Applied Biochemistry and Biotechnology

, Volume 187, Issue 3, pp 708–723 | Cite as

Therapeutic Potential of DNAzyme Loaded on Chitosan/Cyclodextrin Nanoparticle to Recovery of Chemosensitivity in the MCF-7 Cell Line

  • Elham Zokaei
  • Arastoo Badoei-dalfradEmail author
  • Mehdi AnsariEmail author
  • Zahra Karami
  • Touba Eslaminejad
  • Seyed Noureddin Nematollahi-Mahani


Commonly, acquired resistances to anticancer drug are mediated by overexpression of a membrane-associated protein that encode via multi-drug resistance gene-1 (MDR1). Herein, the mRNA-cleaving DNAzyme that targets the mRNA of MDR1 gene in doxorubicin-resistant breast cancer cell line (MCF-7/DR) loaded on the chitosan β-cyclodextrin complexes was used as a tropical agent. Chitosan/β-cyclodextrin complexes were used to deliver DNAzymes into cancer cells. Determination of the physicochemical characteristics of the particles was done by photon correlation spectroscopy and scanning electron microscopy. The encapsulation efficiency of the complexes was tested by using gel retardation assay. Positively charged nanoparticles interacted with DNAzyme that could perform as an efficient DNAzyme transfection system. The rationale usage of this platform is to sensitize MCF-7/DR to doxorubicin by downregulating the drug-resistance gene MDR1. Results demonstrated a downregulation of MDR1 mRNAs in MCF-7/DR/DNZ by real-time PCR, compared to the MCF-7/DR as control. WST1 assay showed the 22-fold decrease in drug resistance on treated cells 24 h after transfection. Results showed the intracellular accumulation of Rh123 increased in the treated cells with DNAzyme. Results suggested a potential platform in association with chemotherapy drug for cancer therapy and indicated extremely efficient at delivery of DNAzyme in restoring chemosensitivity.


