Partial Acetylation of Polyethylenimine Enhances In Vitro Gene Delivery

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

Research output: Contribution to journalArticlepeer-review

231 Scopus citations


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.0 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-Ac 43, 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.

Original languageEnglish
Pages (from-to)365-371
Number of pages7
JournalPharmaceutical Research
Issue number2
StatePublished - Feb 2004

Bibliographical note

Funding Information:
The work was supported by awards from the American Heart Association and the National Science Foundation (BES-0120101 and BES-0134163). In addition, acknowledgment is made to the donors of The Petroleum Research Fund, administered by the ACS, for partial support of this research. We thank Steven C. Zimmerman and Jennifer A. Lewis (University of Illinois, Urbana, IL) for the generous use of equipment and facilities.


  • Buffering capacity
  • Gene delivery
  • Polyethylenimine
  • Proton-sponge

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)


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