Antimicrobial photodynamic therapy with methylene blue and its derivatives in animal studies: Systematic review.

BACKGROUND: Infections are complications in the wound healing process, and their treatment can lead to antibiotic overuse and bacterial resistance. Antimicrobial photodynamic therapy (aPDT) is used to treat infectious diseases caused by fungi, viruses, or bacteria. Methylene blue (MB) and its derivatives are commonly used dyes in antimicrobial photodynamic therapy (aPDT-MB). METHODS: This study is a PRISMA systematic review of animal models used to discuss the usefulness and therapeutic parameters of aPDT-MB or its derivatives for treating infected skin wounds. RESULTS: After an extensive literature review, 13 controlled trials totaling 261 animals were selected to evaluate skin infection by leishmaniasis and cutaneous bacterial and fungal infections. All studies found results favoring the use of aPDT-MB. Great variability in parameters was found for radiant exposure from 12 to 360 J/cm2, MB diluted in saline solution or distilled water, irradiation time from 40 to 3600 s, irradiance most commonly at a maximum of 100 mW/cm2, and wavelength used mainly in the 630-670 nm range. CONCLUSION: MB is a safe and promising agent used as a photosensitizer in aPDT for skin-infected lesions. There is great variability in the parameters found. Comparisons concerning concentration, irradiation time, and light intensity need to be performed.

Photodynamic therapy repairs medication-related osteonecrosis of the jaw by reducing NF-kB protein in rats.

OBJECTIVE: To evaluate whether antimicrobial photodynamic therapy (aPDT) repairs bisphosphonate-related osteonecrosis of the jaw (BRONJ) modulated by the reduction of NF-kB protein in a murine model. METHODOLOGY: Male Wistar rats (N=30) were divided into the following groups (n=6/group): negative control (NC); experimental osteonecrosis (ONE); ONE + photosensitizer (PS); ONE + photobiomodulation (PBM); and ONE + aPDT. Over 8 weeks, ONE was induced by zoledronic acid 250 µg/kg injections, except in the NC group, which received sterile 0.9% saline, followed by extraction of the lower left first molar. Red light laser irradiation (wavelength ~660 nm, power 50 mW, energy of 2 J, energy dose of 66.67 J/cm2 for 40 s) was performed once a week for 4 weeks. Methylene blue 0.3% was used as PS. The animals were euthanized and examined macroscopically for the presence of exposed bone and epithelial repair and microscopically by histochemical (hematoxylin-eosin and Masson's trichrome staining) and immunohistochemical (anti-NF-kB) methods. Macroscopic and histomorphometric data were analyzed by one-way ANOVA and Tukey's post-test (p<0.05). RESULTS: Mucosal repair, viable osteocytes, and NF-kB immunostaining were observed in the NC, ONE+PS, ONE+PBM, and ONE+aPDT groups. The ONE group showed no mucosal repair, showing empty lacunae and multifocal immunostaining for NF-kB. The ONE+PBM and ONE+aPDT groups had greater deposition of extracellular matrix and less necrotic bone tissue (p<0.05). CONCLUSION: PBM and aPDT treatments for BRONJ were effective for bone and epithelial repair, in addition to reducing inflammation mediated by the decrease of NF-kB protein in the irradiated regions.

Reduction of microorganisms in carious dentin by photodynamic therapy mediated by potassium iodide added to methylene blue and red laser.

OBJECTIVE: To evaluate the effect of the association of potassium iodide to antimicrobial photodynamic therapy on human carious dentin produced with a microcosm biofilm model. METHODS: A microcosm biofilm model was used to generate a caries lesion on human dentin. Pooled human saliva diluted with glycerol was used as an inoculum on specimens immersed on McBain artificial saliva enriched with 1 % sucrose (24 h at 37 °C in 5 % CO2). After refreshing culture media for 7 days, the dentin specimens were divided in 5 groups (3 specimens per group, in triplicate; n = 9): C (NaCl 0.9 %), CX (2 % chlorhexidine), PKI (0.01 % methylene blue photosensitizer+50 mM KI), L (laser at 15 J, 180 s, 22.7 J/cm2), and PKIL (methylene blue + KI + Laser). After the treatments, dentin was collected, and a 10-fold serial dilution was performed. The number of total microorganisms, total lactobacilli, total streptococci, and Streptococcus mutans was analyzed by microbial counts (CFU/mL). After normality and homoscedasticity analysis, the Welch's ANOVA and Dunnett's tests were used for CFU. All tests used a 5 % significance level. RESULTS: CX and PKIL groups showed significant bacterial decontamination of dentin, compared to group C (p < 0.05) reaching reductions up to 3.8 log10 for CX for all microorganisms' groups and PKIL showed 0.93, 1.30, 1.45, and 1.22 log10 for total microorganisms, total lactobacilli, total streptococci, and S. mutans, respectively. CONCLUSION: aPDT mediated by the association of KI and methylene blue with red laser reduced the viability of microorganisms from carious dentin and could be a promising option for cavity decontamination.

