The intriguing dose-dependent effect of selected amphiphilic compounds on insulin amyloid aggregation: Focus on a cholesterol-based detergent, Chobimalt

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,andSAXS.WhileatthelowestChobimaltconcentration(0.1 µM;insulinto 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. Wehypothesize thatthedifferent 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 whichundergoestheprocessoffibrilsformation;i.e.,certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates

Structural reorganization of fullerene C70_{70} in N-methyl-2-pyrrolidone/toluene mixtures

The changes in the cluster state of fullerene C$_{70}$ dissolved in N-methyl-2-pyrrolidone/toluene mixture after toluene addition have been studied. In order to measure the wide range of particle sizes, including sub-single-molecule region and respectively large clusters, the Small-Angle X-ray Scattering, Dynamic Light Scattering were used. The main task was to find out correlation between the fullerene clusterization state and a volume concentration of the second nonpolar solvent. It was observed a common dependence of the structure state of fullerene C$_{70}$ on the polarity of the liquid medium; an increase in toluene content led to further dissolution of existing large agglomerates. The obtained data were analyzed in comparison with the previous results on the inverse colloidal system, C$_{70}$/toluene/N-methyl-2-pyrrolidone, and also with semi-empirical calculations, UV–vis spectroscopy measurements and experimental measurements on similar solutions based on fullerene C$_{60}$

On a specific state of C60_{60} fullerene in N-methyl-2-pyrrolidone solution: Mass spectrometric study

A solution of fullerene C$_{60}$ in N-methylpyrrolidone (NMP) presents a suitable system for obtaining fullerene's clusters with the tunable size. However, the mechanism of fullerenes interaction with polar NMP molecules is still elusive. Herein, we present the measured laser desorption/ionization mass spectra (LDI MS) of the precipitates produced from C$_{60}$/NMP solutions of different age in comparison with the typical spectra of C$_{60}$ crystallized from toluene and benzene. The distinctive characteristics of the C$_{60}$/NMP mass spectra were identified and carefully examined. The number of characteristic peaks and their relative intensities in the spectra strongly depend on the age of initial C$_{60}$/NMP solutions. This effect was attributed to the specific C$_{60}$-NMP interactions in the solution, namely to the formation of charge-transfer complexes of C$_{60}$ with NMP molecules followed by fullerene cluster formation. The results of additional measurements carried out by means of small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), UV–Vis absorption spectroscopy together with the density functional theory (DFT) calculations are in accord with the proposed hypothesis

Cluster-cluster interaction in nanodiamond hydrosols by small-angle scattering

The structure and interaction of nanodiamond fractal clusters were studied by small-angle X-ray and neutron scattering including contrast variation. The density of nanodiamonds, the aggregation number, and the ratio of aggregated/non-aggregated particles were determined. The analysis of the structure-factor made it possible to obtain the effective potential of cluster–cluster interaction. Branched fractal colloids behave to a certain extent like hard spheres, but the effective correlation length decreases with increasing concentration up to the sol–gel transition. The proposed approach for analyzing cluster–cluster correlations can be used in wider class of liquid systems with nano-sized non-compact inclusions

C60\mathrm{C_{60}} fullerene enhances cisplatin anticancer activity and overcomes tumor cell drug resistance

We formulated and analyzed a novel nanoformulation of the anticancer drug cisplatin (Cis) with C$_{60}$ fullerene (C$_{60}$+Cis complex) and showed its higher toxicity toward tumor cell lines in vitro when compared to Cis alone. The highest toxicity of the complex was observed in HL-60/adr and HL-60/vinc chemotherapy-resistant human leukemia cell sublines (resistant to Adriamycin and Vinculin, respectively). We discovered that the action of the C$_{60}$+Cis complex is associated with overcoming the drug resistance of the tumor cell lines through observing an increased number of apoptotic cells in the Annexin V/PI assay. Moreover, in vivo assays with Lewis lung carcinoma (LLC) C$_{57}$BL/6J male mice showed that the C$_{60}$+Cis complex increases tumor growth inhibition, when compared to Cis or C$_{60}$ fullerenes alone. Simultaneously, we conducted a molecular docking study and performed an Ames test. Molecular docking specifies the capability of a C$_{60}$ fullerene to form van der Waals interactions with potential binding sites on P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP-1), and multidrug resistance protein 2 (MRP-2) molecules. The observed phenomenon revealed a possible mechanism to bypass tumor cell drug resistance by the C$_{60}$+Cis complex. Additionally, the results of the Ames test show that the formation of such a complex diminishes the Cis mutagenic activity and may reduce the probability of secondary neoplasm formation. In conclusion, the C$_{60}$+Cis complex effectively induced tumor cell death in vitro and inhibited tumor growth in vivo, overcoming drug resistance likely by the potential of the C$_{60}$ fullerene to interact with P-gp, MRP-1, and MRP-2 molecules. Thus, the C$_{60}$+Cis complex might be a potential novel chemotherapy modification

Diluted and concentrated organosols of fullerene C 60 in the toluene–acetonitrile solvent system as studied by diverse experimental methods

In this article, we examined the state of fullerene C$_{60}$ in toluene and its mixtures with acetonitrile in both diluted, (4.0 to 6.3)×10$^{−6}$ M, and concentrated, (0.23 to 1.9)×10$^{−3}$ М solutions, prepared by either equilibrium or non-equilibrium procedures. Typically, the working solutions were prepared by diluting stock solutions of fullerene in toluene. Some specific features of solid fullerene interaction with atmospheric oxygen were revealed using the LDI mass-spectrometry. A combination of electron absorption spectra of the fullerene in C$_6$H$_5$CH$_3$–CH$_3$CN mixtures with the analysis of the particle size distribution using the DLS method demonstrated that even in acetonitrile-rich media, where diluted C$_{60}$ exists in colloidal state, some features of the molecular absorption spectra are still present. Such effect is in line with the formation of the large solvation shells of an aromatic solvent around fullerenes. The TEM images of the dried colloidal solutions demonstrate a loose floc configuration of the aggregates, contrary to the crystal structure of the species in a toluene-free C$_{60}$ dispersion obtained by hand-grinding. In solution, the spectrum of the last-named is a monotonous curve increasing toward ultraviolet. The LDI measurements proved the tendency of C$_{60}$ toward forming negative species under contact with acetonitrile. Electrophoretic studies state that a universal property of the negatively charged colloidal species is their expressed ability to overcharging in the presence of inorganic cations, which are poorly solvated by acetonitrile. In concentrated (oversaturated) fullerene solutions, where the SAXS and SANS methods are applicable, fractal-type aggregates of fullerenes were revealed in solutions. The analysis of aggregates structure indicates that their packing density is increased with growth of fullerene concentration and/or amount of acetonitrile in the mixture. Thus, branched aggregates were observed in toluene solution, while fullerenes form dense clusters with diffusive surface in mixtures with acetonitrile