Sustainable Nano-Hydroxyapatite from Eggshell Waste for pH-Responsive Doxycycline Delivery: In Situ Loading, Biphasic Release Kinetics, and Enhanced Antibacterial Activity against Pathogens

Abstract In this study, we prepared nano-hydroxyapatite (nHAp) from waste chicken eggshells using a wet chemical method and loaded it in situ with doxycycline hyclate (DOXh) at concentrations of 5, 10, and 15 mg/mL. X-ray diffractometry (XRD) confirmed the formation of the nHAp phase and demonstrated that its structural integrity was maintained after drug loading. The crystallite size of pure nHAp was 5.33 nm, which varied with different DOXh concentrations. FTIR and Raman spectroscopy confirmed the presence of HAp functional groups, although the Raman peaks were masked following DOXh loading. Field emission scanning electron microscopy (FESEM) revealed rod-like particles of sizes within 32–34 nm, and energy dispersive X-ray Spectroscopy (EDX) identified elements associated with DOXh in the loaded samples. The loading study indicated that the entrapment efficiency (EE%) exceeded 99% for all DOXh concentrations, with both drug loading capacity (DLC) and adsorption capacity (Qe) increasing proportionally. Density functional theory (DFT) analysis indicated strong binding between nHAp and DOXh via electrostatic and hydrogen bonds. In vitro drug release studies in simulated body fluid at pH levels of 3, 5, and 7.4 demonstrated that acidic conditions facilitated enhanced drug release, showcasing pH-responsive behavior. The release profile showed an initial burst followed by a slower diffusion-controlled phase. The highest cumulative release reached 46.74% for the 10 mg/mL DOXh-loaded sample at pH 3 after two weeks. The release patterns followed the Higuchi and Ritger–Peppas models, suggesting a Fickian diffusion mechanism. Antibacterial tests against Staphylococcus aureus and Escherichia coli revealed strong inhibition, with a maximum zone of inhibition of 32.24 mm against E. coli for the 15 mg/mL DOXh-loaded nHAp, surpassing that of kanamycin. These findings suggest that nHAp-DOXh formulations could serve as efficient, pH-sensitive carriers for targeted drug delivery at infection sites.