aPDT 的增效作用。通过多种技术对 MPP-Ce6 进行理化性质表征,并探究该纳米颗粒是否增强了游离 Ce6 对致龋性细菌和生物膜的吸附渗透能力,同时评估 MPP-Ce6 介导的 aPDT 对致龋性细菌和生物膜的抗菌、抗生物膜能力以及大鼠体内抗龋作用Scheme


Scheme: Illustration of the bioresponsive polymeric nanoparticles loaded with Ce6 (MPP-Ce6) in mediating aPDT for preventing dental caries. Dental caries has multifactorial etiologies, including host factors, bacteria, substrate, and time. Cariogenic microbiota-mediated biofilms, the main factor of dental caries, create an acidic microenvironment and lead to the demineralization of teeth. The bioresponsive nanosized MPP-Ce6 could specifically release Ce6 in the microenvironment of acidic biofilms. After irradiation with a 660-nm laser, multispecies cariogenic bacteria were significantly killed, resulting in the excellent prevention efficiency of dental caries.


一、基于致龋性生物膜理化特征设计合成 pH 响应性纳米颗粒(Fig 1,2)

针对龋病的酸性微环境,作者成功合成了酸响应聚合物纳米颗粒 MPP,并将光敏剂 Ce6 自组装过程中载入 MPP 内部,形成 MPP-Ce6 新型载药纳米颗粒。MPP-Ce6粒径较小且均一,经 660 nm 激光照射后,高效产生 ROS。实验表明,经两亲性聚合物纳米载体 MPP 的修饰,显著提高游离 Ce6 的溶解性、分散性及稳定性,并将 Ce6 的 Zeta 电位转换成正电荷。MPP-Ce6 在生理条件下形成结构稳定,但在酸性条件下,通过叔胺基团的质子化,聚合物DPA 嵌段的疏水-亲水转换,引起纳米颗粒的破裂,释放 Ce6。基于以上纳米化改性的优点,作者证实了 MPP-Ce6 显著增强了浮游致龋菌对游离 Ce6 的内吞摄入作用,同时也提高了游离 Ce6 对致龋性生物膜的吸附渗透能力,保证了致龋性病变部位 Ce6 的有效浓度, 为提高 aPDT 的特异性抗菌及抗龋提供了基础。


Fig. 1. Characterization of multispecies cariogenic bacteria and MPP-Ce6. A) Illustration of bioresponsive release and interaction of MPP-Ce6 with cariogenic biofilm (electrostatic attraction and acidic trigger). B) pH changes of three representative cariogenic bacteria as a function of time (Medium, brain heart infusion broth; S.m, S. mutans; S.s, S. sobrinus; S.a, S. sanguinis; Mix, the mixture of the abovementioned bacterial suspension). C) Zeta potentials of three representative cariogenic bacteria. D) UV–vis spectra of MPP, MPP-Ce6, and Free-Ce6. E) TEM image of MPP-Ce6. F) Size distributions of MPP and MPP-Ce6 as detected by DLS. G) Zeta potentials of MPP, MPP-Ce6, and Free-Ce6. H) Photographs of MPP-Ce6 and Free-Ce6 suspensions at 0 h and 48 h. I) ROS generation of MPP-Ce6 under the 660-nm laser as measured by UV absorption of DPBF. J) Release profiles of Ce6 from MPP-Ce6 at different pH values (pH 5.0, 6.5, and 7.4).


Fig. 2. Internalization and penetration of MPP-Ce6 into planktonic bacteria and biofilms. A) Uptake efficiency of MPP-Ce6 and Free-Ce6 into S. mutans, S. sobrinus, and S. sanguinis. B) 3D images of multispecies biofilms detected by CLSM after incubation with PBS, Free-Ce6, and MPP-Ce6 (green fluorescence, multispecies biofilm labeled by SYTO 9; red fluorescence, Ce6; scale bar, 200 μm). C) Quantification analysis to measure the proportion of fluorescence intensity of Ce6 (red) versus biofilm (green). D) The penetration depth of MPP-Ce6 and Free-Ce6 in the biofilms.


