RESUMO
A new chemical conjugate between protoporphyrin IX (PPIX) and chitosan oligosaccharides (CH) was prepared and evaluated in vitro as an antifungal agent against Penicillium digitatum. Chemical characterization and photophysical/photochemical studies were conducted. The antifungal effect of the CH-PPIX conjugate was compared to its components (PPIX and CH) and a physical mixture of both, under dark and illuminated conditions. The CH-PPIX conjugate was photostable and inhibited fungal growth with 100% efficiency at a dose of 0.005% w/v under visible light irradiation, while no antifungal activity was observed in the dark. Under the same conditions, CH and PPIX did not display any fungicidal activity, demonstrating the improved properties of the conjugate. Insights into the mechanism of fungal inactivation revealed an efficient spore uptake and photoinduced membrane damage through singlet oxygen generation. This new bioconjugate, which is based on natural components, represents a promising agent for fungicidal formulations based on antimicrobial photodynamic therapy.
Assuntos
Quitosana , Fármacos Fotossensibilizantes , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Quitosana/química , Quitosana/farmacologia , Oligossacarídeos/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/química , Protoporfirinas/farmacologiaRESUMO
The current photodynamic therapy (PDT) is majorly hindered by the shallow penetration depth and oxygen dependency, limiting its application to deep-seated solid hypoxic tumors. Thus, it is meaningful to develop efficient X-ray mediated PDT system capable of generating reactive oxygen species (ROS) under both the normoxic and hypoxic conditions. Herein, we report the synthesis and characterization of nanocomposite, YAG:Pr@ZnO@PpIX with an amalgamation of UV-emitting Y2.99Pr0.01Al5O12 (YAG:Pr) nanoscintillator, and zinc oxide (ZnO) and protoporphyrin IX (PpIX) as photosensitizers. YAG:Pr surface was coated with a ZnO layer (â¼10â¯nm) by atomic layer deposition, and then PpIX was covalently conjugated via a linker to give YAG:Pr@ZnO@PpIX. The photo- and cathodoluminescence analyses gave the evidences of efficient energy transfer from YAG:Pr to ZnO at â¼320â¯nm, and YAG:Pr@ZnO to PpIX at Soret region (350-450â¯nm). The nanohybrid was able to produce both, Type I and Type II ROS upon direct and indirect photoactivation with UV365nm and UV290nm, respectively. In vitro cytotoxicity of non-activated YAG:Pr@ZnO@PpIX in mouse melanoma cells revealed low toxicity, which significantly enhanced upon photoactivation with UV365nm indicating the photokilling property of the nanohybrid. Overall, our preliminary studies successfully demonstrate the potential of YAG:Pr@ZnO@PpIX to overcome the limited penetration and oxygen-dependency of traditional PDT.
Assuntos
Nanocompostos/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Alumínio/química , Alumínio/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Estrutura Molecular , Tamanho da Partícula , Fármacos Fotossensibilizantes/química , Praseodímio/química , Praseodímio/farmacologia , Protoporfirinas/química , Protoporfirinas/farmacologia , Propriedades de Superfície , Células Tumorais Cultivadas , Ítrio/química , Ítrio/farmacologia , Óxido de Zinco/química , Óxido de Zinco/farmacologiaRESUMO
Many heme-containing proteins with a histidine in the distal E7 (HisE7) position can form sulfheme in the presence of hydrogen sulfide (H2S) and a reactive oxygen species such as hydrogen peroxide. For reasons unknown, sulfheme derivatives are formed specifically on solvent-excluded heme pyrrole B. Sulfhemes severely decrease the oxygen-binding affinity in hemoglobin (Hb) and myoglobin (Mb). Here, use of hybrid quantum mechanical/molecular mechanical methods has permitted characterization of the entire process of sulfheme formation in the HisE7 mutant of hemoglobin I (HbI) from Lucina pectinata. This process includes a mechanism for H2S to enter the solvent-excluded active site through a hydrophobic channel to ultimately form a hydrogen bond with H2O2 bound to Fe(III). Proton transfer from H2O2 to His64 to form compound (Cpd) 0, followed by hydrogen transfer from H2S to the Fe(III)-H2O2 complex, results in homolytic cleavage of the O-O and S-H bonds to form a reactive thiyl radical (HS(â¢)), ferryl heme Cpd II, and a water molecule. Subsequently, the addition of HS(â¢) to Cpd II, followed by three proton transfer reactions, results in the formation of a three-membered ring ferric sulfheme that avoids migration of the radical to the protein matrix, in contrast to that in other peroxidative reactions. The transformation of this three-membered episulfide ring structure to the five-membered thiochlorin ring structure occurs through a significant potential energy barrier, although both structures are nearly isoenergetic. Both three- and five-membered ring structures reveal longer NB-Fe(III) bonds compared with other pyrrole nitrogen-Fe(III) bonds, which would lead to decreased oxygen binding. Overall, these results are in agreement with a wide range of experimental data and provide fertile ground for further investigations of sulfheme formation in other heme proteins and additional effects of H2S on cell signaling and reactivity.
