Applied Biochemistry and Biotechnology

, Volume 177, Issue 1, pp 1–17 | Cite as

PEGylation of Polypropylenimine Dendrimer with Alkylcarboxylate Chain Linkage to Improve DNA Delivery and Cytotoxicity

  • Maryam Hashemi
  • Sara Ayatollahi
  • Hamideh Parhiz
  • Ahad Mokhtarzadeh
  • Soheila Javidi
  • Mohammad RamezaniEmail author


One of the major limitations of effective nonviral gene carriers is their potential high cytotoxicity. Conjugation of polyethylene glycol (PEG) to polymers is a common approach to decrease toxicity and improve biodistribution. The aim of this study was to evaluate the effect of PEGylation on generation 5 polypropylenimine (PPI) dendrimer by using PEG moieties or alkyl-PEG groups. Polymers were synthesized by grafting of 5 and 10 % primary amines of PPI to NH2–PEG–COOH or Br–(CH2)9–CO–NH–PEG–COOH through Amide bond formation or nucleophilic substitution, respectively. Transfection efficiency and cytotoxicity were analyzed after 4 and 24 h exposure of neuroblastoma cell line (Neuro-2a) with synthesized vectors. Among all of the PEG-PPI derivatives, 5 % PEG-conjugated G5 PPI with alkyl chain (PPI-alkyl-PEG 5 %) resulted in the most efficient gene expression. This vector also significantly decreased the in vitro cytotoxicity and sub-G1 peak in flow cytometry histogram after 24 h incubation. Our results indicate that modification of 5 % primary amines of G5 PPI with PEG using alkyl chain as linker produces a promising vector combining low cytotoxicity, appropriate biodegradability, and high gene transfection efficiency.


Alkyl chain Cytotoxicity Polypropylenimine (PPI) PEG Gene delivery 



This study was funded by Mashhad University of Medical Sciences, Mashhad, Iran.

Conflict of Interest

The authors declare that they have no competing interests.


