New insights in photodynamic inactivation of Leishmania amazonensis: A focus on lipidomics and resistance.

The emergence of drug resistance in cutaneous leishmaniasis (CL) has become a major problem over the past decades. The spread of resistant phenotypes has been attributed to the wide misuse of current antileishmanial chemotherapy, which is a serious threat to global health. Photodynamic therapy (PDT) has been shown to be effective against a wide spectrum of drug-resistant pathogens. Due to its multi-target approach and immediate effects, it may be an attractive strategy for treatment of drug-resistant Leishmania species. In this study, we sought to evaluate the activity of PDT in vitro using the photosensitizer 1,9-dimethyl methylene blue (DMMB), against promastigotes of two Leishmania amazonensis strains: the wild-type (WT) and a lab induced miltefosine-resistant (MFR) strain. The underlying mechanisms of DMMB-PDT action upon the parasites was focused on the changes in the lipid metabolism of both strains, which was conducted by a quantitative lipidomics analysis. We also assessed the production of ROS, mitochondrial labeling and lipid droplets accumulation after DMMB-PDT. Our results show that DMMB-PDT produced high levels of ROS, promoting mitochondrial membrane depolarization due to the loss of membrane potential. In addition, both untreated strains revealed some differences in the lipid content, in which MFR parasites showed increased levels of phosphatidylcholine, hence suggesting this could also be related to their mechanism of resistance to miltefosine. Moreover, the oxidative stress and consequent lipid peroxidation led to significant phospholipid alterations, thereby resulting in cellular dysfunction and parasite death. Thus, our results demonstrated that DMMB-mediated PDT is effective to kill L. amazonensis MFR strain and should be further studied as a potential strategy to overcome antileishmanial drug resistance.

New Insights in Phenothiazinium-Mediated Photodynamic Inactivation of Candida Auris.

In recent years, Candida auris has emerged as a hazardous hospital-acquired pathogen. Its resistance to antifungal treatments makes it challenging, requiring new approaches to manage it effectively. Herein, we aimed to assess the impact of photodynamic inactivation mediated by methylene blue (MB-PDI) or 1,9-dimethyl MB (DMMB-PDI) combined with a red LED against C. auris. To evaluate the photoinactivation of yeasts, we quantified colony-forming units and monitored ROS production. To gain some insights into the differences between MB and DMMB, we assessed lipid peroxidation (LPO) and mitochondrial membrane potential (ΔΨm). After, we verified the effectiveness of DMMB against biofilms by measuring metabolic activity and biomass, and the structures were analyzed through scanning electron microscopy and optical coherence tomography. We also evaluated the cytotoxicity in mammalian cells. DMMB-PDI successfully eradicated C. auris yeasts at 3 µM regardless of the light dose. In contrast, MB (100 µM) killed cells only when exposed to the highest dose of light. DMMB-PDI promoted higher ROS, LPO and ΔΨm levels than those of MB. Furthermore, DMMB-PDI was able to inhibit biofilm formation and destroy mature biofilms, with no observed toxicity in fibroblasts. We conclude that DMMB-PDI holds great potential to combat the global threat posed by C. auris.

Pluronic F-127 Hydrogels Containing Copper Oxide Nanoparticles and a Nitric Oxide Donor to Treat Skin Cancer.

Melanoma is a serious and aggressive type of skin cancer with growing incidence, and it is the leading cause of death among those affected by this disease. Although surgical resection has been employed as a first-line treatment for the early stages of the tumor, noninvasive topical treatments might represent an alternative option. However, they can be irritating to the skin and result in undesirable side effects. In this context, the potential of topical polymeric hydrogels has been investigated for biomedical applications to overcome current limitations. Due to their biocompatible properties, hydrogels have been considered ideal candidates to improve local therapy and promote wound repair. Moreover, drug combinations incorporated into the polymeric-based matrix have emerged as a promising approach to improve the efficacy of cancer therapy, making them suitable vehicles for drug delivery. In this work, we demonstrate the synthesis and characterization of Pluronic F-127 hydrogels (PL) containing the nitric oxide donor S-nitrosoglutathione (GSNO) and copper oxide nanoparticles (CuO NPs) against melanoma cells. Individually applied NO donor or metallic oxide nanoparticles have been widely explored against various types of cancer with encouraging results. This is the first report to assess the potential and possible underlying mechanisms of action of PL containing both NO donor and CuO NPs toward cancer cells. We found that PL + GSNO + CuO NPs significantly reduced cell viability and greatly increased the levels of reactive oxygen species. In addition, this novel platform had a huge impact on different organelles, thus triggering cell death by inducing nuclear changes, a loss of mitochondrial membrane potential, and lipid peroxidation. Thus, GSNO and CuO NPs incorporated into PL hydrogels might find important applications in the treatment of skin cancer.

