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Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells


Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by 30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection.





Dulbecco’s modified Eagle’s MEM medium


fetal bovine serum




HEPES-buffered saline


head and neck squamous cell carcinoma

HSV-tk :

Herpes Simplex Virus thymidine kinase gene


oral squamous cell carcinoma


relative light units


  1. 1.

    Goepfert, H. Squamous cell carcinoma of the head and neck: past progress and future promise. CA Cancer J. Clin. 48 (1998) 195–198.

  2. 2.

    Parkin, D.M., Pisani, P. and Ferlay, J. Global cancer statistics. CA Cancer J. Clin. 49 (1999) 33–64.

  3. 3.

    Harras, A., Edwards, B.K., Blot, W.J. and Ries, L.A. Cancer Rates and Risks. National Institutes of Health, Bethesda, MD, (1996) NIH Publication No. 96-691.

  4. 4.

    Hong, W.K., Bromer, R.H., Amato, D.A., Shapsky, S., Vincent, M., Vaughan, C., Willett, B., Katz, A., Welch, J., Fotonoff, S., et al. Patterns of relapse in locally advanced head and neck cancer patients who achieved complete remission after combined modality therapy. Cancer 56 (1985) 1242–1245.

  5. 5.

    Silverman, S. Jr. Oral cancer: complications of therapy. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 88 (1999) 122–126.

  6. 6.

    Shillitoe, E.J. Gene therapy for oral cancer: recent progress in research. Oral Oncol. 34 (1998) 157–160.

  7. 7.

    O’Malley, B.W. and Couch, M.E. Gene therapy principles and strategies for head and neck cancer. Adv. Otorhinolaryngol. 56 (2000) 279–288.

  8. 8.

    Xi, S. and Grandis, J.R. Gene therapy for the treatment of oral squamous cell carcinoma. J. Dent. Res. 82 (2003) 11–16.

  9. 9.

    Simões, S., Pires, P., Düzgünes, N. and Pedroso de Lima, M.C. Cationic liposomes as gene transfer vectors: Barriers to successful application in gene therapy. Curr. Opin. Mol. Ther. 1 (1999) 147–157.

  10. 10.

    Pedroso de Lima, M.C., Simões, S., Pires, P., Faneca, H. and Düzgünes, N. Cationic lipid-DNA complexes in gene delivery: From biophysics to biological applications. Adv. Drug Deliv. Rev. 47 (2001) 277–294.

  11. 11.

    Zuber, G., Dauty, E., Nothisen, M., Belguise, P. and Behr, J.-P. Towards synthetic viruses. Adv. Drug Deliv. Rev. 52 (2001) 245–253.

  12. 12.

    Nabel, G.J., Nabel, E.G., Yang, Y.Y., Fox, B.A., Plautz, G.E., Gao, X., Huang, L., Shu, S., Gordon, D. and Chang, A.E. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biological activity, and lack of toxicity in humans. Proc. Natl. Acad. Sci. USA 90 (1993) 11307–11311.

  13. 13.

    Zhu, N., Liggitt, D., Liu, Y. and Debs, R. Systemic gene expression after intravenous DNA delivery into adult mice. Science 261 (1993) 209–211.

  14. 14.

    Liu, Y., Mounkes, L.C., Liggitt, H.D., Brown, C.S., Solodin, I., Heath, T.D. and Debs, R.J. Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat. Biotechnol. 15 (1997) 167–173.

  15. 15.

    Templeton, N.S., Lasic, D.D., Frederik, P.M., Strey, H.H., Roberts, D.D. and Pavlakis, G.N. (1997) Improved DNA:liposome complexes for increased systemic delivery and gene expression. Nat. Biotechnol. 15 (1997) 647–652.

  16. 16.

    Xu, L., Pirollo, K.F., Tang, W.H., Rait, A. and Chang, E.H. Transferrin liposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck cancer xenografts. Hum. Gene Ther. 10 (1999) 2941–2952.

  17. 17.

    Simões, S., Slepushkin, V., Pires, P., Gaspar, R., Pedroso de Lima, M.C. and Düzgünes, N. Enhanced gene delivery by lipoplexes associated with human serum albumin. Biochim. Biophys. Acta 1463 (2000) 459–469.

  18. 18.

    Vitadello, M., Schiaffino, M., Picard, A., Scarpa, M. and Schiaffino, S. Gene transfer in regenerating muscle. Hum. Gene Ther. 5 (1994), 11–18.

  19. 19.

    Zabner, J., Fasbender, A.J., Moninger, T., Poellinger, K.A. and Welsch, M.J. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270 (1995) 18997–19007.

