Hydrophobic gentamicin-loaded nanoparticles are effective against Brucella melitensis infection in mice

Abstract

The clinical management of human brucellosis is still challenging and demands in vitro active antibiotics capable of targeting the pathogen-harboring intracellular compartments. A sustained release of the antibiotic at the site of infection would make it possible to reduce the number of required doses and thus the treatment-associated toxicity. In this study, a hydrophobically modified gentamicin, gentamicin-AOT [AOT is bis(2-ethylhexyl) sulfosuccinate sodium salt], was either microstructured or encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The efficacy of the formulations developed was studied both in vitro and in vivo. Gentamicin formulations reduced Brucella infection in experimentally infected THP-1 monocytes (>2-log10 unit reduction) when using clinically relevant concentrations (18 mg/liter). Moreover, in vivo studies demonstrated that gentamicin-AOT-loaded nanoparticles efficiently targeted the drug both to the liver and the spleen and maintained an antibiotic therapeutic concentration for up to 4 days in both organs. This resulted in an improved efficacy of the antibiotic in experimentally infected mice. Thus, while 14 doses of free gentamicin did not alter the course of the infection, only 4 doses of gentamicin-AOT-loaded nanoparticles reduced the splenic infection by 3.23 logs and eliminated it from 50% of the infected mice with no evidence of adverse toxic effects. These results strongly suggest that PLGA nanoparticles containing chemically modified hydrophobic gentamicin may be a promising alternative for the treatment of human brucellosis.

Fig 1

Efficacy of 1 mg/liter (horizontal lines) or 18 mg/liter (diagonal lines) of gentamicin in the different formulations against intracellular Brucella melitensis infection in THP-1 human macrophages. Results are expressed as the log₁₀ intracellular CFU per well and are presented as means value ± standard deviations of at least three independent assays performed in triplicate. The dotted line indicates the intracellular Brucella inoculum at the beginning of the treatment. Statistical analysis: **, P < 0.01 compared to control cells; a, P < 0.05, and b, P < 0.01 compared to gentamicin-treated cells; c, P < 0.05 compared to cells treated with gentamicin-AOT, microstructured gentamicin-AOT, or gentamicin-AOT PLGA 502H nanoparticles (Mann-Whitney U test). Abbreviations: GEN, gentamicin; GEN-AOT, gentamicin-AOT; PCA GEN-AOT, PCA microstructured gentamicin-AOT; GEN-AOT 502H NP, gentamicin-AOT loaded PLGA 502H nanoparticles; GEN-AOT 752H NP, gentamicin-AOT loaded PLGA 752H nanoparticles; 502H NP, PLGA 502H nanoparticles; 752H NP, PLGA 752H nanoparticles.

Fig 2

Pharmacokinetics profiles. Gentamicin concentrations (means ± standard deviations; n = 6) in the spleen (A), liver (B), and kidneys (C) after the administration of a single intraperitoneal dose of gentamicin (GEN), PCA microstructured gentamicin-AOT (PCA GEN-AOT), or gentamicin-AOT-loaded PLGA 752H nanoparticles (GEN-AOT 752H NP) equivalent to 5 mg of gentamicin/kg.

Fig 3

Kidney section obtained after administration (n = 6) of gentamicin (A), a gentamicin and doxycycline combination (B), PCA gentamicin-AOT (C), or gentamicin-AOT PLGA 752H nanoparticles (D) during 14 days (hematoxylin and eosin stain). Magnification, ×400. The kidney showed cytoplasmic vacuolation of the tubular cells (arrows) (A), degeneration and focal tubular necrosis with cellular debris in the lumen of the renal tubules (arrows) (B), or no histopathological changes, with normal structure for the glomerulus and renal tubules (C and D).