Intravital Microscopy Evidence That Methylene Blue Should Be a Vasopressor-Sparing Agent in Sepsis Vasoplegia.

Microvasculature failure is expected in sepsis and at higher amine concentrations. Therefore, special attention focused individually on microcirculation is needed. Here, we present that methylene blue can prevent leukocytes from adhering to the endothelium in a rat model of lipopolysaccharide-induced endotoxemia. As hypothesis evidence, an intravital microscopy image is presented.

Methylene blue@silver nanoprisms conjugates as a strategy against Candida albicans isolated from balanoposthitis using photodynamic inactivation.

Balanoposthitis can affect men in immunocompromised situations, such as HIV infection and diabetes. The main associated microorganism is Candida albicans, which can cause local lesions, such as the development of skin cracks associated with itching. As an alternative to conventional treatment, there is a growing interest in the photodynamic inactivation (PDI). It has been shown that the association of photosensitizers with metallic nanoparticles may improve the effectiveness of PDI via plasmonic effect. We have recently shown that the association of methylene blue (MB), a very known photosensitizer, with silver prismatic nanoplatelets (AgNPrs) improved PDI of a resistant strain of Staphylococcus aureus. To further investigate the experimental conditions involved in PDI improvement, in the present study, we studied the effect of MB concentration associated with AgNPrs exploring spectral analysis, zeta potential measurements, and biological assays, testing the conjugated system against C. albicans isolated from a resistant strain of balanoposthitis. The AgNPrs were synthesized through silver anisotropic seed growth induced by the anionic stabilizing agent poly(sodium 4-styrenesulfonate) and showed a plasmon band fully overlapping the MB absorption band. MB and AgNPrs were conjugated through electrostatic association and three different MB concentrations were tested in the nanosystems. Inactivation using red LED light (660 nm) showed a dose dependency in respect to the MB concentration in the conjugates. Using the highest MB concentration (100 µmol⋅L-1) with AgNPr, it was possible to completely inactivate the microorganisms upon a 2 min irradiation exposure. Analyzing optical changes in the conjugates we suggest that these results indicate that AgNPrs are enhancers of MB photodynamic action probably by a combined mechanism of plasmonic effect and reduction of MB dimerization. Therefore, MBAgNPrs can be considered a suitable choice to be applied in PDI of resistant microorganisms.

Adjuvant effect of antimicrobial photodynamic therapy (aPDT) in the treatment of diabetic foot ulcers: A case series.

This study aimed to evaluate the clinical evolution of patients with diabetic foot ulcer treated with antimicrobial photodynamic therapy (aPDT) using the Bates-Jensen (BJ) scale. A total of 21 patients were monitored, with an average age of 58 years. Patients underwent the standard treatment protocol of the institution, supplemented with aPDT utilizing 0.01% methylene blue (MB) and laser irradiation (660 nm, 100 mW, 6 J per point). Following aPDT, the lesions were protected with hydrofiber dressings containing silver. The Bates-Jensen Scale was employed at pre-treatment and post-aPDT sessions to assess lesion progression. The results demonstrated a significant difference between pre- and post-treatment values in the overall BJ score. The use of MB in aPDT proved to be an effective, safe, well-tolerated treatment with high patient adherence and the potential for implementation in the care of diabetic foot conditions.

Association of Papacarie Duo® and low-level laser in antimicrobial photodynamic therapy (aPDT).

