Evaluation of dual application of photodynamic therapy-PDT in Candida albicans.

This study aimed to evaluate, in vitro, the efficacy of photodynamic therapy - PDT using dimethyl methylene blue zinc chloride double salt (DMMB) and red LED light on planktonic cultures of Candida albicans. The tests were performed using the ATCC 90,028 strain grown at 37 °C for 24 h, according to a growth curve of C. albicans. The colonies were resuspended in sterile saline adjusted to a concentration of 2 × 108 cells / mL, with three experimental protocols being tested (Protocol 1, 2 and 3) with a fixed concentration of 750 ɳg/mL obtained through the IC50, and energy density 20 J/cm2. Protocol 1 was carried out using conventional PDT, Protocol 2 was applied double PDT in a single session, and Protocol 3 was applied double PDT in two sessions with a 24 h interval. The results showed logarithmic reductions of 3 (4.252575 ± 0.068526) and 4 logs (2.669533 ± 0.058592) of total fungal load in protocols 3 and 2 respectively in comparison to the Control (6.633547 ± 0.065384). Our results indicated that double application in a single session of PDT was the most effective approach for inhibiting the proliferation of Candida albicans (99.991% inhibition).

Histological evaluation of skin lesions induced by Leishmania braziliensis treated by PACT using Laser light and 1.9 dimethyl-methylene blue.

This study aimed to perform a histological evaluation in skin lesions caused by Leishmania braziliensis after PACT treatment using Laser associated with 1.9. dimethyl methylene blue BALB/c mouse ear infection model was used. A total of 40 animals were assigned into two groups considering time intervals at 5 and 10 weeks and subdivided into four subgroups: Control, Photosensitizer, Laser and PACT. Two therapeutic interventions were performed after the 5th week of infection at 48 h intervals. 1.9 Dimethyl methylene blue was used as a photosensitizer at the concentration of 7 ng/mL, with a non-invasive topical administration method associated with Laser (λ = 660 nm, 40 mW, 12 J/cm2). Sample collection occurred 5 or 10 weeks after therapeutic interventions. The main histological findings were observed in the laser and PACT groups at the 10-week evaluation. The Laser group showed reduced lymphoplasmacytic inflammation and histiocytes (p = 0.0079). The PACT group showed reductions in lymphoplasmacytic inflammation at 5 and 10 weeks, discrete reduction of histiocytes and a higher percentage of tissue remodeling. PACT with non-invasive topical administration of the photosensitizer was able to reduce lymphoplasmacytic inflammation and increase tissue remodeling in leishmaniosis skin lesions. This protocol may be easily used in humans and clinical trial shall be carried out to confirm the animal's findings.

Enhancement of photodynamic inactivation of planktonic cultures of Staphylococcus aureus by DMMB-AuNPs.

Photodynamic inactivation is a promising method for the treatment of infectious diseases. Nanotechnology through gold nanoparticles, as a tool to improve the delivery of photosensitizer is an attractive approach to enhance photodynamic inactivation of bacteria. Moreover, gold nanoparticles enchance the absorption of light due to their plasmon resonance. The aim of this study was to evaluate in vitro photodynamic inactivation effects of 1.9-Dimethyl-Methylene Blue (DMMB)-AuNPs associated with the red LED (λ630 ηm ± 20 ηm, 125 mW, 12 J / cm², 192 s) on S. aureus strain. Eight experimental groups were studied: Control, LED, AuNPs, AuNPs + LED, DMMB, DMMB + LED, DMMB + AuNPs, DMMB + AuNPs + LED. After incubation, the number of bacteria surviving each treatment was determined and then enumerated by viable counting (CFU / mL). The logarithm of CFU / mL (CFU/mL log10) was calculated. All experiments realized in triplicate. The statistical analyses included one-way ANOVA tests, Tukey's multiple comparisons and nonlinear regression, p values <0.05 were considered statistically significant. According to results, the photodynamic inactivation of S. aureus on groups DMMB + LED and DMMB-AuNPs + LED, showed a significant reduction of the microbial load (p < 0.0001) when compared to the Control group. The decimal reduction (RD) of these groups were 99.96 % (RD = 3) and 99.994 % (RD = 4) respectively. In conclusion, these findings demonstrated that photodynamic inactivation is enhanced by using DMMB-AuNPs on S. aureus.

aPDT using nanoconcentration of 1,9-dimethylmethylene blue associated to red light is efficacious in killing Enterococcus faecalis ATCC 29212 in vitro.

