Hydrophobic Small-Molecule Loading and Cellulase-Responsive Degradation in Lyophilized Cellulose Nanofiber/Pectin Pickering Microcapsules

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Inflammatory bowel disease requires long-term anti-inflammatory therapy, yet systemic corticosteroid exposure remains a significant limitation of current oral treatments. This work presents cellulose nanofiber/pectin Pickering microcapsules with oleic acid cores as enzyme-responsive polysaccharide-based carriers for hydrophobic molecules, using rhodamine 101 as a fluorescent model compound. The formulation process was optimized to yield a dry, redispersible powder while retaining encapsulated fluorescence and morphologically intact microcapsules after cryoprotectant-free lyophilization. A high-throughput flow cytometry protocol, adapted from the International Society for Extracellular Vesicles guidelines, provided single-particle measurements of scatter-based size distributions and fluorescence across formulation stages, revealing minor changes in the size-gate distribution before and after freeze-drying and rehydration. Marangoni flow-driven accumulation of microcapsules at the center of the sessile drop enabled imaging at the population level and real-time monitoring of cellulase-induced enzymatic degradation. Microcapsule counts decreased under cellulase exposure, whereas controls remained comparatively stable, demonstrating the susceptibility of the polysaccharide shell to degradation under the defined in vitro conditions. Together, these results establish a cryoprotectant-free, freeze-dried cellulose nanofiber/pectin Pickering microcapsule platform with a hydrophobic oleic acid core and an integrated flow cytometry and imaging workflow for its characterization. This approach provides a basis for future evaluation of enzyme-responsive polysaccharide-based oral delivery formulations.