Dental caries is a multifactorial, non-communicable disease. Effective treatment options for minimally invasive removal of carious tissue include Papacarie Duo® gel and antimicrobial photodynamic therapy (aPDT). aPDT involves a combination of a light source and photosensitizer. Given that Papacarie Duo® contains a percentage of blue dye, this study aims to explore the antimicrobial potential of Papacarie Duo® when associated with a light source against Streptococcus mutans strains. The chosen light source was a low-power diode laser (λ = 660 nm, E = 3 J, P = 100 mW, t = 30 s). To assess antimicrobial capacity, planktonic suspensions of Streptococcus mutans were plated on Brain Heart Infusion Agar (BHI) to observe the formation of inhibition halos. The studied groups included methylene blue (0.005%), Papacarie Duo®, distilled water (negative control), 2% chlorhexidine (positive control), Papacarie Duo® + laser, and methylene blue (0.005%) + laser. Following distribution onto plates, each group was incubated at 37 °C for 48 h under microaerophilic conditions. Inhibition halos were subsequently measured using a digital caliper. The results showed that chlorhexidine had the greatest antimicrobial effect followed by the group of irradiated methylene blue and irradiated Papacarie Duo®. All experimental groups demonstrated antimicrobial potential, excluding the negative control group. The study concludes that Papacarie Duo® exhibits antimicrobial properties when associated with a low-power diode laser.

Methylene blue as an adjuvant during cardiopulmonary resuscitation: an experimental study in rats.

INTRODUCTION: Methylene Blue (MB) has been shown to attenuate oxidative, inflammatory, myocardial, and neurological lesions during ischemia-reperfusion and has great potential during cardiac arrest. This study aimed to determine the effects of MB combined with epinephrine during cardiac arrest on myocardial and cerebral lesions. METHOD: Thirty-eight male Wistar rats were randomly assigned to four groups: the sham group (SH, n = 5), and three groups subjected to cardiac arrest (n = 11/group) and treated with EPI 20 µg.kg-1 (EPI), EPI 20 µg.kg-1 + MB 2 mg.kg-1 (EPI + MB), or saline 0.9% 0.2 ml (CTL). Ventricular fibrillation was induced by direct electrical stimulation in the right ventricle for 3 minutes, and anoxia was maintained for 5 minutes. Cardiopulmonary Resuscitation (CPR) consisted of medications, ventilation, chest compressions, and defibrillation. After returning to spontaneous circulation, animals were observed for four hours. Blood gas, troponin, oxidative stress, histology, and TUNEL staining measurements were analyzed. Groups were compared using generalized estimating equations. RESULTS: No differences in the Returning of Spontaneous Circulation (ROSC) rate were observed among the groups (EPI: 63%, EPI + MB: 45%, CTL: 40%, p = 0.672). The mean arterial pressure immediately after ROSC was higher in the EPI+MB group than in the CTRL group (CTL: 30.5 [5.8], EPI: 63 [25.5], EPI+MB: 123 [31] mmHg, p = 0.007). Serum troponin levels were high in the CTL group (CTL: 130.1 [333.8], EPI: 3.70 [36.0], EPI + MB: 43.7 [116.31] ng/mL, p < 0.05). CONCLUSION: The coadministration of MB and epinephrine failed to yield enhancements in cardiac or brain lesions in a rodent model of cardiac arrest.

Iontophoresis use for increasing drug penetration into root canals and dentinal tubules: A proof-of-concept study.

INTRODUCTION: The success of endodontic treatment depends on the significant disinfection of the root canal system, its irregularities, and dentinal tubules. However, achieving complete disinfection remains challenging, with frequent failures and occurrence of secondary infections. Here, we propose using iontophoresis to increase the penetration and distribution of disinfecting agents into root canals, using methylene blue for proof-of-concept. METHODS: The marker was applied in bovine root canals, and the radial distribution of the dye in the dentinal tubules was evaluated by optical microscopy. Iontophoresis was applied at 0.5 and 1.5 mA for 5 and 15 min. RESULTS: A significant statistical difference (p < 0.05) was observed in the marker penetration between passive and iontophoretic applications. Both current density and application time had an important effect on methylene blue distribution, with a greater efficacy delivery to the apical region achieved after 1.5 mA for 5 min or 0.5 mA for 15 min, showing longer application time can compensate for lower application current. CONCLUSION: Iontophoresis increases the penetration and distribution of methylene blue into bovine root canals and dentinal tubules, including its innermost portions. CLINICAL SIGNIFICANCE: Iontophoresis has shown to be a promising technique for root canal and dentinal tubule disinfection.

Enhancing antimicrobial photodynamic therapy with phenothiazinium dyes and sodium dodecyl sulfate against Candida albicans at various growth stages.

