Modified Polyethylenimine: Self Assemble Nanoparticle Forming Polymer for pDNA Delivery

Document Type : Original Article


1 Department of Biotechnology, Pharmaceutical and Biotechnology Research Centers, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Medicinal Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN 55455, USA


Polyethylenimine (PEI), a readily available synthetic polycation which has high transfection efficiency owing to its buffering capacity was introduced for transfection a few years ago. But it has been reported that PEI is cytotoxic in many cell lines. In this study, in order to enhance the transfection efficiency of 10 kDa PEI and reduce its toxicity, hydrophobic residues were grafted on PEI.
Materials and Methods
PEI polymers were modified by adding hydrophobic chains to the primary amines of PEI in different degrees of grafting using bromoacetic acid derivatives with different lengths. These polymers were complexed with plasmid DNA at different C/P ratios and the resulting nanoparticles were characterized by dynamic light scattering and EtBr-DNA binding assay to determine particle sizes and complex formation, respectively. Cytotoxicity and transfection efficiency of the polymers were also tested in cultured Neuro2a cell line.
DNA condensation measurement revealed that the resulted polymers could form polyplexes with plasmid DNA and they have the ability to condense DNA in relatively low amounts of polymers. Particle size measurement of polyplexes showed that they form particles in the size range of below 190 nm. Transfection experiments showed that polymers which have been modified with hexanoic derivative could transfect pDNA as good as 25 kDa PEI with the advantage of being much less toxic.
Results indicate that the structure modifications of PEI accomplished in this study play a significant role in increasing the transfection efficiency and without inducing the cytotoxicity compared to PEI itself.


1. Cavazzano-Calvo M, Thrasher A, Mavilio F. The future of gene therapy. Nature 2004; 427: 779-781.
2. Forrest LM, Meister EG, Koerber J, Pack D. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm Res 2004; 21: 365-371.
3. Lungwitz U, Breunig MT, Blunk Gopferich A. Polyethylenimine-based non-viral gene delivery systems. Eur J Pharm Biopharm 2005; 247-266.
4. El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release 2004; 94: 1-14.
5. Lehrman S. Virus treatment questioned after gene therapy death. Nature 1999; 401:517-518.
6. Liu Q, Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 2003; 10: 935-940.
7. Sun JY, Anand-Jawa V, Chatterjee S, Wong K K. Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther 2003; 10:964-976.
8. Merdan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv Drug Deliver Rev 2002; 715-758.
9. Zhang S, Xu Y, Wang B, Qiao W, Liu D, Li Z. Cationic compounds used in lipoplexes and polyplexes for gene delivery. J Control Release 2004; 100:165-180.
10. Boussif O, Lezoualc’h F, Zanta MA. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995; 92:7297–7301.
11. Cho YW, Kim J, Park K. Polycation gene delivery systems: escape from endosomes to cytosol. J Pharm Pharmacol 2003; 55:721-734.
11. Kursa M, Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R, et al. Novel shielded transferrin–polyethylene glycol–polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer. Bioconjugate Chem 2003; 14: 222-231.
12. Kircheis R, Schuller S, Brunner S, Ogris M, Heide KH, Zauner W, et al. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med 1999; 1:111-120.
13. Ogris M, Walker G, Blessing T, Kircheis R, Wolschek M, Wagner E. Tumor-targeted gene therapy: strategies for the preparation of ligand–polyethylene glycol–polyethylenimine/DNA complexes. J Control Release 2003; 91:173-181.
14.  Kircheis R, Wightman L, Schreiber A, Robitza B, Rossler V, Kursa M, et al. Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Ther 2001; 8:28-40.
15. Tseng WC, Jong CM. Improved stability of polycationic vector by dextran-grafted branched polyethylenimine. Biomacromolecules 2003; 4:1277-1284.
16. Kim S, Choi JS, Jang HS, Suh H, Park J. Hydrophobic modification of polyethylenimine for gene transfectants. Bull Korean Chem Soc 201; 22:1069-1075.
17. Thomas M, Klibanov AM. Non-viral gene therapy: polycation mediated DNA delivery. Appl Microbiol Biotechnol 2003; 62:27-3.
18. Thomas M, Klibanov AM. Enhancing polyethylenimine’s delivery of plasmid DNA into mammalian cells. Proc Natl Acad Sci USA 2002; 99:14640–14645.
19. Brownlie A, Uchegbu A, Schatzlein AG. PEI-based vesicle-polymer hybrid gene delivery system with improved biocompatibility. Int J Pharm 2004; 274:41–52.
20. Snyder SL, Sobocinski PZ. An improved 2, 4, 6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem 1975;64:284-288.
21. Kichler A, Leborgne Ch, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3:135-144.
22. Liu D, Ren T, Gao X. Cationic transfection lipids. Curr Med Chem 2003; 10: 1307-1315.
23. Neu M, Fischer D, Kissel T. Recent advances in rational gene transfer vector design based on poly (ethylene imine) and its derivatives. J Gene Med 2005; 7:992–1009.
24. Doody AM, Korley JN, Dang KP, Zawaneh PN, Putnam D. Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro. J Control Release 2006; 116:227-237.
25. Chen DJ, Majors BS, Zelikin A, Putnam D. Structure–function relationships of gene delivery vectors in a limited polycation library. J Control Release 2005; 103:273-283.
26. Gabrielson NP, Pack DW. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules 2006; 7:2427-2435.
27. Fischer D, Bieber T, Li Y, Elsasser HP, Kissel T. 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 1999; 16:1273-1279.
28. Godbey WT, Wu KK, Mikos AG. Size matters: molecular weight affects the efficiency of poly (ethylenimine) as a gene delivery vehicle. J Biomed Mater Res 1999; 45: 268-275.
29. Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A two-stage poly (ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 2005; 11:990-995.
30. Hunter AC. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Adv Drug Deliver Rev 2006; 58:1523-1531.
31. Thomas M, Lu JJ, Ge O, 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 USA 2005; 102:5679-5684.