Gelation Of Droplets Research Articles

Methylene blue accelerates liquid-to-gel transition of tau condensates impacting tau function and pathology

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer’s disease and other tauopathies. Recently, liquid–liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid–liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Microfluidically-generated Encapsulated Spheroids (μ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation.

Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.

Experimental Studies and Condition Monitoring of Auxiliary Processes in the Production of Al2O3 by Sol–Gel Technology

Powders and granules of heavy metal oxides produced through condition monitoring are in high demand as intermediate products for obtaining fine-grained ceramics for a wide range of applications, i.e., nuclear fuel and fuel elements for nuclear power plants. Sol–gel technology to produce nuclear fuel (UO2), as well as catalysts (ThO2) for organic synthesis in the form of granules from pressed microspheres, is a promising method to obtain powders and granules of heavy metal oxides (fine-graded ceramics). Al2O3 was selected as the model analog at the stages of obtaining a solution of heavy metal and sol, the formation and gelation of droplets, and the preparation of gel spheres and their further washing and drying, as well as recovery and firing of particles. In the study, the main parameters were substantiated, e.g., the diameter and angle of inclination of the axis for the holes in the perforated shell, the multiplicity of sol circulation before the holes, the coefficients of liquid (sol) flow rate, the oscillation frequency of the disperser, and the concentration of surfactant and acid in sol. All of these parameters affect the characteristics of the granules that are obtained by sol–gel technology. Moreover, recommendations to increase productivity and the energy efficiency of production were also given. In particular, it was found that oscillation frequency in a range of 70–80 Hz leads to a granulometric composition of the obtained granules of 2.0–2.2 mm. A hole of 0.85 mm and a frequency of 100 Hz slightly change this range to 1.2–2.0 mm, while maintaining monodispersity.

Development of a mist-based printhead for droplet-based bioprinting of ionically crosslinking hydrogel bioinks

In this paper, a novel droplet-based printhead is developed for 3D bioprinting of ionically crosslinking hydrogel bioinks. Contrary to previous approaches to droplet-based bioprinting, many of which use a liquid crosslinking agent to construct scaffolds, the developed system delivers the crosslinker in mist form within the printhead. A mechanism is developed to remove excess mist within the printhead, which prevents scaffold instability caused by the accumulation of liquid on the printing stage. The developed printhead is compatible with commercial systems and offers good droplet gelation and co-droplet adhesion. It is shown that the gelation rate can be controlled by adjusting the delivery rate of the mist. As a result, it is shown that the delivery rate of the mist has an impact on the shape fidelity and mechanical properties of the printed constructs. The printability and swelling properties of printed constructs crosslinked using different mist delivery rates are studied. Moreover, the impacts of printing parameters including printhead height, mist outlet pressure and printhead channel dimensions on the mist distribution and therefore the gelation rate within the printhead is investigated. Additionally, using high-speed imaging, the effects of mist concentration and droplet velocity on the dynamics of droplet impact onto the surface is characterized. Finally, it is shown that the printed constructs using the developed printhead show a low level of cytotoxicity and high level of cell viability. This development advances the applicability of droplet-based bioprinting to construct complex and biocompatible scaffolds for tissue and organ regeneration.

Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based Microfluidics.

