Phasor-FLIM for a direct investigation of Transportan 10 interactions with model membranes

Transportan 10 (TP10), a short and positive charged peptide, belonging to the family of the cell penetrating peptides has gained increasing attention for its antimicrobial and anticancer activity but also for its applications in drug delivery as it is able to translocate therapeutic molecules in cellular environment. Due to the complexity of the phenomena involved in cellular uptake and following processes, which strongly depend on the membrane lipid composition, structural details of the peptide (e.g., charge, hydrophobicity, steric hindrance) and environmental conditions, it is not easy to understand the general rules governing them. Here, we combine spectroscopic techniques and fluorescence lifetime imaging microscopy (FLIM) to investigate (i) the fate of the TP10 in the presence of model membranes, analyzing its conformational changes occurring at membrane interface and distinguishing peptide adsorption from insertion into the lipid bilayer (ii) the changes of the fluidity of the membrane and the formation of pores into the latter induced by TP10 interaction. In addition, thanks to the use of the environment sensitive fluorescence dyes, Laurdan and di-4-ANEPPDHQ, and of the phasor approach to analyze FLIM data, we were able to monitor in real time fine events at different depths of phospholipid bilayers

Deciphering amyloid fibril molecular maturation through FLIM-phasor analysis of thioflavin T

The investigation of amyloid fibril formation is paramount for advancing our understanding of neurodegenerative diseases and for exploring potential correlated therapeutic strategies. Moreover, the self-assembling properties of amyloid fibrils show promise for the development of advanced protein-based biomaterials. Among the methods employed to monitor protein aggregation processes, fluorescence has emerged as a powerful tool. Its exceptional sensitivity enables the detection of early-stage aggregation events that are otherwise challenging to observe. This research underscores the pivotal role of fluorescence analysis, particularly in investigating the aggregation processes of hen egg white lysozyme, a model protein extensively studied for insights into amyloid fibril formation. By combining classical spectroscopies with fluorescence microscopy and by exploiting the fluorescence properties (intensity and lifetime) of the thioflavin T, we were able to noninvasively monitor key and complex molecular aspects of the process. Intriguingly, the fluorescence lifetime imaging-phasor analysis of thioflavin T fluorescence lifetime on structures at different stages of aggregation allowed to decipher the complex fluorescence decay behavior, highlighting that their changes rise from the combination of specific binding to amyloid typical cross-Beta structures and of the rigidity of the molecular environment

Surface-catalyzed liquid–liquid phase separation and amyloid-like assembly in microscale compartments

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process

Different a-casein association states and their interaction with lipid vesicles to study antibacterial activity

The interactions between caseins and lipid membranes are fundamental for the physiological function of these proteins. Moreover, the understanding of the underlying molecular mechanisms is of great interest for the development of new casein derived antimicrobial peptides. Indeed, it was already shown that peptides derived from caseins possess antibacterial activity but their mechanisms of action is still debated. Here, we present an experimental study on the interaction between model lipid membranes and a-casein by means of spectroscopy and fluorescence microscopy techniques. a-casein is an unfolded protein that due to its amphiphilic nature is known to self-assembly into non-stable micellar structures whose presence, diameter and compactness depend on environmental conditions. Presented experiments are aimed at assessing the effects of this protein in different states (monomeric, micellar and aggregated) on the membranes highlighting the role of micelles.The association state of a-casein at different pH and temperatures was analysed by fluorescence spectroscopy, circular dichroism and dynamic light scattering. Then, a-casein in different states was added to giant lipid vesicles and fluorescence microscopy and spectroscopy techniques were used to map and quantify induced modifications on the membrane. Our results indicate that, depending on the specific properties of the added protein state, different membrane structure and morphology changes occur. Interestingly, the most effective species in altering membranes is constituted by highly hydrophobic oligomers originating from larger aggregates disassembly

Oxidation effects in antiaggregogenic properties of Epigallocatechingallate

Epigallocatechin-gallate (EGCG), the most abundant flavonoid in green tea, has been extensively studied for its potential in the treatment of amyloid related disorders. This molecule was found to modulate abnormal protein self-assembly, reducing resulting cellular toxicity. EGCG is known to suppress or to slow down the aggregation processes of several proteins, thus supporting the idea that general mechanisms regulate its anti-aggregogenic effects and, interestingly, in the oxidised form it demonstrated an higher efficiency in reducing protein aggregation with respect to intact molecule. We here investigate the effects of intact and oxidized EGCG the thermal aggregation pathway of Bovine Serum Albumin (BSA), a well-known model protein whose aggregation processes are known in details. By means of different spectroscopic methods, we evaluate similarities and differences of the two molecules during protein aggregation. Different solution conditions are investigated, close and away from the isoelectric point of the protein, with the aim of eliciting the role of electrostatics in the observed EGCG-Protein interaction and in the supramolecular assembly which are dramatically dependent on solution conditions

Electrostatics regulate Epigallocatechin-Gallate effects on Bovine Serum Albumin aggregation

