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Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, we link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events, providing a general tool for a comprehensive analysis of self-assembly reactions.Table of Contents

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

Proteins, and in particular whey proteins, have recently been introduced as a promising excipient class for stabilizing amorphous solid dispersions. However, despite the efficacy of the approach, the molecular mechanisms behind the stabilization of the drug in the amorphous form are not yet understood. To investigate these, we used experimental and computational techniques to study the impact of drug loading on the stability of protein-stabilized amorphous formulations. β-Lactoglobulin, a major component of whey, was chosen as a model protein and indomethacin as a model drug. Samples, prepared by either ball milling or spray drying, formed single-phase amorphous solid dispersions with one glass transition temperature at drug loadings lower than 40-50%; however, a second glass transition temperature appeared at drug loadings higher than 40-50%. Using molecular dynamics simulations, we found that a drug-rich phase occurred at a loading of 40-50% and higher, in agreement with the experimental data. The simulations revealed that the mechanisms of the indomethacin stabilization by β-lactoglobulin were a combination of (a) reduced mobility of the drug molecules in the first drug shell and (b) hydrogen-bond networks. These networks, formed mostly by glutamic and aspartic acids, are situated at the β-lactoglobulin surface, and dependent on the drug loading (>40%), propagated into the second and subsequent drug layers. The simulations indicate that the reduced mobility dominates at low (

Salt ions are considered among the major determinants ruling protein folding, stability, and self-assembly in the context of amyloid-related diseases, protein drug development, and functional biomaterials. Here, we report that Hofmeister ions not only determine the rate constants of the aggregation reaction for human insulin and hen egg white lysozyme but also control the generation of a plethora of amyloid-like morphologies ranging from the nanoscale to the microscale. We anticipate that the latter is a result of a balance between colloidal and conformational stability combined with an ion-specific effect and highlight the importance of salt ions in controlling the biological functions of protein aggregates.

Protein-based materials have found applications in a wide range of biomedical fields because of their biocompatibility, biodegradability and great versatility. Materials of different physical forms including particles, hydrogels, films, fibers and microneedles have been fabricated e.g. as carriers for drug delivery, factors to promote wound healing and as structural support for the generation of new tissue. This review aims at providing an overview of the current scientific knowledge on protein-based materials, and selected preclinical and clinical studies will be reviewed in depth as examples of the latest progress within the field of protein-based materials, specifically focusing on non- and minimally invasive strategies mainly for topical application.

Protein-based materials recently emerged as good candidates for water cleaning applications, due to the large availability of the constituent material, their biocompatibility and the ease of preparation. In this work, new adsorbent biomaterials were created from Soy Protein Isolate (SPI) in aqueous solution using a simple environmentally friendly procedure. Protein microsponge-like structures were produced and characterized by means of spectroscopy and fluorescence microscopy methods. The efficiency of these structures in removing Pb2+ ions from aqueous solutions was evaluated by investigating the adsorption mechanisms. The molecular structure and, consequently, the physico-chemical properties of these aggregates can be readily tuned by selecting the pH of the solution during production. In particular, the presence of β-structures typical of amyloids as well as an environment characterized by a lower dielectric constant seem to enhance metal binding affinity revealing that hydrophobicity and water accessibility of the material are key features affecting the adsorption efficiency. Presented results provide new knowledge on how raw plant proteins can be valorised for the production of new biomaterials. This may offer extraordinary opportunities towards the design and production of new tailorable biosorbents which can also be exploited for several cycles of purification with minimal reduction in performance. Synopsis: Innovative, sustainable plant-protein biomaterials with tunable properties are presented as green solution for water purification from lead(II) and the structure-function relationship is discussed.

The interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane.

Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.

Protein aggregates, hereunder amyloid fibrils, can undergo a maturation process, whereby early formed aggregates undergo a structural and physicochemical transition leading to more mature species. In the case of amyloid-related diseases, such maturation confers distinctive biological properties of the aggregates, which may account for a range of diverse pathological subtypes. Here, we present a protocol for the preparation of α-synuclein amyloid fibrils differing in the level of their maturation. We utilize widely accessible biophysical techniques to characterize the structure and morphology and a simple thermal treatment procedure to test their thermodynamic stability. Their biological properties are probed by means of binding to native plasma membrane sheets originating from mammalian cell lines.