二、MPP-Ce6 介导的 aPDT 体外抗菌及抗生物膜作用研究 (Fig 3,4)

MPP-Ce6 纳米颗粒具有较低的细胞暗毒性。MPP-Ce6 介导的 aPDT 对浮游致龋菌具有优异的抗菌作用,显著提高了游离 Ce6 的抗菌能力,其抗菌作用可能是通过破坏细菌的结构和形态,进而导致细菌的死亡。MPP-Ce6 介导的 aPDT 不能清除已形成的生物膜,但是能够有效灭活生物膜中的细菌,从而降低致龋性生物膜的致病性。MPP-Ce6 介导的 aPDT 通过抑制细菌的生长和 EPS 的产生,有效抑制短期和长期生物膜的形成。


Fig. 3. Biocompatibility and antibacterial efficiency of MPP-Ce6-mediated aPDT in vitro. A) Cell viability of normal human oral cells (HOKs and HGFs) upon treatment with MPP-Ce6 at different concentrations (0–5 μg mL−1 Ce6 equivalent; ns, p > 0.05). B) Antibacterial efficiency of MPP-Ce6-mediated aPDT against S. mutans, S. sobrinus, and S. sanguinis (0–5 μg mL−1 Ce6 equivalent; the dotted line represents 99% inhibition rate). C) Antibacterial efficiency of different treatments against S. mutans, S. sobrinus and S. sanguinis (Control, treatment with BHI only; MPP, treatment with MPP only; Free-Ce6, treatment with free Ce6 only; MPP-Ce6, treatment with MPP-Ce6 only; L, treatment with laser only; MPP+L, treatment with MPP upon laser irradiation; Free-Ce6+L, treatment with free Ce6 upon laser irradiation; MPP-Ce6+L, treatment with MPP-Ce6 upon laser irradiation; concentration of MPP-Ce6 and free Ce6, 0.1 μg mL−1 Ce6 equivalent for S. mutans, 0.2 μg mL−1 Ce6 equivalent for S. sobrinus and S. sanguinis; ∗∗, p < 0.01; ∗∗∗, p < 0.001). D) Representative images of three species of bacterial colonies in the Control, Free-Ce6+L, and MPP-Ce6+L groups (dilution factors of 1:104 for S. mutans, 1:103 for S. sobrinus, and 1:106 for S. sanguinis). E) Morphological changes in bacteria treated with or without MPP-Ce6 upon laser irradiation by TEM (red arrows indicate the destruction of the bacterial structure; framed areas are enlarged on the right).


Fig. 4. Antibiofilm efficiency of MPP-Ce6-mediated aPDT in vitro. A) Construction of multispecies biofilms detected by FISH analysis (scale bar, 200 μm; green, S. mutans; blue, S. sobrinus; red, S. sanguinis). B) Scheme of dual-mode antibiofilm efficiency (biofilm eradication test and biofilm inhibition test). C) Live/dead bacterial staining of multispecies biofilms after different treatments for the biofilm eradication test (scale bar, 200 μm; green, live bacteria; red, dead bacteria; Control, treatment with BHI only; L, treatment with laser only; MPP-Ce6, treatment with MPP-Ce6 only; MPP-Ce6+L, treatment with MPP-Ce6 upon laser irradiation). D) FE-SEM images, E) crystal violet staining, F) crystal violet staining's quantification results, and G) water-insoluble polysaccharide measurements of multispecies biofilms after different treatments for the biofilm inhibition test (scale bar, 2 μm; ∗, p < 0.05; ∗∗∗, p < 0.001).


、MPP-Ce6 介导的 aPDT 体内抗龋作用研究(Fig 5-6

在体内,MPP-Ce6 介导的 aPDT 显著提高游离 Ce6 对大鼠磨牙表面致龋菌的抑制作用,具有较强的抗菌能力。同时显著降低平滑面和窝沟处龋损的范围及深度,具有优异的抗龋能力。未观察到 MPP-Ce6 介导的aPDT 对大鼠造成任何不良影响,具有较好的生物相容性。


Fig. 5. In vivo assessment of MPP-Ce6-mediated aPDT for dental caries. A) Schedule of rodent caries model construction, therapeutic approaches with different treatments, and assessments of antibacterial/anti-caries efficacy. B) Representative images of surviving bacterial colonies on MSB agar plates for different treatment groups at 17, 20, and 47 days (Control, treatment with BHI only; L, treatment with laser only; MPP-Ce6, treatment with MPP-Ce6 only; Free-Ce6+L, treatment with free Ce6 upon laser irradiation; MPP-Ce6+L, treatment with MPP-Ce6 upon laser irradiation). C) CFU counting for surviving bacterial colonies at 17, 20, 23, 29, 35, 41, and 47 days. D) Statistical analysis of surviving bacterial colonies after MPP-Ce6-mediated aPDT (**, p < 0.01; ***, p < 0.001). E) Representative photographs upon stereoscopic microscopy of the occlusal surface of rodent teeth treated as noted at day 47.


