Fluid dynamics alters liquid-liquid phase separation in confined aqueous two-phase systems.

Liquid-liquid phase separation is key to understanding aqueous two-phase systems (ATPS) arising throughout cell biology, medical science, and the pharmaceutical industry. Controlling the detailed morphology of phase-separating compound droplets leads to new technologies for efficient single-cell analysis, targeted drug delivery, and effective cell scaffolds for wound healing. We present a computational model of liquid-liquid phase separation relevant to recent laboratory experiments with gelatin-polyethylene glycol mixtures. We include buoyancy and surface-tension-driven finite viscosity fluid dynamics with thermally induced phase separation. We show that the fluid dynamics greatly alters the evolution and equilibria of the phase separation problem. Notably, buoyancy plays a critical role in driving the ATPS to energy-minimizing crescent-shaped morphologies, and shear flows can generate a tenfold speedup in particle formation. Neglecting fluid dynamics produces incorrect minimum-energy droplet shapes. The model allows for optimization of current manufacturing procedures for structured microparticles and improves understanding of ATPS evolution in confined and flowing settings important in biology and biotechnology.

Interfacial Membranization of Regenerated Cellulose Nanoparticles and a Protein Renders Stable Water-in-Water Emulsion.

Pickering water-in-water (W/W) emulsions stabilized by biobased colloids are pertinent to engineering biomaterials with hierarchical and confined architectures. In this study, stable W/W emulsions are developed through membranization utilizing biopolymer structures formed by the adsorption of cellulose II nanospheres and a globular protein, bovine serum albumin (BSA), at droplet surfaces. The produced cellulose II nanospheres (NPcat, 63 nm diameter) bearing a soft and highly accessible shell, endow rapid and significant binding (16 mg cm- 2) with BSA. NPcat and BSA formed complexes that spontaneously stabilized liquid droplets, resulting in stable W/W emulsions. It is proposed that such a system is a versatile all-aqueous platform for encapsulation, (bio)catalysis, delivery, and synthetic cell mimetics.

Cinnamaldehyde-Treated Bone Marrow Mesenchymal-Stem-Cell-Derived Exosomes via Aqueous Two-Phase System Attenuate IL-1ß-Induced Inflammation and Catabolism via Modulation of Proinflammatory Signaling Pathways.

Osteoarthritis (OA) is a degenerative joint disorder that is distinguished by inflammation and chronic cartilage damage. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine that plays an important role in the catabolic processes that underlie the pathogenesis of OA. In this study, we investigate the therapeutic efficacy of exosomes derived from untreated bone-marrow-derived mesenchymal stem cells (BMMSC-Exo) and those treated with cinnamaldehyde (BMMSC-CA-Exo) for preventing the in vitro catabolic effects of IL-1ß on chondrocytes. We stimulated chondrocytes with IL-1ß to mimic the inflammatory microenvironment of OA. We then treated these chondrocytes with BMMSC-Exo and BMMSC-CA-Exo isolated via an aqueous two-phase system and evaluated their effects on the key cellular processes using molecular techniques. Our findings revealed that treatment with BMMSC-Exo reduces the catabolic effects of IL-1ß on chondrocytes and alleviates inflammation. However, further studies directly comparing treatments with BMMSC-Exo and BMMSC-CA-Exo are needed to determine if CA preconditioning can provide additional anti-inflammatory benefits to the exosomes beyond those of CA preconditioning or treatment with regular BMMSC-Exo. Through a comprehensive molecular analysis, we elucidated the regulatory mechanisms underlying this protective effect. We found a significant downregulation of proinflammatory signaling pathways in exosome-infected chondrocytes, suggesting the potential modulation of the NF-κB and MAPK signaling cascades. Furthermore, our study identified the molecular cargo of BMMSC-Exo and BMMSC-CA-Exo, determining the key molecules, such as anti-inflammatory cytokines and cartilage-associated factors, that may contribute to their acquisition of chondroprotective properties. In summary, BMMSC-Exo and BMMSC-CA-Exo exhibit the potential as therapeutic agents for OA by antagonizing the in vitro catabolic effects of IL-1ß on chondrocytes. The regulation of the proinflammatory signaling pathways and bioactive molecules delivered by the exosomes suggests a multifaceted mechanism of action. These findings highlight the need for further investigation into exosome-based therapies for OA and joint-related diseases.

Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation.

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K2HP04) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the Km (4.5 ± 0.8 mg/mL), Vmax (0.04 ± 0.01 U) and kcat (0.001 s-1) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na+, K+, Ca2+ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration.

Spontaneous Formation of Uniform Cell-Sized Microgels through Water/Water Phase Separation.

In this study, a one-step method is discussed for producing uniform cell-sized microgels using glass capillaries filled with a binary polymer blend of polyethylene glycol (PEG) and gelatin. Upon decreasing temperature, phase separation of the PEG/gelatin blends and gelation of gelatin occur, and then the polymer blend forms linearly aligned, uniformly sized gelatin microgels in the glass capillary. When DNA is added to the polymer solution, gelatin microgels entrapping DNA are spontaneously formed, and the DNA prevents the coalescence of the microdroplets even at temperatures above the melting point. This novel method to form uniform cell-sized microgels may be applicable to other biopolymers. This method is expected to contribute to diverse materials science via biopolymer microgels and biophysics and synthetic biology through cellular models containing biopolymer gels.

3D Printable Gelatin Methacryloyl (GelMA)-Dextran Aqueous Two-Phase System with Tunable Pores Structure and Size Enables Physiological Behavior of Embedded Cells In Vitro.

The restricted porosity of most hydrogels established for in vitro 3D tissue engineering applications limits embedded cells with regard to their physiological spreading, proliferation, and migration behavior. To overcome these confines, porous hydrogels derived from aqueous two-phase systems (ATPS) are an interesting alternative. However, while developing hydrogels with trapped pores is widespread, the design of bicontinuous hydrogels is still challenging. Herein, an ATPS consisting of photo-crosslinkable gelatin methacryloyl (GelMA) and dextran is presented. The phase behavior, monophasic or biphasic, is tuned via the pH and dextran concentration. This, in turn, allows the formation of hydrogels with three distinct microstructures: homogenous nonporous, regular disconnected-pores, and bicontinuous with interconnected-pores. The pore size of the latter two hydrogels can be tuned from ≈4 to 100 µm. Cytocompatibility of the generated ATPS hydrogels is confirmed by testing the viability of stromal and tumor cells. Their distribution and growth pattern are cell-type specific but are also strongly defined by the microstructure of the hydrogel. Finally, it is demonstrated that the unique porous structure is sustained when processing the bicontinuous system by inkjet and microextrusion techniques. The proposed ATPS hydrogels hold great potential for 3D tissue engineering applications due to their unique tunable interconnected porosity.

Water-in-Water Emulsions Stabilized by Silica Janus Nanosheets.

Water-in-water (w/w) emulsions have been recognized for their broad applications in foods, cosmetics, and biomedical engineering. In this work, silica Janus nanosheets (JNs) with polyacrylic acid (PAA) chains grafted on one surface via crushing functional silica foams, and used silica JNs as Pickering stabilizer to produce stable water-in-water (w/w) emulsions from the aqueous two-phase system (ATPS) containing methacrylic acid (MAA) and NaCl are prepared. The interfacial area of w/w emulsions increases linearly with the concentration of silica JNs, and the interfacial coverage of nanosheets is calculated to be about 98%. After polymerizing w/w emulsions prepared from MAA/NaCl ATPS, it is found that silica JNs are entrapped at the interface of w/w emulsions with the smooth PAA-grafted surface located toward MAA-rich phase due to their specific interaction. These results show that functional silica JNs can be used as a promising amphiphilic Pickering stabilizer to produce well-defined w/w emulsions for numerous application fields.

