Department of Oral Pathology and Microbiology, Institute of Dental Sciences, Siksha'O'Anusandhan Deemed to be University, Bhubaneswar, Odisha, India.
Institute of Dental Sciences, Siksha'O'Anusandhan Deemed to be University. Bhubaneswar, Odisha, India.
PLoS One. 2024 Oct 30;19(10):e0312521. doi: 10.1371/journal.pone.0312521. eCollection 2024.
Streptococcus mutans is a major contributor to dental caries due to its ability to produce acid and survive in biofilms. Microbial resistance towards common antimicrobial agents like chlorhexidine and triclosan has shifted the research towards antimicrobial Photodynamic therapy (PDT). In this context, Toluidine Blue O (TBO) is being explored for its photosensitizing properties against Streptococcus mutans. There is a huge variation in the effective concentration of TBO among the current studies owing to the differences in source of and delivery system TBO as well as the time, power and energy densities of light.
The primary objectives of this study are to encapsulate improved Toluidine Blue O (ITBO) in nanoliposomes (NLITBO), characterize it, and evaluate its antibacterial photosensitizing potential against Streptococcus mutans suspensions in vitro.
ITBO was synthesised as per Indian patent (number -543908). NLITBO was prepared using the thin-film hydration method. Dynamic light scattering experiment determined the vesicle size, polydispersity index (PDI), and zeta potential. Surface features were characterized by Scanning and Transmission Electron microscopy. ITBO release from NLITBO was assessed using the extrapolation method. The antibacterial activity of the NLITBO was determined by evaluating the zone of inhibition (ZOI) in the Streptococcus mutans culture and comparing with 2% chlorhexidine gluconate. The minimum inhibitory concentration (MIC) of NLITBO as a photosensitizer with red light (wavelength 650nm, power density 0.1 W/cm2, energy density 9-9.1 J/ cm2, 90seconds time) was evaluated against Streptococcus mutans cells by colorimetric method in 96 well plate.
Percentage drug loading, loading efficiency, yield percentage, vesicle size, PDI, Zeta potential of NLTBO was reported as 9.3±0.4%, 84.4±7.6%, 73.5%, 123.52 nm, 0.57, -39.54mV respectively. Clusters of uni-lamellar nanovesicles with smooth non-perforated surfaces were observed in SEM and TEM. The size of the vesicle was within 100 nm. At 24 hours, a cumulative 79.81% of ITBO was released from NLITBO. Mean ZOI and MIC of NLITBO (1 μg /ml) were found to be 0.7±0.2 mm, 0.6μg/ml respectively.
We have synthesized and encapsulated improved Toluidine Blue O (ITBO) in nanoliposomes (NLITBO) and thoroughly characterized the formulation. The antibacterial efficacy of NLITBO without light was demonstrated by ZOI which is similar to 2% chlorhexidine gluconate. MIC of NLITBO as a photosensitiser along with the optimal light parameter was also proposed in this study. These findings suggested that NLITBO could serve as an effective alternative to conventional antibacterial treatments in managing Streptococcus mutans rich biofilms. It can have potential pharmaceutical application in oral health care.
变形链球菌由于其产生酸和在生物膜中存活的能力，是导致龋齿的主要原因。由于微生物对常见抗菌剂如洗必泰、三氯生的耐药性，研究转向了抗菌光动力疗法（PDT）。在这种情况下，甲苯胺蓝 O（TBO）因其对变形链球菌的光敏特性而被探索。由于 TBO 的来源和输送系统以及光的时间、功率和能量密度的不同，目前研究中 TBO 的有效浓度差异很大。
本研究的主要目的是将改良的甲苯胺蓝 O（ITBO）包封在纳米脂质体（NLITBO）中，对其进行表征，并评估其对体外变形链球菌悬浮液的抗菌光敏潜力。
根据印度专利（编号-543908）合成 ITBO。NLITBO 采用薄膜水化法制备。动态光散射实验测定了囊泡的粒径、多分散指数（PDI）和zeta 电位。表面特征通过扫描和透射电子显微镜进行了表征。通过外推法评估 NLITBO 中 ITBO 的释放情况。通过评估变形链球菌培养物中的抑菌区（ZOI）并与 2%洗必泰葡萄糖酸盐进行比较，来确定 NLITBO 的抑菌活性。通过比色法在 96 孔板中评估 NLITBO 作为光敏剂与红光（波长 650nm，功率密度 0.1 W/cm2，能量密度 9-9.1 J/cm2，90 秒时间）的最小抑菌浓度（MIC）对变形链球菌细胞的影响。
NLITBO 的药物载量百分比、载药量、产率百分比、囊泡大小、PDI、Zeta 电位分别为 9.3±0.4%、84.4±7.6%、73.5%、123.52nm、0.57、-39.54mV。SEM 和 TEM 观察到的是具有光滑非穿孔表面的单层纳米囊泡的簇。囊泡的大小在 100nm 以内。24 小时时，NLITBO 中 79.81%的 ITBO 被释放。NLITBO（1μg/ml）的平均 ZOI 和 MIC 分别为 0.7±0.2mm 和 0.6μg/ml。
我们已经合成并将改良的甲苯胺蓝 O（ITBO）包封在纳米脂质体（NLITBO）中，并对制剂进行了全面的表征。通过 ZOI 证实了 NLITBO 在没有光照的情况下具有抗菌作用，与 2%洗必泰葡萄糖酸盐相似。本研究还提出了 NLITBO 作为光敏剂的 MIC 以及最佳的光照参数。这些发现表明，NLITBO 可以作为传统抗菌治疗管理富含变形链球菌的生物膜的有效替代品。它在口腔保健方面具有潜在的药用应用前景。
