The AAPS Journal

, 21:54 | Cite as

In Vitro and In Vivo Co-delivery of siRNA and Doxorubicin by Folate-PEG-Appended Dendrimer/Glucuronylglucosyl-β-Cyclodextrin Conjugate

  • Ahmed Fouad Abdelwahab Mohammed
  • Taishi HigashiEmail author
  • Keiichi Motoyama
  • Ayumu Ohyama
  • Risako Onodera
  • Khaled Ali Khaled
  • Hatem Abdelmonsef Sarhan
  • Amal Kamal Hussein
  • Hidetoshi ArimaEmail author
Research Article


We have previously reported the utility of folate-polyethylene glycol-appended dendrimer conjugate with glucuronylglucosyl-β-cyclodextrin (Fol-PEG-GUG-β-CDE) (generation 3) as a tumor-selective carrier for siRNA against polo-like kinase 1 (siPLK1) in vitro. In the present study, we evaluated the potential of Fol-PEG-GUG-β-CDE as a carrier for the low-molecular antitumor drug doxorubicin (DOX). Further, to fabricate advanced antitumor agents, we have prepared a ternary complex of Fol-PEG-GUG-β-CDE/DOX/siPLK1 and evaluated its antitumor activity both in vitro and in vivo. Fol-PEG-GUG-β-CDE released DOX in an acidic pH and enhanced the cellular accumulation and cytotoxic activity of DOX in folate receptor-α (FR-α)-overexpressing KB cells. Importantly, the Fol-PEG-GUG-β-CDE/DOX/siPLK1 ternary complex exhibited higher cytotoxic activity than a binary complex of Fol-PEG-GUG-β-CDE with DOX or siPLK1 in KB cells. In addition, the cytotoxic activity of the ternary complex was reduced by the addition of folic acid, a competitor against FR-α. Furthermore, the ternary complex showed a significant antitumor activity after intravenous administration to the tumor-bearing mice. These results suggest that Fol-PEG-GUG-β-CDE has the potential of a tumor-selective co-delivery carrier for DOX and siPLK1.


PAMAM dendrimer doxorubicin siRNA folate tumor-selective drug delivery 



The authors thank Ensuiko Sugar Refining for donating GUG-β-CyD.

Author Contributions

AFAM, TH, KM, AO, RO, and HA had participated in the research design. AFAM and AO had conducted the experiments. AFAM, TH, KM, AO, and RO had performed the data analysis. AFAM and TH had drafted or contributed to the writing of the manuscript. TH, KM, KAK, HAS, AKH and HA had supervised the experiments.

Compliance with Ethical Standards

All animal procedures were carried out in accordance with the approved guidelines and with the approval of the Ethics Committee for Animal Care and Use of Kumamoto University (approval no.: C29-162).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12248_2019_327_MOESM1_ESM.pdf (304 kb)
ESM 1 (PDF 304 kb)


