Biochimica et Biophysica Acta, 1064 (1991) 165-168 © 1991 ElsevierScience Publishers B.V. 0005-2736/91/$03.50 ADONIS 0005273691001827 165 BBAMEM 70676 BBA Report A kinetic method for measuring functional delivery of amphotericin B by drug delivery systems R.P. Peterson, S.K. Benz, B.S. Whyte and S.C. Hartsel Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, WI (U.S.A.) (Received 27 August 1990) Key words: Amphotericin B: Pyranine: Ion current; Drug delivery; Liposome The human toxicity of amphotericin B can be considerably reduced by associating the drug with liposomcs of varying lipid compositions. Some lipid compositions are much more effective than others. We show that a simple kinetic fluorescence assay using pyranine as an indirect probe of amphotericin-induced K + currents may be used to study different iiposomal drug delivery systems in vitro. We find that lipid mixtures composed of DMPC/DMPG/ampho- tericin at a 7:3:1 mole ratio show very slow functional delivery with a preference for ergosterol over cholesterol-con- taining membrane vesicles. On the other hand, amphotericin delivered from egg phosphatidyicholine iiposomes lead to 100-fold increases in K + leakage at one-fifth the amphotericin concentration of the 7:3:1 system. The egg phosphatidyicholine system as well as miceilar amphotericin also show a slight selectivity towards cholesterol-containing vesicles over ergosterol. These results are consistent with previous clinical and in vitro cellular studies and this technique may prove valuable in screening of other delivery systems. The polyene antibiotic amphotericin B (AmB) is the most commonly used drug to treat systemic fungal infections, particularly those that occur in patients with lymphomas, leukemias, AIDS, or other immune system comprorrfising diseases [1]. It forms ion channels, caus- ing lethal changes in membrane permeability to many univalent and divalent cations (see Ref. 2 for a compre- hensive review). It also leads to cellular oxidative damage [3]. The antibiotic is selectively toxic towards organisms whose membranes contain ergosterol such as fungi [2]. However, the commercial form of AmB, fungizone, also causes acute and chronic side effects in patients taking it. This is especially true when the drug is administered in the high doses and long courses of therapy necessary to fight most fungal infections [1]. To improve the fungal selectivity of AmB, derivatives of the drug as well as new liposomal vehicles for solubi- lizing and delivering it have been developed. Some of these lipid/AmB preparations have already been tested clinically with good success (see Ref. 4 for a recent review). When AmB was incorporated into a lipid mix- ture containing dimyristoylphosphatidylcholine and di- myristoylphosphatidylglycerol (DMPC/DMPG) at a Correspondence: S.C. Hartsel, Department of Chemistry, University of Wisconsin, Eau Claire, WI 54702, U.S.A. 7:3 molar ratio, it retained its antifungal activity with fewer of the side effects characteristics of Fungizone [5]. Likewise, in vitro studies have shown that this and other liposomal AmB delivery systems retain their toxicity towa,d fungal cells while reducing AmB's activity on cholesterol-containing cells [1,6-8]. This improved selectivity for ergosterol-containing organisms may result from a preferential vehicle-to- target transfer of AmB that relies on the characteristics of both the delivery system and the target cell [1]. We show that such transfer from a DMPC/DMPG/AmB 7 : 3 : 1 (mol/mol) lipid mixture (7 : 3 : 1 L-AmB) to target small unilamellar vesicles (SUV) is very slow using an in vitro fluorescence assay. Our navel func- tional assay, which uses kinetic measurements of trans- membrane K + current induced by transfer of AmB to target SUV containing cholesterol or ergosterol, also allows us to examine the question of sterol selectivity. Egg phosphatidylcholine (EPC) was isolated from fresh eggs using the Singleton procedure [9]. Di- myristoyl PC and dimyristoylphosphatidylglycerol were purchased from Avanti Polar lipids, Alabaster, AL. Purified amphotericin B was a gift from the Squibb Institute for Medical Research (Princeton, N J). FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was obtained from the Sigma Chemical Company (St. Louis, MO). Laser grade pyranine (1,3,6-pyrenetri-