Liquid-like State Research Articles

Phase-separating peptide coacervates with programmable material properties for universal intracellular delivery of macromolecules

Phase-separating peptides (PSPs) self-assembling into coacervate microdroplets (CMs) are a promising class of intracellular delivery vehicles that can release macromolecular modalities deployed in a wide range of therapeutic treatments. However, the molecular grammar governing intracellular uptake and release kinetics of CMs remains elusive. Here, we systematically manipulate the sequence of PSPs to unravel the relationships between their molecular structure, the physical properties of the resulting CMs, and their delivery efficacy. We show that a few amino acid alterations are sufficient to modulate the viscoelastic properties of CMs towards either a gel-like or a liquid-like state as well as their binding interaction with cellular membranes, collectively enabling to tune the kinetics of intracellular cargo release. We also demonstrate that the optimized PSPs CMs display excellent transfection efficiency in hard-to-transfect cells such as primary fibroblasts and immune cells. Our findings provide molecular guidelines to precisely program the material properties of PSP CMs and achieve tunable cellular uptake and release kinetics depending on the cargo modality, with broad implications for therapeutic applications such as protein, gene, and immune cell therapies.

Enhanced characterization and utilization of cowpea protein isolate: rheological and textural properties of heat-induced gels for plant-based egg omelet formulation

This study aimed to evaluate the potential of cowpea isolate (CPI) as an effective plant-based egg substitute by comparing its composition and physicochemical properties with those of commercial isolates, including mung bean, soy, and pea proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis identified a distinctive vicilin-like globulin in CPI that is critical for the unique functional properties of CPI. CPI has a smaller particle size, resulting in superior gelation capacity and water retention, which are essential for egg substitute applications. Thermal analysis revealed the high stability of CPI, which is advantageous for food processing. Notably, the unique heat-induced gelation behavior of CPI, characterized by a smooth transition from solid- to liquid-like states, provided a strength not observed in its commercial counterparts. Microstructural analysis showed that the CPI gel closely resembled the egg gel, underscoring the potential of CPI to mimic the texture of eggs, particularly in omelets.

Engineering water-shut-off operations: Application of rheological time-temperature superposition approach for organically-crosslinked polymer gel systems

Polymer-based gels have been extensively utilized in reservoirs to prevent excessive water production and improve oil recovery. In this study, a sulfonated-hydrolyzed polyacrylamide polymer (HPAM-S) and low-toxic organic crosslinkers (hydroquinone and hexamethylenetetramine) were mixed in brine at specified concentrations to formulate the polymer-crosslinker fluid system. The quantitative rheological analysis, along with the TTS (Time-Temperature Superposition) approach, was examined for the first time to achieve the water shut-off operations in high-temperature reservoirs (90 °C–150 °C). The TTS approach was defined as modelling of the viscosity vs. time data obtained at various temperatures using an appropriate empirical shift factor (at) such that a single master curve is established for the viscosity evolution of the given fluid system. This approach facilitates the prediction of the gelation behavior of polymer-crosslinker fluid systems as a function of time at varied reservoir temperatures especially over time scales that are not experimentally accessible. The time-dependent rheological evolution of the fluid system was experimentally investigated using batch-wise bottle testing methods as well as viscosity and viscoelastic measurements by employing an advanced rheometer. The TTS approach was applied to the obtained rheological data at varied temperatures. The model was validated for an unknown set of temperature vs. viscosity data. Based on the TTS approach, the term ‘gelation time’ was defined as the time required to reach a pre-specified viscosity to signify a transition from the liquid-like state to the solid-like state. The gelation time decreases with increasing temperatures. After the gelation time, at each temperature, a swift increase in the fluid viscosity was observed, indicating the initiation of the rapid crosslinking in the fluid systems. It was found that beyond the gelation time, the batch-wise bottle testing showed (G-I) gel strength (qualitative). Analogously, the visco-elasticity test demonstrated a rapid rise in elastic modulus (G′) beyond the gelation time. An underlying mechanism based on polymer hydrolysis was utilized to explain the evolution of distinct viscous and viscoelastic properties as a function of temperature. Moreover, the modeling predictions for the fluid systems were examined for in-situ rock plugging with the help of core-flood equipment. The core-flooding tests confirmed a 99% reduction in the rock permeability beyond the gelation time. The gelation time values obtained from both the batch-wise bottle testing method and rheological tests for the polymer-crosslinker fluid systems were found in agreement with each other, with a tolerance of (±5%).

