Andrey Musatov

Blending carbon particles (CPs) and nanoscale bioactive cerium dioxide is a promising approach for designing composites for biomedical applications, combining the sorption and antioxidant potentials of each individual component. To address this issue, it is crucial to assess the correlation between the components' ratio, physicochemical parameters, and biofunctionality of the composites. Thus, the current research was aimed at fabricating C@CeO₂ composites with different molar ratios and the examination of how the parameters of the composites affect their bioactivity. XRD, X-ray photoelectron spectroscopy, and electron microscopy data verified the formation of C@CeO₂ composites. CeO₂ nanoparticles (NPs) of 4-6 nm are highly dispersed on the surfaces of amorphous CPs. The presence of CeO₂ NPs on the carbon surface decreased its adsorption potential in a dose-dependent manner. Besides, the coexistence of carbon and CeO₂ in a single composite promotes some redox interactions between O-functionalities and Ce³⁺/Ce⁴⁺ species, resulting in changes in the chemical state of the surface of the composites. These observations suggest the strong connection between these parameters and the biofunctionality of the composites. The presence of CeO₂ NPs on the surface of carbon led to a significant increase in the stability of the prepared composites in their aqueous suspensions. The enhancement of bioactivity of the newly prepared C@CeO₂ compared to bare carbon and CeO₂ was validated by testing their pseudomimetic (catalase/peroxidase-like and superoxide dismutase-like), antiamyloid, and radioprotective activities.

"Core/shell" nanocomposites based on magnetic magnetite (Fe₃O₄) and redox-active cerium dioxide (CeO₂) nanoparticles (NPs) are promising in the field of biomedical interests because they can combine the ability of magnetic NPs to heat up in an alternating magnetic field (AMF) with the pronounced antioxidant activity of CeO₂ NPs. Thus, this report is devoted to Fe₃O₄/CeO₂ nanocomposites (NCPs) synthesized by precipitation of the computed amount of "CeO₂-shell" on the surface of prefabricated Fe₃O₄ NPs. The X-ray diffraction, X-ray photoelectron spectroscopy, and high-resolution transmission electron microscopy data validated the formation of Fe₃O₄/CeO₂ "core/shell"-like NCPs, in which ultrafine CeO₂ NPs with an average size of approximately 3-3.5 nm neatly surround Fe₃O₄ NPs. The presence of a CeO₂ "shell" significantly increased the stability of Fe₃O₄/CeO₂ NCPs in aqueous suspensions: Fe₃O₄/CeO₂ NCPs with "shell thicknesses" of 5 and 7 nm formed highly stable magnetic fluids with ζ-potential values of >+30 mV. The magnetization values of Fe₃O₄/CeO₂ NCPs decreased with a growing CeO₂ "shell" around the magnetic NPs; however, the resulting composites retained the ability to heat efficiently in an AMF. The presence of a CeO₂ "shell" generates a possibility to precisely regulate tuning of the maximum heating temperature of magnetic NCPs in the 42-50 °C range and stabilize it after a certain time of exposure to an AMF by changing the thickness of the "CeO₂-shell". A great improvement was observed in both antioxidant and antiamyloidogenic activities. It was found that inhibition of insulin amyloid formation, expressed in IC₅₀ concentration, using NCPs with a "shell thickness" of 7 nm was approximately 10 times lower compared to that of pure CeO₂. For these NCPs, more than 2 times higher superoxide dismutase-like activity was observed. The coupling of both Fe₃O₄ and CeO₂ results in higher bioactivity than either of them individually, probably due to a synergistic catalytic mechanism.

The structure and interaction parameters of the water-soluble cholesterol-based surfactant, Chobimalt, are investigated by small-angle neutron and X-ray scattering techniques. The obtained data are analyzed by a model-independent approach applying the inverse Fourier transformation procedure as well as considering a model fitting procedure, using a core-shell form factor and hard-sphere structure factor. The analysis reveals the formation of the polydisperse spherical or moderately elongated ellipsoidal shapes of the Chobimalt micelles with the hard sphere interaction in the studied concentration range 0.17–6.88 mM. The aggregation numbers are estimated from the micelle geometry observed by small-angle scattering and are found to be in the range of 200–300. The low pH of the solution does not have a noticeable effect on the structure of the Chobimalt micelles. The critical micelle concentrations of the synthetic surfactant Chobimalt in water and in H₂O-HCl solutions were obtained according to fluorescence measurements as ~3 μM and ~2.5 μM, respectively. In-depth knowledge of the basic structural properties of the detergent micelles is necessary for further applications in bioscience and biotechnology.

The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril’s ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.

Oxidative stress is known to be associated with a number of degenerative diseases. A better knowledge of the interplay between oxidative stress and amyloidogenesis is crucial for the understanding of both, aging and age-related neurodegenerative diseases. Cerium dioxide nanoparticles (CeO₂ NPs, nanoceria) due to their remarkable properties are perspective nanomaterials in the study of the processes accompanying oxidative-stress-related diseases, including amyloid-related pathologies. In the present work, we analyze the effects of CeO₂ NPs of different sizes and Ce⁴⁺/Ce³⁺ ratios on the fibrillogenesis of insulin, SOD-like enzymatic activity, oxidative stress, biocompatibility, and cell metabolic activity. CeO₂ NPs (marked as Ce1–Ce5) with controlled physical–chemical parameters, such as different sizes and various Ce⁴⁺/Ce³⁺ ratios, are synthesized by precipitation in water–alcohol solutions. All synthesized NPs are monodispersed and exhibit good stability in aqueous suspensions. ThT and ANS fluorescence assays and AFM are applied to monitor the insulin amyloid aggregation and antiamyloid aggregation activity of CeO₂ NPs. The analyzed Ce1–Ce5 nanoparticles strongly inhibit the formation of insulin amyloid aggregates in vitro. The bioactivity is analyzed using SOD and MTT assays, Western blot, fluorescence microscopy, and flow cytometry. The antioxidative effects and bioactivity of nanoparticles are size- or valence-dependent. CeO₂ NPs show great potential benefits for studying the interplay between oxidative stress and amyloid-related diseases, and can be used for verification of the role of oxidative stress in amyloid-related diseases.

Monitoring the aggregation of amyloid-prone proteins is critical for understanding the mechanism of amyloid fibril formation. Insulin, when dissolved in low pH buffer, has a surface tension of 61–64 mN m⁻¹, as measured by the pendant drop technique. Formation of insulin amyloid fibrils resulted in the increase of the surface tension values up to 71.2–73.5 mN m⁻¹. The kinetics of fibril formation and fibril morphology were validated by ThT fluorescence and AFM, respectively. The results demonstrate that monitoring the surface tension by the pendant drop technique is a valuable tool for the detection of insulin amyloid aggregation.

Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The pK_a value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased pK_a (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c. The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.