Guanidinium/Phenyl-Rich Amphiphilic Cationic Polymer for Efficient Cytosolic Protein Delivery and Cancer Immunotherapy.

Protein drugs have garnered increasing attention in biomedical applications due to their high specificity for target receptors and minimal side effects. However, the macromolecular and hydrophilic nature of proteins severely hinders their ability to penetrate cell membranes, restricting their intracellular applications. Herein, we present an innovative amphiphilic cationic polymer, p(PG420-co-HP15), capable of forming stable nanoparticles with diverse proteins and facilitating efficient cytosolic delivery. By incorporating guanidinium and phenyl ligands, p(PG420-co-HP15) effectively complexes with various proteins via hydrogen bonding, salt bridges, and hydrophobic and π-π interactions. Meanwhile, the phenyl ligands further enhance cellular uptake and promote endosomal escape by inducing membrane perturbations, likely through disruption of phospholipid packing and increased membrane fluidity. Consequently, p(PG420-co-HP15) enables efficient cytosolic delivery of 6 proteins, each with unique molecular weights, isoelectric points, and biological functions across different cell lines, surpassing the commercial reagent PULSin in delivery efficiency. Furthermore, p(PG420-co-HP15) effectively constructed nanovaccines with ovalbumin (OVA), significantly boosting T cell-mediated antitumor immunity in a B16-OVA melanoma mouse model. These findings emphasize the potential of p(PG420-co-HP15) as a versatile and efficient cytosolic protein delivery platform with broad applications in disease treatment, vaccine development, and biological research.