In vitro study of the gel cohesivity and persistence to hyaluronidase degradation of a novel stabilized composition of 26 mg/mL of high molecular weight HA

Aim: Hyaluronic acid (HA) is extensively used in injectable skin quality products due to its documented role in skin rejuvenation. The rapid in vivo degradation of HA by the enzyme hyaluronidase necessitates the development of advanced formulations to ensure the efficacy and longevity of the treatments. In this context, a novel 2.6% high molecular weight HA (H-HA)/3.2% sorbitol composition has been introduced, featuring stabilization through hydrogen bonds rather than traditional crosslinking. Methods: The stabilized composition was evaluated through two in vitro enzymatic degradation tests. In the first test, the efficiency on the gel degradation was followed by rheology and compared with two crosslinked HA products available on the market. In the second test, the effect on the gel structure of a less diluted hyaluronidase dose was followed by rheological and cohesivity measurements. Results: In vitro study demonstrates that, before its complete degradation into a liquid-like state, the stabilized composition exhibits high elasticity and cohesivity during the enzymatic degradation process, surpassing traditional crosslinked HA products. Conclusion: The stabilization provided by the sorbitol in the stabilized composition effectively enhances product properties and protects them during gel degradation. These attributes indicate significant potential for improved clinical outcomes in skin quality treatments.

Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in Escherichia coli Cells.

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.

Development of low-molecular-weight polysaccharide-based wound dressings for full-thickness cutaneous wound healing via coacervate formation

The development of polysaccharide-based wound dressings that are easy to prepare, adhere to tissue, adapt to diverse shapes and exhibit tunable mechanical properties holds significant clinical interest. This study introduced a simple spontaneous liquid-liquid phase separation technique employing low-molecular-weight and high polyion concentration of chitosan (CS) and hyaluronic acid (HA) to fabricate CS/HA coacervates. Upon increasing the molecular weight of chitosan from 7 kDa to 250 kDa, a transition in the CS/HA coacervates from liquid-like state to an elastic liquid and eventually to a solid-like state was observed. The resulting CS/HA coacervates demonstrated robust water resistance and adhesion to skin tissue. Notably, the molecular weight of chitosan significantly influenced the mechanical properties and hydration levels of the CS/HA coacervates. Moreover, in vivo studies using a full-thickness cutaneous defect model revealed that the CS/HA coacervates, prepared using 100 kDa chitosan, markedly accelerate wound healing. The coacervates' ease of preparation, wet adhesion, heterogeneous structure, suitability for irregularly shaped wounds, and exceptional wound healing promotion of the coacervates qualify them as an optimal wound dressing.

Aggregation of the amyloid-β peptide (Aβ40) within condensates generated through liquid–liquid phase separation

The deposition of the amyloid-β (Aβ) peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Recently, it has been reported that some proteins can aggregate and form amyloids through an intermediate pathway involving a liquid-like condensed phase. These observations prompted us to investigate the phase space of Aβ. We thus explored the ability of Aβ to undergo liquid–liquid phase separation, and the subsequent liquid-to-solid transition that takes place within the resulting condensates. Through the use of microfluidic approaches, we observed that the 40-residue form of Αβ (Αβ40) can undergo liquid–liquid phase separation, and that accessing a liquid-like intermediate state enables Αβ40 to self-assemble and aggregate into amyloid fibrils through this pathway. These results prompt further studies to investigate the possible role of Αβ liquid–liquid phase separation and its subsequent aggregation in the context of Alzheimer’s disease and more generally on neurodegenerative processes.

Fractal-Dependent Growth of Solidlike Condensates.

