Cell Compatible Arginine Containing Cationic Polymer: One-Pot Synthesis and Preliminary Biological Assessment

  • Nino ZavradashviliEmail author
  • Tamar Memanishvili
  • Nino Kupatadze
  • Lucia Baldi
  • Xiao Shen
  • David Tugushi
  • Christine Wandrey
  • Ramaz Katsarava
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 807)


Synthetic cationic polymers are of interest as both nonviral vectors for intracellular gene delivery and antimicrobial agents. For both applications synthetic polymers containing guanidine groups are of special interest since such kind of organic compounds/polymers show a high transfection potential along with antibacterial activity. It is important that the delocalization of the positive charge of the cationic group in guanidine significantly decreases the toxicity compared to the ammonium functionality. One of the most convenient ways for incorporating guanidine groups is the synthesis of polymers composed of the amino acid arginine (Arg) via either application of Arg-based monomers or chemical modification of polymers with derivatives of Arg. It is also important to have biodegradable cationic polymers that will be cleared from the body after their function as transfection or antimicrobial agent is fulfilled. This chapter deals with a two-step/one-pot synthesis of a new biodegradable cationic polymer—poly(ethylene malamide) containing l-arginine methyl ester covalently attached to the macrochains in β–position of the malamide residue via the α-amino group. The goal cationic polymer was synthesized by in situ interaction of arginine methyl ester dihydrochloride with intermediary poly(ethylene epoxy succinimide) formed by polycondensation of di-p-nitrophenyl-trans-epoxy succinate with ethylenediamine. The cell compatibility study with Chinese hamster ovary (CHO) and insect Schneider 2 cells (S2) within the concentration range of 0.02–500 mg/mL revealed that the new polymer is not cytotoxic. It formed nanocomplexes with pDNA (120–180 nm in size) at low polymer/DNA weight ratios (WR = 5–10). A preliminarily transfection efficiency of the Arg-containing new cationic polymer was assessed using CHO, S2, H5, and Sf9 cells.


l-arginine Polyamides Biodegradable polycations pDNA Complex formation Cytotoxicity 



This study was supported by the Swiss National Science Foundation (SNF), grants SCOPES IZ73Z0_128071 and SCOPES IZ76ZO_147554. We thank Mrs. P. Toidze for DLS measurements.