  20. 20.

    Mortimer, I., Tam, P., MacLachlan, I., Graham, R.W., Sravolec, E.G. and Joshi, P. Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther. 6 (1999) 403–411.

  21. 21.

    O’Malley, B.W., Cope, K.A., Chen, S.-H., Li, D., Schwartz, M.R. and Woo, S.L.C. Combination gene therapy for oral cancer in a murine model. Cancer Res. 56 (1996) 1737–1741.

  22. 22.

    O’Malley, B.W., Cope, K.A., Johnson, C.S. and Schwartz, M.R. A new immunocompetent murine model for oral cancer. Arch. Otolaryngol. Head Neck Surg. 123 (1997) 20–24.

  23. 23.

    O’Malley, B.W., Sewell, D.A., Li, D., Kosai, K., Chen, S.H., Woo, S.L. and Duan, L. The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer. Mol. Endocrinol. 11 (1997) 667–673.

  24. 24.

    Day, K.V., Li, D., Liu, S., Guo, M. and O’Malley, B.W. Granulocytemacrophage colony-stimulating factor in a combination gene therapy strategy for head and neck cancer. Laryngoscope 111 (2001) 801–806.

  25. 25.

    Li, D., Shugert, E., Guo, M., Bishop, J.S. and O’Malley Jr., B.W. Combination nonviral interleukin 2 and interleukin 12 gene therapy for head and neck squamous cell carcinoma. Arch. Otolaryngol. Head Neck Surg. 127 (2001) 1319–1324.

  26. 26.

    Konopka, K., Overlid, N. and Düzgünes, N. Efficient, serum-resistant transfection of murine squamous cell carcinoma cells by Metafectene and GeneJammer: Application to HSV-tk/ganciclovir gene therapy. 8th Meeting of the Am. Soc. Gene Ther., St. Louis, MO, 2005, Mol. Ther. Vol. 11(Suppl.1) Abstr. 590, S228.

  27. 27.

    Fu, K.K., Rayner, P.A. and Lam, K.N. Modification of the effects of continuous low dose irradiation by concurrent chemotherapy infusion. Int. J. Radiat. Oncol. Biol. Phys. 10 (1984) 1473–1478.

  28. 28.

    Konopka, K., Fallah, B., Monzon-Duller, J., Overlid, N. and Düzgünes, N. Serum-resistant gene transfer to oral cancer cells by Metafectene and GeneJammer: Application to HSV-tk/ganciclovir-mediated cytotoxicity. Cell. Mol. Biol. Lett. 10 (2005) 455–470.

  29. 29.

    Konopka, K., Lee, A., Moser-Kim, N., Saghezchi, S., Kim, A., Lim, A., Suzara, V.V. and Düzgünes, N. Gene transfer to human oral cancer cells via non-viral vectors and HSV-tk/ganciclovir-mediated cytotoxicity; Potential for suicide gene therapy. Gene Ther. Mol. Biol. 8 (2004) 307–318.

  30. 30.

    Fields, R.D. and Lancaster, M.V. Dual-attribute continuous monitoring of cell proliferation/cytotoxicity. Am. Biotechnol. Lab. 11 (1993) 48–50.

  31. 31.

    Konopka, K., Pretzer, E., Felgner, P.L. and Düzgünes, N. Human immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes. Biochim. Biophys. Acta 1312 (1996) 186–196.

  32. 32.

    Escriou, V., Ciolina, C., Lacroix, F., Byk, G., Scherman, D. and Wils, P. Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fate of lypopolyamine/DNA complexes. Biochim. Biophys. Acta 1368 (1998) 276–288.

  33. 33.

    Zelphati, O., Uyechi, L.S., Barron, L.G. and Szoka, F.C. Jr. Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim. Biophys. Acta 1390 (1998) 119–133.

  34. 34.

    Yang, J-P. and Huang, L. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther. 4 (1997) 950–960.

  35. 35.

    Yang, J-P. and Huang, L. Time-dependent maturation of cationic liposome-DNA complex for serum resistance. Gene Ther. 5 (1998) 380–387.

  36. 36.

    Audouy, S., Molema, G., de Leij, L. and Hoekstra, D. Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. J. Gene Med. 2 (2000) 465–476.

  37. 37.

    Almofti, M.R., Harashima, H., Shinohara, Y., Almofti, A., Li, W. and Kiwada, H. Lipoplex size determines lipofection efficiency with or without serum. Mol. Membr. Biol. 20 (2003) 35–43.

  38. 38.

    Zhang, Y. and Anchordoquy, T.J. The role of lipid charge density in the serum stability of cationic lipid/DNA complexes. Biochim. Biophys. Acta 1663 (2004) 143–157.