  1. 1.
    Hacein-Bey-Abina, S., Hauer, J., Lim, A., Picard, C., Wang, G. P., Berry, C. C., Martinache, C., Rieux-Laucat, F., Latour, S., & Belohradsky, B. H. (2010). Efficacy of gene therapy for X-linked severe combined immunodeficiency. New England Journal of Medicine, 363, 355–364.CrossRefGoogle Scholar
  2. 2.
    Tuszynski, M. H., Thal, L., Pay, M., & Salmon, D. P. (2005). A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nature Medicine, 11, 551–555.CrossRefGoogle Scholar
  3. 3.
    Mannucci, P. M., & Tuddenham, E. G. (2001). The hemophilias—from royal genes to gene therapy. New England Journal of Medicine, 344, 1773–1779.CrossRefGoogle Scholar
  4. 4.
    Lee, T., Matthews, D., & Blair, G. (2005). Novel molecular approaches to cystic fibrosis gene therapy. The Biochemical Journal, 387, 1–15.CrossRefGoogle Scholar
  5. 5.
    Yamada, M., Mizuno, Y., & Mochizuki, H. (2005). Parkin gene therapy for α-synucleinopathy: a rat model of Parkinson’s disease. Human Gene Therapy, 16, 262–270.CrossRefGoogle Scholar
  6. 6.
    Li, S., & Huang, L. (2000). Nonviral gene therapy: promises and challenges. Gene Therapy, 7, 31–34.CrossRefGoogle Scholar
  7. 7.
    Luten, J., van Nostrum, C. F., De Smedt, S. C., & Hennink, W. E. (2008). Biodegradable polymers as nonviral carriers for plasmid DNA delivery. Journal of Controlled Release, 126, 97–110.CrossRefGoogle Scholar
  8. 8.
    Mintzer, M. A., & Simanek, E. E. (2008). Nonviral vectors for gene delivery. Chemical Reviews, 109, 259–302.CrossRefGoogle Scholar
  9. 9.
    Choi, Y. J., Kang, S. J., Kim, Y. J., Lim, Y.-B., & Chung, H. W. (2010). Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells. Drug and Chemical Toxicology, 33, 357–366.CrossRefGoogle Scholar
  10. 10.
    Šebestík, J., Reiniš, M., Ježek, J., Dendrimers in gene delivery. In Biomedical Applications of Peptide-, Glyco-and Glycopeptide Dendrimers, and Analogous Dendrimeric Structures, Springer: 2012; pp 141–147.Google Scholar
  11. 11.
    Yuan, Q., Yeudall, W. A., & Yang, H. (2010). PEGylated polyamidoamine dendrimers with bis-aryl hydrazone linkages for enhanced gene delivery. Biomacromolecules, 11, 1940–1947.CrossRefGoogle Scholar
  12. 12.
    Taratula, O., Garbuzenko, O. B., Kirkpatrick, P., Pandya, I., Savla, R., Pozharov, V. P., & Minko, T. (2009). Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery. Journal of Controlled Release, 140, 284–293.CrossRefGoogle Scholar
  13. 13.
    Dehshahri, A., Oskuee, R. K., Shier, W. T., Hatefi, A., & Ramezani, M. (2009). Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials, 30, 4187–4194.CrossRefGoogle Scholar
  14. 14.
    Hashemi, M. S. F., H. Amel, F.S. Parhiz, H. Ramezani, M. (2013). Gene transfer enhancement by alkylcarboxylation of poly(propylenimine). Nanomedicine Journal, 1, 55-62.Google Scholar
  15. 15.
    Oskuee, R. K., Dehshahri, A., Shier, W. T., & Ramezani, M. (2009). Alkylcarboxylate grafting to polyethylenimine: a simple approach to producing a DNA nanocarrier with low toxicity. The Journal of Gene Medicine, 11, 921–932.CrossRefGoogle Scholar
  16. 16.
    Dutta, T., Jain, N. K., McMillan, N. A., & Parekh, H. S. (2010). RETRACTED: dendrimer nanocarriers as versatile vectors in gene delivery. Nanomedicine: Nanotechnology, Biology and Medicine, 6, 25–34.CrossRefGoogle Scholar
  17. 17.
    Guillaudeu, S. J., Fox, M. E., Haidar, Y. M., Dy, E. E., Szoka, F. C., & Fréchet, J. M. (2008). PEGylated dendrimers with core functionality for biological applications. Bioconjugate Chemistry, 19, 461–469.CrossRefGoogle Scholar
  18. 18.
    Luo, D., Haverstick, K., Belcheva, N., Han, E., & Saltzman, W. M. (2002). Poly (ethylene glycol)-conjugated PAMAM dendrimer for biocompatible, high-efficiency DNA delivery. Macromolecules, 35, 3456–3462.CrossRefGoogle Scholar
  19. 19.
    Fitzsimmons, R., & Uludağ, H. (2012). Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: interplay between PEG substitution and N/P ratio. Acta Biomaterialia, 8, 3941–3955.CrossRefGoogle Scholar
  20. 20.
    Qi, R., Gao, Y., Tang, Y., He, R.-R., Liu, T.-L., He, Y., Sun, S., Li, B.-Y., Li, Y.-B., & Liu, G. (2009). PEG-conjugated PAMAM dendrimers mediate efficient intramuscular gene expression. The AAPS Journal, 11, 395–405.CrossRefGoogle Scholar
  21. 21.
