Double pH-responsive lipo-xenopeptides combine polar oligoamino acids (OAAs) such as succinoyl tetraethylene pentamine (Stp) with apolar lipoamino fatty acid (LAF) domains. The resulting potent mRNA carriers undergo a profound shift in their polarity upon endosomal protonation. In the current chemical evolution approach, three top-performing xenopeptides based on a U-shaped (LAFStp, LAFStp) or bundle (LAFStp) topology were modified by replacing Stp with a variety of hydrophobized OAAs. Octanol/water distribution (log) was determined by a novel label-free mass spectrometry-based method. At neutral pH, the new xenopeptide analogues displayed an up to 85-fold higher hydrophobicity (log> 2) than Stp-based carriers, with a sharp pH-responsive decrease at endosomal pH (log-1). Transfection efficacies were influenced by the amine count of the OAAs, with tetraethylene pentamine outperforming the OAAs with fewer amines. For both topologies, a strong correlation between mRNA expression and endosomal escape, as quantified by calcein release, was found. For U-shaped carriers (LAF/OAA = 2:1), lipophilic OAAs improved endosomal escape and mRNA transfection efficiency. In contrast, for bundle carriers (LAF/OAA = 4:1), lipophilic OAA incorporation did not further enhance endosomal escape or transfection. Mechanistic studies revealed that LAF-xenopeptide polyplexes can adopt both endosomal pH-dependent and pH-independent cytosolic entry mechanisms, with hydrophobization accelerating expression kinetics and enhancing pH-independent entry. Optimized U-shaped xenopeptides were identified with improved mRNA transfection in tumor cells, a dendritic cell line, and low-passage human colon carcinoma cells. In vivo, enhanced mRNA expression was found upon either intramuscular or intravenous administration in several organs and tumor tissue. 2 1 4 2 4 1 7.4 5.5D D D
