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Abstract Phase-separating peptides (PSPs) self-assembling into coacervate microdroplets (CMs) are a promising class of intracellular delivery vehicles that can release macromolecular modalities deployed in a wide range of therapeutic treatments. However, the molecular grammar governing intracellular uptake and release kinetics of CMs remains elusive. Here, we systematically manipulate the sequence of PSPs to unravel the relationships between their molecular structure, the physical properties of the resulting CMs, and their delivery efficacy. We show that a few amino acid alterations are sufficient to modulate the viscoelastic properties of CMs towards either a gel-like or a liquid-like state as well as their binding interaction with cellular membranes, collectively enabling to tune the kinetics of intracellular cargo release. We also demonstrate that the optimized PSPs CMs display excellent transfection efficiency in hard-to-transfect cells such as primary fibroblasts and immune cells. Our findings provide molecular guidelines to precisely program the material properties of PSP CMs and achieve tunable cellular uptake and release kinetics depending on the cargo modality, with broad implications for therapeutic applications such as protein, gene, and immune cell therapies.

Abstract Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a “memory” of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

Abstract Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non‐zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed‐curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed‐curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.

Increased temperature and high pressure are applied to β‐lactoglobulin fibrils in the autoclave, resulting in the acquisition of a composite material comprised of partially disassembled amyloid fibrils and carbon dots. Confirmation of the preservation of the β‐sheet motif attributed to amyloids in the hydrothermally treated fibrils is obtained through wide‐angle X‐ray scattering and ThT assay. Z‐scan analysis reveals a two‐photon absorption (2PA) enhancement in the low‐lying transition band (La) of tyrosine, while quantum chemical calculations demonstrate a correlation between the yield of 2PA and the interspace distance between aromatic residues. Overall, the intrinsic optical properties of amyloid fibrils treated in a subcritical water environment are found to be linked with the π‐conjugation of tyrosine units and their through‐space coupling. The resulting composite material is employed as a coating for a commercial ultraviolet light‐emitting diode lamp, showcasing the potential utility of sustainable biomaterials with improved optical properties for photonics applications.

Abstract Food protein amyloid fibrils have superior technological, nutritional, sensorial, and physical properties compared to native monomers, but there is as yet insufficient understanding of their digestive fate and safety for wide consumption. By combining SDS-PAGE, ELISA, fluorescence, AFM, MALDI-MS, CD, microfluidics, and SAXS techniques for the characterization of β-lactoglobulin and lysozyme amyloid fibrils subjected to in-vitro gastrointestinal digestion, here we show that either no noticeable conformational differences exist between amyloid aggregates and their monomer counterparts after the gastrointestinal digestion process (as in β-lactoglobulin), or that amyloid fibrils are digested significantly better than monomers (as in lysozyme). Moreover, in-vitro exposure of human cell lines and in-vivo studies with C. elegans and mouse models, indicate that the digested fibrils present no observable cytotoxicity, physiological abnormalities in health-span, nor accumulation of fibril-induced plaques in brain nor other organs. These extensive in-vitro and in-vivo studies together suggest that the digested food amyloids are at least equally as safe as those obtained from the digestion of corresponding native monomers, pointing to food amyloid fibrils as potential ingredients for human nutrition.

Abstract RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

Liquid-liquid phase separation is vital in the formation of many biomolecular condensates, including cellular membraneless organelles. When semiflexible macromolecules are involved, the process switches from mostly enthalpy- to dominantly entropy-driven, changing the intrinsic nature of the interfaces and bringing new physical traits. Here, we present a concept to address the complexity of entropic interfaces where laser trapping and microscopy are combined to capture the behavior of probe colloidal particles onto liquid crystalline droplet surfaces. Hertz-Frank-Oseen contact theory is extended to calculate the elasticity of the droplet based on particle trajectories. We show that this method is sensitive enough to resolve the anisotropic elasticity originating from the collective molecular orientation of constitutive semiflexible mesogens, introducing a possible tool to assess biomechanics of cellular and molecular condensates.

Abstract Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments.

Liquid-liquid phase separation offer a strategy for targeting biomaterial scaffolds, but controlling assembly to form functional materials is challenging. Here, the authors transform native proteins into amyloid fibres for assembly, and use the materials as a gastric ulcer protective agent.

