Melanin pigmented gingival tissue impairs red-light lateral scattering for antimicrobial photodynamic therapy.

Gingival melanin pigmentation is present in many African and Oriental descendant people and its occurrence in patients may interfere with the absorption and scattering of therapeutic doses of light. Antimicrobial photodynamic therapy (aPDT) is used as an adjunctive treatment for periodontitis and light irradiation may be impaired by tissue size and its melanin content. The aim of this clinical study was to measure the red-light attenuation in gingival tissue naturally pigmented with melanin. Ten patients with melanized gingival tissue were selected and irradiated by 100 mW red laser. The patients were photographed in frontal and incisal regions with a T2i camera (Canon, Japan) with 100 mm macro lens, 35 mm focal length, aperture f22, 1/100 shutter speed and ISO 200. Three randomly selected sites of each patient were used for evaluations and the irradiation values were assessed in the IMAGEJ software (NIH, Wayne Rasband, USA). Intensity in pixels was quantified in relation to the distance from the light incident point. Data were normalized and the results were presented as relative light intensity as a function of distance. The results demonstrated that red laser light is exponentially attenuated as a function of lateral distance and loses approximately 50 % of its intensity by 2.23 mm. On the other hand, the light travels 3 mm in depth to decay 50 %. In conclusion, our data suggest that melanin presence decreases optical pathway and irradiation protocols for antimicrobial photodynamic therapy in gingival tissue should consider light attenuation and depth of periodontal pockets so that efficient illumination of the target tissue occurs. Periodontal pockets bigger than 6 mm should be irradiated with more than one point.

Antimicrobial photodynamic therapy mediated by methylene blue in surfactant vehicle on periodontopathogens.

BACKGROUND: Periodontal disease (PD) is a chronic inflammatory disease caused by the presence of microbial biofilm. The aim of this study was to evaluate antimicrobial effect of antimicrobial photodynamic therapy (A-PDT) mediated by methylene blue (MB) in monomer form on A. actinomycetemcomitans and P. gingivalis. METHODS: A. actinomycetemcomitans ATCC 29523 and P. gingivalis ATCC 33577 were cultured on anaerobic jars at 37 °C for 48 h, and we tested APDT in the presence of 0.25% sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS) or in PBS alone. APDT was carried out with 100 µM MB under laser radiation (PhotolaseIII, DMC, Brazil) at ʎ =660 nm and parameters as following (P =100 mW; I =250 mW/cm2, and doses of 15, 45 and 75 J/cm2). RESULTS: Following A-PDT, PBS groups of A. actinomycetemcomitans presented 4 Logs of microbial death after 5 min irradiation. However, there was no bacterial reduction in SDS groups. On the other hand, P. gingivalis was sensitive to APDT in the presence of 0.25% SDS with 2 logs reduction from dark toxicity. CONCLUSION: The presence of 0.25% SDS can lead to different responses depending on the different microbial species.

Photodynamic inactivation assisted by localized surface plasmon resonance of silver nanoparticles: In vitro evaluation on Escherichia coli and Streptococcus mutans.

Localized surface plasmon resonance (LSPR) of gold nanoparticles has been reported to increase the antimicrobial effect of the photodynamic therapy. Although silver nanoparticles (AgNPs) are an efficient growth inhibitor of microorganisms, no studies exploring LSPR of AgNPs to enhance the photodynamic inactivation (PDI) have been related. In this work, we described the LSPR phenomenon of AgNP sand investigated its interaction with riboflavin, a natural photosensitizer. We evaluated the use of AgNPs coated with pectin (p-AgNP) in riboflavin (Rb)-mediated PDI of Escherichia coli (Gram- bacteria) and Streptococcus mutans (Gram + bacteria) using a blue light-emitting diode (λ = 455 ±â€¯20 nm) of optical power 200 mW. Irradiance was 90 mW/cm2 and radiant exposure varied according to the time exposure. Uptake of Rb and p-AgNP by the cells was evaluated by measuring the supernatant absorption spectra of the samples. We observed that LSPR of p-AgNPs was able to enhance the riboflavin photodynamic action on S. mutans but not on E. coli, probably due to the lower uptake of Rb by E. coli. Taken together, our results provide insights to explore the use of the LPRS promoted by silver nanostructures to optimize antimicrobial PDI protocols.

