The Effect of Size, Dose, and Administration Route on Zein Nanoparticle Immunogenicity in BALB/c Mice

Background: Zein-based carriers are a promising delivery system for biomedical applications. However, few studies involve systematic investigation on their in vivo biocompatibility and immunogenicity. Purpose: The objective of this study was to identify the immunogenicity, type of immune response, biocompatibility and systemic recall immune response of zein nanoparticles administrated via different routes in mice. Animals and methods: Female Balb/c mice were selected as the animal model in this paper. The effect of particle size, dose and inoculation routes on immunogenicity were systematically explored. The mice were challenged at week 50 via intramuscular and subcutaneous routes to investigate the systemic recall immune responses of zein nanoparticles. Hematoxylin and eosin staining was performed to investigate the biocompatibility of zein nanoparticles at injection sites. Results: The administration of zein particles by parenteral routes led to a long-term systemic immune response. Particle size did not affect zein-specific IgG antibody titers. IgG antibody titers and inflammatory cell infiltration at the injection sites resulting from intramuscular zein particle injection were significantly higher than those from subcutaneous injection of the same dose. For intramuscular inoculation, dose-dependent IgG antibody titers were observed after the third inoculation, while no significant difference was found via the subcutaneous route. For both routes, IgG titer showed a time-dependent decrease at all dose levels from week 5 onward, and finally plateaued at week 28. The IgG subtype assay showed a predominant Th2-type immune response for both administration routes. Challenge with zein nanoparticles at week 50 led to a significant increase in specific IgG titer at all dose levels, indicating systemic recall immune responses. Interestingly, IgG antibody levels in the subcutaneous groups showed a delayed decrease compared to those of the intramuscular injection groups at all dose levels. Conclusion: This study indicated that immunogenicity may be one of the key challenges of using zein nanoparticles as carriers via parenteral administration. Further investigation is needed to illustrate zein immunogenicity in other forms, and the possible effect of systemic recall immune response on in vivo pharmacokinetic characteristics.