Pharmaceutical Research

, Volume 22, Issue 6, pp 972–980 | Cite as

N4,N9-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

  • Osama A. A. Ahmed
  • Noppadon Adjimatera
  • Charareh Pourzand
  • Ian S. Blagbrough
Research Paper


To study the effect of synthesized N4,N9-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.


The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.


The synthesized N4,N9-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/− ammonium/phosphate) 2.5.


N4,N9-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.

Key words

FEK4 gene delivery lipopolyamine N4,N9-dioleoyl spermine transfection 









enhanced green fluorescent protein


Earle’s minimal essential medium


ethidium bromide


fetal calf serum




nuclear pore complex







We acknowledge the financial support of the Egyptian Government studentship to O.A.A. A. and Universities U.K. for an ORS award to N. A. We are grateful to Prof R.M. Tyrrell (University of Bath) for FEK4 and HtTA cell lines.


  1. 1.
    Friedmann, T., Roblin, R. 1972Gene therapy for human genetic disease?Science175949955PubMedGoogle Scholar
  2. 2.
    Felgner, P. L., Barenholz, Y., Behr, J. P., Cheng, S. H., Cullis, P., Huang, L., Jessee, J. A., Seymour, L., Szoka, F., Thierry, A. R., Wagner, E., Wu, G. 1997Nomenclature for synthetic gene delivery systemsHum. Gene Ther.8511512PubMedGoogle Scholar
  3. 3.
    Verma, I. M., Somia, N. 1997Gene therapy—promises, problems and prospectsNature389239242CrossRefPubMedGoogle Scholar
  4. 4.
    Barr, D., Tubb, J., Ferguson, D., Scaria, A., Lieber, A., Wilson, C., Perkins, J., Kay, M. A. 1995Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in-vivo—comparisons between immunocompetent and immunodeficient inbred strainsGene Ther.2151155PubMedGoogle Scholar
  5. 5.
    Bishop, J. M. 1987The molecular-genetics of cancerScience235305311PubMedGoogle Scholar
  6. 6.
    Donahue, R. E., Kessler, S. W., Bodine, D., Mcdonagh, K., Dunbar, C., Goodman, S., Agricola, B., Byrne, E., Raffeld, M., Moen, R., Bacher, J., Zsebo, K. M., Nienhuis, A. W. 1992Helper virus-induced T-cell lymphoma in nonhuman-primates after retroviral mediated gene-transferJ. Exp. Med.17611251135PubMedGoogle Scholar
  7. 7.
    Russell, S. J., Brandenburger, A., Flemming, C. L., Collins, M. K. L., Rommelaere, J. 1992Transformation-dependent expression of interleukin genes delivered by a recombinant parvovirusJ. Virol.6628212828PubMedGoogle Scholar
  8. 8.
    Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., Danielsen, M. 1987Lipofection—a highly efficient, lipid-mediated DNA- transfection procedureProc. Natl. Acad. Sci. USA8474137417PubMedGoogle Scholar
  9. 9.
    Smedt, S. C., Demeester, J., Hennink, W. E. 2000Cationic polymer based gene delivery systemsPharm. Res.17113126CrossRefPubMedGoogle Scholar
  10. 10.
    Noguchi, A., Hirashima, N., Nakanishi, M. 2002Cationic cholesterol promotes gene transfection using the nuclear localization signal in protaminePharm. Res.19933938PubMedGoogle Scholar
  11. 11.
    Wagner, E. 2004Strategies to improve DNA polyplexes for in vivo gene transfer: will “artificial viruses” be the answer?Pharm. Res.21814PubMedGoogle Scholar
  12. 12.
    Cryan, S. A., O’Driscoll, C. M. 2003Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loopPharm. Res.20569575PubMedGoogle Scholar
  13. 13.
    Wiethoff, C. M., Middaugh, R. 2003Barriers to nonviral gene deliveryJ. Pharm. Sci.92203217PubMedGoogle Scholar
  14. 14.
    Behr, J. P. 1994Gene-transfer with synthetic cationic amphiphiles— prospects for Gene-therapyBioconjug. Chem.5382389PubMedGoogle Scholar
  15. 15.
