Multivalency: Key Feature in Overcoming Drug Resistance with a Cleavable Cell-Penetrating Peptide-Doxorubicin Conjugate

  • Marco Lelle
  • Christoph Freidel
  • Stefka Kaloyanova
  • Klaus Müllen
  • Kalina Peneva


Multivalency is often used in biological systems, to increase affinity and specificity through avidity. This inspired us to prepare a synthetic bioconjugate that mimics natural multivalent systems. It is composed of doxorubicin and two octaarginine cell-penetrating peptides, to strengthen the electrostatic interactions between the negatively charged glycosaminoglycans of the plasma membrane and the guanidinium groups of the arginine residues. The multivalent conjugate has improved cellular uptake and cytotoxicity, compared to a peptide-drug conjugate with only one polyarginine and as a result it can overcome drug resistance in Kelly-ADR cells. The synthetic approach and the multivalent structure reported here can be used further as model systems, to gain insight into the biological interaction of cell-penetrating peptides with artificial membranes or for the preparation of more complex multimers.


Cell-penetrating peptide Multivalency Drug-peptide conjugate MDR Doxorubicin Drug resistance 



The authors are thankful to Dr. Alexander Schramm, Clinic for Pediatrics III, University Hospital Essen for providing the Kelly-WT and Kelly-ADR cells.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Research Involving Human and Animal Rights

This article does not contain any studies with animals and human participants performed by any of the authors.

Supplementary material

10989_2017_9622_MOESM1_ESM.docx (1.9 mb)
Supplementary material 1 (DOCX 1908 KB)


