Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil.
Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil.
Photodiagnosis Photodyn Ther. 2019 Sep;27:124-131. doi: 10.1016/j.pdpdt.2019.05.038. Epub 2019 May 29.
Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes β-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5 min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5 min, followed by incubation with PDZ for 20 min and exposure to LED light (660 nm, 50 J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZ + DNase, light only, light + DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (p = 0.000) and eDNA amounts (p ≤ 0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT.
抗菌光动力疗法（aPDT）已被提议用于治疗念珠菌感染。然而，与浮游培养物相比，生物膜中的微生物对 aPDT 的敏感性较低，这可能是因为基质限制了光敏剂的渗透。因此，本研究的目的是：（1）使用水解酶β-葡聚糖酶和 DNase 单独或联合靶向氟康唑敏感（S）和氟康唑耐药（R）白念珠菌（Ca）菌株的生物膜基质成分；（2）将最佳酶方案应用于 Photodithazine®（PDZ）介导的 aPDT 中；（3）在共聚焦显微镜下验证在不同孵育期用或不用 DNase 预处理生物膜后 PDZ 的渗透情况。将 CaS 和 CaR 48h 龄生物膜与水解酶孵育（5min），并通过细胞活力、生物量和基质成分进行评估。DNase 通过显著降低两种菌株基质中的细胞外 DNA（eDNA）和可溶性蛋白以及 CaR 基质中的水溶性多糖，显示出最佳效果。随后，将 48h 龄生物膜用 DNase 孵育 5min，然后用 PDZ 孵育 20min，并暴露于 LED 光（660nm，50J/cm²）。对照组仅用 aPDT 处理（无 DNase）、仅用 PDZ、PDZ+DNase、仅用光、光+DNase 和未经处理的生物膜。DNase 的预处理允许 PDZ 渗透到更深的生物膜层，并且 aPDT 效果增强，细胞活力（p=0.000）和 eDNA 量（p≤0.047）显著降低。DNase 影响基质组成，改善光敏剂的渗透，从而提高后续 aPDT 的效果。
