Fabrication and characterization of functionalized chitosan nanoparticles with Lactoferrin as a multifunctional nanocarrier for anticancer applications.

This study aimed to evaluate the anticancer efficacy of multifunctional lactoferrin-coated, 5-fluorouracil-encapsulated chitosan nanoparticles (cLF/5-FU/CS-NPs) by investigating their cytotoxicity and underlying mechanisms of action. Both 5-FU/CS-NPs and cLF/5-FU/CS-NPs were fabricated and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) analysis, Fourier transform infrared spectroscopy (FTIR) and zeta potential measurements. The developed cLF/5-FU/CS-NPs were designed to enhance 5-FU delivery and overcome its pharmacological limitations in cancer therapy through conjugation with cLF. The nanoparticles exhibited a polydisperse, regular, and stable morphology, with zeta potentials of +5 mV and + 47 mV for 5-FU/CS-NPs and cLF/5-FU/CS-NPs, respectively. cLF/5-FU/CS-NPs demonstrated enhanced in vitro cytotoxicity (IC50 range 5.31 to 7.34 μg/mL) and improved selectivity (SI up to 7.71) compared with free 5-FU, alongside reduced hemolytic activity in vitro. The treated cancer cells showed pronounced morphological alterations, increased sub-G1 populations, and intense nuclear fluorescence, indicating apoptosis induction. Moreover, cLF/5-FU/CS-NPs significantly downregulated the expression of pro-inflammatory genes, suggesting immunomodulatory activity. In silico analyses, including network pharmacology and protein-protein docking, were employed to elucidate the molecular mechanisms of cLF and provide structural insights into its anticancer potential. Collectively, these findings indicate that cLF/5-FU/CS-NPs represent a safe and selective antitumor candidate that induces apoptosis-mediated cell death, promotes DNA damage, and enhances immune response.