Grzegorz Czernel

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 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.

Spectroscopic studies of amphotericin B–Cu2+ complexes

The aim of this research is to investigate amphotericin B (AmB)–Cu2+ 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 Cu2+ ions in solution leads to formation of complexes with stoichiometry of 2:1 and 1:1. The formation of AmB–Cu2+ 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 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.

Effectiveness of magnetic fluid hyperthermia against Candida albicans cells

Abstract 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.

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

&NA; 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 0.35–0.7 &mgr;g/ml for the AmB‐Cu (II) complex, and 0.45–0.9 &mgr;g/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 ± 1.53 &mgr;g/ml for the AmB‐Cu(II) complex and 17.46 ± 1.24 &mgr;g/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. Graphical Abstract Figure. No caption available.

Synthesis and characterization of iron oxide magnetic nanoparticles

Abstract Small particles of magnetite, i.e. 7.5, 13.4 and 14.1 nm in diameter, were obtained by the method of co-precipitation. The crystal structure and size distributions were determined by means of transmission electron microscopy and X-ray diffraction. The magnetic properties of the nanoparticles were tested by Mössbauer spectroscopy within the temperature range from 3 K to room temperature (RT). The Mössbauer study of magnetic nanoparticles reveals relaxation behaviour related to the existence of the superparamagnetic phase. The blocking temperature depends on the sizes of the nanoparticles and the ammonia concentration.

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 patients 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-β). 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-β 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-β family members and related genes was determined using oligonucleotide microarrays. TGF-β1 protein level was determined using ELISA method. The mRNA level of TGF-β isoforms, TGF-β receptors and differentiating genes was evaluated by real-time RT-qPCR. RESULTS AmB-Cu2+ increased the mRNA levels of TGF-β1 and TGF-β2 isoforms and two genes encoding receptors: TGFBR1 and TGFBR2. TGF-β1 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.