Simple Hydrophobic Modification of bPEI Confers Potent, STING-Dependent Innate Immunostimulatory Activity

Macromolecular agonists are promising immunomodulators due to multivalency and tunable architectures. Previously, we showed that branched polyethylenimine (bPEI) conjugated with nitrogen-containing heterocycles activates the STING pathway, with branching being pivotal. Here we ask whether nitrogen is indispensable. We synthesized a library of bPEI conjugates bearing non-nitrogenous ligands (heterocycles, nonheterocycles, and aliphatic chains). While bPEI or ligands alone were inactive, >60% of conjugates induced robust IFN-β secretion across multiple cell types. Representative conjugates promoted STING clustering and TBK1/IRF3 phosphorylation, and genetic ablation of STING or cGAS markedly reduced IFN-β induction. Conjugation mitigated PEI cytotoxicity and minimized NF-κB-driven pro-inflammatory cytokines. Docking, log <i>P</i> analysis, and time-resolved uptake profiling established a physicochemical structure-activity relationship linking ligand rigidity, aromaticity, and hydrophobicity to STING binding, internalization, and IFN-β induction. Thus, nitrogen is not essential; simple hydrophobic modifications suffice within a macromolecular scaffold.