RESUMO
BACKGROUND: Although Candida albicans is the most frequent etiological agent of candidiasis, it has been reported a sizable number of infections related to the non-albicans Candida (NAC) species, Candida krusei. In addition, dual biofilms (biofilms composed by two species) may easily occur in vivo, becoming even more challenging the treatment of an infection. The fungicide effect of Photodynamic Therapy (PDT), using toluidine blue O (TBO) on both C. albicans and C. krusei development has been demonstrated. Thus, the objective of this study was to investigate the effects of PDT on dual-species biofilms of Candida albicans and Candida krusei. METHODS: The effect of PDT was observed on the metabolic activity of mature dual-species biofilms of Candida albicans and Candida krusei by a metabolic assay based on the reduction of XTT (2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt) assay and the identification of Candida albicans and Candida krusei was performed on CHROMagar Candida medium. RESULTS: it was observed a reduction of â¼30% in the metabolic activity of a mature biofilm treated with PDT, using 0.05 mg·mL-1 TBO and during biofilm formation a predominance of C. albicans on C. krusei was observed. The inhibition observed was related to reduction in the number of Colony Forming Units (CFU) of Candida albicans from 31.33 ± 3.7 to 17.0 ± 1.5. The number of CFU of C. krusei was not significantly modified. CONCLUSIONS: These results demonstrated the efficiency of PDT in inhibiting the dual-species biofilms of Candida albicans and Candida krusei by reducing C. albicans development.
Assuntos
Anti-Infecciosos , Fotoquimioterapia , Candida albicans , Cloreto de Tolônio/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Candida , Anti-Infecciosos/farmacologia , BiofilmesRESUMO
Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the FoF1-ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40 µM. Additionally, TBO (40 µM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Mitocôndrias Hepáticas , Ratos , Animais , Mitocôndrias Hepáticas/metabolismo , Cloreto de Tolônio/metabolismo , Cloreto de Tolônio/farmacologia , Metabolismo Energético , Fármacos Fotossensibilizantes/farmacologia , Trifosfato de Adenosina/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismoRESUMO
Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.
Assuntos
Coccidiostáticos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Redutase/efeitos dos fármacos , Azul de Metileno/análogos & derivados , Neospora/efeitos dos fármacos , Cloreto de Tolônio/farmacologia , Animais , Citoplasma/enzimologia , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Cinética , Masculino , Azul de Metileno/farmacologia , Camundongos Endogâmicos BALB C , Neospora/enzimologia , Neospora/genética , Neospora/crescimento & desenvolvimentoRESUMO
BACKGROUND: There are investigations on multiple photosensitizers for modulation of caries-related biofilms using PDT. However, much controversy remains about recommended parameters mostly on the selection of an efficient photosensitizer. OBJECTIVE: The study performed a systematic review to identify the answer to the following question: What photosensitizers present high bactericidal efficacy against cariogenic biofilms? METHODS: Systematic review with meta-analyses were carried out for English language articles from October to December 2019 (PRISMA standards) using MEDLINE, Scopus, Biomed Central, EMBASE, LILACS, and Web of Science. Information on study design, biofilm model, photosensitizer, light source, energy delivery, the incubation time for photosensitizer, and bacterial reduction outcomes were recorded. We performed two meta-analyses to compare bacterial reduction, data was expressed by (1) base 10 Logarithm values and (2) Log reduction RESULTS: After the eligibility criteria were applied (PEDro scale), the selected studies showed that toluidine Blue Ortho (TBO) and methylene blue (MBO) (5-min incubation time and 5-min irradiation) demonstrated better bacterial reduction outcomes. For the data expressed by Log TBO, MBO, curcumin, and Photogem® presented a significant bacterial decrease in comparison to the control (p = 0.042). For the data represented by Log reduction, the bacterial reduction toward S.mutans was not significant for any photosensitizer (p = 0.679). CONCLUSION: The lack of methodological standardization among the studies still hinders the establishment of photosensitizer and bactericidal efficiency. TBO, MBO, curcumin, and photogem generate greater PDT-based bacterial reduction on caries-related bacteria.. Further clinical studies are necessary in order to obtain conclusive results.