Multi-drug resistance DNAzyme β-Cyclodextrin Chitosan Chemosensitization 


Funding Information

This study received a financial support from the Research Council of the Shahid Bahonar University of Kerman (Iran).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Zhao, J. (2016). Cancer stem cells and chemoresistance: the smartest survives the raid. Pharmacology & Therapeutics, 160, 145–158.CrossRefGoogle Scholar
  2. 2.
    Huang, Y., & Sadée, W. (2006). Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells. Cancer Letters, 239(2), 168–182.CrossRefGoogle Scholar
  3. 3.
    Smalley, M., Piggott, L., & Clarkson, R. (2013). Breast cancer stem cells: obstacles to therapy. Cancer Letters, 338(1), 57–62.CrossRefGoogle Scholar
  4. 4.
    Motomura, S., Motoji, T., Takanashi, M., Wang, Y. H., Shiozaki, H., Sugawara, I., et al. (1998). Inhibition of P-glycoprotein and recovery of drug sensitivity of human acute leukemic blast cells by multidrug resistance gene (mdr1) antisense oligonucleotides. Blood, 91(9), 3163–3171.Google Scholar
  5. 5.
    Gao P, Zhou G, Zhang Q, Li H, Mu K, Yuan Y, et al. (2006). Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA. The Chinese Journal of Physiology, 49(2):96.Google Scholar
  6. 6.
    Du, B., & Shim, J. S. (2016). Targeting epithelial–mesenchymal transition (EMT) to overcome drug resistance in cancer. Molecules, 21(7), 965.CrossRefGoogle Scholar
  7. 7.
    Ebrahimian, M., Taghavi, S., Mokhtarzadeh, A., Ramezani, M., & Hashemi, M. (2017). Co-delivery of doxorubicin encapsulated PLGA nanoparticles and Bcl-xL shRNA using alkyl-modified PEI into breast cancer cells. Biotechnology and Applied Biochemistry, 183(1), 126–136.CrossRefGoogle Scholar
  8. 8.
    Thierry, A. R., & Dritschilo, A. (1992). Liposomal delivery of antisense oligodeoxynucleotides. Annals of the New York Academy of Sciences, 660(1), 300–302.CrossRefGoogle Scholar
  9. 9.
    Xing, A. Y., Shi, D. B., Liu, W., Chen, X., Sun, Y. L., Wang, X., et al. (2013). Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme. Experimental and Molecular Pathology, 94(3), 481–485.CrossRefGoogle Scholar
  10. 10.
    Fokina, A. A., Stetsenko, D. A., & François, J. C. (2015). DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes. Expert Opinion on Biological Therapy, 15(5), 689–711.CrossRefGoogle Scholar
  11. 11.
    Sett, A., Das, S., & Bora, U. (2014). Functional nucleic-acid-based sensors for environmental monitoring. Applied Biochemistry and Biotechnology, 174(3), 1073–1091.CrossRefGoogle Scholar
  12. 12.
    Xu, Z., Yang, L., Sun, L., & Cao, Y. (2012). Use of DNAzymes for cancer research and therapy. Chinese Science Bulletin, 57(26), 3404–3408.CrossRefGoogle Scholar
  13. 13.
    Nikzad, N., & Karami, Z. (2018). Label-free colorimetric sensor for sensitive detection of choline based on DNAzyme-choline oxidase coupling. International Journal of Biological Macromolecules, 115, 1241–1248.CrossRefGoogle Scholar
  14. 14.
    Mahdiannasser, M., & Karami, Z. (2018). An innovative paradigm of methods in microRNAs detection: highlighting DNAzymes, the illuminators. Biosensors & Bioelectronics, 107, 123–144.CrossRefGoogle Scholar
  15. 15.
    Kuznetsova, M., Fokina, A., Lukin, M., Repkova, M., Venyaminova, A., & Vlassov, V. (2003). Catalytic DNA and RNA for targeting MDR1 mRNA. Nucleosides, Nucleotides & Nucleic Acids, 22(5–8), 1521–1523.CrossRefGoogle Scholar
  16. 16.
    Dass, C. R., Choong, P. F., & Khachigian, L. M. (2008). DNAzyme technology and cancer therapy: cleave and let die. Molecular Cancer Therapeutics, 7(2), 243–251.CrossRefGoogle Scholar
  17. 17.
    Fokina, A. A., Kuznetsova, M. A., Repkova, M. N., & Venyaminova, A. G. (2004). Two-component 10–23 DNA enzymes. Nucleosides, Nucleotides & Nucleic Acids, 23(6–7), 1031–1035.CrossRefGoogle Scholar
  18. 18.
    Gao, P., Wei, J. M., Li, P. Y., Zhang, C. J., Jian, W. C., Zhang, Y. H., et al. (2011). Screening of deoxyribozyme with high reversal efficiency against multidrug resistance in breast carcinoma cells. Journal of Cellular and Molecular Medicine, 15(10), 2130–2138.CrossRefGoogle Scholar
  19. 19.
    Karnati, H. K., Yalagala, R. S., Undi, R., Pasupuleti, S. R., & Gutti, R. K. (2014). Therapeutic potential of siRNA and DNAzymes in cancer. Tumor Biology, 35(10), 9505–9521.CrossRefGoogle Scholar
  20. 20.
