Intracellular protein delivery: New insights into the therapeutic applications and emerging technologies

The inability to cross the plasma membranes traditionally limited the therapeutic use of recombinant proteins. However, in the last two decades, novel technologies made delivering proteins inside the cells possible. This allowed researchers to unlock intracellular targets, once considered 'undruggable', bringing a new research area to emerge. Protein transfection systems display a large potential in a plethora of applications. However, their modality of action is often unclear, and cytotoxic effects are elevated, whereas experimental conditions to increase transfection efficacy and cell viability still need to be identified. Furthermore, technical complexity often limits in vivo experimentation, while challenging industrial and clinical translation. This review highlights the applications of protein transfection technologies, and then critically discuss the current methodologies and their limitations. Physical membrane perforation systems are compared to systems exploiting cellular endocytosis. Research evidence of the existence of either extracellular vesicles (EVs) or cell-penetrating peptides (CPPs)- based systems, that circumvent the endosomal systems is critically analysed. Commercial systems, novel solid-phase reverse protein transfection systems, and engineered living intracellular bacteria-based mechanisms are finally described. This review ultimately aims at finding new methodologies and possible applications of protein transfection systems, while helping the development of an evidence-based research approach.