Aqueous Two-Phase Systems Based on Cationic and Anionic Surfactants Mixture for Rapid Extraction and Colorimetric Determination of Synthetic Food Dyes.

In this study, aqueous two-phase systems (ATPSs) containing a cationic and anionic surfactants mixture were used for the preconcentration of the synthetic food dyes Allura Red AC, Azorubine, Sunset Yellow, Tartrazine, and Fast Green FCF. A rapid, simple, low cost, affordable, and environmentally friendly methodology based on microextraction in ATPSs, followed by spectrophotometric/colorimetric determination of the dyes, is proposed. The ATPSs are formed in mixtures of benzethonium chloride (BztCl) and sodium N-lauroylsarcosinate (NaLS) or sodium dihexylsulfosuccinate (NaDHSS) under the molar ratio close to equimolar at the total surfactant concentration of 0.01-0.20 M. The density, viscosity, polarity, and water content in the surfactant-rich phases at an equimolar ratio BztCl:NaA were determined. The effects of pH, total surfactant concentration, dye concentration, and time of extraction/centrifugation were investigated, and the optimum conditions for the quantitative extraction of dyes were established. The smartphone-based colorimetric determination was employed directly in the extract without separating the aqueous phase. The analytical performance (calibration linearity, precision, limits of detection and quantification, reproducibility, and preconcentration factor) and comparison of the spectrophotometric and smartphone-based colorimetric determination of dyes were evaluated. The method was applied to the determination of dyes in food samples and food-processing industrial wastewater.

Study on Extraction and Purification of Acanthopanax senticosus Polyphenols by an Ionic Liquid-Assisted Aqueous Two-Phase System.

This study aimed to extract and purify polyphenols from Acanthopanax senticosus. A new green method was developed, in which ionic liquids (ILs) were used as aqueous two-phase (ATP) adjuvants to extract the polyphenols from A. senticosus. An ionic liquid-assisted aqueous two-phase system (IL-ATPS) was established. The purification of the polyphenols from the extraction fluid by AB-8 macroporous resin was conducted, and the kinetic mechanisms were studied. The reuse of ionic liquids was executed. The results showed that an [OMIM]Br-assisted ethanol/NaH2PO4 system (IL-ATPS) was the best extraction solvent. In this study, the following optimal extraction conditions were determined: 32 wt.% ethanol, 25 wt.% NaH2PO4, 9 wt.% additional ionic liquid, a solid-liquid ratio of 1:40 g/mL, an extraction temperature of 50 °C, a pH of 4.0, an extraction time of 50 min, and an extraction rate of the polyphenols at 15.90 mg/g. The optimum adsorption parameters of the macroporous resin AB-8 were as follows: a flow rate of 3.5 BV·h-1, a sample volume of 40 mL, an elution flow rate of 3.5 BV·h-1, an eluent volume of 80 mL, and an eluant that was constituted by an 85% volume fraction of ethanol. The decolorization effect of 4% activated carbon was better than the other amounts; in addition, a decolorization rate of 76.81% and an ionic liquid recovery rate of 81.12% were found to be the most optimal. Compared with the traditional extraction methods, IL-ATPS has the advantages of requiring simple operation, saving time, and high efficiency. In addition, it can be used for the extraction of the polyphenolic compounds.

Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges.

The advancement of recombinant virus-like particle-based vaccines has attracted global attention owing to substantially safety and high efficacy in provoking a protective immunity against various chronic and infectious diseases in humans and animals. A robust, low-cost, and scalability separation and purification technology is of utmost importance in the downstream processing of recombinant virus-like particles to produce affordable and safe vaccines. Being a relatively simple, environmentally friendly, and efficient biomolecules recovery approach, aqueous two-phase systems have received great attention from researchers worldwide. This review aims to highlight the challenges and outlook in addition to the current applications of aqueous two-phase systems in downstream processing of virus-like particles. The efforts will confidently reinforce scholars' knowledge and fill in the valuable research gap in the aspect of concerning recombinant virus-like particle-based vaccines development, particularly related to the virus-like particles downstream production processes.

