The AAPS Journal

, 11:445 | Cite as

Influence of the Molecular Weight of Bioreducible Oligoethylenimine Conjugates on the Polyplex Transfection Properties

  • Haijun YuEmail author
  • Verena Russ
  • Ernst Wagner
Research Article


The purpose of the present study was to investigate the influence of molecular weights on the chemical, biophysical, and biological properties of bioreducible oligoethylenimine conjugates. The cationic conjugates were synthesized by polyaddition between branched oligoethylenimine 800 Da (OEI) and the disulfide bond containing N,N′-cystamine bisacrylamide (CBA) linker. A correlation between the copolymer molecular weights and the polyplex transfection properties was found. The OEI–CBA copolymers differing in molecular weights (from 8.6 to 37 kDa) showed good plasmid DNA binding ability resulting in compact 90- to 150-nm-sized polyplexes. Colloidal stability of the polyplexes was lost in reductive environment. A low concentration of dithiothreitol of 5 µM was sufficient to render polyplexes unstable in size. Reducing conditions at physiological salt concentration triggered polyplex dissociation. The bioreducible polymers displayed much lower cytotoxicity (IC50 ∼ 100 μg/mL in cell culture) than branched polyethylenimine 25 kDa (BPEI) and linear polyethylenimine 22 kDa (LPEI). Reporter gene transfection experiments were done with CHO-K1 and B16-F10 cells. The largest (37 kDa) copolymer HC-6-8 demonstrated highest transfection levels among all the bioreducible copolymers, which was comparable with LPEI and much more effective than BPEI.

Key words

bioreducible gene delivery PEI polyplexes synthetic vectors 



Branched PEI 25 kDa


N,N′-cystamine bisacrylamide


Dynamic light scattering




Gel permeation chromatograph


High concentration


High molecular weight


Low concentration


Low molecular weight


Linear PEI 22 kDa


Molecular weight cutoff


Oligoethylenimine 800 Da


Plasmid DNA



We thank Ms Olga Brück for skilful assistance in preparing the manuscript, Ms. Terese Magnusson and Dr. Michael Günther for their assistance in FACS measurement. We acknowledge the financial support by DFG SPP1230, excellence cluster NIM and EC project GIANT.


