Processing of gelatin-based cryogels with improved thermomechanical resistance, pore size gradient, and high potential for sustainable protein drug release.

Porous gelatin (GEL) cryogels were processed by spatiotemporal and temperature-controlled gelation and freezing-lyophilizaton process, followed by zero-length crosslinking, using different molarities of reagents (EDC and NHS) and reaction media (100% PBS or 20/80% PBS/EtOH mixture) for variable time extensions (1-24 h). In this way, tuneable cryogels with gradient microporosity (from 100 µm to 1000 µm) were formed, being mainly influenced by crosslinkers' concentration and EtOH addition. Later affect the pore morphology (from round to ellipsoid), consequently modulating the steady-state physiological swelling profile toward twice lower values (∼ 600%) comparing to stepwise swelling of in 100% PBS media crosslinked cryogels. While the presence of EtOH decelerate the crosslinking kinetic by retaining cryogels' microstructure formed during freezing, the 100% PBS and higher EDC molarity resulted in approximately 40% crosslinking degree, being expressed as a thermal resistance of cryogels up to approximately 73°C. Finally, the tuneable enzymatic resistance allow time-dependent poly-L-Lysine (pL) release profile in up to month period. The processed GEL cryogels have potential in broad range biomedical applications, especially as sustainable, protein-based drug delivery systems.