São Paulo State University (Unesp), School of Dentistry, Araçatuba, Department of Preventive and Restorative Dentistry, 16015-050 Araçatuba/São Paulo, Brazil.
School of Dentistry, Presidente Prudente, University of Western São Paulo (UNOESTE), 19050-920 Presidente Prudente/São Paulo, Brazil.
J Dent. 2023 Nov;138:104699. doi: 10.1016/j.jdent.2023.104699. Epub 2023 Sep 14.
This study assembled and characterized a dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluated its antibiofilm and cytotoxic effects.
CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS) and characterized by physical-chemical analyses. Biofilms from human saliva supplemented with Candida species were grown (72 h) on glass discs and treated (24 h) with IONPs-CS carrying CHX (at 39, 78, or 156 µg/mL) and FLZ (at 156, 312, or 624 µg/mL) in three growing associations. IONPs and CS alone, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, microbiological analyses were performed. The viability of human oral keratinocytes (NOKsi lineage) was also determined (MTT reduction assay). Data were submitted to ANOVA or Kruskal-Wallis, followed by Fisher's LSD or Tukey's tests (α=0.05).
Nanocarriers with spherical-like shape and diameter around 6 nm were assembled, without compromising the crystalline property and stability of IONPs. Nanocarrier at the highest concentrations was the most effective in reducing colony-forming units of Streptococcus mutans, Lactobacillus spp., Candida albicans, and Candida glabrata. The other carriers and CHX156-FLZ624 showed similar antibiofilm effects, and significantly reduced lactic acid production (p<0.001). Also, a dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier. IONPs-CS-CHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations ≥7.8 and 31.25 µg/mL, respectively (p<0.05).
The nanotherapy developed outperformed the effect of the combination CHX-FLZ on microcosm biofilms, without increasing the cytotoxic effect of the antimicrobials administered.
The dual nanocarrier is a promising topically-applied therapy for the management of oral candidiasis considering that its higher antibiofilm effects allow the use of lower concentrations of antimicrobials than those found in commercial products.
本研究组装并表征了一种载有洗必泰（CHX）和氟康唑（FLZ）的双纳米载体，并评估了其抗生物膜和细胞毒性作用。
将 CHX 和 FLZ 加入到先前用壳聚糖（CS）涂覆的氧化铁纳米粒子（IONPs）中，并通过物理化学分析进行表征。在玻璃载玻片上培养（72 h）含有口腔唾液来源的 Candida 物种的生物膜，并分别用载有 CHX（39、78 或 156 μg/mL）和 FLZ（156、312 或 624 μg/mL）的 IONPs-CS（3 种生长关联）进行处理（24 h）。IONPs 和 CS 单独，以及 156 μg/mL CHX + 624 μg/mL FLZ（CHX156-FLZ624）作为对照进行测试。然后进行微生物分析。还测定了人口腔角质形成细胞（NOKsi 系）的活力（MTT 还原测定）。数据采用方差分析或 Kruskal-Wallis 检验，然后采用 Fisher's LSD 或 Tukey's 检验（α=0.05）。
组装了具有球形且直径约为 6nm 的纳米载体，而不会损害 IONPs 的结晶性能和稳定性。纳米载体在最高浓度时，对变异链球菌、乳杆菌属、白色念珠菌和光滑念珠菌的集落形成单位的减少最为有效。其他载体和 CHX156-FLZ624 显示出相似的抗生物膜作用，并显著降低了乳酸的产生（p<0.001）。此外，双纳米载体对口腔角质形成细胞表现出剂量依赖性的细胞毒性作用。IONPs-CS-CHX-FLZ 和 CHX-FLZ 分别在 CHX 和 FLZ 浓度≥7.8 和 31.25μg/mL 时显著降低角质形成细胞活力（p<0.05）。
与联合使用 CHX-FLZ 相比，开发的纳米疗法在微宇宙生物膜中表现出更好的效果，而不会增加所施用的抗菌药物的细胞毒性。
鉴于双纳米载体的较高抗生物膜作用允许使用低于商业产品中发现的浓度的抗菌药物，因此该双纳米载体是一种有前途的局部应用疗法，可用于治疗口腔念珠菌病。
