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) (PM2VP 128 - b -PEO 477 ), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS 215 ). 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.

The stability of complex coacervate core micelles (C3Ms) against salt and pH changes is improved by chemical crosslinking of the micelle core. Depending on the crosslinking agent, the resulting covalent network is reversible or irreversible.

No abstract

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.

Chapter 6 : Core-crosslinking enhances the salt resistance of enzyme-containing complex coacervate core micelles .