Formation of aggregated neutrophil extracellular traps in tissues is determining the efficacy of particulate nanoadjuvants

Neutrophils are essential for innate immunity, using mechanisms like Neutrophil Extracellular Trap (NET) formation to fight pathogens. Aggregated NETs (aggNETs) help resolve inflammation by cleaving pro-inflammatory cytokines, while scattered NETs can exacerbate inflammation, leading to tissue damage. Co-injection of 10 nm nanodiamonds (ND10) with peptide antigens boosts immune responses, including anti-SARS-CoV-2 immunity, due to transient immune responses induced by aggNETs around ND10 particles. Diamond nanoparticles in adjuvant mixtures enhance vaccines, though the optimal dose is uncertain. Our study aimed to find the minimal ND10 amount needed for effective aggNETs formation and a robust immune response with minimal long-term tissue damage. In vivo experiments revealed 1 mg of ND10 per injection significantly enhances immune responses, forming granulomas rich in neutrophil elastase. Lower doses left scattered nanoparticles, insufficient for aggNETs formation. The effective ND10 dose for mice, 1 mg per injection, can be extrapolated to other organisms.