Formulation development of nanostructured lipid carriers for oral delivery of recombinant protein-based vaccine antigens in aquatic species

Recombinant proteins have emerged as a safe and effective method in the field of veterinary vaccines. However, the use of oral and stable recombinant antigen vaccines for aquatic species remains underexplored. To investigate the potential use of nanostructured lipid carriers (NLCs) as a strategy to protect recombinant proteins from gastrointestinal degradation and damage, and to provide good protection for fish against Aeromonas hydrophila, we employed high pressure homogenisation and ultrasonication methods for nanoencapsulation using glycerol distearate, propylene glycol dicaprylate as solid/liquid lipids, and polysorbate 80 as the surfactant, in varying ratios. The physicochemical properties of resulting nanostructured lipid carriers (size, homogeneity, charge, miscibility, shape) were assessed using dynamic light scattering (DLS), zeta potential, differential scanning calorimetry (DSC) and transmission electronic microscopy (TEM). The antigen loading efficiency and release in a gastric buffer mimicking fish conditions was measured by micro BCA assay. NLC colloidal stability was assessed at pH values encountered in the fish gastrointestinal tract and storage conditions evaluated at 4 and 42 ℃. Our results showed that while ultrasonication time influenced NLC size and dispersity, neither high pressure homogenisation nor ultrasonication impacted protein integrity. Formulation composition (solid and liquid lipid ratio and surfactant concentration) was crucial to the resulting nanoparticle properties and influenced protein loading capacity with an increase in solid lipid content leading to the highest antigen incorporation (58 ± 10.9 %). All formulations with 90:10, 80:20 and 70:30 ratios of glycerol distearate to propylene glycol dicaprylate and 5 % polysorbate 80 resulted in NLCs with low dispersity, and sizes below 200 nm. However, in vitro release and stability improved with high higher solid lipid, suggesting that the balanced solid and liquid oil composition of the 80:20 is a promising formulation for vaccine delivery in aquatic environment.