Helical quaternary amine polypeptide programs membrane stress to drive immunogenic cell death and cytosolic gene delivery for cancer immunotherapy

Abstract

Nanomaterials can shape antitumor immunity; however, the design rules that link molecular features to immune outcomes remain unclear. Here, we demonstrate that helical polypeptides with quaternary amine sidechains physically disrupt phospholipid membranes, triggering the release of damage-associated molecular patterns (DAMPs) and enabling cytosolic gene delivery. Screening various sidechain amines and using mechanistic assays with a racemic control identifies quaternary amines, along with helicity, as key factors in immune responses. Mechanistically, physical membrane disruption damages membrane-based organelles—including mitochondria, the endoplasmic reticulum, and endosomes—leading to immunogenic cell death and facilitating endosomal escape of nucleic acids. Guided by this mechanism, guanidinium substitution strengthened phosphate engagement and reduced the apparent cationic density, thereby improving polyplex stability and immune activation. In mouse models of melanoma and colon cancer, local transfection with a PD-L1 knockout plasmid using a helical polypeptide reduced tumor burden by 70-80% and boosted effector T cell-mediated immunity. These findings illustrate how sidechain chemistry and helicity affect immune activation, offering a non-viral platform for antitumor immune priming.