Assuntos
Heme/análogos & derivados , Heme/química , Peróxido de Hidrogênio/química , Sulfeto de Hidrogênio/química , Animais , Bivalves/metabolismo , Domínio Catalítico , Hemoglobinas/química , Hemoglobinas/genética , Hemoglobinas/metabolismo , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Protoporfirinas/química , Protoporfirinas/metabolismo , Teoria QuânticaRESUMO
AIMS: To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. METHODS AND RESULTS: Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. CONCLUSIONS: Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. SIGNIFICANCE AND IMPACT OF THE STUDY: These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Heme/farmacologia , Metaloporfirinas/farmacologia , Protoporfirinas/farmacologia , Febre Amarela/virologia , Vírus da Febre Amarela/efeitos dos fármacos , Antivirais/química , Dengue/tratamento farmacológico , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Heme/química , Humanos , Metaloporfirinas/química , Protoporfirinas/química , RNA Viral/genética , Inativação de Vírus/efeitos dos fármacos , Febre Amarela/tratamento farmacológico , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/fisiologiaRESUMO
Nanodispersions of liquid-crystalline phases (NLPs) composed of monoolein and oleic acid were chosen as nanocarriers to improve the topical retention of the photosensitizer protoporphyrin IX (PpIX) and thereby optimize photodynamic therapy (PDT) using this photosensitizer. The nanodispersions were characterized by polarized light microscopy, small-angle X-ray diffraction and dynamic light scattering. The stability and encapsulation efficiency (EE%) of the nanodispersions were also evaluated. In vitro and in vivo skin penetration studies were performed to determine the potential of the nanodispersions for cutaneous application. In addition, skin penetration and skin irritancy (in an animal model) after in vivo application were visualized by fluorescence light microscopy. The nanodispersion obtained was characterized as a monodisperse system (~150.0 nm) of hexagonal liquid-crystalline phase, which provided a high encapsulation efficiency of PpIX (~88%) that remained stable over 90 days of investigation. Skin penetration studies demonstrated that the nanodispersion enhanced PpIX skin uptake 11.8- and 3.3-fold (in vitro) and 23.6- and 20.8-fold (in vivo) compared to the PpIX skin uptake of control formulations, respectively. In addition, the hexagonal phase nanodispersion did not cause skin irritation after application for two consecutive days. Overall, the results show that the nanocarrier developed is suitable for use in topical PDT with PpIX.
Assuntos
Portadores de Fármacos/administração & dosagem , Glicerídeos/administração & dosagem , Nanopartículas/administração & dosagem , Ácido Oleico/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Protoporfirinas/administração & dosagem , Administração Tópica , Animais , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Feminino , Glicerídeos/química , Glicerídeos/farmacologia , Técnicas In Vitro , Cristais Líquidos/química , Camundongos Pelados , Nanopartículas/química , Ácido Oleico/química , Ácido Oleico/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/química , Protoporfirinas/farmacologia , Pele/anatomia & histologia , Pele/efeitos dos fármacos , Pele/metabolismo , Suspensões , SuínosRESUMO
In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Artrópodes/química , Heme/química , Intestinos/fisiologia , Rhipicephalus/fisiologia , Acaricidas/química , Trifosfato de Adenosina/química , Animais , Anticorpos/química , Bovinos , Cromatografia Líquida de Alta Pressão , Ciclosporina/química , Feminino , Metaloporfirinas/química , Metaloporfirinas/farmacocinética , Ligação Proteica , Estrutura Terciária de Proteína , Protoporfirinas/química , Interferência de RNA , Rodamina 123/química , Rodamina 123/farmacocinética , Infestações por Carrapato/tratamento farmacológico , Toluidinas/química , Toluidinas/farmacocinéticaRESUMO
Poly [Ni-Protoporphyrin] film (pNiPP), containing multiwall carbon nanotubes (MWCNT) was used to cover a glassy carbon electrode. The hybrid material (pNiPP/MWCNT) successfully combines the permselectivity of pNiPP with the high conductivity of MWCNT. The modified electrode was used to perform amperometric detection of long chain aliphatic amines (LCAA) in order to prevent the passivation effect of the aliphatic chain. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) demonstrated that the pNiPP/MWCNT facilitates the electron transfer reaction. The charge transfer resistance (Rct) values were significantly lower by up to one order of magnitude compared to the bare electrode. Differential pulse polarography (DPP) showed a marked decrease of the overpotential generated by the aliphatic chain. The calibration of the amperometric peak area vs. concentrations of derivatized LCAA exhibits a linear response within the range of 0.018 and 28 µM and correlation coefficient (R(2)) higher than 0.999 (n=5). The quantitation limit of the pNiPP/MWCNT electrode is about 400 times lower than the UV-visible detection. RSD of 7.2%, 5.8%, 2.5% and 2.3% was obtained for concentrations of 0.028, 0.28, 2.8 and 28 µM of ferrocenyl octadecylamine. A solution of sphingosine, 0.23 µM, was exclusively detected with HPLC-ECD with pNiPP/MWCNT electrode.
