Abstract A highly efficient transfection agent is reported that is based on terpolymer consisting of N ‐(2‐hydroxypropyl)methacrylamide (HPMA), N ‐(3‐guanidinopropyl) methacrylamide (GPMA), and N ‐(2‐indolethyl)methacrylamide monomers (IEMA) by analogy to the amphipathic cell‐penetrating peptides containing tryptophan and arginine residues. The incorporation of the indole‐bearing monomer leads to successful plasmid DNA condensation even at a nitrogen‐to‐phosphate (N/P) ratio of 1. The hydrodynamic diameter of polyplexes is determined to be below 200 nm for all N/P ratios. The transfection studies demonstrate a 200‐fold increase of the transgene expression in comparison to P(HPMA‐co‐GPMA) with the same guanidinium content. This study reveals the strong potential of the indole group as a side‐chain pendant group that can increase the cellular uptake of polymers and the transfection efficiency of the respective polyplexes
