Katarzyna Cieślik-Boczula

Protein fibril formation is of considerable interest due to the association of fibrils with many neurodegenerative diseases as well as non-neuropathic amyloidosis. In this study, the effects of individual phenolic compounds and their mixtures—including caffeic acid (CC), quercetin (Que), and resveratrol (Res)—on the fibrillation of lysozyme (Lys) were investigated using Fourier transform infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy. These phenolic compounds are known as potent modulators, particularly inhibitors, of fibrillation in various proteins and peptides. While their anti-fibrillar activity has been extensively studied via the Thioflavin T (ThT) fluorescence assay, here FTIR and VCD spectroscopy are employed for a comprehensive, label-free investigation of their effects. The results indicate that CC, Que, and Res influence Lys fibrillogenesis in ways that depend on the phenol concentration, their combination (mixing ratios), and the experimental conditions. In particular, Lys was observed to form left-handed fibrils under these conditions in the presence of these phenolic compounds, as evidenced by the VCD signal. Furthermore, a comparison was made of the inhibitory effects of the individual phenols and their mixtures on Lys fibril formation: CC and Res were more effective inhibitors than Que. Interestingly, an equimolar mixture of CC and Res exhibited a strong synergistic inhibitory effect on fibrillation, whereas the combination of CC with Que showed an antagonistic effect. Overall, this study provides deeper insights into how specific polyphenolic compounds influence protein fibrillation processes, offering valuable information for developing therapeutic interventions targeting protein aggregation-associated disorders.

Background: Phytosterols are bioactive lipids susceptible to oxidation, particularly under thermal stress. Incorporation into liposomes may enhance their stability, while resveratrol—a natural antioxidant—could further limit thermal degradation. Stigmasterol esters, which contain fatty acid residues prone to oxidation, require additional characterization to understand their behavior under heating. Methods: Liposomes composed of dipalmitoylphosphatidylcholine (DPPC) were enriched with free stigmasterol (ST), stigmasteryl myristate (ME), or stigmasteryl oleate (OE), with or without resveratrol (RES). Liposomal systems were characterized using transmission electron microscopy, zeta potential, and hydrodynamic diameter analyses. Samples were heated at 60 °C and 180 °C for 8 h to evaluate stigmasterol degradation, oxyphytosterol (SOP) formation, and decomposition of fatty acid residues in the esters. Results: Liposomes remained structurally stable at 60 °C but underwent marked alterations at 180 °C. ST formed the smallest particles, while ME and OE systems exhibited larger hydrodynamic diameters. Incorporation of resveratrol enhanced thermal and oxidative stability, reducing stigmasterol degradation (7.73–18.86% at 60 °C; 29.66–35.28% at 180 °C) and limiting SOP formation. Differences in the breakdown of myristic versus oleic acid residues highlighted the role of fatty acid type in determining thermal resistance. Conclusions: Resveratrol effectively improves the stability of liposomes containing stigmasterol or its esters and mitigates oxidative damage under thermal stress. Protective effects were particularly evident at moderate temperatures, indicating the potential of resveratrol–phytosterol liposomes as thermally stable delivery systems.

Biomembranes, which are the structural and functional basis of the cells of all living organisms, have been an extremely interesting research object for biology and chemistry scientists for years. The multitude of elements constituting the components of natural lipid membranes, however, is associated with interpretation difficulties regarding the nature of the processes taking place in them. A useful research object that is a model of bilamellar biosystems with a significantly simplified composition and at the same time retaining properties that can be a reference point in relation to natural membranes are lipid membranes in the form of one or several component liposomes. It is precisely such systems built of molecules of dipalmitoyl phosphatidylcholine (DPPC) or dipalmitoyl phosphatidylglycerol (DPPG), and analogous systems with the addition of cholesterol (Chol), that were the subject of research in this work. Near-infrared (NIR) vibrational spectroscopy provides a suitable method for the study of the hydrated samples. In most cases it can be alternatively adopted instead of commonly used mid-infrared (MIR) vibrational spectroscopy. This technique was applied for the first time to identify the spectral changes associated with the conformational changes in the hydrophobic region of model lipid bilayers. Trans/gauche isomerization of CH2 groups of lipid hydrocarbon chains is accompanied by characteristic changes in spectral parameters of both νas,s CH2 bands and their first overtones (2νas,s CH2). The heating of all types of analyzed liposomes results in main phase transition (Tm) accompanied by trans to gauche isomerization of CH2 groups of lipid hydrocarbon chains. The NIR-spectroscopy was able to describe in proper way (similar to MIR results) the character of Tm in studied bilayers.