Background: The demand for an effective vaccine delivery system that drives a suitable immune response is increasing. The oxidized carbon nanosphere (OCN), a negatively charged carbon nanoparticle, has the potential to fulfill this requirement because it can efficiently deliver macromolecules into cells and allows endosomal leakage. However, fundamental insights into how OCNs are taken up by antigen-presenting cells, and the intracellular behavior of delivered molecules is lacking. Furthermore, how immune responses are stimulated by OCN-mediated delivery has not been investigated. Purpose: In this study, the model protein antigen ovalbumin (OVA) was used to investigate the uptake mechanism and intracellular fate of OCN-mediated delivery of protein in macrophages. Moreover, the immune response triggered by OVA delivered by OCNs was characterized. Methods: Bone-marrow-derived macrophages (BMDMs) from mice were used to study antigen uptake and intracellular trafficking. Mice were immunized using OCN-OVA combined with known adjuvants, and the specific immune response was measured. Results: OCNs showed no cytotoxicity against BMDMs. OCN-mediated delivery of OVA into BMDMs was partially temperature independent process. Using specific inhibitors, it was revealed that intracellular delivery of OCN-OVA does not rely on phagocytosis or the clathrin- and lipid raft/caveolae-mediated pathways. Delivered OVA was found to colocalize with compartments containing MHC class I, but not with early endosomes, lysosomes, and autophagosomes. Immunization of OVA using OCNs in combination with the known adjuvant monophosphoryl lipid A specifically enhanced interferon gamma (IFNγ)- and granzyme B-producing cytotoxic T cells (CTLs). Conclusion: OCNs effectively delivered protein antigens into macrophages that localized with compartments containing MHC class I partially by the temperature independent, but not clathrin- and lipid raft/caveolae-mediated pathways. Increased CD8⁺ T-cell activity was induced by OCN-delivered antigens, suggesting antigen processing toward antigen presentation for CTLs. Taken together, OCNs are a potential protein antigen delivery system that stimulates the cell-mediated immune response.

#	Concept	H-Index	Publications	Citations
1	Engineering	1	1	19
2	Chemical Engineering	1	1	19
3	Chemistry	1	1	19
4	Biophysics	1	1	19
5	Nanomaterials	1	1	19

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