Detailed splenic single-cell biodistribution of phosphatidylglycerol-containing liposomes

Antigen-specific tolerance induction is a promising therapeutic strategy for autoimmune and chronic inflammatory diseases. This can be achieved by targeted activation of regulatory T and B cells via antigen-presenting cells (APCs) in a tolerogenic context. Anionic antigen-carrying liposomes have shown potential, however, their efficacy is highly dependent on the administration route and liposomal rigidity. Here, we investigate the biodistribution and splenic APC subset-specific uptake of rigid DSPC:DSPG:CHOL liposomes compared to flexible DOPC:DOPG:CHOL liposomes using high-parameter flow cytometry. We developed a panel enabling identification of rare splenic APC subsets involved in immune tolerance, including CD169 ⁺ and MARCO ⁺ marginal zone macrophages, red pulp macrophages, and conventional/plasmacytoid dendritic cells. Our findings confirm that rigid liposomes predominantly accumulate in the liver and spleen following IV injection, with negligible uptake in lymph nodes or lungs. Importantly, systemic distribution is significantly inhibited by subcutaneous administration, which is essential for tolerance induction. Among splenic APCs, macrophage subsets are major contributors to liposome uptake, though the liver remains the primary site of accumulation and may play a more dominant role in tolerance induction. This study underscores the importance of both liposomal design and delivery route in optimizing nanoparticle-based immune modulation strategies.