Thermally sensitive polypeptide-based copolymer for DNA complexation into stable nanosized polyplexes

  • Emilya Ivanova
  • Ivaylo DimitrovEmail author
  • Rahila Kozarova
  • Sevdalina Turmanova
  • Margarita Apostolova
Research Paper


Gene therapy based on non-viral synthetic delivery vectors has attracted much attention in the past two decades. However, it is still in clinical trial stages, mainly due to the lack of safe and efficient delivery vehicles. Herein, we report on the synthesis and DNA complexation ability of novel, hybrid copolymer comprising poly(N-isopropylacrylamide) (PNIPAm) block with poly(ethylene glycol) (PEG) side chains and a polycationic block of poly(l-lysine) (PLLys). The copolymer was synthesized in a two-step procedure. In the first step, a thermally sensitive PNIPAm-g-PEG copolymer with terminal ammonium hydrochloride group was prepared. The second step involves controlled ring-opening polymerization of Z-l-lysine N-carboxyanhydride initiated by the PNIPAm-g-PEG macroinitiator. The hybrid copolymer obtained show high ability to condense DNA into stable polyplexes with sizes below 100 nm. Cytotoxicity evaluation of both hybrid copolymer and its polyplex with DNA indicates that it might be a good candidate for gene-delivery applications.


DNA Polyplexes Stimuli-sensitive polymers Synthesis 



This study was financially supported by the Bulgarian National Science Fund through a project “Ideas” DO 02-247/2008.

Supplementary material

11051_2012_1358_MOESM1_ESM.doc (98 kb)
Electronic supplementary material (DOC 98 kb)