Human immune cells intrinsically exist as heterogenous populations. To understand cellular heterogeneity, both cell culture and analysis should be executed with single-cell resolution to eliminate juxtacrine and paracrine interactions, as these can lead to a homogenized cell response, obscuring unique cellular behavior. Droplet microfluidics has emerged as a potent tool to culture and stimulate single cells at high throughput. However, when studying adherent cells at single-cell level, it is imperative to provide a substrate for the cells to adhere to, as suspension culture conditions can negatively affect biological function and behavior. Therefore, we combined a droplet-based microfluidic platform with a thermo-reversible polyisocyanide (PIC) hydrogel, which allowed for robust droplet formation at low temperatures, whilst ensuring catalyzer-free droplet gelation and easy cell recovery after culture for downstream analysis. With this approach, we probed the heterogeneity of highly adherent human macrophages under both pro-inflammatory M1 and anti-inflammatory M2 polarization conditions. We showed that co-encapsulation of multiple cells enhanced cell polarization compared to single cells, indicating that cellular communication is a potent driver of macrophage polarization. Additionally, we highlight that culturing single macrophages in PIC hydrogel droplets displayed higher cell viability and enhanced M2 polarization compared to single macrophages cultured in suspension. Remarkably, combining phenotypical and functional analysis on single cultured macrophages revealed a subset of cells in a persistent M1 state, which were undetectable in conventional bulk cultures. Taken together, combining droplet-based microfluidics with hydrogels is a versatile and powerful tool to study the biological function of adherent cell types at single-cell resolution with high throughput.

Gelatin Beads/Hemp Hurd as pH Sensitive Devices for Delivery of Eugenol as Green Pesticide

In this paper gelatin beads reinforced with natural hemp hurd have been produced as pH sensitive devices for the release of eugenol, as green pesticide. The composites beads, with a mean diameter of about 1 mm, were obtained by polymer droplet gelation in sunflower oil. Thermal properties were evaluated showing no noticeable difference after the introduction of hemp hurd. Barrier properties demonstrated an improvement of hydrophobization. The introduction of 5% w/w of hemp hurd led to a reduction of sorption coefficient of about 85% compared to unloaded gelatin beads. Besides, the diffusion coefficient decreased, introducing 5% w/w of hemp hurd, from 8.91 × 10−7 to 0.77 × 10−7 cm2/s. Swelling and dissolution phenomena of gelatin beads were studied as function of pH. The swelling of gelatin beads raised as pH increased up to 2.3 g/g, 9.1 g/g and 27.33 g/g at pH 3, 7 and 12, respectively. The dissolution rate changed from 0.034 at pH 3 to 0.077 h−1 at pH 12. Release kinetics of eugenol at different pH conditions were studied. The released eugenol after 24 h is 98%, 91%, 81 and 63% w/w (pH 3), 87%, 62%, 37 and 32 wt% (pH 7) and 81%, 68%, 60 and 52 wt% (pH 12) for unloaded gelatin beads and gelatin beads with 1%, 3 and 5% of hemp hurd, respectively. The eugenol release behavior was demonstrated to be highly sensitive to the pH release medium, which allows to tune such devices as green pesticide release systems in soils with different level of acidity/basicity.

Technologies and Formulation Design of Polysaccharide-Based Hydrogels for Drug Delivery.

Polysaccharide-based hydrogel particles (PbHPs) are very promising carriers aiming to control and target the release of drugs with different physico-chemical properties. Such delivery systems can offer benefits through the proper encapsulation of many drugs (non-steroidal and steroidal anti-inflammatory drugs, antibiotics, etc) ensuring their proper release and targeting. This review discusses the different phases involved in the production of PbHPs in pharmaceutical technology, such as droplet formation (SOL phase), sol-gel transition of the droplets (GEL phase) and drying, as well as the different methods available for droplet production with a special focus on prilling technique. In addition, an overview of the various droplet gelation methods with particular emphasis on ionic cross-linking of several polysaccharides enabling the formation of particles with inner highly porous network or nanofibrillar structure is given. Moreover, a detailed survey of the different inner texture, in xerogels, cryogels or aerogels, each with specific arrangement and properties, which can be obtained with different drying methods, is presented. Various case studies are reported to highlight the most appropriate application of such systems in pharmaceutical field. We also describe the challenges to be faced for the breakthrough towards clinic studies and, finally, the market, focusing on the useful approach of safety-by-design (SbD).

Homogeneous gelation leads to nanowire forests in the transition between electrospray and electrospinning

We demonstrate that homogeneous gelation of droplets in electrospray leads to the generation of nanowire forests and foams.

