Scalable Production of Enzyme-Loaded Gelatin Microgels Using Water-in-Water Emulsion

Enzyme-loaded spherical microgels composed of cross-linked polymer networks that swell in water and provide an excellent platform for encapsulating enzymes. They have large surface area and colloidal stability of microgels which make them ideal scaffolds for anchoring enzymes where they can protect the enzymes, enhancing their stability and maintain their correct folding within supportive microenvironments and make them suitable for applications in biosensors, drug delivery systems, biological scavengers, therapeutic microreactors, and industrial catalysis. Unfortunately, traditional methods for preparing enzyme-loaded microgels have significant challenges and conventional preparation strategies typically involve water-in-oil (W/O) emulsions or flow chemistry techniques. The W/O emulsion method, while capable of producing monodispersed spherical microgels it compromises enzyme activity due to the use of organic solvents. Additionally, residual solvents pose potential toxicity risks in biomedical applications. On the other hand, flow chemistry techniques are expensive, complex and difficult to scale up.  To this end, new study published in Journal Soft Matter and led by Professors Yota Okuno and Yasuhiko Iwasaki from the Department of Chemistry and Materials Engineering at Kansai University in Japan, developed a novel and simpler strategy for creating enzyme-loaded gelatin-based microgels and provided a scalable, efficient solution that maintains enzyme activity and encapsulation efficiency while being cost-effective and straightforward to implement.

The first step the research synthesized methacryloyl gelatin (GelMA) derived from cold-water fish. They chose GelMA due to its lower risk of disease transfection compared to mammalian gelatin, making it suitable for therapeutic applications. They also introduced Methacryloyl groups to the gelatin by conjugating methacrylic anhydride to the amino groups in gelatin and resulted in a methacryloyl modification ratio of 72%. This modified gelatin was confirmed by 1H-NMR spectroscopy and size-exclusion chromatography, indicating successful synthesis with a molecular weight of 46,000 Da. Afterward, the researchers mixed a 20 wt% GelMA solution with a 10 wt% polyvinylpyrrolidone (PVP) solution in a specific ratio to create the W/W emulsion. The mixture was vigorously stirred to form a stable emulsion, with GelMA as the dispersed phase. Fluorescein-labeled GelMA-1 was used to confirm the presence of the GelMA phase within the PVP solution. Professors Okuno and Iwasaki verified the emulsion’s stability and remained intact for several hours and allowed sufficient time for subsequent processing. Moreover, the researchers investigated the encapsulation of various enzymes, including bovine serum albumin (BSA), horseradish peroxidase (HRP), glucose oxidase (GOD), and β-galactosidase (β-Gal). Fluorescein-labeled BSA (FITC-BSA) was first added to the emulsion, with 92% partitioning into the GelMA phase.

The enzyme-loaded emulsion was passed through microporous glass filters with pore sizes of 5 and 10 μm and then exposed to ultraviolet light for crosslinking. This process yielded spherical microgels. Confocal laser scanning microscopy confirmed the homogeneous distribution of FITC-BSA within the microgels. These microgels remained stable for over a week, as evidenced by fluorescence measurements. With high encapsulation efficiency of multiple enzymes simultaneously.  Additionally, the researchers evaluated the enzymatic activity of the encapsulated enzymes. β-Gal encapsulated in GelMA-1 microgels was tested using o-nitrophenyl-β-D-galactopyranoside as a substrate. The microgels retained β-Gal activity, and worked well as microreactors. It is noteworthy to mention that smaller microgels were 21% faster initial reaction speeds due to their larger specific surface areas, which highlights the importance of microgel size in enzymatic reactions. They also analyzed HRP and GOD-loaded microgels for enzymatic activity and found smaller microgels to have higher initial reaction speeds, especially for GOD-loaded microgels due to easier substrate permeation and diffusion in the gel network and this confirmed that specific surface area played an important role in the reaction speed of GOD-loaded microgels.

Furthermore, the authors incubated β-Gal encapsulated in GelMA-1 microgels in citrate phosphate buffer at pH 6.0 and 37°C for 24 hours to test the stability of the enzyme-loaded microgels, the encapsulated β-Gal retained its activity, whereas naked β-Gal lost approximately 60% of its activity under the same conditions. This demonstrated the protective effect of the gelatin network against enzyme denaturation which indicates the potential industrial application of these microgels in producing hydrolyzed lactose for dairy intolerance. In conclusion, the study conducted by Professors Yota Okuno and Yasuhiko Iwasaki addressed key limitations of enzyme-loaded microgels traditional preparation methods. The new method, avoided organic solvents and used full aqueous system which ensured that enzyme activity is preserved during the encapsulation process which is vital for biosensors and therapeutic microreactors applications. They successfully demonstrated encapsulation efficiencies of over 70%, even when multiple enzymes were used. According to the authors, the innovative method is scalable and produce microgels on a 10 g scale in a single batch. The enzyme-loaded microgels can be used in therapeutic microreactors for drug synthesis and delivery, in biosensor and also can be employed as biocatalysts in the synthesis of chemicals.  In a statement to advances in Engineering, the authors said “By adjusting the mixing ratio of GelMA and PVP (reducing the proportion of GelMA), we have achieved an encapsulation efficiency of over 95% for BSA, HRP, and GOD when encapsulated individually.”