Protein Post-Translational Modification-Inspired Regulation of Peptide Coacervation in Living Cells for Pro-Necroptotic Kinase Delivery

Protein post-translational modifications (PTMs) play pivotal roles in modulating protein structures and functions in a complex biological environment; however, creation of artificial biomaterials resembling PTM processes remains challenging. Here, we report a PTM-inspired arginine methylation-regulated complex peptide coacervate R8-PD in living cells as delivery vehicles for pro-necroptotic protein receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and cancer therapy. Inspired by arginine methylation catalyzed by protein arginine methyltransferases (PRMTs), we create the peptide coacervate via complex liquid–liquid phase separation of an arginine-rich peptide R8 and charge-complementary poly(aspartic acid). The coacervate undergoes PRMT1-induced methylation, thus triggering coacervate dissolution and RIPK1 release. Cellular studies reveal the enhanced cellular uptake of the coacervate formulation RIPK1@R8-PD through caveolae-mediated endocytosis with dissolution occurring specifically in PRMT1-overexpressing cancer cells. Mechanistic studies directly illustrate activation of the necroptotic pathway involving phosphorylation of RIPK3 and mixed lineage kinase domain-like pseudokinase (MLKL) based on the PRMT1 overexpression-induced RIPK1 release from RIPK1@R8-PD, thus leading to cellular membrane permeabilization and death. Animal studies confirm that administration of the coacervate formulation significantly enhances the efficacy of tumor growth inhibition with an excellent biosafety profile. These findings in this study elucidate an innovative approach for the creation of PTM-inspired enzyme-responsive peptide coacervates to deliver proteins and necroptotic induction, thus providing a new strategy for drug delivery and cancer therapy in the future.