Nanoconfined water differs significantly from bulk water and challenges our common understanding of liquid water in both its most fundamental features, as well as in many applied aspects which stem out from its peculiar behavior. This brief perspective pinpoints both challenges associated with the study of water under soft nanoconfinement as well as some opportunities which arise from it, and which would not be at reach with standard bulk water. A special focus is given to the strong nanoconfinement (∼1–10 nm) offered by inverse lipidic mesophases, viewed as a natural soft nanoconfinement environment for water.

In heterogeneous colloidal systems, composition, shape, structure and physical properties result from the trade-off between thermodynamic and kinetic effects during nucleation and growth. Here, the authors demonstrate that kinetic and thermodynamic effects can be disentangled by careful selection of a colloidal systems and controlling phase separation in microfluidic devices

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

Here the authors report the formation of toxic clumps of protein, similar to amyloid assemblies found in Alzheimer’s disease and suggest their possible role for some of the neurological symptoms of long-COVID.

Tactoids, consisting of micro-confined liquid crystalline colloids with self-selected shape, bear both fundamental and technological significance. The authors show that the shape relaxation of tactoids follows an exponential decay and develop a model to predict this out-of-the-equilibrium process.

Abstract Amyloid fibrils have generated steadily increasing traction in the development of natural and artificial materials. However, it remains a challenge to construct bulk amyloid films directly from amyloid fibrils due to their intrinsic brittleness. Here, a facile and general methodology to fabricate macroscopic and tunable amyloid films via fast electrostatic self‐assembly of amyloid fibrils at the air–water interface is introduced. Benefiting from the excellent templating properties of amyloid fibrils for nanoparticles (such as conductive carbon nanotubes or magnetic Fe3O4 nanoparticles), multifunctional amyloid films with tunable properties are constructed. As proof‐of‐concept demonstrations, a magnetically oriented soft robotic swimmer with well‐confined movement trajectory is prepared. In addition, a smart magnetic sensor with high sensitivity to external magnetic fields is fabricated via the combination of the conductive and magnetic amyloid films. This strategy provides a convenient, efficient, and controllable approach for the preparation of amyloid‐based multifunctional films and related smart devices.

The ongoing COVID-19 pandemic has revealed that developing effective therapeutics against viruses might be outpaced by emerging variants, waning immunity, vaccine skepticism/hesitancy, lack of resources, and the time needed to develop virus-specific therapeutics, emphasizing the importance of non-pharmaceutical interventions as the first line of defense against virus outbreaks and pandemics. However, fighting the spread of airborne viruses has proven extremely challenging, much more if this needs to be achieved on a global scale and in an environmentally-friendly manner. Here, we introduce an aerosol filter media made of granular material based on whey protein nanofibrils and iron oxyhydroxides nanoparticles. The material is environmentally-friendly, biodegradable, and composed mainly of a dairy industry byproduct. It features filtration efficiencies between 95.91% and 99.99% for both enveloped and non-enveloped viruses, including SARS-CoV-2, the influenza A virus strain H1N1, enterovirus 71, bacteriophage Φ6, and bacteriophage MS2. While the filtration efficiencies were relatively high, they came at the cost of high pressure drop (≈0.03 bar). We believe that the methods and results presented here will contribute to advancing our understanding of granular-based aerosol filters, hopefully helping the design of highly-efficient granular media with low-pressure drops.

Peptides possess high chemical diversity at the amino acid sequence level, which further translates into versatile functions. Peptides with self-assembling properties can be processed into diverse formats giving rise to bio-based materials. Peptide-based spun fibers are an interesting format due to high surface-area and versatility, though the field is still in its infancy due to the challenges in applying the synthetic polymer spinning processes to protein fibers to peptides. In this work we show the use of solution blow-spinning to produce peptide fibers. Peptide fiber formation was assisted by the polymer poly (vinyl pyrrolidone) (PVP) in two solvent conditions. Peptide miscibility and further self-assembling propensity in the solvents played a major role in fiber formation. When employing acetic acid as solvent, peptide fibers (0.5 μm) are formed around PVP fibers (0.75 μm), whereas in isopropanol only one type of fibers are formed, consisting of mixed peptide and PVP (1 μm). This report highlights solvent modulation as a mean to obtain different peptide sub-microfibers via a single injection nozzle in solution blow spinning. We anticipate this strategy to be applied to other small peptides with self-assembly propensity to obtain multi-functional proteinaceous fibers.