  1. 1.
    Luo D, Saltzman WM (2000) Synthetic DNA delivery systems. Nat Biotechnol 18:33–37CrossRefGoogle Scholar
  2. 2.
    Ryser H, Hancock R (1965) Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 150:501–503CrossRefGoogle Scholar
  3. 3.
    Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56(5):318–325CrossRefGoogle Scholar
  4. 4.
    Calnan BJ, Tidor B, Biancalana S, Hudson D, Frankel AD (1991) Arginine-mediated RNA recognition: the arginine fork. Science 252:1167–1171CrossRefGoogle Scholar
  5. 5.
    Rothbard JB, Garlington S, Lin Q, Kirschberg T, Kreider E, Wender PA, Khavari PA (2000) Conjugation of arginine oligomers to cyclosporin a facilitates topical delivery and inhibition of inflammation. Nat Med 6:1253–1257CrossRefGoogle Scholar
  6. 6.
    Holowka EP, Sun VZ, Kamei DT, Deming TJ (2007) Polyarginine segments in block copolypeptides drive both vesicular assembly and intracellular delivery. Nat Mater 6:52–57CrossRefGoogle Scholar
  7. 7.
    Rothbard JB, Jessop TC, Wender PA (2005) Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells. Adv Drug Deliv Rev 57:495–504CrossRefGoogle Scholar
  8. 8.
    Futaki S (2005) Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv Drug Deliv Rev 57:547–558CrossRefGoogle Scholar
  9. 9.
    Brooks H, Lebleu B, Vives E (2005) Tat peptide-mediated cellular delivery: back to basics. Adv Drug Deliv Rev 57:559–577CrossRefGoogle Scholar
  10. 10.
    Torchilin VP (2006) Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng 8:343–375CrossRefGoogle Scholar
  11. 11.
    Yamanouchi D, Wu J, Lazar AN, Kent KC, Chu CC, Liu B (2008) Biodegradable arginine-based poly(ester-amide)s as non-viral gene delivery reagents. Biomaterials 29(22):3269–3277CrossRefGoogle Scholar
  12. 12.
    Weber E, Keana J (1993) N, N’—disubstituted guanidines and their use as excitatory amino acid antagonists. US. Patents 3,252,816 (1990); 5,190,976 (1993)Google Scholar
  13. 13.
    Graham JD, Lain-Yen H, Sharad M (1999) Therapeutic substituted guanidines. US Patent 5,922,772Google Scholar
  14. 14.
    Goldin SM (1997) Substituted adamantyl guanidines and methods of use there of US patent No.5,637623Google Scholar
  15. 15.
    Manetti F, Castagnolo D, Raffi F, Zizzari AT, Rajamäki S, D’Arezzo S, Visca P, Cona A, Fracasso ME, Doria D, Posteraro B, Sangui netti M, Fadda G, Botta M (2009) Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp. J Chem Med 52(23):7376–7379Google Scholar
  16. 16.
    Hensler ME, Bernstein G, Nizet V, Nefzi A (2006) Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant gram-positive pathogens identified from a mixture-based combinatorial library. Bioorg Med Chem Lett 16(19):5073–5079CrossRefGoogle Scholar
  17. 17.
    Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55(1):27–55CrossRefGoogle Scholar
  18. 18.
    Zasloff M (1992) Antibiotic peptides as mediators of innate immunity. Curr Opin Immunol 4:3–7CrossRefGoogle Scholar
  19. 19.
    Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature (London) 415:389–395Google Scholar
  20. 20.
    Sitaram N, Nagaraj R (2002) Host-defense antimicrobial peptides: importance of structure for activity. Curr Pharm Des 8(9):727–742CrossRefGoogle Scholar
  21. 21.
    Hancock RE, Chapple DS (1999) Peptide antibiotics. Antimicrob Agents Chemother 43:1317–1323Google Scholar
  22. 22.
    Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88CrossRefGoogle Scholar
  23. 23.
    Sivov NS (2006) Biocide guanidine containing polymers: synthesis, structure and properties. CRC Press, Taylor & Francis Group, Boca ratonGoogle Scholar
  24. 24.
    Kasymova GF, Burichenko VK, Meitus ÉE, Poroshin KT (1973) Lysine- and arginine-containing polypeptides of regular structure and their bactericidal properties. Chem Nat Compd 9(1):79–81CrossRefGoogle Scholar
  25. 25.
    Lu H, Zhang S, Wang B, Cui S, Yan JJ (2006) Inhibition of protein kinase C by cationic amphiphiles. J Controll Rel 114(1):100–109CrossRefGoogle Scholar
  26. 26.
