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Conjugating Peptides for Effective Trans-membrane Drug Delivery

16 November 2015

The research group of Prof. Jonel P. Saludes at Washington State University used Biotage® Initiator+ SP Wave and Biotage® Initiator+ Alstra™ synthesizers in developing cell penetrating peptides (CPPs) for drug delivery across the human cell membrane. 


About 90% of druggable targets are located inside the cell, and so an efficient delivery vector is highly desirable to transport active compounds across the cell membrane. The use of short peptide sequences as cell penetrating peptides (CPPs) is a promising area of research, and stems from the discovery of the protein transduction domain in HIV-1 Tat. Since then, a large array of protein-derived guanidinium containing CPPs have been defined including octaarginine, penetratin, pVEC, and VP22 among others. TATp and related protein-derived CPPs show great promise as delivery agents but face the limitations of poor translocation efficiency, lack of target specificity, cytotoxicity, and penurious biostability. Multimers and dendrimers of CPPs have shown higher cellular uptake compared to their parent monomers. Branched or multivalent designs often leads to increased uptake, which in turn increases efficacy at much lower concentrations. Further, CPP multimers can be designed to be proteolysis resistant compared to their linear precursors and are generally not cytotoxic at therapeutic concentrations.

The study utilized a solid phase peptide synthesis method for preparing multimeric CPPs and investigated the effects of multivalency on cell membrane permeability. The hypothesis was that simple dimerization through a C-terminal Lys residue keeping the main peptide sequence unaltered would lead to increased cell uptake efficiency, hence requiring less amount of peptide to effect the desired intracellular delivery. A dimer of TATp was prepared, as it is the prototypical CPP that is widely studied to carry a variety of cargoes.

TATp-D peptide structure

The branched dimeric Tat peptide (TATp-D) synthesized by microwave-assisted solid phase peptide synthesis. X = Cyanine 5 (Cy5). 

The authors write: "This work demonstrates the utility of multimerization and represents a simple approach to multivalent CPPs that show great promise in improving existing CPP technologies. TATp-D showed a six- to seven-fold fluorescence increase versus TATp at 0.25 μM. TATp-D also translocated in neuronal cells with no apparent increase in cytotoxicity. This orthogonally protected design enabled the selective conjugation of different cargoes at the N- and C-termini, on- or off-resin. Furthermore, our results suggest that the method presented herein is applicable for the preparation of higher valency CPPs to improve their membrane permeability at lower concentrations, which is beneficial in the delivery of high-value therapeutics."


A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations.

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I. Abrrey Monreal, Qian Liu, Katherine Tyson, Tyler Bland, Doralyn S. Dalisay, Erin V. Adams, Gary A. Wayman, Hector C. Aguilarb and Jonel P. Saludes, 2015Branched dimerization of Tat peptide improves Q1 Q2 permeability to HeLa and hippocampal neuronal cells. Chem. Commun., 2015, 51, 5463-5466.