Summary: Reactive amyloid oligomers are responsible for cytotoxicity in amyloid pathologies and because of their unstable nature characterizing their behavior is a challenge. The physics governing the self-assembly of proteins in crowded conditions is extremely complex and its comprehension, despite its paramount relevance to understanding molecular mechanisms inside cells and optimizing pharmaceutical processes, remains inconclusive. Here, we focus on the amyloid oligomerization process in self-crowded lysozyme aqueous solutions in acidic conditions. We reveal that the amyloid oligomers form at high protein concentration and low pH. Through multi-length scale spectroscopic investigations, we find that amyloid oligomers can further interconnect with each other by weak and non-specific interactions forming an extended network that leads to the percolation of the whole system. Our multi-length scale structural analysis follows the thermal history of amyloid oligomers from different perspectives and highlights the impact of hierarchical self-assembly of biological macromolecules on functional properties.

Water scarcity is a rapidly spreading global challenge but water purification technologies are often not sustainable. Here, the authors review the research on water purification technologies based on protein nanofibrils as a green and affordable solution to alleviate a water crisis.

Tactoids are liquid crystal droplets with nearly vanishing interfacial tension. Almohammadi et al. show using a microfluidic focusing device how to manipulate them gently enough to facilitate the study of amyloid liquid crystal phase transitions subject to non-equilibirum forcing and shape changes.

Relaxation of liquid crystals is crucial in a broad area, e.g. biomimicry and stimuli-responsive material design. Here Khadem et al explore distinct relaxations, reveal the corresponding physics and propose a novel method for estimating viscoelastic properties without applying external fields.

Abstract Amyloid functional materials from amyloid fibril building blocks, produced in vitro from amyloidogenic natural proteins or synthetic peptides, show diverse functionalities ranging from environmental science and biomedicine, to nanotechnology and biomaterials. However, sustainable and affordable sources of amyloidogenic proteins remain the bottleneck for large‐scale applications, and to date, interest remains essentially limited to fundamental studies. Plant‐derived proteins would be an ideal source due to their natural abundance and low environmental impact. Hereby oat globulin, the primary protein of oat plant (Avena sativa), is utilized to yield high‐quality amyloid fibrils and functional materials based thereof. These fibrils show a rich multistranded ribbon‐like polymorphism and a fibrillization process with both irreversible and reversible pathways. The authors furthermore fabricate oat‐amyloid aerogels, films, and membranes for possible use in water purification, sensors, and patterned electrodes. The sustainability footprint of oat‐amyloids against other protein sources is demonstrated, anticipating an environmentally‐efficient platform for advanced materials and technologies.

We study the conditions necessary for the onset of liquid-liquid crystalline phase separation (LLCPS) in a system of dynamically evolving amyloid fibrils whose kinetics is controlled by nucleation, growth and fragmentation. In the case of thin, rigid rods, the volume fraction does not influence the emergence of LLCPS, which is always possible. Beyond the constraints imposed by rigid rods, for semiflexible fibrils, LLCPS stems out instead from a trade-off between the contour and persistence length. Fibrils fragmentation affects LLCPS in a similar manner by delaying the concentration needed for LLCPS onset.

Water purification from per- and poly-fluoroalkyl substances (PFASs), as a group of persistent and mobile fluoro-organic contaminants, is receiving increasing attention worldwide due to the ubiquitous presence of these highly toxic compounds. To reduce the risk of exposure of human life to PFASs and their dispersion in the environment, various techniques, primarily based on membrane technologies, have been rapidly developed. Here we critically review and analyze the current state-of-the-art of membrane-based techniques for PFASs removal, including direct membrane filtrations, adsorption-based membranes, and hybrid membrane processes. Membranes performance, treatment efficiencies, characteristic parameters and mechanisms for PFASs removal are discussed in detail. We highlight and discuss advantages and limitations, as well as challenges and prospects of individual membrane-based PFASs treatments, pointing towards the practical and sustainable application of these technologies.

