Modulation of α-synuclein in vitro aggregation kinetics by its alternative splice isoforms.

The misfolding and aggregation of α-synuclein is linked to a family of neurodegenerative disorders known as synucleinopathies, the most prominent of which is Parkinson's disease (PD). Understanding the aggregation process of α-synuclein from a mechanistic point of view is thus of key importance. SNCA, the gene encoding α-synuclein, comprises six exons and produces various isoforms through alternative splicing. The most abundant isoform is expressed as a 140-amino acid protein (αSyn-140), while three other isoforms, αSyn-126, αSyn-112, and αSyn-98, are generated by skipping exon 3, exon 5, or both exons, respectively. In this study, we performed a detailed biophysical characterization of the aggregation of these four isoforms. We found that αSyn-112 and αSyn-98 exhibit accelerated aggregation kinetics compared to αSyn-140 and form distinct aggregate morphologies, as observed by transmission electron microscopy. Moreover, we observed that the presence of relatively small amounts of αSyn-112 accelerates the aggregation of αSyn-140, significantly reducing the aggregation half-time. These results indicate a potential role of alternative splicing in the pathological aggregation of α-synuclein and provide insights into how this process could be associated with the development of synucleinopathies.

Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH.

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson's disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Adsorption free energy predicts amyloid protein nucleation rates.

Primary nucleation is the fundamental event that initiates the conversion of proteins from their normal physiological forms into pathological amyloid aggregates associated with the onset and development of disorders including systemic amyloidosis, as well as the neurodegenerative conditions Alzheimer's and Parkinson's diseases. It has become apparent that the presence of surfaces can dramatically modulate nucleation. However, the underlying physicochemical parameters governing this process have been challenging to elucidate, with interfaces in some cases having been found to accelerate aggregation, while in others they can inhibit the kinetics of this process. Here we show through kinetic analysis that for three different fibril-forming proteins, interfaces affect the aggregation reaction mainly through modulating the primary nucleation step. Moreover, we show through direct measurements of the Gibbs free energy of adsorption, combined with theory and coarse-grained computer simulations, that overall nucleation rates are suppressed at high and at low surface interaction strengths but significantly enhanced at intermediate strengths, and we verify these regimes experimentally. Taken together, these results provide a quantitative description of the fundamental process which triggers amyloid formation and shed light on the key factors that control this process.

Kinetic Analysis Reveals the Role of Secondary Nucleation in Regenerated Silk Fibroin Self-Assembly.

Silk proteins obtained from the Bombyx mori silkworm have been extensively studied due to their remarkable mechanical properties. One of the major structural components of this complex material is silk fibroin, which can be isolated and processed further in vitro to form artificial functional materials. Due to the excellent biocompatibility and rich self-assembly behavior, there has been sustained interest in such materials formed through the assembly of regenerated silk fibroin feedstocks. The molecular mechanisms by which the soluble regenerated fibroin molecules self-assemble into protein nanofibrils remain, however, largely unknown. Here, we use the framework of chemical kinetics to connect macroscopic measurements of regenerated silk fibroin self-assembly to the underlying microscopic mechanisms. Our results reveal that the aggregation of regenerated silk fibroin is dominated by a nonclassical secondary nucleation processes, where the formation of new fibrils is catalyzed by the existing aggregates in an autocatalytic manner. Such secondary nucleation pathways were originally discovered in the context of polymerization of disease-associated proteins, but the present results demonstrate that this pathway can also occur in functional assembly. Furthermore, our results show that shear flow induces the formation of nuclei, which subsequently accelerate the process of aggregation through an autocatalytic amplification driven by the secondary nucleation pathway. Taken together, these results allow us to identify the parameters governing the kinetics of regenerated silk fibroin self-assembly and expand our current understanding of the spinning of bioinspired protein-based fibers, which have a wide range of applications in materials science.

Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.

Biomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area.

Label-Free Protein Analysis Using Liquid Chromatography with Gravimetric Detection.

The detection and analysis of proteins in a label-free manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 µg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.

pH-Responsive Capsules with a Fibril Scaffold Shell Assembled from an Amyloidogenic Peptide.

Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.

Correction: Correction: Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions.

Correction for 'Correction: Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions' by Zenon Toprakcioglu et al., Soft Matter, 2020, 16, 3586-3586, DOI: .

Biocompatible Hybrid Organic/Inorganic Microhydrogels Promote Bacterial Adherence and Eradication in Vitro and in Vivo.

Self-assembling peptides and proteins have the potential to serve as multifunctional building blocks for the generation of versatile materials for a wide range of biomedical applications. In particular, supramolecular hydrogels comprised of self-assembled protein nanofibrils, have been used in contexts ranging from tissue engineering to drug delivery. Due to the rapid emergence of multidrug resistant bacteria, development of biomaterials with intrinsic antimicrobial properties has been continuously increasing. Here, we describe hybrid organic/inorganic nanofibrillar silk microgels decorated with silver nanoparticles that display potent antimicrobial activity in vitro and in vivo and are able to adhere bacterial cells to their surfaces while subsequently eradicating them, through a two-step mechanism of action. Importantly, in contrast to treatments involving conventional silver, these silk-silver microgels are nonhemolytic and noncytotoxic toward mammalian cell lines. Finally, we show that these hybrid microgels display substantial efficacy as topical antimicrobial agents in a murine model of surgical site infections.

Modulating the Mechanical Performance of Macroscale Fibers through Shear-Induced Alignment and Assembly of Protein Nanofibrils.

Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.

A Microfluidic Co-Flow Route for Human Serum Albumin-Drug-Nanoparticle Assembly.

Nanoparticles are widely studied as carrier vehicles in biological systems because their size readily allows access through cellular membranes. Moreover, they have the potential to carry cargo molecules and as such, these factors make them especially attractive for intravenous drug delivery purposes. Interest in protein-based nanoparticles has recently gained attraction due to particle biocompatibility and lack of toxicity. However, the production of homogeneous protein nanoparticles with high encapsulation efficiencies, without the need for additional cross-linking or further engineering of the molecule, remains challenging. Herein, we present a microfluidic 3D co-flow device to generate human serum albumin/celastrol nanoparticles by co-flowing an aqueous protein solution with celastrol in ethanol. This microscale co-flow method resulted in the formation of nanoparticles with a homogeneous size distribution and an average size, which could be tuned from ≈100 nm to 1 µm by modulating the flow rates used. We show that the high stability of the particles stems from the covalent cross-linking of the naturally present cysteine residues within the particles formed during the assembly step. By choosing optimal flow rates during synthesis an encapsulation efficiency of 75±24 % was achieved. Finally, we show that this approach achieves significantly enhanced solubility of celastrol in the aqueous phase and, crucially, reduced cellular toxicity.

Lipid-Stabilized Double Emulsions Generated in Planar Microfluidic Devices.

Microemulsions have found a wide range of applications exploiting their chemical and physical properties. Development of microfluidic-based approaches has allowed for the controlled production of highly monodispersed emulsions, including the formation of multiple and hierarchical emulsions. Conventional poly(dimethylsiloxane)-based microfluidic systems require tight spatial control over the surface chemistry when used for double emulsion generation, which can be challenging to achieve on the micrometer scale. Here, we present a two-dimensional device design, which can selectively be surface-treated in a straightforward manner and allows for the formation of uniform water/oil/water double emulsions by combining two distinct hydrophilic and hydrophobic surface properties. These surfaces are sufficiently separated in space to allow for imparting their functionalization without the requirement for lithographic approaches or complex flow control. We demonstrate that a mismatch between the wettability requirements of the continuous phase and the channel wall inherent in this approach can be tolerated over several hundreds of micrometers, opening up the possibility to use simple pressure-driven flows to achieve surface functionalization. The design architecture exhibits robust efficiency in emulsion generation while retaining simple device fabrication. We finally demonstrate the potential of this approach by generating water in oil in water emulsions with lipid molecules acting as surfactants.

Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions.