    Remy, J. S., Sirlin, C., Vierling, P., Behr, J. P. 1994Gene-transfer with a series of lipophilic DNA-binding moleculesBioconjug. Chem.5647654PubMedGoogle Scholar
  16. 16.
    Bloomfield, V. A. 1997DNA condensation by multivalent cationsBiopolymers44269282PubMedGoogle Scholar
  17. 17.
    Blessing, T., Remy, J. S., Behr, J. P. 1998Monomolecular collapse of plasmid DNA into stable virus-like particlesProc. Natl. Acad. Sci. USA9514271431PubMedGoogle Scholar
  18. 18.
    Montigny, W. J., Houchens, C. R., Illenye, S., Gilbert, J., Coonrod, E., Chang, Y. C., Heintz, N. H. 2001Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cellsNucleic Acids Res.2919821988PubMedGoogle Scholar
  19. 19.
    Radler, J. O., Koltover, I., Salditt, T., Safinya, C. R. 1997Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimesScience275810814PubMedGoogle Scholar
  20. 20.
    Niidome, T., Ohmori, N., Ichinose, A., Wada, A., Mihara, H., Hirayama, T., Aoyagi, H. 1997Binding of cationic alpha-helical peptides to plasmid DNA and their gene transfer abilities into cellsJ. Biol. Chem.2721530715312PubMedGoogle Scholar
  21. 21.
    P L. Felgner, C. N. Sridhar, C. J. Wheeler, and J. Felgner. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Cell Biochem. 206 (1993).Google Scholar
  22. 22.
    Tseng, W. C., Huang, L. 1998Liposome-based gene therapyPharm. Sci. Technol. Today1206213Google Scholar
  23. 23.
    Farhood, H., Serbina, N., Huang, L. 1995The role of dioleoyl phosphatidylethanolamine in cationic liposome-mediated gene-transferBiochim. Biophys. Acta, Biomembr.1235289295Google Scholar
  24. 24.
    Behr, J. P., Demeneix, B., Loeffler, J. P., Mutul, J. P. 1989Efficient gene-transfer into mammalian primary endocrine-cells with lipopolyamine-coated DNAProc. Natl. Acad. Sci. USA8669826986PubMedGoogle Scholar
  25. 25.
    Byk, G., Dubertret, C., Escriou, V., Frederic, M., Jaslin, G., Rangara, R., Pitard, B., Crouzet, J., Wils, P., Schwartz, B., Scherman, D. 1998Synthesis, activity, and structure-activity relationship studies of novel cationic lipids for DNA transferJ. Med. Chem.41224235Google Scholar
  26. 26.
    Buchberger, B., Fernholz, E., v.d.Eltz, H., Hinzepeter, M. 1996DOSPER liposomal transfection reagent: a reagent with unique transfection propertiesBiochem. Inform.982729Google Scholar
  27. 27.
    Blagbrough, I. S., Geall, A. J., Neal, A. P. 2003Polyamines and novel polyamine conjugates interact with DNA in ways that can be exploited in non-viral gene therapyBiochem. Soc. Trans.31397406PubMedGoogle Scholar
  28. 28.
    Geall, A. J., Eaton, M. A. W., Baker, T., Catterall, C., Blagbrough, I. S. 1999The regiochemical distribution of positive charges along cholesterol polyamine carbamates plays significant roles in modulating DNA binding affinity and lipofectionFEBS Lett.459337342PubMedGoogle Scholar
  29. 29.
    Feuerstein, B. G., Williams, L. D., Basu, H. S., Marton, L. J. 1991Implications and concepts of polyamine-nucleic acid interactionsJ. Cell. Biochem.463747PubMedGoogle Scholar
  30. 30.
    A. J. Geall, D. Al-Hadithi, and I. S. Blagbrough. Spermine and thermine conjugates of cholic acid condense DNA, but lithocholic acid polyamine conjugates do so more efficiently. Chem. Commun. 2035–2036 (1998).Google Scholar
  31. 31.
    Heyes, J. A., Niculescu-Duvaz, D., Cooper, R. G., Springer, C. J. 2002Synthesis of novel cationic lipids: effect of structural modification on the efficiency of gene transferJ. Med. Chem.4599114PubMedGoogle Scholar
  32. 32.
    G. Ronsin, C. Perrin, P. Guedat, A. Kremer, P. Camilleri, and A. J. Kirby. Novel spermine-based cationic gemini surfactants for gene delivery. Chem Commun. 2234–2235 (2001).Google Scholar
  33. 33.
