Green synthesis of <i>Ceratonia siliqua</i> –mediated calcium hydroxide–chitosan biohybrid nanocomposite: Structural characterization and biological performance

The development of sustainable polymer–inorganic nanocomposites has gained increasing attention as environmentally friendly alternatives for advanced biomedical applications. This study reports the synthesis of a biohybrid calcium hydroxide–chitosan nanocomposite through a co-precipitation approach, employing Ceratonia siliqua (carob fruit) extract as a green reducing and stabilizing agent. The resulting CS–Ca(OH)₂–Ch nanocomposite was characterized using spectroscopic and microscopic techniques to evaluate its structural, compositional, and morphological properties. Cytocompatibility was evaluated in the human telomerase reverse transcriptase–immortalized (hTERT-20) cell line using the Alamar blue assay. Antibacterial evaluation of G +ve and G -ve strains was assessed using agar diffusion for zones of inhibition (ZOI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. A characteristic UV–vis absorption peak at 290 nm suggested nanocomposite formation, while X-ray diffraction analysis (XRD) analysis verified the crystalline incorporation of Ca(OH)₂ within the chitosan matrix. Fourier transform infrared (FTIR) spectra validated the existence of functional groups in both the extract and the nanocomposite. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS) results confirmed the non-aggregated morphology, spherical shapes, and size (less than 100 nm), respectively. The nanocomposite demonstrated favorable in vitro cytocompatibility, with cell viability exceeding 90% at concentrations ≤50 µg/mL and remaining above 70% at 100 µg/mL. The nanocomposite exhibited concentration-dependent antibacterial performance at 100 µg/mL, with inhibition zones of 18.2 ± 0.3 mm ( E. faecalis ) to 21.2 ± 0.3 mm ( S. aureus ) for G +ve strains, and 19.7 ± 0.6 mm ( E. coli ) to 21.0 ± 1.0 mm ( P. aeruginosa ) for G -ve strains. MIC values were recorded between 6.25 and 50 µg/mL, while MBC values ranged from 12.5 to 100 µg/mL, collectively confirming bactericidal activity. Overall, the biohybrid nanocomposite integrates structural stability, cytocompatibility, and antibacterial efficacy, supporting its potential as a functional polymer-based material for biomedical applications.