Effect of methylene blue-induced photodynamic therapy on a Streptococcus mutans biofilm model

BackgroundSeveral studies have reported the use of antimicrobial photodynamic therapy (aPDT) to control biofilm but its efficacy depends on several factors, such as biofilm model used. This study aims to examine whether exposure to diode laser combined with methylene blue affects the bacterial viability and polysaccharide content in a Streptococcus mutans cariogenic biofilm model, which simulated ‘feast-famine’ episodes of exposure to sucrose that occur in the oral cavity.Materials and methodsS. mutans biofilms were formed on acrylic resin discs and exposed to a 10% sucrose solution for 1 min, eight times/day. After growing for 48 h, the biofilms were submitted to the following treatments, twice daily (n = 4): (i) 0.9% NaCl (NaCl) as the negative control; (ii) 0.12% chlorhexidine digluconate (CHX) as the positive antibacterial control; (iii) diode laser combined with methylene blue, using an energy density of 320 J/cm² (aPDT). After 120 h of growth, the biofilm formed on each disc was collected to determine the viable bacterial counts and concentration of insoluble exopolysaccharides (IEPS) and intracellular polysaccharides (IPS).ResultsBacterial counts in the biofilms formed differed among the treatments. Compared with NaCl, aPDT significantly destabilized biofilm (p < 0.0001). aPDT and CHX equally lowered the concentration of IEPS and IPS in biofilms.ConclusionUnder the experimental conditions assessed, our findings indicate that a twice-daily treatment with diode laser combined with methylene blue effectively decreased bacterial viability and the intra- and extracellular polysaccharide concentration in biofilms of S. mutans, a cariogenic bacterium.     

Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis

Antimicrobial photodynamic therapy (aPDT) has shown to exert a bactericidal effect against Streptococcus sanguinis and Streptococcus mutans. However, this efficacy has been reported for either type of bacteria separately. Bacterial suspensions of both strains, separately or together, were treated with concentrations of methylene blue (MB) and rose bengal (RB). Suspensions were irradiated with a light–emitting diode lamp (λ center at 625 nm for MB and λ center at 515 nm for RB) using a fluence of 18 J/cm². RB-aPDT at concentrations of 0.16–0.62 and 0.16–0.31 μg/mL, and MB-aPDT at concentrations of 0.62–1.25 and 0.31–1.25 μg/mL inhibited the growth of S. mutans and S. sanguinis respectively by 6 log₁₀. In suspensions of both strains together, the same 6 log₁₀ reduction in bacterial growth was achieved using the same concentrations of each photosensiziser.In conclusion, RB-aPDT and MB-aPDT appear to exert the same bactericidal effect against suspensions of S. sanguinis and S. mutans either for single strain treatment or for samples constituted by both bacteria mixed together. RB shows to be slightly more efficient than MB.     

Effect of different wavelengths and dyes on Candida albicans: In vivo study using Galleria mellonella as an experimental model

BackgroundStudies on photodynamic inactivation against microorganisms had a great development in recent years. The aim of this work was to test the application of different laser wavelengths with or without different photosensitizing dyes on Candida albicans cells in vitro and in photodynamic therapy protocols in vivo in larvae of Galleria mellonella.MethodsLaser application was realized on C. albicans cells suspended in saline solution or cultured on solid medium for the in vitro study, and in a model of G. mellonella candidal infection for the in vivo study. Three wavelengths (650, 405, and 532 nm) were used in continuous mode with different values of applied fluences: 10, 20 and 30 J/cm² for the in vitro study and 10 J/cm² for the in vivo study, without and with photosensitizing dyes.ResultsNo growth inhibition was obtained on yeast cells in saline solution without photosensitizers. The maximum inhibition of growth (100%) was obtained with 405 nm diode laser and curcumin at any used fluence. No growth inhibition was observed for yeast cells cultured on solid medium after laser application without dyes. An inhibition was observed after laser application when curcumin and erythrosine were added to the medium.The survival curves of G. mellonella larvae infected with C. albicans with or without the different dyes and after laser application showed a statistically significant difference (p < 0.001) in comparison with the proper control groups.ConclusionsThese results show the efficacy of photodynamic inactivation exploiting a suitable combination of light and dyes against C. albicans and the potential of photodynamic therapy for the treatment of candidal infections.     

