Pharmaceutical Research

, 25:2807

Chitosan-Thiamine Pyrophosphate as a Novel Carrier for siRNA Delivery

  • Theerasak Rojanarata
  • Praneet Opanasopit
  • Sunee Techaarpornkul
  • Tanasait Ngawhirunpat
  • Uracha Ruktanonchai
Research Paper



A novel siRNA carrier was formulated between chitosan (CS) and thiamine pyrophosphate (TPP). Their ability to deliver siRNA were evaluated in stable and constitutive EGFP-expressing HepG2 cells.


CS-TPP was prepared by dissolving CS in TPP solution at a CS:TPP molar ratio of 1.5:1. Complexes of CS-TPP/siRNA were formed at varying weight ratios and characterized using gel electrophoresis. Their morphologies and particle sizes were evaluated, and the transfection efficiency and cytotoxicity of CS-TPP/siRNA complexes were examined in stable and constitutive EGFP-expressing HepG2 cells.


Gel electrophoresis results indicated that binding of CS-TPP and siRNA depended on the molecular weight (MW) and weight ratio of CS, and the particle sizes of CS-TPP/siRNA complexes were in nano-size. The CS-TPP-mediated siRNA silencing of the endogenous EGFP gene occurred maximally with 70–73% efficiency. The CS-TPP/siRNA complex with the lowest MW of CS (20 kDa) at a weight ratio of 80 showed the strongest inhibition of gene expression, which was higher than Lipofectamine 2000™. Over 90% the average cell viabilities of the complexes were observed by MTT assay.


This study suggests that CS-TPP is straightforward to prepare, safe and exhibits significantly improved siRNA delivery potential in vitro.


