Optimization of Maduramicin Ammonium-Loaded Nanostructured Lipid Carriers Using Box-Behnken Design for Enhanced Anticoccidial Effect against Eimeria tenella in Broiler Chickens

Abstract

Maduramicin ammonium (MAD) is one of the most frequently used anticoccidial agents in broiler chickens. However, the high toxicity and low solubility of MAD limit its clinical application. In this study, MAD-loaded nanostructured lipid carriers (MAD-NLCs) were prepared to overcome the defects of MAD by using highly soluble nanostructured lipid carriers (NLCs). The formulation was optimized via a three-level, three-factor Box-Behnken response surface method. Then, the optimal MAD-NLCs were evaluated according to their hydrodynamic diameter (HD), zeta potential (ZP), crystal structure, encapsulation efficiency (EE), drug loading (DL), in vitro release, and anticoccidial effect. The optimal MAD-NLCs had an HD of 153.6 ± 3.044 nm and a ZP of -41.4 ± 1.10 mV. The X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the MAD was encapsulated in the NLCs in an amorphous state. The EE and DL were 90.49 ± 1.05% and 2.34 ± 0.04%, respectively, which indicated that the MAD was efficiently encapsulated in the NLCs. In the in vitro study, the MAD-NLCs demonstrated a slow and sustained drug release behavior. Notably, MAD-NLCs had an excellent anticoccidial effect against Eimeria tenella in broiler chickens. In summary, MAD-NLCs have huge potential to form a new preparation administered via drinking water with a powerful anticoccidial effect.

Keywords: Box–Behnken design; anticoccidial effect; maduramicin ammonium; nanostructured lipid carriers.

Figure 1

Screening of the solid and liquid lipids for the solubility of MAD. (A) Solubility of MAD was determined in different solid lipids. (B) Solubility of MAD was analyzed in various liquid lipids. (C) Solubility of MAD was detected in varied ratios of stearic acid and oleic acid.

Figure 2

Three-dimensional response surface images of the solid lipid to lipid ratio (SL), emulsifier to lipid ratio (EL), and MAD to lipid ratio (ML) on (A) the hydrodynamic diameter (HD) and (B) zeta potential (ZP).

Figure 3

Characterization of the optimized MAD–NLCs. (A) Hydrodynamic diameter (HD) and (B) zeta potential (ZP) of the MAD–NLCs were determined by DLS. (C) The morphology of the MAD–NLCs was observed by TEM, and (D) the size distribution was obtained via analysis of the particles from several TEM images. (E) XRD patterns and (F) FTIR spectra for (a) MAD–NLCs, (b) blank NLCs, (c) the physical mixture, (d) MAD, and (e) SA were shown.

Figure 4

Cumulative release rates of the MAD–NLCs and MAD in PBS solution.

Figure 5

Histopathological analysis of chicken ceca (n = 3). (A) Uninfected–untreated group. (B) Infected–untreated group (black solid arrow represents coccidia oocysts). (C) MAD premix group(black solid arrow represents structural disorders). (D) Low-dose group of MAD–NLCs (black solid arrow represents coccidia oocysts). (E) Medium-dose group of MAD–NLCs. (F) High-dose group of MAD–NLC structural disorders.