The effect of indocyanine green loaded on a novel nano-graphene oxide for high performance of photodynamic therapy against Enterococcus faecalis

BackgroundRecently developed photodynamic therapy (PDT) has gained attention for achieving effective root canal disinfection. Using an optimized nontoxic photosensitizer (PS), such as indocyanine green (ICG), is an imperative part of this technique. Therefore, the objective of the current study was to improve ICG photodynamic properties through incorporation of ICG into nano-graphene oxide (NGO) in order to produce NGO-ICG as a new PS and also to assess the antimicrobial effects of NGO-ICG against Enterococcus faecalis after photodynamic therapy.Materials and methodsNGO-ICG was synthesized based on oxidation of graphite flakes and direct loading of ICG onto NGO. NGO-ICG formation was confirmed using the Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), and UV–vis spectrometry. The antimicrobial and anti-biofilm potential of NGO-ICG-PDT against E. faecalis was assessed via colony forming unit and crystal violet assays, respectively.ResultsFT-IR, SEM and UV–vis spectrometry confirmed successful synthesis of NGO-ICG containing 200 μg/mL of ICG. NGO-ICG-PDT at an energy density of 31.2 J/cm² showed a significant reduction (2.81 log) in the count of E. faecalis (P < 0.05). NGO-ICG-PDT significantly reduced the biofilm formation ability of E. faecalis up to 99.4% (P < 0.05). The overall antimicrobial and anti-biofilm potential of NGO-ICG-PDT was higher than PDT based on ICG (1000 μg/mL) (47% and 21%, respectively).ConclusionBecause NGO-ICG-PDT showed a significant reduction in the number and biofilm formation ability of E. faecalis at low ICG concentrations (200 μg/mL), it could be a new approach to adjuvant treatment of endodontic infections.     

金银花主要活性成分对烟曲霉生物膜的体外影响

目的 探讨金银花主要活性成分绿原酸（CRA）对烟曲霉（A.f）生物膜（BF）的体外影响。方法扫描电镜（SEM）鉴定体外烟曲霉BF;二倍稀释法测定绿原酸的最低抑菌浓度（MIC）及最低杀真菌浓度（MFC）;各浓度的CRA分别作用于BF 48h后, XTT减低法测定生物膜内菌细胞的代谢活力;结晶紫（CV）法定量生物膜。结果受试菌株经过培养24h、48h后可形成稳定的BF。药物作用48h后,各浓度CRA对菌体代谢活力与空白组相比差异均无统计学意义（P＞0.05）;较高浓度的CRA组（256、512、1024μg/ml）生物膜量与空白组相比,差异有统计学意义（P〈0.05）。结论烟曲霉体外经培养24h、48h后可形成稳定生物膜;金银花主要活性成分绿原酸不能杀死烟曲霉生物膜细胞,但可以抑制生物膜胞外基质的形成,其抑制作用呈浓度依赖性。     

Synthesis,characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by ¹H NMR, UV–vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2 mW cm⁻²) of red light (643 nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC₅₀ values. The obtained IC₅₀ values for 5 were 1.4 μM at 15 min irradiation time and 0.7 μM when the time of irradiation was 30 min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.     

