Overcoming cellular barriers with cell-penetrating peptides: functional roles of individual amino acids

The delivery efficiency of cell-penetrating peptides (CPPs) is much lower than that of other non-viral delivery systems, mainly because of cellular barriers such as the plasma membrane, endosomal acidification, cytosolic degradation, and nuclear entry. We focus on how the functional properties of individual amino acids help overcome cellular barriers, thereby improving biomolecular delivery, and we also discuss CPPs that have advanced to clinical trials and highlight their strengths. We discuss how the plasma membrane acts as a major extracellular barrier that regulates biomolecule entry into cells, a challenge that can be addressed with peptides containing functional groups, such as basic amino acids, and how, after internalisation, CPPs cross intercellular barriers, and how their ability to do so mainly depends on the specific amino acids making up the peptide. This includes analyzing how histidine helps with endosomal escape, and conversely, how the presence of a nuclear localisation signal increases the likelihood of CPPs entering the nucleus. Specific amino acid residues successfully tackle these cellular bottlenecks. Incorporating basic amino acids effectively regulates entry across the plasma membrane, histidine assists with endosomal escape after internalisation, and the presence of a nuclear localisation signal increases the likelihood of CPPs entering the nucleus. Overcoming these barriers depends heavily on the presence and position of specific amino acid residues within a CPP. Sequence optimisation and rational design enhance delivery efficiency, supporting the translational potential of advanced CPPs currently evaluated in clinical trials.