Background. Enzymes are fundamental biological catalysts that have many promising biomedical applications. Since most enzymes are derived from non-human sources, they face two main challenges: limited bioactivity mostly due to rapid degradation by proteases and immunogenic activity. Therefore, there is a need for a versatile enzyme-encapsulation strategy to prevent their interaction with plasma proteins and immune cells to increase their biological half-life and effectiveness.
Hypothesis. We developed a novel method to encapsulate enzymes in nanoporous silica nanoparticles that shield the enzyme from large macromolecules while allowing the entrance of small molecule substrates only. We hypothesized that this versatile encapsulation method will improve the stability of the enzyme and eliminate immune responses from the body. We used catalase as a model enzyme in our experiments.
Materials & Methods. We directly modified the surface of the enzyme with N-acryloyloxysuccinimide and (3-aminopropyl)trimethoxysilane to generate a silica precursor, then coated it with silica by hydrolyzing tetraethyl orthosilicate in basic conditions. Lastly, we conjugated polyethylene glycol groups to improve biocompatibility and half-life. We used dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy to characterize the particles, and we measured catalase activity electrochemically.
Results. Catalase-loaded silica nanoparticles (CAT-SiNPs) demonstrated high retention of activity, optimal protection from proteolysis by proteinase K and trypsin, and excellent stability over time. The formulation yielded nanoparticles with a mean diameter of 230 ± 10 nm and a concentration of 1.3 ± 0.8 × 10¹² NPs/mL. We also developed a new electrochemical assay to measure catalase activity in a rapid, simple and accurate manner. Lastly, the ability of CAT-SiNPs to generate oxygen when exposed to H₂O₂ was demonstrated in vivo using a rat model.
Conclusion. We demonstrated that we encapsulated catalase in small nanoporous silica nanoparticles with high activity and optimal protection. Additionally, the ability of these CAT-SiNPs to generate oxygen has the potential to relieve hypoxia in tissues and potentially sensitize tumors against radiation therapy. We are currently continuing in vivo studies with CAT-SiNPs and applying this technology to the encapsulation of other enzymes.

Robert Mattrey

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