    Tranchant, I., Thompson, B., Nicolazzi, C., Mignet, N., Scherman, D. 2004Physicochemical optimisation of plasmid delivery by cationic lipidsJ. Gene Med.6S24S35PubMedGoogle Scholar
  34. 34.
    Lima, M. C. P., Neves, S., Filipe, A., Duzgunes, N., Simoes, S. 2003Cationic liposomes for gene delivery: from biophysics to biological applicationsCurr. Med. Chem.1012211231PubMedGoogle Scholar
  35. 35.
    Blagbrough, I. S., Geall, A. J. 1998Practical synthesis of unsymmetrical polyamine amidesTetrahedron Lett.39439442Google Scholar
  36. 36.
    Geall, A. J., Blagbrough, I. S. 2000Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate-DNA interactions for the analysis of lipoplex formation in gene therapyJ. Pharm. Biomed. Anal.22849859PubMedGoogle Scholar
  37. 37.
    Tyrrell, R. M., Pidoux, M. 1986Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsyCancer Res.4626652669PubMedGoogle Scholar
  38. 38.
    Fischer, D., Bieber, T., Li, Y. X., Elsasser, H. P., Kissel, T. 1999A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicityPharm. Res.1612731279PubMedGoogle Scholar
  39. 39.
    Wu, J. Y., Wu, C. H. 1987Receptor-mediated in vitro gene transformation by a soluble DNA carrier systemJ. Biol. Chem.26244294432PubMedGoogle Scholar
  40. 40.
    Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., Behr, J. P. 1995A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimineProc. Natl. Acad. Sci. USA9272977301PubMedGoogle Scholar
  41. 41.
    Benns, J. M., Mahato, R. I., Kim, S. W. 2002Optimization of factors influencing the transfection effeciency of folate-PEG-folate-graft-polyethylenimineJ. Control. Release79255269PubMedGoogle Scholar
  42. 42.
    Kunath, K., Harpe, A., Fischer, D., Peterson, H., Bickel, U., Voigt, K., Kissel, T. 2003Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high- molecular-weight polyethylenimineJ. Control. Release89113125PubMedGoogle Scholar
  43. 43.
    Remy, J. S., Abdallah, B., Zanta, M. A., Boussif, O., Behr, J. P., Demeneix, B. 1998Gene transfer with lipospermines and polyethyleniminesAdv. Drug Deliv. Rev.308595PubMedGoogle Scholar
  44. 44.
    Gosule, L. C., Schellman, J. A. 1976Compact form of DNA induced by spermidineNature259333335PubMedGoogle Scholar
  45. 45.
    Wilson, R. W., Bloomfield, V. A. 1979Counterion-induced condesation of deoxyribonucleic acid. a light-scattering studyBiochemistry1821922196PubMedGoogle Scholar
  46. 46.
    Basu, H. S., Marton, L. J. 1987The interaction of spermine and pentamines with DNABiochem. J.244243246PubMedGoogle Scholar
  47. 47.
    Gosule, L. C., Schellman, J. A. 1978DNA condensation with polyamines I. Spectroscopic studiesJ. Mol. Biol.121311326PubMedGoogle Scholar
  48. 48.
    Mosmann, T. 1983Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assaysJ. Immunol. Methods655563PubMedGoogle Scholar
  49. 49.
    Lu, Q. L., Bou-Gharios, G., Partridge, T. A. 2003Non-viral gene delivery in skeletal muscle: a protein factoryGene Ther.10131142PubMedGoogle Scholar
  50. 50.
    Niidome, T., Huang, L. 2002Gene therapy progress and prospects: nonviral vectorsGene Ther.916471652PubMedGoogle Scholar
  51. 51.
    Liu, L., Zern, M. A., Lizarzaburu, M. E., Nantz, M. H., Wu, J. 2003Poly(cationic lipid)-mediated in vivo gene delivery to mouse liverGene Ther.10180187PubMedGoogle Scholar
  52. 52.
    Faneca, H., Simoes, S., Lima, M. C. P. 2002Evaluation of lipid-based reagents to mediate intracellular gene deliveryBiochim. Biophys. Acta, Biomembr.15672333Google Scholar
  53. 53.
    Pantazatos, S. P., MacDonald, R. C. 2003Real-time observation of lipoplex formation and interaction with anionic bilayer vesiclesJ. Membr. Biol.19199112PubMedGoogle Scholar
