Phase transitions of protein molecules are central to biological function and malfunction. One such transition commonly encountered in nature is the conversion of soluble monomeric states into solid ...phases, which include crystals and amyloid fibrils, the latter of which are associated with the onset and development of neurodegenerative diseases. Monitoring aggregate formation and protein phase behavior is essential in gaining mechanistic insights into these fundamental processes. Fluorescence techniques have proven invaluable in observing biological molecules; yet, most such approaches rely on the use of an extrinsic fluorophore that binds to the molecule of interest, the installation of which can perturb the molecular systems under study. However, most proteins also possess aromatic amino acids within their peptide sequence and therefore exhibit intrinsic fluorescence. Here, we show that by measuring in space and time tryptophan autofluorescence for three proteins, reconstituted silk fibroin, β-lactoglobulin, and lysozyme, fibrillar self-assembly can be monitored accurately and without the need for extrinsic dyes. When fibrillar protein self-assembly takes place, hydrophobic burial occurs, resulting in the minimization of exposed tryptophan residues to the solvent and consequently leading to an increase in protein autofluorescence. Moreover, by employing a droplet-microfluidic approach to confine protein self-assembly in space, we demonstrate that intrinsic fluorescence can be used to image protein nanofibrils in a label-free manner and that the microstructural analysis obtained from intrinsic fluorescence microscopy correlates well with that from samples treated with extrinsic dyes. Finally, our results show that protein autofluorescence is not limited to the observation of β-sheet-rich structures, but can also be used to distinguish between different types of solid phases including spherulites and crystals, making this approach suitable for overall characterization of protein phase transition phenomena.
Nanoparticles are increasingly being used for biological applications, such as drug delivery and gene transfection. Different biological and bioinspired building blocks have been used for generating ...such particles, including lipids and synthetic polymers. Proteins are an attractive class of material for such applications due to their excellent biocompatibility, low immunogenicity, and self-assembly characteristics. Stable, controllable, and homogeneous formation of protein nanoparticles, which is key to successfully delivering cargo intracellularly, has been challenging to achieve using conventional methods. In order to address this issue, we employed droplet microfluidics and utilized the characteristic of rapid and continuous mixing within microdroplets in order to produce highly monodisperse protein nanoparticles. We exploit the naturally occurring vortex flows within microdroplets to prevent nanoparticle aggregation following nucleation, resulting in systematic control over the particle size and monodispersity. Through combination of simulation and experiment, we find that the internal vortex velocity within microdroplets determines the uniformity of the protein nanoparticles, and by varying parameters such as protein concentration and flow rates, we are able to finely tune nanoparticle dimensional properties. Finally, we show that our nanoparticles are highly biocompatible with HEK-293 cells, and through confocal microscopy, we determine that the nanoparticles fully enter into the cell with almost all cells containing them. Due to the high throughput of the method of production and the level of control afforded, we believe that the approach described in this study for generating monodisperse protein-based nanoparticles has the potential for intracellular drug delivery or for gene transfection in the future.
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.
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.
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.
Microscale hydrogels consisting of macromolecular networks in aqueous continuous phases have received increasing attention because of their potential use in tissue engineering, cell encapsulation and ...for the storage and release of cargo molecules. However, for applications targeting intracellular delivery, their micrometer-scale size is unsuitable for effective cellular uptake. Nanoscale analogs of such materials are thus required for this key area. Here, we describe a microfluidics/nanofluidics-based strategy for generating monodisperse nanosized water-in-oil emulsions with controllable sizes ranging from 2500 ± 110 nm down to 51 ± 6 nm. We demonstrate that these nanoemulsions can act as templates to form protein nanogels stabilized by supramolecular fibrils from three different proteins. We further show that these nanoparticles have the ability to penetrate mammalian cell membranes and deliver intracellular cargo. Due to their biocompatibility and lack of toxicity, natural protein-based nanoparticles present advantageous characteristics as vehicles for cargo molecules in the context of pharmaceutical and biomedical applications.
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.
The self-assembly of peptide and protein molecules into nanoscale filaments is a process associated with both biological function and malfunction. Microfluidic techniques can provide powerful tools ...in the study of such aggregation phenomena while providing access to exploring the role of molecular interactions in disease development. Yet, a common challenge encountered in the study of protein aggregation is the difficulty in achieving spatial and temporal control of the underlying processes. Here, we present a planar (2-D) device allowing for both the generation and confinement of 10 000 monodisperse water-in-oil droplets in an array of chambers with a trapping efficiency of 99%. Due to the specific geometry of the device, droplets can be formed and immediately trapped on the same chip, without the need for continuous flow of the oil phase. Furthermore, we demonstrate the capability of this device as a platform to study the aggregation kinetics and determine stochastic molecular nanoscale self-assembly events in a highly parallel manner for the aggregation of the dipeptide, diphenylalanine, the core recognition motif of the Aβ-42 peptide associated with Alzheimer's disease. The ability to reproducibly generate and confine monodisperse water-in-oil droplets with an extremely high trapping efficiency while maintaining entrapment under zero-flow conditions, on timescales compatible with observing molecular self-assembly events, renders it promising for numerous potential further applications in the biological and biophysical fields.
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.
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