Trojan vectors with clinical and therapeutic potential

What are CPPs?

Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are typically 5-30 amino acid residues in length. Whether they occur naturally or are synthetic, CPPs are designed to breach cell membranes and deliver bioactive cargo to the intracellular space [1].  Like other vectors (i.e., polyplexes, liposomes, gold nanoparticles, and bioengineered viruses and bacteria), CPPs can translocate nucleic acids, small molecules, synthetic drugs, monoclonal antibodies and proteins into cells [2].  However, what sets them apart from the rest, is their unique chemical backbone and conformation, and the ease with which their side groups can be modified.  These attributes potentially allow greater cell-penetrating ability and diversity of cargo that can be delivered [3].  Cyclization and/or modification of the CPP backbone have further improved the uptake efficiency, metabolic stability and targeted delivery of CPPs and their cargo [4, 5, 6].  Notably, CPPs were the first reported vectors to ferry imaging agents into target cells, a feat that has fuelled studies into their potential use in clinical and therapeutic medicine [2].

The first CPPs to be discovered

The first naturally-occurring CPP to be identified and characterized was the 12 amino acid, Transactivator of Transcription (Tat) peptide, encoded in the Human Immunodeficiency Virus Type 1 (HIV-1) genome.  Tat was observed to cross the plasma membrane of cultured cells, and translocate to the nucleus where it trans-activated virus gene expression [7, 8].   Later, in 1991, a 16 amino acid peptide, encoded within the third helix of the Antennapedia homeodomain of Drosophila melanogaster, was found to be necessary and sufficient to translocate cargo across cell membranes, using an energy-independent mechanism that did not involve endocytosis [9].   While CPPs continue to be sourced from naturally-occurring proteins and chimeric peptides, their analogs can be synthesized in vitro.  Where there is no natural counterpart, a CPP can be designed in silico, and synthesised and tested in vitro.

Classification of CPPs

CPPs have been classified according to (i) their physicochemical properties, (ii) their origin, or (iii) their ability to target and permeate the nuclear membrane.  The so-called Nuclear Localization Sequences (NLS), are short, cationic CPPs, containing poly-lysine, -arginine, or -proline motifs. NLS enter the nucleus through the nuclear pore, a multimeric complex that contains 50-100 proteins [1].

Table 1 lists some CPPs that have been experimentally validated so far.  An extensive list can be viewed at “CPPsite 2.0 Database of Cell-Penetrating Peptides” ( cppsite/index.html) [10].  This, and other sources in the peer-reviewed literature, provide detailed information of more than 1,700 experimentally validated CPPs.

How do CPPs penetrate cell membranes?

Broadly speaking, cells internalize CPPs and their cargo by (i) Endocytosis, an energy-dependent mechanism, or (ii) Direct Translocation, an energy-independent mechanism. Which one is used, is determined by a host of factors, such as the CPP sequence, its conformation and net charge, the temperature, the chemical composition and charge of the cell’s lipid membrane, the size and chemical composition of the cargo, and how the cargo is conjugated to the CPP.

 - Endocytosis - Endocytosis of bioactive molecules occurs either by phagocytosis (uptake of large macromolecules) or pinocytosis (ingestion of fluids and solutes). Whereas phagocytosis is restricted to macrophages and leukocytes, two specialized cell types of the immune system, pinocytosis occurs in every cell, and subsumes four different types of uptake mechanisms, i.e., (i) macropinocytosis, (ii) clathrin-dependent, (ii) caveolin-dependent, or (iii) clathrin- and caveolin-independent endocytic pathways [11].

