IONOPHORE-MEDIATED UPTAKE OF CIPROFLOXACIN AND VINCRISTINE INTO LARGEUNILAMELLAR VESICLES EXHIBITING TRANSMEMBRANE ION GRADIENTS

A new method, based on the ion-translocating properties of the ionopho res nigericin and A23187, is described for loading large unilamellar v esicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs comp osed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 m ol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a trans membrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presenc e of drug and, for experiments involving divalent cations, the chelato r EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying th e vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown th at both nigericin and A23187 facilitate the rapid uptake of vincristin e and ciprofloxacin, with entrapment levels approaching 100% and excel lent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ sy stem exhibits superior drug retention over the nigericin/K+ system, an d compares favorably with vesicles loaded by the standard Delta pH or amine methods. The unique features of this methodology and possible be nefits are discussed. (C) 1998 Elsevier Science B.V. All rights reserv ed.