The effect of fatty acid substitution on the in vitro release of amphotericin B from micelles composed of poly(ethylene oxide)-block-poly(N-hexyl stearate-l-aspartamide)

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Abstract

The effect of fatty acid substitution on the in vitro release of amphotericin B (AmB) from micelles composed of poly(ethylene oxide)-block-poly[N-(6-hexyl stearate)-l-aspartamide] (PEO-b-PHSA) was investigated. PEO-b-PHSA at 11, 50 and 70% of stearic acid substitution self assembled into micelles that effectively encapsulate AmB by solvent evaporation and dialysis methods. The sustained release of AmB from PEO-b-PHSA micelles was evidenced, by measuring the transfer of the drug to lipid vesicles [dipalmitoyl phosphatidylcholine:cholesterol:dimyristoyl phosphatidyglycerol (3:1:0.25)]. The release of AmB for PEO-b-PHSA micelles was markedly influenced by the degree of fatty acid substitution—as it increased, the release of AmB slowed. Accordingly, drug release was found to correlate with haemolysis induced by AmB encapsulated in PEO-b-PHSA micelles. At 11% stearic acid substitution, encapsulation of AmB had little effect on the drug’s ability to induce untoward haemolysis. In contrast, AmB stably encapsulated in PEO-b-PHSA micelles at 50 and 70% caused no hemolysis up to 20 μg/ml. Lastly, PEO-b-PHSA micelles at 50 and 70% were able to elute entirely as micelles during size-exclusion chromatography, indicating their stability toward dissociation after dilution. The results point to a nanoscopic drug depot that may release AmB at controlled rates.

Introduction

Micelles made of poly(ethylene oxide)-block-poly(l-aspartic acid) (PEO-b-PLAA) have recently emerged as novel drug delivery systems for many water insoluble drugs [1]. In this context, the chemical conjugation and the physical encapsulation of therapeutic molecules have been pursued with the goal of solubilization as well as sustained drug release properties [2], [3], [4], [5], [6].

We have reported on a polymeric micelle formulation for amphotericin B (AmB) [7], [8]. AmB is the most potent antifungal agent in clinical practice [9]. Despite great efficacy, severe side effects associated with the administration of its water-soluble formulation, Fungizone®, have restricted the clinical application of AmB. While three lipid-based carriers are available for AmB that circumvent its poor water solubility and reduce its toxicity [10], there are still concerns about high cost and high dose requirements and on whether the therapeutic index of the drug has been increased [11]. Recently, AmB encapsulated in long circulating liposomes has been found to be more effective than AmB in standard liposomes in a murine model of candidiasis [12]. Thus, long circulating carriers of AmB may be able to increase the therapeutic index of AmB at low dose, low risk of toxicity, and low cost.

With this in mind, we have prepared micelles composed of fatty acid esters of PEO-b-PLAA for the delivery of AmB. The goal is to prepare polymeric micelles that can effectively encapsulate AmB and circulate for long periods in blood, while functioning as a depot for AmB release. In other words, we aim to mimic biological transport systems, i.e., low-density serum lipoprotein, but with a favorable effect on the biodistribution of AmB. We have synthesized poly(ethylene oxide)-block-poly[N-(6-hexyl stearate)-l-aspartamide] (PEO-b-PHSA), which self-assembles into nanoscopic micelles at very low concentration [8]. Owing to favorable interaction between AmB and the fatty acid ester core, AmB encapsulated in PEO-b-PHSA micelles is non-toxic toward red blood cells, but active against fungal cells [13]. A low rate of drug release perhaps with the drug in a monomeric state has been hypothesized to be the basis of the reduced toxicity of encapsulated AmB.

We have studied the release of AmB from PEO-b-PHSA micelles and the stability of the micelles with respect to dissociation. The novel results show the sustained release of AmB by PEO-b-PHSA micelles, which is dependent on the level of fatty acid substitution. A dependence of micelle stability on the level of fatty acid ester substitution has also been revealed. Owing to high micelle stability and the sustained release of AmB, we speculate that PEO-b-PHSA micelles at a high level of fatty acid substitution may act as a long-circulating depot of AmB for increased drug efficacy.

Section snippets

Synthesis of PEO-b-PHSA

PEO-b-PHSA was prepared from poly(ethylene oxide)-block-poly(β-benzyl-l-aspartate) (PEO-b-PBLA) as described previously [7]. The molecular weight of PEO and the number of BLA monomers of PEO-b-PBLA were 12 000 g/mol and 24, respectively. Briefly, PEO-b-PBLA (0.10 mmol BLA units) underwent aminolysis with 6-aminohexanol (10 equiv.) at room temperature in the presence of 2-hydroxypyridine (0.3 mmol). The product, PEO-block-poly(hydroxyhexyl-l-aspartamide) (PEO-b-PHHA) (0.003 mmol HAA units), was

Results

The chemical structure of PEO-b-PHSA is shown in Fig. 1. AmB was encapsulated at three levels of stearic acid substitution (Table 1) and the release of drug for PEO-b-PHSA micelles assessed by measuring the transfer of AmB to lipid vesicles. The lipid vesicles were used to maintain sink conditions for poorly soluble AmB and to separate the released drug from encapsulated drug. We assume the rate-determining step is the release of AmB from PEO-b-PHSA micelles. While the method needs to be

Discussion

The long-term objective of this study is to develop a novel long circulating nanoscopic carrier based on PEO-b-PLAA for AmB. To enhance the encapsulation of AmB, we attached a compatible moiety, i.e., fatty acid, to the core-forming block (Fig. 1). Through a solvent evaporation method PEO-b-PLAA micelles effectively encapsulated AmB, resulting in reduced toxicity (hemolysis), particularly in comparison to the more common dialysis method of drug loading [13]. The degree of fatty acid

Conclusions

PEO-b-PHSA micelles can gradually release AmB for prolonged periods. The release of encapsulated AmB from PEO-b-PHSA micelles as well as the stability of the micelles can be controlled, by varying the degree of stearic acid substitution. Lastly, the tailoring the structure of PEO-b-PHSA might lead to micelles that can improve the efficacy of AmB in the treatment of systemic fungal diseases.

Acknowledgements

This work was supported in part by NIH grant AI43346-01. We thank Nanocarrier Inc. for providing PEO-b-PBLA.

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