Polarity-sensitive nanocarrier for oral delivery of Sb(V) and treatment of cutaneous leishmaniasis

Abstract

There is a great need for orally active drugs for the treatment of the neglected tropical disease leishmaniasis. Amphiphilic Sb(V) complexes, such as 1:3 Sb-N-octanoyl-N-methylglucamide complex (SbL8), are promising drug candidates. It has been previously reported that SbL8 forms kinetically stabilized nanoassemblies in water and that this simple dispersion exhibits antileishmanial activity when given by oral route to a murine model of visceral leishmaniasis. The main objective of the present work was to interfere in the structural organization of these nanoassemblies so as to investigate their influence on the oral bioavailability of Sb, and ultimately, optimize an oral formulation of SbL8 for the treatment of cutaneous leishmaniasis. The structural organization of SbL8 nanoassemblies was manipulated through addition of propylene glycol (PG) to the aqueous dispersion of SbL8. The presence of 50% (v/v) PG resulted in the loss of hydrophobic microenvironment, as evidenced by fluorescence probing. However, nanostructures were still present, as demonstrated by dynamic light scattering, small-angle X-ray scattering, and atomic force microscopy (AFM). A remarkable property of these nanoassemblies, as revealed by AFM analysis, is the flexibility of their supramolecular organization, which showed changes as a function of the solvent and substrate polarities. The formulation of SbL8 in 1:1 water:PG given orally to mice promoted significantly higher and more sustained serum levels of Sb, when compared to SbL8 in water. The new formulation, when given as repeated doses (200 mg Sb/kg/day) to BALB/c mice infected with Leishmania amazonensis, was significantly more effective in reducing the lesion parasite burden, compared to SbL8 in water, and even, the conventional drug Glucantime(®) given intraperitoneally at the same dose. In conclusion, this work introduces a new concept of polarity-sensitive nanocarrier that was successfully applied to optimize an oral formulation of Sb(V) for treating cutaneous leishmaniasis.

Keywords: AFM; Leishmania amazonensis; SAXS; amphiphilic complex; antimony; propylene glycol.

Figure 1

Change in the hydrophobic environment of SbL8 and L8 aqueous dispersions upon addition of PG. Notes: The graph shows fluorescence of DPH in SbL8 (30 mM of L8) or L8 (75 mM) aqueous dispersions as a function of the proportion of added PG. Fluorescence intensities were subtracted from those of DPH in the respective mixed solvents. DPH final concentration was 0.5 μM. Fluorescence measurements were carried out using a Cary Eclipse fluorescence spectrometer after 24-hour incubation at 25°C. Excitation and emission wavelengths were 360 and 428 nm, respectively. Abbreviations: SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; L8, N-octanoyl-N-methylglucamide; PG, propylene glycol; DPH, 1,6-diphenylhexatriene; au, arbitrary units.

Figure 2

Topographical AFM images of SbL8 dispersion in water. Notes: A dispersion of SbL8 in water (L8 at 30 mM) was deposited and partially dried onto cleaved mica (A, B, and D) or paraffin substrate (C), obtained in the tapping mode. Magnification in (A), scan size 2.0×2.0 μm; image (B) is a magnified inset of (A), scan size 300×300 nm; magnification in (C), scan size 2.0×2.0 μm; and magnification of the detailed 3D image in (D), scan size 663.6×663.6 nm. Abbreviations: 3D, three-dimensional; AFM, atomic force microscopy; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; L8, N-octanoyl-N-methylglucamide.

Figure 3

Topographical AFM images of SbL8 dispersion in 1:1 W:PG. Notes: A dispersion of SbL8 in 1:1 W:PG (L8 at 30 mM) was deposited and partially dried onto cleaved mica (A) or paraffin substrate (B–D), obtained in the tapping mode. Magnification in (A) and (B): scan size 5.0×5.0 μm; Magnification in (C and D): scan size 500×500 nm, high-resolution 2D and 3D images, respectively. Abbreviations: 2D, two-dimensional; 3D, three-dimensional; AFM, atomic force microscopy; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water:propylene glycol; L8, N-octanoyl-N-methylglucamide.

