Intrinsic Adjuvanticity of Branched Polyethylenimine in Vitro and Subcutaneously

Branched polyethylenimine (BPEI) is a cationic polymer and has been applied as an antigen carrier in vaccines. However, its intrinsic adjuvanticity and underlying mechanism remain unclear. Here, we systematically explored the direct stimulation effects of a series of BPEI on bone-marrow-derived dendritic cells (BMDCs) in vitro. Only high concentrations of BPEI with molecular weight (M-W) equal to or greater than 25 kDa showed an immune-activating effect, indicating that both M-W and cationic degree contribute to this effect. In a charge neutralization experiment, the intrinsic adjuvanticity of BPEI also demonstrated a cationic degree-dependent pattern. Moreover, we identified reactive oxygen species (ROS) as the crucial messengers in BPEI-mediated BMDC activation. However, high M-W and cationic charges from the BPEI exerted a direct toxicity on dendritic cells, which would restrict its in vivo applications. Therefore, we introduced fibroblasts as bystander cells and found that BPEI-treated fibroblasts passed stimulation effects to BMDCs with undetectable cytotoxicity. During subcutaneous stimulation, which corresponded to the fibroblast-mediated coculture system, BPEI induced an inflammatory microenvironment and local immune cell infiltration without obvious cell death. To the best of our knowledge, this is the first systematic and incisive investigation of BPEI's intrinsic adjuvanticity and its functional exploration in the presence of bystander cells. This finding paves the way to advance the BPEI-based vaccines and will give insights into the use of other immunoactive biomaterials with similar properties.