Efficiency of Antimicrobial Photodynamic Therapy with Photodithazine® on MSSA and MRSA Strains.

Staphylococccus aureus is a ubiquitous and opportunistic bacteria associated with high mortality rates. Antimicrobial photodynamic therapy (aPDT) is based on the application of a light source and a photosensitizer that can interact with molecular oxygen, forming Reactive Oxygen Species (ROS) that result in bacterial inactivation. This study aimed to analyze, in vitro, the action of aPDT with Photodithazine® (PDZ) in methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. The strains were incubated with PDZ at 25, 50, 75, and 100 mg/L for 15 min and irradiated with fluences of 25, 50, and 100 J/cm2. The internalization of PDZ was evaluated by confocal microscopy, the bacterial growth by counting the number of colony-forming units, as well as the bacterial metabolic activity post-aPDT and the production of ROS. In both strains, the photosensitizer was internalized; the production of ROS increased when the aPDT was applied; there was a bacterial reduction compared to the control at all the evaluated fluences and concentrations; and, in most parameters, it was obtained complete inactivation with significant difference (p < 0.05). The implementation of aPDT with PDZ in clinical strains of S. aureus has resulted in its complete inactivation, including the MRSA strains.

CELLULAR AND METABOLIC CHANGES AFTER PHOTODYNAMIC THERAPY IN LEISHMANIA PROMASTIGOTES.

Leishmaniasis is a zoonotic disease, regarded by WHO as a public health problem that has presented a significant increase in the recent years. Conventional treatment is toxic and leads to serious side effects. Photodynamic therapy has been studied as a treatment to cutaneous leishmaniasis. This study aimed to evaluate the cell viability, morphological changes, type of cell death, production of reactive oxygen species, and changes in the mitochondrial membrane and DNA fragmentation in Leishmania braziliensis and Leishmania major promastigotes. Confocal microscopy was used to quantify the fluorescence emitted by JC-1, Annexin V, and propidium iodide reagents. The trypan blue exclusion test was used to evaluate the viability of the cells, the mitochondrial activity was verified with MTT, and the morphological changes were analyzed for SEM and DNA damage using the comet assay. PDT using curcumin at 500, 125, and 31,25 µg/mL decreased the viability of the parasites and induced changes in the mitochondrial membrane potential. The production of reactive oxygen species was dose-dependent and was observed only in the groups submitted to PDT. DNA damage was also observed in the parasite cells. The morphology of the cells was affected mainly at the highest curcumin concentration, resulting in rounded cells with a shortened flagellum. When the type of cell death was analyzed, the prevalence of apoptosis was noted. The results support the use of curcumin as photosensitizer in PDT against Leishmania promastigotes in the treatment for cutaneous leishmaniasis.

Evaluation of the Photodynamic Therapy with Curcumin on L. braziliensis and L. major Amastigotes.

Cutaneous leishmaniasis (CL) is a neglected disease prevalent in tropical countries with the ability to cause skin lesions. Photodynamic therapy (PDT) represents a specific and topical option for the treatment of these lesions. This study evaluated the response of macrophages infected with L. braziliensis and L. major to PDT with curcumin. Curcumin concentrations were evaluated in serial dilutions from 500.0 to 7.8 µg/mL using LED (λ = 450 ± 5 nm), with a light dose of 10 J/cm2. The Trypan blue viability test, ultrastructural analysis by scanning electron microscopy (SEM), mitochondrial polarity by Rhodamine 123 (Rho 123), curcumin internalization by confocal microscopy, and counting of the recovered parasites after the PDT treatment were performed. The lowest concentrations of curcumin (15.6 and 7.8 µg/mL) presented photodynamic inactivation. Cell destruction and internalization of curcumin in both macrophages and intracellular parasites were observed in microscopy techniques. In addition, an increase in mitochondrial membrane polarity and a decrease in the number of parasites recovered was observed in the PDT groups. This study indicates that PDT with curcumin has the potential to inactivate infected macrophages and might act as a basis for future in vivo studies using the parameters herein discussed.

Antimicrobial effects of photodynamic therapy with Fotoenticine on Streptococcus mutans isolated from dental caries.