Protein aggregation processes are complex phenomena often involved in the etiology of several pathologies. It is now assessed that all proteins, in suitable conditions, may undergo supramolecular assembly. Aggregation pathways are known to be controlled by solution conditions which regulate protein-protein and protein-solvent interactions affecting binding mechanisms, morphology and inherent toxicity of the aggregate species. In this context, the presence of small molecules was indicated as a promising method to modulate protein-protein interactions reducing pathogenic aggregation. In the light of the idea that common mechanisms regulate anti-aggregogenic properties of small molecules, we here investigate Epigallocatechin-Gallate (EGCG) effects on the thermal aggregation pathway of Bovine Serum Albumin (BSA), a well-known model protein. EGCG is a small molecule extracted from green tea, which is known to reduce aggregation of key proteins involved in neurodegenerative diseases [1]. Fundamental mechanisms which regulate EGCG effectiveness as therapeutic molecule are still not clearly elucidated. The interaction of EGCG with BSA and its effects on thermal aggregation pathway were investigated by means of spectroscopic methods and Isothermal Titration calorimetry as a function of solution conditions. Results show that electrostatic forces modulated by pH play a key role in regulating EGCG interactions with BSA. Data shows that close to the isoelectric point of the protein, EGCG is found to promote the supramolecular assembly, whilst away from the isoelectric point, EGCG is found to reduce aggregation mechanisms increasing protein conformational stability. These results reveal the large impact of electrostatics in small molecules effects on the protein aggregation phenomena requiring larger investigation aimed at rationalizing their effects on related pathogenic mechanisms

Effect of cholesterol on the interaction between amphyphylic peptides and liposomes

With the rise of antibiotic resistance, antimicrobial peptides (AMPs) have been proposed as an alternative novel class of therapeutic agents. They are polycationic, with a net positive charge of more than +2, and they are characterized by amphipathic structures, with both a hydrophobic and a hydrophilic domain. These characteristics allow them to selectively bind to negatively charged lipids (largely present in bacteria, not in mammalian cells), via hydrophobic and electrostatic interactions. Moreover, mammalian cells are characterized by a high content of cholesterol. For this reason, here we present an experimental study on the effect of the presence of cholesterol on the capability of amphyphylic peptide Trasportant 10 (TP10) to interact with model membranes with selected composition. The study was performed by means of fluorescence spectroscopy and fluorescence confocal microscopy measurements also exploiting the advantages of phasor plot analysis of Fluorescence Lifetime Imaging (FLIM) measurements. Our results show that the presence of cholesterol inhibits TP-10 interaction with lipid vesicles, the extent of the observed effect being dependent on the cholesterol concentration in the membrane

Freestanding Cellulose Acetate/ZnO Flowers Composites for Solar Photocatalysis and Controlled Zinc Ions Release

The versatile properties of ZnO micro- and nano- structures have resulted in many applications in piezotronics, biosensors and photocatalysis. However, ZnO can easily dissolve in aqueous fluids, potentially resulting in the release of reactive oxygen species and zinc ions at toxic concentrations. Such an issue can be solved by dispersing ZnO within biocompatible polymeric matrices to reduce the direct exposure to the aqueous fluid and control the release of zinc ions. Herein, this work explores tailored ZnO flowers/cellulose acetate photocatalytic composites at different ZnO weight percentages (1-15 wt%). The photocatalytic degradation of methylene blue dye under simulated solar light is studied, finding an optimal value of ZnO filler loading in the polymer (10 wt %), resulting from a compromise between the photodegradation efficiency and the hydrophobicity induced by ZnO flowers. The reusability of the composites is investigated, finding a surprising improvement in the photodegradation efficiency after the first cycle. Simulated solar light stimulation induces the controllable release of zinc ions in aqueous solution at ppm-levels from the composites at the optimal ZnO filler loading. Finally, the release of ionic species in the absence of light stimulation is found to be directly proportional to the ZnO-loading in the composite, as a result of its degradation in aqueous environments

Improved Photocatalytic Activity of Polysiloxane TiO2 Composites by Thermally Induced Nanoparticle Bulk Clustering and Dye Adsorption

Fine control of nanoparticle clustering within polymeric matrices can be tuned to enhance the physicochemical properties of the resulting composites, which are governed by the interplay of nanoparticle surface segregation and bulk clustering. To this aim, out-of-equilibrium strategies can be leveraged to program the multiscale organization of such systems. Here, we present experimental results indicating that bulk assembly of highly photoactive clusters of titanium dioxide nanoparticles within an in situ synthesized polysiloxane matrix can be thermally tuned. Remarkably, the controlled nanoparticle clustering results in improved degradation photocatalytic performances of the material under 1 sun toward methylene blue. The resulting coatings, in particular the 35 wt % TiO2-loaded composites, show a photocatalytic degradation of about 80%, which was comparable to the equivalent amount of bare TiO2 and two-fold higher with respect to the corresponding composites not subjected to thermal treatment. These findings highlight the role of thermally induced bulk clustering in enhancing photoactive nanoparticle/polymer composite properties