BACKGROUND: Sarcosine is an amino acid that is formed by methylation of glycine and is present in trace amounts in the body. Increased sarcosine concentrations in blood plasma and urine are manifested in sarcosinemia and in some other diseases such as prostate cancer. For this purpose, sarcosine detection using the nanomedicine approach was proposed. In this study, we have prepared superparamagnetic iron oxide nanoparticles (SPIONs) with different modified surface area. Nanoparticles (NPs) were modified by chitosan (CS), and sarcosine oxidase (SOX). SPIONs without any modification were taken as controls. Methods and Results: The obtained NPs were characterized by physicochemical methods. The size of the NPs determined by the dynamic light scattering method was as follows: SPIONs/Au/NPs (100⁻300 nm), SPIONs/Au/CS/NPs (300⁻700 nm), and SPIONs/Au/CS/SOX/NPs (600⁻1500 nm). The amount of CS deposited on the NP surface was found to be 48 mg/mL for SPIONs/Au/CS/NPs and 39 mg/mL for SPIONs/Au/CS/SOX/NPs, and repeatability varied around 10%. Pseudo-peroxidase activity of NPs was verified using sarcosine, horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. For TMB, all NPs tested evinced substantial pseudo-peroxidase activity at 650 nm. The concentration of SPIONs/Au/CS/SOX/NPs in the reaction mixture was optimized to 0⁻40 mg/mL. Trinder reaction for sarcosine detection was set up at 510 nm at an optimal reaction temperature of 37 °C and pH 8.0. The course of the reaction was linear for 150 min. The smallest amount of NPs that was able to detect sarcosine was 0.2 mg/well (200 µL of total volume) with the linear dependence y = 0.0011x - 0.0001 and the correlation coefficient r = 0.9992, relative standard deviation (RSD) 6.35%, limit of detection (LOD) 5 µM. The suggested method was further validated for artificial urine analysis (r = 0.99, RSD 21.35%, LOD 18 µM). The calculation between the detected and applied concentrations showed a high correlation coefficient (r = 0.99). NPs were tested for toxicity and no significant growth inhibition was observed in any model system (S. cerevisiae, S. aureus, E. coli). The hemolytic activity of the prepared NPs was similar to that of the phosphate buffered saline (PBS) control. The reaction system was further tested on real urine specimens. Conclusion: The proposed detection system allows the analysis of sarcosine at micromolar concentrations and to monitor changes in its levels as a potential prostate cancer marker. The whole system is suitable for low-cost miniaturization and point-of-care testing technology and diagnostic systems. This system is simple, inexpensive, and convenient for screening tests and telemedicine applications.
- MeSH
- chitosan chemie MeSH
- Escherichia coli účinky léků růst a vývoj MeSH
- hemolýza účinky léků MeSH
- individualizovaná medicína MeSH
- koncentrace vodíkových iontů MeSH
- křenová peroxidasa chemie MeSH
- lidé MeSH
- limita detekce MeSH
- magnetické nanočástice chemie ultrastruktura MeSH
- nádorové biomarkery moč MeSH
- nádory prostaty diagnóza moč MeSH
- oxidace-redukce MeSH
- reprodukovatelnost výsledků MeSH
- Saccharomyces cerevisiae účinky léků růst a vývoj MeSH
- sarkosin moč MeSH
- sarkosinoxidasa chemie MeSH
- Staphylococcus aureus účinky léků růst a vývoj MeSH
- velikost částic MeSH
- železité sloučeniny chemie MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Doxorubicin (DOX) is one of the most frequently used anticancer drugs in breast cancer treatment. However, clinical applications of DOX are restricted, largely due to the fact that its action disturbs the pro/antioxidant balance in both cancerous and non-cancerous cells. The aim of this study was to investigate the influence of fullerene (C60) in cell treatment by DOX on the proliferation of human breast cancer cells (MCF-7), concentration of metallothionein (MT) and superoxide dismutase (SOD), and SOD activity in these cells. The use of C60 in complexes with DOX causes a change in the level of cell proliferation of about 5% more than when caused by DOX alone (from 60⁻65% to 70%). The use of C60 as a DOX nanotransporter reduced the MT level increase induced by DOX. C60 alone caused an increase of SOD1 concentration. On the other hand, it led to a decrease of SOD activity. C60 in complex with DOX caused a decrease of the DOX-induced SOD activity level. Exposure of MCF-7 cells to DOX-C60 complexes results in a decrease in viable cells and may become a new therapeutic approach to breast cancer. The effects of C60 in complexes with DOX on MCF-7 cells included a decreased enzymatic (SOD activity) and nonenzymatic (MT) antioxidant status, thus indicating their prooxidant role in MCF-7 cells.
- MeSH
- antitumorózní látky farmakologie MeSH
- doxorubicin farmakologie MeSH
- fullereny chemie MeSH
- lidé MeSH
- metalothionein metabolismus MeSH
- MFC-7 buňky MeSH
- nanočástice chemie MeSH
- proliferace buněk účinky léků MeSH
- superoxid dismutáza 1 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. OBJECTIVES: The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. MATERIAL AND METHODS: Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. RESULTS: In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. CONCLUSIONS: The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.
