Engineering Protein‐Polymer Hybrid Multilayer Microreactors as Antioxidant and Anti‐Inflammatory Therapeutics: Effect of Single Enzyme Nanogel Localization Within the Multilayer Systems

Proteins have become increasingly prominent compared with conventional small molecule-based medicines in the realm of therapeutics. However, they are often prone to denaturation, instability, and aggregation, which limit their transferability to clinics. To exploit their extraordinary characteristics, emerging strategies have aimed at incorporating them into microcarriers. In this work, two complementary approaches are combined to protect catalase (CAT) from degradation and develop multilayer microreactors: the synthesis of single enzyme nanogels (SENs) and the layer-by-layer (LbL) methodology. CAT is first wrapped within an acrylamide-based thin polymeric mantle, increasing both its stability and negative surface charge. The obtained SENs-CAT are then integrated either into the core of multilayer microreactors or assembled as building blocks at different positions of the multilayer membrane. The antioxidant and therapeutic effects of the synthesized microreactor configurations were assessed to explore the potential effect of the spatial location of the enzyme. The results highlight that the antioxidant activity of SENs-CAT is preserved within multilayer microreactors regardless of their location within the multilayer structure. Furthermore, the ability to attenuate the release of inflammatory cytokines in an inflamed biological microenvironment makes these microreactors promising candidates for therapeutic applications.