The immunogenicity risk of therapeutic protein aggregates has been extensively investigated over the past decades. While it is established that not all aggregates are equally immunogenic, the specific aggregate characteristics, which are most likely to induce an immune response, remain ambiguous. The aim of this study was to perform comprehensive in vitro and in vivo immunogenicity assessment of human insulin aggregates varying in size, structure and chemical modifications, while keeping other morphological characteristics constant. We found that flexible aggregates with highly altered secondary structure were most immunogenic in all setups, while compact aggregates with native-like structure were found to be immunogenic primarily in vivo. Moreover, sub-visible (1–100 µm) aggregates were found to be more immunogenic than sub-micron (0.1–1 µm) aggregates, while chemical modifications (deamidation, ethylation and covalent dimers) were not found to have any measurable impact on immunogenicity. The findings highlight the importance of utilizing aggregates varying in few characteristics for assessment of immunogenicity risk of specific morphological features and may provide a workflow for reliable particle analysis in biotherapeutics.

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of SignificanceLocalized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and...

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight loss up to 40% within minutes, and a faster nicotine release (26.1 ± 4.6% after 1 min of incubation) of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable nicotine dose (i.e., 7.9 ± 5.1 and 2.2 ± 0.3% nicotine was released from a lozenge and a sublingual tablet, respectively). Model-fitting of the release data indicated that the release mechanism of nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more efficient permeation of the nicotine released from the nanofibers [flux of 1.06 ± 0.22 nmol/(cm2·min)] compared to when dosing even a ten-fold concentrated nicotine solution [flux of 0.17 ± 0.14 nmol/(cm2·min)]. Moreover, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MS) imaging of ex vivo porcine buccal mucosa exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing, and biocompatible delivery system for nicotine, which can overcome the limitations of the currently marketed NRTs.

Protein (mis)folding, stability and aggregation are of interest in numerous fields, such as food sciences, biotechnology, and health sciences, and efforts are directed towards the elucidation of the underlying molecular mechanisms. Through an integrative approach, we show that a subtle balance between hydrogen bond formation and hydrophobic interactions defines protein self-assembly pathways. Hydrophobic co-solvents, such as monohydric alcohols, modulate these two forces through a combination of direct solvent-protein and solvent-mediated interactions, depending on the size of the alcohol. This affects the initial conformation of the model protein α-lactalbumin, which can be linked to variations of its fibrillation propensity, as well as the morphology of the final structures. These findings pave the way towards a better understanding of the forces governing protein self-assembly, allowing the development of strategies to suppress unwanted aggregation and control the growth of tuneable protein-based biomaterials.

Electrospun nanofibers have gained great interest in many fields of research from water and air filtration or food packaging to medical use as scaffolds for tissue engineering, drug delivery systems and wound bandages. Proteins such as soy protein isolate (SPI) are biodegradable and safe polymers that can be purified from renewable and sustainable sources, and are thus interesting as building blocks for sustainable and biocompatible nanofiber-based materials. Sufficient solvent evaporation and intermolecular entanglement are required for efficient nanofiber formation. Electrospinning of proteins is therefore often achieved using organic solvents, strong bases or surfactants, which limits the safety, sustainability and usability of protein-based nanofibers. In this work, green and biocompatible electrospun nanofibers with a high content of SPI (up to 75% (w/w)) were fabricated with polyethylene oxide (PEO) as a co-spinning polymer and with water being the only solvent, thus avoiding use of any organic solvent, strong base or surfactant. A thorough biophysical assessment based on microscopy and spectroscopies, and a rheological profiling of SPI suggested that disassembly of larger structures into smaller in the SPI suspension and increase of SPI solubility improved the electrospinnability of SPI. The content of SPI in the nanofibers affected the morphology as visualized by scanning electron microscopy, brittleness assessed by dynamic mechanical analysis, and aqueous stability of the nanofibers, which are key parameters for the future use of SPI-based nanofibers. The biocompatibility of the electrospun SPI/PEO nanofibers was demonstrated by exposure of human epithelial cell monolayers (TR146) to the nanofibers without loss of cell viability. We propose that the presented strategies can serve as a universal workflow for waterborne electrospinning of other proteins or protein isolates.

Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated beta-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

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The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (alpha-La). alpha-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize alpha-La-membrane mechanisms of interaction and alpha-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of alpha-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.

