Synthetic Membraneless Droplets for Synaptic-Like Clustering of Lipid Vesicles

In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.     

Protein/polysaccharide complexes and coacervates in food systems

Since the pioneering work of Bungenberg de Jong and co-workers on gelatin-acacia gum complex coacervation in the 1920-40s, protein/polysaccharide complexes and coacervates have received increasing research interest in order to broaden the possible food applications. This review focuses on the main research streams followed in this field during the last 12 years regarding: i) the parameters influencing the formation of complexes and coacervates in protein-polysaccharide systems; ii) the characterization of the kinetics of phase separation and multi-scale structure of the complexes and coacervates; and iii) the investigation of the functional properties of complexes and coacervates in food applications. This latter section encompasses various technological aspects, namely: the viscosifying and gelling ability, the foaming and emulsifying ability and finally, the stabilization and release of bioactives or sensitive compounds.     

Coacervate formation studied by explicit solvent coarse-grain molecular dynamics with the Martini model

Complex coacervates are liquid–liquid phase separated systems, typically containing oppositely charged polyelectrolytes. They are widely studied for their functional properties as well as their potential involvement in cellular compartmentalization as biomolecular condensates. Diffusion and partitioning of solutes into a coacervate phase are important to address because their highly dynamic nature is one of their most important functional characteristics in real-world systems, but are difficult to study experimentally or even theoretically without an explicit representation of every molecule in the system. Here, we present an explicit-solvent, molecular dynamics coarse-grain model of complex coacervates, based on the Martini 3.0 force field. We demonstrate the accuracy of the model by reproducing the salt dependent coacervation of poly-lysine and poly-glutamate systems, and show the potential of the model by simulating the partitioning of ions and small nucleotides between the condensate and surrounding solvent phase. Our model paves the way for simulating coacervates and biomolecular condensates in a wide range of conditions, with near-atomic resolution.

Martini 3 force field can capture the experimental trends of complex coacervates and can be extended to gain physical insight on the mechanisms that drive the formation of LLPS.     

Protein–polysaccharide complexes and coacervates

Recent advances on protein–polysaccharide electrostatic complex and coacervates' formation, structure and properties are presented. The first section emphasises on the thermodynamics of complexes formation, the contribution of enthalpic and entropic effects is specifically discussed. Then, their structure is described at different length scales and recent findings on the phase-ordering dynamics of complex and coacervates formation are covered. Finally, the most relevant functional properties of protein–polysaccharide complexes and coacervates for food applications are described.     

Studying phase separation in confinement

Cells organize their interior through membrane-bound organelles and through membraneless condensates that are formed by liquid–liquid phase separation (LLPS). The complex process of coacervation that is involved in LLPS is challenging to study in living cells. Hence, studying coacervation in cell-mimicking synthetic containers can yield valuable insights. Here, we review recent progress with respect to studying LLPS (particularly coacervation) in artificial compartments, from water-in-oil droplets to membranous liposomes. We describe different strategies to form and control coacervates in microconfinements and to study their physicochemical and biological characteristics. We also describe how coacervation can itself be used in container formation. This review highlights the importance of in vitro coacervate studies for understanding cellular biology and for designing synthetic cells.     

Molecular determinants of protein-based coacervates

Protein–polyelectrolyte coacervates have gained interest for their potential to stabilize proteins or function as adhesives and their biological implications in the formation of membraneless organelles. To effectively design these materials or predict their biological formation, knowledge of the macromolecular properties that dictate phase separation is required. This review highlights recent advances in the understanding of molecular determinants of protein–polyelectrolyte phase behavior. Properties that promote the phase separation of protein–polyelectrolyte pairs are covered from the perspective of synthetic systems and simplified biological condensates. Prominent factors that determine coacervate formation and material properties include nonspecific intermolecular interactions, as well as specific biological interactions and structures. Here, we summarize the essential roles of electrostatics, including charge magnitude and distribution, (bio)polymer chemistry and structure, and post-translational modifications to protein phase separation in both a synthetic and cellular context.     

Preparation of zinc sodium polyphosphates glasses from coacervates precursors. Characterisation of the obtained glasses, and their applications

A soft chemistry route is described to obtain glasses in the P₂O₅–Na₂O–ZnO–H₂O. It is based on the addition of zinc salts to coacervates prepared from sodium polyphosphate. The processing of these coacervates leads to polyphosphate glasses with the same properties as those of glasses prepared in the classical way. So far, little work has been implemented in this system using ‘coacervate route’. However, it makes an attractive method for coating and joining processes on the industrial scale. As the anion associated to zinc may take part in the adhesion mechanism, coacervate formation has been studied using zinc chloride, nitrate and sulphate as starting materials. The physical properties of the glasses obtained by this method are reported and potential applications of zinc and silver coacervate are described.     

Glutaraldehyde-crosslinked O-carboxymethyl chitosan–gum Arabic coacervates: Characteristics versus complexation acidity

Glutaraldehyde-crosslinked O-carboxymethyl chitosan (O-CMC)–gum Arabic (GA) coacervates were characterized against coacervation acidity. As the coacervation pH increased from 3.0 to 6.0, the crosslinking degree of the coacervates and its sensitivity to glutaraldehyde concentration variation declined gradually, but the elasticity increased markedly. Crosslinking improved the structure compactness and thermal stability of the coacervates and high coacervation pH favored the increase of the two parameters, but a reverse trend was observed regarding swelling ratio in the simulated gastric fluid. It was concluded that glutaraldehyde-crosslinked O-CMC–GA coacervates with required properties could be tailored by selecting an appropriate complexation acidity.     

Microstructure of beta-lactoglobulin/pectin coacervates studied by small-angle neutron scattering

Small-angle neutron scattering (SANS) has been used to investigate the microstructure of beta-lactoglobulin/pectin coacervates prepared by different initial protein/polysaccharide weight ratio (r), sodium chloride concentration (C(NaCl)), and pectin charge density. The higher r and higher pectin charge density lead to higher scattering intensity at small q range (0.007 Angstrom(-1) < q < 0.02 Angstrom(-1)), suggesting that the charges of pectin chains are screened significantly by the binding of oppositely charged protein molecules, leading to a tighter aggregation of pectin chains. On the other hand, the appearance of a shoulder peak at intermediate q range (0.04 Angstrom(-1) < q < 0.2 Angstrom(-1)) is used to interpret the formation of protein domains in beta-lactoglobulin/pectin coacervates. At C(NaCl) = 0.1 M, the coacervate of beta-lactoglobulin and pectin A does not show a shoulder peak at intermediate q range at r = 10:1, suggesting that protein molecules are separately bound on pectin chains. However, a shoulder peak appears at intermediate q range at r = 20:1 and 30:1, and the average protein domain size estimated from the shoulder peak position is 7.2 and 8.5 nm, respectively, for these two coacervates. When C(NaCl) increases from 0.05 to 0.2 M, the shoulder peak shifts toward smaller q and becomes broader, indicating that the addition of a higher amount of salt leads to a more heterogeneous coacervate structure. Pectin B with a lower linear charge density favors the formation of larger protein domains. The formation of protein domains in beta-lactoglobulin/pectin coacervates is partially ascribed to the self-aggregation of beta-lactoglobulin molecules. Two kinds of microstructures of beta-lactoglobulin/pectin coacervates with and without observable protein domains have been proposed.     

Fabrication and characterization of Grass pea (Lathyrus sativus) protein isolate-Alyssum homolocarpum seed gum complex coacervate

The present work was carried out to fabricate Grass pea (Lathyrus sativus) protein isolate (GPPI) and Alyssum homolocarpum seed gum (AHSG) complex coacervate and investigate its morphological and structural properties. Formation of complex coacervates were studied by zeta potentiometry and turbidimetric analysis as function of different pHs (7.0–2.0) and GPPI to AHSG ratios (3:1 to 1:3). The critical pH values associated with the formation of soluble (pH_c) and insoluble (pH_φ1) complexes, and complete dissociation (pH_φ2) at GPPI to AHSG ratio (1:1) were found to be 4.8, 4.0, and 2.5, respectively. Formation of insoluble complex coacervate was maximum at pH 3.2 and GPPI to AHSG ratio of 1:1, where the highest yield (69%) was observed. Scanning electron microscopic (SEM) demonstrated that GPPI-AHSG complex had a rough branched-like structure. Results also showed that the complexation between GPPI and AHSG were formed through the electrostatic interaction and hydrogen bonding. Circular dichroism spectroscopy (CD) indicated that the β-sheet and random coil content in GPPI increased when AHSG molecules were added to protein solution. The fading of pure peaks of GPPI and AHSG in X-Ray diffraction (XRD) patterns of GPPI-AHSG complexes confirmed the alterations in the physical state of mixture from crystalline to amorphous. GPPI-AHSG complex coacervates had lower weight loss compared to individual GPPI and AHSG.     

Sequestration of Proteins by Fatty Acid Coacervates for Their Encapsulation within Vesicles

Encapsulating biological materials in lipid vesicles is of interest for mimicking cells; however, except in some particular cases, such processes do not occur spontaneously. Herein, we developed a simple and robust method for encapsulating proteins in fatty acid vesicles in high yields. Fatty acid based, membrane‐free coacervates spontaneously sequester proteins and can reversibly form membranous vesicles upon varying the pH value, the precrowding feature in coacervates allowing for protein encapsulation within vesicles. We then produced enzyme‐enriched vesicles and show that enzymatic reactions can occur in these micrometric capsules. This work could be of interest in the field of synthetic biology for building microreactors.     

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Coacervate microdroplets produced by liquid–liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane‐free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light‐responsive coacervate droplets prepared from mixtures of double‐stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light‐activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.     

A Water‐Borne Adhesive Modeled after the Sandcastle Glue of P. californica

Polyacrylate glue protein analogs of the glue secreted by Phragmatopoma californica, a marine polycheate, were synthesized with phosphate, primary amine, and catechol sidechains with molar ratios similar to the natural glue proteins. Aqueous mixtures of the mimetic polyelectrolytes condensed into liquid complex coacervates around neutral pH. Wet cortical bone specimens bonded with the coacervates, oxidatively crosslinked through catechol sidechains, had bond strengths nearly 40% of the strength of a commercial cyanoacrylate. The unique material properties of complex coacervates may be ideal for development of clinically useful adhesives and other biomaterials.

     

Effects of dilute acid and steam explosion pretreatments on the cellulose structure and kinetics of cellulosic fraction hydrolysis by dilute acids in lignocellulosic materials

Worldwide, yellows diseases impact plants important in human nutrition, the natural environment, and the culture and commerce of humans. Since the presumed pathogens, mycoplasma-like organisms (MLOs), have not been isolated in pure culture in vitro, their study must proceed by other experimental approaches. In a study of disease affecting grapevines in Europe and North America, polymerase chain reactions (PCR) and restriction analyses of PCR-amplified DNA were used to detect and differentiate strains of MLOs associated with grapevine yellows. MLOs were detected both in naturally diseased grapevines and in experimentally inoculated host plants. The data indicated an unexpected genomic diversity among grapevine-infecting MLOs, and supported their classification with MLOs in the aster yellows, X-disease, and elm yellows groups. The presence of diverse MLOs in grapevines provokes consideration that these MLOs may be present in overlapping geographic ranges and that multiple MLO infections may occur in individual plants, increasing the complexity of grapevine yellows epidemiology and control and the significance of sensitive MLO detection in planting stock and phytosanitary-regulated germplasm.     

Complexation and coacervation of polyelectrolytes with oppositely charged colloids

Polyelectrolyte-colloid coacervation could be viewed as a sub-category of complex coacervation, but is unique in (1) retaining the structure and properties of the colloid, and (2) reducing the heterogeneity and configurational complexity of polyelectrolyte-polyelectrolyte (PE-PE) systems. Interest in protein-polyelectrolyte coacervates arises from preservation of biofunctionality; in addition, the geometric and charge isotropy of micelles allows for better comparison with theory, taking into account the central role of colloid charge density. In the context of these two systems, we describe critical conditions for complex formation and for coacervation with regard to colloid and polyelectrolyte charge densities, ionic strength, PE molecular weight (MW), and stoichiometry; and effects of temperature and shear, which are unique to the PE-micelle systems. The coacervation process is discussed in terms of theoretical treatments and models, as supported by experimental findings. We point out how soluble aggregates, subject to various equilibria and disproportionation effects, can self-assemble leading to heterogeneity in macroscopically homogeneous coacervates, on multiple length scales.     