- Direct Translocation - All cells have the potential to internalize CPPs and their cargo by various energy-independent mechanisms, i.e., (i) pore formation [12], (ii) carpet-like internalization [13], (iii) membrane thinning [14], or (iv) inverted micelle formation [15]. In every case, internalization is initiated by membrane interaction, followed by membrane permeation, and ends with the release of the CPP and its cargo into the cytosol (see:


TABLE 1 – CPPs and their classification

Basis for Classification

Type of CPP

Name of CPP





HIV-1 TAT48-60 >


7, 8

Penetratin [pAntp43-58] >







kalata B1 (cysteine knot cyclic peptide)







Pept 1



Pept 2















Amphiphilic model peptide










HIV-1 TAT48-60 >


7, 8

Penetratin   [pAntp43-58] >





SV40 T-antigen NLS (Monopartite)



Nucleoplasmin (Bipartite)





1, 21



1, 21



1, 21



1, 21



1, 21


Therapeutic applications of CPPs and current obstacles to their clinical translation

CPPs are among the most efficient and effective vectors of bioactive molecules tested to date, making them attractive candidates for use in clinical and therapeutic biomedicine.  Pre-clinical studies show they are able to cross the blood-brain barrier, intestinal mucosa, nasal mucosa and skin [22]. Elsewhere, CPPs have demonstrated therapeutic benefit in a range of experimental models, including acute cochlear injury [23], labour, multiple sclerosis, cancer, chronic pain, obesity and cardiovascular disease [18]. Pre-clinical experiments have also revealed some real and potential obstacles to their clinical translation. Some of these are listed in Table 2, together with strategies to mitigate the underlying causes.

 TABLE 2 - Potential Obstacles To The Clinical Usefulness Of CPPs And Their Solutions






1. Design dormant CPP that cannot penetrate cell plasma membrane (e.g. fusion peptide; side-chain modification).  2. Activate CPP by external trigger (e.g. Δ pH; Δ temp.; UV exposure; proteolysis).

6, 24, 25, 26, 27













Consider physico-chemical properties of CPP:

. Guanidinium content

. Hydrophobicity

. Amphipathicity

. Charge

. Chirality (L- and D- combinations)

. Secondary structure

. Folding capacity in the presence of membranes

. Affinity of CPP for membranes


Possible modifications to CPP scaffold:

. Use of cell-penetrating polydisulfides

. Alpha vs beta helices; cyclization via triazole bridge; reversible bicyclization via paired S-S bonds

. N-alkylated proline spacers in-between R residues in Arginine-rich CPPs (R-X-R).

. Diketopiperazine-based oligo peptide.

. Gamma-aminoproline-based hexapeptide

. Octapeptides

. Filamentous CPPs

. Triple helical CPPs

. Supramolecular CPPs based on intermolecular interaction of repeating units

. Tryptophan-rich CPPs that assemble into spherical aggregates

6, 28, 29, 30, 31, 32




1. Covalent conjugation:

. Chemical (disulphide bonds, amine bonds, specific linkers).

. Expression host (E. coli or S. cerevisiae) produces the CPP-cargo conjugate.

2. Reversible, non-covalent coupling:

. Calmodulin plus calmodulin-binding motif.

. EF hand adaptor proteins (calmodulin-like protein 3 (CALML3), troponin).

2. Physical complexation

. Electrostatic and/or hydrophobic interactions between CPP and its cargo, achieved by simple bulk-mixing.

33, 34





1. Direct CPP and its cargo to cytosol instead of endosomes:

. Cyclization of CPP

. Arginine-rich CPP plus fusogenic lipids, membrane-disruptive peptides, membrane-disruptive polymers, lysosomotropic agents, photochemical internalization.

. PolyArginine CPP fused to the translocation domain of Pseudomonas aeruginosa exotoxin A.

2. Incorporate subcellular targeting sequences into CPP backbone.

4, 5, 6, 35



. Affecting cell and organelle membranes


. Resulting from specific interactions between CPP and cell components

1. Measure cell membrane integrity as an indirect indicator of cytotoxicity:

. Trypan Blue exclusion

. MTT assay

. Fluorescein leakage

. 2-deoxyglucose-6-phosphate leakage


2. Test whether peptide concentration, cargo molecule or coupling strategy is the likely cause of cytotoxicity.

36, 37



1. Modify backbone (e.g. adjust length; test different alpha > beta peptide substitutions).

2. Introduce chirality (L- and D- combinations).

3. Cyclization (e.g. via triazole bridge).

4. Reversible bicyclization (paired S-S bonds).

28, 29, 30, 31, 32


1. Incorporate targeting sequences into CPP sequence

2. Synthesise activatable CPPs.

3. Adjust MW.

4. Test different routes of administration (nasal, pulmonary, transdermal delivery).






1. Covalent conjugation:

. Chemical (disulphide bonds, amine bonds, specific linkers)