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Biofilmes , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Streptococcus mutans , Cloreto de Tolônio/farmacologia , TriazenosRESUMO
Toluidine blue O (TBO) is a water-soluble photosensitizer that has been used in photodynamic antimicrobial and anticancer treatments, but suffers from limited solubility in hydrophobic media. In an effort to incrementally increase TBO's hydrophobicity, we describe the synthesis of hexanoic (TBOC6) and myristic (TBOC14) fatty acid derivatives of TBO formed in low to moderate percent yields by condensation with the free amine site. Covalently linking 6 and 14 carbon chains led to modifications of not only TBO's solubility, but also its photophysical and photochemical properties. TBOC6 and TBOC14 derivatives were more soluble in organic solvents and showed hypsochromic shifts in their absorption and emission bands. The solubility in phosphate buffer solution was low for both TBOC6 and TBOC14, but unexpectedly slightly greater in the latter. Both TBOC6 and TBOC14 showed decreased triplet excited-state lifetimes and singlet oxygen quantum yields in acetonitrile, which was attributed to heightened aggregation of these conjugates particularly at high concentrations due to the hydrophobic "tails." While in diluted aqueous buffer solution, indirect measurements showed similar efficiency in singlet oxygen generation for TBOC14 compared to TBO. This work demonstrates a facile synthesis of fatty acid TBO derivatives leading to amphiphilic compounds with a delocalized cationic "head" group and hydrophobic "tails" for potential to accumulate into biological membranes or membrane/aqueous interfaces in PDT applications.
Assuntos
Ácidos Graxos/química , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Cloreto de Tolônio/análogos & derivados , Estrutura Molecular , Fármacos Fotossensibilizantes/química , Oxigênio Singlete/química , Espectrometria de Fluorescência , Cloreto de Tolônio/síntese química , Cloreto de Tolônio/farmacologiaRESUMO
Chagas disease is endemic in Latin America and increasingly found in non-endemic countries. Its treatment is limited due to the variable efficacy and several side effects of benznidazole. Photodynamic antimicrobial chemotherapy (PACT) may be an attractive approach for treating Chagas disease. Here, the trypanocidal activity of PACT was investigated in vitro using phenothiazine derivatives. The cytotoxicity of both, methylene blue (MB) and toluidine blue (TBO), was determined on macrophages cultures using AlamarBlue method. The trypanocidal activity of the two photosensitizers was initially evaluated by determining their IC50 values against trypomastigote forms. After this, the trypanocidal effect was evaluated in cultures of infected macrophages using an automatized image analysis protocol. All experiments were performed in the dark and in the clear phase (after a photodynamic exposure). The compounds showed no cytotoxicity in both phases at the tested concentrations. The IC50 values for the sole use of MB and TBO were 2.6 and 1.2 µM, respectively. The photoactivation of the compounds using a fixed energy density (J/cm2) caused a reduction of the IC50 values to 1.0 and 0.9 µM, respectively. It was found that, on infected macrophage, the use of TBO significantly reduced the number of infected cells and parasitic load, and this effect was increased in the presence of light. The results of the present study are indicative that PACT may be considered as both selective and effective therapeutic intervention for treating Chagas disease.