Controlling the surface area, pore size and pore volume of microcapsules is crucial for modulating their activity in applications including catalytic reactions, delivery strategies or even cell culture assays, yet remains challenging to achieve using conventional bulk techniques. Here we describe a microfluidics-based approach for the formation of monodisperse silica-coated micron-scale porous capsules of controllable sizes. Our method involves the generation of gas-in water-in oil emulsions, and the subsequent rapid precipitation of silica which forms around the encapsulated gas bubbles resulting in hollow silica capsules with tunable pore sizes. We demonstrate that by varying the gas phase pressure, we can control both the diameter of the bubbles formed and the number of internal bubbles enclosed within the silica microcapsule. Moreover, we further demonstrate, using optical and electron microscopy, that these silica capsules remain stable under particle drying. Such a systematic manner of producing silica-coated microbubbles and porous microparticles thus represents an attractive class of biocompatible material for biomedical and pharmaceutical related applications.

Correction: Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions.

Correction for 'Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions' by Zenon Toprakcioglu et al., Soft Matter, 2020, DOI: 10.1039/c9sm02274k.

Programmable On-Chip Artificial Cell Producing Post-Translationally Modified Ubiquitinated Protein.

In nature, intracellular microcompartments have evolved to allow the simultaneous execution of tightly regulated complex processes within a controlled environment. This architecture serves as the blueprint for the construction of a wide array of artificial cells. However, such systems are inadequate in their ability to confine and sequentially control multiple central dogma activities (transcription, translation, and post-translational modifications) resulting in a limited production of complex biomolecules. Here, an artificial cell-on-a-chip comprising hierarchical compartments allowing the processing and transport of products from transcription, translation, and post-translational modifications through connecting channels is designed and fabricated. This platform generates a tightly controlled system, yielding directly a purified modified protein, with the potential to produce proteoform of choice. Using this platform, the full ubiquitinated form of the Parkinson's disease-associated α-synuclein is generated starting from DNA, in a single device. By bringing together all central dogma activities in a single controllable platform, this approach will open up new possibilities for the synthesis of complex targets, will allow to decipher diverse molecular mechanisms in health and disease and to engineer protein-based materials and pharmaceutical agents.

Fabrication and Characterization of Reconstituted Silk Microgels for the Storage and Release of Small Molecules.

Silk fibroin is a natural protein obtained from the Bombyx mori silkworm. In addition to being the key structural component in silkworm cocoons, it also has the propensity to self-assemble in vitro into hierarchical structures with desirable properties such as high levels of mechanical strength and robustness. Furthermore, it is an appealing biopolymer due to its biocompatability, low immunogenicity, and lack of toxicity, making it a prime candidate for biomedical material applications. Here, it is demonstrated that nanofibrils formed by reconstituted silk fibroin can be engineered into supramolecular microgels using a soft lithography-based microfluidic approach. Building on these results, a potential application for these protein microgels to encapsulate and release small molecules in a controlled manner is illustrated. Taken together, these results suggest that the tailored self-assembly of biocompatible and biodegradable silk nanofibrils can be used to generate functional micromaterials for a range of potential applications in the biomedical and pharmaceutical fields.

Hierarchical Biomolecular Emulsions Using 3-D Microfluidics with Uniform Surface Chemistry.

Microfluidic devices can be used to produce single, double and higher order emulsions, where droplet sizes can be precisely controlled and modulated. Such emulsions have great potential for the storage and study of biomolecules, including peptides and proteins. However, advancement of this technique has remained challenging due to the tendency of various biomolecules to adhere to the surface of the formed channels, resulting in changes in surface wetting and fouling on the micrometer scale. Thus, precise control of surface wettability plays a crucial role in the processes that govern droplet formation. Here, we report an approach for producing both water-oil-water (w/o/w) and oil-water-oil (o/w/o) double emulsions without any need for surface modification, an enabling feature for biomolecular encapsulation. Using this strategy, we show that the number of monodisperse encapsulated internal droplets can be controlled systematically and reproducibly by suitable adjustment of the relevant flow rates, and ranges from 1 to 40 in the case of w/o/w emulsions. We further demonstrate that the number of internal droplets scales linearly with the reciprocal flow rate of the outer continuous phase, when the inner and middle phase flow rates are kept constant. We demonstrate that this approach is suitable for forming double emulsions where the inner phase consists of reconstituted silk protein solution whereby incubation of the internal droplets can be induced to form a gel resulting in silk fibroin microgels surrounded by an external oil shell. Finally, for o/w/o emulsions, we show that single or multiple monodisperse internal droplets can be encapsulated with a size that ranges over 1 order of magnitude, from ca. 10 µm to >100 µm. Moreover, o/w/o emulsions where the middle phase consists of silk fibroin solution were prepared and by allowing the protein to aggregate, a core-shell structure was formed. This microfluidic strategy allows for multiple emulsions to be generated drop by drop for biomolecular solutions with potential applications in the biomedical and pharmaceutical fields.