    Khan, A., Benboubetra, M., Sayyed, P. Z., Wooi Ng, K., Fox, S., Beck, G., et al. (2004). Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies. Journal of Drug Targeting, 12(6), 393–404.CrossRefGoogle Scholar
  21. 21.
    Lin Tan, M., Choong, P. F., & Dass, C. R. (2009). DNAzyme delivery systems: getting past first base. Expert Opinion on Drug Delivery, 6(2), 127–138.CrossRefGoogle Scholar
  22. 22.
    Fokina, A. A., Chelobanov, B. P., Fujii, M., & Stetsenko, D. A. (2017). Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes): from cell culture studies to clinical trials. Expert Opinion on Drug Delivery, 14(9), 1077–1089.CrossRefGoogle Scholar
  23. 23.
    Li, G. F., Wang, J. C., Feng, X. M., Liu, Z. D., Jiang, C. Y., & Yang, J. D. (2015). Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles. Biotechnology and Applied Biochemistry, 175(7), 3244–3257.CrossRefGoogle Scholar
  24. 24.
    Alexakis T, Boadi DK, Quong D, Groboillot A, O’neill I, Poncelet D, et al. (1995). Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application. Biotechnology and Applied Biochemistry 50(1):93–106.Google Scholar
  25. 25.
    Csaba, N., Köping-Höggård, M., & Alonso, M. J. (2009). Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery. International Journal of Pharmaceutics, 382(1), 205–214.CrossRefGoogle Scholar
  26. 26.
    Csaba, N., Köping-Höggård, M., Fernandez-Megia, E., Novoa-Carballal, R., Riguera, R., & Alonso, M. J. (2009). Ionically crosslinked chitosan nanoparticles as gene delivery systems: effect of PEGylation degree on in vitro and in vivo gene transfer. Journal of Biomedical Nanotechnology, 5(2), 162–171.CrossRefGoogle Scholar
  27. 27.
    Trapani, A., Garcia-Fuentes, M., & Alonso, M. (2008). Novel drug nanocarriers combining hydrophilic cyclodextrins and chitosan. Nanotechnology, 19(18), 185101.CrossRefGoogle Scholar
  28. 28.
    Krauland, A. H., & Alonso, M. J. (2007). Chitosan/cyclodextrin nanoparticles as macromolecular drug delivery system. International Journal of Pharmaceutics, 340(1-2), 134–142.CrossRefGoogle Scholar
  29. 29.
    Challa, R., Ahuja, A., Ali, J., & Khar, R. K. (2005). Cyclodextrins in drug delivery: an updated review. AAPS PharmSciTech, 6(2), E329–EE57.CrossRefGoogle Scholar
  30. 30.
    Liu, Y., Zhai, Y., Han, X., Liu, X., Liu, W., Wu, C., et al. (2014). Bioadhesive chitosan-coated cyclodextrin-based superamolecular nanomicelles to enhance the oral bioavailability of doxorubicin. Journal of Nanoparticle Research, 16(10), 2587.CrossRefGoogle Scholar
  31. 31.
    Trapani, A., Lopedota, A., Franco, M., Cioffi, N., Ieva, E., Garcia-Fuentes, M., et al. (2010). A comparative study of chitosan and chitosan/cyclodextrin nanoparticles as potential carriers for the oral delivery of small peptides. European Journal of Pharmaceutics and Biopharmaceutics, 75(1), 26–32.CrossRefGoogle Scholar
  32. 32.
    Thanh Nguyen, H., & Goycoolea, F. M. (2017). Chitosan/cyclodextrin/TPP nanoparticles loaded with quercetin as novel bacterial quorum sensing inhibitors. Molecules, 22(11), 1975.CrossRefGoogle Scholar
  33. 33.
    Teijeiro-Osorio, D., Remuñán-López, C., & Alonso, M. J. (2009). Chitosan/cyclodextrin nanoparticles can efficiently transfect the airway epithelium in vitro. European Journal of Pharmaceutics and Biopharmaceutics, 71(2), 257–263.CrossRefGoogle Scholar
  34. 34.
    Eslaminejad, T., Nematollahi-Mahani, S. N., & Ansari, M. (2016). Cationic β-cyclodextrin–chitosan conjugates as potential carrier for pmCherry-C1 gene delivery. Molecular Biotechnology, 58(4), 287–298.CrossRefGoogle Scholar
  35. 35.
    AbuHammad, S., & Zihlif, M. (2013). Gene expression alterations in doxorubicin resistant MCF7 breast cancer cell line. Genomics, 101(4), 213–220.CrossRefGoogle Scholar
  36. 36.
    Yin, L. M., Wei, Y., Wang, Y., Xu, Y. D., & Yang, Y. Q. (2013). Long term and standard incubations of WST-1 reagent reflect the same inhibitory trend of cell viability in rat airway smooth muscle cells. International Journal of Medical Sciences, 10(1), 68.CrossRefGoogle Scholar
  37. 37.
    Zhao, Q. Q., Chen, J. L., Lv, T. F., He, C. X., Tang, G. P., Liang, W. Q., et al. (2009). N/P ratio significantly influences the transfection efficiency and cytotoxicity of a polyethylenimine/chitosan/DNA complex. Biological & Pharmaceutical Bulletin, 32(4), 706–710.CrossRefGoogle Scholar
