Liquid-liquid Phase Separation Phenomenon Research Articles

The liquid-liquid phase separation in programmed cell death

In recent years, the physical phenomenon of liquid-liquid phase separation has been widely introduced into biological research. Membrane-free organelles have been found to exist in cells that were driven by liquid-liquid phase separation. Intermolecular multivalent interactions can drive liquid-liquid phase separation to form condensates that are independent of other substances in the environment and thus can play an effective role in regulating multiple biological processes in the cell. The way of cell death has also long been a focus in multiple research. In the face of various stresses, cell death-related mechanisms are crucial for maintaining cellular homeostasis and regulating cell fate. With the in-depth study of cell death pathways, it has been found that the process of cell death was also accompanied by the regulation of liquid-liquid phase separation and played a key role. Therefore, this review summarized the roles of liquid-liquid phase separation in various cell death pathways, and explored the regulation of cell fate by liquid-liquid phase separation, with the expectation that the exploration of the mechanism of liquid-liquid phase separation would provide new insights into the treatment of diseases caused by regulated cell death.

Screening of liquid–liquid phase separation conditions for proteins with a mixed solution kit of biomacromolecular crowding agents

Protein liquid–liquid phase separation (LLPS) is a significant process in biology and material sciences. Finding suitable conditions for protein LLPS is essential for its in-depth and systematic study. Here, we provide a mixed solution screening kit based on biomacromolecule crowding agents to find protein LLPS conditions. The screening kit consists of 96 chemical formulations, in which the biomacromolecule crowding agents are used as the key components. The aqueous solution of the target protein is mixed with each chemical agent in the kit and then placed in an incubator for some time. The LLPS conditions for the target protein can be found by examining the LLPS phenomena in the droplets. We used nine proteins as model target proteins—lysozyme, bovine serum albumin, bovine hemoglobin, ovalbumin, catalase, FUS, Aβ, Tau, and α-Synuclein—to evaluate the performance of the screening kit. We found that utilization of the screening kit is efficient and useful to find different conditions for LLPS of proteins. The screening results reveal two types of conditions: those that promote and those that inhibit LLPS. Both conditions are extremely valuable because those that promote LLPS can be used to do extensive studies on the LLPS processes of the proteins of interest, whereas those that inhibit LLPS can be utilized as leads for developing novel medications to treat LLPS-related disorders. Furthermore, novel applications for the kit were investigated by utilizing the screening products.

Liquid-Liquid Phase Separation-Mediated Photocatalytic Subcellular Hybrid System for Highly Efficient Hydrogen Production.

Plant chloroplasts have a highly compartmentalized interior, essential for executing photocatalytic functions. However, the construction of a photocatalytic reaction compartment similar to chloroplasts in inorganic-biological hybrid systems (IBS) has not been reported. Drawing inspiration from the compartmentalized chloroplast and the phenomenon of liquid-liquid phase separation, herein, a new strategy is first developed for constructing a photocatalytic subcellular hybrid system through liquid-liquid phase separation technology in living cells. Photosensitizers and in vivo expressed hydrogenases are designed to coassemble within the cell to create subcellular compartments for synergetic photocatalysis. This compartmentalization facilitates efficient electron transfer and light energy utilization, resulting in highly effective H2 production. The subcellular compartments hybrid system (HM/IBSCS) exhibits a nearly 87-fold increase in H2 production compared to the bare bacteria/hybrid system. Furthermore, the intracellular compartments of the photocatalytic reactor enhance the system's stability obviously, with the bacteria maintaining approximately 81% of their H2 production activity even after undergoing five cycles of photocatalytic hydrogen production. The research brings forward visionary prospects for the field of semi-artificial photosynthesis, offering new possibilities for advancements in areas such as renewable energy, biomanufacturing, and genetic engineering.

Control of Liquid–Liquid Phase Separation, Nucleation, and Growth of L‐Menthol Single Crystals from Water–Ethanol Solvent Mixtures

AbstractL‐Menthol is a commonly used material in food production for flavor enhancement and fragrances, as well as in pharmaceuticals. However, during the solution crystallization process of L‐menthol, there is a major issue of oiling out or liquid–liquid phase separation (LLPS) in most organic solvents and their mixtures. The objective of the current work is to prevent the LLPS phenomenon and grow high‐quality single crystals of L‐menthol. A water–ethanol mixed solvent with nine different mixing ratios is used, ranging from 0.99W:0.01E to 0.91W:0.09E, through the conventional slow evaporation process. The findings indicate that the mixing composition of the solvent plays a significant role in the variation of solubility, refractive index, LLPS, nucleation, and crystallization. This is clearly demonstrated in the phase diagram. The occurrence of LLPS phenomenon, nucleation, growth behavior, and morphology of L‐menthol single crystals through optical microscopy is identified. Additionally, the internal structure and thermal stability of the grown polymorphs using PXRD, SCXRD, and DSC analyses are confirmed.