The presence of αSN fibrils indisputably associates with the development of synucleinopathies. However, while certain fibril morphologies have been linked to downstream pathological phenotypes, others appear less harmful, leading to the concept of fibril strains, originally described in relation to prion disease. Indeed, the presence of fibrils does not associate directly with neurotoxicity. Rather, it has been suggested that the toxic compounds are soluble amyloidogenic oligomers, potentially co-existing with fibrils. Here, combining synchrotron radiation circular dichroism, transmission electron microscopy and binding assays on native plasma membrane sheets, we reveal distinct biological and biophysical differences between initial and matured fibrils, transformed within the timespan of few days. Immature fibrils are reservoirs of membrane-binding species, which in response to even gentle experimental changes release into solution in a reversible manner. In contrast, mature fibrils, albeit macroscopically indistinguishable from their less mature counterparts, are structurally robust, shielding the solution from the membrane active soluble species. We thus show that particular biological activity resides transiently with the fibrillating sample, distinct for one, but not the other, spontaneously formed fibril polymorph. These results shed new light on the principles of fibril polymorphism with consequent impact on future design of assays and therapeutic development.

Background: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. Methods: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. Results: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. General significance: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins.

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications.

International audience; Proteins can misfold and form either amorphous or organized aggregates with different morphologies and features. Aggregates of amyloid nature are pathological hallmarks in so-called protein conformational diseases, including Alzheimer's and Parkinson's. Evidence prevails that the transient early phases of the reaction determine the aggregate morphology and toxicity. As a consequence, real-time monitoring of protein aggregation is of utmost importance. Here, we employed time-resolved neutron backscattering spectroscopy to follow center-of-mass self-diffusion and nano- to picosecond internal dynamics of lysozyme during aggregation into a specific β-sheet rich superstructure, called particulates, formed at the isoelectric point of the protein. Particulate formation is found to be a one-step process, and protein internal dynamics, to remain unchanged during the entire aggregation process. The time-resolved neutron backscattering spectroscopy approach developed here, in combination with standard kinetics assays, provides a unifying framework in which dynamics and conformational transitions can be related to the different aggregation pathways.

Skin wound especially burn injury is a major threat for public health. One of the pursuits in the current wound healing research is to identify new promising biological materials, which can not only promote tissue repair but also reduce scar formation. In this current study, the potentials of α-lactalbumin (ALA), a tryptophan-rich dietary protein acting as a precursor of neurotransmitter serotonin, to promote the burn wound healing and reduce the scar formation were investigated. The ALA was initially electrospun with polycaprolactone (PCL) to accomplish electrospun nanofibrous mats (ENMs), subsequently assessed for their physicochemical attributes and wound healing efficiency on a burn rat model, and then their healing mechanisms at cellular and molecular levels were explored. The results showed that ALA and PCL were physicochemically compatible in ENMs. The average diameter of various nanofibers was within 183-344 nm. Their wettability and mechanical properties could be readily modulated by adjusting the mass ratios of ALA and PCL from 1/9 to 1/2. The selected ENMs exhibited negligible cytotoxicity and satisfactory adhesion to fibroblasts and promoting the proliferation of the fibroblasts. As compared to pristine PCL based ENMs, the composite scaffolds could accelerate the wound healing process and exhibit effects comparable to a marketed wound dressing over 16 days. Moreover, the ALA/PCL based ENMs could increase the synthesis of type I collagen and decrease the expression of α-smooth muscle actin, conferring that the novel wound dressings could reduce the formation of scars. Collectively, this study demonstrates that the ALA is a promising biological material and could promote the regeneration of burn skins with reduced scar formation, when being loaded on ultrafine fibrous scaffolds, mimicking the structure of the natural extra cellular matrix.

The reliable preparation of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent molecular recognition of nucleic acids, along with their facile synthesis and breadth of available functionalizations, DNA nanotechnology is widely acknowledged as a prime route for the rational design of nanostructured materials. Yet, the preparation of crystalline DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of simple amphiphilic DNA motifs, dubbed “C-stars”, as a versatile platform for the design of programmable DNA crystals. In contrast to all-DNA materials, in which structure depends on the precise molecular details of individual building blocks, the self-assembly of C-stars is controlled uniquely by their topology and symmetry. Exploiting this robust self-assembly principle, we design a range of topologically identical, but structurally and chemically distinct C-stars that following a one-pot reaction self-assemble into highly porous, functional, crystalline frameworks. Simple design variations allow us to fine-tune the lattice parameter and thus control the partitioning of macromolecules within the frameworks, embed responsive motifs that can induce isothermal disassembly, and include chemical moieties to capture target proteins specifically and reversibly.

Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable beta-structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by beta-structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, we offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues. (C) 2020 Elsevier Inc. All rights reserved.

