Modification of Short Non‐Permeable Peptides to Increase Cellular Uptake and Cytostatic Activity of Their Conjugates

Abstract

Cell‐penetrating peptides (CPPs) are well‐known delivery vectors, oligoarginines are an important class of CPPs, which was modified to increase the cellular uptake and change the internalization mechanism. A minimum number of four Arg residues was used and tetrarginine sequence was modified by 4‐((4‐(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp(s). The Dabcyl group was attached to the N‐terminus, while peptides were different in the number and position of Trp in their sequence. Their internalization was studied on breast cancer cells (MCF‐7 and MDA‐MB‐231), and Chinese hamster ovary cells (CHO) using different fluorescence techniques. Our most active CPPs, Dabcyl‐RRWRRK and Dabcyl‐WRRRRK were conjugated to antitumor drugs, resulting in considerable cytostatic activity, especially on MDA‐MB‐231 cell lines. These newly developed cell‐penetrating tetrarginine derivatives may enter cells very efficiently, mainly by direct translocation and caveolae‐mediated endocytosis, thus they may be promising delivery vehicle. Tetragrinines are considered inefficient CPPs, but with our modifications, we rendered them effective.

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

“…This has prompted the synthesis of Trp-containing, Arg-rich CPPs with improved internalization property [ 10 , 26 , 36 ]. We described that Dabcyl group increases the cellular uptake of hexa- and tetraarginine [ 32 ] and its effect may be further enhanced by Trp [ 33 ]. Two tetraarginine derivatives (Dabcyl-RRWRRK and Dabcyl-WRRRRK) were proven to be effective.…”

“…Another method to accurately measure the internalized peptide is the lysis of cells after accurate removal of extracellular and membrane-bound peptides, and subsequently measure the fluorescence intensity. Such a method can overcome bias related to the Dabcyl quenching of the fluorophore since the peptides are degraded by the cell-lysis released and added (trypsin) enzymes [ 32 , 33 ]. The internalization was quantified at 37 °C and 4 °C.…”

“…Recently, we extended our approach to include Trp into the sequence of Dabcyl-containing tetraarginines. The presence of Dabcyl and Trp further increased the uptake of tetraarginines, where the structure directly impacted the activity [ 33 ].…”

Membrane Internalization Mechanisms and Design Strategies of Arginine-Rich Cell-Penetrating Peptides

“…In recent research, 4-(dimethylaminoazo) benzene-4-carboxylic acid (DABCYL) was used as a new kind of modification because of its structural simplicity, availability along with hydrophobicity, which could assist cell permeability. It has been demonstrated that utilizing the DABCYL group to modify arginine-rich CPPs effectively increases cellular uptake efficacy [ 104 , 105 , 106 ].…”

Redesigning of Cell-Penetrating Peptides to Improve Their Efficacy as a Drug Delivery System

“…Although Dabcyl could enhance the internalization of tetraarginine, this effect was modest. For further improvement of cellular uptake, tryptophan was introduced into the Dabcyl-modified tetraarginine [ 156 ]. In spite of the fact that tryptophan insertion alone did not affect the internalization, the presence of Dabcyl highly increased the uptake.…”