Selenium-silk microgels as antifungal and antibacterial agents.

Antimicrobial resistance is a leading threat to global health. Alternative therapeutics to combat the rise in drug-resistant strains of bacteria and fungi are thus needed, but the development of new classes of small molecule therapeutics has remained challenging. Here, we explore an orthogonal approach and address this issue by synthesising micro-scale, protein colloidal particles that possess potent antimicrobial properties. We describe an approach for forming silk-based microgels that contain selenium nanoparticles embedded within the protein scaffold. We demonstrate that these materials have both antibacterial and antifungal properties while, crucially, also remaining highly biocompatible with mammalian cell lines. By combing the nanoparticles with silk, the protein microgel is able to fulfill two critical functions; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while simultaneously serving as a carrier for microbial eradication. Furthermore, since the antimicrobial activity originates from physical contact, bacteria and fungi are unlikely to develop resistance to our hybrid biomaterials, which remains a critical issue with current antibiotic and antifungal treatments. Therefore, taken together, these results provide the basis for innovative antimicrobial materials that can target drug-resistant microbial infections.

Pharmacological inhibition of α-synuclein aggregation within liquid condensates.

Aggregated forms of α-synuclein constitute the major component of Lewy bodies, the proteinaceous aggregates characteristic of Parkinson's disease. Emerging evidence suggests that α-synuclein aggregation may occur within liquid condensates formed through phase separation. This mechanism of aggregation creates new challenges and opportunities for drug discovery for Parkinson's disease, which is otherwise still incurable. Here we show that the condensation-driven aggregation pathway of α-synuclein can be inhibited using small molecules. We report that the aminosterol claramine stabilizes α-synuclein condensates and inhibits α-synuclein aggregation within the condensates both in vitro and in a Caenorhabditis elegans model of Parkinson's disease. By using a chemical kinetics approach, we show that the mechanism of action of claramine is to inhibit primary nucleation within the condensates. These results illustrate a possible therapeutic route based on the inhibition of protein aggregation within condensates, a phenomenon likely to be relevant in other neurodegenerative disorders.

Selenium Silk Nanostructured Films with Antifungal and Antibacterial Activity.

The rapid emergence of drug-resistant bacteria and fungi poses a threat for healthcare worldwide. The development of novel effective small molecule therapeutic strategies in this space has remained challenging. Therefore, one orthogonal approach is to explore biomaterials with physical modes of action that have the potential to generate antimicrobial activity and, in some cases, even prevent antimicrobial resistance. Here, to this effect, we describe an approach for forming silk-based films that contain embedded selenium nanoparticles. We show that these materials exhibit both antibacterial and antifungal properties while crucially also remaining highly biocompatible and noncytotoxic toward mammalian cells. By incorporating the nanoparticles into silk films, the protein scaffold acts in a 2-fold manner; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while also providing a template for bacterial and fungal eradication. A range of hybrid inorganic/organic films were produced and an optimum concentration was found, which allowed for both high bacterial and fungal death while also exhibiting low mammalian cell cytotoxicity. Such films can thus pave the way for next-generation antimicrobial materials for applications such as wound healing and as agents against topical infections, with the added benefit that bacteria and fungi are unlikely to develop antimicrobial resistance to these hybrid materials.