  1. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM (1999) Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol 39:361–398CrossRefGoogle Scholar
  2. Aroui S, Ram N, Appaix F, Ronjat M, Kenani A, Pirollet F, De Waard M (2009) Maurocalcine as a non toxic drug carrier overcomes doxorubicin resistance in the cancer cell line MDA-MB 231. Pharm Res-Dordr 26(4):836–845CrossRefGoogle Scholar
  3. Balendiran GK, Dabur R, Fraser D (2004) The role of glutathione in cancer. Cell Biochem Funct 22(6):343–352CrossRefPubMedGoogle Scholar
  4. Böhme D, Beck-Sickinger AG (2015) Drug delivery and release systems for targeted tumor therapy. J Pept Sci 21(3):186–200CrossRefPubMedGoogle Scholar
  5. Dean M, Hamon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42(7):1007–1017PubMedGoogle Scholar
  6. Deneke SM, Fanburg BL (1989) Regulation of cellular glutathione. Am J Physiol 257(4):L163–L173PubMedGoogle Scholar
  7. Dubikovskaya EA, Thorne SH, Pillow TH, Contag CH, Wender PA (2008) Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters. Proc Natl Acad Sci USA 105(34):12128–12133CrossRefPubMedGoogle Scholar
  8. Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8(7):848–866CrossRefPubMedGoogle Scholar
  9. Fasting C, Schalley CA, Weber M, Seitz O, Hecht S, Koksch B, Dernedde J, Graf C, Knapp EW, Haag R (2012) Multivalency as a chemical organization and action principle. Angew Chem Int Ed 51(42):10472–10498CrossRefGoogle Scholar
  10. Furedi A, Szebenyi K, Toth S, Cserepes M, Hamori L, Nagy V, Karai E, Vajdovich P, Imre T, Szabo P, Szuts D, Tovari J, Szakacs G (2017) Pegylated liposomal formulation of doxorubicin overcomes drug resistance in a genetically engineered mouse model of breast cancer. J Control Release 261:287–296CrossRefPubMedGoogle Scholar
  11. Gamcsik MP, Millis KK, Colvin M (1995) noninvasive detection of elevated glutathione levels in Mcf-7 cells resistant to 4-hydroperoxycyclophosphamide. Cancer Res 55(10):2012–2016PubMedGoogle Scholar
  12. Gatlik-Landwojtowicz E, Aanismaa P, Seelig A (2006) Quantification and characterization of P-glycoprotein-substrate interactions. BioChemistry 45(9):3020–3032CrossRefPubMedGoogle Scholar
  13. Gewirtz DA (1999) A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol 57(7):727–741CrossRefPubMedGoogle Scholar
  14. Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2(1):48–58CrossRefPubMedGoogle Scholar
  15. Gottlieb HE, Kotlyar V, Nudelman A (1997) NMR chemical shifts of common laboratory solvents as trace impurities. J Org Chem 62(21):7512–7515CrossRefPubMedGoogle Scholar
  16. Gu YJ, Cheng J, Man CW, Wong WT, Cheng SH (2012) Gold-doxorubicin nanoconjugates for overcoming multidrug resistance. Nanomedicine 8(2):204–211CrossRefPubMedGoogle Scholar
  17. Iyer AK, Singh A, Ganta S, Amiji MM (2013) Role of integrated cancer nanomedicine in overcoming drug resistance. Adv Drug Deliv Rev 65(13–14):1784–1802CrossRefPubMedGoogle Scholar
  18. Jones AT, Sayers EJ (2012) Cell entry of cell penetrating peptides: tales of tails wagging dogs. J Control Release 161(2):582–591CrossRefPubMedGoogle Scholar
  19. Kalia J, Raines RT (2008) Hydrolytic stability of hydrazones and oximes. Angew Chem Int Ed 47(39):7523–7526CrossRefGoogle Scholar
  20. Kratz F, Beyer U, Roth T, Tarasova N, Collery P, Lechenault F, Cazabat A, Schumacher P, Unger C, Falken U (1998) Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy. J Pharm Sci 87(3):338–346CrossRefPubMedGoogle Scholar
  21. Kunjachan S, Blauz A, Mockel D, Theek B, Kiessling F, Etrych T, Ulbrich K, van Bloois L, Storm G, Bartosz G, Rychlik B, Lammers T (2012) Overcoming cellular multidrug resistance using classical nanomedicine formulations. Eur J Pharm Sci 45(4):421–428CrossRefPubMedGoogle Scholar
  22. Lelle M, Frick SU, Steinbrink K, Peneva K (2014) Novel cleavable cell-penetrating peptide-drug conjugates: synthesis and characterization. J Pept Sci 20(5):323–333CrossRefPubMedGoogle Scholar
  23. Lelle M, Kaloyanova S, Freidel C, Theodoropoulou M, Musheev M, Niehrs C, Stalla G, Peneva K (2015) Octreotide-mediated tumor-targeted drug delivery via a cleavable doxorubicin-peptide conjugate. Mol Pharm 12(12):4290–4300CrossRefPubMedGoogle Scholar
  24. Lelle M, Freidel C, Kaloyanova S, Tabujew I, Schramm A, Musheev M, Niehrs C, Mullen K, Peneva K (2017) Overcoming drug resistance by cell-penetrating peptide-mediated delivery of a doxorubicin dimer with high DNA-binding affinity. Eur J Med Chem 130:336–345CrossRefPubMedGoogle Scholar
  25. Leslie EM, Deeley RG, Cole SPC (2005) Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol Appl Pharmacol 204(3):216–237CrossRefPubMedGoogle Scholar
  26. Liang JF, Yang VC (2005) Synthesis of doxorubicin-peptide conjugate with multidrug resistant tumor cell killing activity. Bioorg Med Chem Lett 15(22):5071–5075CrossRefPubMedGoogle Scholar
  27. Madani F, Lindberg S, Langel Ü, Futaki S, Gräslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011:1–10CrossRefGoogle Scholar
