Enhanced stability of complex coacervate core micelles following different core-crosslinking strategies

Complex coacervate core micelles (C3Ms) are formed by mixing aqueous solutions of a charged (bio)macromolecule with an oppositely charged-neutral hydrophilic diblock copolymer. The stability of these structures is dependent on the ionic strength of the solution; above a critical ionic strength, the micelles will completely disintegrate. This instability at high ionic strengths is the main drawback for their application in, e.g., drug delivery systems or protein protection. In addition, the stability of C3Ms composed of weak polyelectrolytes is pH-dependent as well. The aim of this study is to assess the effectiveness of covalent crosslinking of the complex coacervate core to improve the stability of C3Ms. We studied the formation of C3Ms using a quaternized and amine-functionalized cationic-neutral diblock copolymer, poly(2-vinylpyridine)-block-poly(ethylene oxide) (QP2VP-b-PEO), and an anionic homopolymer, poly(acrylic acid) (PAA). Two different core-crosslinking strategies were employed that resulted in crosslinks between both types of polyelectrolyte chains in the core (i.e., between QP2VP and PAA) or in crosslinks between polyelectrolyte chains of the same type only (i.e., QP2VP). For these two strategies we used the crosslinkers 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dimethyl-3,3′-dithiopropionimidate dihydrochloride (DTBP), respectively. EDC provides permanent crosslinks, while DTBP crosslinks can be broken by a reducing agent. Dynamic light scattering showed that both approaches significantly improved the stability of C3Ms against salt and pH changes. Furthermore, reduction of the disulphide bridges in the DTBP core-crosslinked micelles largely restored the original salt-stability profile. Therefore, this feature provides an excellent starting point for the application of C3Ms in controlled release formulations

Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles

[Image: see text] Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP(128)-b-PEO(477)) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems

Balancing Enzyme Encapsulation Efficiency and Stability in Complex Coacervate Core Micelles

Encapsulation of charged proteins into complex coacervate core micelles (C3Ms) can be accomplished by mixing them with oppositely charged diblock copolymers. However, these micelles tend to disintegrate at high ionic strength. Previous research showed that the addition of a homopolymer with the same charge sign as the protein improved the stability of protein-containing C3Ms. In this research, we used fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to study how the addition of the homopolymer affects the encapsulation efficiency and salt stability of the micelles. We studied the encapsulation of laccase spore coat protein A (CotA), a multicopper oxidase, using a strong cationic-neutral diblock copolymer, poly(N-methyl-2-vinyl-pyridinium iodide)-block-poly(ethylene oxide) (PM2VP128-b-PEO477), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS215). DLS indeed showed an improved stability of this three-component C3M system against the addition of salt compared to a two-component system. Remarkably, FCS showed that the release of CotA from a three-component C3M system occurred at a lower salt concentration and over a narrower concentration range than the dissociation of C3Ms. In conclusion, although the addition of the homopolymer to the system leads to micelles with a higher salt stability, CotA is excluded from the C3Ms already at lower ionic strengths because the homopolymer acts as a competitor of the enzyme for encapsulation

Дослідження енергоефективності будівлі філії "Білопільський РЕМ" ПАТ "Сумиобленерго"

Однією із інноваційних технологій в енергоефективному будівництві є «пасивний будинок», схема обладнання якого була запропонована у 1988 році доктором В. Файстом та професором Б. Адамсоном. «Пасивний будинок» – це споруда, яка не має потреби в опаленні або ж її енергоспоживання становить менше 10 % від енергії на одиницю об’єму, яка споживається більшістю сучасних будівель. Тепло у такому будинку генерується пасивно, тобто лише засобами внутрішніх джерел тепла, сонячної енергії, яка потрапляє через вікна, та шляхом підігрівання повітря, що надходить через вентиляцію. На основі такої схеми обладнання доцільно не лише будувати нові будинки, але й модернізувати старі

Amphifunctionally Electrified Interfaces: Coupling of Electronic and Ionic Surface-Charging Processes

International audienc

Adhesion of emulsified oil droplets to hydrophilic and hydrophobic surfaces-effect of surfactant charge, surfactant concentration and ionic strength

