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

Bovine beta-casein micelles as delivery systems for hydrophobic flavonoids

The milk protein β-casein (β-CN) is an intrinsically unstructured amphiphilic protein that self-assembles into micelles. Naringenin is the main hydrophobic flavanone in grapefruit and has several beneficial biological effects: it exhibits, for example, antioxidant, anticancer and anti-inflammatory activity. This paper shows that naringenin can be encapsulated in β-CN micelles. Fluorescence spectroscopy, molecular docking modelling, dynamic light scattering (DLS), static light scattering (SLS) and isothermal titration calorimetry (ITC) were applied to characterize the effect of naringenin on the protein association behavior and properties of the resulting micelles. Naringenin binds to β-CN at both pH 7 and pH 2, promotes the formation of micelles with a well-defined size distribution and stabilizes the micelles. It was found that naringenin-containing β-CN micelles have a lower critical micelle concentration (CMC) and a larger aggregation number (Nagg) compared to pure β-CN micelles. SLS and multi-angle DLS results suggest considerable differences between the structures of pure β-CN micelles and naringenin-containing β-CN micelles. In the presence of naringenin spherical micelles were formed with a relatively loose core (“hollow sphere”), while the pure β-CN micelles are smaller and seem to be elliptic. Notably, by uptake of naringenin in the micelles, the concentration of naringenin in aqueous solution could be raised considerably. These findings lead to the conclusion that β-CN micelles are very promising as effective delivery nano-vehicles for hydrophobic bioactive compounds.</p

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

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