A Microfluidic System for One-Chip Harvesting of Single-Cell-Laden Hydrogels in Culture Medium.

Single-cell analysis has shown great potential to fully quantify the distribution of cellular behaviors among a population of individuals. Through isolation and preservation of single cells in the aqueous phase, droplet encapsulation followed by gelation enables high-throughput analysis in biocompatible microgels. However, the lack of control over the number of cells encapsulated and complicated gelation processes significantly limit its efficiency. Here, a microfluidic system for one-chip harvesting of single-cell-laden microgels is presented. Through ultraviolet irradiation, an on-chip gelation technique is seamlessly combined with droplet generation to realize high-throughput fabrication of microscale hydrogels in microfluidic channel. Moreover, a sorting module is introduced to simultaneously complete cell-laden microgel selection and transfer into culture medium. To demonstrate the efficiency of this method, two types of single cells are respectively encapsulated and collected, showing desirable single-cell encapsulation and cell viability. This technique realizes integrated droplet gelation, microgel sorting, and transfer into culture medium, allowing high-throughput analysis of single cells and comprehensive understanding of the cellular specificity.

Effect of Droplet Size in Acrylamide-Based Microgel Formation by Microfluidics

Polymer microgels with sizes of some tens to hundreds of micrometers can be formed with exquisite control by droplet-based microfluidic templating. This study presents a systematic assessment of the effect of the premicrogel droplet size on the ability of production of such microgels. The focus is on two popular acrylamide-derivatives at a fixed monomer concentration and external polymerization temperature. An exponential dependence of the success of droplet gelation on the droplet size is found, which can be rationalized in view of the balance between production and transfer of heat within and from the droplets on basis of a simple Arrhenius argument.

Preparation of microspheres of carbon black dispersion in uranyl-ascorbate gels as precursors for uranium carbide

A novel technique to fabricate precursors of carbide-ceramic nuclear fuel via synthesis of spherical particles (with a diameter below 150 μm) of carbon black dispersion in uranyl-ascorbate gels by a combination of the Complex Sol–Gel Process and the Double Extraction Process has been elaborated. Applying of ascorbic acid as complexing agent in the Complex Sol–Gel Process allows to obtain stable and uniform dispersion of carbon in an uranyl-ascorbate sol. Traditionally, the dispersability is improved by employment of dispersing agents – surfactants. In the presented Complex Sol–Gel Process, a dispersing agent is needless. The ascorbate sols perfectly wet carbon black and do not form agglomerates. In the Double Extraction Process, dispersion of carbon black in sols is emulsified by drops in the organic phase consisting of 2-ethylhexanol-1 and 1 volume % of Primene JM-T. The microspheres are created by gelation of droplets, through simultaneous extraction of water with 2-ethylhexanol-1 and nitrate ions with Primene JM-T. The elaboration of the optimal gelation conditions requires considering many issues which are described in this paper.