Measurement report: New insights into the mixing structures of black carbon on the eastern Tibetan Plateau – soot redistribution and fractal dimension enhancement by liquid–liquid phase separation

Abstract. Black carbon (BC; i.e. soot) absorbs radiation and contributes to glacier retreat over the Tibetan Plateau (TP). A lack of comprehensive understanding of the actual mixing state leads to large controversies in the climatic simulation of soot over the TP. In this study, ground-based sampling, electron microscopy analyses, and theoretical calculations were used to investigate the interactions among the liquid–liquid phase separation (LLPS), soot redistribution in secondary particles, and fractal dimension (Df) of soot particles on the eastern rim of the TP. We found that more than half of the total analysed particles were soot-containing particles. One-third of soot-containing particles showed a core shell structure that probably formed the LLPS phenomenon after long-range transport. Particle size and the ratio of organic coating thickness to soot size are two of the major possible factors that likely induce soot redistribution between organic matter and inorganic aerosols in individual particles. The Df sequence is ranked as externally mixed soot (1.79±0.09)< sulfate-coated soot (1.84±0.07)< organic-coated soot (1.95±0.06). We concluded that the soot redistribution process promoted the morphological compaction of soot particles. This study indicates that soot-containing particles experienced consistent ageing processes that induced a more compact morphology and soot redistribution in the LLPS particles on the remote eastern rim of the TP. Understanding the microscopic changes in aged soot particles could further improve the current climate models and evaluations of BC's radiative impacts on the eastern TP and similar remote air.

Multicompartment calcium alginate microreactors to reduce substrate inhibition in enzyme cascade reactions.

The formation of macromolecularly enriched condensates through associative or segregative liquid-liquid phase separation phenomena is known to play a central role in controlling various cellular functions in nature. The potential to spatially and temporally modulate multistep chemical reactions and pathways has inspired the use of phase-separated systems for the development of various synthetic colloidal micro- and nanoreactor systems. Here, we report a rational and synthetically minimal design strategy to emulate intended spatiotemporal functions in morphologically intricate and structurally defined calcium alginate hydrogel microreactors possessing multicompartmentalized internal architectures. Specifically, we implement a thermal phase separation protocol to achieve fine-control over liquid-liquid phase separation inside complex aqueous emulsion droplet templates that are loaded with hydrophilic polymer mixtures. Subsequent gelation of alginate-containing droplet templates using a novel freeze-thaw approach that can be applied to both scalable batch production or more precise microfluidic methods yields particle replicas, in which subcompartmentalized architectures can be retained. Larger active components can be enriched in the internal compartments due to their preferential solubility, and we show that selective sequestration of enzymes serves to create desired microenvironments to control and tune the reaction kinetics of a multistep enzyme cascade by reducing their mutual interference. This demonstration of mitigating substrate inhibition that is based primarily on optimizing the multicompartmentalized hydrogel particle morphology offers new opportunities for the simple and synthetically-minimal batch generation of hydrogel-based synthesis microreactors.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

Liquid-Liquid Phase Separation of an Intrinsically Disordered Region of a Germ Cell-Specific Protein Modulates the Stability and Conformational Exchange Rate of SH3 Domain.

The phenomenon of liquid-liquid phase separation is found in numerous biological processes. The biomolecules enveloped in the phase-separated droplets experience an obviously different environment from those in cellular or aqueous solution. Herein, we quantitatively characterized the thermodynamics and exchange kinetics of a model protein SH3 domain in the condensed phase of an intrinsically disordered region of a germ cell-specific protein DDX4N1 by using 19F-NMR spectroscopy. The stability and exchange rate of the SH3 domain are different from those in buffer and macromolecular crowding conditions. Our finding indicates that the local transient ordered microstructure and heterogeneity in the condensates play significant roles in modulating the biophysical properties of the enveloped proteins, and this finding may be essential to further our understanding how phase separation regulates the function of proteins in cells.

