RESUMO
Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. Experiments were carried out to examine the susceptibility of C. albicans to PDI. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15J/cm2) than in non-irradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by photodynamic inactivation. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains.
Assuntos
Laranja de Acridina/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Lipossomos/química , Fármacos Fotossensibilizantes/química , Triclosan/química , Laranja de Acridina/metabolismo , Laranja de Acridina/farmacologia , Antifúngicos/química , Candida albicans/efeitos da radiação , Liberação Controlada de Fármacos/efeitos da radiação , Lasers , Lipossomos/metabolismo , Microscopia de Força Atômica , Microscopia de Fluorescência , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Triclosan/metabolismo , Triclosan/farmacologiaRESUMO
Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.
Assuntos
Ácidos/metabolismo , Laranja de Acridina/metabolismo , Autofagia , Técnicas Citológicas/métodos , Organelas/metabolismo , Animais , Tamanho Celular , Citometria de Fluxo , Fluorescência , Células HEK293 , Humanos , Microscopia Confocal , Ratos Wistar , Espectrometria de FluorescênciaRESUMO
We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.
Assuntos
Antimaláricos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Venenos de Crotalídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Venenos de Serpentes/química , Laranja de Acridina/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/isolamento & purificação , Transporte Biológico , Carbocianinas/química , Peptídeos Penetradores de Células/isolamento & purificação , Células Cultivadas , Cloroquina/farmacologia , Venenos de Crotalídeos/isolamento & purificação , Crotalus/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração Inibidora 50 , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Coloração e Rotulagem , Vacúolos/efeitos dos fármacos , Vacúolos/parasitologiaRESUMO
This study was conducted to investigate the modulating effects of green tea polyphenols on genotoxic damage and apoptotic activity induced by hexavalent chromium [Cr (VI)] in CD-1 mice. Animals were divided into the following groups: (i) injected with vehicle; (ii) treated with green tea polyphenols (30 mg/kg) via gavage; (iii) injected with CrO3 (20 mg/kg) intraperitoneally; (iv) treated with green tea polyphenols in addition to CrO3. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCEs) obtained from peripheral blood at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. Treatment of green tea polyphenols led to no significant changes in the MN-PCEs. However, CrO3 treatment significantly increased MN-PCEs at 24 and 48 h after injection. Green tea polyphenols treatment prior to CrO3 injection led to a decrease in MN-PCEs compared to the group treated with CrO3 only. The average of apoptotic cells was increased at 48 h after treatment compared to control mice, suggesting that apoptosis could contribute to eliminate the DNA damaged cells induced by Cr (VI). Our findings support the proposed protective effects of green tea polyphenols against the genotoxic damage induced by Cr (VI).
Assuntos
Laranja de Acridina/metabolismo , Apoptose/efeitos dos fármacos , Células Sanguíneas/metabolismo , Cromo/toxicidade , Dano ao DNA , Polifenóis/farmacologia , Chá/química , Animais , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/patologia , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Etídio/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Masculino , Camundongos , Testes para Micronúcleos , Extratos Vegetais/farmacologia , Coloração e RotulagemRESUMO
The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.
Assuntos
Fibroblastos/citologia , Gases em Plasma/farmacologia , Polimetil Metacrilato/farmacologia , Poliestirenos/farmacologia , Laranja de Acridina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Eletricidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Camundongos , Microscopia de Força Atômica , Termodinâmica , Água/química , Molhabilidade/efeitos dos fármacosRESUMO
When cultured cells are treated with fluorescent organelle probes or photosensitizer agents, a characteristic redistribution of fluorescence in cell structures occurs frequently after light irradiation. It is currently assumed that such changes, referred to as relocalizations of the fluorescent compounds, represent an important aspect of the photodynamic process, which is based on the excitation of photosensitizers by light in the presence of oxygen. As cell damage and death result from the oxidative stress induced by photodynamic treatments, we have studied here the redistribution of acridine orange (AO) and 3,3'-dimethyl-oxacarbocyanine (DiOC(1)(3)) fluorescence after incubation of HeLa cell cultures with these compounds followed by blue light irradiation to achieve lethal effects. The relocalization of dyes from their original labeling sites (AO: lysosomes, DiOC(1)(3): mitochondria) to nucleic acid-containing structures (cytoplasm, nuclei and nucleoli) appeared clearly associated with cell death. Therefore, the relocalization phenomenon simply reflects fluorescence changes due to the different affinity of these dyes for living and damaged or dead cells. As fluorescent probes are often photosensitizers, prolonged light exposures using fluorescence microscopy will produce lethal photodynamic effects with relocalization of the fluorescent signal and changes in the cell morphology.