. Expression host (E. coli or S. cerevisiae) produces the CPP-cargo conjugate

2. Reversible, non-covalent coupling:

. Calmodulin plus calmodulin-binding motif

. EF hand adaptor proteins (calmodulin-like protein 3 (CALML3), troponin)

2. Physical complexation

. Electrostatic and/or hydrophobic interactions between CPP and its cargo, achieved by simple bulk-mixing

33, 34

 Concluding remarks

CPPs, by virtue of their physico-chemical properties and their structural and conformational versatility, offer promise as vehicles for the delivery of bioactive molecules to sub-cellular compartments that hitherto, could not be targeted precisely, efficiently, nor with an acceptably low level of toxicity.  Continued efforts to overcome all obstacles to their use, should pave the way for CPPs to be included in tailored treatments for a range of diseases and disorders.

 Do you require CPPs in your research?

Our experienced team at Genaxxon bioscience can provide technical and practical advice regarding:

-  Selection and purchase of CPPs

-  Our Peptide Synthesis Service > that is tailored to suit your research needs and your budget

- Here you can find more CPPs from Genaxxon: >


1. BORRELLI, A, TORNESELLO, AL, TORNESELLO, ML, et al. “Cell penetrating peptides as molecular carriers for anticancer agents”. Molecules 2018; 23: 295.

2. DERAKHSHANKHAH, H & JAFARI, S. “Cell penetrating peptides: a concise review with emphasis on biomedical applications”. Biomed Pharmacother 2018; 108: 1090-1096.

3. CASCALES, L, HENRIQUES, ST, KERR, MC, et al. “Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides”. J Biol Chem 2011; 286(42): 36932-36943.

4. EL-SAYED, A, FUTAI, S, HARASHIMA, H. “Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment”. AAPS J 2009; 11(1): 13-22.

5. MICKAN, A, SARKO, D, HABERKORN, U, et al. “Rational design of CPP-based drug delivery systems: considerations from pharmacokinetics”. Curr Pharm Biotechnol 2014; 15(3): 200-209.

6. BODE, SA & LÖWIK, DW.“Constrained cell penetrating peptides” Drug Discov Today Technol 2017; 26: 33-42.

7. FRANKEL, AD & PABO, CO. “Cellular uptake of the tat protein from human immunodeficiency virus”. Cell 1988; 55: 1189-1193.

8. GREEN, M & LOEWENSTEIN, PM. “Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein”. Cell 1988; 55: 1179-1188.

9. DEROSSI, D, JOLIOT, AH, CHASSAING, G, et al. “The third helix of the Antennapedia homeodomain translocates through biological membranes”. J Biol Chem 1994; 269(14): 10444-10450.

10. AGRAWAL, P, BHALLA, S, USMANI, SS, et al. “CPPsite 2.0: A repository of experimentally validated cell-penetrating peptides.” Nucleic Acids Res 2016; 44(D1): D1098-D1103.

11. MADANI, F, LINDBERG, S, LANGEL, Ü, et al. “Mechanisms of cellular uptake of cell-penetrating peptides”. J Biophys 2011;  doi: 10.1155/2011/414729.

12. MATSUZAKI, K, YONEYAMA, S, MURASE, O, et al. “Transbilayer transport of ions and lipids coupled with mastoparan X translocation”. Biochemistry 1996; 35(25): 8450-8456.

13. POUNY, Y, RAPAPORT, D, MOR, A, et al. “Interaction of antimicrobial dermaseptin and its fluorescently labelled analogues with phospholipid membranes”. Biochemistry 1992; 31(49): 12416-12423.

14. LEE, MT, HUNG, WC, CHEN, FY, et al. “Many-body effect of antimicrobial peptides on the correlation between lipid’s spontaneous curvature and pore formation”. Biophys J 2005; 89(6): 4006-4016.

15. DEROSSI, D, CALVET, S, TREMBLEAU, A, et al. “Cell internalization of the third helix of the antennapedia homeodomain is receptor-independent”. J Biol Chem 1996; 271(30): 18188-18193.