  1. Baum C, Kustikova O, Modlich U, Li Z, Fehze B (2006) Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors. Hum Gene Ther 17:253–263CrossRefGoogle Scholar
  2. Check E (2002) Gene therapy: a tragic setback. Nature 420:116–118CrossRefGoogle Scholar
  3. Cho YW, Kim Y-D, Park K (2003) Polycation gene delivery systems: escape from endosomes to cytosol. J Pharm Pharmacol 55:721–734CrossRefGoogle Scholar
  4. Choi YH, Liu F, Kim J-S, Choi YK, Park YS, Kim SW (1998) Polyethylene glycol-grafted poly-l-lysine as polymeric gene carrier. J Control Release 54:39–48CrossRefGoogle Scholar
  5. Dash PR, Read ML, Barrett LB, Wolfert MA, Seymour LW (1999) Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Ther 6:643–650CrossRefGoogle Scholar
  6. de Ilarduya CT, Sun Y, Düzgüneş N (2010) Gene delivery by lipoplexes and polyplexes. Eur J Pharm Sci 40:159–170CrossRefGoogle Scholar
  7. Dimitrov I, Schlaad H (2003) Synthesis of nearly monodisperse polystyrene-polypeptide block copolymers via polymerisation of N-carboxyanhydrides. Chem Commun 23:2944–2945CrossRefGoogle Scholar
  8. Dimitrov I, Berlinova I, Vladimirov N (2006) Synthesis of poly(oxyethylene)-poly(Z-l-lysine) hybrid graft copolymers. Macromolecules 39:2423–2426CrossRefGoogle Scholar
  9. Dimitrov I, Trzebicka B, Müller AHE, Dworak A, Tsvetanov C (2007) Thermosensitive water-soluble copolymers with doubly responsive reversibly interacting entities. Prog Polym Sci 32:1275–1343CrossRefGoogle Scholar
  10. Dimitrov I, Berlinova I, Iliev P, Vladimirov N (2008) Controlled synthesis of peptide-based amphiphilic copolymers. Macromolecules 41:1045–1049CrossRefGoogle Scholar
  11. Dimitrov I, Petrova E, Kozarova R, Apostolova M, Tsvetanov C (2011) A mild and versatile approach for DNA encapsulation. Soft Matter 7:8002–8004CrossRefGoogle Scholar
  12. Du F-S, Wang Y, Zhang R, Li Z-C (2010) Intelligent nucleic acid delivery systems based on stimuli-responsive polymers. Soft Matter 6:835–848CrossRefGoogle Scholar
  13. Gil ES, Hudson SM (2004) Stimuli-responsive polymers and their bioconjugates. Prog Polym Sci 29:1173–1222CrossRefGoogle Scholar
  14. Grigsby CL, Leong KW (2010) Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery. J R Soc Interface 7:S67–S82CrossRefGoogle Scholar
  15. Grisham J (2000) Inquiry into gene therapy widens. Nat Biotechnol 18:254–255CrossRefGoogle Scholar
  16. Hinrichs WLJ, Schuurmans-Nieuwenbroek NME, van de Wetering P, Hennink WE (1999) Thermosensitive polymers as carriers for DNA delivery. J Control Release 60:249–259CrossRefGoogle Scholar
  17. Itaka K, Harada A, Nakamura K, Kawaguchi H, Kataoka K (2002) Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions. Biomacromolecules 3:841–845CrossRefGoogle Scholar
  18. Kakizawa Y, Kataoka K (2002) Block copolymer micelles for delivery of gene and related compounds. Adv Drug Deliv Rev 54:203–222CrossRefGoogle Scholar
  19. Karmali PP, Chaudhuri A (2007) Cationic liposomes as non-viral carriers of gene medicines: resolved issues, open questions, and future promises. Med Res Rev 27:696–722CrossRefGoogle Scholar
  20. Kurisawa M, Yokoyama M, Okano T (2000) Gene expression control by temperature with thermo-responsive polymeric gene carriers. J Control Release 69:127–137CrossRefGoogle Scholar
  21. Lavigne MD, Pennadam SS, Ellis J, Yates LL, Alexander C, Górecki DC (2007) Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors. J Gene Med 9:44–54CrossRefGoogle Scholar
  22. LePecq J-B, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical–chemical characterization. J Mol Biol 27:87–106CrossRefGoogle Scholar
  23. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  24. O’Rorke S, Keeney M, Pandit A (2010) Non-viral polyplexes: scaffold mediated delivery for gene therapy. Prog Polym Sci 35:441–458CrossRefGoogle Scholar
  25. Oupický D, Reschel T, Koňak Č, Oupická L (2003) Temperature-controlled behavior of self-assembly gene delivery vectors based on complexes of DNA with poly(l-lysine)-graft-poly(N-isopropylacrylamide). Macromolecules 36:6863–6872CrossRefGoogle Scholar
  26. Poché D, Moore M, Bowles J (1999) An unconventional method for purifying the N-carboxyanhydride derivatives of γ-alkyl-l-glutamates. Synth Commun 29:843–854CrossRefGoogle Scholar
  27. Roques C, Fattal E, Fromes Y (2009) Comparison of toxicity and transfection efficiency of amphiphilic block copolymers and polycationic polymers in striated muscles. J Gene Med 11:240–249CrossRefGoogle Scholar
  28. Schaffer DV, Fidelman NA, Dan N, Lauffenburger DA (2000) Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol Bioeng 67:598–606CrossRefGoogle Scholar
  29. Shen Z, Shi B, Zhang H, Bi J, Dai S (2012) Exploring low-positively charged thermosensitive copolymers as gene delivery vectors. Soft Matter 8:1385–1394CrossRefGoogle Scholar
  30. Soliman M, Allen S, Davies MC, Alexander C (2010) Responsive polyelectrolyte complexes for triggered release of nucleic acid therapeutics. Chem Commun 46:5421–5433CrossRefGoogle Scholar
  31. Trentin D, Hubbell J, Hall H (2005) Non-viral gene delivery for local and controlled DNA release. J Control Release 102:263–275CrossRefGoogle Scholar
  32. Vinogradov SV, Bronich TK, Kabanov AV (1998) Self-assembly of polyamine-poly(ethylene glycol) copolymers with phosphorothioate oligonucleotides. Bioconjug Chem 9:805–812CrossRefGoogle Scholar
  33. Wong SY, Pelet JM, Putnam D (2007) Polymer systems for gene delivery—past, present, and future. Prog Polym Sci 32:799–837CrossRefGoogle Scholar
  34. Zintchenko A, Ogris M, Wagner E (2006) Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer. Bioconjug Chem 17:766–772CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Emilya Ivanova
    • 1
    • 3
  • Ivaylo Dimitrov
    • 1
    Email author
  • Rahila Kozarova
    • 2
  • Sevdalina Turmanova
    • 3
  • Margarita Apostolova
    • 2
  1. 1.Institute of Polymers, Bulgarian Academy of SciencesSofiaBulgaria
  2. 2.Institute of Molecular Biology, Bulgarian Academy of SciencesSofiaBulgaria
  3. 3.Department of Materials ScienceProf. Assen Zlatarov UniversityBurgasBulgaria

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