Assuntos
Antiparasitários/farmacologia , Fenotiazinas/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Antiparasitários/uso terapêutico , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Doença de Chagas/tratamento farmacológico , Humanos , Luz , Azul de Metileno/química , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Camundongos Endogâmicos BALB C , Carga Parasitária , Fenotiazinas/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Cloreto de Tolônio/química , Cloreto de Tolônio/farmacologia , Cloreto de Tolônio/uso terapêutico , Trypanosoma cruzi/efeitos da radiaçãoRESUMO
The aim of this study was to perform a systematic review of the literature followed by a meta-analysis about the efficacy of photodynamic therapy (PDT) on the microorganisms responsible for dental caries. The research question and the keywords were constructed according to the PICO strategy. The article search was done in Embase, Lilacs, Scielo, Medline, Scopus, Cochrane Library, Web of Science, Science Direct, and Pubmed databases. Randomized clinical trials and in vitro studies were selected in the review. The study was conducted according the PRISMA guideline for systematic review. A total of 34 articles were included in the qualitative analysis and four articles were divided into two subgroups to perform the meta-analysis. Few studies have achieved an effective microbial reduction in microorganisms associated with the pathogenesis of dental caries. The results highlight that there is no consensus about the study protocols for PDT against cariogenic microorganisms, although the results showed the PDT could be a good alternative for the treatment of dental caries.
Assuntos
Infecções por Bacteroidaceae/tratamento farmacológico , Candidíase/tratamento farmacológico , Cárie Dentária/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções por Bacteroidaceae/microbiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida/efeitos dos fármacos , Candida/crescimento & desenvolvimento , Candida/patogenicidade , Candidíase/microbiologia , Curcumina/farmacologia , Cárie Dentária/microbiologia , Humanos , Azul de Metileno/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/patogenicidade , Corantes de Rosanilina/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus/efeitos dos fármacos , Streptococcus/crescimento & desenvolvimento , Streptococcus/patogenicidade , Cloreto de Tolônio/farmacologia , Resultado do TratamentoRESUMO
Chagas disease is a tropical illness caused by the protozoan Trypanosoma cruzi. The disease affects populations of the Americas and has been spread to other continents due to the migration process. The disease is partially controlled by two drugs, Benznidazole and Nifurtimox. These molecules are active in the acute phase of the infection but are usually ineffective during the symptomatic chronic phase. Several research groups have developed novel candidates to control Chagas disease; however, no novel commercial formulation is available. In this article, we described the anti-T. cruzi effects of phenothiazinium dyes in amastigote and trypomastigote forms of the parasite. Methylene Blue, New Methylene Blue, Toluidine Blue O, and 1,9-Dimethyl Methylene Blue inhibited the parasite proliferation at nanomolar concentrations and also demonstrated low toxicity in host cells. Moreover, combinations of phenothiazinium dyes indicated a synergic pattern against amastigotes compared to the Benznidazole counterparts. Phenothiazinium dyes levels of reactive oxygen species (ROS) and decreased the mitochondrial potential in trypomastigotes, indicating the mechanism of action of the dyes in T. cruzi. Our article offers a basis for future strategies for the control of Chagas disease using low-cost formulations, an important point for endemic underdeveloped regions.
Assuntos
Proliferação de Células/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Fenotiazinas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Doença de Chagas/parasitologia , Corantes/farmacologia , Humanos , Azul de Metileno/análogos & derivados , Azul de Metileno/farmacologia , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Cloreto de Tolônio/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/patogenicidadeRESUMO