The Enterococcus faecalis is a microorganism that causes multiple forms of resistance to a wide range of drugs used clinically. aPDT is a technique in which a visible light activates photosensitizer (PS), resulting in generation of reactive oxygen species that kill bacteria unselectively via an oxidative burst. aPDT is an alternative to antibiotics with the advantage of not causing resistance. The search for an alternative treatment of infections caused by E. faecalis, without using antibiotics, is off great clinical importance. The aim of present investigation was to assess the efficacy of using 3.32 ηg/mL of 1,9-dimethylmethylene blue (DMMB) as photosensitizer associated with the use of either Laser (λ660 nm) or LED (λ632 ±â€¯2 nm) using different energy densities (6, 12 and 18 J/cm2) to kill E. faecalis in vitro. Under different experimental conditions, 14 study groups, in triplicate, were used to compare the efficacy of the aPDT carried out with either the laser or LED lights using different energy densities associated to DMMB. The most probable number method (MPN) was used for quantitative analysis. Photodynamic antimicrobial effectiveness was directly proportional to the energy density used, reaching at 18 J/cm2, 99.999998% reduction of the counts of E. faecalis using both light sources. The results of this study showed that the use of 3.32 ηg/mL of DMMB associated with the use 18 J/cm2 of LED light (λ632 ±â€¯2 nm) reduced >7-log counts of planktonic culture of E. faecalis.

Effects of photostimulation on the catabolic process of xenobiotics.

Light biotechnology is a promising tool for enhancing recalcitrant compounds biodegradation. Xenobiotics can cause a significant impact on the quality of the results achieved by sewage treatment systems due to their recalcitrance and toxicity. The optimization of bioremediation and industrial processes, aiming to increase efficiency and income is of great value. The aim of this study was to accelerate and optimize the hydrolysis of Remazol Brilliant Blue R by photo stimulating a thermophilic bacterial consortium. Three experimental groups were studied: control group; LED Group and Laser Group. The control group was exposed to the same conditions as the irradiated groups, except exposure to light. The samples were irradiated in Petri dishes with either a Laser device (λ660 nm, CW, θ = 0.04 cm2, 40 mW, 325 s, 13 J/cm2) or by a LED prototype (λ632 ±â€¯2 nm, CW, θ = 0.5 cm2, 145 mW, 44 s, 13 J/cm2). We found that, within 48-h, statistically significant differences were observed between the irradiated and the control groups in the production of RNA, proteins, as well as in the degradation of the RBBR. It is concluded that, both Laser and LED light irradiation caused increased cellular proliferation, protein production and metabolic activity, anticipating and increasing the catabolism of the RBBR. Being the economic viability a predominant aspect for industrial propose our results indicates that photo stimulation is a low-cost booster of bioprocesses.

Photobiological effect of Laser or LED light in a thermophilic microbial consortium.

Cellulose has a highly diversified architecture and its enzymatic complexes are studied for achieving an efficient conversion and a high level of efficiency in the deconstruction of cellulolytic biomass into sugars. The aim of this investigation was to evaluate the effect of Laser or LED light in the cellulolytic activity (CMCase) and on the proliferation of the thermophilic microbial consortium used on the degradation process of a lignocellulosic biomass of green coconut shell. The irradiation protocol consisted of six Laser irradiations (λ660 ηm, 40 mW, 270 s, 13 J/cm2) or LED (λ632 ±â€¯2 ηm, 145 mW, 44 s, 13 J/cm2) with 12- h time intervals in nutrient deprivation conditions. After irradiation, the consortium was inoculated into a lignocellulosic biomass (coconut fibers). Non- irradiated consortium was also inoculated and acted as control. Cell proliferation and endoglucanase activity were quantified during the experimental time. Experiments were carried out in triplicate. The results showed an increase of 250 % of thermo-cellulolytic microorganisms for the LED group and 200% for the Laser group when compared to the control. The enzymatic index (red Congo method), showed a statistically significant difference in the process of degradation of the lignocellulosic biomass between the Laser and LED groups compared to the control group [p < 0.0029; p < 0.029, respectively] 48-hs after the inoculation of the microorganisms. At the end of 72-h, this significant difference was maintained for both irradiated groups (p < 0.0212). Based upon the protocol used on the present study, it is possible to concluded that LED light enhanced cell proliferation of the thermophilic microbial consortium while the Laser light increase the enzymatic index of the lignocellulosic biomass of green coconut shell.

