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Developing protein confinement platforms is an attractive research area that not only promotes protein delivery but also can result in artificial environment mimicking of the cellular one, impacting both the controlled release of proteins and the fundamental protein biophysics. Polymeric nanoparticles (PNPs) are attractive platforms to confine proteins due to their superior biocompatibility, low cytotoxicity, and controllable release under external stimuli. However, loading proteins into PNPs can be challenging due to the potential protein structural perturbation upon contacting the interior of PNPs. In this work, we developed a novel approach to encapsulate proteins in PNPs with the assistance of the zeolitic imidazolate framework (ZIF). Here, ZIF offers an additional protection layer to the target protein by forming the protein@ZIF composite via aqueous-phase cocrystallization. We demonstrated our platform using a model protein, lysozyme, and a widely studied PNP composed of poly(ethylene glycol)-poly(lactic- co -glycolic acid) (PEG-PLGA). A comprehensive study via standard loading and release tests as well as various spectroscopic techniques was carried out on lysozyme loaded onto PEG-PLGA with and without ZIF protection. As compared with the direct protein encapsulation, an additional layer with ZIF prior to loading offered enhanced loading capacity, reduced leaching, especially in the initial stage, led to slower release kinetics, and reduced secondary structural perturbation. Meanwhile, the function, cytotoxicity, and cellular uptake of proteins encapsulated within the ZIF-bound systems are decent. Our results demonstrated the use of ZIF in assisting in protein encapsulation in PNPs and established the basis for developing more sophisticated protein encapsulation platforms using a combination of materials of diverse molecular architectures and disciplines. As such, we anticipate that the protein-encapsulated ZIF systems will serve as future polymer protein confinement and delivery platforms for both fundamental biophysics and biochemistry research and biomedical applications where protein delivery is needed to support therapeutics and/or nutrients.

Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal-organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Jordahl, D.; Armstrong, Z.; Li, Q.; Gao, R.; Liu, W.; Johnson, K.; Brown, W.; Scheiwiller, A.; Feng, L.; Ugrinov, A.; Mao, H.; Chen, B.; Quadir, M.; Pan, Y.; Li, H.; Yang, Z. Expanding the Library of Metal-Organic Frameworks (MOFs) for Enzyme Biomineralization. ACS Appl. Mater. Interfaces 2022, 14 (46), 51619-51629, DOI: 10.1021/acsami.2c12998]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)-electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOFs) to customize enzyme immobilization.

Confining enzymes in well-shaped MOF compartments is a promising approach to mimic the cellular environment of enzymes and determine enzyme structure-function relationship therein. Under the cellular crowding, however, enzymes can also be confined in unstructured spaces that are close to the shapes/outlines of the enzyme. Therefore, for a better understanding of enzymes in their physiological environments, it is necessary to study enzymes in these unstructured spaces. However, practically it is challenging to create compartments that are close to the outline of an enzyme and probe enzyme structural information therein. Here, for proof-of-principle, we confined a model enzyme, lysozyme, in the crystal defects of a MOF via co-crystallization, where lysozyme served as the nuclei for MOF crystal scaffolds to grow on so that unstructured spaces close to the outline of lysozyme are created, and determined enzyme relative orientation and dynamics. This effort is important for understanding enzymes in near-native environments and guiding the rational design of biocatalysts that mimic how nature confines enzymes.

Metal-organic frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multiphase crystals, even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability and were stable under weakly acidic pH values. The stability under weakly basic conditions depended upon the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current "library" of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzyme stability, functionality, and optimal pH.