The phenomenon of droplet growth occurs in various industrial and natural processes. Recently, the discovery of liquidlike condensates within cells has sparked an increasing interest in understanding their growth behaviors. These condensates exhibit varying material properties that are closely related to many cellular functions and diseases, particularly during the phase transition from liquidlike droplets to solidlike aggregates. However, how the liquid-to-solid phase transition affects the growth of condensates remains largely unknown. In this study, we investigate the growth of peptide-RNA condensates, which behave as either liquidlike droplets or solidlike aggregates depending on the RNA sequences. Dynamic light scattering experiments show that solidlike condensates grow surprisingly faster, with their hydrodynamic diameters increasing over time as d_{h}(t)∼t^{1/2}, contrasting with d_{h}(t)∼t^{1/3} for liquidlike droplets. By combining theoretical analysis and simulations, we demonstrate that this accelerated growth is caused by the noncoalescence aggregation of solidlike condensates and thus formation of percolated swollen structures with a decreased fractal dimension. Moreover, we demonstrate that the accelerated growth can be slowed down by introducing agents that can revert solidlike condensates back to their liquidlike states, such as urea or specific RNAs. Together, our work reveals a fractal-dependent growth mechanism of condensates, with useful insights for understanding the aging of condensates and modulating their aggregation behaviors in synthetic and biological systems.

Sequence programmable nucleic acid coacervates.

Nature uses bottom-up self-assembly to build structures with remarkable complexity and functionality. Understanding how molecular-scale interactions translate to macroscopic properties remains a major challenge and requires systems that effectively bridge these two scales. Here, we generate DNA and RNA liquids with exquisite programmability in their material properties. Nucleic acids are negatively charged, and in the presence of polycations, they may condense to a liquid-like state. Within these liquids, DNA and RNA retain sequence-specific hybridization abilities. We show that intermolecular hybridization in the condensed phase cross-links molecules and slows down chain dynamics. This reduced chain mobility is mirrored in the macroscopic properties of the condensates. Molecular diffusivity and material viscosity scale with the intermolecular hybridization energy, enabling precise sequence-based modulation of condensate properties over orders of magnitude. Our work offers a robust platform to create self-assembling programmable fluids and may help advance our understanding of liquid-like compartments in cells.

Dynamical phase transition in models that couple chromatin folding with histone modifications.

Genomic regions can acquire heritable epigenetic states through unique histone modifications, which lead to stable gene expression patterns without altering the underlying DNA sequence. However, the relationship between chromatin conformational dynamics and epigenetic stability is poorly understood. In this paper, we propose kinetic models to investigate the dynamic fluctuations of histone modifications and the spatial interactions between nucleosomes. Our model explicitly incorporates the influence of chemical modifications on the structural stability of chromatin and the contribution of chromatin contacts to the cooperative nature of chemical reactions. Through stochastic simulations and analytical theory, we have discovered distinct steady-state outcomes in different kinetic regimes, resembling a dynamical phase transition. Importantly, we have validated that the emergence of this transition, which occurs on biologically relevant timescales, is robust against variations in model design and parameters. Our findings suggest that the viscoelastic properties of chromatin and the timescale at which it transitions from a gel-like to a liquidlike state significantly impact dynamic processes that occur along the one-dimensional DNA sequence.

Cell Division and Motility Enable Hexatic Order in Biological Tissues.

Biological tissues transform between solid- and liquidlike states in many fundamental physiological events. Recent experimental observations further suggest that in two-dimensional epithelial tissues these solid-liquid transformations can happen via intermediate states akin to the intermediate hexatic phases observed in equilibrium two-dimensional melting. The hexatic phase is characterized by quasi-long-range (power-law) orientational order but no translational order, thus endowing some structure to an otherwise structureless fluid. While it has been shown that hexatic order in tissue models can be induced by motility and thermal fluctuations, the role of cell division and apoptosis (birth and death) has remained poorly understood, despite its fundamental biological role. Here we study the effect of cell division and apoptosis on global hexatic order within the framework of the self-propelled Voronoi model of tissue. Although cell division naively destroys order and active motility facilitates deformations, we show that their combined action drives a liquid-hexatic-liquid transformation as the motility increases. The hexatic phase is accessed by the delicate balance of dislocation defect generation from cell division and the active binding of disclination-antidisclination pairs from motility. We formulate a mean-field model to elucidate this competition between cell division and motility and the consequent development of hexatic order.