  54. 54.
    Templeton, N. S. 2002Liposomal delivery of nucleic acids in vivoDNA Cell Biol.21857867PubMedGoogle Scholar
  55. 55.
    Trubetskoy, V. S., Wong, S. C., Subbotin, V., Budker, V. G., Loomis, A., Hagstrom, J. E., Wolff, J. A. 2003Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene deliveryGene Ther.10261271PubMedGoogle Scholar
  56. 56.
    Simoes, S., Fonseca, C., Faneca, H., Duzgunes, N., Lima, M. C. P. 2002Protein-associated lipoplexes: novel strategies to enhance gene delivery mediated by lipid-based particlesSTP Pharma Sci.12339344Google Scholar
  57. 57.
    Dauty, E., Remy, J. S., Blessing, T., Behr, J. P. 2001Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in cultureJ. Am. Chem. Soc.12392279234PubMedGoogle Scholar
  58. 58.
    LePecq, J. B., Paoletti, C. 1967A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterizationJ. Mol. Biol.2787106PubMedGoogle Scholar
  59. 59.
    Niculescu-Duvaz, D., Heyes, J., Springer, C. J. 2003Structure-activity relationship in cationic lipid mediated gene transfectionCurr. Med. Chem.1012331261PubMedGoogle Scholar
  60. 60.
    Fabio, K., Gaucheron, J., Giorgio, C., Vierling, P. 2003Novel galactosylated polyamine bolaamphiphiles for gene deliveryBioconjug. Chem.14358367PubMedGoogle Scholar
  61. 61.
    Xu, Y., Szoka, F. C.,Jr 1996Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfectionBiochemistry3556165623PubMedGoogle Scholar
  62. 62.
    Vijayanathan, V., Thomas, T., Shirahata, A., Thomas, T. J. 2001DNA condensation by polyamines: a laser light scattering study of structural effectsBiochemistry401364413651PubMedGoogle Scholar
  63. 63.
    Vijayanathan, V., Thomas, T., Thomas, T. J. 2002DNA nanoparticles and development of DNA delivery vehicles for gene therapyBiochemistry411408514094PubMedGoogle Scholar
  64. 64.
    Zhou, X. H., Huang, L. 1994DNA transfection mediated by cationic liposomes containing lipopolylysine – characterization and mechanism of actionBiochim. Biophys. Acta, Biomembr.1189195203Google Scholar
  65. 65.
    Blagbrough, I. S., Adjimatera, N., Ahmed, O. A. A., Neal, A. P., Pourzand, C. 2004Spermine and lipopolyamines as gene delivery agentsBeadle, D. J.Mellor, I. R.Usherwood, P. N. R. eds. Neurotox ‘03: Neurotoxicological targets from functional genomics and proteomicsSCILondon147159Google Scholar
  66. 66.
    Coulberson, A. L., Hud, N. V., Ledoux, J. M., Vilfan, I. D., Prausnitz, M. R. 2003Gene packaging with lipids, peptides and viruses inhibits transfection by electroporation in vitroJ. Control. Release86361370PubMedGoogle Scholar
  67. 67.
    Weyermann, J., Lochmann, D., Zimmer, A. 2004Comparison of antisense oligonucleotide drug delivery systemsJ. Control. Release100411423PubMedGoogle Scholar
  68. 68.
    Sakurai, F., Inoue, R., Nishino, Y., Okuda, A., Matsumoto, O., Taga, T., Yamashita, F., Takakura, Y., Hashida, M. 2000Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expressionJ. Control. Release66255269PubMedGoogle Scholar
  69. 69.
    Bell, H., Kimber, W. L., Li, M., Whittle, I. R. 1998Liposomal transfection efficiency and toxicity on glioma cell lines: in vitro and in vivo studiesNeuroreport9793798PubMedGoogle Scholar
  70. 70.
    Wu, J., Lizarzaburu, M. E., Kurth, M. J., Liu, L., Wege, H., Zern, M. A., Nantz, M. H. 2001Cationic lipid polymerization as a novel approach for constructing new DNA delivery agentsBioconjug. Chem.12251257PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Osama A. A. Ahmed
    • 1
  • Noppadon Adjimatera
    • 1
  • Charareh Pourzand
    • 1
  • Ian S. Blagbrough
    • 1
  1. 1.Department of Pharmacy and PharmacologyUniversity of BathBathUnited Kingdom

Personalised recommendations