pH and reduction dual-bioresponsive nanosized polymersomes based on poly(ethylene glycol)-SS-poly(2-(diethyl amino)ethyl methacrylate) (PEG-SS-PDEA) diblock copolymers were developed for efficient encapsulation and triggered intracellular release of proteins. PEG-SS-PDEA copolymers with PDEA-block molecular weights ranging from 4.7, 6.8, to 9.2 kg/mol were synthesized in a controlled manner via reversible addition fragmentation chain transfer (RAFT) polymerization of 2(diethyl amino)ethyl methacrylate (DEAEMA) using PEG-SS-CPADN (CPADN = 4-c-yanopentanoic acid dithionaphthalenoate; M-n PEG = 1.9 kg/mol) as a macro-RAFT agent. These copolymers existed as unimers in water at mildly acidic pH (<7.2) conditions, but readily formed monodisperse nanosized polymersomes (54.5-66.8 nm) when adjusting solution pH to 7.4. These polymersomes were highly sensitive to intracellular pH and reductive environments, which resulted in fast dissociation and aggregation of polymersomes, respectively. Notably, both fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and cytochrome C (FITC-CC) proteins could facilely be encapsulated into polymersomes with excellent protein-loading efficiencies, likely as a result of electrostatic interactions between proteins and PDEA. The in vitro release studies showed that protein release was minimal (<20% in 8 h) at pH 7.4 and 37 degrees C. The release of proteins was significantly enhanced at pH 6.0 due to collapse of polymersomes. Notably, the fastest protein release was observed under intracellular-mimicking reductive environments (10 mM dithiothreitol, pH 7.4). MTT assays in RAW 264.7 and MCF-7 cells indicated that PEG-SS-PDEA (9.2 k) polymersomes had low cytotoxicity up to a polymer concentration of 300 mu g/mL. Confocal laser scanning microscope (CLSM) observations revealed that FITC-CC-loaded PEG-SS-PDEA (9.2 k) polymersomes efficiently delivered and released proteins into MCF-7 cells following 6 h of incubation. Importantly, flow cytometry assays showed that CC-loaded PEG-SS-PDEA (9.2 k) polymersomes induced markedly enhanced apoptosis of MCF-7 cells as compared to free CC and CC-loaded PEG PDEA (8.9 k) polymersomes (reduction-insensitive control). These dual-bioresponsive polymersomes have appeared to be highly promising for intracellular delivery of protein drugs.