Background: Chitosan (CS) crosslinked with genipin (GNP) provides a mild, non-toxic route to generate nanogels (NGs) with enhanced integrity and colloidal stability. Objectives: To develop and characterise CS-GNP NG as a novel platform for targeted cellular delivery, optimising design through physicochemical characterisation and biocompatibility evaluation. Methods: NGs were synthesised under optimised conditions by adjusting the pH of the CS solution, followed by high-intensity ultrasound (HIUS) to achieve disaggregation. Physicochemical characterisation was carried out using UV-Vis spectroscopy, FTIR, dynamic light scattering (DLS), and scanning electron microscopy (SEM). Rheological studies and SAXS analysis assessed structural properties. Biocompatibility was evaluated via MTT assay, and internalisation was monitored by fluorescence microscopy on mammalian cell lines. Results: NG formation was highly pH-dependent, with optimal configuration at pH 4.5, yielding stable, uniformly sized particles (~200 nm, ζ-potential +29 mV). Kinetic modelling showed a sigmoidal formation pattern, suggesting nucleation, growth, and stabilisation. FTIR confirmed covalent bonding between CS and GNP via primary amide bonds and Schiff bases. Rheology indicated pseudoplastic behaviour, and SAXS revealed a compact network formation. Biocompatibility assays confirmed non-cytotoxicity below 100 µg/mL and efficient cellular uptake. Conclusions: This study presents a rapid, reproducible protocol for generating colloidally stable, biocompatible NGs suitable for drug delivery.

Background: the crosslinking of chitosan (CS) with genipin (GNP) offers a mild, non-toxic strategy to improve the structural integrity and stability of nanogels (NGs). However, generating homogeneous, colloidally stable systems still requires optimisation of parameters such as CS and GNP concentrations to ensure functional performance for cellular uptake. Objectives: To develop and characterise CS-based NGs crosslinked with GNP as a novel platform for targeted delivery at the cellular level. The aim was to optimise NG synthesis and evaluate their physicochemical properties and biocompatibility. Methods: NGs were synthesised under optimised conditions by adjusting the pH of the CS solution, followed by high-intensity ultrasound (HIUS) to achieve disaggregation. Physicochemical characterisation was carried out using UV-Vis spectroscopy, FTIR, dynamic light scattering (DLS), and scanning electron microscopy (SEM). Rheological studies and SAXS analysis assessed structural properties. Biocompatibility was evaluated via MTT assay, and internalisation was monitored by fluorescence microscopy on mammalian cell lines. Results: NG formation was highly pH-dependent, with optimal configuration at pH 4.5, yielding stable, uniformly sized particles (~200 nm, ζ-potential +29 mV). Kinetic modelling showed a sigmoidal formation pattern, suggesting nucleation, exponential growth, and stabilisation. FTIR confirmed covalent bonding between CS and GNP via primary amide bonds and Schiff bases. Rheology indicated pseudo-plastic behaviour, and SAXS revealed a compact network. Biocompatibility assays confirmed non-cytotoxicity below 50 µg/mL and efficient cellular uptake. Conclusions: This study presents a rapid, reproducible protocol for generating colloidally stable, biocompatible NGs suitable for drug delivery and biomedical applications requiring cellular internalisation.