Skip to main content
SpringerLink
Log in

Partial Acetylation of Polyethylenimine Enhances In Vitro Gene Delivery

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose. Polyethylenimine (PEI) is a highly effective gene delivery vector, but because it is an off-the shelf material, its properties may not be optimal. To investigate the effects of the protonation properties of the polymer, we generated PEI derivatives by acetylating varying fractions of the primary and secondary amines to form secondary and tertiary amides, respectively.

Methods. Reaction of PEI with increasing amounts of acetic anhydride at 60°C for 4.5 h yielded polymers with 15%, 27%, and 43% of the primary amines modified with acetyl groups. Polymer-DNA complexes were characterized by dynamic light scattering and ζ potential measurements. Cytotoxicity of the polymers was assessed by XTT assay for metabolic activity, and gene delivery efficiency was determined as the relative expression of a luciferase gene in MDA-MB-231 and C2C12 cell lines.

Results. Acetylation of PEI decreased the “physiological buffering capacity,” defined as the moles of protons absorbed per mole of nitrogen on titration from pH 7.5 to 4.5, from 0.29 mol H+/mol N to 0.17 mol H+/mol N, 0.12 mol H+/mol N, and 0.090 mol H+/mol N for PEI-Ac15, PEI-Ac27, and PEI-Ac43, respectively. In addition, acetylation decreased the ζ potential of polyplexes from 14 mV to 8-11 mV and increased the polyplex diameter by two- to threefold. Surprisingly, acetylation had a negligible effect on cytotoxicity of the polymers and increased gene delivery effectiveness by up to 21-fold compared to unmodified PEI, both in the presence and absence of serum.

Conclusions. Reduction of the buffering capacity of PEI greatly enhanced the gene delivery activity of the polymer. The mechanism is not yet understood, but the enhancement may be caused by more effective polyplex unpackaging, altered endocytic trafficking, and/or increased lipophilicity of acetylated PEI-DNA complexes. Future studies will address these possibilities in more detail.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

references

  1. I. M. Verma and N. Somia. Gene therapy—promises, problems and prospects. Nature 389:239-242 (1997).

    Google Scholar 

  2. R. C. Mulligan. The basic science of gene therapy. Science 260:926-932 (1993).

    Google Scholar 

  3. Protocols by Vector. J. Gene Med. (accessed Apr. 7, 2003) http://www.wiley.co.uk/genetherapy/clinical/

  4. A. Rolland and P. L. Felgner. Non-viral gene delivery systems. Adv. Drug Deliv. Rev. 30:1-3 (1998).

    Google Scholar 

  5. D. W. Pack. Gene delivery polymers. In: J. I. Kroschwitz (ed.), Encyclopedia of Polymer Science and Technology. John Wiley & Sons, New York, 2001.

    Google Scholar 

  6. D. Luo and W. M. Saltzman. Synthetic DNA delivery systems. Nat. Biotechnol. 18:33-37 (2000).

    Google Scholar 

  7. M. E. Davis. Non-viral gene delivery systems. Curr. Opin. Biotechnol. 13:128-131 (2002).

    Google Scholar 

  8. G. Y. Wu and C. H. Wu. Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263:14621-14624 (1988).

    Google Scholar 

  9. E. Wagner, M. Zenke, M. Cotten, H. Beug, and M. L. Birnstiel. Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990).

    Google Scholar 

  10. 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 

  11. B. Abdallah, A. Hassan, C. Benoist, D. Goula, J.-P. Behr, and B. A. Demeneix. A powerful nonviral vector of in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7:1947-1954 (1996).

    Google Scholar 

  12. 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 

  13. D. M. Lynn, D. G. Anderson, D. Putnam, and R. Langer. Accelerated discovery of synthetic transfection vectors: parallel synthesis and screening of a degradable polymer library. J. Am. Chem. Soc. 123:8155-8156 (2001).

    Google Scholar 

  14. A. Kichler, C. Leborgne, E. Coeytaux, and O. Danos. Polyethylenimine-mediated gene delivery: a mechanistic study. J. Gene Med. 3:135-144 (2001).

    Google Scholar 

  15. M. L. Forrest and D. W. Pack. On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design. Mol. Ther. 6:57-66 (2002).

    Google Scholar 

  16. A. Akinc and R. Langer. Measuring the pH environment of DNA delivered using nonviral vectors: implications for lysosomal trafficking. Biotechnol. Bioeng. 78:503-508 (2002).

    Google Scholar 

  17. J.-P. Behr. The proton sponge: A trick to enter cells the viruses did not exploit. Chimia 51:34-36 (1997).

    Google Scholar 

  18. M. Lecocq, S. W.-D. Coninck, N. Laurent, R. Wattiaux, and M. Jadot. Uptake and intracellular fate of polyethylenimine in vivo. Biochem. Biophys. Res. Commun. 278:414-418 (2000).

