Tuning the lipophilicity of a 3,4-dialkoxy-1,8-naphthalimide scaffold to optimise live cell lipid droplet imaging

Lipid droplets (LDs) are dynamic organelles that regulate cellular lipid storage and metabolism, processes that are particularly important in diseases such as obesity and cancer. Confocal fluorescence microscopy has the potential to enable high-resolution visualisation of LD biogenesis, morphology and function in live cells, but is limited by the availability of agents for very specific lipid imaging. Physiochemical properties such as lipophilicity may be exploited as a key design parameter yet most LD probes lack a clear consensus targeting motif, unlike molecular probes for other organelles that exploit well-established targeting groups. Here, using a collection of fluorescent 3,4-dialkoxy-1,8-naphthalimides, we explore the influence that subtle changes in structure and lipophilicity (cLog P ) has on LD staining and imaging. A small series of low molecular weight structural derivatives were synthesised (including structural isomers), which spanned a cLog P range from 2.08 to 3.94. The compounds exhibited excellent photophysical characteristics such as large Stokes shifts (up to 133 nm) and high fluorescent quantum yields in non-polar solvents (0.81–0.98), showed no apparent toxicity at working concentrations, and provided instantaneous LD staining at very low working concentrations (< 1.0 µM). Two compounds from the series ( 2b and 6b ) were used to capture critical differences in LD size and location in prostate cancer cells and to visualise adaptive LD biology in cells treated with metabolic inhibitors or exogenous lipid. This work establishes a structure-lipophilicity relationship for optimising LD targeting and defines a practical lipophilicity window for LD-targeted 1,8-naphthalimides that can probe LD diversity and function.