Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers

Abstract

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).

The Balance between Hydrophobicity/Aromaticity and Positively Charged Residues May Influence the Cell Penetration Ability

“…The interesting cellular uptake-enhancing effect of the Dabcyl group was further investigated in this study. Based on our previous results concerning the possible superior effect of the Dabcyl-AMBA tandem group compared to Dabcyl alone [19], we planned to analyze the effect of this construct on oligoarginines with different lengths. In the case of shorter oligoarginines, one can assume that the aromaticity/hydrophobicity has an enhanced role in driving the internalization compared to the positive character of the peptide, while the uptake of longer Dabcyl-AMBA-oligoarginines has a more pronounced charge-driven factor (ion-pair formation with glycosaminoglycans and with phosphate groups of membrane lipids).…”

“…In these studies, hexa-and tetraarginines were modified to increase their internalization, thus we decided to investigate the effect of the number of arginine residues on the cell penetration of these kinds of constructs. In one of our previous peptide studies both groups were used, thus we planned to compare the effect of AMBA-and Dabcyl-AMBA modification based on the promising findings of our research group concerning the Dabcyl-AMBA-(Arg) 4 -Lys(Cf) peptide, among others [19]. Therefore, peptides with different numbers of arginines in their sequence were prepared, and their N-termini were modified with Dabcyl, AMBA or Dabcyl-AMBA (Figure 1).…”

“…The coupling reagents were DIC and OxymaPure, and the modification of the N-terminus (Dabcyl, AMBA or 5(6)-carboxyfluorescein (Cf)) was performed on the resin using the same coupling reagents. The commercially available AMBA was Fmoc-protected in advance according to a previously established method [19]. For the uptake studies the fluorescent molecule (Cf) was coupled either to the ε-amino group of an inserted Lys at the C-terminus in a solution, or to the N-terminus on a solid phase, such as the Dabcyl group.…”

“…Thus, there is a focus on efforts to improve the direct penetration of CPPs and/or increase their endosomal escape. The direct penetration can be increased e.g., by the modification of well-known CPPs with fatty acid acylation [15] or using the 4-((4-(dimethylamino)phenyl)azo)benzoyl group (Dabcyl) as an internalization enhancer [16,17], or Dabcyl and aromatic residues [18,19]. The endosomal escape may be enhanced by inserting lytic peptides into the sequence [20].…”

“…[18] The insertion of Trp into the N-terminus or middle of the tetraarginine resulted in a higher cellular uptake, while the direct penetration was more pronounced. As the aromatic side chain of the tryptophan had a high impact on the cellular uptake, we replaced it with some other aromatic unnatural amino acids, such as 4-aminobenzoic acid (PABA), 4-(aminomethyl) benzoic acid (AMBA) and 6-amino-2-naphthoic acid (NAPH) [19]. These residues were applied at the N-terminus or in the middle of the peptides instead of the Trp.…”