4D Mapping of ZIF Biocomposites for High Protein Loading and Tunable Release Profiles

Metal-organic framework (MOF) biocomposites consist of MOF matrices into which biomacromolecules (e.g. proteins/enzymes) are immobilized for applications in drug delivery and biocatalysis. Zeolitic Imidazolate Frameworks (ZIFs) based on Zn2+ and 2-methylimidazole are the most studied MOFs for protein encapsulation. We systematically investigate how varying the Zn2+:2-methylimidazole:protein ratio, total precursor concentration, and washing procedure yields distinct Zeolitic Imidazolate Framework (ZIF) phases (ZIF-C, sod, dia) and amorphous forms. We find that each phase strongly influences crucial properties, including encapsulation efficiency (EE%), loading capacity (LC%), and release kinetics. Notably, we achieve unprecedented LC values (up to ~85%), ensuring a minimal carrier fraction while enabling high protein content. Using bovine serum albumin (BSA) as a model protein, we establish the relationships between precursors, crystallographic phase, EE%, LC%, and release profiles. We further encapsulate α-1-antitrypsin (AAT), a protein-based biotherapeutic, in ZIF-C, sod, and dia, verifying retained inhibitor activity upon release. Moreover, we demonstrate that blending rapid-releasing phases (sod) with slower ones (dia) enables multi-step release profiles, which are highly desirable for controlled drug delivery. These results highlight the pivotal role of systematic phase control to tune protein loading and release, offering a solid foundation for the rational design of ZIF-based biocomposites in advanced biomedical applications.