    Parhiz, H., Hashemi, M., Hatefi, A., Shier, W. T., Farzad, S. A., & Ramezani, M. (2013). Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors. Journal of Biomaterials Applications, 28, 112–124.CrossRefGoogle Scholar
  22. 22.
    Mousavi, S. H., Tavakkol-Afshari, J., Brook, A., & Jafari-Anarkooli, I. (2009). Role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells. Food and Chemical toxicology : an International Journal Published for the British Industrial Biological Research Association, 47, 1909–1913.CrossRefGoogle Scholar
  23. 23.
    Zhang, X. D., Gillespie, S. K., & Hersey, P. (2004). Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways. Molecular Cancer Therapeutics, 3, 187–197.Google Scholar
  24. 24.
    Russ, V., Günther, M., Halama, A., Ogris, M., & Wagner, E. (2008). Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. Journal of Controlled Release, 132, 131–140.CrossRefGoogle Scholar
  25. 25.
    Dutta, T., Garg, M., & Jain, N. K. (2008). Poly (propyleneimine) dendrimer and dendrosome mediated genetic immunization against hepatitis B. Vaccine, 26, 3389–3394.CrossRefGoogle Scholar
  26. 26.
    Murugan, E., Geetha Rani, D., & Yogaraj, V. (2014). Drug delivery investigations of quaternised poly (propylene imine) dendrimer using nimesulide as a model drug. Colloids and Surfaces B: Biointerfaces, 114, 121–129.CrossRefGoogle Scholar
  27. 27.
    Wang, W., Xiong, W., Zhu, Y., Xu, H., & Yang, X. (2010). Protective effect of PEGylation against poly(amidoamine) dendrimer-induced hemolysis of human red blood cells. Journal of biomedical materials research. Part B, Applied Biomaterials, 93, 59–64.Google Scholar
  28. 28.
    Russ, V., Gunther, M., Halama, A., Ogris, M., & Wagner, E. (2008). Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. Journal of Controlled release : Official Journal of the Controlled Release Society, 132, 131–140.CrossRefGoogle Scholar
  29. 29.
    Kim, T.-I., Baek, J.-U., Zhe Bai, C., & Park, J.-S. (2007). Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier. Biomaterials, 28, 2061–2067.CrossRefGoogle Scholar
  30. 30.
    Koppu, S., Oh, Y. J., Edrada-Ebel, R., Blatchford, D. R., Tetley, L., Tate, R. J., & Dufès, C. (2010). Tumor regression after systemic administration of a novel tumor-targeted gene delivery system carrying a therapeutic plasmid DNA. Journal of Controlled Release, 143, 215–221.CrossRefGoogle Scholar
  31. 31.
    Kesharwani, P., Tekade, R. K., Gajbhiye, V., Jain, K., & Jain, N. K. (2011). Cancer targeting potential of some ligand-anchored poly (propylene imine) dendrimers: a comparison. Nanomedicine: Nanotechnology, Biology and Medicine, 7, 295–304.CrossRefGoogle Scholar
  32. 32.
    Duncan, R., & Izzo, L. (2005). Dendrimer biocompatibility and toxicity. Advanced Drug Delivery Reviews, 57, 2215–2237.CrossRefGoogle Scholar
  33. 33.
    Shahidi-Hamedani, N., Shier, W. T., Moghadam Ariaee, F., Abnous, K., & Ramezani, M. (2013). Targeted gene delivery with noncovalent electrostatic conjugates of sgc-8c aptamer and polyethylenimine. The Journal of Gene Medicine, 15, 261–269.CrossRefGoogle Scholar
  34. 34.
    Alshamsan, A., Haddadi, A., Incani, V., Samuel, J., Lavasanifar, A., & Uludag, H. (2008). Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine. Molecular Pharmaceutics, 6, 121–133.CrossRefGoogle Scholar
  35. 35.
    Kurisawa, M., Yokoyama, M., & Okano, T. (2000). Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers. Journal of Controlled Release, 68, 1–8.CrossRefGoogle Scholar
  36. 36.
    Liu, Z., Zhang, Z., Zhou, C., & Jiao, Y. (2010). Hydrophobic modifications of cationic polymers for gene delivery. Progress in Polymer Science, 35, 1144–1162.CrossRefGoogle Scholar
  37. 37.
    Santos, J. L., Oliveira, H., Pandita, D., Rodrigues, J., Pêgo, A. P., Granja, P. L., & Tomás, H. (2010). Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. Journal of Controlled Release, 144, 55–64.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Maryam Hashemi
    • 1
  • Sara Ayatollahi
    • 2
  • Hamideh Parhiz
    • 2
  • Ahad Mokhtarzadeh
    • 2
  • Soheila Javidi
    • 2
  • Mohammad Ramezani
    • 2
    Email author
  1. 1.Nanotechnology Research Center, School of PharmacyMashhad University of Medical SciencesMashhadIran
  2. 2.Pharmaceutical Research Center, School of PharmacyMashhad University of Medical SciencesMashhadIran

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