Enhancing the efficiency of 5-aminolevulinic acid-mediated photodynamic therapy using 5-fluorouracil on human melanoma cells

Background5-Aminolevulinic acid-mediated photodynamic therapy (ALA–PDT) is an effective and noninvasive modality for treatment of several types of non-melanoma skin cancers. This in-vitro study attempted to know whether the killing effect of ALA–PDT on the human melanoma cells (Mel-Rm cell line) could be increased by the presence of 5-fluorouracil (5-FU).MethodsTo evaluate the effect of ALA–PDT in combination with 5-FU on viability of human melanoma Mel-Rm cells, the cells incubated with 5-ALA and 5-FU for 3 h in nontoxic concentrations, and subsequently illuminated with a 630 nm light-emitting diode array. The cells viability and cytotoxicity determined by mitochondrial activity and lactate dehydrogenase assays.ResultsCombination of ALA–PDT and 5-FU (FU–ALA–PDT) showed a considerable growth inhibition according to the results of MTT assay compared to ALA–PDT. The results of LDH assay also showed a cytotoxicity effect in ALA–PDT; however, the FU–ALA–PDT showed no significantly enhancement in cytotoxicity compared to ALA–PDT using LDH assay.ConclusionThe Mel-Rm cells incubation with 5-FU before PDT enhances the efficiency of 5-Aminolevulinic acid-mediated photodynamic therapy.     

The effect of ethanol or long-time reaction on the diatom test in water samples using sodium hypochlorite

BackgroundThe diatom test method using sodium hypochlorite (NaClO) was equivalent to the conventional method in water samples. However, the method using NaClO was inferior to the conventional method in lung samples, in which ethanol was used and the reaction with NaClO was longer compared with the method in water samples. Using water samples, we aimed to clarify whether these differences affect the diatom test result.Materials and methodsThirteen water samples from natural water sources were each divided into four parts corresponding to four (2 × 2) digestion methods: 3 “digestion” vs. 1 “digestion” and with ethanol vs. without ethanol. After the base-2 logarithmic transformation, the diatom counts were analyzed using three-way analysis of variance (ANOVA); factor 1 was “digestion times,” factor 2 was “ethanol,” and factor 3 was “sample number,” and the interaction between factors 1 and 2 was also analyzed.ResultsThe geometric means of the diatoms from the 3 “digestion” with ethanol method, the 3 “digestion” without ethanol method, the 1 “digestion” with ethanol method, and the 1 “digestion” without ethanol method were 373.5, 551.8, 436.6, and 522.0, respectively. ANOVA showed a significant difference in factor 2 (P = 1.7 × 10^-4). There was no significant difference in factor 1 (P = 0.46), and no significant interaction between factors 1 and 2 (P = 0.13).ConclusionEthanol may decrease the diatom count in the diatom test using NaClO. In contrast, the diatom frustules do not dissolve through three-times digestion using NaClO.     

Prediction of the response to photodynamic therapy in patients with chronic central serous chorioretinopathy based on optical coherence tomography using deep learning

PurposeTo assess the prediction of the response to photodynamic therapy (PDT) in chronic central serous chorioretinopathy (CSCR) based on spectral-domain optical coherence tomography (SD-OCT) images using deep learning (DL).MethodsRetrospective study including 216 eyes of 175 patients with CSCR and persistent subretinal fluid (SRF) who underwent half-fluence PDT. SD-OCT macular examination was performed before (baseline) and 3 months after treatment. Patients were classified into groups by experts based on the response to PDT: Group 1, complete SRF resorption (n = 100); Group 2, partial SRF resorption (n = 66); and Group 3, absence of any SRF resorption (n = 50). This work proposes different computational approaches: 1st approach compares all groups; 2nd compares groups 1 vs. 2 and 3 together; 3rd compares groups 2 vs. 3.ResultsThe mean age was 55.6 ± 10.9 years and 70.3% were males. In the first approach, the algorithm showed a precision of up to 57% to detect the response to treatment in group 1 based on the initial scan, with a mean average accuracy of 0.529 ± 0.035. In the second model, the mean accuracy was higher (0.670 ± 0.046). In the third approach, the algorithm showed a precision of 0.74 ± 0.12 to detect the response to treatment in group 2 (partial SRF resolution) and 0.69 ± 0.15 in group 3 (absence of SRF resolution).ConclusionDespite the high clinical variability in the response of chronic CSCR to PDT, this DL algorithm offers an objective and promising tool to predict the response to PDT treatment in clinical practice.