Enzymatically Crosslinked Chitosan–Hyaluronic Acid Layer-by-Layer Microcapsules with Controlled Permeability and Enhanced Stability for Cell Encapsulation

Cell encapsulation within semipermeable membranes is a promising strategy for protecting transplanted cells from host immune responses, while permitting the diffusion of nutrients and therapeutic molecules. Although alginate-based microcapsules are commonly used, ionically crosslinked capsules often exhibit limited structural stability and tunability in terms of membrane permeability. In this study, we developed covalently stabilized microcapsules. Alginate microgel beads were first prepared as sacrificial templates and subsequently coated with phenol-modified chitosan and hyaluronic acid (Chitosan–Ph and HA-Ph) via layer-by-layer assembly. The multilayer membrane was then covalently stabilized through horseradish peroxidase (HRP)-mediated oxidative coupling of phenol groups, followed by liquefaction of the alginate core. The crosslinked microcapsules maintained structural integrity after liquefaction, while markedly reducing γ-globulin permeation under in vitro conditions and preserving β-cell viability and glucose responsiveness. The findings of this study demonstrate the feasibility of this system as an in vitro platform for stable cell encapsulation, with potential relevance to cell therapy.