  28. Mazel M, Clair P, Rousselle C, Vidal P, Scherrmann JM, Mathieu D, Temsamani J (2001) Doxorubicin-peptide conjugates overcome multidrug resistance. Anti-Cancer Drug 12(2):107–116CrossRefGoogle Scholar
  29. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760CrossRefPubMedGoogle Scholar
  30. Meyer-Losic F, Quinonero J, Dubois V, Alluis B, Dechambre M, Michel M, Cailler F, Fernandez AM, Trouet A, Kearsey J (2006) Improved therapeutic efficacy of doxorubicin through conjugation with a novel peptide drug delivery technology (Vectocell). J Med Chem 49(23):6908–6916CrossRefPubMedGoogle Scholar
  31. Miklan Z, Orban E, Csik G, Schlosser G, Magyar A, Hudecz F (2009) New daunomycin-oligoarginine conjugates: synthesis, characterization, and effect on human leukemia and human hepatoma cells. Biopolymers 92(6):489–501CrossRefPubMedGoogle Scholar
  32. Milletti F (2012) Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17(15–16):850–860CrossRefPubMedGoogle Scholar
  33. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56(2):185–229CrossRefPubMedGoogle Scholar
  34. Nadas J, Sun DX (2006) Anthracyclines as effective anticancer drugs. Expert Opin Drug Dis 1(6):549–568CrossRefGoogle Scholar
  35. Nakase I, Konishi Y, Ueda M, Saji H, Futaki S (2012) Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug delivery in vivo. J Control Release 159(2):181–188CrossRefPubMedGoogle Scholar
  36. Orban E, Mezo G, Schlage P, Csik G, Kulic Z, Ansorge P, Fellinger E, Moller H, Manea M (2011) In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites. Amino Acids 41(2):469–483CrossRefPubMedGoogle Scholar
  37. Riganti C, Voena C, Kopecka J, Corsetto PA, Montorfano G, Enrico E, Costamagna C, Rizzo AM, Ghigo D, Bosia A (2011) Liposome-encapsulated doxorubicin reverses drug resistance by inhibiting P-glycoprotein in human cancer cells. Mol Pharm 8(3):683–700CrossRefPubMedGoogle Scholar
  38. Rottenberg S, Borst P (2012) Drug resistance in the mouse cancer clinic. Drug Resist Updat 15(1–2):81–89CrossRefPubMedGoogle Scholar
  39. Schlage P, Mezo G, Orban E, Bosze S, Manea M (2011) Anthracycline-GnRH derivative bioconjugates with different linkages: synthesis, in vitro drug release and cytostatic effect. J Control Release 156(2):170–178CrossRefPubMedGoogle Scholar
  40. Siegfried JM, Burke TG, Tritton TR (1985) Cellular-transport of anthracyclines by passive diffusion—implications for drug-resistance. Biochem Pharmacol 34(5):593–598CrossRefPubMedGoogle Scholar
  41. Sorkin A, von Zastrow M (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 3(8):600–614CrossRefPubMedGoogle Scholar
  42. Szabo I, Manea M, Orban E, Antal C, Bosze S, Szabo R, Tejeda M, Gaal D, Kapuvari B, Przybylski M, Hudecz F, Mezo G (2009) Development of an oxime bond containing daunorubicin-gonadotropin-releasing hormone-III conjugate as a potential anticancer drug. Bioconjugate Chem 20(4):656–665CrossRefGoogle Scholar
  43. Szakacs G, Hall MD, Gottesman MM, Boumendjel A, Kachadourian R, Day BJ, Baubichon-Cortay H, Di Pietro A (2014) Targeting the Achilles heel of multidrug-resistant cancer by exploiting the fitness cost of resistance. Chem Rev 114(11):5753–5774CrossRefPubMedPubMedCentralGoogle Scholar
  44. Tabujew I, Lelle M, Peneva K (2015) Cell-penetrating peptides for nanomedicine—how to choose the right peptide. Bionanomaterials 16(1):59–72CrossRefGoogle Scholar
  45. Tannock IF, Rotin D (1989) Acid Ph in tumors and its potential for therapeutic exploitation. Cancer Res 49(16):4373–4384PubMedGoogle Scholar
  46. Tew KD (1994) Glutathione-associated enzymes in anticancer drug-resistance. Cancer Res 54(16):4313–4320PubMedGoogle Scholar
  47. Vargas JR, Stanzl EG, Teng NNH, Wender PA (2014) Cell-penetrating, guanidinium-rich molecular transporters for overcoming efflux-mediated multidrug resistance. Mol Pharm 11(8):2553–2565CrossRefPubMedPubMedCentralGoogle Scholar
  48. Weiss RB (1992) The anthracyclines—will we ever find a better doxorubicin. Semin Oncol 19(6):670–686PubMedGoogle Scholar
  49. William Lown J (1993) Anthracycline and anthraquinone anticancer agents: current status and recent developments. Pharmacol Ther 60(2):185–214CrossRefGoogle Scholar
  50. Wu CP, Hsieh CH, Wu YS (2011) The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol Pharm 8(6):1996–2011CrossRefPubMedGoogle Scholar
  51. Ziegler A, Seelig J (2008) Binding and clustering of glycosaminoglycans: a common property of mono- and multivalent cell-penetrating compounds. Biophys J 94:2142–2149CrossRefPubMedGoogle Scholar
  52. Zorko M, Langel U (2005) Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev 57(4):529–545CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Max Planck Institute for Polymer ResearchMainzGermany
  2. 2.Institute of Organic and Macromolecular Chemistry, Jena Center of Soft MatterFriedrich Schiller University JenaJenaGermany
  3. 3.Institute of Physiology IIUniversity Hospital JenaJenaGermany

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