Morphological evolution during liquid-liquid phase separation governed by composition change pathways

Liquid-liquid phase separation (LLPS) phenomenon are widely recognized to be of vital importance for physics, materials science, and biology. It is highly desired to develop powerful tools to study the LLPS behavior and related physical mechanisms. For this purpose, a phase-field method was developed here which combines the Cahn-Hilliard diffusion equation and the Navier-Stokes equation. The morphological evolution of LLPS behavior with the change in composition was comprehensively investigated under a prototypical ternary theoretical phase diagram. The phase-field simulation results indicated that the microstructural evolution was controlled by the phase diagram and driven by the coupling of diffusion and gravity effect. Moreover, the intermediate morphological microstructures and corresponding interfacial properties during LLPS could be tuned by selecting different composition change pathways. Furthermore, gravity-dependent density overturning and consequent Rayleigh-Taylor instability were observed in a unique LLPS process, demonstrating that the proposed model can capture the critical features of LLPS phenomenon.

Self-assembly in the transparent droplets formed during the screening of protein self-assembly conditions

Protein self-assemblies at the micro- and nano-scale are of great interest because of their morphological diversity and good biocompatibility. High-throughput screening of protein self-assembly at different scales and morphologies using protein crystallization screening conditions is an emerging method. When using this method to screen protein self-assembly conditions, some apparently transparent droplets are often observed, in which it is not clear whether self-assembly occurs. We explored the interaction between β-lactoglobulin and the protein crystallization kit Index™ C10 and observed the presence of micro- and nano-scale protein self-assemblies in the transparent droplets. The diverse morphology of the micro- and nano-scale self-assemblies in the transparent droplets formed by mixing different initial concentrations of β-lactoglobulin and Index™ C10 was further investigated by scanning electron microscope. Self-assembly process of fluorescence-labelled β-lactoglobulin was monitored continuously by laser confocal microscope, allowing real-time observation of the liquid-liquid phase separation phenomenon and the morphology of the final self-assemblies. The internal structure of the self-assemblies was gradually ordered over time by in-situ X-ray diffraction. This indicates that the self-assembly phenomenon within transparent droplets, observed in protein self-assembly condition screening experiments, is worthy of further in-depth exploration.

Investigation of Agglomeration in the Presence of Oiling Out in the Antisolvent Crystallization Process

Oiling out during the crystallization process often generates agglomeration. This research is focused on the control of agglomeration in the antisolvent crystallization of the propylparaben-ethanol-water system in the presence of oiling out, that is, the liquid-liquid phase separation phenomenon. Crystallization trajectories were designed to start from a homogeneous solution with different initial concentrations of propylparaben in ethanol, crossing the liquid-liquid phase separation region in the ternary phase diagram by adding the antisolvent of water. A combination of process analytical technology tools, such as focused beam reflectance measurement, particle visual monitoring, and attenuated total reflectance ultraviolet/visible, enabled the detection of the different stages of the oiling out crystallization process, including the droplet formation of the dispersed phase, prenucleation, nucleation, and crystal growth both in the liquid-liquid phase-separated system and in the homogeneous solution. The crystals tended to form normally at higher initial concentrations of propylparaben while tending to agglomerate at a lower initial concentration. Crystallization experiments were also performed with the addition of sonication, with variation in sonication start time, duration, and power. The results showed that ultrasound could reduce the agglomeration dependent on the initial application time. The mechanism of crystallization and agglomeration in oiling out was discussed.

Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization.

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (kD) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40°C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.

The Bre1/Rad6 machinery: writing the central histone ubiquitin mark on H2B and beyond.

Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity. Recent discoveries have yielded evidence for the phenomenon of liquid-liquid phase separation in the context of H2Bub1, and its regulation by other histone post-translational modifications. This review summarizes current knowledge about Bre1/Rad6-mediated H2B ubiquitination, including the physiological functions and the molecular basis for writing and regulation of this central histone ubiquitin mark. Possible models for the Bre1/Rad6 machinery bound to nucleosomes bearing different modifications in the writing step are also disclosed.

Formulation of Cinnarizine for Stabilization of Its Physiologically Generated Supersaturation.

