Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes.

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.