Effects of PACT using phenothiazine-derived drugs and red light on the macrophage x S. aureus interface.

The aim of this study was to evaluate the lethal potential of macrophages infected with Staphylococcus aureus after PACT (Photochemical Antimicrobial Chemotherapy) using phenothiazine derivatives (a solution containing 1:1 methylene blue and O toluidine blue) and laser (660 nm, 40 mW, 60 s, 12 J/cm2) or LED (632 ±â€¯2 nm, 145 mW, 40 s, 12 J/cm2). Six experimental groups were evaluated: Control Group (untreated); Photosensitizer group (phenothiazines - 12.5 µg/mL); Laser Group; LED Group; Laser PACT Group; and LED PACT Group. The pre-irradiation time used in this study was 5 min. Macrophages and bacteria were cultured in specific culture media and/or allowed interaction between the cell types. Subsequently, tests were carried out to evaluate microbial proliferation, ROS production by macrophages and survival capacity of S. aureus after phagocytosis. Fluorescence microscopy assays were performed with the H2DCFDA probe, after PACT, at the initial time (0 h), 4-h and 12-h. The tests were performed in triplicate and the statistical test used was ANOVA with Tukey post-test. After PACT, a statistically significant difference (p > 0.0001) was observed between the microbial growth of the control group and the PACTs groups. Laser PACT and LED PACT groups presented, respectively, reductions of 84.2% and 81.5% when compared to control and 53.3% and 46% when compared to the photosensitizer group. It is concluded that the therapeutic protocols presented in this study increased the phagocytic capacity, the response rate of the phagocytes and the consequent reduction of the numbers of S. aureus for both PACT protocols, however the increase in ROS production was only observed in the group irradiated with Laser light.

LED antimicrobial photodynamic therapy with phenothiazinium dye against Staphylococcus aureus: An in vitro study.

The objective of this study was to evaluate, in vitro, the bactericidal effect of AmPDT on Staphylococcus aureus (ATCC 25923) using different concentrations (100, 50, 25, 12.5 and 6.25µg/mL) of phenothiazine compound combined with LED light (λ632±2nm) using varied energy densities (12, 9.6, 7.2, 4.8 and 2.4J/cm2). The experiments were carried out in triplicate and the samples were divided into groups: Control, Irradiated (treated only with light at different energy densities), Photosensitizer (treated only in the presence of the dye), AmPDT (treatment with light associated with dye). Counts of the colony forming units and the data obtained were statistically analyzed (ANOVA, Tukey's test, p<0.05). The results showed no difference between irradiated and Control groups. However, using the photosensitizer alone caused significant increased cytotoxicity and consequent reduction on the CFU counts (12.5µg/mL (p<0.001), 25µg/mL, 50µg/mL and 100µg/mL (p<0.0001). When AmPDT was used significant inhibition above 70% were detected for all concentrations of the photosensitize (p<0.0001) except for 6.25µg/mL. The results indicate a dose-response dependent when the photosensitizer is used alone but not for the sole use of the light is used. It is concluded that, a single application of AmPDT, using energy density of 12J/cm2 associated either to 12.5 (81.52%) or 25µg/mL (91.57%) resulted in higher in vitro inhibition of S. aureus.

Effectiveness of antimicrobial photodynamic therapy (AmPDT) on Staphylococcus aureus using phenothiazine compound with red laser.