Protein-based drug carriers are an interesting alternative to traditional polymeric drug delivery systems due to their intrinsic biocompatibility and biodegradability. Electrospinning of neat proteins holds advantages over electrospinning of protein mixtures, e.g., whey isolates, such as better control of the physicochemical and biological function of the resulting nanofiber-based system. In this study, we explore electrospinning of the isolated milk protein α-lactalbumin (ALA), which is a whey protein with important nutritional and pharmacological properties. Via waterborne electrospinning of ALA with a minimum amount of poly(ethylene oxide) (PEO) as a cospininng polymer, nanofibers of high protein content were successfully produced (up to 84% (w/w)). We demonstrate the ability to produce ALA-based nanofibers with a high degree of tunability in terms of size, stability in water, and mechanical properties. The nanofibers displayed excellent biocompatibility in vitro as the viability of cultured TR146 human buccal epithelium and NIH 3T3 murine fibroblast cells was not influenced by exposure to ALA-based nanofibers. ALA-based nanofibers were loaded with up to 6% (w/w) ampicilin, and the nanofibers were capable of maintaining the activity of the antibiotic after electrospinning and cross-linking. Using such a property of the material, we demonstrate that ampicillin-loaded nanofibers successfully inhibit the growth of Gram-negative bacteria in vitro. Importantly, after treatment with ampicillin-loaded nanofibers, no bacterial regrowth was observed, which indicates that this treatment may clear eventual persisters to ampicillin. Finally, the structural properties of the native functional protein were maintained after release of ALA from the nanofibers. This promotes our platform, not only as a sustainable protein-based drug delivery system, but also as an innovative solid form of ALA for food and pharmaceutical applications.

Co-solutes, such as sugars, are used in in vitro protein aggregation experiments to mimic crowding and, in general, complex environments. Sugars often increase the stability of the native protein structure by affecting inter- and intramolecular protein–protein interactions. This, in turn, modifies the protein self-assembly pathways. Using a combination of fluorescence spectroscopy, synchrotron radiation circular dichroism and transmission electron microscopy, we study the kinetics of formation and structural properties of human insulin fibrils in the presence of sucrose. The presence of sucrose results in a delay of the onset of fibrillation. Moreover, it leads to a dramatic change in both the morphology and overall amount of fibrils. Our results emphasize that the detailed composition of protein surroundings likely influences not only the fibrillation kinetics but also the balance between different species, potentially determining fibril strains with different biological activities. This aspect is crucial in the etiology of pathologies associated with amyloidosis.

Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer’s as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aβ peptide deposits characteristic for Alzheimer’s disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target.

The reliable preparation of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent molecular recognition of nucleic acids, along with their facile synthesis and breadth of available functionalizations, DNA Nanotechnology is widely acknowledged as a prime route for the rational design of nanostructured materials. Yet, the preparation of crystalline DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of simple amphiphilic DNA motifs, dubbed C-stars, as a versatile platform for the design of programmable DNA crystals. In contrast to all-DNA materials, in which structure depends on the precise molecular details of individual building blocks, the self-assembly of C-stars is controlled uniquely by their topology and symmetry. Exploiting this robust self-assembly principle we design a range of topologically identical, but structurally and chemically distinct C-stars that following a one-pot reaction self- assemble into highly porous, functional, crystalline frameworks. Simple design variations allow us to fine-tune the lattice parameter and thus control the partitioning of macromolecules within the frameworks, embed responsive mo- tifs that can induce isothermal disassembly, and include chemical moieties to capture target proteins specifically and reversibly.

Protein self-assembly into amyloid fibrils or highly hierarchical superstructures is closely linked to neurodegenerative pathologies as Alzheimer's and Parkinson's diseases. Moreover, protein assemblies also emerged as building blocks for bioinspired nanostructured materials. In both the above mentioned fields, the main challenge is to control the growth and properties of the final protein structure. This relies on a more fundamental understanding of how interactions between proteins can determine structures and functions of biomolecular aggregates. Here, we identify a striking effect of the hydration of the single human insulin molecule and solvent properties in controlling hydrophobicity/hydrophilicity, structures, and morphologies of a superstructure named spherulite, observed in connection to Alzheimer's disease. Depending on the presence of ethanol, such structures can incorporate fluorescent molecules with different physicochemical features and span a range of mechanical properties and morphologies. A theoretical model providing a thorough comprehension of the experimental data is developed, highlighting a direct connection between the intimate physical protein-protein interactions, the growth, and the properties of the self-assembled superstructures. Our findings indicate structural variability as a general property for amyloid-like aggregates and not limited to fibrils. This knowledge is pivotal not only for developing effective strategies against pathological amyloids but also for providing a platform to design highly tunable biomaterials, alternative to elongated protein fibrils.