BACKGROUND: The eradication of C. albicans is difficult due to the organization of the yeast in biofilms. Photodynamic therapy (PDT) has been proposed as an alternative to antifungals. Phenothiazinium dyes, e.g. methylene blue (MB), have been proposed as photosensitizing agents (PS), and their association with sodium dodecyl sulfate (SDS) has recently been shown to improve the effectiveness of PDT in planktonic culture. In this sense, the objective of this work was to evaluate the effect of PDT with phenothiazinium dyes associated to SDS in biofilms at the different stages of growth. METHODS: Experiments were carried out to evaluate the effects of PDT on biofilm formation and on established biofilms of C. albicans ATCC 10231. Samples were exposed to PS 50 mg/L (MB, Azure A - AA, Azure B - AB and dimethyl methylene blue - DMMB) dissolved in water or 0.25% SDS, for 5 min in the dark. After irradiation at 660 nm, 37.3mW/cm2 for 27 min, 60.4J/cm2 colony forming units count assay (CFU/mL) was performed. One or two irradiations were applied. Statistical methods were used to assess effectiveness. RESULTS: PSs showed low toxicity in the dark. An application of PDT irradiation was not able to reduce the CFU/mL both in mature biofilms (24h) and in biofilms in the dispersion phase (48h), only in the adherence phase did PDT prevent the formation of biofilms. With two successive applications of PDT irradiation in the dispersion phase, PDT with MB, AA, and DMMB completely inactivated C. albicans. The similar was not observed with mature biofilms. CONCLUSIONS: Different stages of biofilm growth respond differently to PDT, with the greatest inhibitory effect found in the adhesion stage. Mature and dispersed biofilms are less susceptible to PDT. The use of two successive applications of PDT with PSs associated with SDS may be a useful approach to inactivate C. albicans biofilms.

The activity of methylene blue against asexual and sexual stages of Plasmodium vivax.

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 µM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment.

The combination of methylene blue and sodium dodecyl sulfate enhances the antimicrobial photodynamic therapy of Candida albicans at lower light parameters.

BACKGROUND: The growth of resistant microorganisms has been a challenge for health systems. Antimicrobial Photodynamic Therapy (aPDT) has gained attention due to its effects on resistant strains. Recently, it was shown that the association of methylene blue (MB) and sodium dodecyl sulfate (SDS) are an effective strategy to increase the effect of aPDT; however, it is unknown which are the best light parameters (such as irradiance and radiant exposure, RE), to reach the most effective protocols. This work aimed to evaluate the light parameters, irradiance, and radiant exposure, in aPDT with MB when conveyed in water compared to MB associated with SDS. METHODS: Tests were carried out to quantify the colony-forming units (CFU) of ATCC 10,231 strain of Candida albicans when using MB in different media and with different light parameters: Control (water), SDS (0.25%), MB (20 mg/mL), and the MB/SDS at irradiances of 3.7; 11.2; 18.6, and 26.1 mW/cm2 and varied irradiation times to reach radiant exposures of 4.4; 17.8; 26.7, and 44 J/cm². RESULTS: The results showed that aPDT with MB/SDS had a higher antimicrobial effect than MB when conveyed in water. Furthermore, for the highest irradiance studied (26.1 mW/cm2), CFU decreases exponentially with increasing RE from 4.4 up to 44 J/cm2. Similarly, at a fixed RE, the higher the irradiance used, the higher the antimicrobial effect was observed, except for the lowest RE studied (4.4 J/cm2). CONCLUSIONS: aPDT with MB/SDS had a greater antimicrobial action at the lower light parameters when compared to MB conveyed in water. The authors suggest the use of RE above 18 J/cm2 and irradiance above 26 mW/cm2 since at the mentioned parameters the increase in its value caused a greater antimicrobial effect.

Vaterite microparticle-loaded methylene blue for photodynamic activity in macrophages infected with Leishmania braziliensis.

Calcium carbonate (CaCO3) exhibits a variety of crystalline phases, including the anhydrous crystalline polymorphs calcite, aragonite, and vaterite. Developing porous calcium carbonate microparticles in the vaterite phase for the encapsulation of methylene blue (MB) as a photosensitizer (PS) for use in photodynamic therapy (PDT) was the goal of this investigation. Using an adsorption approach, the PS was integrated into the CaCO3 microparticles. The vaterite microparticles were characterized by scanning electron microscopy (SEM) and steady-state techniques. The trypan blue exclusion method was used to measure the biological activity of macrophages infected with Leishmania braziliensis in vitro. The vaterite microparticles produced are highly porous, non-aggregated, and uniform in size. After encapsulation, the MB-loaded microparticles kept their photophysical characteristics. The carriers that were captured allowed for dye localization inside the cells. The results obtained in this study indicated that the MB-loaded vaterite microparticles show promising photodynamic activity in macrophages infected with Leishmania braziliensis.

