Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

Abstract

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent 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+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.

Liposomal Irinotecan

“…Irinotecan was actively loaded into DSPC/Chol (55:45 mol%) liposomes using an ionophore-mediated proton gradient (31). The divalent cation ionophore A23187 (0.5 g per 1 mg lipid) was incorporated into the liposomal bilayer after incubation at 37°C or 60°C for 10 minutes.…”

In Vitro and in Vivo Characterization of Doxorubicin and Vincristine Coencapsulated within Liposomes through Use of Transition Metal Ion Complexation and pH Gradient Loading

“…To generate the gradients, DSPC/Chol liposomes in MnSO 4 buffer (pH 3.5) were eluted on Sephadex G-50 columns equilibrated with a 300 mM Sucrose/20 mM HEPES/15 mM EDTA (SHE) buffer at pH 7.5. To stabilize the pH gradient across the liposomes, the divalent cation ionophore A23187 was added to some of the indicated liposome preparations (23,24). A23187 is an electroneutral ionophore capable of translocating a divalent cation for the exchange of two hydrogen ions (25).…”

Nanotechnology-based approaches in anticancer research

“…73 Use of the ionophore method of drug loading allows much higher drug:lipid ratios than the conventional citrate method. 74 This formulation has given excellent results in studies comparing the efficacy of conventional CHOP (chemotherapy treatment composed of cyclophosphamide, doxorubicin, vincristine, and prednisone) and CHOP in which the vincristine has been replaced by LNP-vincristine. 75,76 Moreover, increased drug retention of vincristine has been reported to be related to increased drug:lipid ratios.…”