Pharmaceutical Research

, Volume 22, Issue 3, pp 373–380

Cross-linked Small Polyethylenimines: While Still Nontoxic, Deliver DNA Efficiently to Mammalian Cells in Vitro and in Vivo

  • Mini Thomas
  • Qing Ge
  • James J. Lu
  • Jianzhu Chen
  • Alexander Klibanov
Research Papers

No Heading

Purpose.

Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation’s effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.

Methods.

The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing β-galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs.

Results.

Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI.

Conclusions.

Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.

Key Words:

biodegradability COS-7 cells cross-linking cytotoxicity in vitro gene delivery in vivo gene delivery plasmid DNA polyethylenimine 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    1. L. M. Schwiebert. Cystic fibrosis, gene therapy, and lung inflammation: for better or worse? Am. J. Physiol. 286:L715–L716 (2004).Google Scholar
  2. 2.
    2. H. S. Kingdon and R. L. Lundblad. An adventure in biotechnology: the development of haemophilia A therapeutics - from whole-blood transfusion to recombinant DNA to gene therapy. Biotechnol. Appl. Biochem. 35:141–148 (2002).Google Scholar
  3. 3.
    3. C. J. Kuo, F. Farnebo, E. Y. Yu, R. Christofferson, R. A. Swearingen, R. Carter, H. A. von Recum, J. Yuan, J. Kamihara, E. Flynn, R. D’Amato, J. Folkman, and R. C. Mulligan. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer. Proc. Natl. Acad. Sci. USA 98:4605–4610 (2001).Google Scholar
  4. 4.
    4. C.-H. Lecellier and O. Voinnet. RNA silencing: no mercy for viruses? Immunol. Revs. 198:285–303 (2004).Google Scholar
  5. 5.
    5. A. J. Frater, S. J. Fidler, and M. O. McClure. Gene therapy for AIDS and other infectious diseases. Gene Ther. 9:189–213 (2002).Google Scholar
  6. 6.
    6. Q. Ge, L. Filip, A. Bai, N. Tam, H. N. Eisen, and J. Chen. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc. Natl. Acad. Sci. USA 101:8676–8681 (2004).Google Scholar
  7. 7.
    7. Gene therapy clinical trials worldwide provided by the Journal of Gene Medicine. http://www.wiley.co.uk/genmed/clinical/.Google Scholar
  8. 8.
    8. M. E. Davis. Non-viral gene delivery systems. Curr. Opin. Biotechnol. 13:128–131 (2002).Google Scholar
  9. 9.
    9. M. Thomas and A. M. Klibanov. Non-viral gene therapy: polycation-mediated DNA delivery. Appl. Microbiol. Biotechnol. 62:27–34 (2003).Google Scholar
  10. 10.
    10. C. M. Wiethoff and R. C. Middaugh. Barriers to nonviral gene delivery. J. Pharm. Sci. 92:203–217 (2003).Google Scholar
  11. 11.
    11. E. Check. Gene therapy: a tragic setback. Nature 420:116–118 (2002).Google Scholar
  12. 12.
    12. S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, M. P. McCormack, N. Wulffraat, P. Leboulch, A. Lim, C. S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P. Fraser, J.I. Cohen, G. de Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A. Aurias, D. Stoppa-Lyonnet, S. Romana, I. Radford-Weiss, F. Gross, F. Valensi, E. Delabesse, E. Macintyre, F. Siqaux, J. Soulier, L. E. Leiva, M. Wissler, C. Prinz, T. H. Rabbitts, F. Le Deist, A. Fischer, and M. Cavazzana-Calvo. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415–419 (2003).CrossRefPubMedGoogle Scholar
  13. 13.
    13. N. Boyce. Trial halted after gene shows up in semen. Nature 414:677–678 (2001).Google Scholar
  14. 14.
    14. G. Y. Wu and C. H. Wu. Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263:14621–14624 (1988).Google Scholar
  15. 15.
    15. O. Boussif, F. Lezoualc’h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, and J.-P. Behr. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92:7297–7301 (1995).Google Scholar
  16. 16.
    16. W. T. Godbey, K. K. Wu, and A. G. Mikos. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. USA 96:5177–5181 (1999).Google Scholar
  17. 17.
    17. M. Thomas and A. M. Klibanov. Enhancing polyethylenimine’s delivery of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. USA 99:14640–14645 (2002).Google Scholar
  18. 18.
    18. J. Suh, D. Wirtz, and J. Hanes. Efficient active transport of gene nanocarriers to the cell nucleus. Proc. Natl. Acad. Sci. USA 100:3878–3882 (2003).Google Scholar
  19. 19.
    19. N. D. Sonawane, F. C. Szoka Jr., and A. S. Verkman. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J. Biol. Chem. 278:44826–44831 (2003).Google Scholar
  20. 20.
    20. E. Wagner. Strategies to improve DNA polyplexes for in vivo gene transfer: will “artificial viruses” be the answer? Pharm. Res. 21:8–14 (2004).Google Scholar
  21. 21.
    21. A. Kirchler. Gene transfer with modified polyethylenimines. J. Gene Med. 6:S3–S10 (2004).Google Scholar
  22. 22.
    22. M. L. Forrest, G. E. Meister, J. T. Koerber, and D. W. Pack. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm. Res. 21:365–371 (2004).Google Scholar
  23. 23.
    23. Y.-B. Lim, S.-M. Kim, H. Suh, and J.-S. Park. Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and non-toxic gene delivery carrier. Bioconjug. Chem. 13:952–957 (2002).Google Scholar