Antimicrobial photodynamic therapy on Streptococcus mutans is altered by glucose in the presence of methylene blue and red LED.

BACKGROUND: Dental caries are a multifactorial disease that progressively produces tooth destruction as a result of bacterial colonization of enamel surface, especially Streptococcus mutans. The objective of this work was to investigate the role of glucose in antimicrobial photodynamic therapy (aPDT) on S. mutans. METHODS: S. mutans ATCC 25175 were cultured on microaerophilia at 37°C for 48h, and we tested aPDT in the presence of 50mM glucose. Bacterial suspension was used to investigate aPDT with 100µM methylene blue (MB) under LED emitting radiation at ʎ=660nm and parameters as following (P=473 mW; I=166.8 mW/cm2, and doses of 5, 10 and 20J/cm2). A seventy-two hours biofilm was grown on 96 flat buttoned well-plate and irradiation was performed from 10 to 80J/cm2 at similar conditions. RESULTS: There was no dark toxicity nor bacterial death regarding LED irradiation on suspension and on biofilm. Nevertheless, aPDT presented expressive bacterial inactivation following 1 and 2min of irradiation on cell suspension. On the other hand, there was no inactivation in the presence of glucose under the same conditions. Biofilm was completely inactivated by MB-mediated aPDT after 6min of irradiation. However, the presence of glucose delayed the complete inactivation of the biofilm. CONCLUSION: The presence of glucose in the suspension drastically delayed the effect of aPDT on S. mutans and this effect is more pronounced in bacterial suspension than on biofilm.

CdTe quantum dots conjugated to concanavalin A as potential fluorescent molecular probes for saccharides detection in Candida albicans.

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

Effect of virulence factors on the photodynamic inactivation of Cryptococcus neoformans.

Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance.

Antimicrobial photodynamic therapy on drug-resistant Pseudomonas aeruginosa-induced infection. An in vivo study.

Pseudomonas aeruginosa is considered one of the most important pathogens that represent life-threatening risk in nosocomial environments, mainly in patients with severe burns. Antimicrobial photodynamic therapy (aPDT) has been effective to kill bacteria. The purpose of this study was to develop a burn wound and bloodstream infection model and verify aPDT effects on it. In vitro, we tested two wavelengths (blue and red LEDs) on a clinical isolate of P. aeruginosa strain with resistance to multiple antibiotics using HB:La(+3) as photosensitizer. Verapamil(®) associated to aPDT was also studied. In vivo, P. aeruginosa-infected burned mice were submitted to aPDT. Bacterial counting was performed on local infection and bloodstream. Survival time of animals was also monitored. In this study, aPDT was effective to reduce P. aeruginosa in vitro. In addition, Verapamil(®) assay showed that HB:La(+3) is not recognized by ATP-binding cassete (ABC) efflux pump mechanism. In the in vivo study, aPDT was able to reduce bacterial load in burn wounds, delay bacteremia and keep the bacterial levels in blood 2-3 logs lower compared with an untreated group. Mice survival was increased on 24 h. Thus, this result suggests that aPDT may also be a novel prophylactic treatment in the care of burned patients.

Antimicrobial photodynamic therapy as a strategy to arrest enamel demineralization: a short-term study on incipient caries in a rat model.

