In Vitro Assessment of Cellular Interaction and Trans-Buccal Permeability of Exosome-Functionalized Carbonate Apatite Nanoparticles as a Potential Mucosal Adjuvant

Abstract

Background Developing effective oral mucosal adjuvants remains a major challenge in mucosal vaccine design due to the epithelial barrier and limited local immune activation. This study aimed to develop and evaluate exosome-functionalized carbonate apatite (CHA-EXO) nanoparticles (NPs) as a novel immunomodulatory platform for oral mucosal vaccination. Methods CHA nanoparticles were synthesized and functionalized with mesenchymal stem cell-derived exosomes, followed by fluorescence labelling, cytocompatibility assessment, cellular uptake, and immunomodulatory evaluation using TR146 and RAW264.7 cells. A TR146-seeded, fibronectin-modified electrospun poly(lactic acid) (PLA) nanofibrous scaffold was fabricated to establish an in vitro buccal epithelial model for nanoparticle permeability studies. Results CHA-EXO nanoparticles exhibited efficient cellular uptake and induced formulation-dependent IL-6 secretion in both TR146 and RAW264.7 cells, indicating immunomodulatory activity. The fibronectin-functionalized PLA scaffold supported epithelial attachment and multilayer formation resembling non-keratinized oral epithelium. Permeability studies revealed that CHA-EXO at 0.05 mg/mL demonstrated the highest relative translocation across the epithelial model, whereas higher exosome loading was associated with reduced permeability, likely due to enhanced epithelial interaction and intracellular retention. Conclusion Exosome functionalization enhances the biological interaction of carbonate apatite nanoparticles with TR146 and RAW264.7 cells. These findings support the potential of CHA-EXO as a promising immunomodulatory adjuvant platform for oral mucosal vaccine development. Lay Summary This study developed and evaluated exosome-functionalized carbonate apatite (CHA-EXO) nanoparticles as a novel type of mucosal vaccine adjuvant. Using oral epithelial and immune cells for in vitro studies, the nanoparticles showed excellent compatibility, enhanced cellular uptake, and balanced immune activation without inducing excessive inflammation. A fibronectin-coated nanofibrous PLA mesh was further established to mimic the oral mucosal barrier, revealing that CHA-EXO nanoparticles can effectively traverse epithelial layers. Collectively, these findings suggest that CHA-EXO hold strong potential as a safe and efficient delivery platform for future oral mucosal vaccines.