Adhesion of emulsified oil droplets to a surface plays an important role in processes such as crossflow membrane filtration, where the oil causes fouling. We present a novel technique, in which we study oil droplets on a model surface in a flow cell under shear force to determine the adhesive force between droplets and surface. We prepared an emulsion of hexadecane and used hydrophilic and hydrophobic glass slides as model surfaces. Different surfactants were used as emulsifiers: negatively charged sodium dodecyl sulphate (SDS), positively charged hexadecyltrimethylammonium bromide (CTAB) and nonionic Triton X-100. We evaluate the role of the surfactant, the glass surface properties and the ionic strength of the emulsion. We found a minimum in the adhesion force between droplets and surface as a function of surfactant concentration. The charged surfactants cause a lower droplet adhesion compared to the nonionic surfactant. The flow cell technique presented here proved to be very useful in understanding the interaction between oil droplets and a surface

Competition between surface adsorption and folding of fibril-forming polypeptides

Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a ß -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a ß -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded ß -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014)]

Formation and ripening of alginate-like exopolymer gel layers during and after membrane filtration

The properties of biofilm EPS are determined by the multiple interactions between its constituents and the surrounding environment. Because of the high complexity of biofilm EPS, its constituents' characterisation is still far from thorough, and identification of these interactions cannot be done yet. Therefore, we use gels of bacterial alginate-like exopolysaccharides (ALEs) as a model component for biofilm EPS in this work. These gels have been examined for their cohesive properties as a function of CaCl2 and KCl concentration. Hereto, ALE gel layers were formed on membranes by dead-end filtration of ALE solutions. Accumulation of the cations Ca2+ and K+ in the gels could be well predicted from a Donnan equilibrium model based on the fixed negative charges in the ALE. This suggests that there is no specific binding of Ca2+ to the ALE and that on the time scale of the experiments, the Ca2+ ions can distribute freely over the gel and the surrounding solution. The concentration of fixed negative charges in the ALE was estimated around 1 mmol/g VSS (volatile suspended solids, organic mass) from the Donnan equilibrium. Moreover, an accumulation of H+ was predicted. Gels with more CaCl2 in the supernatant were more compact and bore a higher osmotic pressure than those with less CaCl2, revealing the role of Ca2+ ions in the network crosslinking. It is hypothesised that this mechanism later transitions into a rearrangement of the ALE molecules, which eventually leads to a fibrous network structure with large voids.BT/Environmental Biotechnolog

Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10–20 µm) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3–9, the protein (pI ˜ 10) is positive and the gel is negative. Uptake was monitored at different protein concentrations, pH values and ionic strengths. Lysozyme release was triggered by changing the pH and salt concentration and measured during enzymatic degradation of the gel by a-amylase. The release is of importance for potential use of the microgel in gastro-intestine drug delivery applications, while amylase-induced protein release is relevant for antimicrobial applications. To analyze the uptake and release kinetics we used a model based on diffusion, taking into account equilibrium exchange between protein bound to the gel matrix and free protein in the gel. For the uptake process the time-dependent evolution of the protein concentration profile in the gel phase and the medium was computed numerically. The calculated concentration profiles closely resemble the experimentally found profiles. In the case of high affinity between protein and the gel network (at low pH and low ionic strength), the initial uptake rate equals the limiting flux of protein at the gel–solution interface, completely determined by the rate of diffusion in the medium. The diffusion coefficient of free protein in the gel, Dp,g, was found to be on the order of 10-11 m2 s-1, about one order of magnitude lower than the diffusion coefficient of lysozyme in bulk solution. Release experiments were carried out with zero protein concentration in the medium, for which approximate analytic release equations are available. The experimental release curves obtained at pH 7 yielded estimated values for the concentration ratio R of bound and free protein in the gel and, related to this ratio, the effective diffusion coefficient of lysozyme in the gel, Deff. These values are extremely dependent on the ionic strength, ranging from ca. 1000 and 10-14 m2 s-1 at 0.025 M NaCl to 50 and 2 × 10-12 m2 s-1 at 0.05 M, respectively. The results are in line with our earlier absorption and FRAP (fluorescence recovery after photobleaching) studies, showing a sharply decreasing affinity and increasing overall exchange rate with increasing ionic strength. Amylase completely breaks down the oxidized starch microgel, while releasing the embedded protein into solution