Photodynamic therapy mediated by a red LED and methylene blue inactivates resistant Leishmania amazonensis.

Cutaneous leishmaniasis is a neglected parasitic disease that leads to destructive lesions. The emergence of drug resistance has been a global concern over the past years. Photodynamic therapy (PDT) mediated by a red LED and methylene blue (MB) involves the overproduction of oxidative stress, which oxidizes several cellular biomolecules and prevents the selection of resistant strains. Herein, we investigated the potential of PDT mediated by MB against wild-type and miltefosine-resistant strains of Leishmania amazonensis. As a result, both strains were susceptible to PDT, thus encouraging us to seek the best conditions to overcome the drug resistance problem in cutaneous leishmaniasis.

Towards effective natural anthraquinones to mediate antimicrobial photodynamic therapy of cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is an important tropical neglected disease with broad geographical dispersion. The lack of effective drugs has raised an urgent need to improve CL treatment, and antimicrobial photodynamic therapy (APDT) has been investigated as a new strategy to face it with positive outcomes. Natural compounds have emerged as promising photosensitizers (PSs), but their use in vivo remains unexplored. PURPOSE: In this work, we investigated the potential of three natural anthraquinones (AQs) on CL induced by Leishmania amazonensis in BALB/c mice. STUDY DESIGN/METHODS: ANIMALS WERE INFECTED AND RANDOMLY DIVIDED INTO FOUR GROUPS: CG (control, non-treated group), G5ClSor-gL (treated with 5-chlorosoranjidiol and green LED, 520±10 nm), GSor-bL and GBisor-bL (treated with soranjidiol and bisoranjidiol, respectively, exposed to violet-blue LED, 410±10 nm). All AQs were assayed at 10 µM and LEDs delivered a radiant exposure of 45 J/cm2 with an irradiance of 50 mW/cm2. We assessed the parasite burden in real time for three consecutive days. Lesion evolution and pain score were assessed over 3 weeks after a single APDT session. RESULTS: G5ClSor-gL was able to sustain low levels of parasite burden over time. Besides, GSor-bL showed a smaller lesion area than the control group, inhibiting the disease progression. CONCLUSION: Taken together, our data demonstrate that monoAQs are promising compounds for pursuing the best protocol for treating CL and helping to face this serious health problem. Studies involving host-pathogen interaction as well as monoAQ-mediated PDT immune response are also encouraged.

Could Light-Based Technologies Improve Stem Cell Therapy for Skin Wounds? A Systematic Review and Meta-Analysis of Preclinical Studies†.

Several diseases or conditions cause dermatological disorders that hinder the process of skin repair. The search for novel technologies has inspired the combination of stem cell (SC) and light-based therapies to ameliorate skin wound repair. Herein, we systematically revised the impact of photobiomodulation therapy (PBM) combined with SCs in animal models of skin wounds and quantitatively evaluated this effect through a meta-analysis. For inclusion, SCs should be irradiated in vitro or in vivo, before or after being implanted in animals, respectively. The search resulted in nine eligible articles, which were assessed for risk of bias. For the meta-analysis, studies were included only when PBM was applied in vivo, five regarding wound closure, and three to wound strength. Overall, a positive influence of SC + PBM on wound closure (mean difference: 9.69; 95% CI: 5.78-13.61, P < 0.00001) and strength (standardized mean difference: 1.7, 95% CI: 0.68-2.72, P = 0.001) was detected, although studies have shown moderate to high heterogeneity and a lack of information regarding some bias domains. Altogether, PBM seems to be an enabling technology able to be applied postimplantation of SCs for cutaneous regeneration. Our findings may guide future laboratory and clinical studies in hopes of offering wound care patients a better quality of life.