  38. 38.
    Jouan, E., Le Vée, M., Mayati, A., Denizot, C., Parmentier, Y., & Fardel, O. (2016). Evaluation of P-glycoprotein inhibitory potential using a rhodamine 123 accumulation assay. Pharmaceutics, 8(2), 12.CrossRefGoogle Scholar
  39. 39.
    Tsou, S. H., Chen, T. M., Hsiao, H. T., & Chen, Y. H. (2015). A critical dose of doxorubicin is required to alter the gene expression profiles in MCF-7 cells acquiring multidrug resistance. PLoS One, 10(1), e0116747.CrossRefGoogle Scholar
  40. 40.
    Chen, V. Y., Posada, M. M., Zhao, L., & Rosania, G. R. (2007). Rapid doxorubicin efflux from the nucleus of drug-resistant cancer cells following extracellular drug clearance. Pharmaceutical Research, 24(11), 2156–2167.CrossRefGoogle Scholar
  41. 41.
    Rajagopal, A., & Simon, S. M. (2003). Subcellular localization and activity of multidrug resistance proteins. Molecular Biology of the Cell, 14(8), 3389–3399.CrossRefGoogle Scholar
  42. 42.
    Dalton, W. S., & Scheper, R. J. (1999). Lung resistance-related protein: determining its role in multidrug resistance. Journal of the National Cancer Institute, 91(19), 1604–1605.CrossRefGoogle Scholar
  43. 43.
    Ramachandran, C., & Wellham, L. L. (2003). Effect of MDR1 phosphorothioate antisense oligodeoxynucleotides in multidrug-resistant human tumor cell lines and xenografts. Anticancer Research, 23(3B), 2681–2690.Google Scholar
  44. 44.
    Cairns, M. J., Hopkins, T. M., Witherington, C., Wang, L., & Sun, L. Q. (1999). Target site selection for an RNA-cleaving catalytic DNA. Nature Biotechnology, 17(5), 480–486.CrossRefGoogle Scholar
  45. 45.
    Beale, G., Hollins, A. J., Benboubetra, M., Sohail, M., Fox, S. P., Benter, I., et al. (2003). Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system. Journal of Drug Targeting, 11(7), 449–456.CrossRefGoogle Scholar
  46. 46.
    Iversen, P. O., Nicolaysen, G., & Sioud, M. (2001). DNA enzyme targeting TNF-α mRNA improves hemodynamic performance in rats with postinfarction heart failure. American Journal of Physiology. Heart and Circulatory Physiology, 281(5), H2211–H22H7.CrossRefGoogle Scholar
  47. 47.
    Takahashi, H., Hamazaki, H., Habu, Y., Hayashi, M., Abe, T., Miyano-Kurosaki, N., et al. (2004). A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells. FEBS Letters, 560(1–3), 69–74.CrossRefGoogle Scholar
  48. 48.
    Pun, S. H., Bellocq, N. C., Cheng, J., Grubbs, B. H., Jensen, G. S., Davis, M. E., et al. (2004). Biodistribution of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles. Cancer Biology & Therapy, 3, 641–650.CrossRefGoogle Scholar
  49. 49.
    Tack, F., Bakker, A., Maes, S., Dekeyser, N., Bruining, M., Elissen-Roman, C., et al. (2006). Modified poly (propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes). Journal of Drug Targeting, 14(2), 69–86.CrossRefGoogle Scholar
  50. 50.
    Vimal, S., Majeed, S. A., Taju, G., Nambi, K. S. N., Raj, N. S., Madan, N., et al. (2013). RETRACTED: chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp. Acta Tropica, 128(3), 486–493.CrossRefGoogle Scholar
  51. 51.
    Katas, H., & Alpar, H. O. (2006). Development and characterisation of chitosan nanoparticles for siRNA delivery. Journal of Controlled Release, 115(2), 216–225.CrossRefGoogle Scholar
  52. 52.
    Takechi-Haraya, Y., Tanaka, K., Tsuji, K., Asami, Y., Izawa, H., Shigenaga, A., et al. (2015). Molecular complex composed of β-cyclodextrin-grafted chitosan and pH-sensitive amphipathic peptide for enhancing cellular cholesterol efflux under acidic pH. Bioconjugate Chemistry, 26(3), 572–581.CrossRefGoogle Scholar
  53. 53.
    Thews, O., Gassner, B., Kelleher, D. K., Schwerd, G., & Gekle, M. (2006). Impact of extracellular acidity on the activity of P-glycoprotein and the cytotoxicity of chemotherapeutic drugs. Neoplasia, 8(2), 143–152.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biology, Faculty of SciencesShahid Bahonar University of KermanKermanIran
  2. 2.Pharmaceutics Research Centre, Faculty of PharmacyKerman University of Medical SciencesKermanIran
  3. 3.Department of Anatomy, Afzalipour School of MedicineKerman University of Medical SciencesKermanIran
  4. 4.Pharmaceutics Research Centre, Institute of NeuropharmacologyKerman University of Medical SciencesKermanIran

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