title: Partial acetylation of polyethylenimine enhances in vitro gene delivery.
abstract: OBJECTIVE
  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 degrees 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 zeta 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+/mot 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 zeta 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, IL 61801, USA.
authors.forename: M Laird
authors.initials: ML
authors.lastname: Forrest
authors.forename: Glenna E
authors.initials: GE
authors.lastname: Meister
authors.forename: James T
authors.initials: JT
authors.lastname: Koerber
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Pack
mesh_terms: D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry* / Q000633:toxicity; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000089:Acetic Anhydrides; C031800:acetic anhydride; D011094:Polyethyleneimine; D004247:DNA; D008156:Luciferases