Messenger RNA (mRNA) therapeutics formulated in lipid nanoparticles (LNPs) have transformed vaccinology and enabled a versatile platform for protein-based therapy across a wide range of diseases. Despite their clinical success, LNP-encapsulated mRNA (LNP-mRNA) systems are still largely designed through empirical screening due to an incomplete understanding of their in vivo behavior. This review systematically examines how in vivo imaging modalities have been used in the recent literature to interrogate LNP biodistribution, cellular uptake, and functional mRNA expression. Across the studies reviewed, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers have demonstrated that LNP biodistribution, including organ tropism and size-dependent transport, is governed by formulation parameters and route of administration. Liver and spleen accumulation dominate current systemic delivery profiles. Particles in the 20-100 nm range generally favor efficient extravasation, cellular uptake, and lymphatic transport, while larger ones are gradually associated with reduced cellular internalization and increased reliance on cell-mediated transport to reach draining lymph nodes. Quantitative fluorescence and reporter-gene studies identified endosomal escape as the principal intracellular bottleneck, with less than 2% of internalized RNA typically reaching the cytosol. MRI results, although currently limited, have provided coherent evidence while offering a non-invasive approach for longitudinal monitoring of intracellular RNA trafficking. Imaging has also revealed a recurrent disconnect between LNP exposure and protein expression, demonstrating that LNP organ accumulation alone is an inadequate measure of productive delivery. Bioluminescent imaging approaches have enabled tracking of protein expression kinetics, showing that peak expression often occurs within hours post-administration, but varies significantly with ionizable lipid nature. Collectively, the recent literature indicates that in vivo imaging can help connect biodistribution and functional delivery with LNP designs. This review highlights that integrating quantitative in vivo imaging into the LNP-mRNA development pipeline enables mechanistic identification of delivery barriers and supports rational optimization of formulations.