Objective: The goals of this investigation were to compare the effect of photodynamic antimicrobial chemotherapy (PACT) with two different red lights on in vitro Streptococcus mutans biofilms, as well as to assess the temperature variances caused by PACT on human teeth. Methods: S. mutans biofilms (n = 3) were grown on hydroxyapatite disks, and the antimicrobial effect of PACT was evaluated using toluidine blue O (100 µg/mL) associated with Laserbeam® (LB 56.6 J/cm2) and LumaCare™ (LC -56.6, 158.5, 317.0, and 475.6 J/cm2). Pulpal temperature variances were analyzed using a digital thermocouple placed into the pulp chamber and positioned at the cement-enamel junction level of five teeth samples during irradiation times of 300, 600, and 900 sec for LB, and 22, 60, 120, and 180 sec for LC. The mean average temperature variance was calculated for each group. All data were analyzed through analysis of variance. Results: LB (900 sec) and LC (22 sec) induced similar reductions in the viability of microorganisms. LB did not cause statistically significant increase of temperature, regardless of experimental time, and LC caused temperature increase within the safe spectrum up to 60 sec. Conclusions: PACT seems to be a minimal invasive approach for reducing the viability of cariogenic bacteria. Thus, when applied in vitro for times equal or inferior to 900 and 60 sec for LB and LC, respectively, these light sources might be considered harmless to tooth structures.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Cárie Dentária/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Streptococcus mutans/efeitos dos fármacos , Cloreto de Tolônio/farmacologia , Humanos , Técnicas In Vitro , TemperaturaRESUMO
BACKGROUND: The thermo-dimorphic fungus Paracoccidioides brasiliensis is the pathogen of Paracoccidioidomycosis, an important public health problem in Latin American with prevalence in Brazil. Photodynamic Antimicrobial Chemotherapy (PACT) is a process that combines a photosensitizer and light, producing reactive oxygen species (ROS) that can promote damages to treated cells. METHODS: In this work was study the effect of PACT, using Toluidine blue (TBO) on both yeast and mycelial cells of P. brasiliensis. RESULTS: It was observed that PACT decreased P. brasiliensis yeast growth, in a dependent manner of both TBO concentrations and fluence. In the presence of TBO 0.005â¯mg/mL, PACT reduced P. brasiliensis yeast growth in 63, 62 and 86%, using fluences of 20, 30 and 40â¯J/cm2, respectively. After PACT, ROS production increased 2.80, 4.64 and 7.90 times, in the presence of TBO 0.001, 0.002 and 0.005â¯mg/mL, respectively. It was observed that after PACT, the cells are predominantly in mycelia form, indicating that mycelial cells irradiated in the presence of TBO, maintained their filamentous form and absence and/or decreased presence of transition structures. CONCLUSIONS: These results demonstrated the potential of PACT, using TBO to inhibit both yeast and mycelium development of P. brasiliensis.
Assuntos
Paracoccidioides/efeitos dos fármacos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Tolônio/farmacologia , Biofilmes/efeitos dos fármacos , Brasil , Relação Dose-Resposta a DrogaRESUMO
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 (660â¯nm, 40â¯mW, 60â¯s, 12â¯J/cm2) or LED (632⯱â¯2â¯nm, 145â¯mW, 40â¯s, 12â¯J/cm2). Six experimental groups were evaluated: Control Group (untreated); Photosensitizer group (phenothiazines - 12.5⯵g/mL); Laser Group; LED Group; Laser PACT Group; and LED PACT Group. The pre-irradiation time used in this study was 5â¯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 (0â¯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 (pâ¯>â¯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.
Assuntos
Macrófagos/efeitos dos fármacos , Fenotiazinas/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Luz , Macrófagos/microbiologia , Azul de Metileno/farmacologia , Microscopia de Fluorescência , Cloreto de Tolônio/farmacologiaRESUMO
Antimicrobial Photodynamic Inactivation (a-PDI) is based on the oxidative destruction of biological molecules by reactive oxygen species generated by the photo-excitation of a photosensitive molecule. When a-PDT is performed with the use of mathematical models, the optimal conditions for maximum inactivation are found. Experimental designs allow a multivariate analysis of the experimental parameters. This is usually made using a univariate approach, which demands a large number of experiments, being time and money consuming. This paper presents the use of the response surface methodology for improving the search for the best conditions to reduce E. coli survival levels by a-PDT using methylene blue (MB) and toluidine blue (TB) as photosensitizers and white light. The goal was achieved by analyzing the effects and interactions of the three main parameters involved in the process: incubation time (IT), photosensitizer concentration (CPS), and light dose (LD). The optimization procedure began with a full 23 factorial design, followed by a central composite one, in which the optimal conditions were estimated. For MB, CPS was the most important parameter followed by LD and IT whereas, for TB, the main parameter was LD followed by CPS and IT. Using the estimated optimal conditions for inactivation, MB was able to inactivate 99.999999%â¯CFUâ¯mL-1 of E. coli with IT of 28â¯min, LD of 31â¯Jâ¯cm-2, and CPS of 32⯵molâ¯L-1, while TB required 18â¯min, 39â¯Jâ¯cm-2, and 37⯵molâ¯L-1. The feasibility of using the response surface methodology with a-PDT was demonstrated, enabling enhanced photoinactivation efficiency and fast results with a minimal number of experiments.