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems.

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.

A Deep Eutectic Solvent Thermomorphic Multiphasic System for Biocatalytic Applications.

The applicability of a thermomorphic multiphasic system (TMS) composed of a hydrophobic deep eutectic solvent (DES) and an aqueous potassium phosphate buffer with a lower critical solution temperature (LCST) phase change for homogeneous biocatalysis was investigated. A lidocaine-based DES with the fatty acid oleic acid as a hydrogen-bond donor was studied. Phase diagrams were determined and presented within this study. We tested different additional components to the solvent system and observed a decrease in the cloud point of approximately 0.026 °C per concentration unit. Distribution studies revealed a clear distribution of the protein in the aqueous buffer phase (>95 %), whereas the hydrophobic substrate and educt accumulated (>95 %) in the DES-enriched layer. Finally, a reduction catalyzed by horse liver alcohol dehydrogenase was performed in a larger-scale experiment, and the biocatalyst could be recycled by simply removing the DES phase for three recycling runs.

Graphitic Carbon Nitride Stabilized Water-in-Water Emulsions.

Aqueous multiphase systems have attracted a lot of interest recently espeically due to target applications in the biomedical field, cosmetics, and food. In turn, water-in-water Pickering emulsions are investigated frequently. In here, graphitic carbon nitride (g-CN) stabilized water-in-water Pickering emulsions are fabricated via the dextran and poly(ethylene glycol)-based aqueous two-phase system. Five different derivatives of g-CN as the Pickering stabilizer are described and the effect of g-CN concentration on droplet sizes is investigated. Stable emulsions (up to 16 weeks) are obtained that can be broken on purpose via various approaches, including dilution, surfactant addition, and most notably light irradiation. The novel approach of water-in-water emulsion stabilization via g-CN opens up considerable advances in aqueous multiphase systems and may also introduce photocatalytic properties.

Current methods for studying intracellular liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is a ubiquitous process that drives the formation of membrane-less intracellular compartments. This compartmentalization contains vastly different protein/RNA/macromolecule concentrations compared to the surrounding cytosol despite the absence of a lipid boundary. Because of this, LLPS is important for many cellular signaling processes and may play a role in their dysregulation. This chapter highlights recent advances in the understanding of intracellular phase transitions along with current methods used to identify LLPS in vitro and model LLPS in situ.

Microfluidic Generation of All-Aqueous Double and Triple Emulsions.

Higher order emulsions are used in a variety of different applications in biomedicine, biological studies, cosmetics, and the food industry. Conventional droplet generation platforms for making higher order emulsions use organic solvents as the continuous phase, which is not biocompatible and as a result, further washing steps are required to remove the toxic continuous phase. Recently, droplet generation based on aqueous two-phase systems (ATPS) has emerged in the field of droplet microfluidics due to their intrinsic biocompatibility. Here, a platform to generate all-aqueous double and triple emulsions by introducing pressure-driven flows inside a microfluidic hybrid device is presented. This system uses a conventional microfluidic flow-focusing geometry coupled with a coaxial microneedle and a glass capillary embedded in flow-focusing junctions. The configuration of the hybrid device enables the focusing of two coaxial two-phase streams, which helps to avoid commonly observed channel-wetting problems. It is shown that this approach achieves the fabrication of higher-order emulsions in a poly(dimethylsiloxane)-based microfluidic device, and controls the structure of the all-aqueous emulsions. This hybrid microfluidic approach allows for facile higher-order biocompatible emulsion formation, and it is anticipated that this platform will find utility for generating biocompatible materials for various biotechnological applications.