    Bottega R, Epand RM (1992) Inhibition of protein kinase C by cationic amphiphiles. Biochemistry 31(37):9025–9030CrossRefGoogle Scholar
  27. 27.
    Yingyongnarongkul B, Howarth M, Elliott T, Bradley M (2004) Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells. Chem Eur J 10(2):463–473CrossRefGoogle Scholar
  28. 28.
    Katsarava R, Tugushi D, Torchilin VP, Memanishvili T, Gverdtsiteli M, Kupatadze N (2010) Biodegradable l-Arginine based cationic poly(Ether-Ester-Amide)s, poly(Ether-Ester-Urethanes) and poly(Ether-Ester-Ureas) and methods of synthesis thereof. Georgian patent application, AP2009011558. (Positive decision)Google Scholar
  29. 29.
    Memanishvili T, Kupatadze N, Tugushi D, Torchilin VP, Katsarava R (2010) Biodegradable arginine-based polymers with PEG-like backbones as potential non-viral gene delivery system. In: 1st Russian-hellenic symposium with international participation and Yong’s scientist school “Biomaterials and bionanomaterials: recent advances and safety-toxicology issues”, Iraklion, 3–9 May 2010Google Scholar
  30. 30.
    Memanishvili T, Bedinashvili M, Tugushi D, Torchilin VP, Katsarava R (2010) The Study on the cytotoxicity of new biodegradable Arginine-based poly(Ether-Esther-Urethane cation)s on 4T1 cells. Georgian Eng News 4(56):93–98Google Scholar
  31. 31.
    Memanishvili T, Zavradashvili N, Kupatadze N, Tugushi D, Gverdtsiteli M, Torchilin VP, Wandrey C, Baldi L, Manoli SS, Katsarava R. Arginine-based biodegradable ether-ester polymers of low cytotoxicity as potential gene carriers (submitted to Biomacromolecules).Google Scholar
  32. 32.
    Vert M (2009) Degradable and bioresorbable polymers in surgery and in pharmacology: beliefs and facts. J Mater Sci Mater Med 20:437–446CrossRefGoogle Scholar
  33. 33.
    Lynn DM, Langer R (2000) Degradable poly(β-amino esters): synthesis, characterization, and self-assembly with plasmid DNA. J Am Chem Soc 122:10761–10768CrossRefGoogle Scholar
  34. 34.
    Akinc A, Lynn DM, Anderson DG, Langer R (2003) Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery. J Am Chem Soc 125:5316–5323CrossRefGoogle Scholar
  35. 35.
    Forrest M, Koerber J, Pack D (2003) A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug Chem 14:934–940CrossRefGoogle Scholar
  36. 36.
    Cohen T, Lipowitz J (1964) Acid catalyzed amide hydrolysis assisted by neighboring amide group. J Amer Chem Soc 86:5611–5616CrossRefGoogle Scholar
  37. 37.
    Zavradashvili N, Jokhadze G, Gverdtsiteli M, Otinashvili G, Kupatadze N, Gomurashvili Z, Tugushi D, Katsarava R (2013) Amino acid based epoxy-poly(Ester Amide)s—a new class of functional biodegradable polymers: synthesis and chemical transformations. J Macromol Sci Part A Pure Appl Chem 50(5):449–465CrossRefGoogle Scholar
  38. 38.
  39. 39.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398CrossRefGoogle Scholar
  40. 40.
    Kadlecova Z, Baldi L, Hacker D, Wurm FM, Klok HA (2012) Comparative study on the in vitro cytotoxicity of linear, dendritic, and hyperbranched polylysine analogues. Biomacromolecules 13(10):3127–3137CrossRefGoogle Scholar
  41. 41.
    Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM (2011) A simple high-yielding process for transient gene expression in CHO cells. J Biotechnol 153:22–26CrossRefGoogle Scholar
  42. 42.
    Shen X, Michel PO, Xie Q, Hacker DL (2011) Wurm FW (2011) Transient transfection of insect Sf-9 cells in TubeSpin® bioreactor 50 tubes. BMC Proc 5(suppl 8):P37CrossRefGoogle Scholar

Copyright information

© Springer India 2014

Authors and Affiliations

  • Nino Zavradashvili
    • 1
    Email author
  • Tamar Memanishvili
    • 1
  • Nino Kupatadze
    • 1
  • Lucia Baldi
    • 2
  • Xiao Shen
    • 2
  • David Tugushi
    • 1
  • Christine Wandrey
    • 2
  • Ramaz Katsarava
    • 1
  1. 1.Institute of Chemistry and Molecular EngineeringAgricultural University of GeorgiaTbilisiGeorgia
  2. 2.Ecole Polytechnique Federale de LausanneLausanneSwitzerland

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