When Escape Isn’t: Reevaluating LysoTracker for Proton Sponge–Driven Endosomal Escape

Steven Huysecom – Doctoral student, KU Leuven; Thibo Iven – Doctoral student, KU Leuven; Sandra Krzyzowska – Doctoral student, KU Leuven; Beatrice Fortuni – Post doctoral researcher, KU Leuven; Hiroshi Uji-i – Professor, RIES Hokkaido; Susana Rocha – Assistant professor, KU Leuven

Introduction: Endosomal escape is a major barrier in nanoparticle-mediated drug delivery and is often inferred from reduced colocalization with acidotropic dyes such as Lysotracker. However, because Lysotracker accumulation depends on acidic pH, loss of signal may reflect lysosomal alkalinization rather than true escape. Here, we re-evaluated Lysotracker as a readout for endosomal escape in proton sponge-active systems using PEI-coated mesoporous silica nanoparticles as a model.