Bioluminescence imaging using in vivo imaging systems (IVIS) is widely used to assess reporter protein expression after administration of messenger RNA (mRNA) lipid nanoparticles (LNPs). However, methodological variation and prior LNP exposure can complicate interpretation. Here, we evaluated selected parameters affecting IVIS readouts after intramuscular administration of firefly luciferase mRNA formulated in SM-102 or ALC-0315 LNPs in BALB/c mice. We also investigated the effects of repeated administration. D-luciferin delivery, imaging interval and mouse position influenced signal acquisition, while mRNA dose and injection volume affected the magnitude and regional distribution of expression. Background bioluminescence in mice receiving PBS was negligible compared with mRNA-LNP-associated signals, suggesting that dedicated negative-control groups may be unnecessary once baseline signal is established under consistent imaging conditions. Repeated administration of either SM-102 or ALC-0315 LNPs reduced expression following a subsequent mRNA-LNP dose. This occurred after prior administration of LNPs containing either firefly luciferase mRNA or non-coding polyadenylic acid, whereas repeated PBS injections had no comparable effect. Therefore, the reduced expression was associated with prior exposure to the LNP carrier rather than repeated injection or expression of the encoded reporter. These results highlight the need to standardise IVIS acquisition and dosing parameters, including mRNA dose, injection volume, and administration schedule, and demonstrate that prior LNP exposure can attenuate subsequent mRNA-mediated protein expression.