Physiologically generated supersaturation and subsequent crystallization of a weakly basic drug in the small intestine leads to compromised bioavailability. In this study, the pH-induced crystallization of cinnarizine (CNZ) in the presence of different polymers was investigated. Inhibitory effect of Eudragit L100 (Eu) on crystallization of CNZ at varying supersaturation ratios was examined. The effect of Eu on the dissolution behavior of CNZ from CNZ/Eu physical mixtures (PMs) and solid dispersions (SDs) was assessed. Results showed that both Eu and hydroxypropyl methylcellulose (HPMC) have a considerable maintenance effect on supersaturation of CNZ but Eu was more effective than HPMC. When Eudragit was used the phenomenon of liquid-liquid phase separation (formation of colloidal phase) was observed at supersaturation ratio of 20 times above the solubility of the drug. PMs showed a higher area under the dissolution curve (AUDC) compared with plain CNZ. In contrast, SDs showed a lower AUDC than plain CNZ. For SDs, the AUDC was limited by the slow release of the drug from Eu in acidic pH which in turn hindered the creation of CNZ supersaturation following the transition of acidic to neutral pH. From these findings, it can be concluded that the ability of the formulation to generate supersaturation state and also maintain the supersaturation is vital for improving the dissolution of CNZ.

Pressure-Induced Dissolution and Reentrant Formation of Condensed, Liquid-Liquid Phase-Separated Elastomeric α-Elastin.

We investigated the combined effects of temperature and pressure on liquid-liquid phase separation (LLPS) phenomena of α-elastin up to the multi-kbar regime. FT-IR spectroscopy, CD, UV/Vis absorption, phase-contrast light and fluorescence microscopy techniques were employed to reveal structural changes and mesoscopic phase states of the system. A novel pressure-induced reentrant LLPS was observed in the intermediate temperature range. A molecular-level picture, in particular on the role of hydrophobic interactions, hydration, and void volume in controlling LLPS phenomena is presented. The potential role of the LLPS phenomena in the development of early cellular compartmentalization is discussed, which might have started in the deep sea, where pressures up to the kbar level are encountered.

PH-Dependent Liquid–Liquid Phase Separation of Highly Supersaturated Solutions of Weakly Basic Drugs

Supersaturated solutions of poorly aqueous soluble drugs can be formed both in vivo and in vitro. For example, increases in pH during gastrointestinal transit can decrease the aqueous solubility of weakly basic drugs resulting in supersaturation, in particular when exiting the acidic stomach environment. Recently, it has been observed that highly supersaturated solutions of drugs with low aqueous solubility can undergo liquid-liquid phase separation (LLPS) prior to crystallization, forming a turbid solution such that the concentration of the drug in the continuous solution phase corresponds to the amorphous solubility while the colloidal phase is composed of a disordered drug-rich phase. Although it is well established that the equilibrium solubility of crystalline weakly basic drugs follows the Henderson-Hasselbalch relationship, the impact of pH on the LLPS phenomenon or the amorphous solubility has not been explored. In this work, the LLPS concentration of three weakly basic compounds-clotrimazole, nicardipine, and atazanavir-was determined as a function of pH using three different methods and was compared to the predicted amorphous solubility, which was calculated from the pH-dependent crystalline solubility and by estimating the free energy difference between the amorphous and crystalline forms. It was observed that, similar to crystalline solubility, the experimental amorphous solubility at any pH follows the Henderson-Hasselbalch relation and can be predicted if the amorphous solubility of the free base is known. Excellent agreement between the LLPS concentration and the predicted amorphous solubility was observed. Dissolution studies of amorphous drugs showed that the solution concentration can reach the corresponding LLPS concentration at that pH. Solid-state analysis of the precipitated material confirmed the amorphous nature. This work provides insight into the pH-dependent precipitation behavior of poorly water-soluble compounds and provides a fundamental basis with which to understand the performance of supersaturating dosage forms.

Enhancements and Limits in Drug Membrane Transport Using Supersaturated Solutions of Poorly Water Soluble Drugs

Amorphous solid dispersions (ASDs) give rise to supersaturated solutions (solution concentration greater than equilibrium crystalline solubility). We have recently found that supersaturating dosage forms can exhibit the phenomenon of liquid-liquid phase separation (LLPS). Thus, the high supersaturation generated by dissolving ASDs can lead to a two-phase system wherein one phase is an initially nanodimensioned and drug-rich phase and the other is a drug-lean continuous aqueous phase. Herein, the membrane transport of supersaturated solutions, at concentrations above and below the LLPS concentration has been evaluated using a side-by-side diffusion cell. Measurements of solution concentration with time in the receiver cell yield the flux, which reflects the solute thermodynamic activity in the donor cell. As the nominal concentration of solute in the donor cell increases, a linear increase in flux was observed up to the concentration where LLPS occurred. Thereafter, the flux remained essentially constant. Both nifedipine and felodipine solutions exhibit such behavior as long as crystallization is absent. This suggests that there is an upper limit in passive membrane transport that is dictated by the LLPS concentration. These results have several important implications for drug delivery, especially for poorly soluble compounds requiring enabling formulation technologies.