Early adjunctive methylene blue in patients with septic shock: a randomized controlled trial.

PURPOSE: Methylene blue (MB) has been tested as a rescue therapy for patients with refractory septic shock. However, there is a lack of evidence on MB as an adjuvant therapy, its' optimal timing, dosing and safety profile. We aimed to assess whether early adjunctive MB can reduce time to vasopressor discontinuation in patients with septic shock. METHODS: In this single-center randomized controlled trial, we assigned patients with septic shock according to Sepsis-3 criteria to MB or placebo. Primary outcome was time to vasopressor discontinuation at 28 days. Secondary outcomes included vasopressor-free days at 28 days, days on mechanical ventilator, length of stay in ICU and hospital, and mortality at 28 days. RESULTS: Among 91 randomized patients, forty-five were assigned to MB and 46 to placebo. The MB group had a shorter time to vasopressor discontinuation (69 h [IQR 59-83] vs 94 h [IQR 74-141]; p < 0.001), one more day of vasopressor-free days at day 28 (p = 0.008), a shorter ICU length of stay by 1.5 days (p = 0.039) and shorter hospital length of stay by 2.7 days (p = 0.027) compared to patients in the control group. Days on mechanical ventilator and mortality were similar. There were no serious adverse effects related to MB administration. CONCLUSION: In patients with septic shock, MB initiated within 24 h reduced time to vasopressor discontinuation and increased vasopressor-free days at 28 days. It also reduced length of stay in ICU and hospital without adverse effects. Our study supports further research regarding MB in larger randomized clinical trials. Trial registration ClinicalTrials.gov registration number NCT04446871 , June 25, 2020, retrospectively registered.

Photodynamic therapy reduces cell viability, migration and triggers necroptosis in prostate tumor cells.

Prostate cancer is the most common cancer in American men, aside from skin cancer. As an alternative cancer treatment, photodynamic laser therapy (PDT) can be used to induce cell death. We evaluated the PDT effect, using methylene blue as a photosensitizer, in human prostate tumor cells (PC3). PC3 were subjected to four different conditions: DMEM (control); laser treatment (L-660 nm, 100 mW, 100 J.cm-2); methylene blue treatment (MB-25 µM, 30 min), and MB treatment followed by low-level red laser irradiation (MB-PDT). Groups were evaluated after 24 h. MB-PDT treatment reduced cell viability and migration. However, because MB-PDT did not significantly increase the levels of active caspase-3 and BCL-2, apoptosis was not the primary mode of cell death. MB-PDT, on the other hand, increased the acid compartment by 100% and the LC3 immunofluorescence (an autophagy marker) by 254%. Active MLKL level, a necroptosis marker, was higher in PC3 cells after MB-PDT treatment. Furthermore, MB-PDT resulted in oxidative stress due to a decrease in total antioxidant potential, catalase levels, and increased lipid peroxidation. According to these findings, MB-PDT therapy is effective at inducing oxidative stress and reducing PC3 cell viability. In such therapy, necroptosis is also an important mechanism of cell death triggered by autophagy.

Photodynamic antimicrobial therapy (A-PDT) using 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB and λ640 ± 5ηm LED light in patients undertaking orthodontic treatment.

Orthodontic treatment involves the use of apparatuses that impairs oral hygiene making patients susceptible to periodontal diseases and caries. To prevent increased antimicrobial resistance A-PDT has shown itself a feasible option. The aim of this investigation was to assess the efficiency of A-PDT employing 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB as a photosensitizing agent combined with red LED irradiation (λ640 ± 5 ηm) against oral biofilm of patients undertaking orthodontic treatment. Twenty-one patients agreed to participate. Four biofilm collections were carried out on brackets and gingiva around inferior central incisors; first was carried out before any treatment (Control); second followed five minutes of pre-irradiation, the third was immediately after the first AmPDT, and the last after a second AmPDT. Then, a microbiological routine for microorganism growth was carried out and, after 24-h, CFU counting was performed. There was significant difference between all groups. No significant difference was seen between Control and Photosensitizer and AmpDT1 and AmPDT2 groups. Significant differences were observed between Control and AmPDT1 and AmPDT2 groups, Photosensitizer and AmPDT1 and AmPDT2 groups. It was concluded that double AmPDT using DMBB in nano concentration and red LED was capable to meaningfully decrease the number of CFUs in orthodontic patients.

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).

Monoolein-based nanodispersions for cutaneous co-delivery of methylene blue and metformin: Thermal and structural characterization and effects on the cutaneous barrier, skin penetration and cytotoxicity.

