For drugs to exert their biological effects, they must reach their intracellular
targets, requiring efficient membrane permeation, diffusion, and distribution.
We examined the intracellular diffusion and distribution of smallmolecule
fluorescent drugs in comparison with proteins using fluorescence
microscopy and fluorescence recovery after photobleaching (FRAP). While
proteins diffused freely, small molecules showed a strong dependence of diffusion
behavior on their pKa. Weakly basic drugs exhibited markedly reduced
fractional recovery and 10–20-fold slower diffusion in cells than in
aqueous solution. As most pharmaceutical drugs are weak bases, their protonation
in the cytoplasm forms membrane-impermeable ionic species, leading
to ion trapping and reduced mobility under crowded intracellular conditions.
Imaging revealed substantial accumulation of these molecules in acidic
organelles, particularly lysosomes. Inhibition of lysosomal import slightly
improved diffusion, whereas preventing protonation through N-acetylation
greatly enhanced both diffusion and recovery. These findings indicate that
N-acetylation may be an effective strategy to improve the intracellular availability
and distribution of weakly basic small-molecule drugs.