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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cutaneous leishmaniasis (CL) is a neglected disease caused by Leishmania parasites. The oral drug miltefosine is effective, but there is a growing problem of drug resistance, which has led to ...increasing treatment failure rates and relapse of infections. Photodynamic therapy (PDT) combines a light source and a photoactive drug to promote cell death by oxidative stress. Although PDT is effective against several pathogens, its use against drug-resistant Leishmania parasites remains unexplored. Herein, we investigated the potential of organic light-emitting diodes (OLEDs) as wearable light sources, which would enable at-home use or ambulatory treatment of CL. We also assessed its impact on combating miltefosine resistance in Leishmania amazonensis-induced CL in mice. The in vitro activity of OLEDs combined with 1,9-dimethyl-methylene blue (DMMB) (OLED-PDT) was evaluated against wild-type and miltefosine-resistant L. amazonensis strains in promastigote (EC50 = 0.034 μM for both strains) and amastigote forms (EC50 = 0.052 μM and 0.077 μM, respectively). Cytotoxicity in macrophages and fibroblasts was also evaluated. In vivo, we investigated the potential of OLED-PDT in combination with miltefosine using different protocols. Our results demonstrate that OLED-PDT is effective in killing both strains of L. amazonensis by increasing reactive oxygen species and stimulating nitric oxide production. Moreover, OLED-PDT showed great antileishmanial activity in vivo, allowing the reduction of miltefosine dose by half in infected mice using a light dose of 7.8 J/cm2 and 15 μM DMMB concentration. In conclusion, OLED-PDT emerges as a new avenue for at-home care and allows a combination therapy to overcome drug resistance in cutaneous leishmaniasis.
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•A new approach to treating cutaneous leishmaniasis is proposed.•A combination of photodynamic therapy and miltefosine is shown to be effective against drug resistant L. amazonensis.•OLEDs are shown to be novel wearable light sources suitable for at-home care of cutaneous leishmaniasis.•Photodynamic therapy combined with oral miltefosine promotes complete healing.•Photodynamic therapy reduces the miltefosine dose by half, preventing the emergence of drug resistance.
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.
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•We successfully synthesized and characterized NO-loaded chitosan nanoparticles.•NONPs were able to efficiently inactivate Leishmania (L.) amazonensis promastigotes.•NONPs were not toxic to macrophages.•NONPs inactivated intracellular amastigotes following one single application.•NONPs promoted parasitophorous vacuole reduction indicating infection resolution.
ABSTRACT
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.
Skin regeneration still challenges healthcare worldwide. Stem cell and light‐based therapies are novel technologies that have been proposed to ameliorate wound healing. This study systematically assessed whether light‐based therapies could improve cutaneous repair at pre or postexposure of stem cells using preclinical animal models. Although all included studies have shown a lack of information related to selection, performance and detection bias, an overall positive effect on wound closure and strength was noticed through the meta‐analysis. These results may guide future laboratory and clinical studies in hopes of offering wound care patients a better quality of life.
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.
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•NO-releasing chitosan nanoparticles (NONPs) can efficiently inactivate L. amazonensis in infected mice in a dose-dependent manner.•NONPs significantly decreased the parasite burden of treated animals in one single application.•NONPs at 2 mM remarkably reduced the lesion thickness, promoting clinical healing of mice.•NONP positive effects were sustained for at least 21 days after therapy.•NONPs are suitable for topical administration, being an attractive approach to treat CL.
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.
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.
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.
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.
Taking together, our results indicate that Sor presents the highest specificity towards Leishmania cells with no toxicity on fibroblasts.
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•We evaluated 3 AQs to mediate APDT on mouse-induced cutaneous- leishmaniasis.•5-chlorosoranjidiol combined to green LED sustained the levels of parasite load.•Soranjidiol combined to blue LED ...significantly reduced lesion size.•MonoAQs are promising natural photosensitizers to mediate APDT on CL.
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.
In this work, we investigated the potential of three natural anthraquinones (AQs) on CL induced by Leishmania amazonensis in BALB/c mice.
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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.
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.
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.
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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.
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.
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.
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.
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.
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.
•ZnTnHex-2-PyP4+ showed chemical stability in accelerated solvolysis studies.•Photodynamic treatment induced intense mitochondrial depolarization and significant morphological changes on promastigotes.•ZnTnHex-2-PyP4+ efficiently photoinactivated promastigote and amastigote forms.•No noteworthy cytotoxicity was observed on mammalian cells.•ZnTnHex-2-PyP4+ has high potential for pre-clinical studies as an alternative to cutaneous leishmaniasis.
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.
Cutaneous leishmaniasis (CL) is a neglected disease that causes ulcerated lesions. Current treatments are limited due to high cost, resistance and toxicity. Photodynamic therapy has been put forward as an alternative treatment for CL. Here, we investigated methylene blue‐mediated photodynamic therapy (MB‐APDT) on Leishmania (L.) amazonensis in vitro using a red LED at different fluences. In vivo, we compared one and two MB‐APDT sessions at L. amazonensis‐induced CL in mice. Disease progress was evaluated by bioluminescence, lesion size and pain score. Our results indicate that MB‐APDT could be a cost‐effective strategy to apply in public health.