This study evaluated the potential of monoolein (MO)-based nanodispersions to promote the cutaneous co-delivery of metformin (MET) and methylene blue (MB) for the treatment of non-melanoma skin cancer. MO-based nanodispersions were obtained using Kolliphor® P407 (KP) and/or sodium cholate (CH), and characterized concerning the structure, thermal stability, ability to disrupt the skin barrier, cutaneous permeation and retention of MB and MET. Additionally, the cytotoxic effect of MO nanodispersions-mediated combination therapy using MET and MB in A431 cells was evaluated. The nanodispersions exhibited nanometric size (<200 nm) and thermal and physical stability. Small angle X-ray scattering studies revealed multiple structures depending on composition. They were able to interact with stratum corneum lipid structure, increasing its fluidity. The effect of MO-nanodispersions on topical/transdermal delivery of MB and MET was composition-dependent. Nanodispersions with low MO content (5 %) and stabilized with KP and CH (0.05-0.10 %) were the most promising, enhancing the cutaneous delivery of MB and MET by 1.9 to 2.2-fold and 1.4 to 1.7-fold, respectively, compared to control. Cytotoxic studies revealed that the most promising MO nanodispersion-mediated combination therapy using MET and MB (1:1) reduced the IC50 by 24-fold, compared to MB solution, and a further reduction (1.5-fold) was observed by MB photoactivation.

Antimicrobial photodynamic therapy mediated by methylene blue-loaded polymeric micelles against Streptococcus mutans and Candida albicans biofilms.

BACKGROUND: Streptococcus mutans and Candida albicans can colonize the teeth, the oral cavity as biofilm and can cause oral infections. Thus, strategies to prevent and control oral biofilms are requested. The present study aims the development and characterization of methylene blue (MB)-loaded polymeric micelles for antimicrobial photodynamic therapy (aPDT) against Streptococcus mutans and Candida albicans biofilms METHODS: MB-loaded polymeric micelles were produced and characterized by particle size, polydispersity index, morphology, zeta potential, stability, MB release profile, and antimicrobial effect against S. mutans and C. albicans biofilms. RESULTS: MB-loaded polymeric micelles showed a reduced particle size, moderate polydisperse profile, spherical and neutral shape, which demonstrated to be promising features to allow micelles penetration into biofilms. Antimicrobial effect against bacterial and yeast biofilms was demonstrated once MB was irradiated by light under 660 nm (aPDT). Furthermore, MB-loaded polymeric micelles showed significant inhibition of S. mutans and C. albicans biofilms. Furthermore, the treatment with MB-micelles incubated with high pre-incubation times (15 and 30 min) were more effective than 5 min. It can be explained by the time required for this nanosystem to penetrate the innermost layer of biofilms and release MB for aPDT. CONCLUSION: MB-loaded polymeric micelles can effectively decrease the bacteria and yeast viability and it may cause positive impacts in the clinical practice. Thus, the developed formulation showed potential in the treatment to remove oral biofilms, but clinical studies are needed to confirm its potential.

Methylene blue-mediated antimicrobial photodynamic therapy on chicken semen.

BACKGROUND: Artificial insemination is widely employed in poultry, but high degrees of bacterial contamination are often observed in semen because of its passage through the cloaca. Consequently, most semen extenders for birds have antibiotics that could aggravate bacterial resistance. METHODS: We evaluated the potential of antimicrobial photodynamic therapy (PDT) as an alternative to the use of antibiotics, and assessed whether changes in concentration and incubation time with methylene blue (MB), radiant exposure, and irradiance of light affect spermatozoa activity and bacteria in chicken semen. RESULTS: Incubation with MB (< 25 µM) did not alter sperm motility, regardless of the pre-irradiation time (PIT, 1 or 5 min). Following 1 min of PIT with MB at 10 µM, samples were irradiated for 30, 60, 120, and 180 s at irradiances of 44, 29, and 17 mW/ cm² (660 nm LedBox). MB and light alone did not interfere with the analyzed parameters. However, when both factors were associated, increases in light dose led to greater reductions in sperm parameters, regardless of the irradiance used. Besides, PDT conditions that were less harmful to spermatozoa were not able to significantly reduce bacterial colonies in chicken semen. CONCLUSIONS: A failure in MB selectivity could explain unsuccessful bacterial reduction following PDT. Further research involving other photosensitizers or conjugating molecules to MB to target microbial cells is needed for PDT application in poultry breeders.