The aim of this study was to evaluate, in vitro, the bactericidal effect of antimicrobial photodynamic therapy (AmPDT) using phenothiazinium dyes (Toluidine Blue O and methylene blue, 1:1) using different concentrations (100, 50, 25, 12.5, and 6.25 µg/mL) associated to red laser with different energy densities (2.4, 4.8, 7.2, 9.6, and 12 J/cm2) on a strain of Staphylococcus aureus (ATCC 23529). On this study, tests were performed in triplicate and the samples were distributed into 36 test groups: Control and bacterial suspensions were irradiated with the different energy densities, respectively, in the absence of photosensitizer, bacterial suspensions were irradiated with the laser in the different concentrations of the photosensitizer, and finally bacterial suspensions only in the presence of phenothiazinium dye. The pre-irradiation time was 5 min. Therefore, we analyzed the potential of the AmPDT by counting colony-forming units. The logarithm of CFU/mL (log10 CFU/mL) was calculated and the data was analyzed statistically (ANOVA, Tukey's test, p < 0.05). The results showed that the association 50 and 100 µg/mL with 12 J/cm2 showed the highest percentage of inhibition (100 %). Based upon the present results, it may be concluded that the AmPDT was able to enhance the antimicrobial effect of phenothiazines and both concentration of the compound and energy density are important factors for greater effectiveness of therapy.

Effects of imiquimod and low-intensity laser (λ660 nm) in chemically induced oral carcinomas in hamster buccal pouch mucosa.

Squamous cell carcinoma (SCC) is the most common neoplasm of the oral cavity. It is aggressive, highly proliferative, and metastatic. This study aimed to evaluate the effect of LLLT and imiquimod on DMBA chemically induced lesions on the oral mucosa of hamsters. SCCs were induced on 25 hamsters. Animals of G1 (control 1) were killed and the presence of tumors confirmed; G2 (control 2) suffered no interventions for additional 4 weeks; animals of G3 (laser treatment) were irradiated (λ660 nm, 50 mW, CW, Ø=3 mm, 0.07 cm(2), 714.2 mW/cm(2), 133 s, 95 J/cm(2), 6.65 J) at every other day for 4 weeks; animals of G4 (imiquimod treatment) received 5 % imiquimod three times a week for 4 weeks; and animals of G5 (imiquimod and laser treatment) received both treatments for the same period. Samples were taken and underwent histological analysis by light microscopy and were investigated using immunohistochemistry for S-100(+) dendritic cells. In G1, G2, and G3, the evaluations showed malignant tumors and the absence of S-100(+) dendritic cells in the tumor stroma. In G4, 60 % of the animals had no malignant tumors, and S-100(+) dendritic cells were present in the stroma of the tumors as well as dysplasia. In G5, 40 % of the animals presented SCC, with scarce or no S-100(+) dendritic cells. The imiquimod treatment played a direct effect on SCC, demonstrated by the increased number of S-100(+) dendritic cells, which could suggest an important role of immune surveillance against neoplastic proliferation. Furthermore, its association with laser needs to be further investigated.

Influence of laser phototherapy (λ660 nm) on the outcome of oral chemical carcinogenesis on the hamster cheek pouch model: histological study.

PURPOSE: This study aimed to evaluate, histologically, the effect of low-level laser therapy (LLLT) (λ660 nm) on DMBA chemically induced lesions of the oral mucosa of hamsters. BACKGROUND DATA: Squamous cell carcinoma (SCC) is the most common neoplasm of the oral cavity. It is aggressive, highly proliferative, invasive, and metastatic. There is evidence that LLLT similarly affects neoplasic and non-neoplasic cells. METHODS: Cancerous lesions were induced on the cheek pouch of 15 golden Syrian hamsters by using DMBA 3 times a week for 8 weeks. At the end of the cancer induction (8 weeks), animals in G1 were killed and the presence of tumors confirmed. Animals in G3 were irradiated (λ660 nm, 30 mW, CW, Ø=3 mm, area: 0.07 cm(2), 424 mW/cm(2), 133 sec, 56.4(2)J/ cm(2), 4J) at every other day for 4 weeks. G2 received no interventions for the same period. Samples were taken and underwent histological analysis by light microscopy. RESULTS: GI showed 100% well-differentiated SCC. G2 showed 20% moderately differentiated and 80% well-differentiated SCC. G3 showed 40% well-differentiated, 40% poorly differentiated, and 20% moderately differentiated SCC. Significant differences (p=0.02) in the amount of well-differentiated SCC were seen between G1 and G3 and between G3 and G2 (p=0.04). Significant difference was also seen between G3 and G1 and G2 with regard to the amount of poorly differentiated tumors (p=0.04). CONCLUSIONS: It is concluded that LLLT, within the parameters used in the present study, caused a significant progression of the severity of SCC in the oral cavity of hamsters.