Multielement NMR Studies of the Liquid–Liquid Phase Separation and the Metal-to-Nonmetal Transition in Fluid Lithium– and Sodium–Ammonia Solutions

(1)H, (7)Li, (14)N, and (23)Na high resolution nuclear magnetic resonance (NMR) measurements are reported for fluid solutions of lithium and sodium in anhydrous liquid ammonia across the metal-to-nonmetal transition (MNM transition), paying particular attention to the phenomenon of liquid-liquid phase separation which occurs in the composition/temperature region close to the MNM transition. Our results are discussed in terms of the electronic structure of fluid metal-ammonia solutions at low temperatures (ca. 240 K). We find that the electronic phase transition to the metallic state in these solutions, especially at temperatures close to the liquid-liquid critical consolute temperature, occurs from a nonmetallic, electrolytic solution containing a predominance of electron spin-paired, (diamagnetic) charged bosonic states. The possible implications of these observations to the nature of the liquid-liquid phase separation are discussed, both from the views of N. F. Mott, regarding the MNM transition in sodium-ammonia solutions, and those of R. A. Ogg, regarding the possibility of high-temperature superconductivity in these solutions. Similarities between the electronic structure of metal-ammonia solutions and the high-temperature cuprate superconductors are also briefly emphasized.

The Structure Of Liquid Water; Novel Insights From Materials Research; Potential Relevance To Homeopathy

This paper provides an interdisciplinary base of information on the structure of liquid water. It begins with a synthesis built on the information base on the structure 5 of noncrystalline, inorganic, covalently-bonded condensed liquid phases, such as SiO2, S, Se, P, and H2O, which exists in the materials science literature. The data for water are analyzed through the prism of well-established algorithms in materials research: the connection of properties to structure; the pressure-temperature (P-T) phase diagrams; the phenomenon of epitaxy; the phenomenon of liquid-liquid phase separation; the stability of two phase colloids; and, the recently discovered effects of weak magnetic and electric fields on the structure of simple inorganic oxides. A thorough combing of the literature of the condensed matter properties reflecting structural features of essentially pure water obtained via the normal processes of preparing homeopathic remedies, provides another rich data base. The examination of these data through the standard materials science paradigms leads to the following conclusion: Many different structures of liquid water must exist within the range of observations and processes encountered near ambient conditions. A typical sample of water in these experimental ranges no doubt consists of a statistical-mechanical-determined assemblage of monomers and oligomers (clusters) of various sizes up to at least several hundred H2O units. The importance of the structural similarity of SiO2 and OH2 is very relevant to the structure of the latter as well as to the probability of epitaxy in controlling at least the region contiguous to the silicate glass surfaces of many common containers. The most distinctive feature of bonding in liquid water is not only the “well-known hydrogen bonds, but the necessary presence of a wide range” of van der Waals bonds between and among the various oligomeric (cluster) structural units. It is this range of very weak bonds that could account for the remarkable ease of changing the structure of water, which in turn could help explain the half-dozen well-known anomalies in its properties. In its subtler form, such weak bonds would also allow for the changes of structure caused by electric and

Evaluation of thermodynamic parameters for blends of polyethersulfone and poly(methyl methacrylate) or polystyrene in dimethylformamide

Liquid-liquid phase separation phenomena have been investigated for a ternary system containing two polymers and a solvent. Namely, dimethylformamide (DMF)/polyethersulfone (PES)/poly(methyl methacrylate) (PMMA) and DMF/PES/Polystyrene (PS). The composition of the three components in the two phases in equilibrium has been determined by size exclusion chromatographic (s.e.c.) analysis. The lattice-based mean-field theory, first developed by Flory and Huggins, has been modified to adequately describe these systems. In this respect, we have assumed that the parameters depend on the polymer concentration, and we have included a ternary parameter. The phase equilibrium compositions have been used as input data to solve a set of equations raised following four different approximations mainly concerned with the composition dependence of the interaction parameters. Values of the Gibbs free energy of mixing as a function of the blend composition were also evaluated and discussed in terms of the blend stability.