Fig. 6. Anti-caries efficacy after different treatments as evaluated by Keyes' scoring and micro-CT analysis. A) Representative images of carious lesions stained by murexide (60 mg mL−1) on the smooth surface and sulcal surface with teeth of different severities (green arrows, affected enamel only, E; blue arrows, affected slight dentinal, within 1/4 of the dentin, Ds; purple arrows, affected moderate dentinal, 1/4–3/4 of the dentin, Dm; red arrows, affected extensive dentinal, beyond 3/4 of the dentin, Dx; Control, treatment with BHI only; L, treatment with laser only; MPP-Ce6, treatment with MPP-Ce6 only; Free-Ce6+L, treatment with free Ce6 upon laser irradiation; MPP-Ce6+L, treatment with MPP-Ce6 upon laser irradiation). B) and C) Statistical analysis of Keyes' scoring on the smooth surface and sulcal surface based on the depth and extent of carious lesions (total lesions, E+Ds+Dm+Dx; initial lesions, Ds+Dm+Dx; moderate lesions, Dm+Dx; extensive lesions, Dx; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D) 3D reconstruction of micro-CT images of maxillary molars in different groups, separated enamel (blue) by setting the density threshold above 11000 Hounsfield units. E) 2D scale sagittal images of the maxillary molars analyzed by micro-CT (red arrows, caries lesion sites).


02

论文第一/通讯作者简介



第一作者:刘丹枫


武汉大学口腔医学院口腔预防科2019级博士生,现为郑州大学第一附属医院口腔医学中心医师。主要致力于牙体牙髓疾病的预防、诊断及治疗。研究方向为开发新型纳米材料用于口腔疾病的预防。



第一作者:马宪彬


西南大学材料与能源学院硕士研究生,现为北京理工大学医学技术学院博士研究生,研究方向为新型纳米生物材料的设计及生物成像分析。



通讯作者:许志刚


西南大学材料与能源学院教授、硕士生导师。重庆英才·青年拔尖人才入选者(2020年),重庆市优秀创新创业导师(2021年),担任Chinese Chemical Letters和Asian Journal of Pharmaceutical Sciences期刊青年编委。近五年来以通讯作者身份在Chemical Society Reviews、Advanced  Materials、Advanced Functional Materials、Advanced Science等期刊上发表学术论文60余篇,研究论文累计被他人正面引用超过3500次,个人H因子35,ESI高被引论文3篇,授权国家发明专利13项,参编英文书籍1部。主持国家自然科学基金、重庆英才·青年拔尖人才项目等课题10余项。主要研究方向为微环境响应型高分子纳米药物递送系统的设计、构建及应用。成果曾获甘肃省高校科技进步奖一等奖、西南大学教学成果奖二等奖和第六届中国国际“互联网+”大学生创新创业大赛全国总决赛银奖。



通讯作者:杜民权


武汉大学口腔医院教授、博士生导师、口腔预防科主任。从事口腔预防医学的教学、科研和临床工作二十余年,目前担任中华口腔医学会口腔预防专业委员会副主任委员。十三五规划全国高等学校教材《口腔预防医学》副主编。发表SCI论文60余篇,科研成果曾获“湖北省科技进步二等奖”。曾入选Elsevier发布的牙医学“Most Cited Chinese Researchers”榜单。


03

资助信息


该研究获国家自然科学基金(8177108451703187)以及重庆英才·青年拔尖人才(CQYC202005029)的资助。


04

原文信息


Danfeng Liu, Xianbin Ma, Yaoting Ji, Rourong Chen, Shuhui Zhou, Hantao Yao, Zichen Zhang, Mengjie Ye, Zhigang Xu*, Minquan Du*. 

Bioresponsive nanotherapy for preventing dental caries by inhibiting multispecies cariogenic biofilms. 

Bioactive Materials, 14 (2022) 1-14. 




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Bioactive Materials是一本高质量英文期刊,目前已经被SCIE、PubMed Central、Scopus、Embase收录。同时本刊还入选了2019年中国科技期刊卓越行动计划--“高起点新刊”项目。

2022年Bioactive Materials 获得影响因子16.874 ,在Materials Science,Biomaterials领域排名第一

位于《2021年中国科学院文献情报中心期刊分区表》1区TOP期刊

CiteScore 2021: 14.3




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