Abstract
For establishment of cancer immunotherapy, efficient antigen carriers are needed to deliver antigen into cytosol of dendritic cells (DCs) and induce antigen-specific cellular immune responses. Many studies have been done to achieve cytoplasmic delivery of exogenous antigens into the DCs and induction of antigen-specific cellular immune responses.Among these delivery systems, pH-sensitive liposomes are useful as an intracellular delivery system for their ability to transfer their contents into cytosol by pH-responsive fusion with endosomal membrane. We previously reported cytoplasmic delivery using liposomes modified with 3-methylglutarylated linear or hyperbranched poly(glycidol)(MGlu-LPG or MGlu-HPG, respectively). These polymers become fusogenic under weakly acidic condition and hence modification with these polymers provides stable liposomes with pH-sensitive fusion ability. In this study, we applied these pH-sensitive polymer-modified liposomes to antigen delivery system. First, these liposomes were used for delivery of FITC-labeled OVA into bone marrow-derived DCs (BMDCs). Compared with the case of polymerunmodified liposomes, BMDCs treated with polymer-modified liposomes showed more intensive diffuse fluorescence of FITC-OVA within cells, indicating that polymer-modified liposomes delivered their contents into cytosol of BMDCs by efficient fusion with endosomal membranes. To confirm the antigen presentation pathway, BMDCs were treated with these liposomes and co-cultured with two kinds of T cells, CD8-OVA1.3 or OT4H.1D5 cells, which recognize the MHC class I/peptide complexes and MHC class II/peptide complexes, respectively. Highly enhanced IL-2 production was observed from the supernatant of polymer-modified liposome-treated BMDCs co-cultured with CD8-OVA1.3 cells, indicating that polymer-modified liposomes induced MHC class I-restricted immune responses due to their ability to deliver the loaded proteins into cytosol of DCs. These liposomes were administered to mice subcutaneously and cytotoxic T-lymphocyte (CTL) responses in spleen were evaluated. Administration of polymer-modified liposomes induced strong CTL responses against antigen-expressing tumor cells (E.G7-OVA cells), while no CTL response was observed against antigen-non-expressing tumor cells (EL4 cells). These results indicate that polymer-modified liposomes induced antigen-specific and MHC class I-restricted immune responses in vivo. Finally, the OVA-loaded liposomes were administered to E.G7-OVA and EL4 tumor-bearing mice and then tumor growth was monitored. E.G7-OVA-tumor volumes of mice treated with polymer-modified liposomes decreased drastically and disappeared completely. In contrast, EL4 tumor volumes of same mice increased. These results indicate that only E.G7-OVA tumor cells were killed by strong OVA-specific CTL responses induced by immunization with pH-sensitive polymer-modified liposomes. Therefore, they might have potential usefulness as delivery vehicles for cancer immunotherapy.