Assuntos
Escherichia coli/efeitos dos fármacos , Azul de Metileno/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Cloreto de Tolônio/farmacologia , Relação Dose-Resposta a Droga , Azul de Metileno/administração & dosagem , Modelos Teóricos , Fármacos Fotossensibilizantes/administração & dosagem , Fatores de Tempo , Cloreto de Tolônio/administração & dosagemRESUMO
Among non-albicans Candida species, the opportunistic pathogen Candida krusei emerges because of the high mortality related to infections produced by this yeast. The Candida krusei is an opportunistic pathogen presenting an intrinsic resistance to fluconazol. In spite of the reduced number of infections produced by C. krusei, its occurrence is increasing in some groups of patients submitted to the use of fluconazol for prophylaxis. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using toluidine blue, as a photosensitizer on both growth and biofilm formation by Candida krusei. In this work, we studied the effect of the PACT, using TB on both cell growth and biofilm formation by C. krusei. PACT was performed using a light source with output power of 0.068 W and peak wavelength of 630 nm, resulting in a fluence of 20, 30, or 40 J/cm2. In addition, ROS production was determined after PACT. The number of samples used in this study varied from 6 to 8. Statistical differences were evaluated by analysis of variance (ANOVA) and post hoc comparison with Tukey-Kramer test. PACT inhibited both growth and biofilm formation by C. krusei. It was also observed that PACT stimulated ROS production. Comparing to cells not irradiated, irradiation was able to increase ROS production in 11.43, 6.27, and 4.37 times, in the presence of TB 0.01, 0.02, and 0.05 mg/mL, respectively. These results suggest that the inhibition observed in the cell growth after PACT could be related to the ROS production, promoting cellular damage. Taken together, these results demonstrated the ability of PACT reducing both cell growth and biofilm formation by C. krusei.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida/fisiologia , Fotoquimioterapia , Cloreto de Tolônio/farmacologia , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/crescimento & desenvolvimento , Luz , Testes de Sensibilidade Microbiana , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Root demineralization is used in Periodontics as an adjuvant for mechanical treatment. The aim of this study was to evaluate the effects of root surface modification with mechanic, chemical, and photodynamic treatments on adhesion and proliferation of human gingival fibroblasts and osteoblasts. Root fragments were treated by scaling and root planing (C-control group), EDTA (pH 7), citric acid plus tetracycline (CA-pH 1), and antimicrobial photodynamic therapy (aPDT) with toluidine blue O and red laser (pH 4). Cells were seeded (104 cells/well, 6th passage) on root fragments of each experimental group and cultured for 24, 48, and 72 h. Cells were counted in scanning electron microscopy images by a calibrated examiner. For fibroblasts, the highest number of cells were present at 72-h period (p < 0.05). EDTA group showed a very low number of cells in relation to CA group (p < 0.05). CA and aPDT group presented higher number of cells in all periods, but without differences between other treatment groups (p > 0.05). For osteoblasts, there was a significant increase in cell numbers for aPDT group at 72 h (p < 0.05). In conclusion, aPDT treatment provided a positive stimulus to osteoblast growth, while for fibroblasts, aPDT and CA had a tendency for higher cell growth.
