Antibiotic Loaded Phytosomes as a Way to Develop Innovative Lipid Formulations of Polyene Macrolides

Abstract

Background: The threat of antibiotic resistance of fungal pathogens and the high toxicity of the most effective drugs, polyene macrolides, force us to look for new ways to develop innovative antifungal formulations.

Objective: The aim of this study was to determine how the sterol, phospholipid, and flavonoid composition of liposomal forms of polyene antibiotics, and in particular, amphotericin B (AmB), affects their ability to increase the permeability of lipid bilayers that mimic the membranes of mammalian and fungal cells.

Methods: To monitor the membrane permeability induced by various polyene-based lipid formulations, a calcein leakage assay and the electrophysiological technique based on planar lipid bilayers were used.

Key results: The replacement of cholesterol with its biosynthetic precursor, 7-dehydrocholesterol, led to a decrease in the ability of AmB-loaded liposomes to permeabilize lipid bilayers mimicking mammalian cell membranes. The inclusion of plant flavonoid phloretin in AmB-loaded liposomes increased the ability of the formulation to disengage a fluorescent marker from lipid vesicles mimicking the membranes of target fungi. I-V characteristics of the fungal-like lipid bilayers treated with the AmB phytosomes were symmetric, demonstrating the functioning of double-length AmB pores and assuming a decrease in the antibiotic threshold concentration.

Conclusions and perspectives: The therapeutic window of polyene lipid formulations might be expanded by varying their sterol composition. Polyene-loaded phytosomes might be considered as the prototypes for innovative lipid antibiotic formulations.

Keywords: amphotericin B; lipid bilayers; liposomes; membranes; phytosomes; sterols.

Figure 1

Chemical structures of phospholipids (A–D), sterols (E–J), antifungal polyene macrolides (K,L), and flavonoids (M–P) used in this study.

Figure 2

(A) Time dependence of calcein leakage from mammalian-like (black curve) and fungal-like lipid vesicles (red curve) at the subsequent increase in the dose of POPC/CHOL/AmB formulation in bathing solution. The moments of addition of the formulation into the liposomal suspension are indicated by the arrows. The concentrations of the monomeric AmB in µM are shown above the arrows. (B) Time dependence of calcein leakage from fungal-like liposomes at the addition of antibiotic-free POPC/CHOL (blue curve) and POPC/CHOL/phloretin (olive curve) formulations. The addition of the formulations until the same volume concentration as antibiotic-loaded formulations in the bathing solution was carried out at the first moment.

Figure 3

Time dependence of calcein leakage (IF, %) from mammalian-like (A) and fungal-like lipid vesicles (B) induced by different formulations: POPC/CHOL/AmB (1, black curves), POPC/DESM/AmB (2, red curves), POPC/7DCHOL/AmB (3, green curves), POPC/CAMPO/AmB (4, blue curves), POPC/STIGM/AmB (5, cyan curves), and POPC/ERG/AmB (6, pink curves). The addition of formulations in bathing solution up to 2 μM of monomeric AmB was carried out at the first moment.

Figure 4

The voltage dependence of the ratio of membrane conductance produced by one-side (◊) and two-side (*) addition of AmB dissolved in DMSO to bilayer conductance at zero applied voltage (G/G₀). Membranes were made from POPC/CHOL (mammalian-like) (A) and POPC/ERG (fungal-like) (B) and bathed in 2.0 M KCl (pH 7.4).

Figure 5

The voltage dependence of the ratio of membrane conductance produced by one-side addition of different AmB formulations to conductance at zero transmembrane voltage (G/G₀): POPC/CHOL/AmB (1, black curves), POPC/DESM/AmB (2, red curves), POPC/7DCHOL/AmB (3, green curves), POPC/CAMPO/AmB (4, blue curves), POPC/STIGM/AmB (5, cyan curves), and POPC/ERG/AmB (6, pink curves). Mammalian-like (A) and fungal-like model membranes (B) were bathed in 2.0 M KCl (pH 7.4). Insets: The time dependence of the alteration in the transmembrane current induced by formulation 1 at the voltage ramp from −200 to +200 mV per 5 s.

Figure 6

Time dependence of calcein leakage (IF, %) from fungal-like lipid vesicles at the addition of (A) AmB-loaded phytosomes containing different flavonoids, POPC/CHOL/AmB/phloretin (7, dark yellow curve), POPC/CHOL/AmB/biochanin A (8, navy curve), POPC/CHOL/AmB/genistein (9, olive curve), and POPC/CHOL/AmB/quercetin (10, purple curve); (B) AmB-loaded phytosomes with phloretin and composed of different sterols, POPC/DESM/AmB/phloretin (11, wine curve), POPC/7DCHOL/AmB/phloretin (12, violet curve), POPC/CAMPO/AmB/phloretin (13, orange curve), POPC/STIGM/AmB/phloretin (14, dark cyan curve), and POPC/ERG/AmB/phloretin (15, dark grey curve). The formulations were added in bathing solution up to 2 μM of monomeric AmB at the first moment.

Figure 7

(A) The voltage dependence of the ratio of membrane conductance produced by one-side addition of the different phytosomes to conductance at zero transmembrane voltage (G/G₀): POPC/CHOL/AmB/phloretin (7, dark yellow curve), POPC/DESM/AmB/phloretin (11, wine curve), POPC/7DCHOL/AmB/phloretin (12, violet curve), POPC/CAMPO/AmB/phloretin (13, orange curve), POPC/STIGM/AmB/phloretin (14, dark cyan curve), and POPC/ERG/AmB/phloretin (15, dark grey curve). Inset: The time dependence of the alteration in the transmembrane current induced by formulation 7 at the voltage ramp from −200 to +200 mV per 5 s. (B) The voltage dependence of the ratio of membrane conductance produced by one-side addition of different Nys formulations to conductance at zero transmembrane voltage (G/G₀): POPC/CHOL/NyS (16, dark blue curve), POPC/ERG/NyS (17, olive curve), POPC/CHOL/NyS/phloretin (18, blue curve), and POPC/ERG/NyS/phloretin (21, green curve). Fungal-like lipid bilayers were bathed in 2.0 M KCl (pH 7.4).