    Google Scholar 

  19. M. Tang and J. F. C. Szoka. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 4:823-832 (1997).

    Google Scholar 

  20. W. T. Godbey, M. A. Barry, P. Saggau, K. K. Wu, and A. G. Mikos. Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J. Biomed. Mater. Res. 51:321-328 (2000).

    Google Scholar 

  21. D. Fischer, T. Bieber, Y. Li, H.-P. Elsässer, and T. Kissel. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16:1273-1279 (1999).

    Google Scholar 

  22. B. Brissault, A. Kichler, C. Guis, C. Leborgne, O. Danos, and H. Cheradame. Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconj. Chem. 14:581-587 (2003).

    Google Scholar 

  23. S.-M. Zou, P. Erbacher, J.-S. Remy, and J.-P. Behr. Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J. Gene Med. 2:128-134 (2000).

    Google Scholar 

  24. 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 

  25. D. Wang, A. S. Narang, M. Kotb, A. O. Gaber, D. D. Miller, S. W. Kim, and R. I. Mahato. Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery. Biomacromolecules 3:1197-1207 (2002).

    Google Scholar 

  26. D. Fischer, A. von Harpe, K. Kunath, H. Petersen, Y. Li, and T. Kissel. Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes. Bioconj. Chem. 13:1124-1133 (2002).

    Google Scholar 

  27. 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 

  28. M. G. Stevens and S. Olsen. Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity. J. Immunol. Methods 157:225-231 (1993).

    Google Scholar 

  29. D. D. Dunlap, A. Maggi, M. R. Soria, and L. Monaco. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res. 25:3095-3101 (1997).

    Google Scholar 

  30. G. Liu, M. Molas, G. A. Grossmann, M. Pasumarthy, J. C. Perales, M. J. Cooper, and R. W. Hanson. Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation. J. Biol. Chem. 276:34379-34387 (2001).

    Google Scholar 

  31. S. Choksakulnimitr, S. Masuda, H. Tokuda, Y. Takakura, and M. Hashida. In vitro cytotoxicity of macromolecules in different cell culture systems. J. Control. Rel. 34:233-241 (1995).

    Google Scholar 

  32. S.-O. Han, R. I. Mahato, and S. W. Kim. Water-soluble lipopolymer for gene delivery. Bioconj. Chem. 12:337-345 (2001).

    Google Scholar 

  33. D. Y. Furgeson, W. S. Chan, J. W. Yockman, and S. W. Kim. Modified linear polyethylenimine-cholesterol conjugates for DNA complexation. Bioconj. Chem. 14:840-847 (2003).

    Google Scholar 

  34. M. Lee, J. Rentz, S.-O. Han, D. A. Bull, and S. W. Kim. Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium. Gene Ther. 10:585-593 (2003).

    Google Scholar 

  35. M. B. Roufai and P. Midoux. Histidylated polylysine as DNA vector: elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes. Bioconj. Chem. 12:92-99 (2001).

    Google Scholar 

  36. M. Ogris, S. Brunner, S. Schüller, R. Kircheis, and E. Wagner. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6:595-605 (1999).

    Google Scholar 

  37. P. R. Dash, M. L. Read, K. D. Fisher, K. A. Howard, M. Wolfert, D. Oupicky, V. Subr, J. Strohalm, K. Ulbrich, and L. W. Seymour. Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin. J. Biol. Chem. 275:3793-3802 (2000).

    Google Scholar 

  38. I. R. Hill, M. C. Garnett, F. Bignotti, and S. S. Davis. In vitro cytotoxicity of poly(amidoamine)s: relevance to DNA delivery. Biochim. Biophys. Acta 1427:161-174 (1999).

    Google Scholar 

  39. P. Erbacher, A. C. Roche, M. Monsigny, and P. Midoux. The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes. Biochim. Biophys. Acta 1324:27-36 (1997).

    Google Scholar 

  40. M. G. Banaszczyk, C. P. Lollo, D. Y. Kwoh, A. T. Phillips, A. Amini, D. P. Wu, P. M. Mullen, C. C. Coffin, S. W. Brostoff, and D. J. Carlo. Poly-L-lysine-graft-PEG-comb-type polycation copolymers for gene delivery, J. Macromol. Sci. Pure Appl. Chem. 36:1061-1084 (1999).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, IL, 61801

    M. Laird Forrest, Glenna E. Meister, James T. Koerber & Daniel W. Pack

Authors
  1. M. Laird Forrest
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Glenna E. Meister
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James T. Koerber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel W. Pack
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel W. Pack.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Forrest, M.L., Meister, G.E., Koerber, J.T. et al. Partial Acetylation of Polyethylenimine Enhances In Vitro Gene Delivery. Pharm Res 21, 365–371 (2004). https://doi.org/10.1023/B:PHAM.0000016251.42392.1e

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:PHAM.0000016251.42392.1e

Search

Navigation