Pharmaceutical Research

, Volume 30, Issue 8, pp 1968–1978 | Cite as

Bioreducible Polypeptide Containing Cell-Penetrating Sequence for Efficient Gene Delivery

  • Si Chen
  • Kai Han
  • Juan Yang
  • Qi Lei
  • Ren-Xi Zhuo
  • Xian-Zheng Zhang
Research Paper



To design excellent polypeptide-based gene vectors and determine the gene delivery efficiency.


Polypeptides (designated as xPolyK6, xPolyK6-R81 and xPolyK6-R82), comprising the DNA condensing and buffering peptide HK6H as well as cell penetrating peptide (CPP) R8 were obtained by the oxidative polymerization of CHK6HC and CR8C at different molar ratios in 4 mL phosphate-buffered saline (PBS) containing 30% (v/v) DMSO at room temperature for 96 h. The cytotoxicity of vectors was studied by MTT assay. Moreover, particle size, zeta potential and morphology along with the in vitro transfection efficiency and cellular uptake of vector/plasmid DNA (pDNA) complexes were characterized at various w/w ratios to determine their potential in gene therapy.


All the vectors presented excellent ability of binding and condensing pDNA, additionally with low cytotoxicity. Simultaneously, transfection efficiency of the vectors appeared apparent dependence on the vector composition. The distinct correlation between the content of CR8C with the transfection efficiency demonstrated the effective improvement in transfection efficacy by the oxidative polymerization. Particularly, xPolyK6-R82 possessed the highest transfection efficiency at a w/w ratio of 50. Furthermore, xPolyK6-R82 also presented the best cellular uptake capability demonstrated by confocal microscopy and flow cytometry.


Bioreducible polypeptides incorporating with proper amount of CPP are promising as effective non-viral gene vectors in gene therapy.


cell-penetrating peptide disulfide bond gene delivery polypeptide 



This work was financially supported by National Natural Science Fundodation of China (51125014, 51233003) and the Ministry of Science and Technology of China (2011CB606202).


