Redox-activated cholesterol-dependent cytolysin enables cytosolic release of liposomal cargo.

Precise intracellular delivery of biologic therapeutics remains a major challenge due to endosomal entrapment and inefficient delivery systems. Here, we develop a bioinspired platform that uses Streptolysin O (SLO), a member of the cholesterol-dependent cytolysin (CDC) family, for cytosolic cargo delivery. This delivery system incorporates an affibody for selective targeting and endocytosis and a redox-cleavable PEG-conjugated dithiol-ethyl carbonate linker (PEG-DEC) that reversibly inactivates SLO extracellularly. After endosomal uptake, the reductive intracellular environment removes the PEG layer, reactivating SLO to induce localized endosomal disruption and cargo release. This mechanism minimizes off-target toxicity while promoting efficient cytosolic delivery of diverse cargo, including doxorubicin (DOX), the fluorescent protein GFP and mApple, and the enzyme NanoLuciferase (NanoLuc) and lactate oxidase (LOX). PEGylated SLO exhibited significantly improved cytosolic release efficiency compared with conventional liposomal formulations, confirming the advantage as a controllable intracellular delivery module.