Tailoring Hyperbranched Poly(β‐amino ester) as a Robust and Universal Platform for Cytosolic Protein Delivery

Abstract Cytosolic protein delivery is a prerequisite for protein‐based biotechnologies and therapeutics on intracellular targets. Polymers that can complex with proteins to form nano‐assemblies represent one of the most important categories of materials, because of the ease of nano‐fabrication, high protein loading efficiency, no need for purification, and maintenance of protein bioactivity. Stable protein encapsulation and efficient intracellular liberation are two critical yet opposite processes toward cytosolic delivery, and polymers that can resolve these two conflicting challenges are still lacking. Herein, hyperbranched poly(β‐amino ester) (HPAE) with backbone‐embedded phenylboronic acid (PBA) is developed to synchronize these two processes, wherein PBA enhanced protein encapsulation via nitrogen–boronate (N–B) coordination while triggered polymer degradation and protein release upon oxidation by H 2 O 2 in cancer cells. Upon optimization of the branching degree, charge density, and PBA distribution, the best‐performing A2‐B3‐C2‐S 2 ‐P 2 is identified, which mediates robust delivery of various native proteins/peptides with distinct molecular weights (1.6–430 kDa) and isoelectric points (4.1–10.3) into cancer cells, including enzymes, toxins, antibodies, and CRISPR‐Cas9 ribonucleoproteins (RNPs). Moreover, A2‐B3‐C2‐S 2 ‐P 2 mediates effective cytosolic delivery of saporin both in vitro and in vivo to provoke remarkable anti‐tumor efficacy. Such a potent and universal platform holds transformative potentials for protein pharmaceuticals.