In recent years, protein therapeutics plays a promising role in the field of medicine. However, intrinsic properties of proteins, including short plasma half-life and hydrolytic stability in vivo, are severely limits their application. To surmount these issues, we developed an anionic injectable hydrogel based on temperature- and pH-sensitive poly(ethylene glycol)-poly(sulfamethazine carbonate urethane) (PEG-PSMCU) copolymers for the sustained delivery of cationic model protein, lysozyme. The PEG-PSMCU copolymers exhibit pH and temperature-induced sol-to-gel phase transition in aqueous solutions. The mechanical properties of PEG-PSMCU copolymers, such as viscosity, gelation rate, and mechanical strength, were controllably tunable by varying the polymer weight, pH and temperature. An in vitro biocompatibility test indicated that PEG-PSMCU-based copolymers, even at high polymer concentrations (up to 2000 mu g/ml), was not toxic to fibroblast cells. The in vivo gel formation was confirmed by subcutaneous injection of PEG-PSMCU-based copolymer solutions (20 wt%) into Sprague-Dawley (SD) rats, which indicated in situ gel formation with uniform porous structure. Furthermore, an in vivo biodegradation study of the PEG-PSMCU anionic hydrogels showed a surface-controlled degradation of the gel matrix. Lysozyme, chosen as a cationic model protein, was loaded into an anionic hydrogel through ionic and hydrophobic interactions. Lysozyme-loaded PEG-PSMCU copolymers readily formed an in situ hydrogel after subcutaneous injection in SD rats, which markedly retarded the initial burst and led to the sustained release of lysozyme for 7 days. Overall, injectable anionic hydrogels prepared in this study can act as a localized hydrogel depot of cationic proteins, which inhibited initial burst while facilitating sustained release, and open a new paradigm for sustained delivery of cationic proteins. (C) 2016 Elsevier Ltd. All rights reserved.