  1. 1.
    Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther. 2008;15:1131–8.PubMedCrossRefGoogle Scholar
  2. 2.
    Ferrari S, Moro E, Pettenazzo A, Behr JP, Zacchello F, Scarpa M. ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 1997;4:1100–6.PubMedCrossRefGoogle Scholar
  3. 3.
    Bonnet ME, Erbacher P, Bolcato-Bellemin AL. Systemic delivery of DNA or siRNA mediated by linear polyethylenimine (L-PEI) does not induce an inflammatory response. Pharm Res. 2008;25:2972–82.PubMedCrossRefGoogle Scholar
  4. 4.
    Kichler A, Leborgne C, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med. 2001;3:135–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Sonawane ND, Szoka FC Jr, Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine–DNA polyplexes. J Biol Chem. 2003;278:44826–31.PubMedCrossRefGoogle Scholar
  6. 6.
    Kloeckner J, Wagner E, Ogris M. Degradable gene carriers based on oligomerized polyamines. Eur J Pharm Sci. 2006;29:414–25.PubMedCrossRefGoogle Scholar
  7. 7.
    Petersen H, Merdan T, Kunath K, Fischer D, Kissel T. Poly(ethylenimine-co-l-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery. Bioconjug Chem. 2002;13:812–21.PubMedCrossRefGoogle Scholar
  8. 8.
    Forrest ML, Koerber JT, Pack DW. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug Chem. 2003;14:934–40.PubMedCrossRefGoogle Scholar
  9. 9.
    Kloeckner J, Bruzzano S, Ogris M, Wagner E. Gene carriers based on hexanediol diacrylate linked oligoethylenimine: effect of chemical structure of polymer on biological properties. Bioconjug Chem. 2006;17:1339–45.PubMedCrossRefGoogle Scholar
  10. 10.
    Ahn CH, Chae SY, Bae YH, Kim SW. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release. 2002;80:273–82.PubMedCrossRefGoogle Scholar
  11. 11.
    Arote R, Kim TH, Kim YK, et al. A biodegradable poly(ester amine) based on polycaprolactone and polyethylenimine as a gene carrier. Biomaterials. 2007;28:735–44.PubMedCrossRefGoogle Scholar
  12. 12.
    Russ V, Elfberg H, Thoma C, Kloeckner J, Ogris M, Wagner E. Novel degradable oligoethylenimine acrylate ester-based pseudodendrimers for in vitro and in vivo gene transfer. Gene Ther. 2008;15:18–29.PubMedCrossRefGoogle Scholar
  13. 13.
    Lee Y, Mo H, Koo H, et al. Visualization of the degradation of a disulfide polymer, linear poly(ethylenimine sulfide), for gene delivery. Bioconjug Chem. 2007;18:13–8.PubMedCrossRefGoogle Scholar
  14. 14.
    Peng Q, Zhong Z, Zhuo R. Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors. Bioconjug Chem. 2008;19:499–506.PubMedCrossRefGoogle Scholar
  15. 15.
    Gosselin MA, Guo W, Lee RJ. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjug Chem. 2001;12:989–94.PubMedCrossRefGoogle Scholar
  16. 16.
    Wang Y, Chen P, Shen J. The development and characterization of a glutathione-sensitive cross-linked polyethylenimine gene vector. Biomaterials. 2006;27:5292–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Lin C, Zhong Z, Lok MC, et al. Novel bioreducible poly(amido amine) s for highly efficient gene delivery. Bioconjug Chem. 2007;18:138–45.PubMedCrossRefGoogle Scholar
  18. 18.
    Zhong Z, Song Y, Engbersen JF, Lok MC, Hennink WE, Feijen J. A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine) s. J Control Release. 2005;109:317–29.PubMedCrossRefGoogle Scholar
  19. 19.
    Hoon JJ, Christensen LV, Yockman JW, et al. Reducible poly(amido ethylenimine) directed to enhance RNA interference. Biomaterials. 2007;28:1912–7.CrossRefGoogle Scholar
  20. 20.
    Lin C, Blaauboer CJ, Timoneda MM, et al. Bioreducible poly(amido amine) s with oligoamine side chains: synthesis, characterization, and structural effects on gene delivery. J Control Release. 2008;126:166–74.PubMedCrossRefGoogle Scholar
  21. 21.
    Sun YX, Zeng X, Meng QF, Zhang XZ, Cheng SX, Zhuo RX. The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes. Biomaterials. 2008;29:4356–65.PubMedCrossRefGoogle Scholar
  22. 22.
    Saito G, Swanson JA, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Adv Drug Deliv Rev. 2003;55:199–215.PubMedCrossRefGoogle Scholar
  23. 23.
    Chen CP, Kim JS, Steenblock E, Liu D, Rice KG. Gene transfer with poly-melittin peptides. Bioconjug Chem. 2006;17:1057–62.PubMedCrossRefGoogle Scholar
  24. 24.
    Blacklock J, Handa H, Soundara MD, Mao G, Mukhopadhyay A, Oupicky D. Disassembly of layer-by-layer films of plasmid DNA and reducible TAT polypeptide. Biomaterials. 2007;28:117–24.PubMedCrossRefGoogle Scholar
  25. 25.
    Plank C, Zatloukal K, Cotten M, Mechtler K, Wagner E. Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjug Chem. 1992;3:533–9.PubMedCrossRefGoogle Scholar
  26. 26.
    Boeckle S, von Gersdorff K, van der Piepen S, Culmsee C, Wagner E, Ogris M. Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med. 2004;6:1102–11.PubMedCrossRefGoogle Scholar
  27. 27.
    Zou SM, Erbacher P, Remy JS, Behr JP. Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med. 2000;2:128–34.PubMedCrossRefGoogle Scholar
  28. 28.
    Chollet P, Favrot MC, Hurbin A, Coll JL. Side-effects of a systemic injection of linear polyethylenimine–DNA complexes. J Gene Med. 2002;4:84–91.PubMedCrossRefGoogle Scholar
  29. 29.
    Wagner E. The silent (R) evolution of polymeric nucleic acid therapeutics. Pharm Res. 2008;25:2920–3.PubMedCrossRefGoogle Scholar
  30. 30.
    Lynn DM, Langer R. Degradable poly(ß-amino esters): synthesis, characterization, and self-assembly with plasmid DNA. J Am Chem Soc. 2000;122:10761–8.CrossRefGoogle Scholar
  31. 31.
    Hong CY, You YZ, Wu DC, Liu Y, Pan CY. Thermal control over the topology of cleavable polymers: from linear to hyperbranched structures. J Am Chem Soc. 2007;129:5354–5.PubMedCrossRefGoogle Scholar
  32. 32.
    Breunig M, Lungwitz U, Liebl R, Goepferich A. Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci U S A. 2007;104:14454–9.PubMedCrossRefGoogle Scholar
  33. 33.
    Read ML, Singh S, Ahmed Z, et al. A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids. Nucleic Acids Res. 2005;33:e86.PubMedCrossRefGoogle Scholar
  34. 34.
    Lim YB, Kim SM, Suh H, Park JS. Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjug Chem. 2002;13:952–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Oupicky D, Parker AL, Seymour LW. Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors. J Am Chem Soc. 2002;124:8–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci U S A. 2005;102:5679–84.PubMedCrossRefGoogle Scholar
  37. 37.
    Mandel R, Ryser HJ, Ghani F, Wu M, Peak D. Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase. Proc Natl Acad Sci U S A. 1993;90(9):4112–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Feener EP, Shen WC, Ryser HJ. Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J Biol Chem. 1990;265:18780–5.PubMedGoogle Scholar
  39. 39.
    Neu M, Germershaus O, Mao S, Voigt KH, Behe M, Kissel T. Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice. J Control Release. 2007;118:370–80.PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2009

Authors and Affiliations

  1. 1.Pharmaceutical Biology–Biotechnology, Center of Drug Research, Department of Pharmacy, and Center for Nanoscience (CeNS)Ludwig-Maximilians UniversitätMunichGermany

Personalised recommendations