Results are presented as the mean standard deviation of three separate experiments. At a 20:5:1 ratio, the onset and degree of hemolysis for AmB co-loaded with ergosterol, cholesterol, or 7-DHC in PEG-DSPE micelles was reduced (Determine 6B). the absence of sterol or with cholesterol, but aggregated in the presence of ergosterol or 7-dehydrocholesterol. The fraction of aggregated AmB in PEG-phospholipid micelles was lower at the 20:5:1 ratio. In an aggregated state or in the absence of sterol, AmB caused rapid and complete hemolysis. In contrast, deaggregated AmB co-loaded with cholesterol caused slower and incomplete hemolysis, especially at a 20:5:1 ratio. == Conclusions == The aggregation state of AmB in PEG-phospholipid micelles was sterol dependant. AmB/cholesterol co-loaded PEG-phospholipid micelles caused lowin vitrohemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region. Keywords:Amphotericin B, aggregation state, hemolysis, micelle, sterol == INTRODUCTION == Over the past two decades, the frequency of sepsis has increased significantly in the United States, with systemic candidiasis accounting for the fourth most common contamination in immunocompromised patients.(1,2) Amphotericin B (AmB) is a common choice for the treatment of systemic fungal diseases despite causing severe toxic side effects.(3) In a drug delivery approach, liposomal AmB has reduced the dose-limiting renal toxicity over AmB deoxycholate (AmB-D), its standard formulation, but still, mortality rates for systemic fungal diseases remain largely unchanged and high, ca. 40%, providing solid rationale for progress in the delivery of this potent, membrane-active antifungal agent. In an alternative strategy based on the aggregation state hypothesis, monomeric (i.e. deaggregated) AmB is usually selective for ergosterol in fungal cell membranes, forming channels by the classic barrel stave motif (Physique 1. B). In contrast, soluble aggregated species of AmB are non-selective and form channels in both fungal and mammalian cell membranes, causing host toxicity. Due to Rabbit Polyclonal to EDG4 its amphiphilic nature, Flopropione AmB has a high propensity towards self-aggregation in water and for poor water solubility. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (PEG-DSPE) micelles solubilized AmB in a largely monomeric state, evidenced by a loss of a peak in the UV-Vis spectrum at 328 nm, characteristic of aggregated AmB (Physique 1. C). As a result, AmB caused lowin vitrohemolysis relative to free drug.(4) In contrast, serum proteins quickly disrupted the integrity of PEG-DSPE micelles, resulting in the rapid release of AmB, its reaggregation, and potential for host toxicity.(5) More recently, co-incorporation of cholesterol along with AmB enhanced the structural stability of PEG-DSPE micelles in the presence of serum albumin, owing to cholesterol/phospholipid interactions that increase core viscosity.(6) == Physique Flopropione 1. == PEG-DSPE, cholesterol, and AmB assembly into micelles (A), aggregation state hypothesis for AmB in ergosterol and cholesterol made up of membranes showing pore formation of monomeric AmB Flopropione in ergosterol made up of mambranes and pore formation by aggregated AmB in cholesterol made up of membranes (B), and absorption spectra of aggregated and deaggregated AmB (C) The goal of this work was to characterize the effects of various co-loaded sterols around the aggregation state and hemolysis of AmB incorporated in PEG-DSPE micelles: Ergosterol, cholesterol, and 7-dehydrocholesterol (7-DHC) (Physique 2). Ergosterol and cholesterol were chosen because they represent the native membrane sterols in fungal cells and mammalian cells, respectively. AmB has been shown to preferentially interact with ergosterol over cholesterol in lipid environments, when AmB is in a deaggregated state, while losing conversation specificity at higher concentration, when AmB begins to self-associate.(7,8) 7-DHC has been previously shown to bind AmB in a deaggregated state in a co-solvent system.(9) == Physique 2. == Chemical structure of cholesterol, ergosterol, and 7-dehydrocholesterol == MATERIALS AND METHODS == == Materials == AmB was a gift from X-GEN Pharmaceuticals Inc. (Big Flats, NY). Glacial acetic acid, phosphoric acid, HPLC-grade acetonitrile, methanol, and chloroform were purchased Flopropione from Sigma-Aldrich Inc. (St. Louis, MO) and used as received. Phosphate buffered saline 1 (pH 7.4) (PBS) was purchased from Cellgro (Mediatech Inc., Manassas, VA). Ergosterol, cholesterol, and 7-DHC were purchased from Sigma-Aldrich Inc. Flopropione (St. Louis, MO) and used as received. Sterols were stored at 20C..