Metrics & MoreArticle Recommendations CONSPECTUS: Protein therapeutics holds enormous promise for the treatment of various diseases but is limited to extracellular targets because of the membrane impermeable nature of most proteins. Cytosolic protein delivery systems are of great importance in the development of next-generation protein therapeutics. Since proteins are biomacromolecules characterized with limited binding sites, chemical modification or genetic engineering of cargo proteins is usually required to strengthen their binding affinity with the delivery carriers, which, however, may irreversibly impair their bioactivities. As thus, protein delivery systems that can efficiently transport native proteins into the cytosol of living cells with uncompromised bioactivities are highly desired.In this Account, we summarize recent advances by our two research groups in the rational design of cytosolic protein delivery systems. To enhance the binding affinity between the carriers and cargo proteins, protein binding ligands, such as guanidium, boronate, catechol, piperazine, and metal chelates, were introduced into the delivery systems. The obtained carriers showed robust efficiency in the cytosolic delivery of various cargo proteins with maintained bioactivities. Alternatively, hydrophobic ligands such as lipids and fluorolipids were conjugated to cationic polymers to yield amphiphiles that can encapsulate cargo proteins via electrostatic interaction and supramolecular assembly. Furthermore, carrier-free protein delivery strategies based on L-type amino acid transporter 1 (LAT1)-assisted direct transport and metabolic glycoengineering-mediated cell labeling were developed for targeted delivery of proteins into cancer cells, mitigating the materials toxicity commonly associated with nanocarriers. To achieve efficient protein delivery in serum, several strategies including reversible covalent cross-linking, responsive phase transition, and surface coating were proposed. These methods effectively enhanced the stability of polymer/protein nanocomplexes in a serum-containing medium.Besides protein binding, efficient intracellular protein release is another critical step for the cargo proteins to exert their bioactivities, while carriers with excessively high protein binding capacity may hinder the release of cargo proteins in the cytosol. To solve this dilemma, protein delivery systems were engineered with stimuli-responsiveness to adopt on-demand drug release profiles. After clarifying the rational design principles of cytosolic delivery systems, we describe the applications of our developed systems for genome editing, cancer vaccines, anti-inflammation therapies, etc. The key challenges and future directions of protein delivery systems are discussed. This Account will provide new insights into the rational design of cytosolic protein delivery systems and facilitate the development of next-generation protein therapeutics acting on intracellular targets.