  24. 24.
    24. A. Akinc, D. M. Lynn, D. G. Anderson, and R. Langer. Parallel synthesis and characterization of a degradable polymer library for gene delivery. J. Am. Chem. Soc. 125:5316–5323 (2003).Google Scholar
  25. 25.
    25. A. Kichler, C. Leborgne, J. Marz, O. Danos, and B. Bechinger. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc. Natl. Acad. Sci. USA 100:1564–1568 (2003).Google Scholar
  26. 26.
    26. Y. Liu, L. Wenning, M. Linch, and T. M. Reineke. New poly(D-glucaramidoamine)s induce DNA nanoparticle formation and efficient gene delivery into mammalian cells. J. Am. Chem. Soc. 126:7422–7423 (2004).Google Scholar
  27. 27.
    27. S.-O. Han, R. I. Mahato, and S. W. Kim. Water-soluble lipopolymer for gene delivery. Bioconjug. Chem. 12:337–345 (2001).Google Scholar
  28. 28.
    28. S. Kim, J. S. Choi, H. S. Jang, H. Suh, and J. Park. Hydrophobic modification of polyethyleneimine for gene transfectants. Bull. Korean Chem. Soc 22:1069–1075 (2001).Google Scholar
  29. 29.
    29. C. L. Gebhart, S. Sriadibhatla, S. Vinogradov, P. Lemieux, V. Alakhov, and A. V. Kabanov. Design and formulation of polyplexes based on pluronic-polyethylenimine conjugates for gene transfer. Bioconjug. Chem. 13:937–944 (2002).Google Scholar
  30. 30.
    30. N. Oku, Y. Yamazaki, M. Matsuura, M. Sugiyama, M. Hasegawa, and M. Nango. A novel non-viral gene transfer system, polycation liposomes. Adv. Drug Deliv. Revs. 52:209–218 (2001).Google Scholar
  31. 31.
    31. M. A. Gosselin, W. Guo, and R. J. Lee. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjug. Chem. 12:989–994 (2001).Google Scholar
  32. 32.
    32. H. Petersen, K. Kunath, A. L. Martin, S. Stolnik, C. J. Roberts, M. C. Davies, and T. Kissel. Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines. Biomacromolecules 3:926–936 (2002).Google Scholar
  33. 33.
    33. M. Thomas and A. M. Klibanov. Conjugation to gold nanoparticles enhances polyethylenimine’s transfer of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. USA 100:9138–9143 (2003).Google Scholar
  34. 34.
    34. J. Panyam and V. Labhasetwar. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55:329–347 (2003).Google Scholar
  35. 35.
    35. J. Wang, H.-Q. Mao, and K. W. Leong. A novel biodegradable gene carrier based on polyphosphoester. J. Am. Chem. Soc. 123:9480–9481 (2001).Google Scholar
  36. 36.
    36. H. Peterson, T. Merdan, K. Kunath, D. Fisher, and T. Kissel. Poly(ethylenimine-co-L-lactamide-co-succinimide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery. Bioconjug. Chem. 13:812–821 (2002).Google Scholar
  37. 37.
    37. C.-H. Ahn, S. Y. Chae, Y. H. Bae, and S. W. Kim. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J. Control. Rel. 80:273–282 (2002).Google Scholar
  38. 38.
    38. Y.-B. Lim, S.-O. Han, H.-U. Kong, Y. Lee, J.-S. Park, B. Jeong, and S. W. Kim. Biodegradable polyester, poly[(α-(4-aminobutyl)-L-glycolic acid], as a non-toxic gene carrier. Pharm. Res. 17:811–816 (2000).Google Scholar
  39. 39.
    39. Y.-B. Lim, Y. H. Choi, and J.-S. Park. A self-destroying polycationic polymer: biodegradable poly(4-hydroxy-L-proline ester). J. Am. Chem. Soc. 121:5633–5639 (1999).Google Scholar
  40. 40.
    40. M. L. Forrest, J. T. Koerber, and D. W. Pack. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug. Chem. 14:934–940 (2003).Google Scholar
  41. 41.
    41. A. von Harpe, H. Petersen, Y. Li, and T. Kissel. Characterization of commercially available and synthesized polyethylenimines for gene delivery. J. Control. Rel. 69:309–322 (2000).Google Scholar
  42. 42.
    42. Y. Tan and L. Huang. Overcoming the inflammatory toxicity of cationic gene vectors. J. Drug Target 10:153–160 (2002).Google Scholar
  43. 43.
    43. D. V. Schaffer, N. A. Fidelman, N. Dan, and D. A. Lauffenburger. Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol. Bioeng. 67:598–606 (2000).Google Scholar
  44. 44.
    44. C. Moon, Y. Oh, and J. A. Roth. Current status of gene therapy for lung cancer and head and neck cancer. Clin. Cancer Res. 9:5055–5067 (2003).Google Scholar
  45. 45.
    45. A. C. Willis and X. Chen. The promise and obstacle of p53 as a cancer therapeutic agent. Curr. Mol. Med. 2:329–345 (2002).Google Scholar
  46. 46.
    46. Y. Zhang, T. Li, L. Fu, C. Yu, Y. Li, X. Xu, Y. Wang, H. Ning, S. Zhang, W. Chen, L. A. Babiuk, and Z. Chang. Silencing SARS-CoV spike protein expression in cultured cells by RNA interference. FEBS Lett. 560:141–146 (2004).Google Scholar
  47. 47.
    47. D. J. Weiss. Delivery of gene transfer vectors to lung: obstacles and the role of adjunct techniques for airway administration. Mol. Ther. 6:148–152 (2002).Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Mini Thomas
    • 1
  • Qing Ge
    • 2
  • James J. Lu
    • 2
  • Jianzhu Chen
    • 2
  • Alexander Klibanov
    • 1
  1. 1.Department of Chemistry and Division of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Center for Cancer Research and Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations