Coacervate Formation Research Articles

Monte Carlo Simulation on Complex Formation of Proteins and Polysaccharides

In protein-polysaccharide complex systems, how nonspecific interactions such as electrostatic and van der Waals interactions affect complex formation has not been clearly understood. On the basis of a coarse-grained model with the specificity of a target system, we have applied Monte Carlo (MC) simulation to illustrate the process of complex coacervate formation from the association of proteins and polysaccharides. The coarse-grained model is based on serum albumin and a polycation system, and the MC simulation of pH impact on complex coacervation has been carried out. We found that complex coacervates could form three ways, but the conventional association through electrostatic attraction between the protein and polysaccharide still dominated the complex coacervation in such systems. We also observed that the depletion potential always participated in protein crowding and was weakened in the presence of strong electrostatic interactions. Furthermore, we observed that the sizes of polysaccharide chains nonmonotonically increased with the number of bound proteins. Our approach provides a new way to understand the details during protein-polysaccharide complex coacervation at multiple length scales, from interaction and conformation to aggregation.

Turbidity and rheological properties of bovine serum albumin/pectin coacervates: Effect of salt concentration and initial protein/polysaccharide ratio

Abstract The turbidity and rheological properties of bovine serum albumin (BSA)/pectin coacervates show correlations with sodium chloride concentration ( C NaCl ) and initial protein/polysaccharide ratio ( r ). Increasing C NaCl from 0.01 to 0.4 M shifts the critical pH (pH φ1 ), which designates as the critical pH for BSA/pectin coacervate formation, to lower values, and the storage modulus ( G ′) values of BSA/pectin coacervates tend to be smaller, which can be explained by a salt screening effect in the BSA/pectin coacervates. Moreover, an increase of r from 1:1 to 10:1 favors the formation of BSA/pectin coacervates, as indicated by the increase in pH φ1 and the decrease in pH φ2 . The values of G ′ increase simultaneously. With further increase of r to 20:1, the pH φ1 changes negligibly and G ′ values become much lower. These results reflect that a balance between the positive charges of BSA and the negative charges of pectin favors the formation of BSA/pectin coacervates with more compact network structures.

Ethylcellulose microparticles containing chitosan and gelatin: pH-dependent release caused by complex coacervation

Ethylcellulose microparticles containing chitosan and gelatin were prepared by spray drying water-in-oil (W/O) emulsion. Water phase was chitosan:gelatin solution in distilled water, and oil phase was ethylcellulose solution in dichloromethane. FITC-dextran was included in water phase as a fluorescence dye. The maximum coacervation was observed at pH 6.0 when the ratio of chitosan to gelatin was 1:15. Microparticles containing chitosan and gelatin in the ratio showed a higher release under acidic condition (e.g. pH 4.0) and neutral condition (e.g. pH 7.0), but a lower release at pH 5.0 and pH 6.0. The complex coacervate composed of chitosan and gelatin was efficiently formed at those pHs, and the formation of coacervate would be responsible for the lower release.

Secretion of elastin-like polypeptides with different transition temperatures by Pichia pastoris

Like natural tropoelastin, polypeptides based on an elastin-like VPGXG repeat have a characteristic inverse temperature response, which leads to coacervate formation above a certain transition temperature and which could be useful for a variety of applications. The key advantage of elastin-like polypeptides (ELPs) over (tropo)elastin is a full control over this temperature response by adjustment of either the amino acid composition or the chain length, according to insights provided by extensive research. Future application of ELPs will require efficient ELP production systems, and in a previous article, we described the successful use of Pichia pastoris for secreted production of an ELP, with an overall yield of ≈ 200 mg L(-1). In this study, we investigated the influence of changed amino acid composition and chain length on the yield of secreted ELP. We have found that both parameters have a distinct impact on the overall yield, with higher yield for shorter and more hydrophilic ELPs. Because yield and transition temperature (Tt) thus appear to be positively correlated, we hypothesize that good solubility of ELP below the Tt promotes the secreted production and coacervate formation above Tt decreases it.

Complex coacervation of pea protein isolate and alginate polysaccharides

Complex coacervation between pea protein isolate (PPI) and alginate (AL) was investigated as a function of pH (1.50–7.00) and mixing ratio (1:1–20:1 PPI:AL) by turbidimetric analysis and electrophoretic mobility during an acid titration. Conformational changes to the secondary structures during coacervation were also studied by Raman spectroscopy. Critical structure-forming events associated with the formation of soluble (pH 5.00) and insoluble (pH 2.98) complexes were found for a 1:1 PPI–AL mixture, with optimal biopolymer interactions occurring at pH 2.10 (pHopt). As mixing ratios increased between 4:1 and 8:1, critical pHs shifted towards higher pH. Maximum coacervate formation at pHopt occurred at a mixing ratio of 4:1. Electrophoretic mobility measurements showed a shift in net neutrality from pH 4.00 in homogenous PPI solutions, to pH 1.55 for the 1:1 mixture. As biopolymer ratios increased towards 8:1 PPI:AL, net neutrality shifted to higher pHs (∼3.80). Raman spectroscopy revealed minimal complexation-induced conformational changes. Findings could aid in the design of pH-sensitive biopolymer carriers for use in functional food and bio-material applications.

Role of pH in the coacervation of the systems: gelatin-water-ethanol and gelatin-water-sodium sulphate.

Abstract Phase boundary determination, coacervate volume measurements and analysis of the phases have been made to assess the influence of pH on the coacervation of gelatin solutions by ethanol and sodium sulphate. Coacervation was found to be pH dependent. In the ethanol system coacervation was noticeable only within a pH range in the vicinity of the isoionic point; at other pH values either a viscous gel phase or floccules occurred. In the sodium sulphate system, coacervation occurred at all pH values examined. The effect of pH in changing the overall charge on the gelatin molecule is explained in relation to the formation of gelatin coacervates. Finally, the role of the coacervate phase in the microencapsulation of oil and solid particulates is discussed.

Altered ocular absorption and disposition of sodium cromoglycate upon ion-pair and complex coacervate formation with dodecylbenzyldimethylammonium chloride.

Ion-pair formation between the dianionic drug sodium cromoglycate and dodecylbenzyldimethylammonium chloride has been found to alter the extent and rate of corneal penetration of both large ions upon their coadministration. Additionally, using a complex coacervate of the two large ions as the applied dose form (in which the complex is in equilibrium with the ion pairs), a change in the overall disposition of both large ions to the various components of the eye is observed.

New insights into the pathogenesis of autosomal‐dominant cutis laxa with report of fiveELNmutations

Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28-34 of the ELN gene in five probands with ADCL features and found five de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5), and c.2124del25 (CL-6). Four probands (CL-1,-2,-3,-6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGF-β) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications, associated with increased TGF-β signaling and mutation-specific differences in endoplasmic reticulum stress and apoptosis.

Effect of Chitosan Degradation on Its Interaction with β-Lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.

Ion-specific weak adsorption of salts and water/octanol transfer free energy of a model amphiphilic hexapeptide

An amphiphilic hexapeptide has been used as a model to quantify how specific ion effects induced by addition of four salts tune the hydrophilic/hydrophobic balance and induce temperature-dependant coacervate formation from aqueous solution. The hexapeptide chosen is present as a dimer with low transfer energy from water to octanol. Taking sodium chloride as the reference state in the Hofmeister scale, we identify water activity effects and therefore measure the free energy of transfer from water to octanol and separately the free energy associated to the adsorption of chaotropic ions or the desorption of kosmotropic ions for the same amphiphilic peptide. These effects have the same order of magnitude: therefore, both energies of solvation as well as transfer into octanol strongly depend on the nature of the electrolytes used to formulate any buffer. Model peptides could be used on separation processes based on criteria linked to "Hofmeister" but different from volume and valency.

Preparation and Characterization of Oil Containing Microcapsules Obtained by an Interaction Induced Coacervation

A novel method for microencapsulation of oil by coacervation is presented. The method employs segregative phase separation between sodium carboxymethyl cellulose (NaCMC) and a complex of hydroxypropylmethyl cellulose (HPMC) and sodium dodecylsulfate (SDS), which results in coacervate formation. Microstructural properties of the coacervate can be varied by tuning NaCMC-HPMC/SDS interaction, which is achieved by changing SDS concentration. Microcapsules preparation route is presented. Encapsulation efficiency and dispersion properties of microcapsules with coacervate shell of different properties and different oil content were tested. Microcapsules with smallest droplet size, the narrowest droplet size distribution, and with lowest extractability of encapsulated oil were obtained when NaCMC-HPMC/SDS interaction results in formation of the most compact coacervate shell, no matter of the encapsulated oil.

Evaluation of novel synthetic conditioning polymers for shampoos

Cationic polymers have traditionally been used in shampoo formulations to impart conditioning properties to hair. In this study, commercial synthetic cationic polymers were investigated using coacervate formation, objective wet comb analysis, silicone deposition and panel studies to determine structure function properties with the goal of developing novel conditioning polymers. New polymers were synthesized and, based on criteria determined in the first part of the study, found to have marginal improvement over existing synthetic cationic conditioning polymers. A novel experimental polymer developed for a different industry was also investigated for conditioning properties. This polymer showed significant enhancement of silicone deposition over current commercial polymers, including cationic guar, even at significantly reduced silicone and polymer concentrations. The experimental polymer exhibited parity or improvement over benchmark polymers in panel studies, and similar performance to other synthetic polymers in objective wet comb studies.

Modifications in stability and structure of whey protein-coated o/w emulsions by interacting chitosan and gum arabic mixed dispersions

The influence of chitosan and gum arabic mixtures on the behaviour of o/w emulsions has been investigated at pH = 3.0. The emulsion behaviour, properties and microstructure were found to be greatly dependent on the precise gum arabic to chitosan ratio. Mixing of gum arabic with chitosan leads to the formation of coacervates of a size dependent on their ratio. Incorporation of low gum arabic to chitosan weight ratios into whey protein-coated emulsions causes depletion flocculation and gravity-induced phase separation. Increasing the polysaccharide weight ratio further, a droplet network with a rather high viscosity (at low shear stress) is generated, which prevents or even inhibits phase separation. At even higher gum arabic to chitosan ratios, the emulsion droplets were immobilised into clusters of an insoluble ternary matrix. Although the emulsion droplet charge had the same sign as that of the coacervates, clusters of oil droplets in a ternary matrix were generated. A mechanism to explain the behaviour of the whey protein-stabilised o/w emulsions is described on the basis of confocal and phase contrast microscopic observations, rheological data, zeta potential measurements, particle size analysis and visual assessment of the macroscopic phase separation events.

Physicochemistry of the Interaction between Inulin and Alkyltrimethylammonium Bromides in Aqueous Medium and the Formed Coacervates

Inulin, a polydisperse reserve polysaccharide, has prospective uses in food, pharmacy, and industry. Its uses and applications often encounter interactions with lipids and amphiphiles. Reports on such interactions are scarcely found in literature. In the present study, we have examined the nature of interactions between inulin and cationic amphiphiles, alkyltrimethylammonium bromides (CnTAB: n=12, 14, 16, 18), over a fair range of concentrations for both the polymer and the amphiphile. At low concentration, small induced amphiphile aggregates form complexes with inulin; at moderate concentration, the complexed inulin self-aggregates leading to coacervate formation, and at higher concentration, the amphiphile forms free micelles in solution. Tensiometric, conductometric, viscometric, and turbidimetric methods have been employed to study the above phenomena. The isolated coacervates of inulin with C18TAB were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), thermogravimetry (TG), and differential thermal analysis (DTA) to ascertain their morphology, structure, and thermal stability.

Coacervate formation of α-lactalbumin–chitosan and β-lactoglobulin–chitosan complexes

α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) are important whey proteins with isoelectric points of pH 4.80 and 5.34, respectively, and evidence negative charge over a range of pHs. Chitosan exhibits a cationic property under pH 6.5. In an effort to determine the physicochemical properties of mixtures of 0.5% α-La and 0.1% chitosan, and 0.5% β-Lg and 0.1% chitosan, optical structure, turbidity, electrophoresis, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were assessed in a pH range of 2.0–8.0. The results demonstrated that α-La, β-Lg, and chitosan precipitated at pH values of approximately 5.0, 5.0, and 7.0, respectively. The mixtures of α-La and chitosan as well as β-Lg and chitosan coacervated at a pH range of 6.0–6.5. The turbidity of α-La and α-La–chitosan achieved a maximum at pH 5.0, whereas those of β-Lg and β-Lg–chitosan achieved maximum values at a pH of 6.5. The electrophoregram showed a large band with high molecular weight in increasing pH values from 5.0 to 6.0, which suggested that α-La and β-Lg form polymers with chitosan. The denaturation temperature and enthalpy of α-La were shown to increase, whereas those of β-Lg were reduced. The SEM images demonstrated that the α-La was characterized by uneven and associated cluster morphology, whereas that of β-Lg was even, globular, and harbored dense particles, whereas the chitosan evidenced a flat morphology. Our assessment of the complex demonstrated that α-La and β-Lg attached to the surface of the chitosan. The α-La–chitosan and β-Lg–chitosan complexes evidenced opposite charges at a pH range of 5.0–6.0, and formed coacervates. It appears, therefore, that the α-La–chitosan and β-Lg–chitosan coacervates might be applied as a delivery system for foods, nutraceuticals, cosmetics, and drugs.

Kinetics of Protein−Protein Complex Coacervation and Biphasic Release of Salbutamol Sulfate from Coacervate Matrix

Turbidimetric titration was used to initiate associative intermolecular interactions between a pair of protein molecules, gelatin-A and gelatin-B, having complementary charges that led to pH-induced liquid-liquid phase separation and the formation of complex coacervate. The stoichiometric binding ratio was found to be [gelatin-A]/[gelatin-B]=3:2. The size of soluble intermolecular aggregates present in the supernatant exhibited interesting time-dependent coacervation because of residual electrostatic interactions. Dynamic light scattering and turbidity studies provided a systematic account of coacervation behavior. Rheology studies attributed the softening of the coacervate matrix to the presence of encapsulated salbutamol sulfate. The in vitro drug release kinetics was probed in simulated gastric fluid medium at physiological temperature (37 degrees C), which showed biphasic behavior. The initial release kinetics exhibited an exponential growth to saturation behavior, followed by a slower logarithmic release process.

Structural study of coacervation in protein-polyelectrolyte complexes

Coacervation is a dense liquid-liquid phase separation and herein we report coacervation of protein bovine serum albumin (BSA) in the presence of polyelectrolyte sodium polystyrene sulfonate (NaPSS) under varying solution conditions. Small-angle neutron scattering (SANS) measurements have been performed on above protein-polyelectrolyte complexes to study the structural evolution of the process that leads to coacervation and the phase separated coacervate as a function of solution pH , protein-polyelectrolyte ratio and ionic strength. SANS study prior to phase separation on the BSA-NaPSS complex shows a fractal structure representing a necklace model of protein macromolecules randomly distributed along the polystyrene sulfonate chain. The fractal dimension of the complex decreases as pH is shifted away from the isoelectric point ( approximately 4.7) of BSA protein, which indicates the decrease in the compactness of the complex structure due to increase in the charge repulsion between the protein macromolecules bound to the polyelectrolyte. Concentration-dependence studies of the polyelectrolyte in the complex suggest coexistence of two populations of polyelectrolytes, first one fully saturated with proteins and another one free from proteins. Coacervation phase has been obtained through the turbidity measurement by varying pH of the aqueous solution containing protein and polyelectrolyte from neutral to acidic regime to get them to where the two components are oppositely charged. The spontaneous formation of coacervates is observed for pH values less than 4. SANS study on coacervates shows two length scales related to complex aggregations (mesh size and overall extent of the complex) hierarchically branched to form a larger network. The mesh size represents the distance between cross-linked points in the primary complex, which decreases with increase in ionic strength and remains the same on varying the protein-polyelectrolyte ratio. On the other hand, the overall extent of the complex shows a similar structure irrespective of varying ionic strength and protein-polyelectrolyte ratio. A large fraction ( approximately 50%) of protein-polyelectrolyte complexes is also found to be free in the supernatant after the coacervation.

Glycosaminoglycan-Mediated Coacervation of Tropoelastin Abolishes the Critical Concentration, Accelerates Coacervate Formation, and Facilitates Spherule Fusion: Implications for Tropoelastin Microassembly

Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems that have been studied. To address the apparent paradox of a paucity of tropoelastin for cell surface microassembly, we examined the effects of the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfate B, on tropoelastin aggregate formation through coacervation. We found a significant effect, particularly of heparin, on the minimum or critical concentration of tropoelastin, which was required for microassembly, lowering critical concentration to a point that it was no longer detectable. The assemblies resulted in protein droplet formation that was visually indistinguishable from the spherules that typify coacervation. The spherules readily coalesced in the presence of heparin and higher concentrations of tropoelastin, resulting in an almost continuous layer of coacervated tropoelastin. Four stages of droplet behavior were observed: early droplet formation, approximately 6 mum droplet formation, and fusion of droplets followed by the formation of a coalesced layer. We conclude that glycosaminoglycans in the extracellular matrix have the capacity to promote coacervation at low concentrations of tropoelastin.

Formation of Biopolymer‐Coated Liposomes by Electrostatic Deposition of Chitosan

The purpose of this study was to prepare stable biopolymer-coated liposome suspensions using an electrostatic deposition method. Liposome suspensions were produced by homogenizing 1% soy lecithin in acetate buffer (0.1 M, pH 3). Cationic chitosan (Mw approximately 200 kDa) solutions were mixed with anionic liposome suspensions (d approximately 100 and 200 nm), and the effect of phospholipid concentration, chitosan concentration, and liposome size on the properties of the particles formed was determined. The particle size and charge (zeta-potential) were measured using dynamic light scattering and particle electrophoresis. The particle charge changed from -38 mV in the absence of chitosan to +60 mV in the presence of chitosan, indicating complex formation between the anionic liposomes and cationic chitosan molecules. Below a minimum critical chitosan concentration (c(min)), large aggregates were formed that phase separated within minutes, whose origin was attributed to formation of coacervates. On the other hand, above a maximum critical chitosan concentration (c(max)), large flocs were formed that sedimented within hours, whose formation was attributed to depletion flocculation. Minimum and maximum critical chitosan concentrations depended on liposomal concentration and size. At c(min) < c < c(max'), chitosan-coated liposomes were formed that did not aggregate and were stable to sedimentation. Coated liposomes had better stability to aggregation than uncoated liposomes when stored at ambient temperatures for 45 d. This study indicates that chitosan can be used to form biopolymer-coated liposomes with enhanced stability over uncoated liposomes.

Single-drop coacervative microextraction of organic compounds prior to liquid chromatography: Theoretical and practical considerations

Coacervates made of surfactant aggregates, namely aqueous and reverse micelles and vesicles, were firstly used as solvents in single-drop microextraction (SDME) and proposed for the extraction and concentration of chlorophenols prior to liquid chromatography. The formation of coacervate drops in the needle tip of conventional microsyringes depended on the type of intermolecular forces established between the surfactant headgroups making up the supramolecular aggregates; hydrogen bond interactions were strong enough to permit the formation of spherical drops. Stability of 1–50 μL coacervate drops was achieved by introducing the microsyringe needle tip in a PTFE rod, the end of which had been machined out with a heated flanging-tool to get circular flanges (diameters in the range 3.5–6 mm). The parameters affecting the efficiency of single-drop coacervative microextraction (SDCME) were investigated using vesicular coacervates as a solvent and 2-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) as model analytes. Coacervative microextraction dynamics fit to the general rate equation of liquid–liquid extraction. The effect of variables such as extraction time, drop volume, stirring rate, pH and temperature, on the extraction of chlorophenols was similar to that described for organic solvent drops. Electrolyte concentrations above 0.1 M caused drop instability. Under the optimum conditions, detection limits were in the range 0.1–0.3 μg L −1. The relative standard deviation was between 4.3 and 5.6 at 20 μg L −1 spiked level. The method was applied to the determination of the four chlorophenols in wastewater, superficial water from a reservoir and groundwater and the recoveries were in the range 79 and 106% at 5–20 μg L −1 spiked level.