Figure 4

Transmission electron photomicrograph of SbL8 dispersions. Notes: Dispersions of SbL8 in water (A) or 1:1 W:PG (B) (L8 at 30 mM) were stained with osmium tetroxide and then deposited and dried onto a 300-mesh Formvar copper grid. The images were obtained using a transmission electron microscope operating at an acceleration voltage of 120 kV, a line resolution of 0.34 nm, a point resolution of 0.49 nm, and a Cs of 6.3 (spherical aberration), implying high contrast. Abbreviations: SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water:propylene glycol; L8, N-octanoyl-N-methylglucamide.

Figure 5

SAXS curves of SbL8 and L8 dispersions. Notes: Samples consist of SbL8 at 30 mM of L8 and L8 at 140 mM in water or 1:1 W:PG. In (A) and (C), SAXS curves were registered with q in the range of 0–0.4 and 0–0.25 nm⁻¹, respectively. (B) The spherical core–shell model is presented that shows best adjustment, when fitting the data from curve in (A), using the SASfit software (excluding the low-angle part of the curves). I(q) represents the intensity function, and q the scattering vector amplitude. Abbreviations: SAXS, small-angle X-ray scattering; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; L8, N-octanoyl-N-methylglucamide; W:PG, water:propylene glycol.

Figure 6

Influence of PG on the serum pharmacokinetics of Sb after oral administration of SbL8. Notes: Swiss mice received by gavage SbL8 in either water or 1:1 W:PG at 200 mg Sb/kg. The concentration of Sb was determined by GFAAS technique. Data are shown as mean ± SEM (n=6–12). ^*P<0.05 and ^**P<0.01 for comparisons between formulations by two-way ANOVA with Bonferroni posttest. Abbreviations: PG, propylene glycol; SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water:propylene glycol; GFAAS, graphite furnace atomic absorption spectroscopy; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 7

Size lesion evolution in a murine model of cutaneous leishmaniasis submitted to treatment with oral formulations of SbL8 in either water or W:PG mixture. Notes: The graph shows the change in the size of lesion from the first day of treatment in BALB/c mice infected with Leishmania amazonensis, following oral treatment with SbL8 (200 mg/kg/day for 30 days), either dissolved in water or 1:1 W:PG, in comparison to saline-treated control and positive control treated with intraperitoneal Glucantime^® (200 mg/kg/day for 30 days). Data are shown as mean ± SEM. ^****P<0.0001 for comparison between treated and saline groups; two-way repeated measures ANOVA and Dunnett’s multiple comparison test. The inset shows images of animal lesions in saline and SbL8 W:PG groups at 1, 15, and 30 days of treatment, top to bottom. Abbreviations: SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water: propylene glycol; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 8

Antileishmanial efficacy in a murine model of cutaneous leishmaniasis submitted to treatment with oral formulations of SbL8 in either water or W:PG mixture. Notes: The graph shows the parasite load in the lesion of BALB/c mice infected with Leishmania amazonensis following oral treatment with SbL8 (200 mg/kg/day for 30 days) dissolved in either water or 1:1 W:PG, in comparison with saline-treated control and positive control treated with intraperitoneal Glucantime^® (200 mg/kg/day for 30 days). Parasite burden was determined after 30 days of treatment by the limiting dilution assay. Points show individual results of log(parasite burden/mg). Mean ± SEM are also shown. ^**P<0.01, ^***P<0.001, and ^****P<0.0001, in comparison with saline; ^###P<0.001 in comparison with SbL8 in W:PG; one-way ANOVA and Tukey’s multiple comparison test. Abbreviations: SbL8, 1:3 Sb–N-octanoyl-N-methylglucamide complex; W:PG, water: propylene glycol; SEM, standard error of the mean; ANOVA, analysis of variance.