Polymer chitosan nanoparticles functionalized with Ulva ohnoi extracts boost in vitro ulvan immunostimulant effect in Solea senegalensis macrophages

The potential of chitosan-based nanoparticles to incorporate ulvan was investigated and the immunostimulant properties of ulvan polysaccharide as nanocarrier in Solea senegalensis macrophage were evaluated. Ulvan native extract (UL) and ulvan fractionated by chemical (ULQ) or enzymatic (ULE) methods were obtained from Ulva ohnoi macroalgae in culture. These different ulvan types were tested for their ability to form polymeric nanoparticles. Nanoparticles including either unaltered or fractionated ulvan were prepared by ionotropic gelation and characterized. Activity of the ulvan-loaded nanoparticles was tested in vitro on fish macrophages against free unaltered or fractioned ulvan extract. Intracellular and extracellular reactive oxygen species (ROS) production from the different ulvan products was determined in S. senegalensis macrophages using oxidative burst assay. The native ulvan extract (UL) as well as the fractioned form (ULQ and ULE) successfully yielded nanoparticles with a size of 250-300 nm and with a Z-potential of 30-40 mV. Highest nanoparticle production was achieved using ulvan native extract (NPsUL). Our results indicate that the configuration of the structure of ulvan molecules influence the immune response; in this way, unaltered ulvan is required for successful stimulation of Solea macrophages by Ulva ohnoi polysaccharides. This immune response significantly increases when unaltered ulvan is nanoencapsulated. We conclude that the here developed hybrid polysaccharide nanoparticles composed of chitosan and ulvan are functional. This might open the way for production of nanocarriers that can be used for oral administration of active compounds in aquaculture.