Synergistic Antifungal Interactions between Antibiotic Amphotericin B and Selected 1,3,4-thiadiazole Derivatives, Determined by Microbiological, Cytochemical, and Molecular Spectroscopic Studies.

In recent years, drug-resistant and multidrug-resistant fungal strains have been more frequently isolated in clinical practice. This phenomenon is responsible for difficulties in the treatment of infections. Therefore, the development of new antifungal drugs is an extremely important challenge. Combinations of selected 1,3,4-thiadiazole derivatives with amphotericin B showing strong synergic antifungal interactions are promising candidates for such formulas. In the study, microbiological, cytochemical, and molecular spectroscopy methods were used to investigate the antifungal synergy mechanisms associated with the aforementioned combinations. The present results indicate that two derivatives, i.e., C1 and NTBD, demonstrate strong synergistic interactions with AmB against some Candida species. The ATR-FTIR analysis showed that yeasts treated with the C1 + AmB and NTBD + AmB compositions, compared with those treated with single compounds, exhibited more pronounced abnormalities in the biomolecular content, suggesting that the main mechanism of the synergistic antifungal activity of the compounds is related to a disturbance in cell wall integrity. The analysis of the electron absorption and fluorescence spectra revealed that the biophysical mechanism underlying the observed synergy is associated with disaggregation of AmB molecules induced by the 1,3,4-thiadiazole derivatives. Such observations suggest the possibility of the successful application of thiadiazole derivatives combined with AmB in the therapy of fungal infections.

Classification of Honey Powder Composition by FTIR Spectroscopy Coupled with Chemometric Analysis.

Fourier transform infrared spectroscopy (FTIR) in connection with chemometric analysis were used as a fast and direct approach to classify spray dried honey powder compositions in terms of honey content, the type of diluent (water or skim milk), and carrier (maltodextrin or skim milk powder) used for the preparation of feed solutions before spray drying. Eleven variants of honey powders containing different amounts of honey, the type of carrier, and the diluent were investigated and compared to pure honey and carrier materials. Chemometric discrimination of samples was achieved by principal component analysis (PCA), hierarchical clustering analysis (HCA), linear discriminant analysis (LDA), and partial least squares-discriminant analysis (PLS-DA) modelling procedures performed on the FTIR preprocessed spectral data for the fingerprint region (1800-750 cm-1) and the extended region (3600-750 cm-1). As a result, it was noticed that the type of carrier is a significant factor during the classification of different samples of powdered multifloral honey. PCA divided the samples based on the type of carrier, and additionally among maltodextrin-honey powders it was possible to distinguish the type of diluent. The result obtained by PCA-LDA and PLS-DA scores yielded a clear separation between four classes of samples and showed a very good discrimination between the different honey powder with a 100.0% correct overall classification rate of the samples.

Biodirected Synthesis of Silver Nanoparticles Using Aqueous Honey Solutions and Evaluation of Their Antifungal Activity against Pathogenic Candida Spp.

Silver nanoparticles (AgNPs) were synthesized using aqueous honey solutions with a concentration of 2%, 10%, and 20%-AgNPs-H2, AgNPs-H10, and AgNPs-H20. The reaction was conducted at 35 °C and 70 °C. Additionally, nanoparticles obtained with the citrate method (AgNPs-C), while amphotericin B (AmB) and fluconazole were used as controls. The presence and physicochemical properties of AgNPs was affirmed by analyzing the sample with ultraviolet-visible (UV-Vis) and fluorescence spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). The 20% honey solution caused an inhibition of the synthesis of nanoparticles at 35 °C. The antifungal activity of the AgNPs was evaluated using opportunistic human fungal pathogens Candida albicans and Candida parapsilosis. The antifungal effect was determined by the minimum inhibitory concentration (MIC) and disc diffusion assay. The highest activity in the MIC tests was observed in the AgNPs-H2 variant. AgNPs-H10 and AgNPs-H20 showed no activity or even stimulated fungal growth. The results of the Kirby-Bauer disc diffusion susceptibility test for C. parapsilosis strains indicated stronger antifungal activity of AgNPs-H than fluconazole. The study demonstrated that the antifungal activity of AgNPs is closely related to the concentration of honey used for the synthesis thereof.

ESIPT-Related Origin of Dual Fluorescence in the Selected Model 1,3,4-Thiadiazole Derivatives.

