A Covalent Brush-on-Brush Polymer Programs Ordered Intracellular Activation of a Mitochondria-Disrupting Peptide

Can the order in which a material encounters distinct biological environments be encoded directly into its covalent architecture? Intracellular peptide effectors acting at defined subcellular sites require activation logic that follows this order, yet many responsive carriers couple multiple functional changes to one trigger. Here, we report sequential unmasking peptide release architecture (SUPRA), a covalent brush-on-brush (BOB) bottlebrush polymer that distributes two orthogonal biochemical inputs across two structural layers: a mitochondria-disrupting KLAK peptide inner brush densely grafted onto a glutathione-cleavable polydisulfide backbone, and an acid-labile PEG outer brush that masks the construct during circulation. Acidity first removes the outer mask to expose a membrane-interactive peptide-brush state, whereas cytosolic reduction deconstructs the backbone and relieves brush confinement. Architecture-disrupted control and intracellular perturbation/FRET studies support a sequential pathway of acid-triggered endolysosomal escape followed by reductive scaffold deconstruction and mitochondrial accumulation of KLAK-bearing species. SUPRA extends circulation, accumulates in tumors, induces tumor-cell death with immunogenic features, sensitizes tumors to PD-1 blockade, and produces no detectable anti-PEG antibody response under the tested dosing schedule. These results establish covalent BOB architectures as a design strategy for programming the sequence, not only the occurrence, of intracellular activation events.