The subtle interplay between long range electrostatic forces, hydrophobic interactions and short range protein-protein interactions regulates the onset/evolution of protein aggregation processes as well as the stability of protein supramolecular structures. Using a combination of FTIR spectroscopy, light scattering and advanced imaging, we present evidence on the main role of electrostatic forces in the formation and stability of amyloid-like fibrils formed from concanavalin A (ConA), a protein showing structural homology with the human serum amyloid protein. At high protein concentration, where protein-protein interactions cannot be neglected, we highlight a thermal-induced aggregation pathway in which amyloid-like aggregates are readily formed. When dissolved in solutions at different pHs, these aggregates show either a reduced β-sheet content keeping the same morphology (3 < pH < 10) or they are promptly dissolved leaving in solution non-native oligomers (pH > 10). The latter result can be ascribed to the change of the charge state for the ConA amino acid side chain with high pKa values. Our results support the idea of fibrils as a reservoir of oligomeric species that can be released if changes or discontinuities in the aggregate microenvironment occur.

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1-40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1-40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1-40) aggregation processes.

Understanding the mechanism behind protein aggregation is a challenging task that requires a combined use of both experimental and computational approaches. In this work, we present a 2D model for the formation of amyloid-like fibrils. The model allows one to explicitly consider the structural change of the native state of a protein into the aggregation-prone state together with the overall charge of the molecules. By means of Metropolis Monte Carlo and dynamic Monte Carlo, we simulate both the equilibrium and kinetic behavior of an ensemble of model proteins. Our results show the effect of the charge and protein concentration on both the conformational equilibrium and the kinetics of the aggregation process. Specifically, the model is capable of capturing the self-catalytic conversion of native proteins into aggregation-prone ones in the presence of preexisting aggregates, naturally reproducing some peculiar scaling laws observed for a class of amyloidogenic systems.

Amyloid fibrils are characterized by a structural arrangement of cross β-sheet as a common motif. However they can also experience a more complicated packing into a variety of 3D supramolecular structures (polymorphism). Confinement and flow rate play a crucial role in protein aggregation in living systems, but controlling such parameters during in vitro experiments still remains an unsolved problem. Here we present evidence of the effect of flow rate on the aggregation process in a confined environment using microfluidics. Specifically, we show that a gradual transition from spherical aggregates, that is, spherulites, to thick fiber-like structures takes place as a result of increasing the flow rate. Such results have implications both for a basic understanding of the mechanism behind aggregation phenomena and in the development of novel biomaterials.

Extra virgin olive oil (EVOO) is recognized as one of the healthiest foods for its high content of antioxidants, which forestall and slow down radical formation. Free radical-initiated oxidation is considered one of the main causes of rancidity in fats and oils. As a consequence, reliable protocols for the investigation of oil oxidation based on selective, noninvasive, and fast methods are highly desirable. Here we report an experimental approach based on UV-Vis absorbance, steady-state fluorescence, and electron paramagnetic resonance (EPR) spectroscopy for studying oxidation processes induced by temperature for a period up to 35 d on Sicilian EVOO samples. We followed the decrease in β-carotene content during incubation time and observed changes in polyphenols and tocopherols during the oxidation processes, focusing on the time scale of those changes. Using EPR spectroscopy, the free radical formation in different oil samples is reported, providing a fingerprint for both the antioxidant content and temporal features of the oxidation process at its early stage. Practical Application: We monitor β-carotene and chlorophyll in an auto-oxidation process. A protocol based on spectroscopic measurements is presented and can be used for the quality control process of commercial olive oil.

DNA nanostructures with programmable shape and interactions can be used as building blocks for the self-assembly of crystalline materials with prescribed nanoscale features, holding a vast technological potential. Structural rigidity and bond directionality have been recognised as key design features for DNA motifs to sustain long-range order in 3D, but the practical challenges associated with prescribing building-block geometry with sufficient accuracy have limited the variety of available designs. We have recently introduced a novel platform for the one-pot preparation of crystalline DNA frameworks supported by a combination of Watson-Crick base pairing and hydrophobic forces (Brady et al 2017 Nano Lett. 17 3276-81). Here we use small angle x-ray scattering and coarse-grained molecular simulations to demonstrate that, as opposed to available all-DNA approaches, amphiphilic motifs do not rely on structural rigidity to support long-range order. Instead, the flexibility of amphiphilic DNA building-blocks is a crucial feature for successful crystallisation.