Abstract Nanomechanical properties of amyloid fibrils and nanocrystals depend on their secondary and quaternary structure, and the geometry of intermolecular hydrogen bonds. Advanced imaging methods based on atomic force microscopy (AFM) have unravelled the morphological and mechanical heterogeneity of amyloids, however a full understanding has been hampered by the limited resolution of conventional spectroscopic methods. Here, it is shown that single molecule nanomechanical mapping and infrared nanospectroscopy (AFM‐IR) in combination with atomistic modelling enable unravelling at the single aggregate scale of the morphological, nanomechanical, chemical, and structural transition from amyloid fibrils to amyloid microcrystals in the hexapeptides, ILQINS, IFQINS, and TFQINS. Different morphologies have different Young's moduli, within 2–6 GPa, with amyloid fibrils exhibiting lower Young's moduli compared to amyloid microcrystals. The origins of this stiffening are unravelled and related to the increased content of intermolecular β‐sheet and the increased lengthscale of cooperativity following the transition from twisted fibril to flat nanocrystal. Increased stiffness in Young's moduli is correlated with increased density of intermolecular hydrogen bonding and parallel β‐sheet structure, which energetically stabilize crystals over the other polymorphs. These results offer additional evidence for the position of amyloid crystals in the minimum of the protein folding and aggregation landscape.

Abstract The gut–brain axis has attracted increasing attention in recent years, fueled by accumulating symptomatic, physiological, and pathological findings. In this study, the aggregation and toxicity of amyloid beta (Aβ), the pathogenic peptide associated with Alzheimer's disease (AD), seeded by FapC amyloid fragments (FapCS) of Pseudomonas aeruginosa that colonizes the gut microbiome through infections are examined. FapCS display favorable binding with Aβ and a catalytic capacity in seeding the peptide amyloidosis. Upon seeding, twisted Aβ fibrils assume a much‐shortened periodicity approximating that of FapC fibrils, accompanied by a 37% sharp rise in the fibrillar diameter, compared with the control. The robust seeding capacity for Aβ by FapCS and the biofilm fragments derived from P. aeruginosa entail abnormal behavior pathology and immunohistology, as well as impaired cognitive function of zebrafish. Together, the data offer the first concrete evidence of structural integration and inheritance in peptide cross‐seeding, a crucial knowledge gap in understanding the pathological correlations between different amyloid diseases. The catalytic role of infectious bacteria in promoting Aβ amyloidosis may be exploited as a potential therapeutic target, while the altered mesoscopic signatures of Aβ fibrils may serve as a prototype for molecular assembly and a biomarker for screening bacterial infections in AD.

In meso crystallization of membrane proteins is limited to proteins with small extracellular domains (ECDs). Here, authors combine monoacylglycerols and phospholipids to design stable ultra-swollen bicontinuous cubic phases that allow in meso crystallization of proteins with large ECDs.

Aggregation of amyloidogenic peptides into fibrils and crystals has incidence in several amyloid-related diseases. Here, the authors investigate the origins of the fibril-to-crystal conversion in amyloidogenic hexapeptides pointing to the amyloid crystals as the ground state in the protein folding energy landscape.

Silk fibres currently used in biotechnology are chemically reconstituted silk fibroins (RSF), which are more stable than native silk fibroin (NSF) but possess different biophysical properties. Here, the authors use microfluidic droplets to encapsulate and store NSF, preserving their native structure.

The isotropic and nematic (I + N) coexistence for rod-like colloids is a signature of the first-order thermodynamics nature of this phase transition. However, in the case of amyloid fibrils, the biphasic region is too small to be experimentally detected, due to their extremely high aspect ratio. Herein, we study the thermophoretic behaviour of fluorescently labelled β-lactoglobulin amyloid fibrils by inducing a temperature gradient across a microfluidic channel. We discover that fibrils accumulate towards the hot side of the channel at the temperature range studied, thus presenting a negative Soret coefficient. By exploiting this thermophoretic behaviour, we show that it becomes possible to induce a continuous I-N transition with the I and N phases at the extremities of the channel, starting from an initially single N phase, by generating an appropriate concentration gradient along the width of the microchannel. Accordingly, we introduce a new methodology to control liquid crystal phase transitions in anisotropic colloidal suspensions. Because the induced order-order transitions are achieved under stationary conditions, this may have important implications in both applied colloidal science, such as in separation and fractionation of colloids, as well as in fundamental soft condensed matter, by widening the accessibility of target regions in the phase diagrams.

