Phospholipid-Conjugated PEG- b-PCL Copolymers as Precursors of Micellar Vehicles for Amphotericin B

Abstract

Amphotericin B (AmB) is a widely used antifungal that presents a broad action spectrum and few reports on the development of resistance. However, AmB is highly toxic, causing renal failure in a considerable number of treated patients. Although when AmB is transported via polymer micelles (PMs) as delivery vehicles its nephrotoxicity has been successfully attenuated, this type of nanoparticle has limitations, such as low encapsulation capacity and poor stability in aqueous media. In this research, the effect of modifying polyethyleglicol-block-poly(ε-caprolactone) (PEG-b-PCL) with 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) on the performance of PMs as vehicles for AmB was studied. PEG-b-PCL with two different lengths of a PCL segment was prepared via ring opening polymerisation and modified with DSPE at a post-synthesis stage through amidation. Upon modification with DSPE, a copolymer was self-assembled, thereby producing particles with hydrodynamic diameters below 100 nm and a lower critical micelle concentration than that of the raw copolymers. Likewise, in the presence of DSPE, the loading capacity of AmB increased because of the formed intermolecular interactions, such as hydrogen bonds, which also caused a lower aggregation of this drug. The assessment of in vitro toxicity against red blood cells indicated that the toxicity of AmB decreased upon encapsulation; however, its antifungal action against clinical yeasts was maintained and enhanced, as indicated by a decrease in its minimum inhibitory concentration.

Keywords: amphotericin B; phospholipid-modified copolymer; polymer micelle.

Figure 1

Spectral characterisation of a representative DSPE-terminated copolymer (PP3-DSPE). (a) XPS and (b) ¹NMR spectra.

Scheme 1

CL polymerisation starting from mPEG as an initiator via ROP (a), reaction of PEG-b-PCL with succinic anhydride (b) and conjugation of carboxylated copolymers with DSPE through amidation (c).

Figure 2

Excitation spectra of pyrene dissolved in PP3-DSPE solutions with different concentrations (a) and plot of the ratio of the fluorescence intensity of pyrene at 335 and 332 nm as a function of copolymer concentrations for PP3-DSPE and PP6-DSPE samples (b).

Figure 3

High-resolution XPS spectrum of P_2p in PP3-DSPE (a) and AmB/PP3-DSPE (b).

Figure 4

Comparison of the distribution plots of the hydrodynamic diameter of empty micelles and AmB-loaded particles obtained from PP3-DSPE.

Figure 5

Characterisation of lyophilised formulations based on PP3 and PP3-DSPE. (a) DRX profiles and (b) DSC traces.

Scheme 2

(a) representative structure of AmB-loaded polymer micelles composed of PEG-b-PCL and (b) PEG-b-PCL-DSPE. In raw copolymers, hydrophobic PCL segment forming the nucleus was highly amorphous allowing for AmB dissolutions. Upon conjugation, PCL crystallized and AmB encapsulation was enabled by interactions with DSPE moiety.

Figure 6

Release of AmB from micellar formulations measured for 100 h.

Figure 7

Erythrocyte toxicity of AmB-loaded PMs in comparison with commercial AmB formulation (a). (b) Aggregation state of AmB in Fungizone ^® and a micellar formulation obtained using PP3-DSPE as a polymeric precursor.