In our previous work, we discussed the emergence of the dual fluorescence phenomenon in selected compounds from the group of 1,3,4-thiadiazoles. The results obtained in a number of experimental studies, supported by [TD]DFT calculations, clearly indicated that the phenomenon of dual fluorescence stemmed from an overlap of several factors, including the correct conformation of the analyzed molecule and, very significantly in this context, aggregation effects. Where those two conditions were met, we could observe the phenomenon of intermolecular charge transfer (CT) and the emergence of electronic states responsible for long wave emissions. However, in light of the new studies presented in this paper, we were able, for the first time, to provide a specific theory for the effect of dual fluorescence observed in the analyzed group of 1,3,4-thiadiazoles. We present the results of spectroscopic measurements conducted for two selected analogues from the 1,3,4-thiadiazole group, both in polar and non-polar solvents, which clearly evidence (as we have already suspected in the past, albeit have not shown in publications to date) the possibility of processes related to emission from the tautomer formed in the process of excited state intramolecular proton transfer, which is responsible for the long-wavelength emissions observed in the selected analogues. The presented results obtained with the use of UV-Vis, fluorescence (stationary and time-resolved), FTIR, and Raman spectroscopy, as well as from calculations of dipole moment changes between the ground and excited state with the use of two derivatives with different structures of the resorcylic system, corroborated our standing hypothesis. At the same time, they excluded the presence of ground state keto forms of the analyzed analogues unless necessitated by the structure of the molecule itself. In this case, aggregation factors enhance the observed effects related to the dual fluorescence of the analyzed compounds (by way of AIE-aggregated induced emissions).

Spectroscopic Studies of Dual Fluorescence in 2-(4-Fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole: Effect of Molecular Aggregation in a Micellar System.

The article presents the results of spectroscopic studies focused on a selected compound from the 1,3,4-thiadiazole group-2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thia-diazole (FABT)-in a micellar system formed by Triton X-100, a non-ionic detergent. Fluorescence measurements revealed the phenomenon of dual fluorescence whose emergence is related to the particular molecular organisation of the compound, which depends both on the concentration of the detergent and, most of all, the concentration of the compound itself. Dual fluorescence of FABT in a micellar system was observed for the compound dissolved in a methanol aqueous system, i.e., an environment wherein the dual fluorescence of the compound had never been reported before. Based on the interpretation of UV-Vis electronic absorption, resonance light scattering (RLS), emission and excitation fluorescence spectra, as well as measurements of dynamic light scattering (DLS) and Principal Component Analysis (PCA), we were able to relate the occurrence of this effect to the process of molecular aggregation taking place between FABT molecules in the micellar system in question. Results of fluorescence spectra measurements and time-correlated single photon counting (TCSPC) indicate that dual fluorescence occurs at detergent concentrations necessary to form micellar systems, which in turn facilitate the process of aggregation of FABT molecules. The correlation between the observed fluorescence effects and the previous measurements performed for analogues from this group suggests the possibility of charge transfer (CT) within the range of detergent concentrations wherein the aforementioned fluorescence effects are observed. It ought to be emphasised that this type of fluorescence effects are relatively easy to induce, which predisposes this groups of fluorophores as ideal fluorescence probes in the context of biological samples.

Effectiveness of magnetic fluid hyperthermia against Candida albicans cells.

Candida albicans is one of the most frequently isolated fungal pathogens causing opportunistic infections in humans. Targeted magnetic fluid hyperthermia (MFH) is a promising method in thermal therapy facilitating selective heating of pathogen cells like C. albicans. In the paper, we used meso-2,3-dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles (MNPs) and functionalised anti-C. albicans immunomagnetic nanoparticles (IMNPs) to investigate the potential of MFH in combating C. albicans cells in vitro. Using Mössbauer spectroscopy it was found that synthesised MNPs exhibited superparamagnetic phenomena. On the basis of calorimetric experiments, the maximum SAR (specific absorption rate) was found and a proper concentration of MNPs was established to control the temperature. MFH based on both DMSA-coated MNPs and functionalised anti-C. albicans IMNPs was more effective in combating C. albicans cells in vitro than thermostat hyperthermia. Especially promising results were obtained using functionalised IMNPs, which eradicated most of the pathogen colonies at the temperature of 43 °C.

Effect of cholesterol and ergosterol on the antibiotic amphotericin B interactions with dipalmitoylphosphatidylcholine monolayers: X-ray reflectivity study.