16. LINDBERG, M, BIVERSTÅHL, H, GRÄSLUND, A, et al. “Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR”. Eur J Biochem 2003; 270(14): 3055-3063.

17. POOGA, M, HÄLLBRINK, M, ZORKO, M, et al. “Cell penetration by transportan”. FASEB J 1998; 12(1): 67-77.

18. WEIDMANN, J & CRAIK, DJ. “Discovery, structure, function, and applications of cyclotides: circular proteins from plants”. J Exp Botany 2016; 67(16): 4801-4812.

19. KALDERON, D, ROBERTS, BL, RICHARDSON, WD, et al. “A short amino acid sequence able to specify nuclear location”. Cell 1984; 39: 499-509.

20. ROBBINS, J, DILWORTH, SM, LASKEY, RA, et al.  “Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 1991; 20, 615–623.

21. MILLETTI, F. “Cell-penetrating peptides: classes, origin, and current landscape”.  Drug Discov Today 2012; 17: 850-860.

22. SHI, NQ, QI, XR, XIANG, B, et al. “A survey on “Trojan Horse” peptides: opportunities, issues and controlled entry to “Troy”. J Control Release 2014; 194: 53-70.

23. ESHRAGHI, AA, ARANKE, M, SALVI, R, et al. “Preclinical and clinical otoprotective applications of cell-penetrating peptide D-JNKI-1 (AM-111)”. Hear Res 2018; 368: 86-91.

24. JIANG, T, OLSON, ES, NHUYEN, QT, et al. “Tumour imaging by means of proteolytic activation of cell-penetrating peptides”.  Proc Natl Acad Sci USA 2004; 101(51): 17867-17872.

25. ZHU, L, KATE, P, TORCHILIN, VP. “Matrix metalloprotease 2-responsive multifunctional liposomal nanocarrier for enhanced tumor targeting”. ACS Nano 2012; 6(4): 3491-3498.

26. ZHU, L, WANG, T, PERCHE, F, et al. “Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety”. Proc Natl Acad Sci USA 2013; 110(42): 17047-17052.

27. SETHURAMAN, VA & BAE, YH. “TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors”. J Control Release 2007; 118(2): 216-224.

28. de LUCIO, H, GAMO, AM, RUIZ-SANTAQUITERIA, M, et al. “Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides”. Eur J Med Chem 2017; 140: 615-623.

29. KALAFATOVIC, D & GIRALT, E. “Cell-penetrating peptides: design strategies beyond primary structure and amphipathicity”. Molecules 2017; 22(11): doi: 10.3390/molecules22111929.

30. NAJJAR, K, ERAZO-OLIVERAS, A, BROCK, DJ, et al. ‘An I- to d-amino acid conversion in an endosomolytic analog of the cell-penetrating peptide TAT influences proteolytic stability, endocytic uptake, and endosomal escape”.  J Biol Chem 2017; 292(3): 847-861.

31. REICHART, F, HORN, M & NEUNDORF, I. “Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery”.  J Pept Sci 2016; 22(6): 421-426.

32. QIAN, Z, RHODES, CA, McCROSKEY, LC, et al. “Enhancing the cell permeability and metabolic stability of peptidyl drugs by reversible bicyclization”.  Agnew Chem Int Ed Engl 2017; 56(6): 1525-1529.

33. KRISTENSEN, M, BIRCH, D & NIELSEN, HM. “Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos”. Int J Mol Sci 2016; 17(2): doi: 10.3390/ijms17020185.

34. NGWA, VM, AXFORD, DS, HEALEY, AN, et al. “A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations”. PLoS One 2017; 12(5): doi: 10.1371/journal.pone.0178648.

35. MOHAMMED, AF, ABDUL-WAHID, A, HUANG, EH, et al. “The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells”. J Control Release 2012; 164(1): 58-64.

36. ZORKO, M & LANGEL, U. “Cell-penetrating peptides: mechanism and kinetics of cargo delivery”. Adv Drug Deliv Rev 2005; 57(4): 529-545.

37. EL-ANDALOUSSI, S, JÄRVER, P, JOHANSSON, HJ, et al. “Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study”. Biochem J 2007; 407(Pt 2): 285-292.

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