Toxicological evaluation of fluorescent 11-mercaptoundecanoic gold nanoclusters as promising label-free bioimaging probes in different cancer cell lines.

Due to advancement in nanomaterials and increasing use of functionalized gold nanoclusters (AuNCs) in different biomedical applications, better understanding of their potential cytotoxicity is necessary. Interactions of ultra-small fluorescent AuNCs with mammalian cells remains up to this day poorly understood, therefore, cytotoxic evaluation of thoroughly characterized ca. 2.5 nm spherical water-soluble 11-mercaptoundecanoic acid coated AuNCs (AuNC@M) with diverse fluorescent properties in variety of mammalian cancer cell lines was performed. Cell viability was assessed by traditional MTT assay and xCELLigence real time cell analyzer. Cell apoptosis was evaluated via an Annexin V-FITC/propidium iodide (PI) assay. Confocal fluorescence imaging confirmed that tested AuNC@M entered live cells and were homogeneously distributed in their cytoplasm. The results suggested that the cytotoxicity of tested nanoclusters was very low, or near the control level at concentrations 0.1 and 0.5 mg/mL in the cell lines after 24 h exposition. The purity of tested AuNC@M had no relevant effect on cell viability and no differences were observed after 24 h in our study. The low toxicity toward cancer cells further strengthens our view that AuNC@M are promising label-free fluorescent probes for bio-labelling and bio-imaging, or they can even serve as platforms for antitumor drug delivery systems.

In vitro study of disodium cromoglicate as a novel effective hydrotrope solvent for hypericin utilisation in photodynamic therapy.

Hypericin (HY) is a naphthodianthrone that naturally occurs in Hypericum perforatum L. It is a promising photosensitiser used in photodynamic therapy for and diagnosis of oncological diseases. However, its hydrophobic character is an obstacle that has prevented its efficient use. The commonly used solvent, dimethyl sulfoxide (DMSO), is a controversial constituent of HY formulations and its use has been rejected by many researchers studying HY both in vitro and in vivo. In this study, we propose the utilisation of hydrotropy to solubilise HY in an aqueous environment. Cromolyn (DSCG) is a non-toxic, well-tolerated, antiallergic drug that has been employed in clinical practice since 1970, and in aqueous solution it acts as a hydrotrope. At a molecular ratio of 1:12,000 HY to DSCG, the compound is able to solubilise HY in aqueous environment. In an HT-29 cell suspension, DSCG (1.8 mmol L-1) considerably enhances the interaction between HY (150 nmol L-1) and HT-29 cells, which leads to an HY fluorescence emission increase with a half-time approximately 2 min compared to 29 min for samples that lack DSCG. Studies using HT-29 adenocarcinoma cells showed that DSCG at a given concentration significantly improved accumulation of HY within cells compared to DMSO (p < 0.05) despite the relative resistance of the HT-29 cell line to HY-PDT. Though no significant difference between total reactive oxygen species production was observed for photoactivated HY dissolved in DMSO and DSCG, significant singlet oxygen generation by photoactivated HY dissolved in a DSCG-containing water solution at the studied molecular ratio was confirmed. We also clarified that DSCG does not act as a scavenger of ABTS and galvinoxyl free radicals. The results from an MTT assay showed that DSCG also significantly enhanced the cytotoxicity of photoactivated HY compared to DMSO (p < 0.05). This study has demonstrated the ability of DSCG to act as a solvent of HY and enhance the effectiveness of HY-PDT compared to the commonly used organic solvent, DMSO.

Preparation of CuO/ZnO nanocomposite and its application as a cysteine/homocysteine colorimetric and fluorescence detector.

Cysteine and homocysteine play a crucial role in many biological functions but abnormal levels of these amino acids may lead to various forms of pathogenesis. Therefore, selective and easy-to-use methods for the detection of cysteine and homocysteine are essential for the early diagnosis of developing diseases. In this paper we report on a rapid, straightforward and highly selective method for the detection of cysteine (Cys) and homocysteine (Hcy) which uses a CuO/ZnO nanocomposite as a dual colorimetric and fluorometric assay. The presence of Cys and Hcy in a solution of these nanorods (NRs) induces a change in its color from light blue to dark grey which is visible to the naked eye. This is accompanied by a blue shift in the absorption spectra from 725 nm to 650 nm and a decrease in the intensity of CuO/ZnO nanocomposite emission. These changes are ascribed to the reduction of Cu(II) to Cu(0), and the oxidation of cysteine (homocysteine) and subsequent formation of the disulfide bond. This novel assay method does not respond to any other amino-acid which is present in living organisms; therefore the selective determination of cysteine (homocysteine) with a lower analyte limit of 40 µM (4.8 µg mL(-1)) can be carried out in aqueous solutions without the need for any sophisticated instrumentation, fluorophore molecules or complicated procedures.

Cytochrome c conjugated to ZnO-MAA nanoparticles: the study of interaction and influence on protein structure.

Nanoparticle-protein conjugates have potential for numerous applications due to the combination of the properties of both components. In this paper we studied the conjugation of horse heart cytochrome c with ZnO nanoparticles modified by mercaptoacetic acid (MAA) which may be a material with great potential in anticancer therapy as a consequence of synergic effect of both components. Cyt c adsorption to the ZnO-MAA NPs surface was studied by UV-vis spectroscopy and by a dynamic light scattering in various pH. The results indicate that the optimal pH for the association of protein with modified nanoparticles is in range 5.8-8.5 where 90-96% of cytochrome c was assembled on ZnO-MAA nanoparticles. The interaction of proteins with nanoparticles often results in denaturation or loss of protein function. Our observations from UV-vis spectroscopy and circular dichroism performed preserved protein structure after the interaction with modified nanoparticles.

Structural and thermodynamic behavior of cytochrome c assembled with glutathione-covered gold nanoparticles.

The conformational changes of horse heart ferricytochrome c (cyt c) after association of gold nanoparticles have been studied by electronic absorption spectroscopy and circular dichroism (CD). Our results show that the structural stability around the heme of complexed cyt c was increased successfully. Glutathione-layered gold nanoparticles caused a significant increase of the apparent pK values of the cyt c alkaline transition. Similarly, the heme crevice became more stable to heat after assembly of cyt c with gold nanoparticles. In contrast, gold nanoparticles weaken the overall thermal stability of the cyt c by decreasing the denaturation temperature estimated from far-UV CD measurements. Similar behavior has previously been reported for cyt c complexed with physiological redox partners as well as hydrophilic polyanions.

Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond.

The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, approximately 530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be approximately 21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, T(trs), independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, E(a), and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min(-1), indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.