Assuntos
Laranja de Acridina/metabolismo , Corantes Fluorescentes/metabolismo , Carbocianinas/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismoRESUMO
This study evaluated the potential cytotoxicity of the natural diterpenoids kauren-19-oic acid (KA), 14-hydroxy-kaurane (1) and xylopic acid (2), and semi-synthetic derivatives of KA (3-5) towards human cancer cell lines (K562, HL60, MDA-MB435 and SF295) and lymphocytes. Mouse erythrocytes were used to verify a possible hemolytic activity Cytotoxicity mechanisms were investigated in HL60 cells. KA showed a moderate antiproliferative effect in MTT assay towards all cancer cells (IC(50), 9.1-14.3 microg ml(-1)). However, KA appeared not selective to cancer cells, since it also inhibited the lymphocytes proliferation (IC(50), 12.6 microg ml(-1)). Unlike KA, compounds 1-5 displayed no cytotoxicity and were also free from antiproliferative and hemolytic effects, suggesting that the exocyclic double bond (C16) unit may be the active pharmacophore of KA cytotoxicity. KA-treated HL60 cells displayed decreased proliferation (5-bromo-2';-deoxyuridine incorporation assay) and topoisomerase I activity (DNA relaxation assay). These assays revealed that KA primarily intercalates with DNA and not with topoisomerase I. Fluorescence microscopy using AO/EB (acridine orange/ethidium bromide) staining indicated that KA can induce both apoptosis and necrosis in HL-60 cell cultures, which corroborate the findings with MTT. From these findings, we conclude that KA, although demonstrating cytotoxic potential, may have a limited or poor therapeutic potential due to lack of selectivity to tumor cells. Further studies on the structure modification of KA and the mechanism of the new derivatives are currently in progress.
Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA , Diterpenos/toxicidade , Substâncias Intercalantes/toxicidade , Leucemia/metabolismo , Laranja de Acridina/metabolismo , Bromodesoxiuridina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , DNA Topoisomerases Tipo I/metabolismo , Diterpenos/química , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Etídio/metabolismo , Corantes Fluorescentes/metabolismo , Glioblastoma/patologia , Células HL-60 , Humanos , Concentração Inibidora 50 , Células K562 , Melanoma/patologia , Estrutura Molecular , Necrose/induzido quimicamenteRESUMO
OBJECTIVES: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction of beta-lapachone with benzaldehyde, which among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes. METHODS: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation of potential targets in the parasite using electron microscopy and flow cytometry techniques. RESULTS: N1 presented the following order of activity: amastigotes > trypomastigotes > epimastigotes. The effect on intracellular forms was approximately 25 times higher than on macrophages and heart muscle cells. N1-treated parasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes, alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density of acidocalcisomes was observed. In epimastigotes, the naphthoimidazole inhibited the activity of succinate cytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both forms treated with N1. CONCLUSIONS: The results suggest that epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply.
Assuntos
Organelas/efeitos dos fármacos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Laranja de Acridina/metabolismo , Animais , Benzaldeídos/química , Células Cultivadas , Cromatina/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/parasitologia , Naftoquinonas/síntese química , Naftoquinonas/química , Naftoquinonas/farmacologia , Naftoquinonas/toxicidade , Rodamina 123/metabolismo , Succinato Citocromo c Oxirredutase/antagonistas & inibidores , Tripanossomicidas/síntese química , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/toxicidade , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 microm diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warren's medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.
Assuntos
Vesículas Citoplasmáticas/ultraestrutura , Leishmania mexicana/crescimento & desenvolvimento , Laranja de Acridina/metabolismo , Animais , Cálcio/análise , Meios de Cultura/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/enzimologia , Microanálise por Sonda Eletrônica , Imageamento Tridimensional , Pirofosfatase Inorgânica/análise , Pirofosfatase Inorgânica/efeitos dos fármacos , Ferro/análise , Ferro/metabolismo , Leishmania mexicana/citologia , Leishmania mexicana/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Nigericina/farmacologiaRESUMO
To determine the ability of monocyte phagosomal acidification in chronic paracoccidioidomycosis, 13 patients were recruited at different times during follow-up and compared with 18 normal controls. Eight patients were studied at diagnosis, six of them also during treatment and five additional patients after ending treatment. Phagosomal acidification of monocytes, triggered by challenge with opsonized zymosan, was evaluated with acridine orange and expressed as percentage of orange-stained intracellular particles, as mean +/- SE. In comparison with controls, acidification was severely impaired before treatment (33 +/- 11% vs. 67 +/- 6%) and reached values similar to controls during treatment (73 +/- 6%, n = 6). In addition, phagosomal acidification of the patients studied after treatment (63 +/- 4%) had no difference when compared with controls. This study demonstrates that phagosomal acidification is perturbed among chronic paracoccidioidomycosis patients and reverses with antifungal treatment.