Ultralow two-channel thermal conductivity in aikinite

Realizing glass-like ultralow lattice thermal conductivity κL in an ordered crystalline material is overwhelmingly appealing for various applications in thermoelectrics and thermal barrier coatings. Here, based on first-principles calculations, we take CuPbBiS3 as an example and systematically study the underlying physical mechanism of the ultralow and glass-like κL in aikinite. It is found that the strong anharmonic renormalization induces temperature-driven stiffening of the acoustic modes and low-lying optical modes from Pb, Cu, and Bi atoms, while leading to the softening of high-frequency optical phonon modes from S atoms. Further analyses highlight that CuPbBiS3 hosts metavalent bonding, stereochemical lone pair activity, and loosely bonded rattling atoms. These characteristics arouse partially liquid-like state, synergistically contribute to strong anharmonicity, and result in unexpectedly low thermal conductivity. Finally, our calculated κL at 300 K is 0.68 Wm−1K−1 with a non-standard dependency of κL ∝ T−0.7, which reasonably agrees well with experimental values in both magnitude (0.52 ± 0.05 Wm−1K−1 at 300 K) and temperature-dependence trend (∝ T−0.2). We demonstrate that the anomalous thermal transport originates from the dual particle-wave behavior of the heat-carrying phonons, in which wavelike tunneling contribute over 18 % to the total κL when T exceeds 300 K. Our work underpins the microscopic origins of ultralow κL in CuPbBiS3, providing crucial insights into designing highly efficient aikinite materials for thermoelectric conversion.

Pseudofermion functional renormalization group study of dipolar-octupolar pyrochlore magnets

Motivated by recent experiments on Ce2Zr2O7 that reveal a dynamic, liquidlike ground state, we study the nearest-neighbor XYZ Hamiltonian of dipolar-octupolar pyrochlore magnets with the pseudofermion functional renormalization group (PFFRG), which is numerically implemented by the software. Taking the interaction between the octupolar components to be dominant and antiferromagnetic, we map out the phase diagram demarcating the quantum disordered and magnetically ordered states. We identify four distinct phases, namely, the 0-flux and π-flux quantum spin ices and the all-in-all-out magnetic orders along the local z and x axes. We further use the static two-spin correlation output by the PFFRG algorithm to compute the polarized neutron scattering cross sections, which are able to capture several qualitative features observed experimentally, in the materially relevant parameter regime that stabilizes the π-flux quantum spin ice. Our results provide support for a quantum spin-liquid ground state in Ce2Zr2O7. Published by the American Physical Society 2024

Thermodynamic crossovers in supercritical fluids

Can liquid-like and gas-like states be distinguished beyond the critical point, where the liquid-gas phase transition no longer exists and conventionally only a single supercritical fluid phase is defined? Recent experiments and simulations report strong evidence of dynamical crossovers above the critical temperature and pressure. Despite using different criteria, many existing theoretical explanations consider a single crossover line separating liquid-like and gas-like states in the supercritical fluid phase. We argue that such a single-line scenario is inconsistent with the supercritical behavior of the Ising model, which has two crossover lines due to its symmetry, violating the universality principle of critical phenomena. To reconcile the inconsistency, we define two thermodynamic crossover lines in supercritical fluids as boundaries of liquid-like, indistinguishable, and gas-like states. Near the critical point, the two crossover lines follow critical scalings with exponents of the Ising universality class, supported by calculations of theoretical models and analyses of experimental data from the standard database. The upper line agrees with crossovers independently estimated from the inelastic X-ray scattering data of supercritical argon, and from the small-angle neutron scattering data of supercritical carbon dioxide. The lower line is verified by the equation of states for the compressibility factor. This work provides a fundamental framework for understanding supercritical physics in general phase transitions.