  1. 1.
  2. 2.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–11.CrossRefGoogle Scholar
  3. 3.
    Liu Z, Sun Q, Wang X. PLK1, a potential target for cancer therapy. Transl Oncol. 2017;10(1):22–32.CrossRefGoogle Scholar
  4. 4.
    Uekama K, Hirayama F, Irie T. Cyclodextrin drug carrier systems. Chem Rev. 1998;98(5):2045–76.CrossRefGoogle Scholar
  5. 5.
    Ceborska M. Folate appended cyclodextrins for drug, DNA, and siRNA delivery. Eur J Pharm Biopharm. 2017;120:133–45.CrossRefGoogle Scholar
  6. 6.
    Zhao F, Yin H, Zhang Z, Li J. Folic acid modified cationic γ-cyclodextrin-oligoethylenimine star polymer with bioreducible disulfide linker for efficient targeted gene delivery. Biomacromolecules. 2013;14(2):476–84.CrossRefGoogle Scholar
  7. 7.
    Zhao F, Yin H, Li J. Supramolecular self-assembly forming a multifunctional synergistic system for targeted co-delivery of gene and drug. Biomaterials. 2014;35(3):1050–62.CrossRefGoogle Scholar
  8. 8.
    Li J, Loh XJ. Cyclodextrin-based supramolecular architectures: syntheses, structures, and applications for drug and gene delivery. Adv Drug Deliv Rev. 2008;60(9):1000–17.CrossRefGoogle Scholar
  9. 9.
    Zhang J, Ma PX. Cyclodextrin-based supramolecular systems for drug delivery: recent progress and future perspective. Adv Drug Deliv Rev. 2013;65(9):1215–33.CrossRefGoogle Scholar
  10. 10.
    Higashi T, Iohara D, Motoyama K, Arima H. Supramolecular pharmaceutical sciences: a novel concept combining pharmaceutical sciences and supramolecular chemistry with a focus on cyclodextrin-based supermolecules. Chem Pharm Bull. 2018;66(3):207–16.CrossRefGoogle Scholar
  11. 11.
    Anno T, Higashi T, Motoyama K, Hirayama F, Uekama K, Arima H. Possible enhancing mechanisms for gene transfer activity of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate. Int J Pharm. 2012;426(1–2):239–47.CrossRefGoogle Scholar
  12. 12.
    Mohammed AFA, Higashi T, Motoyama K, Ohyama A, Onodera R, Khaled KA, et al. Targeted siRNA delivery to tumor cells by folate-PEG-appended dendrimer/glucuronylglucosyl-β-cyclodextrin conjugate. J Incl Phenom Macrocycl Chem. 2019:in press;93:41–52.CrossRefGoogle Scholar
  13. 13.
    Silber JH, Barber G. Doxorubicin-induced cardiotoxicity. N Engl J Med. 1995;333(20):1359–60.CrossRefGoogle Scholar
  14. 14.
    Wang Y, Cao X, Guo R, Shen M, Zhang M, Zhu M, et al. Targeted delivery of doxorubicin into cancer cells using a folic acid–dendrimer conjugate. Polym Chem. 2011;2(8):1754–60.CrossRefGoogle Scholar
  15. 15.
    Al-Jamal KT, Al-Jamal WT, Wang JT, Rubio N, Buddle J, Gathercole D, et al. Cationic poly-L-lysine dendrimer complexes doxorubicin and delays tumor growth in vitro and in vivo. ACS Nano. 2013;7(3):1905–17.CrossRefGoogle Scholar
  16. 16.
    Choi SK, Thomas T, Li MH, Kotlyar A, Desai A, Baker JR Jr. Light-controlled release of caged doxorubicin from folate receptor-targeting PAMAM dendrimer nanoconjugate. Chem Commun. 2010;46(15):2632–4.CrossRefGoogle Scholar
  17. 17.
    Han L, Huang R, Liu S, Huang S, Jiang C. Peptide-conjugated PAMAM for targeted doxorubicin delivery to transferrin receptor overexpressed tumors. Mol Pharm. 2010;7(6):2156–65.CrossRefGoogle Scholar
  18. 18.
    Mohammed AFA, Ohyama A, Higashi T, Motoyama K, Khaled KA, Sarhan HA, et al. Preparation and evaluation of polyamidoamine dendrimer conjugate with glucuronylglucosyl-β-cyclodextrin (G3) as a novel carrier for siRNA. J Drug Target. 2014;22(10):927–34.CrossRefGoogle Scholar
  19. 19.
    Ohyama A, Higashi T, Motoyama K, Arima H. In vitro and in vivo tumor-targeting siRNA delivery using folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugates. Bioconjug Chem. 2016;27(3):521–32.CrossRefGoogle Scholar
  20. 20.
    Arima H, Chihara Y, Arizono M, Yamashita S, Wada K, Hirayama F, et al. Enhancement of gene transfer activity mediated by mannosylated dendrimer/α-cyclodextrin conjugate (generation 3, G3). J Control Release. 2006;116(1):64–74.CrossRefGoogle Scholar
  21. 21.
    Corbett TH, Griswold DP Jr, Roberts BJ, Peckham JC, Schabel FM Jr. Biology and therapeutic response of a mouse mammary adenocarcinoma (16/C) and its potential as a model for surgical adjuvant chemotherapy. Cancer Treat Rep. 1978;62(10):1471–88.PubMedGoogle Scholar
  22. 22.