Assuntos
Anti-Infecciosos/farmacologia , Ácido Cítrico/farmacologia , Ácido Edético/farmacologia , Fibroblastos/citologia , Gengiva/citologia , Osteoblastos/citologia , Fotoquimioterapia , Raiz Dentária/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Aplainamento Radicular , Tetraciclina/farmacologia , Cloreto de Tolônio/farmacologiaRESUMO
BACKGROUND: Candida albicans is an opportunistic fungus producing both superficial and systemic infections, especially in immunocompromised individuals. It has been demonstrated that C. albicans ability to form biofilms is a crucial process for colonization and virulence. Furthermore, a correlation between the development of drug resistance and biofilm maturation at Candida biofilms has been shown. Photodynamic Antimicrobial Chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a non-toxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using Toluidine Blue O (TBO) on the viability of biofilms produced by C. albicans at different stages of development. METHODS: In this study, the effects of PACT on both biofilm formation and viability of the biofilm produced by C. albicans were studied. Biofilm formation and viability were determined by a metabolic assay based on the reduction of XTT assay. In addition, the morphology of the biofilm was observed using light microscopy. RESULTS: PACT inhibited both biofilm formation and viability of the biofilm produced by C. albicans. Furthermore, PACT was able to decrease the number of both cells and filamentous form present in the biofilm structure. This inhibitory effect was observed in both early and mature biofilms. CONCLUSIONS: The results obtained in this study demonstrated the potential of PACT (using TBO) as an effective antifungal therapy, including against infections associated with biofilms at different stages of development.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Cloreto de Tolônio/farmacologia , Sobrevivência Celular , Humanos , Espécies Reativas de OxigênioRESUMO
The purpose of this study was to assess, for the very first time, the effects of photodynamic therapy (PDT) on Schistosoma mansoni in vitro by measuring reactive oxygen species (ROS) generation throughout the treatment, as well as the behavior of the parasites (mating, motility and contraction/shortening), and damage to their tegument and excretory systems. The parasites were divided into 4 groups: control, photosensitizer, laser and PDT. Light irradiation was delivered with an InGaAlP low-level laser device operating at 660nm, with 40mW and 100J/cm2. For PDT, different toluidine blue dye (TBO) concentrations and times of exposure were utilized. Interestingly, TBO-mediated PDT was able to kill S. mansoni (P<0.001) due to the significant amount of ROS released that inflicted damages in the tegument and excretory system, as well as contraction and cessation of motility. In addition, males of S. mansoni were shown to be more sensitive to PDT if compared to their corresponding females when the optimal TBO concentration of 31.2µL was considered (P=0.0126). PDT presents two major advantages: not inducing microbial resistance and also lacking adverse effects. Therefore, PDT may become a promising therapeutic alternative for schistosomiasis in the near future, especially for cases of allergy and resistance to praziquantel.
Assuntos
Schistosoma mansoni/efeitos dos fármacos , Cloreto de Tolônio/farmacologia , Animais , Feminino , Lasers , Masculino , Microscopia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Schistosoma mansoni/metabolismo , Cloreto de Tolônio/químicaRESUMO
Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm2) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.
Assuntos
Biofilmes/crescimento & desenvolvimento , Raios Infravermelhos , Fotoquimioterapia , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/efeitos da radiação , Endopeptidase K/farmacologia , Queratinócitos/efeitos da radiação , Camundongos , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/efeitos da radiação , Staphylococcus aureus/ultraestrutura , Temperatura , Cloreto de Tolônio/farmacologiaRESUMO
The antifungal drug therapy often employed to treat paracoccidiodomycosis (PCM), an important neglected fungal systemic infection, leads to offensive adverse effects, besides being very long-lasting. In addition, PCM compromises the oral health of patients by leading to oral lesions that are very painful and disabling. In that way, photodynamic therapy (PDT) arises as a new promising adjuvant treatment for inactivating Paracoccidioides brasiliensis (Pb), the responsible fungus for PCM, and also for helping the patients to deal with such debilitating oral lesions. PDT has been linked to an improved microbial killing, also presenting the advantage of not inducing immediate microbial resistance such as drugs. For the present study, we investigated the generation of reactive oxygen species (ROS) by using the fluorescent probes hydroxyphenyl fluorescein (HPF) and aminophenyl fluorescein (APF) after toluidine blue (TBO-37.5 mg/L)-mediated PDT (660 nm, 40 mW, and 0.04 cm2 spot area) and the action of TBO-PDT upon Pb cultures grown for 7 or 15 days in semisolid Fava Netto's culture medium; we also targeted oral PCM manifestations by reporting the first clinical cases (three patients) to receive topic PDT for such purpose. We were able to show a significant generation of hydroxyl radicals and hypochlorite after TBO-PDT with doses around 90 J/cm2; such ROS generation was particularly useful to attack and inactivate Pb colonies at 7 and 15 days. All three patients reported herein related an immediate relief when it came to pain, mouth opening, and also the ability to chew and swallow. As extracted from our clinical results, which are in fact based on in vitro outcomes, TBO-PDT is a very safe, inexpensive, and promising therapy for the oral manifestations of PCM.