- MeSH
- elektroforéza kapilární metody MeSH
- králíci MeSH
- lidé MeSH
- metalothionein analýza MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
The clinical use of doxorubicin (DOX) is limited by dose-related cardiomyopathy, which becomes more prevalent with increasing cumulative doses of the drug. Complexes of fullerene with DOX were designed and studied using biophysical methods. The ability of DOX to release from fullerene at different pHs was analyzed. It has been shown that the size of the fullerene-DOX complexes was ∼280 nm. The zeta potential for fullerene was -30 mV, for DOX -8 mV, and for fullerene-DOX conjugates -24 mV. Drug release was studied by CE with LIF detection. When fullerene-DOX conjugates were separated in a pH 7.5 buffer, 43% of all DOX signals were derived from free DOX, with the signal increasing as pH decreased. At pH 5.25, all DOX had been released and 100% of the signal was derived from free DOX. The release of DOX from complexes with fullerene at lower pH can be used in targeted antineoplastic therapy, resulting in lower toxicity for less acidic non-target tissue.
- MeSH
- antibiotika antitumorózní terapeutické užití MeSH
- doxorubicin terapeutické užití MeSH
- elektroforéza kapilární MeSH
- fluorescenční barviva chemie MeSH
- fullereny chemie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádory prsu farmakoterapie MeSH
- nanočástice chemie MeSH
- nosiče léků chemie MeSH
- optické zobrazování MeSH
- povrchové vlastnosti MeSH
- uvolňování léčiv MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Metallothionein (MT) plays the important role in the detoxification of heavy metals, protection against oxidative compounds and as a prognostic marker in the development of tumors. It is important to find selective, stable and sensitive tools and probes to evaluate the presence of MT in biological fluids or tissues. QDs linked with ligands such as peptides or small molecules are a promising tool for selective, fast, and sensitive tagging and imaging in medicine. In previous findings, the authors proved the possibility of interaction with QDs (particularly with CdTe) and analyzed the stability of the formed complexes between CdTe and MT during incubation over time. Following that, an initial analysis of the interactions between CdTe quantum dots (QDs) and human metallothionein (MT) was performed. Complexes of mercaptosuccinic acid-covered CdTe QDs + MT were investigated using fluorescence intensity changes along a timeline, quenching analysis, stability interpretation based on zeta potential, and quenching intensity. Based on the preliminary results, it appears as though the possible interactions depend on the size of the CdTe QDs. Additionally, the formation of complexes between CdTe and human MT likely depends mostly on structural changes and conformational reorganization rather than on electrostatic interactions. Both types of interactions are responsible for complex creation and stabilization.
- MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- kvantové tečky chemie MeSH
- lidé MeSH
- metalothionein chemie MeSH
- povrchové vlastnosti MeSH
- sloučeniny kadmia chemie MeSH
- telur chemie MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Thanks to quantum dots' (QDs) properties, they can be used as selective and sensitive biomarkers in molecular imaging. In a previous paper, we confirmed the possibility of interaction between mercaptosuccinic acid-capped cadmium telluride QDs (MSA-CdTe) and human metallothionein (MT). The aim of this study was to expand on our previous research with an evaluation of the stability of the formed complexes between human MT and four CdTe compounds of the following sizes: 3.4nm (blue QDs), 3.8nm (green QDs), 4.5nm (yellow QDs), and 5.2nm (red QDs). Complexes were evaluated over time using fluorescence intensity and differential pulse voltammetry. Differences between the voltammograms obtained for standard solutions and for CdTe+MT show that complexes were formed. An increase in fluorescence intensity was observed for blue (Δ%≈40 for t=1→120min) and red (Δ%≈30 for t=1→120min) CdTe-MT complexes than CdTe alone, whereas green and yellow CdTe-MT complexes had a lower fluorescence intensity than CdTe alone. A stronger time dependence of the mercaptosuccinic acid (MSA) peak height on the timeline and differences in the MSA peak shape (in CdTe, and CdTe+MT complexes) were also observed by voltammetry. Authors noticed a decrease in the Cat2 signal of the red and green CdTe+MT complexes at the time of conjugation. Our results reveal that the size of QDs has an impact on the interaction between CdTe and human MT, as well as on the stability of complexes formed during these interactions. The bioconjugates' stability was also found to depend on the time of interaction.
- MeSH
- časové faktory MeSH
- elektrochemické techniky metody MeSH
- fluorescence MeSH
- fluorescenční spektrometrie metody MeSH
- kinetika MeSH
- kvantové tečky * MeSH
- lidé MeSH
- metalothionein chemie metabolismus MeSH
- sloučeniny kadmia chemie metabolismus MeSH
- telur chemie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mammalian metallothioneins (MTs) are a group of low molecular weight (6-7 kDa) proteins. They consist of a single polypeptide chain of 61-68 amino acid residues, including 20 cysteinyl residues. In the human, there are four isoforms: MT-1, MT-2, MT-3, MT-4. Heterogeneity of isoforms results also from post-translational acetylation and/or variations in metal compositions. MTs are involved in the metabolism of heavy metals and constitute an intracellular reservoir of trace elements. Furthermore, they neutralize electrophilic compounds, have a protective function against DNA damage and an ability to inhibit apoptosis. Due to the characteristics, such as low molecular weight and unique primary structure, elaborating the isolation, separation and determination conditions of MTs analysis poses many problems, especially in achieving a satisfactory sensitivity and specificity. In addition to traditional analytical techniques, such as gel electrophoresis, liquid chromatography, and size exclusion chromatography, capillary electrophoresis (CE) has proven to be a powerful separation technique for MT analysis. Numerous studies devoted to the optimization of CE conditions as capillary surface, electrolyte background or modifiers or kind of detectors. Coupling CE with mass spectrometry creates a powerful analytical tool for the characterization of MT.
- MeSH
- elektroforéza kapilární * metody MeSH
- lidé MeSH
- metalothionein * analýza MeSH
- protein - isoformy analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
