In situ nanoformulation of amino acid complexed drug loaded eggshell derived hydroxyapatite: A promising strategy to combat Staphylococcus aureus biofilm

Abstract

Chronic osteomyelitis is a persistent bone infection frequently complicated by biofilm formation, most commonly associated with Staphylococcus aureus (S. aureus). Current treatment relies on a combination of surgical intervention, defect filling, and prolonged antibiotic therapy. In this study, we report the in situ fabrication of doxycycline (Dox) loaded hydroxyapatite nanoparticles (HAp NPs) complexed with amino acids, arginine (Arg) and aspartic acid (Asp), using a simple wet precipitation method, as a defect filling and antibiofilm therapeutic platform. The prepared formulations were systematically characterized for their phase composition, morphology, antibacterial efficacy, and cytocompatibility. Amino acid complexation significantly enhanced drug loading and promoted sustained drug release under both physiological and pathological pH conditions compared to non-complexed formulations. The amino acid complexed drug loaded samples exhibited ~ 6.5 log and ~ 1 log reduction in planktonic colony growth when compared to control and drug alone loaded sample, respectively. All the drug loaded samples effectively inhibited the sessile bacterial growth for up to 14 days, without detectable colony formation. Confocal laser scanning microscopy revealed that the untreated control biofilm exhibited an average thickness of ~ 30 µm, whereas treatment with Arg and Asp complexed drug loaded formulations significantly reduced the biofilm thickness to 16.6 µm and 15.0 ± 1.6 µm, respectively, accompanied by extensive bacterial cell death (93% and 90%, respectively). The developed formulations also demonstrated temperature dependent storage stability, with the powder form being more stable than the slurry. At 5 °C, amino acid containing samples retained > 90 % Dox after one month and > 80 % after two months, whereas for the amino acid free formulation the stability of Dox decreased to ~ 64 % after two months. Overall, these results highlight the potential of amino acid complexed Dox-loaded HAp nanoparticles as an effective therapeutic treatment options for biofilm associated and drug resistant S. aureus infections in chronic osteomyelitis.