Affinity switching for lysozyme and dual-responsive microgels by stopped-flow technique: Kinetic control and activity evaluation

Abstract

The use of proteins as therapeutics in nanomedicine is an emerging research field and has developed rapidly. However, proteins are always vulnerable to renal excretion or digestion by the proteolytic system in vivo, which limits their usage to a large extent. Although biocompatible polymers have been covalently linked to proteins to protect them from recognition by the immune system and prolong their circulation time, the biological activity of them is sometimes decreased. To fill this gap, physical isolation, wrapping, or encapsulation techniques are employed. Up to now, various mature examples were reported, but the whole time scales for guest molecules loading and releasing, especially the initial rapid loading process, were rarely mentioned. Herein, a series of dual-responsive poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAM-co-MAA)) microgels were synthesized and employed to investigate the kinetics of in situ complexation and release of lysozyme under external stimuli modulation upon a stopped-flow apparatus, which was suitable for rapid dynamic monitoring. Close inspection of the adsorption kinetics during the early stages (< 50 s) revealed that the initial microgel collapse occurred within ~1 s, with more rapid transitions being observed when higher lysozyme concentrations were targeted. All the dynamic traces could be well fitted with a double exponential function, suggesting a fast (τ 1) and a slow (τ 2) relaxation time, respectively. Then, the kinetics of releasing bound lysozyme from microgels was carried on by utilizing the pH-responsive property, and the evaluation of the activity of released lysozyme was synchronously measured in a Micrococcus lysodeikticus (M. lysodeikticus) cell suspension. The corresponding relaxation time (τ) was also calculated by fitting the recorded dynamic traces. We speculate that this work can provide basic dynamics data and theoretical basis for microgels based nanocarriers to be used for protein delivery, controlled release, and possible chemical separation.