Amphotericin B is a Streptomyces nodosus metabolite and one of the oldest polyene antibiotics used in the treatment of invasive systemic fungal infections. Despite its over 50-year existence in clinical practice and the recognition of amphotericin B as the gold standard in the treatment of serious systemic mycosis, it still remains one of the most toxic pharmaceuticals. Understanding of the processes at the molecular levels and the interactions between amphotericin B with lipid membranes containing sterols should elucidate the mechanisms of the action and toxicity of this widely used antibiotic. In this work, we use X-ray reflectivity to study the structural changes on a molecular scale after amphotericin B incorporation. These changes are accompanied by an increase in monolayer surface pressure which is more pronounced for ergosterol - rather than cholesterol-rich membranes. The data indicate that this difference is not due to the higher affinity of amphotericin B towards ergosterol-containing membranes but is rather due to a ~3Angstrom corrugation of the monolayer. Furthermore, the total quantity of amphotericin B incorporated into lipid monolayers containing cholesterol and ergosterol is the same.

Spectroscopic studies of amphotericin B-Cu²+ complexes.

The aim of this research is to investigate amphotericin B (AmB)-Cu(2+) complexes in aqueous solution at different pH values. Electronic absorption, circular dichroism (CD), Raman and FTIR spectroscopies were used in this study. We found that different concentrations of AmB and Cu(2+) ions in solution leads to formation of complexes with stoichiometry of 2:1 and 1:1. The formation of AmB-Cu(2+) complexes at physiological pH values is accompanied by changes of the molecular organization of AmB especially disaggregation. These observed effects might be significant from a medical point of view.

Effect of Fe3O4 nanoparticles on germination of seeds and concentration of elements in Helianthus annuus L. under constant magnetic field.

The aim of the study was to investigate the effect of the Fe3O4 nanoparticles (Fe-NPs) on the germination of sunflower seeds, early growth of seedlings and the concentration of selected elements in seedlings. The influence of constant magnetic fields in systems with and without Fe-NPs was investigated. Experiments were done on seeds subjected to germination under constant magnetic field (0 (control), 5, 25 and 120 mT) for 7 days in the presence of solution containing 0, 50 or 500 ppm Fe-NPs. No significant effect of Fe-NPs and the magnetic field on germination of seeds and the growth of seedlings has been demonstrated. In most cases, a decrease in germination parameters was observed. For the majority of samples the relative decrease in the concentrations of elements was demonstrated mainly for samples without Fe-NPs. Interestingly, a significant decrease in the concentrations of trivalent (including iron - Fe) and toxic elements in samples containing Fe-NPs in relation to control samples was observed. The authors suggest that in this case the binding (adsorption) of these elements in the roots and seeds of the sunflower by Fe-NPs took place. This explains the lower iron content in seedlings than in seeds prior to sowing.

Physical assessment, spectroscopic and chemometric analysis of starch-based foils with selected functional additives.

The paper presents the results of studies related to the impact of functional additives in the form of polylactide (PLA), polyvinyl alcohol (PVA), and keratin hydrolysate (K) on the physical characteristics of biopolymer foils. TPS granulate was obtained using a TS-45 single-screw extruder with L/D = 16. Foil was produced with the use of an L/D = 36 extruder with film-blowing section. The impact of the quantity and type of the functional additives on the processing efficiency and energy consumption of granulate extrusion, as well as the physical characteristics of the foil produced: thickness, basis weight, and colour were determined. By measuring the FTIR spectra it was determined the type and origin of the respective functional groups. It was observed that foils produced from granulates with the addition of 3% PVA were characterised by the lowest thickness and basis weight. Addition of 2 and 3% of PLA increased thickness and basis weight of starch-based foils significantly. Increasing the content of keratin in SG/K samples resulted in a decrease of brightness and intensify the yellow tint of foils, especially when 2 and 3% of keratin was used. In terms of the other samples, it was observed that the colour remained almost unchanged irrespective of the percentage content of the additive used. Infrared analyses conducted on foil containing PVA, PLA, and K revealed a change in spectra intensity in the frequency range associated with-OH groups originating from the forming free, intra- and intermolecular hydrogen bonds. Based on an analysis of the respective bands within the IR range it was also concluded that considerable structural changes took place with respect to the glycosidic bonds of starch itself. The application of the mentioned additives had a significant structural impact on the produced starch-based foils. Furthermore, the conducted UV-Vis analyses revealed a substantial increase in absorbance and a related reduction of the permeability (colour change) of the obtained materials in the range of ultraviolet and visible light.

Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: implications for the biological action.

Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the therapeutic action and the toxic side effects of this drug are dependent on its molecular organization. AmB appears as a zwitterion at neutral pH owing to NH(3)(+) and COO(-) groups. The results obtained with electronic absorption, fluorescence, resonance light scattering and infrared absorption spectroscopic analyses show that in the aqueous medium at pH above 10 AmB appears in the monomeric form owing to the negative net electric charge of the molecule. On the contrary, anomalously high aggregation level has been observed at pH below 2, despite the positive net electric charge. The effect is interpreted in terms of the permanent polarization of the polyene chain at low pH, associated with relative rotational freedom of the charged mycosamine fragment of the molecule. The pH-dependent aggregation of AmB is discussed in aspect of pharmacological action of the drug.

Amphotericin B-copper(II) complex shows improved therapeutic index in vitro.

The AmB-Cu(II) complex has recently been reported as an antifungal agent with reduced aggregation of AmB in aqueous solutions, increased anti C. albicans activity and lower toxicity against human cells in vitro. In the present work, investigations of the activity of the AmB-Cu (II) complex against fungal pathogens with varying susceptibility, including C. albicans and C. parapsilosis strains and intrinsically resistant A. niger, and cytotoxicity in normal human dermal fibroblasts (NHDF) in vitro were performed. For better understanding of the mechanism of reduced cytotoxicity and increased fungicidal activity, the influence of the AmB-Cu (II) complex on membrane integrity and accumulation of cellular reactive oxygen species (ROS) and mitochondrial superoxide was compared with that of conventional AmB. In the sensitive C. albicans and C. parapsilosis strains, the AmB-Cu(II) complex showed higher fungicidal activity (the MIC value was 0.35-0.7µg/ml for the AmB-Cu (II) complex, and 0.45-0.9µg/ml for Fungizone) due to increased induction of oxidative damage with rapid inhibition of the ability to reduce tetrazolium dye (MTT). In the NHDF cell line, the CC50 value was 30.13±1.53µg/ml for the AmB-Cu(II) complex and 17.46±1.24µg/ml for (Fungizone), therefore, the therapeutic index (CC50/MIC90) determined in vitro was 86.09-43.04 for the AmB-Cu(II) complex and 38.80-19.40 for Fungizone. The lower cytotoxicity of the AmB-Cu(II) complex in human cells resulted from lower accumulation of cellular and mitochondrial reactive oxygen species. This phenomenon was probably caused by the induction of successful antioxidant defense of the cells. The mechanism of the reduced cytotoxicity of the AmB-Cu(II) complex needs further investigation, but the preliminary results are very promising.

Amphotericin B-copper (II) complex alters transcriptional activity of genes encoding transforming growth factor-beta family members and related proteins in renal cells.

BACKGROUND: Several chemical modifications have been developed to overcome the toxicity of amphotericin B (AmB). Oxidized forms of AmB (AmB-ox), which may occur in patient's circulation during therapy, are as toxic as AmB. Complexes with copper (II) ions (AmB-Cu2+) have been reported to be less toxic to human cells. Previous studies showed that AmB changed the expression of transforming growth factor-beta (TGF-ß). Therefore, the objective of this study was to investigate the influence of AmB and its modified forms on the expression of genes encoding for TGF-ß family members and related proteins in renal cells. METHODS: Human renal proximal tubule cells (RPTEC) were treated with AmB-Cu2+, AmB, or the oxidized form AmB-ox. The expression of TGF-ß family members and related genes was determined using oligonucleotide microarrays. TGF-ß1 protein level was determined using ELISA method. The mRNA level of TGF-ß isoforms, TGF-ß receptors and differentiating genes was evaluated by real-time RT-qPCR. RESULTS: AmB-Cu2+ increased the mRNA levels of TGF-ß1 and TGF-ß2 isoforms and two genes encoding receptors: TGFBR1 and TGFBR2. TGF-ß1 protein level in culture medium was not increased after stimulation with AmB-Cu2+. Microarray analysis revealed changes in both pro-fibrotic and anti-fibrotic genes. CONCLUSIONS: These results suggest that AmB-Cu2+ may induce repair mechanisms in renal proximal tubule cells via changes in the expression of genes involved in intracellular signaling.

A new form of amphotericin B - the complex with copper (II) ions - downregulates sTNFR1 shedding and changes the activity of genes involved in TNF-induced pathways: AmB-Cu2+ downregulates sTNFR1 shedding and changes the activity of genes involved in TNF-induced pathways.

