Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes

Abstract The cationizable nature of ‘proton-sponge’ transfection agents facilitates pDNA delivery in several steps. Protonated amines account for electrostatic DNA binding and cellular uptake, buffering amines mediate polyplex escape from acidifying intracellular vesicles. As demonstrated with a sequence-defined library of oligo(ethanamino)amides containing selected oligoethanamino acids and histidines, the total protonation capacity as well as the cationization pH profile within the endolysosomal range have critical impact on gene transfer. Building blocks with even numbered amine groups (Gtt, Sph) exhibited higher total endolysosomal buffer capacity than odd number (Stp) analogs. Within the endolysosomal range, Gtt has the highest buffer capacity around pH 5, whereas Stp has its maximum around pH 7. Histidines increased the total buffer capacity, resulted in a more continuous cationization pH profile and greatly improved transgene expression in vitro and in vivo. Using receptor targeted and polyethylene glycol shielded polyplexes, better endosomal escape and > 100-fold enhanced transfection was detected. From the Clinical Editor Proton-sponge transfection agents for pDNA delivery are characterized in this study, demonstrating over 100-fold enhanced transection and better endosomal escape by using receptor targeted and polyethylene glycol shielded polyplexes.