Transport of Hemicyanine Dyes with Varied Alkyl Chains through the Lipid Membrane and the Effect of Cholesterol

Abstract Second-harmonic-generation (SHG)-active dyes have attracted increasing attention as interfacial probes for evaluating molecular dynamics at membrane interfaces through their nonlinear optical response. However, how subtle structural variations regulate membrane retention and transport behavior remains insufficiently understood. Herein, four hemicyanine dyes, THC1, THC3, THC6, and THC8, based on the (3-alkyl-2-(4-hydroxy-3,5-dimethoxystyryl))benzo[d]thiazol-3-ium scaffold and bearing different N-alkyl-chain lengths, were synthesized and investigated in DOPG (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) vesicles using time-dependent SHG spectroscopy. All THC dyes produced notably enhanced membrane-associated SHG signals, indicating adsorption and insertion into the lipid bilayer. THC1 and THC3 exhibited efficient transport, whereas THC6 and THC8, which contain longer N-alkyl chains, showed markedly slower SHG decay. The influence of membrane composition on THC dyes with different alkyl-chain lengths was further examined by incorporating 30 mol % cholesterol into DOPG vesicles. Although cholesterol reduced the membrane transport of all four dyes, its effect was more pronounced for THC1 and THC3 than for THC6 and THC8. The difference in the interactions between the alkyl chains and the lipid membranes was analyzed to understand the different membrane retention of the THC dyes.