BACKGROUND: A new form of amphotericin B (AmB)- complex with copper (II) ions (AmB-Cu2+) - is less toxic to human renal cells. Cytokines, including Tumor Necrosis Factor (TNF), are responsible for nephrotoxicity observed in patients treated with AmB. Another problem during therapy is the occurrence of oxidized forms of AmB (AmB-ox) in patients' circulation. To elucidate the molecular mechanism responsible for the reduction of the toxicity of AmB-Cu2+, we evaluated the expression of genes encoding TNF and its receptors alongside encoding proteins involved in TNF-induced signalization. METHODS: Renal cells (RPTECs) were treated with AmB, AmB-Cu2+ or AmB-ox. The expression of TNF and its receptors was evaluated by ELISA tests and real-time RT-qPCR. The expression of TNF-related genes was appointed using oligonucleotide microarrays. RESULTS: Only sTNFR1 was detected, and its level was lower in AmB-Cu2+- and AmB-ox-treated cells. TNFR1 mRNA was downregulated in AmB-ox, while TNFR2 mRNA was upregulated in AmB and AmB-Cu2+. Several changes in the expression of TNF-related genes coincided with changes in the expression of TNF receptors. CONCLUSIONS: The lower toxicity of AmB-Cu2+ could result from the changes in the expression of TNF receptors, which coincided with the changes in the expression of genes encoding proteins involved in TNF-induced pathways. This situation might subsequently result in a changes in intracellular signalization and influence the toxicity of tested forms of AmB on renal cells.

Effect of the Amphotericin B and Its Copper Complex on a Model of the Outer Leaflet of Human Erythrocyte Membrane.

A comparative study on the effect of an antibiotic amphotericin B (AmB) and its copper complex on the multicomponent monolayers imitating the outer leaflet of human erythrocyte membrane has been performed by means of the Langmuir monolayer technique. The properties of mixed films were analyzed by thermodynamic description of the interactions between the molecules complemented with the morphology of monolayers established by Brewster angle microscopy (BAM). The results revealed differences in the molecular organization of the two antibiotic forms at the air/water interface, which were explained by the different spatial structure of the complex. The lipophilicity of the complex contributed to considerably more effective interactions with the components of the model membrane, compared to pure antibiotic, expressed by negative values of the excess of free energy of mixing ΔGexc in the whole range of mole fractions. The mixed films with AmB were more stable as the proportion of lipids in the mixture increased. BAM images demonstrated that the addition of antibiotic at high content into the lipid mixture led to the formation of crystallite structures within the film, probably caused by the expelling of AmB molecules from mixed monolayers. These findings could help to explain the mechanism of the hemolytic activity of polyene antibiotics.

In vitro evaluation of antifungal and cytotoxic activities as also the therapeutic safety of the oxidized form of amphotericin B.

The aim of this study was to evaluate the antifungal and cytotoxic activities of the oxidized form of amphotericin B (AmB-Ox) as well as to determine whether oxidation process of AmB is therapeutically beneficial in vitro. The antifungal activity was estimated against Candida albicans ATCC 10231 and Candida parasilosis ATCC 22019 by broth microdilution method according to the NCCLS M27-A2 standards. The in vitro cytotoxicity was evaluated using normal green monkey kidney cells (GMK) by MTT assay. The obtained results demonstrated that AmB-Ox possesses 16-fold decreased antifungal properties against the two Candida strains and 5-fold lower cytotoxic activity towards GMK cells in comparison with AmB. The therapeutic safety in vitro assessed by calculating the ratio between cytotoxicity (CC50 value) to antifungal activity (MIC value) showed that oxidation of AmB is a very unfavourable process in vitro, because leads to formation of derivative (AmB-Ox) that lost antifungal properties much more rapidly than cytotoxic activity. Thus, the process of the oxidation of AmB in vivo (if it occurs) can be also highly harmful for patient.

Amphotericin B-copper(II) complex as a potential agent with higher antifungal activity against Candida albicans.

Amphotericin B (AmB) is a polyene antibiotic produced by Streptomyces nodosus used for more than 50 years in the treatment of acute systemic fungal infections. It exhibits a broad spectrum of activity against fungal and protozoan pathogens with relatively rare resistance. The aim of this study was to prepare and evaluate the utility of the AmB-Cu(2+) complex as a potential compound with a high fungicidal activity at lower concentrations, compared with conventional AmB. It was hypothesized that insertion of copper ions into fungal cell membranes, together with the AmB-Cu(2+) complex bypassing the natural homeostatic mechanisms of this element, may contribute to the increased fungicidal activity of AmB. The analysis of results indicates the increased antifungal activity of the AmB-Cu(2+) complex against Candida albicans in comparison with the pure AmB and Fungizone. Additionally, it was stated that the increased antifungal activity of the AmB-Cu(2+) complex is not the sum of the toxic effects of AmB and Cu(2+) ions, but is a result of the unique structure of this compound.