Development of a Water-in-Oil Microemulsion Template for Chitosan Nanogel Fabrication via Genipin Crosslinking

This study presents a promising strategy for the fabrication of a novel chitosan-based nanogel-in-oil system by integrating the development of a water-in-oil (W/O) microemulsion containing chitosan as a template, followed by crosslinking with genipin, a natural crosslinking agent, via emulsion crosslinking. To develop the W/O microemulsion template, nanometer-sized internal aqueous droplets were successfully formed in cottonseed oil, a vegetable oil, using a blend of nonionic surfactants, polysorbate 80 and sorbitan monooleate. A pseudoternary phase diagram was constructed to investigate the phase behavior of systems composed of chitosan solution, mixed surfactant, and cottonseed oil. Compositions falling within the monophasic region were selected for further formulation optimization. The microemulsions were characterized for droplet size, size distribution, electrical conductivity, and viscosity. The optimal microemulsion exhibited W/O characteristics with the lowest viscosity. Dynamic light scattering (DLS) analysis confirmed the presence of uniformly distributed nanometer-sized droplets, as evidenced by a Z-average diameter of 92.9 ± 2.3 nm and a PDI of 0.100 ± 0.072. The microemulsion system demonstrated physical stability, as confirmed by centrifugal testing. Crosslinking of chitosan with genipin was monitored by fluorescence intensity measurements of the crosslinking products. Fourier transform infrared spectroscopy further confirmed the formation of genipin-crosslinked chitosan structure. DLS and transmission electron microscopy revealed that the nanogels possessed nanoscale dimensions and discrete spherical morphologies. Overall, this approach demonstrates a viable route for producing a nanogel-in-oil system by combining microemulsion templating with emulsion crosslinking.