In this study we developed a rat model of incipient caries to investigate the short-term effects of antimicrobial photodynamic therapy (aPDT) on oral microbiota regulation and demineralization arrestment. Twenty-nine male rats were submitted to caries induction. Early carious lesion was confirmed by optical coherence tomography (OCT) 5 days after experiment beginning in five animals. The remaining animals (n = 24) were randomly divided into two groups: control (n = 12), animals were untreated; and aPDT (n = 12), animals were treated with 100 µM of methylene blue for 5 min and irradiated by a light emitting diode at λ = 645 ± 30 nm, fluence rate of 480 mW cm(-2) and exposure time of 3 min. Bacterial burden was evaluated before, immediately after, 3, 7 and 10 days following treatment, and total number of microaerophilic bacteria was counted. OCT was also used to quantify teeth demineralization. A significant bacterial decrease of about 1.6 log was observed immediately after aPDT. Besides, bacterial load in aPDT group remained lower than control until 10 days post-treatment (P < 0.05) and variation of optical attenuation coefficient before and after aPDT was 15%, corroborating to caries arrestment. Put together, these findings suggest that aPDT was competent to reduce cariogenic bacteria and to avoid further mineral loss.

Influence of multidrug efflux systems on methylene blue-mediated photodynamic inactivation of Candida albicans.

OBJECTIVES: To investigate whether the major fungal multidrug efflux systems (MESs) affect the efficiency of methylene blue (MB)-mediated antimicrobial photodynamic inactivation (APDI) in pathogenic fungi and test specific inhibitors of these efflux systems to potentiate APDI. METHODS: Candida albicans wild-type and mutants that overexpressed two classes of MESs [ATP-binding cassette (ABC) and major facilitator superfamily (MFS)] were tested for APDI using MB as the photosensitizer with and without addition of MES inhibitors. The uptake and cytoplasm localization of photosensitizer were achieved using laser confocal microscopy. RESULTS: ABC MES overexpression reduced MB accumulation and APDI killing more than MFS MES overexpression. Furthermore, by combining MB APDI with the ABC inhibitor verapamil, fungal killing and MB uptake were potentiated, while by combining MB APDI with the MFS inhibitor INF(271), fungal killing and MB uptake were inhibited. This latter surprising finding may be explained by the hypothesis that the MFS channel can also serve as an uptake mechanism for MB. CONCLUSIONS: The ABC pumps are directly implicated in MB efflux from the cell cytoplasm. Both the influx and efflux of MB may be regulated by MFS systems, and blocking this gate before incubation with MB can decrease the uptake and APDI effects. An ABC inhibitor could be usefully combined with MB APDI for treating C. albicans infections.

Effects of 960-nm diode laser irradiation on calcium solubility of dental enamel: an in vitro study.

OBJECTIVE: The aim of this study was to investigate the effects of 960 nm diode laser and acidulated phosphate fluoride on calcium solubility of human dental enamel. BACKGROUND DATA: Interest in diode lasers has grown steadily since its invention due to its inherent advantages and its range of applications. Several other laser types have shown good results in caries prevention; however, there are few studies on dental tissue interactions using diode lasers. METHODS: Acid resistance was evaluated using 65 enamel specimens, divided into five groups: control (C), fluoride (F), laser (L), laser + fluoride (LF), and fluoride + laser (FL). The laser was operated using the parameters of 6.5-W peak power, 5-msec pulse duration, 10-Hz repetition rate, and 33-mJ pulse energy. These parameters were previously tested regarding pulpal temperature rise and enamel morphology, and were determined to be safe. The amount of calcium lost during demineralization was measured. RESULTS: The calcium solubility of the laser group was 12% higher than of the control group (p > 0.05). Group F showed a 33.6% increase of acid resistance (p < 0.05). When laser was associated with fluoride, the calcium solubility increased significantly (p < 0.05) compared to both the control group and the laser group. Groups treated with fluoride showed the same results (p > 0.05). CONCLUSION: The 960-nm diode laser promoted a slight increase in calcium solubility. A statistically significant reduction on calcium solubility was achieved with the three treatments that involve fluoride (F, FL, and LF). The additional application of laser irradiation did not cause any significant increase or decrease in calcium solubility.