45 research outputs found
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Shear-mediated sol-gel transition of regenerated silk allows the formation of Janus-like microgels
Abstract: Microcapsules and microgels consisting of macromolecular networks have received increasing attention due to their biomedical and pharmaceutical applications. Protein microgels and in particular silk-based microcapsules have desirable properties due to their biocompatibility and lack of toxicity. Typically such structures formed through emulsion templating are spherical in geometry due to interfacial tension. However, approaches to synthesis particles with more complex and non-spherical geometries are sought due to their packing properties and cargo release characteristics. Here, we describe a droplet-microfluidic strategy for generating asymmetric tubular-like microgels from reconstituted silk fibroin; a major component of native silk. It was determined using fluorescence microscopy, that the shear stress within the microchannel promotes surface protein aggregation, resulting in the asymmetric morphology of the microgels. Moreover, the structural transition that the protein undergoes was confirmed using FTIR. Crucially, the core of the microgels remains liquid, while the surface has fully aggregated into a fibrillar network. Additionally, we show that microgel morphology could be controlled by varying the dispersed to continuous phase flow rates, while it was determined that the radius of curvature of the asymmetric microgels is correlated to the wall shear stress. By comparing the surface fluorescence intensity of the microgels as a function of radius of curvature, the effect of the shear stress on the amount of aggregation could be quantified. Finally, the potential use of these asymmetric microgels as carriers of cargo molecules is showcased. As the core of the microgel remains liquid but the shell has gelled, this approach is highly suitable for the storage of bio-active cargo molecules such as antibodies, making such a delivery system attractive in the context of biomedical and pharmaceutical applications
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Attoliter protein nanogels from droplet nanofluidics for intracellular delivery.
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
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Shear-mediated sol-gel transition of regenerated silk allows the formation of Janus-like microgels
Abstract: Microcapsules and microgels consisting of macromolecular networks have received increasing attention due to their biomedical and pharmaceutical applications. Protein microgels and in particular silk-based microcapsules have desirable properties due to their biocompatibility and lack of toxicity. Typically such structures formed through emulsion templating are spherical in geometry due to interfacial tension. However, approaches to synthesis particles with more complex and non-spherical geometries are sought due to their packing properties and cargo release characteristics. Here, we describe a droplet-microfluidic strategy for generating asymmetric tubular-like microgels from reconstituted silk fibroin; a major component of native silk. It was determined using fluorescence microscopy, that the shear stress within the microchannel promotes surface protein aggregation, resulting in the asymmetric morphology of the microgels. Moreover, the structural transition that the protein undergoes was confirmed using FTIR. Crucially, the core of the microgels remains liquid, while the surface has fully aggregated into a fibrillar network. Additionally, we show that microgel morphology could be controlled by varying the dispersed to continuous phase flow rates, while it was determined that the radius of curvature of the asymmetric microgels is correlated to the wall shear stress. By comparing the surface fluorescence intensity of the microgels as a function of radius of curvature, the effect of the shear stress on the amount of aggregation could be quantified. Finally, the potential use of these asymmetric microgels as carriers of cargo molecules is showcased. As the core of the microgel remains liquid but the shell has gelled, this approach is highly suitable for the storage of bio-active cargo molecules such as antibodies, making such a delivery system attractive in the context of biomedical and pharmaceutical applications
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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.This study was supported by the Adelis Foundation. S.Z.T. was supported by the Daniel Turnberg Travel Fellowship
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From Protein Building Blocks to Functional Materials
Proteins are the fundamental building blocks for high performance materials in nature. Such materials fulfil structural roles, as in the case of silk and collagen, and can generate active structures including the cytoskeleton. Attention is increasingly turning to this versatile class of molecules for the synthesis of next generation green functional materials for a range of applications. Protein nanofibrils are a fundamental supramolecular unit from which many macroscopic protein materials are formed. In this review, we focus on the multiscale assembly of such protein nanofibrils formed from naturally occurring proteins into new supramolecular architectures and discuss how they can form the basis of material systems ranging from bulk gels, films, fibers, micro/nanogels, condensates and active materials. We review current and emerging approaches to process and assemble these building blocks in a manner which is different to their natural evolutionarily selected role, but allows the generation of tailored functionality, with a focus on microfluidic approaches. We finally discuss opportunities and challenges for this class of materials, including applications that can be involved in this material system which consists of fully natural, biocompatible and biodegradable feedstocks yet has the potential to generate materials with performance and versatility rivalling that of the best synthetic polymers
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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.ERC
EPSRC
Frances and Augustus Newman Foundation
Oppenheimer Early Career Fellowship
Nanotechnologies Doctoral Training Centre
Fluidic Analytics Lt
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
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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
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.ERC
EPSRC
Frances and Augustus Newman Foundation
Oppenheimer Early Career Fellowship
Nanotechnologies Doctoral Training Centre
Fluidic Analytics Lt
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Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.
Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Biotechnology and Biological Sciences Research CouncilFunder: Centre for Misfolding Diseases, University of CambridgeFunder: Wellcome TrustBiomimetics 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