Diels-Alder "click" crosslinked matrices within calcium alginate templated beads

Event Abstract Back to Event Diels-Alder "click" crosslinked matrices within calcium alginate templated beads Alison Stewart1, Nicholas A. Burke1 and Harald D. Stöver1 1 McMaster University, Department of Chemistry & Chemical Biology, Canada Introduction: Covalently crosslinkable polymer hydrogels are a promising approach for the treatment of hormone and enzyme-deficiency disorders[1]. These scaffolds help provide protection from the immune system for encapsulated therapeutic cells. This presentation describes the use of the Diels-Alder reaction between furan and maleimide as a crosslinking reaction to form synthetic hydrogel scaffolds within a calcium alginate template. Materials and Methods: Poly(methyl vinyl ether-alt-maleic anhydride) was functionalized with furan and maleimide to give PMM-FFA/MAL. Beads were formed via gelation of droplets of sodium alginate and PMM-FFA/MAL in NaCl gelling bath. Distribution of PMM-FFA/MAL within the bead core was determined using fluorescence microscopy. In-diffusion of fluorescently-labeled dextrans was used to probe the pore-size of the beads. To determine crosslinking time and degree, the alginate scaffold was liquefied using sodium citrate, following various curing times. Results and Discussion: It was found that the presence of PMM-FFA/MAL does not interfere with alginate gelation, and low polymer loss (3-12%) during gelation indicates efficient trapping within the beads. To investigate crosslinking time, matrix beads were exposed to sodium citrate after set crosslinking times and observed via confocal microscopy. It was shown that as incubation time increased, the degree of swelling subsequently decreased, due to extended time for the Diels-Alder reaction to form covalent crosslinks. Figure 1 shows the swelling of Alg-PMM-FFA25/MAL24 beads in sodium citrate at A) 10 minutes, B) 2.5h and C) 6h post-bead formation. To characterize degree of crosslinking, beads were incubated for 24h, followed by exposure to excess citrate for 6h. All bead compositions remained intact post-citrate extraction, indicating sufficient Diels-Alder crosslinking. The beads composed of Alg-PMM-FFA12/MAL9 exhibited the highest degree of swelling, due to their low number of functional groups. Surprisingly, Alg-PMM-FFA18/MAL17 and Alg-PMM-FFA25/MAL23 beads had identical swell ratios. It is hypothesized that once a critical number of crosslink points are achieved, chain mobility is sufficiently decreased such that no more crosslinks form. In-diffusion studies of fluorescently-labeled dextran showed that 10 and 70kDa dextrans are able to readily diffuse into all beads. The 250 and 500kDa dextrans are partially excluded from the beads, demonstrating that Alg-PMM-FFA/MAL matrix beads show differential permeability for lower and higher-MW species. As expected, citrate treated beads exhibit higher permeability compared with untreated beads, due to swelling. Conclusions: Diels-Alder coupling between diene and dienophile-modified polyanions was used to form covalently crosslinked beads within an alginate-mediated template. The beads exhibited sufficient crosslinking to withstand alginate liquefaction, while pore-size and swell ratio could be successfully controlled via polymer and functional group loading. On-going work is focused on characterization of physical properties, and applications in cell encapsulation. We would like to thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for supporting this work through discovery and CREATE grants

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.

Microgel Capsules Tailored by Droplet‐Based Microfluidics

Microgel capsules are micrometer-sized particles that consist of a cross-linked, solvent-swollen polymer network complexed with additives. These particles have various applications, such as drug delivery, catalysis, and analytics. To optimize the performance of microgel capsules, it is crucial to control their size, shape, and content of encapsulated additives with high precision. There are two classes of microgel-capsule structures. One class comprises bulk microcapsules that consist of a polymer network spanning the entire particle and entrapping the additive within its meshes. The other class comprises core-shell structures; in this case, the microgel polymer network just forms the shell of the particles, whereas their interior is hollow and hosts the encapsulated payload. Both types of structures can be produced with exquisite control by droplet-based microfluidic templating followed by subsequent droplet gelation. This article highlights some early and recent achievements in the use of this technique to tailor soft microgel capsules; it also discusses applications of these particles. A special focus is on the encapsulation of living cells, which are very sensitive and complex but also very useful additives for immobilization within microgel particles.

Monodisperse alginate microgel formation in a three-dimensional microfluidic droplet generator

Droplet based microfluidic systems provide an ideal platform for partitioning and manipulating aqueous samples for analysis. Identifying stable operating conditions under which droplets are generated is challenging yet crucial for real-world applications. A novel three-dimensional microfluidic platform that facilitates the consistent generation and gelation of alginate-calcium hydrogel microbeads for microbial encapsulation, over a broad range of input pressures, in the absence of surfactants is described. The unique three-dimensional design of the fluidic network utilizes a height difference at the junction between the aqueous sample injection and organic carrier channels to induce droplet formation via a surface tension enhanced self-shearing mechanism. Combined within a flow-focusing geometry, under constant pressure control, this arrangement facilitates predictable generation of droplets over a much broader range of operating conditions than that of conventional two-dimensional systems. The impact of operating pressures and geometry on droplet gelation, aqueous and organic material flow rates, microbead size, and bead generation frequency are described. The system presented provides a robust platform for encapsulating single microbes in complex mixtures into individual hydrogel beads, and provides the foundation for the development of a complete system for sorting and analyzing microbes at the single cell level.