Feline sporotrichosis successfully treated with methylene blue-mediated antimicrobial photodynamic therapy and low doses of itraconazole.

Sporotrichosis is a mycotic infection of humans and animals caused by different fungal species of the genus Sporothrix. Feline sporotrichosis presents a broad spectrum of clinical manifestations and its treatment with classic antifungal drugs is often long and frustrating. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) comes to light as an interesting approach against fungal infections, including sporotrichosis. In this case report, a 1-year-old male cat was diagnosed with sporotrichosis, being confirmed by fungal culture. The cat was treated by MB-APDT combined with oral administration of itraconazole. Following 2 weeks after the end of treatment, the animal was clinically cured, and an additional fungal culture was negative for Sporothrix spp., confirming the total remission of sporotrichosis. No side effects and recurrences were observed after a 3-moth follow-up. MB-APDT is a promising strategy against feline sporotrichosis, however large-scale studies are welcome to confirm its potential.

Methylene blue-mediated antimicrobial photodynamic therapy for canine dermatophytosis caused by Microsporum canis: A successful case report with 6 months follow-up.

Dermatophytosis is a superficial skin infection that widely effects companion animals. Miscrosporum canis is one of the most prevalent species isolated from dogs and cats, and because of the serious zoonotic potential, short-term treatment regimens are preferred to prevent the spread of disease either by direct contact or through contamination of the environment. Antimicrobial photodynamic therapy (APDT) has emerged as a promising strategy able to kill effectively a wide range of pathogens in a short period with minimal morbidity . In this case report, a 7-year-old male dog was diagnosed with dermatophytosis caused by M. canis. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) was applied over the lesions in two sessions with an interval of 7 days. The dog successfully healed, achieving a complete clinical cure after 21 days, without reports of recurrence after a follow-up period of 6 months. Therefore, MB-APDT could be a potential ally of small animal clinicians to treat superficial fungal diseases and should be further explored in Veterinary Medicine.

A systematic scoping review of ultraviolet C (UVC) light systems for SARS-CoV-2 inactivation.

A significant amount of epidemiological evidence has underlined that human-to-human transmission due to close contacts is considered the main pathway of transmission, however since the SARS-CoV-2 can also survive in aerosols, water, and surfaces, the development and implementation of effective decontamination strategies are urgently required. In this regard, ultraviolet germicidal irradiation (UVGI) using ultraviolet C (UVC) has been proposed to disinfect different environments and surfaces contaminated by SARS-CoV-2. Herein, we performed a systematic scoping review strictly focused on peer-reviewed studies published in English that reported experimental results of UVC-based technologies against the SARS-CoV-2 virus. Studies were retrieved from PubMed and the Web of Science database. After our criterious screening, we identified 13 eligible articles that used UVC-based systems to inactivate SARS-CoV-2. We noticed the use of different UVC wavelengths, technologies, and light doses. The initial viral titer was also heterogeneous among studies. Most studies reported virus inactivation in well plates, even though virus persistence on N95 respirators and different surfaces were also evaluated. SARS-CoV-2 inactivation reached from 90% to 100% depending on experimental conditions. We concluded that there is sufficient evidence to support the use of UVC-based technologies against SARS-CoV-2. However, appropriate implementation is required to guarantee the efficacy and safety of UVC strategies to control the COVID-19 pandemic.

Nitric-oxide releasing chitosan nanoparticles towards effective treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a major public health problem caused by Leishmania parasites that produce destructive and disfiguring skin conditions. There is an urgent need for alternative topical therapies due to the limitations of current systemic treatments. Recently, we have synthesized nitric oxide-releasing chitosan nanoparticles (NONPs) and shown their potential in vitro against Leishmania amazonensis. Herein we evaluated the application of NONPs for the treatment of CL on infected BALB/c mice. Mice were treated with topical administration of increasing concentrations of NONPs and disease progression was investigated regarding parasite load, lesion thickness, and pain score. As a result, we observed a dose-dependent NONPs effect. Parasite burden and lesion thickness were substantially lower on animals receiving NONPs at a 2 mM concentration compared to untreated control. Moreover, the clinical presentation of the lesions did not show any visible signs of ulcer, suggesting clinical healing in these animals. This successful outcome was sustained for at least 21 days after therapy even in one single dose. Thus, we demonstrate that NONPs are suitable for topical administration, and represent an attractive approach to treat CL.

Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4.

BACKGROUND: Photodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP4+ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites. METHODS: ZnTnHex-2-PyP4+ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed. RESULTS: ZnTnHex-2-PyP4+, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells. CONCLUSIONS: ZnTnHex-2-PyP4+ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP4+, indicating readiness for evaluation in in vivo studies as an alternative approach to CL. GENERAL SIGNIFICANCE: This report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.

Preclinical Investigation of Methylene Blue-mediated Antimicrobial Photodynamic Therapy on Leishmania Parasites Using Real-Time Bioluminescence.

Cutaneous leishmaniasis (CL) is a neglected disease that promotes destructive lesions. Difficulties in treatment are related to accessibility of drugs, resistance and toxicity. Antimicrobial photodynamic therapy (APDT) has been emerging as a promising treatment for CL. In this work, we evaluated methylene blue (MB)-mediated APDT (MB-APDT) on Leishmania amazonensis in vitro and in vivo by bioluminescence technique. In vitro, MB-APDT was performed using a red LED (λ = 660 ± 11 nm, 100 mW cm-2 ) and MB (100 µm) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into three groups: control, APDT 1 (single session) and APDT 2 (two sessions of MB-APDT). MB was used at 100 µm and energy dose was established at 150 J cm-2 . Parasite burden, lesion size and pain were evaluated weekly for 4 weeks. In vitro, lethal dose for 90% parasite inactivation was achieved at 48.8 J cm-2 . In vivo, although APDT 1 and APDT 2 groups have showed similar parasite burden after 4 weeks, two sessions were clinically better, especially considering the inflammatory process associated to CL. Our findings reinforce MB-APDT as a cost-effective treatment to combat CL.

Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

Natural anthraquinones as novel photosentizers for antiparasitic photodynamic inactivation.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne disease caused by obligate protist parasites from the genus Leishmania. The potential toxicity as well as the increased resistance of standard treatments has encouraged the development of new therapeutical strategies. Photodynamic inactivation (PDI) combines the use of a photosensitizer and light to generate reactive oxygen species and kill cells, including microorganisms. Vegetal kingdom constitutes an important source of bioactive compounds that deserve to be investigated in the search of naturally occurring drugs with leishmanicidal activity. PURPOSE: The purpose of this study was to test the antiparasitic activity of PDI (ApPDI) of five natural anthraquinones (AQs) obtained from Heterophyllaea lycioides (Rusby) Sandwith (Rubiacae). To support our results, effect of AQ mediated-PDI on parasite´s morphology and AQ uptake were studied. Cytotoxicity on fibroblasts was also evaluated. STUDY DESIGN/METHODS: Two monomers, soranjidiol (Sor) and 5-chlorosoranjidiol (5-ClSor) plus three bi-anthraquinones (bi-AQs), bisoranjidiol (Bisor), 7-chlorobisoranjidiol (7-ClBisor) and Lycionine (Lyc) were selected for this study. Recombinant L. amazonensis promastigote strain expressing luciferase was subjected to AQs and LED treatment. Following irradiation with variable light parameters, cell viability was quantified by bioluminescence. Alteration on parasite's morphology was analyzed by scanning electron microscopy (SEM). In addition, we verified the AQ uptake in Leishmania cells by fluorescence and their toxicity on fibroblasts by using MTT assay. RESULTS: Bisor, Sor and 5-ClSor exhibited photodynamic effect on L. amazonensis. SEM showed that promastigotes treated with Bisor-mediated PDI exhibited a significant alteration in shape and size. Sor and 5-ClSor presented higher uptake levels than bi-AQs (Bisor, Lyc and 7-ClBisor). Finally, Sor and Bisor presented the lowest toxic activity against fibroblasts. CONCLUSION: Taking together, our results indicate that Sor presents the highest specificity towards Leishmania cells with no toxicity on fibroblasts.