Nanoparticles as a novel delivery system for vitamin C administration in aquaculture

The potential of chitosan-based nanoencapsulation as a tool for delivering ascorbic acid (AA) to marine and freshwater organisms was investigated. Polymeric non-loaded and loaded vitamin C nanoparticles (NPs) were made by ionic gelation and the particles were characterized. In vitro performance of nanoparticles was evaluated in a zebrafish liver cell-line (ZFL) and in vivo studies were carried out in fish (post-metamorphic larvae of Solea senegalensis) and rotifers (Brachionus plicatilis) to assess the potential use of these NPs to be used as a tool in nutritional aquaculture studies. The results showed that NPs are suitable to trap hydrosoluble compounds such as AA by forming positively charged complexes (30-35 mV), in a nanosize range (<300 nm), with encapsulation efficiency (EE) higher than 15% and high stability (>90% of loaded AA remained within nanoparticles after 2 h in seawater). The potential cytotoxicity of the NPs was evaluated in ZFL cells and no decrease in cell viability was noted up to 2.5 mg/ml of nanoparticle concentration. The NP uptake was analyzed in ZFL cells by FACS cytometry and confocal laser scanning microscopy (CLSM). Time course and dose-response experiments were performed using fluorescein isothiocyanate labeled NPs (FITC-NPs). The in vitro endocytosis assays with ZFL cells showed a maximum uptake after 6 h of incubation and a dose-dependent increase of fluorescence intensity directly proportional to the FITC-NP concentration. The antioxidant properties of vitamin C nanoparticles (AA-NPs) were also analyzed in ZFL cell extracts. Lipopolysaccharide (LPS) was added to ZFL cells to induce oxidative stress. The total antioxidant capacity of the AA-NP-treated cells showed a statistically significant increase with respect to the control with non-loaded nanoparticles (71.00 +/- 9.6 and 25.36 +/- 3.96 mu M Trolox equivalent; p < 0.05 respectively). The NPs' ability to penetrate fish intestinal epithelium was also evaluated. After 2 h, NPs were able to penetrate through intestinal epithelium in post-metamorphic larvae of S. senegalensis as detected by CLSM. The potential use of NPs as additive to rotifers was analyzed using AA-loaded or non-loaded NPs in rotifer enrichment for 2 h. Rotifers fed with AA-NPs increased up to 2-fold of their ascorbic acid levels in comparison to control groups. As a whole, results show that these polymeric NPs might represent an interesting vehicle for oral administration of AA and other active compounds in aquaculture. (C) 2014 Elsevier B.V. All rights reserved.