From colloids in liquid crystals to colloidal liquid crystals

ABSTRACT

Liquid crystals in combination with nanoparticles are a fascinating topic of research, because of the wealth of aspects and questions to study. These range from simple effects of nanoparticles on phase transitions and phase diagrams, to the tuning of physical properties, adding of novel functionalities, all the way to the formation of spontaneous order by nanoparticles themselves and the possibilities that templating has for future materials design and applications. This article intends to provide a flavour of the multiplicity, variety and diversity that these thermotropic and lyotropic systems have to offer in the area of materials development, which we believe will become increasingly important, especially for switchable non-display applications and nanotechnology. It is not intended to provide a conclusive overview, which would be a presumptuous attempt considering the limited space available, but rather to place our own work into a wider context and to point out some more recent developments and trends in liquid crystal – nanoparticle dispersions.     

Characterization of O-Carboxymethyl Chitosan – Gum Arabic Coacervates as a Function of Degree of Substitution

Three O-carboxymethylated chitosans (O-CMCs) with degrees of substitution (DS) 0.41, 0.51, and 0.83 were synthesized and the effects of the DS of O-CMC on its coacervation with gum Arabic (GA) and partial characteristics of resultant coacervates were investigated. All the O-CMCs exhibited the same optimum pH of 3.0 for coacervation with GA and the O-CMC–GA coacervates displayed similar differential scanning calorimetry (DSC) patterns, thermogravity (TG) profiles, and porous microstructures, but differed markedly in their rheological properties. As the O-CMC DS increased, the elasticity of the coacervates raised accordingly. It was concluded that O-CMC–GA coacervates with desired viscoelasticity can be obtained by varying the DS of O-CMC.     

Protein–polysaccharide associative interactions in the design of tailor-made colloidal particles

This article reviews some recent advances in the use of diverse protein–polysaccharide associative interactions in the design of colloidal particles having potential to be used for both fortification of food colloids with health-promoting bioactive compounds with better control of their physical stability and breakdown within the gastrointestinal tract. Protein–polysaccharide associative interactions are discussed in the following aspects: (i) the formation of micro- and nanoparticles for the delivery of health promoting ingredients (nutraceuticals); (ii) the controlled gastrointestinal fate of colloidal particles; (iii) the formation of biopolymer-based particles as fat replacers; and (iv) the behavior of colloidal particles as stabilizers of emulsions and foams. The first aspect concerns soluble protein–polysaccharide complex particles (electrostatic nanocomplexes, complex coacervates, covalent conjugates), mixed hydrogel particles, and nanoemulsion-based delivery systems.     

Can coacervation unify disparate hypotheses in the origin of cellular life?

Here, we review the recent progress in the characterisation and utilisation of coacervates as protocell models in the origin of life studies. We provide evidence that coacervation could have played a unique role during the origin of life, based on its ability to form from a range of different prebiotically relevant molecules; partition solutes; support and alter RNA catalysis and readily deform its shape. We discuss how these properties could have been important for the formation of the first membrane-bound cells, supporting RNA-peptide evolution and primitive metabolism, and in replicating and proliferating by growth and division processes.     

Protein–polysaccharide interactions and aggregates in food formulations

The protein–polysaccharide combinations that lead to electrostatic complex and coacervate formation are the object of extensive research using both layer-by-layer and mixed emulsion approaches. The protein–polysaccharide conjugates demonstrated interesting physicochemical properties as stabilizers and emulsifiers, as well as texture modifiers in food products. Furthermore, they are potential optimal nutrient delivery systems. Their complex behavior due to several factors such as pH, ionic strength, concentration, heat, and mechanical treatments is the main reason behind the continuous growth of the research field. The review is reporting some recent advances on the topic, along with an overview of the possible interactions between protein and polysaccharide, from Maillard reaction to enzymatic cross-linking passing through coacervates.