Aqueous two-phase systems based on deep eutectic solvents and their application in green separation processes.

As a new environmentally friendly separation technology, deep eutectic solvent based aqueous two-phase systems are extensively applied in various fields. Herein, we review recent advances in this field and highlight the possible directions of future developments. This article focuses on the effects of deep eutectic solvent and inorganic salts on the phase equilibrium, the microstructure of deep eutectic solvent based aqueous two-phase systems, the applications of deep eutectic solvent based aqueous two-phase systems in separation (proteins, biopolymers, saponins, and organic acids), and removal and recovery technologies for deep eutectic solvent from aqueous two-phase systems.

Production, extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems.

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (MPEG), concentrations of PEG (CPEG) and citrate (CCIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°m (-10.89 kJ mol-1), ΔHm (-5.0 kJ mol-1) and partition ΔSm (19.74 J mol-1 K-1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.

Determination of Aqueous Two-Phase System Binodals and Tie-Lines by Electrowetting-on-Dielectric Droplet Manipulation.

Handling the aqueous two-phase systems (ATPSs) formed by liquid-liquid phase separation (LLPS) relies on the accurate construction of binodal curves and tie-lines, which delineate the polymer concentrations required for phase separation and depict the properties of the resulting phases, respectively. Various techniques to determine the binodal curves and tie-lines of ATPSs exist, but most rely on manually pipetting relatively large volumes of fluids in a slow and tedious manner. We describe a method to determine ATPS binodals and tie-lines that overcomes these disadvantages: microscale droplet manipulation by electrowetting-on-dielectric (EWOD). EWOD enables automated handling of droplets in an optically transparent platform that allows for in situ droplet observation. Separated phases are clearly visible, and the volumes of each phase are readily determined. Additionally, in considering the molecular crowding present in living cells, this work examines the role of a macromolecule in prompting LLPS. These results show that EWOD-driven droplet manipulation effectively interrogates the phase dynamics of ATPSs and macromolecular crowding in LLPS.

On-line electroextraction in capillary electrophoresis: Application on the determination of glutamic acid in soy sauces.

We present an on-line, single step coupling between liquid-liquid extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection, which allows an efficient analysis of complex food matrices with high sodium content. The sodium depletion was demonstrated using an aqueous two-phase system. The aqueous two-phase system enables the electrically driven extraction of the target compounds. The sample was prepared in Dextran-rich phase (8% w/v 500 kDa Dextran, DEX). The background electrolyte (acetic acid 5.0 mol/L) contained 6% w/v of 6 kDa PEG. As proof of applicability, we employed the developed method for glutamic acid quantification on soy sauces. The peak area of glutamic acid presents no significant difference (α = 0.05), while the peak area of the sodium presented a reduction of 11.7 ± 0.2 and 19 ± 3% for premium and low-cost soy sauce samples analyzed. The glutamic acid concentration for premium soy sauce sample was 2.7 ± 0.8 and 4.8 ± 0.4 g/L, and for low-cost soy sauce sample, the concentration was 9.9 ± 0.9 g/L, which agreed with those obtained by other analytical techniques.

Complex Extraction of Metals in an Aqueous Two-Phase System Based on Poly(Ethylene Oxide) 1500 and Sodium Nitrate.

This article presents an ecologically safe aqueous two-phase system based on poly(ethylene oxide) with a molecular weight of 1500, designed for complex extraction of Ni(II), Co(II), Fe(III), Mn(II), Zn(II), Cu(II), and Al(III) from nitrate solutions. A kinetic dependence has been investigated for a distribution ratio for the metals examined. The influence of pH-values, temperature, initial metal concentration, and nitric acid content have on the extraction of a wide range of metals in the heterogeneous poly(ethylene oxide) 1500-NaNO3-H2O system has been discovered. As a result, the complex extraction of metals (EMe > 60%) was achieved in one step of extraction without introducing additional chemicals into the system.