Unveiling the contribution of histidine-end-terminal pBAEs to endosomal escape using super-resCLEM imaging.

The success of gene therapy focuses on the development of efficient and safe carriers capable of overcoming cellular barriers to deliver genetic material to the nucleus. Poly(beta-amino ester) nanoparticles (pβAE NPs) are promising cationic polymers that can complex nucleic acids to form polyplexes, facilitating intracellular delivery. Among the key intracellular hurdles, endosomal escape remains one of the most critical and debated steps. While the incorporation of histidine into polyplex formulations has been proposed to enhance endosomal escape via proton buffering, definitive evidence of this process has remained elusive. To address this gap, a super-resolution correlative light and electron microscopy (CLEM) approach was employed to decipher the intracellular trafficking of pβAE NPs. Specifically, we compared two pβAE formulations, one incorporating a histidine-arginine end-capping structure (RH) and a control containing only arginine end-capping (R). Our findings revealed that RH polyplexes efficiently escaped endosomes at early timepoints and achieved cytosolic localization, whereas R polyplexes predominantly remained trapped within endo-lysosomal compartments, ultimately undergoing exocytosis or degradation, as confirmed by lysosome-specific staining. This study provides, for the first time, direct high-resolution evidence of histidine-mediated endosomal escape in gene therapy, underscoring the role of histidine in overcoming intracellular barriers. Moreover, it highlights the power of super-resolution correlative imaging techniques in elucidating the nanoscale trafficking of gene delivery systems, enabling more rational design of next generation nanocarriers.