Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery.

Cytosolic protein delivery is important for the development of protein therapeutics towards intracellular targets. Guanidyl polymers exhibit high binding affinity with cargo proteins, and thus were designed as carriers for intracellular protein delivery. However, the structure-activity relationship and mechanism of these polymers in cytosolic protein delivery remained to be investigated. In this study, we synthesized a total number of eighteen guanidyl-rich polymers by grafting various guanidyl containing compounds onto a polyethylenimine scaffold. The investigated guanidyl analogues were consisted of a guanidyl group and a hydrophobic component including cyclohexane, benzene, and alkanes with various chain lengths. It is surprising that only the polymers with both benzene and guanidyl possessed high efficiency in cytosolic protein delivery. Further results showed that all the synthesized polymers have efficient protein binding in water and high cellular uptake, but these polymers except the benzene-guanidyl based one enter the cytosol of cells without carrying their cargo proteins, suggesting poor stability of the polymer/protein complexes in culture medium. Paired guanidinium-π interactions between the ligands on benzene-guanidyl polymers are critical to the stabilization of polymer/protein complexes. In addition, a lead polymer in the library exhibited robust delivery efficacy to various cargo proteins, while maintaining their bioactivity after cell internalization. The results suggest that complex stability is a critical factor in polymer-mediated intracellular protein delivery systems, and provide new insights to guide design of polymeric protein vehicles.