  39. 39.

    Gebhart, C.L. and Kabanov, A.V. Evaluation of polyplexes as gene transfer agents. J. Control. Release. 73 (2001) 401–416.

  40. 40.

    Uchida, E., Mizuguchi, H., Ishii-Watabe, A. and Hayakawa, T. Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells. Biol. Pharm. Bull. 25 (2002) 891–8977.

  41. 41.

    Li, W., Ishida, T., Tachibana, R., Almofti, M.R., Wang, X. and Kiwada, H. Cell-type specific gene expression, mediated by TFL-3, a cationic liposomal vector, is controlled by a post-transcription process of delivered plasmid DNA. Int. J. Pharm. 276 (2004) 67–74.

  42. 42.

    Almofti, M.R., Harashima, H., Shinohara, Y., Almofti, A., Baba, Y. and Kiwada, H. Cationic liposome-mediated gene delivery: Biophysical study and mechanism of internalization. Arch. Biochem. Biophys. 419 (2003) 246–253.

  43. 43.

    Faneca, H., Simões, S. and Pedroso de Lima, M.C. Evaluation of lipid-based reagents to mediate intracellular gene delivery. Biochim. Biophys. Acta 1567 (2002) 23–33.

  44. 44.

    Li, S., Tseng, W.C., Stolz, D.B., Wu, S.P., Watkins, S.C. and Huang, L. Dynamic changes in the characteristics of cationic lipid vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther. 4 (1999) 585–594.

  45. 45.

    Tandia, B-M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J-M. and Elouahabi, A. Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency: implications for intravenous gene therapy. Mol. Ther. 8 (2003) 264–273.

  46. 46.

    Tandia, B-M., Lonez, C., Vandenbranden, M., Ruysschaert, J-M. and Elouahabi, A. Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids. J. Biol. Chem. 280 (2005) 12255–12261.

  47. 47.

    Ross, P.C. and Hui, S.W. Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther. 6 (1999) 651–659.

  48. 48.

    Faneca, H., Simões, S. and Pedroso de Lima, M.C. Association of albumin and protamine to lipoplexes: enhancement of transfection and resistance to serum. J. Gene. Med. 6 (2004) 681–692.

  49. 49.

    Pires, P., Simões, S., Nir, S., Gaspar, R., Düzgünes, N. and Pedroso de Lima, M.C. Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. Biochim. Biophys. Acta 1418 (1999) 71–84.

  50. 50.

    Goebel, E.A., Davidson, B.L., Zabner, J., Graham, S.M. and Kern, J.A. Adenovirus-mediated gene therapy for head and neck squamous cell carcinomas. Ann. Otol. Rhinol. Laryngol. 105 (1996) 562–567.

  51. 51.

    Goebel, E.A., Davidson, B.L., Graham, S.M. and Kern, J.A. Tumor reduction in vivo after adenoviral mediated gene transfer of the herpes simplex virus thymidine kinase gene and ganciclovir treatment in human head and neck squamous cell carcinoma. Otolaryngol. Head Neck Surg. 119 (1998) 331–336.

  52. 52.

    Fukui, T., Hayashi, Y., Kagmi, H., Yamamoto, N., Fukuhara, H., Tohnai, I., Ueda, M., Mizuno, M. and Yoshida, J. Suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector. Oral Oncol. 37 (2001) 211–215.

  53. 53.

    Fukuhara, H., Hayashi, Y., Yamamoto, N., Fukui, T., Nishikawa, M., Mitsudo, K., Tohnai, I., Ueda, M., Mizuno, M. and Yoshida, J. Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines. Oral Oncol. 39 (2003) 601–609.

  54. 54.

    Sewell, D.A., Li, D., Duan, L., Schwartz, M.R. and O’Malley Jr., B.W. Optimizing suicide gene therapy for head and neck cancer. Laryngoscope 107 (1997) 1490–1495.

  55. 55.

    Sewell, D.A., Li, D., Duan, L., Westra, W.H. and O’Malley Jr., B.W. Safety of in vivo adenovirus-mediated thymidine kinase treatment of oral cancer. Arch. Otolaryngol. Head Neck Surg. 123 (1997) 1298–1302.

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Correspondence to Krystyna Konopka.

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Konopka, K., Overlid, N., Nagaraj, A.C. et al. Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells. Cell. Mol. Biol. Lett. 11, 171–190 (2006).

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Key words

  • Transfection
  • Metafectene
  • GeneJammer
  • Serum inhibition
  • HSV-tk gene
  • SCCVII murine squamous cell carcinoma cells