Real-time in vivo tracking of nanoparticulate vaccines in inguinal lymph nodes.

Understanding the kinetics of nanoparticle vaccine drainage to the lymph nodes and their residence time in these tissues is critical in vaccine development. Evaluating these parameters using conventional techniques is expensive, time-consuming, and often leads to variable data, as several animals at different time points need to be sacrificed, and lymph nodes need to be harvested for characterization. Here, we adapted an abdominal window chamber for intravital imaging of the lymph nodes, enabling real-time tracking of ionizable lipid nanoparticles (LNPs) and polymeric nanoparticles in live mice following intramuscular administration. To mimic delivery of vaccine payloads, these nanoparticles were encapsulated with luciferase mRNA or a small molecule fluorophore, indocyanine green, and tracked for bioluminescence and fluorescence, respectively, in the lymph nodes. The results demonstrated differential lymph node drainage kinetics and retention for LNPs and polymeric nanoparticles. Notably, variations in the ionizable lipid components of LNPs resulted in changes in the mRNA translation ability within the lymph nodes. Taken together, this study introduces a valuable tool to optimize nanoparticle vaccine design in preclinical studies. Graphical Abstract: Real-time in vivo tracking of the lymph node drainage kinetics of nanoparticles encapsulating mRNA or small molecule fluorescent dye, facilitated by the use of a lymph node window chamber. Created in BioRender. https://BioRender.com/786dnai. Supplementary Information: The online version contains supplementary material available at 10.1186/s41120-026-00163-5.