chitosan salts EGFP siRNA delivery thiamine pyrophosphate 


  1. 1.
    P. A. Sharp. RNA interference. Genes Dev. 15:485–490 (2001) doi:10.1101/gad.880001.PubMedCrossRefGoogle Scholar
  2. 2.
    Y. Dorsett, and T. Tuschl. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3:318–329 (2004) doi:10.1038/nrd1345.PubMedCrossRefGoogle Scholar
  3. 3.
    R. C. Ryther, A. S. Flynt, J. A. Phillips, and J. G. Patton. siRNA therapeutics big potential from small RNAs. Gene Ther. 1:25–11 (2005).Google Scholar
  4. 4.
    B. Urban-Klein, S. Werth, S. Abuharbeid, F. Czubayko, and A. Aigner. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 12:461–466 (2005) doi:10.1038/ Scholar
  5. 5.
    F. Takeshita, Y. Minakuchi, S. Nagahara, K. Honma, H. Sasaki, K. Hirai, T. Teratani, N. Namatame, Y. Yamamoto, K. Hanai, T. Kato, A. Sano, and T. Ochiya. Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. Proc. Natl. Acad. Sci. U. S. A. 102:12177–12182 (2005) doi:10.1073/pnas.0501753102.PubMedCrossRefGoogle Scholar
  6. 6.
    C. N. Landen Jr., A. Chavez-Reyes, C. Bucana, R. Schmandt, M. T. Deavers, L.opez-G. Berestein, and A.K. Sood. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res. 65:6910–6918 (2005) doi:10.1158/0008-5472.CAN-05-0530.PubMedCrossRefGoogle Scholar
  7. 7.
    A. Hassan, Y. Tian, W. Zheng, H. Ji, K. Sandberg, and J. G. Verbalis. Small interfering RNA-mediated functional silencing of vasopressin V2 receptors in the mouse kidney. Physiol. Genomics. 21:382–388 (2005) doi:10.1152/physiolgenomics.00147.2004.PubMedCrossRefGoogle Scholar
  8. 8.
    B. I. Florea, M. Thanou, H. E. Junginger, and G. Borchard. Enhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro/in vivo correlation. J. Control. Release. 110:353–361 (2006) doi:10.1016/j.jconrel.2005.10.001.PubMedCrossRefGoogle Scholar
  9. 9.
    M. Lee, J.W. Nah, Y. Kwon, J. J. Koh, K. S. Ko, and S. W. Kim. Water-soluble and low molecular weight chitosan-based plasmid DNA delivery. Pharm. Res. 18:427–431 (2001) doi:10.1023/A:1011037807261.PubMedCrossRefGoogle Scholar
  10. 10.
    T. Ishii, Y. Okahata, and T. Sato. Mechanism of cell transfection with plasmid/chitosan complexes. Biochim. Biophys. Acta. 1514:51–64 (2001) doi:10.1016/S0005-2736(01)00362-5.PubMedCrossRefGoogle Scholar
  11. 11.
    W. Weecharangsan, P. Opanasopit, T. Ngawhirunpat, T. Rojanarata, and A. Apirakaramwong. Chitosan lactate as a nonviral gene delivery vector in COS-1 cells. AAPS PharmSciTech. 7:E1–E6 (2006) doi:10.1208/pt070366.CrossRefGoogle Scholar
  12. 12.
    X. Zhao, S. B. Yu, F. L. Wu, Z. B. Mao, and C. L. Yu. Transfection of primary chondrocytes using chitosan-pEEGFP nanoparticles. J. Control. Release. 112:223–228 (2006) doi:10.1016/j.jconrel.2006.01.016.PubMedCrossRefGoogle Scholar
  13. 13.
    F. C. MacLaughlin, R.J. Mumper, J. Wang, J.M. Tagliaferri, I. Gill, M. HinCHyiffe, and A.P. Rolland. Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J. Control. Release. 56:259–272 (1998) doi:10.1016/S0168-3659(98)00097-2.PubMedCrossRefGoogle Scholar
  14. 14.
    M. Huang, C. W. Fong, E. Khorc, and L. Y. Lim. Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation. J. Control. Release. 106:391–406 (2005) doi:10.1016/j.jconrel.2005.05.004.PubMedCrossRefGoogle Scholar
  15. 15.
    T. Kiang, J. Wen, H. W. Lim, and K. W. Leong. The effect of the degree of chitosan deacetylation on the efficiency of gene transfection. Biomaterials. 25:5293–5301 (2004) doi:10.1016/j.biomaterials.2003.12.036.PubMedCrossRefGoogle Scholar
  16. 16.
    M. Lavertu, S. Méthot, N. Tran-Khanh, and M.D. Buschmann. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation. Biomaterials. 27:4815–4824 (2006) doi:10.1016/j.biomaterials.2006.04.029.PubMedCrossRefGoogle Scholar
  17. 17.
    W. B. Tan, S. Jiang, and Y. Zhang. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. Biomaterials. 28:1565–1571 (2007) doi:10.1016/j.biomaterials.2006.11.018.PubMedCrossRefGoogle Scholar
  18. 18.
    X. Liu, K. A. Howard, M. Dong, M. O. Andersen, U. L. Rahbek, M. G. Johnsen, O. C. Hansen, F. Besenbacher, and J. Kjems. The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing. Biomaterials. 28:1280–1288 (2007) doi:10.1016/j.biomaterials.2006.11.004.PubMedCrossRefGoogle Scholar
  19. 19.
    J. Y. Pille, H. Li, E. Blot, J. R. Bertrand, L. L. Pritchard, P. Opolon, A. Maksimenko, H. Lu, J. P. Vannier, J. Soria, C. Malvy, and C. Soria. Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer. Hum. Gene Ther. 17:1019–1026 (2006) doi:10.1089/hum.2006.17.1019.PubMedCrossRefGoogle Scholar
  20. 20.
    H. Katas, and H. O. Alpar. Development and characterisation of chitosan nanoparticles for siRNA delivery. J. Control. Release. 115:216–225 (2006).PubMedCrossRefGoogle Scholar
  21. 21.
    K. A. Howard, U. L. Rahbek, X. Liu, C. K. Damgaard, S. Z. Glud, M. O. Andersen, M. B. Hovgaard, A. Schmitz, J. R. Nyengaard, F. Besenbacher, and J. Kjems. RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. Mol. Ther. 14:476–484 (2006) doi:10.1016/j.ymthe.2006.04.010.PubMedCrossRefGoogle Scholar
  22. 22.
    W. Weecharangsan, P. Opanasopit, T. Ngawhirunpat, A. Apirakaramwong, T. Rojanarata, U. Ruktanonchai, and R. J. Lee. Evaluation of chitosan salts as non-viral gene vectors in CHO-K1 cells. Int. J. Pharm. 348:161–168 (2008) doi:10.1016/j.ijpharm.2007.07.011.PubMedCrossRefGoogle Scholar
  23. 23.
    P. C. Engel. Enzyme cofactors. In P. C. Engel (ed.), Enzymology LabFax, Academic, California, 1996, pp. 244–245.Google Scholar
  24. 24.
    M. D. Smith, J. C. Barbenel, J. M. Courtney, and M. H. Grant. Novel quantitative methods for the determination of biomaterial cytotoxicity. Int. J. Artif. Organs. 15:191–184 (1992).PubMedGoogle Scholar
  25. 25.
    A. C. Grayson, A. M. Doody, and D. Putnam. Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro. Pharm. Res. 23:1868–1876 (2006) doi:10.1007/s11095-006-9009-2.PubMedCrossRefGoogle Scholar
  26. 26.
    M. O. Andersen, K. A. Howard, S. R. Paludan, F. Besenbacher, and J. Kjems. Delivery of siRNA from lyophilized polymeric surfaces. Biomaterials. 29:506–512 (2008) doi:10.1016/j.biomaterials.2007.10.003.PubMedCrossRefGoogle Scholar
  27. 27.
    K. Romóren, S. Pedersen, G. Smistad, O. Evensen, and B.J. Thu. The influence of formulation variables on in vitro transfection efficiency and physicochemical properties of chitosan-based polyplexes. Int. J. Pharm. 261:115–127 (2003) doi:10.1016/S0378-5173(03)00301-6.PubMedCrossRefGoogle Scholar
  28. 28.
    T. Sato, T. Ishii, and Y. Okahata. In vitro gene delivery mediated by chitosan: effect of pH, serum, and molecular mass of chitosan on the transfection efficiency. Biomaterials. 22:2075–2080 (2001) doi:10.1016/S0142-9612(00)00385-9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Theerasak Rojanarata
    • 1
  • Praneet Opanasopit
    • 1
  • Sunee Techaarpornkul
    • 1
  • Tanasait Ngawhirunpat
    • 1
  • Uracha Ruktanonchai
    • 2
  1. 1.Faculty of PharmacySilpakorn UniversityNakhonpathomThailand
  2. 2.National Nanotechnology CenterPathumthaniThailand

Personalised recommendations