Influence of cellulose nanofibers on the behavior of Pickering emulsions. Part II: Thixotropy and dynamic-mechanical tests

Nonlinear rheology of Pickering emulsions is used to further investigate the nonlinear and unrecoverable transformation of inner structures, which is beyond the linear viscoelastic regime of tiny structural disturbances. Exploring various rheological methods plays a vital role in emulsion applications, such as simulating the macroscopic structural transformation between static and liquidlike flow states, strain overshoot, and regeneration for broken structures. According to our previous studies, cellulose nanofibers (CNFs) Pickering emulsions are a typical system for investigating polymer-based emulsions with the auxiliary surfactant [didodecyldimethylammonium bromide (DDAB)] for enhancing CNF absorption. To further study different rheological properties by varying CNF or DDAB contents, multiple interval thixotropic test, large amplitude oscillatory shear, and concentration-time-dependent superposition are employed to study the linear viscoelasticity and structural transformation of nonlinear range. This research was conducted based on the previous published works [Cui et al., Materials 15, 8285 (2022)] as a further characterization for the same sample series.

Topological balance of cell distributions in plane monolayers

Most of normal proliferative epithelia of plants and metazoans are topologically invariant and characterized by similar cell distributions according to the number of cell neighbors (DCNs). Here we study peculiarities of these distributions and explain why the DCN obtained from the location of intercellular boundaries and that based on the Voronoi tessellation with nodes located on cell nuclei may differ from each other. As we demonstrate, special microdomains where four or more intercellular boundaries converge are topologically charged. Using this fact, we deduce a new equation describing the topological balance of the DCNs. The developed theory is applied for a series of microphotographs of non-tumoral epithelial cells of the human cervix (HCerEpiC) to improve the image processing near the edges of microphotographs and reveal the topological invariance of the examined monolayers. Special contact microdomains may be present in epithelia of various natures, however, considering the well-known vertex model of epithelium, we show that such contacts are absent in the usual solid-like state of the model and appear only in the liquid-like cancer state. Also, we discuss a possible biological role of special contacts in context of proliferative epithelium dynamics and tissue morphogenesis.

Lipid droplets as substrates for protein phase separation

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

Influence of repulsion on entropy scaling and density scaling of monatomic fluids.

Entropy scaling is applied to the shear viscosity, self-diffusion coefficient, and thermal conductivity of simple monatomic fluids. An extensive molecular dynamics simulation series is performed to obtain these transport properties and the residual entropy of three potential model classes with variable repulsive exponents: n, 6 Mie (n = 9, 12, 15, and 18), Buckingham's exponential-six (α = 12, 14, 18, and 30), and Tang-Toennies (αT = 4.051, 4.275, and 4.600). A wide range of liquid and supercritical gas- and liquid-like states is covered with a total of 1120 state points. Comparisons to equations of state, literature data, and transport property correlations are made. Although the absolute transport property values within a given potential model class may strongly depend on the repulsive exponent, it is found that the repulsive steepness plays a negligible role when entropy scaling is applied. Hence, the plus-scaled transport properties of n, 6 Mie, exponential-six, and Tang-Toennies fluids lie basically on one master curve, which closely corresponds with entropy scaling correlations for the Lennard-Jones fluid. This trend is confirmed by literature data of n, 6 Mie, and exponential-six fluids. Furthermore, entropy scaling holds for state points where the Pearson correlation coefficient R is well below 0.9. The condition R > 0.9 for strongly correlating liquids is thus not necessary for the successful application of entropy scaling, pointing out that isomorph theory may be a part of a more general framework that is behind the success of entropy scaling. Density scaling reveals a strong influence of the repulsive exponent on this particular approach.

The in-cell liquid-like state of the nuclear pore complex permeability barrier

The in-cell liquid-like state of the nuclear pore complex permeability barrier

2D Ionic Liquid-Like State of Charged Rare-Earth Clusters on a Metal Surface.

Rare-earth complexes are vital for separation chemistry and useful in many advanced applications including emission and energy upconversion. Here, 2D rare-earth clusters having net charges are formed on a metal surface, enabling investigations of their structural and electronic properties on a one-cluster-at-a-time basis using scanning tunneling microscopy. While these ionic complexes are highly mobile on the surface at ≈100 K, their mobility is greatly reduced at 5 K and reveals stable and self-limiting clusters. In each cluster, a pair of charged rare-earth complexes formed by electrostatic and dispersive interactions act as a basic unit, and the clusters are chiral. Unlike other non-ionic molecular clusters formed on the surfaces, these rare-earth clusters show mechanical stability. Moreover, their high mobility on the surface suggests that they are in a 2D liquid-like state.