Amphotericin B/sterol co-loaded PEG-phospholipid micelles: effects of sterols on aggregation state and hemolytic activity of amphotericin B.

PURPOSE: To elucidate the effect of sterols on the aggregation of amphotericin B (AmB) in PEG-phospholipid micelles and its consequences on the hemolytic activity of AmB. METHODS: AmB-incorporated PEG-phospholipid micelles co-loaded with ergosterol, cholesterol, or 7-dehydrocholesterol were prepared at 4:1:1 and 20:5:1 ratios of polymer-to-sterol-to-AmB. The aggregation state of AmB was elucidated by UV-vis spectroscopy. AmB/sterol co-loaded PEG-phospholipid micelles were incubated with red blood cells and the hemolytic activity of AmB assessed by measurement of free hemoglobin. RESULTS: AmB in PEG-phospholipid micelles stayed mostly in a deaggregated state in 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 low in vitro hemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region.

Pharmacokinetics and nephrotoxicity of amphotericin B-incorporated poly(ethylene glycol)-block-poly(N-hexyl stearate l-aspartamide) micelles.

The purpose of this investigation was to study the pharmacokinetics and nephrotoxicity of amphotericin B (AmB), incorporated in poly(ethylene glycol)-block-poly(N-hexyl stearate l-aspartamide) (PEG-b-PHSA) micelles (AmB/PEG-b-PHSA). After AmB/PEG-b-PHSA or AmB for injection, United States Pharmacopeia (USP), was dosed intravenously in rats (0.8 mg/kg), serum was collected over 72 h, and organs collected at 72 h for AmB analysis. To test for the nephrotoxicity caused by AmB, renal markers of damage were assessed 24 h after a single injection of AmB/PEG-b-PHSA or AmB for injection, USP, focusing on detection of urinary enzymes. PEG-b-PHSA micelles caused a significantly lower area under serum concentration curve and higher clearance relative to AmB for injection, USP. PEG-b-PHSA micelles lowered the distribution of AmB in liver and lung tissues, but did not significantly lower the level of AmB in the kidneys relative to AmB for injection, USP. However, urine levels of N-acetyl-ß-glucosaminidase and γ-glutamyltransferase were significantly lower for AmB/PEG-b-PHSA relative to AmB for injection, USP. In summary, PEG-b-PHSA micelles reduced the nephrotoxicity of AmB, the dose-limiting toxicity of this important antifungal agent.

Enhanced stability of PEG-block-poly(N-hexyl stearate l-aspartamide) micelles in the presence of serum proteins.

Polyethylene glycol-phospholipid micelles form a major class of nanocarriers in pharmacy and medicine due to proven capability in drug solubilization, sustained drug release, and evidence for targeted drug delivery in vivo. In this report, we have prepared micelles composed of PEG-block-poly(N-hexyl stearate l-aspartamide) (PEG-b-PHSA), having nine stearic acid side chains, and have studied their stability in the presence of serum proteins by Forster resonance energy transfer (FRET) experiments. In the presence of serum albumin, alpha and beta globulins, or gamma globulins, there are minimal changes in FRET over two hours in vitro, indicating integrity of PEG-b-PHSA micelles. In contrast, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (PEG-DSPE) micelles lose FRET over two hours in vitro, especially in the presence of alpha and beta globulins, indicating the disruption of PEG-DSPE micelles and leakage of fluorescent probes. Owing to the aliphatic nature of DSPE and PHSA, both PEG-b-PHSA and PEG-DSPE micelles efficiently solubilize amphotericin B (AmB), a poorly water-soluble antifungal agent used to combat systemic mycoses. However, only PEG-b-PHSA micelles gradually liberate AmB in the presence of alpha and beta globulins, based on time-dependent changes in the self-aggregation state of AmB, monitored by UV/vis spectroscopy. PEG-b-PHSA micelles are remarkably stable in the presence of serum proteins and a more stable alternative for poorly water-soluble drugs, which have been solubilized by PEG-DSPE micelles.

Mixed polymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents.

As a novel drug nanocarrier for combination delivery of multiple anticancer agents, mixed polymeric micelles were developed and characterized in this study. A DNA-damaging anthracycline agent doxorubicin (DOX) and a phosphatidylinositol-3 kinase inhibitor wortmannin (WOR) were conjugated alone or combination onto poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers through a hydrazone bond. Polymer-drug conjugates assembled into a unimodal micelle structure with a <100 nm particle size, in which the drug mixing ratios between DOX/WOR were precisely controlled. Cytotoxicity assay against a human breast cancer MCF-7 cell line showed that drug nanocarrier based on mixed polymeric micelles can reduce DOX required for cytotoxicity while maintaining the biological activity of the independent polymeric micelles. It is postulated that WOR enhances the efficacy of DOX through an efficient combination delivery accompanied by synergistic drug action. These findings, therefore, bring an effective drug delivery methodology that might reduce the effective dose as well as toxicity in vivo compared to the conventional drug formulations.