Mycoplasma infections pose significant challenges in the poultry industry, necessitating effective therapeutic interventions. Tiamulin, a veterinary antibiotic, has demonstrated efficacy against Mycoplasma species. However, the emergence of resistant Mycoplasma species could dramatically reduce the therapeutic potential, contributing to economic losses. Optimizing the tiamulin’s pharmacokinetic profile via nanocarrier incorporation could enhance its therapeutic potential and reduce the administration frequency, ultimately reducing the resistant strain emergence. Niosomes, a type of self-assembled non-ionic surfactant-based nanocarrier, have emerged as a promising drug delivery system, offering improved drug stability, sustained release, and enhanced bioavailability. In this study, niosomal nanocarriers encapsulating tiamulin were prepared, characterized and assessed in Mycoplasma-inoculated broilers following oral administration. Differential scanning colorimetry (DSC) confirmed the alterations in the crystalline state following components integration into the self-assembled structures formed during the formulation procedure. Transmission electron microscopy (TEM) showed the spherical nanostructure of the formed niosomes. The formulated nanocarriers exhibited a zeta potential and average hydrodynamic diameter of −10.65 ± 1.37 mV and 339.67 ± 30.88 nm, respectively. Assessment of the pharmacokinetic parameters following oral administration to Mycoplasma gallisepticum-infected broilers revealed the ability of the niosomal nanocarriers to increase the tiamulin’s bioavailability and systemic exposure, marked by significantly higher area under the curve (AUC) (p < 0.01) and prolonged elimination half-life (T1/2) (p < 0.05). Enhanced bioavailability and prolonged residence time are crucial factors in maintaining therapeutic concentrations at reduced doses and administration frequencies. This approach provides a viable strategy to decrease the risk of subtherapeutic levels, thereby mitigating the development of antibiotic resistance. The findings presented herein offer a sustainable approach for the efficient use of antibiotics in veterinary medicine.
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