  1. 1.
    Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov. 2005;4:581–93.PubMedCrossRefGoogle Scholar
  2. 2.
    Maheshri N, Koerber JT, Kaspar BK, Schaffer DV. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol. 2006;24:198–204.PubMedCrossRefGoogle Scholar
  3. 3.
    Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol. 2000;18:33–7.PubMedCrossRefGoogle Scholar
  4. 4.
    Park TG, Jeong JH, Kim SW. Current status of polymeric gene delivery systems. Adv Drug Deliv Rev. 2006;58:467–86.PubMedCrossRefGoogle Scholar
  5. 5.
    Jeong JH, Kim SW, Park TG. Molecular design of functional polymers for gene therapy. Prog Polym Sci. 2007;32:1239–74.CrossRefGoogle Scholar
  6. 6.
    Neu M, Fischer D, Kissel TJ. Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. Gene Med. 2005;7:992–1009.CrossRefGoogle Scholar
  7. 7.
    Wong SY, Pelet JM, Putnam D. Polymer systems for gene delivery—past, present, and future. Prog Polym Sci. 2007;32:799–837.CrossRefGoogle Scholar
  8. 8.
    Sun XL, Liu CX, Liu DH, Li P, Zhang N. Novel biomimetic vectors with endosomal-escape agent enhancing gene transfection efficiency. Int J Pharm. 2012;425:62–72.PubMedCrossRefGoogle Scholar
  9. 9.
    Wiethoff CM, Middaugh CR. Barriers to nonviral gene delivery. J Pharm Sci. 2003;92:203–17.PubMedCrossRefGoogle Scholar
  10. 10.
    Newland B, Zheng Y, Jin Y, Abu-Rub M, Cao HL, Wang WXA. Single cyclized molecule versus single branched molecule: a simple and efficient 3D “Knot” polymerstructure for nonviral gene delivery. J Am Chem Soc. 2012;134:4782–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Shan YB, Luo T, Peng C, Sheng RL, Cao A, Cao XY, et al. Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors. Biomaterials. 2012;33:3025–35.PubMedCrossRefGoogle Scholar
  12. 12.
    Zhou JB, Liu J, Cheng CJ, Patel TR, Weller CE, Piepmeier JM, et al. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery. Nat Mater. 2012;11:82–90.CrossRefGoogle Scholar
  13. 13.
    Shim MS, Kwon YJ. Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications. Adv Drug Deliv Rev. 2012. doi: 10.1016/j.addr.2012.01.018.PubMedGoogle Scholar
  14. 14.
    Hu JM, Zhang GQ, Liu SY. Enzyme-responsive polymeric assemblies, nanoparticles and hydrogels. Chem Soc Rev. 2012. doi: 10.1039/C2CS35103J.Google Scholar
  15. 15.
    Nam HY, Nam K, Lee M, Kim SW, Bull DA. Dendrimer type bio-reducible polymer for efficient gene delivery. J Control Release. 2012;160:592–600.PubMedCrossRefGoogle Scholar
  16. 16.
    Ho YC, Liao ZX, Panda N, Tang DW, Yu SH, Mi FL, et al. Self-organized nanoparticles prepared by guanidine- and disulfide-modified chitosan as a gene delivery carrier. J Mater Chem. 2011;21:16918–27.CrossRefGoogle Scholar
  17. 17.
    Kima T, Lee M, Kima SW. A guanidinylated bioreducible polymer with high nuclear localization ability for gene delivery systems. Biomaterials. 2010;31:1798–804.CrossRefGoogle Scholar
  18. 18.
    Lehto T, Mager I, Sork H, Copolovici DM, Oskolkov N, Suhorutsenko J. A peptide-based vector for efficient gene transfer in vitro and in vivo. Mol Ther. 2011;19:1457–67.PubMedCrossRefGoogle Scholar
  19. 19.
    Ma DX, Shi NQ, Qi XR. Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells. Int J Pharm. 2011;419:200–8.PubMedCrossRefGoogle Scholar
  20. 20.
    McKenzie DL, Smiley E, Kwok KY, Rice KG. Low molecular weight disulfide cross-Linking peptides as nonviral gene delivery carriers. Bioconjug Chem. 2000;11:901–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Martin ME, Rice EG. Peptide-guided gene delivery. AAPS J. 2007;9:E18–29.PubMedCrossRefGoogle Scholar
  22. 22.
    Chen CS, Ji TJ, Xu XD, Zhang XZ, Zhuo RX. Nanofibers Self-assembled from Structural Complementary Borono-decapeptides. Rapid Commun. 2010;31:1903–8.CrossRefGoogle Scholar
  23. 23.
    Chen JX, Wang HY, Quan CY, Xu XD, Zhang XZ, Zhuo RX. Amphiphilic cationic lipopeptides with RGD sequences as gene vectors. Org Biomol Chem. 2010;8:3142–8.PubMedCrossRefGoogle Scholar
  24. 24.
    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
  25. 25.
    Lo SL, Wang S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials. 2008;2:2408–14.CrossRefGoogle Scholar
  26. 26.
    Manickam DS, Bisht HS, Wan L, Mao GZ, Oupicky D. Influence of TAT-peptide polymerization on properties and transfection activity of TAT/DNA polyplexes. J Control Release. 2005;102:293–306.CrossRefGoogle Scholar
  27. 27.
    Han K, Yang J, Chen S, Chen JX, Liu CW, Li C, et al. Novel gene transfer vectors based on artificial recombinant multi-functional oligopeptides. Int J Pharm. 2012;436:555–63.PubMedCrossRefGoogle Scholar
  28. 28.
    Yang J, Lei Q, Han K, Gong YH, Chen S, Cheng H, et al. Reduction-sensitive polypeptides incorporated with nuclear localization signal sequences for enhanced gene delivery. J Mater Chem. 2011. doi: 10.1039/c2jm32223d.PubMedGoogle Scholar
  29. 29.
    Sun YX, Zhang XZ, Cheng H, Cheng SX, Zhuo RX. A low-toxic and efficient gene vector: Carboxymethyl dextran-graft-polyethylenimine. J Biomed Mater Res Part A. 2008;84:1102–10.CrossRefGoogle Scholar
  30. 30.
    McKenzie DL, Kwok KY, Rice KG. A potent new class of reductively activated peptide gene delivery agents. J Biol Chem. 2000;275:9970–7.PubMedCrossRefGoogle Scholar
  31. 31.
    Wang HY, Chen JX, Sun YX, Deng JZ, Li C, Zhang XZ, et al. Construction of cell penetrating peptide vectors with N-terminal steaylated nuclear localization signal for targeted delivery of DNA into the cell nuclei. J Control Release. 2011;155:26–33.PubMedCrossRefGoogle Scholar
  32. 32.
    Godbey WT, Wu KK, Mikos AG. Characterization of a multifunctional PEG-based gene delivery systemcontaining nuclear localization signals and endosomalescape peptides. J Control Release. 1999;60:149–60.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Si Chen
    • 1
  • Kai Han
    • 1
  • Juan Yang
    • 1
  • Qi Lei
    • 1
  • Ren-Xi Zhuo
    • 1
  • Xian-Zheng Zhang
    • 1
  1. 1.Key Laboratory of Biomedical Polymers (The Ministry of Education) Department of ChemistryWuhan UniversityWuhanChina

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