Photodynamic therapy (PDT) is a promising strategy to control cariogenic pathogens, such as Streptococcus mutans. Seeking to reach the total bacterial elimination from dental surfaces, novel photosensitizers have been investigated, such as Fotoenticine (FTC) derived from chlorin e6. The objective of this study was to investigate the photodynamic effects of FTC against several clinical strains of S. mutans. Clinical isolates were obtained from patients with active carious lesions, identified by molecular analysis and subjected to PDT using laser irradiation (660 nm and 39.5 J/cm2) in planktonic and biofilm stages. We identified 11 S. mutans strains from cervical, occlusal and proximal caries. PDT mediated by FTC has totally eliminated the S. mutans cells in planktonic growth for all analyzed strains. In biofilms, PDT with FTC reached statistically significant reductions compared with the non-treated control group, at 5.4, 5.5 and 6.5 Log10 (CFU/mL), respectively, for the strains from proximal, occlusal and cervical caries. The scanning electron microscopy evaluations confirmed that PDT mediated by FTC was able to disaggregate and kill the S. mutans cells adhered to enamel surface, suggesting its potential to disinfect the dental tissues.

Analysis of the effect of photodynamic therapy with Fotoenticine on gliosarcoma cells.

Gliosarcoma is a highly aggressive malignant neoplasm and a histopathological variant of wild-type glioblastoma multiforme isocitrate dehydrogenase (HDI). The current standard treatment consists of chemotherapy, radiotherapy and surgical resection, however, despite advances in these techniques, the patient's prognosis remains unfavorable. Photodynamic therapy (PDT) is a noninvasive technique that has been highlighted as an alternative form of cancer treatment because it does not present the side effects associated with systemic treatments. The objective of this study was to evaluate the cell viability and the intracellular localization of photosensitizer (PS) chlorin e6 Fotoenticine in 9L/lacZ cells. Therefore, tests of cytotoxicity, morphology, and location of PS were performed. The viability test showed no cytotoxicity in the dark at all concentrations and 100 % cell death at the highest concentrations after PDT. The mitochondrial activity test showed a reduction in all groups after PDT. The production of reactive oxygen species (ROS) was higher in the PDT groups and dependent on the PS concentration. Morphological analysis after PDT showed apparent cytoplasmic destruction in all the tested concentrations, with the presence of rounded cells due to the loss of their extensions and absence of nuclear alterations. The PS accumulation in the mitochondria and cytoskeleton was observed by the confocal microscopy; however, there is no evidence of its internalization in the lysosomes. It was concluded that PDT with Fotoenticine is a promising alternative therapy showing decreased cell viability, increased ROS production and adequate localization to trigger cell death.

Methylene blue internalization and photodynamic action against clinical and ATCC Pseudomonas aeruginosa and Staphyloccocus aureus strains.

Bacterial infections have been a major challenge to health. Increasing resistance to antimicrobial agents, according to World Health Organization, could be the major cause of death until 2050. Photodynamic therapy emerges as an alternative in microbial inactivation, due to its selectivity and to decreasing or dismissing antibiotic use. This study aimed at evaluating, in vitro, the internalization of the Methylene Blue and its photodynamic activity against a clinical and ATCC strain of Pseudomonas aeruginosa and Staphyloccocus aureus. Thus, the strains were incubated with MB in concentrations of 100, 300 e 500 µg/ml and then irradiated with a LED (±660 nm) at fluence of 10 and 25 J/cm2. The MB internalization was evaluated using a confocal microscope (Zeiss LSM 700), to capture the MB and the DAPI (for DNA staining). It was possible to observe that the MB was internalized by the bacterial cells, in all concentrations tested. The CFU/ml count demonstrated significant reduction (p ≤ 0,01) at the average 5.0 logs comparing with control group for the two species in all the tested concentrations. In conclusion, the strains tested were capable of internalizing the MB. PDT with MB was able to decrease the growth of the tested strains in vitro, being a promising alternative to the future treatment of infections caused by these species.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.

Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes-In vitro study.

BACKGROUND: Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as a treatment for cancer, and it now appears to be a promising therapy for local treatment with fewer side effects of infectious diseases. METHODS: This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received fluence of 10J/cm(2) (660nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. RESULTS: The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. CONCLUSIONS: This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols.