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)
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.
References (18)
- et al.
Micelle-like structures of poly(ethylene oxide)-block-poly(2-hydroxyethyl aspartamide)-methotrexate conjugates
Colloids Surfaces B: Biointerfaces
(1999) - et al.
Improved synthesis of adriamycin-conjugated poly(ethylene oxide)-poly(aspartic acid) block copolymer and formation of unimodal micellar structure with controlled amount of physically entrapped adriamycin
J. Control. Release
(1994) - et al.
Preparation and characterization of the micelle-forming polymeric drug indomethacin-incorporated poly(ethylene oxide)-poly(β-benzyl l-aspartate) block copolymer micelles
Pharm. Sci.
(1996) - et al.
The effect of alkyl core structure on micellar properties of poly(ethylene oxide)-block-poly(l-aspartamide) derivatives
Colloids Surfaces B: Biointerfaces
(2001) - et al.
Lipid-based amphotericin B formulations: from animals to man
Pharm. Sci. Tech. Today
(1999) - et al.
Block copolymer micelles for the encapsulation and delivery of amphotericin b
Pharm. Res.
(2001) - et al.
Transfer of the polyene antibiotic amphotericin B between single walled vesicles of dipalmitoylphosphatidylcholine and egg-yolk phosphatidylcholine
Biochim. Biophys. Acta
(1981) - et al.
Block copolymer micelles for drug delivery: loading and release of doxorubicin
J. Control. Release
(1997) - et al.
Influencing factors on in vitro micelle stability of adriamycin-block copolymer conjugates
J. Control. Release
(1994)
Cited by (81)
In vitro synergistic antifungal evaluation through combination of F127-conjugated amphotericin B and curcumin-loaded micelles
2024, Journal of Drug Delivery Science and TechnologyPolymeric micelles in drug delivery and targeting
2023, Molecular Pharmaceutics and Nano Drug Delivery: Fundamentals and ChallengesPolymeric micelles for theranostic uses
2023, Design and Applications of Theranostic NanomedicinesPolymeric micelles and nanomedicines: Shaping the future of next generation therapeutic strategies for infectious diseases
2021, Journal of Drug Delivery Science and TechnologyPolymeric micelles in cancer therapy: State of the art
2021, Journal of Controlled ReleasepH-responsive ultrasonic self-assembly spinosad-loaded nanomicelles and their antifungal activity to Fusarium oxysporum
2019, Reactive and Functional Polymers