Synthesis, characterization and diffusion properties of biomimetic silica-coated gelatine beads

Several micro-organisms are able to coat their surface with a silica deposit exhibiting tailored diffusion properties. In a biomimetic approach, we studied the deposition of silica on gelatine beads. Gelatine capsules were obtained by polymer droplet gelation at low temperature. Soaking of these beads in sodium silicate dilute aqueous solutions leads to the formation of a mineral membrane consisting of close-packed silica nanoparticles. Studies of the diffusion of Rhodamine B and myoglobin indicate that the presence of the silica coating significantly modify the permeability of the beads, suggesting possible applications in controlled release systems.

Hydrogel beads based on amidated pectins for colon-specific drug delivery: the role of chitosan in modifying drug release

A multiparticulate system with the potential for site-specific delivery to the colon has been investigated. Gelation of droplets of amidated pectin solutions in the presence of calcium is the basis of the method of preparation. Two drugs, indomethacin and sulphamethoxazole were successfully incorporated into the beads. Drug release from the beads was a function of media pH and drug loading. In simulated gastric and small intestinal conditions, drug release was greater with the more soluble sulphamethoxazole, but release of both drugs could be reduced to satisfactory levels by the formation of a chitosan polyelectrolyte complex around the beads. All the preparations released drug in simulated colonic conditions within 135 min.

Calcium alginate matrices for oral multiple unit administration: II. Effect of process and formulation factors on matrix properties

Small calcium alginate matrices were prepared by ionotropic gelation of droplets of an alginate solution containing dispersed theophylline, followed by air-drying of the gel beads. The effect of various production factors on the size, composition and drug release properties was investigated in two separate studies. A 2 3 factorial design and a 2 V 5−1 fractional factorial design were applied. The size of the matrices was controlled mainly by the coaxial airstream applied during droplet production. However, the alginate concentration and the calcium concentration used for gelation also appeared to have a significant influence. The latter two factors, together with the amount of drug dispersed, determined the matrix drug content. The calcium concentration and the amount of drug affected matrix calcium content the most. The amount of drug also affected the moisture content. The calcium and alginate concentrations, the gelling time, the drug addition and the alginate G content affected the drug release rate in water. An increase in the level of all these factors caused a retardation in release. Several synergistic two-factor interactions were also observed.

Reverse micelles as microreactors

In this paper an extensive introduction to reverse micellar properties is given. It is shown that reverse micelles can be used as microreactors to produce either well-defined nanosized crystallites or chemically modified enzymes. Solubilization of macromolecules induces either the gelation of droplets or a percolation process followed by a phase transition. In the latter case this could favor the extraction of product reactions catalyzed by enzymes. Finally, a summary gives some prospective aspects of the use of such water-in-oil droplets

Synthesis of spherical silica particles by spontaneous emulsification

Spherical silica particles are produced by spontaneous emulsification when an ethanolic solution of partially hydrolyzed tetraethoxysilane (TEOS) is mixed with water. The driving force for emulsification is the difference in ethanol concentration between the two solutions. For particle synthesis, the process can be divided into the following stages: (1) emulsification, (2) droplet growth through coalescence and (3) droplet gelation. The physical and interfacial properties of partially hydrolyzed TEOS directly determine the relative importance of each of these steps through their influence on phase equilibria, interfacial behavior, and the rate of droplet gelation after emulsification. It is demonstrated that either solid or hollow spherical particles ranging in size from as small as 0.1 to > 50 μm can be produced by altering the degree of TEOS hydrolysis and condensation before spontaneous emulsification.