Berberine-Loaded Liposomes for the Treatment of Leishmania infantum-Infected BALB/c Mice

Abstract

Berberine (BER)-an anti-inflammatory quaternary isoquinoline alkaloid extracted from plants-has been reported to have a variety of biologic properties, including antileishmanial activity. This work addresses the preparation of BER-loaded liposomes with the aim to prevent its rapid liver metabolism and improve the drug selective delivery to the infected organs in visceral leishmaniasis (VL). BER liposomes (LP-BER) displayed a mean size of 120 nm, negative Z-potential of -38 mV and loaded 6 nmol/μmol lipid. In vitro, the loading of BER in liposomes enhanced its selectivity index more than 7-fold by decreasing its cytotoxicity to macrophages. In mice, LP-BER enhanced drug accumulation in the liver and the spleen. Consequently, the liposomal delivery of the drug reduced parasite burden in the liver and spleen by three and one logarithms (99.2 and 93.5%), whereas the free drug only decreased the infection in the liver by 1-log. The organ drug concentrations-far from IC₅₀ values- indicate that BER immunomodulatory activity or drug metabolites also contribute to the efficacy. Although LP-BER decreased 10-fold-an extremely rapid clearance of the free drug in mice-the value remains very high. Moreover, LP-BER reduced plasma triglycerides levels.

Keywords: Leishmania infantum; berberine; liposomes; visceral leishmaniasis.

Figure 1

Nitrites production by bone marrow-derived macrophages (BMDM) after 48-h treatment with different concentrations of free berberine (BER), LP-BER (berberine liposomes) or blank LP (LP, equivalent to 25-µM BER in BER LP), alone or plus Escherichia coli lipopolysaccharide (LPS) (0.1 μg/mL). Macrophages stimulated with LPS used as positive control. Results expressed as mean ± SD (n = 5). Data analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05.

Figure 2

In vitro release of BER from LP-BER using dialysis bag diffusion method in PBS with 10% (v/v) methanol, at 25 °C. Data presented as mean ± SD (n = 3).

Figure 3

Plasma concentration-time curve of BER in mice after (a) i.v. or (b) i.p. administration of free BER and LP-BER at (a) 7.5 or (b) 15 mg/kg. Data represents mean ± SD, n = 3 mice per group.

Figure 4

BER accumulation in livers and spleens after 8, 24 and 48 h post i.p. administration of 15 mg/kg of free BER or LP-BER. Data presented as mean ± SD, n = 3 mice per group.

Figure 5

Parasite burden in liver and spleen, measured by qRT-PCR, after 10 consecutive days of i.p. treatment with free BER and LP-BER at 15 mg/kg. Results expressed as median ± 95% IC (n = 6 per group). Data analyzed using a nonparametric Kruskal–Wallis test, followed by Dunn’s multiple comparison. * p < 0.05, ** p < 0.01.