    Ke W, Zhao Y, Huang R, Jiang C, Pei Y. Enhanced oral bioavailability of doxorubicin in a dendrimer drug delivery system. J Pharm Sci. 2008;97(6):2208–16.CrossRefGoogle Scholar
  23. 23.
    Chandra S, Dietrich S, Lang H, Bahadur D. Dendrimer–doxorubicin conjugate for enhanced therapeutic effects for cancer. J Mater Chem. 2011;21(15):5729–37.CrossRefGoogle Scholar
  24. 24.
    Yamanoi T, Yoshida N, Oda Y, Akaike E, Tsutsumida M, Kobayashi N, et al. Synthesis of mono-glucose-branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo-β-N-acetylglucosaminidase. Bioorg Med Chem Lett. 2005;15(4):1009–13.CrossRefGoogle Scholar
  25. 25.
    Anno T, Higashi T, Hayashi Y, Motoyama K, Jono H, Ando Y, et al. Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a siRNA carrier for the treatment of familial amyloidotic polyneuropathy. J Drug Target. 2014;22(10):883–90.CrossRefGoogle Scholar
  26. 26.
    Reddy JA, Low PS. Folate-mediated targeting of therapeutic and imaging agents to cancers. Crit Rev Ther Drug Carrier Syst. 1998;15(6):587–627.CrossRefGoogle Scholar
  27. 27.
    Strebhardt K. Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy. Nat Rev Drug Discov. 2010;9(8):643–60.CrossRefGoogle Scholar
  28. 28.
    Spankuch B, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Rational combinations of siRNAs targeting Plk1 with breast cancer drugs. Oncogene. 2007;26(39):5793–807.CrossRefGoogle Scholar
  29. 29.
    Hu K, Law JH, Fotovati A, Dunn SE. Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells. Breast Cancer Res. 2012;14(1):R22.CrossRefGoogle Scholar
  30. 30.
    Chen Y, Wu JJ, Huang L. Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy. Mol Ther. 2010;18(4):828–34.CrossRefGoogle Scholar
  31. 31.
    Wang Y, Gao S, Ye WH, Yoon HS, Yang YY. Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer. Nat Mater. 2006;5(10):791–6.CrossRefGoogle Scholar
  32. 32.
    Xiong XB, Lavasanifar A. Traceable multifunctional micellar nanocarriers for cancer-targeted co-delivery of MDR-1 siRNA and doxorubicin. ACS Nano. 2011;5(6):5202–13.CrossRefGoogle Scholar
  33. 33.
    Taratula O, Kuzmov A, Shah M, Garbuzenko OB, Minko T. Nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and siRNA. J Control Release. 2013;171(3):349–57.CrossRefGoogle Scholar
  34. 34.
    Quintana A, Raczka E, Piehler L, Lee I, Myc A, Majoros I, et al. Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharm Res. 2002;19(9):1310–6.CrossRefGoogle Scholar
  35. 35.
    Singh P, Gupta U, Asthana A, Jain NK. Folate and folate-PEG-PAMAM dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug Chem. 2008;19(11):2239–52.CrossRefGoogle Scholar
  36. 36.
    Salmaso S, Semenzato A, Caliceti P, Hoebeke J, Sonvico F, Dubernet C, et al. Specific antitumor targetable β-cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconjugate. Bioconjug Chem. 2004;15(5):997–1004.CrossRefGoogle Scholar
  37. 37.
    Okamatsu A, Motoyama K, Onodera R, Higashi T, Koshigoe T, Shimada Y, et al. Design and evaluation of folate-appended α-, β-, and γ-cyclodextrins having a caproic acid as a tumor selective antitumor drug carrier in vitro and in vivo. Biomacromolecules. 2013;14(12):4420–8.CrossRefGoogle Scholar
  38. 38.
    Okamatsu A, Motoyama K, Onodera R, Higashi T, Koshigoe T, Shimada Y, et al. Folate-appended β-cyclodextrin as a promising tumor targeting carrier for antitumor drugs in vitro and in vivo. Bioconjug Chem. 2013;24(4):724–33.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2019

Authors and Affiliations

  • Ahmed Fouad Abdelwahab Mohammed
    • 1
    • 2
    • 3
  • Taishi Higashi
    • 1
    • 4
    Email author
  • Keiichi Motoyama
    • 1
  • Ayumu Ohyama
    • 1
    • 3
  • Risako Onodera
    • 5
  • Khaled Ali Khaled
    • 2
  • Hatem Abdelmonsef Sarhan
    • 2
  • Amal Kamal Hussein
    • 2
  • Hidetoshi Arima
    • 1
    • 3
    Email author
  1. 1.Graduate School of Pharmaceutical SciencesKumamoto UniversityKumamotoJapan
  2. 2.Department of Pharmaceutics, Faculty of PharmacyMinia UniversityMiniaEgypt
  3. 3.Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and Glocal Oriented) Program”Kumamoto UniversityKumamotoJapan
  4. 4.Priority Organization for Innovation and ExcellenceKumamoto UniversityKumamotoJapan
  5. 5.School of Pharmacy, Building Regional Innovation EcosystemsKumamoto UniversityKumamotoJapan

Personalised recommendations