Cell-penetrating peptides (CPP) are promising tools for the transport of a broad range of compounds into cells. Since the discovery of the first members of this peptide family, many other peptides have been identified; nowadays, dozens of these peptides are known. These peptides sometimes have very different chemical–physical properties, but they have similar drawbacks; e.g., non-specific internalization, fast elimination from the body, intracellular/vesicular entrapment. Although our knowledge regarding the mechanism and structure–activity relationship of internalization is growing, the prediction and design of the cell-penetrating properties are challenging. In this review, we focus on the different modifications of well-known CPPs to avoid their drawbacks, as well as how these modifications may increase their internalization and/or change the mechanism of penetration.

Peptides are highly potent biological active compounds with excellent selectivity and binding, but they have some drawbacks (e.g., low stability in vivo because of the enzymatic degradation, and fast elimination). To overcome their drawbacks, various peptidomimetics have been gaining ground. Different modifications have been examined, such as the modification of peptide backbone. One such seemingly simple modification is the replacement of the CH^α group by an N atom. These amino acid derivatives are called azaamino acids, and peptides containing azaamino acid are called azapeptides. This exchange results in both steric and electronic differences from the original amino acids, thus affecting the structure and biological activity of the modified peptide. In this review, the synthesis possibilities of azapeptides and the impact of azaamino acid incorporation on the structure and biological activity are presented through examples. Different synthetic solutions for azaamino acid introduction and the various routes to build in the side chain are summarized to illustrate the improvement of the field of azaamino acid chemistry. The influence of the altered electronic and steric properties of N-atom on the structure is described, too. Finally, some examples are given with potent biological activity.

Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure–activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

Getting through the cell membrane is challenging, and transporting a therapeutic agent while entering the cell is even more complicated. Cell-penetrating peptides (CPPs) are valuable tools for solving this problem, although they have drawbacks. In this work, the synthesis and investigation of efficient CPPs are described. We used an aromatic group, 1-pyrene carboxylic acid (PCA), to enhance internalization. We designed oligoarginines to investigate the effect of PCA in different positions at the N-terminus or in the side chain. Our novel peptide derivatives showed remarkable internalization on tumor cell lines, and more than one endocytic pathway plays a role in their internalization mechanism. With this modification, there is an opportunity to design short oligoarginines that can rival well-known CPPs like octaarginine in internalization.