Assuntos
Viabilidade Microbiana/efeitos dos fármacos , Doenças da Boca/tratamento farmacológico , Doenças da Boca/microbiologia , Paracoccidioides/efeitos da radiação , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologia , Fotoquimioterapia , Cloreto de Tolônio/uso terapêutico , Adulto , Antifúngicos/farmacologia , Corantes Fluorescentes/química , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Doenças da Boca/patologia , Paracoccidioides/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Tolônio/farmacologiaRESUMO
OBJECTIVE: The aim of this literature review is to study the effect of photodynamic antimicrobial chemotherapy (PACT) on mono- and multi-species cariogenic biofilms. METHODS: To this purpose, the database, PubMed, was searched using the descriptors, photodynamic therapy, antimicrobial photodynamic chemotherapy, and photoinactivation, associated with the mandatory presence of the word biofilm. A total of 98 references published from 2003 to 2016 were selected. Moreover, literature reviews (15), investigations that did not have biofilms related to dental caries (65), and those that did not have Streptococcus mutans count as an outcome (7) were excluded, yielding a final amount of 11 publications. RESULTS: The results revealed that Toluidine Blue O was the most used photosensitizer. Among the sources of light, light-emitting diode was the choice, and the biofilm models varied between in vitro and in situ. Multi-species biofilms were more resistant to the antimicrobial effects of PACT due to the thickness and complexity they have, which impede the penetration of the photosensitizer. This fact may also be associated with the type of photosensitizer used as well as with the light exposure time since the antimicrobial effect seems to be dose dependent. Despite this, in all the included publications, the therapy was effective in reducing S. mutans count. CONCLUSIONS: This review demonstrated that under different conditions, PACT is effective in reducing S. mutans count in monospecies biofilms. Multi-species biofilms were more resistant to the antimicrobial action of the therapy, possibly due to their thickness and complexity.
Assuntos
Antibacterianos/farmacologia , Cárie Dentária/tratamento farmacológico , Cárie Dentária/microbiologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Biofilmes/efeitos dos fármacos , Humanos , Cloreto de Tolônio/farmacologiaRESUMO
The aim of this study was to evaluate the effect of a low-level laser therapy in combination with toluidine blue on polysaccharides and biofilm of Streptococcus mutans. S. mutans biofilms were formed on acrylic resin blocks. These biofilms were exposed eight times/day to 10 % sucrose, and two times/day, they were subjected to one of the following treatments: G1, 0.9 % NaCl as a negative control; G2, 0.12 % chlorhexidine digluconate (CHX) as a positive antibacterial control; and G3 and G4 antimicrobial photodynamic therapy (aPDT) combined with toluidine blue using dosages of 320 and 640 J/cm(2), respectively. The experiment was performed in triplicate. The biofilm formed on each block was collected for determination of the viable bacteria and concentration of insoluble extracellular polysaccharides (IEPS) and intracellular polysaccharides (IPS). CHX and aPDT treatments were able to inhibit bacterial growth in comparison with negative control (p < 0.05). The aPDT treatment reduced the number of viable bacteria formed in the S. mutans biofilm, in a dose-dependent manner (p < 0.05). The concentration of IEPS and IPS in the biofilms formed in presence of aPDT did not differ each other or in comparison to CHX (p > 0.05). The results suggest that low-level laser therapy presents effects on biofilm bacteria viability and in polysaccharides concentration.