Assuntos
Aminas/análise , Metaloporfirinas/química , Nanocompostos/química , Nanotubos de Carbono/química , Níquel/química , Protoporfirinas/química , Aminas/química , Condutividade Elétrica , EletroquímicaRESUMO
Concentrations of lead (Pb) in breast milk (PbM) and blood (PbB) were measured in a current cohort of lactating mothers living in Andean communities where women of childbearing age engage in the occupational use of Pb, and compared to results obtained in earlier studies. Mean PbM concentration in the current group of breastfeeding mothers tested in 2012/2013 was 3.73 µg/L (SD: 7.3; range: 0.049-28.04), and significantly lower than the 9.83 µg/L (SD: 12.75; range: 0.2-49) previously observed in breastfeeding mothers in the study area from 1999 to 2007. Breastfeeding women in the current cohort showed an average PbM/PbB ratio of 3.6%, which is in agreement with other studies. The mean PbB level obtained for the current cohort was 7.8 µg/dl (SD: 5.2; range: 1.4-21), and significantly lower than the mean PbB level of 20.8 µg/dl (SD: 16.4; range: 4-73) obtained for the comparison group of breastfeeding mothers tested between 1999 and 2007. A correlation of .687 between paired PbM and maternal PbB was found, indicating that maternal PbB level is a significant predictor of PbM. Current PbM levels remain higher than international averages, but indicate that maternal Pb exposure has declined over time in the environmentally Pb-contaminated study area. The current reduction in Pb in milk and blood of breastfeeding mothers may be due to adherence to a Pb-exposure education and prevention program initiated by the authors in the study area years earlier, as well as recent improvements in local health care delivery.
Assuntos
Exposição Ambiental/análise , Chumbo/sangue , Chumbo/química , Leite Humano/química , Adolescente , Adulto , Arsênio/análise , Cádmio/análise , Estudos de Coortes , Equador , Feminino , Humanos , Lactação , Mercúrio/análise , Mães , Protoporfirinas/sangue , Protoporfirinas/química , Adulto JovemRESUMO
The selectivity of MnIII/II porphyrinates toward nitroxyl or nitric oxide donors provides a convenient starting point for the development of new materials for the speciation of these nitrogen-containing redox relatives. In the present report, we describe the insertion of MnIII protoporphyrinate IX in apomyoglobin and its chemical behavior toward HNO or NO donors, either under anaerobic or aerobic conditions. For comparison and discussion, the MnIII porphyrinate, devoid of the protein matrix, was studied in parallel. The MnIII reconstituted globin successfully reacted with the nitroxyl donor trioxodinitrate, while it was unreactive toward NO or NO donors, in good agreement with previously reported data on water soluble MnIII porphyrinates. The estimated association rate constant for the reaction with the nitroxyl donor was of the same order of magnitude for the reconstituted globin and the free porphyrinate, suggesting that the protein environment is not involved in the reaction mechanism. In contrast, the reaction product exhibited enhanced stability in the presence of dioxygen only when the porphyrinate was included in the protein matrix; this feature is ascribed to the role of the distal residues on the metal centered reactivity. This behavior is required for spectroscopic detection under biologically relevant conditions.
Assuntos
Manganês/química , Óxido Nítrico/química , Óxidos de Nitrogênio/química , Protoporfirinas/química , Domínio Catalítico , Cinética , OxirreduçãoRESUMO
The goal of this work was to study the liquid crystalline structure of a nanodispersion delivery system intended to be used in photodynamic therapy after loading with photosensitizers (PSs) and additives such as preservatives and thickening polymers. Polarized light microscopy and light scattering were performed on a standard nanodispersion in order to determine the anisotropy of the liquid crystalline structure and the mean diameter of the nanoparticles, respectively. Small angle X-ray diffraction (SAXRD) was used to verify the influence of drug loading and additives on the liquid crystalline structure of the nanodispersions. The samples, before and after the addition of PSs and additives, were stable over 90 days, as verified by dynamic light scattering. SAXRD revealed that despite the alteration observed in some of the samples analyzed in the presence of photosensitizing drugs and additives, the hexagonal phase still remained in the crystalline phase.