Carboxymethyl chitosan nanoparticles loaded with Ctenopharyngodon idella interferon-γ2 (CiIFN-γ2) enhance protective efficacy against bacterial infection in grass carp

Abstract

Type II interferon may be one of the most important immunomodulatory factors involved in the coordination of immune responses in vertebrates, but the efficacy of protein molecular drugs in treating bacterial infection still needs to be improved. Therefore, stable delivery technologies are required to improve the efficacy of proteins. This work evaluates the feasibility and biological activity of the carboxymethyl chitosan (CMCS) load with the interferon-γ2 of Ctenopharyngodon idella (CiIFN-γ2). Moreover, the resistance to Aeromonas hydrophila infection in grass carp will also be examined. The nanoparticle based on CMCS load with CiIFN-γ2 was constructed and designated as CMCS-CiIFN-γ2 (CMCS-Ciγ2) by ion exchange method. The CMCS-loading protein efficiency was measured at 28.46 ± 2.87%. The hemolytic analysis and cytotoxicity assays were performed to assess the biocompatibility of CMCS-Ciγ2. The cytotoxicity of CMCS-Ciγ2 nanoparticles was 90.8 ± 0.95% at the highest tested concentration (200 μg/mL), which proves that it is safe and non-toxic. Additionally, the findings indicated that the expression levels of pro-inflammatory cytokines (IL-1β, TNF-α, iNOS) in macrophages activated by nanoparticles CMCS-Ciγ2, were significantly enhanced, and nitric oxide (NO) was detected at high levels. In vivo imaging system (IVIS) results showed that the CMCS-Ciγ2 can still be detected at 8 h after oral gavage. This remains inside the host for a longer period of time contributing to the effective uptake of the protein. The challenge test revealed that CMCS-Ciγ2 had a higher relative immune protection rate than other treatment groups. The findings also showed that there was a reduction in tissue bacterial loads and damage. The in-vivo experiment results showed that the CMCS-Ciγ2 elicited higher levels of serum biochemical indexes and increased expressions of several immune-related genes (IL-1β, TNF-α) in grass carp. In turn, these results demonstrated that the CMCS-Ciγ2 can effectively regulate the immune system and facilitate the development of an immune barrier that enables the host to cope with external environment changes. This research will provide critical insights into the development and application of nanomaterials, which can be used to prevent and treat bacterial diseases in aquaculture.