Abstract Biological gels generally require polymeric chains that produce long‐lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad‐hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross‐linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid‐state NMR, circular dichroism, infrared spectroscopy, and X‐ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right‐handed 310‐helices stabilized in solution by π–π stacking. During gelation, the 310‐helices undergo conformational transition into antiparallel β‐sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross β‐sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 310‐helices as transient building blocks for gelation via a 310‐to‐β‐sheet conformational transition.

Gold nanoparticle monolayers provide convenient templates to study charge transport in organic molecules beyond single junction techniques. Conductance is reported to increase by several orders of magnitude following immersion of alkanethiol-stabilized gold nanoparticle monolayers in a solution containing conjugated thiol-functionalized molecules. Typically, this observation is attributed to molecular exchange. Less attention has been paid to the role of the solvent alone. Here, we report on an increase in conductance of dodecanethiol-stabilized gold nanoparticle monolayers on Si/SiO2 by an average factor of 36 and 22 after immersion in pure ethanol (EtOH) and tetrahydrofuran (THF), respectively. Analysis by scanning electron microscopy (SEM) and small-angle X-ray scattering (SAXS) reveals a solvent-induced decrease in lattice constant of close-packed monolayers. We compare the conductance of the monolayer after molecular exchange with two different oligophenylenes to shed light on the respective contribution of the solvent-induced structural change and the molecular exchange itself on the conductance increase.

Transparent and electrically conducting flexible films are in high demand but production can be both time-consuming and expensive. Here, the authors report a method for assembling modified graphene flakes in controlled distributions within polymeric matrices by use of magnetic fields.

Starch, as the major nutritional component of our staple crops and a feedstock for industry, is a vital plant product. It is composed of glucose polymers that form massive semi-crystalline granules. Its precise structure and composition determine its functionality and thus applications; however, there is no versatile model system allowing the relationships between the biosynthetic apparatus, glucan structure and properties to be explored. Here, we expressed the core Arabidopsis starch-biosynthesis pathway in Saccharomyces cerevisiae purged of its endogenous glycogen-metabolic enzymes. Systematic variation of the set of biosynthetic enzymes illustrated how each affects glucan structure and solubility. Expression of the complete set resulted in dense, insoluble granules with a starch-like semi-crystalline organization, demonstrating that this system indeed simulates starch biosynthesis. Thus, the yeast system has the potential to accelerate starch research and help create a holistic understanding of starch granule biosynthesis, providing a basis for the targeted biotechnological improvement of crops.

In a current procedure for periodontal tissue regeneration, enamel matrix derivative (EMD), which is the active component, is mixed with a propylene glycol alginate (PGA) gel carrier and applied directly to the periodontal defect. Exposure of EMD to physiological conditions then causes it to precipitate. However, environmental changes during manufacture and storage may result in modifications to the conformation of the EMD proteins, and eventually premature phase separation of the gel and a loss in therapeutic effectiveness. The present work relates to efforts to improve the stability of EMD-based formulations such as Emdogain™ through the incorporation of arginine, a well-known protein stabilizer, but one that to our knowledge has not so far been considered for this purpose. Representative EMD-buffer solutions with and without arginine were analyzed by 3D-dynamic light scattering, UV-Vis spectroscopy, transmission electron microscopy and Fourier transform infrared spectroscopy at different acidic pH and temperatures, T, in order to simulate the effect of pH variations and thermal stress during manufacture and storage. The results provided evidence that arginine may indeed stabilize EMD against irreversible aggregation with respect to variations in pH and T under these conditions. Moreover, stopped-flow transmittance measurements indicated arginine addition not to suppress precipitation of EMD from either the buffers or the PGA gel carrier when the pH was raised to 7, a fundamental requirement for dental applications.

The original version of this article contained an error in Fig. 5c. The label for the back series of columns was incorrectly given as ‘1.5 mM pH 2’, rather than the correct ‘1.5 mM pH 7’. This has now been corrected in both the PDF and HTML versions of the article.