Skip to main content
Log in

Targeted Delivery to Neuroblastoma of Novel siRNA-anti-GD2-liposomes Prepared by Dual Asymmetric Centrifugation and Sterol-Based Post-Insertion Method

Pharmaceutical Research Aims and scope Submit manuscript

Cite this article



To optimise and simplify preparation of targeted liposomes for efficient siRNA delivery to neuroblastoma, the most common solid tumour in early childhood.


Liposomes containing siRNA were prepared by combining the novel dual asymmetric centrifugation (DAC) method and the recently optimised sterol-based post-insertion technique (SPIT) to couple anti-GD2 antibody for selective interaction with neuroblastoma cells. Cultured human neuroblastoma cell lines were used to evaluate the efficiency of siRNA delivery.


The size of liposomes prepared by DAC ranged from 190 to 240 nm; siRNA encapsulation efficiency was up to 50%. An average of 70 and 100 molecules of anti-GD2 antibody per particle were coupled. A significant association of liposomes with neuroblastoma cells as well as effective siRNA delivery was observed only when anti-GD2 antibody was coupled. Preliminary data suggest delivery of siRNA using anti-GD2-liposomes occurs via GD2-mediated endocytosis. Vascular endothelial growth factor A (VEGF-A) was down-regulated using siRNA delivered by anti-GD2-liposomes.


DAC and SPIT allow for the straightforward preparation of liposomes for the targeted delivery of siRNA. Anti-GD2-liposomes thus produced can serve as versatile carriers of siRNA to neuroblastoma cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others







cryo-transmission electron microscope


dual asymmetric centrifugation


dimethyldioctadecylammonium (bromide salt)

DSPE-PEG2000 :



fetal calf serum



HN buffer:

HEPES/NaCl buffer


hydrogenated soybean phosphatidylcholine




phosphate buffered saline




rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt


short interfering RNA


sterol-based post-insertion technique


total lipid


vascular endothelial growth factor A


  1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–8.

    Article  PubMed  CAS  Google Scholar 

  2. Tiemannand K, Rossi JJ. RNAi-based therapeutics-current status, challenges and prospects. EMBO Mol Med. 2009;1:142–51.

    Article  Google Scholar 

  3. de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 2007;6:443–53.

    Article  PubMed  Google Scholar 

  4. Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, et al. RNAi-mediated gene silencing in non-human primates. Nature. 2006;441:111–4.

    Article  PubMed  CAS  Google Scholar 

  5. Villares GJ, Zigler M, Wang H, Melnikova VO, Wu H, Friedman R, et al. Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA. Cancer Res. 2008;68:9078–86.

    Article  PubMed  CAS  Google Scholar 

  6. Peer D, Park EJ, Morishita Y, Carman CV, Shimaoka M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science. 2008;319:627–30.

    Article  PubMed  CAS  Google Scholar 

  7. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov. 2005;4:145–60.

    Article  PubMed  CAS  Google Scholar 

  8. Hirsch M, Ziroli V, Helm M, Massing U. Preparation of small amounts of sterile siRNA-liposomes with high entrapping efficiency by dual asymmetric centrifugation (DAC). J Control Release. 2009;135:80–8.

    Article  PubMed  CAS  Google Scholar 

  9. Gurney JG, Ross JA, Wall DA, Bleyer WA, Severson RK, Robison LL. Infant cancer in the U.S.: histology-specific incidence and trends, 1973 to 1992. J Pediatr Hematol Oncol. 1997;19:428–32.

    Article  PubMed  CAS  Google Scholar 

  10. Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3:203–16.

    Article  PubMed  CAS  Google Scholar 

  11. Pai SI, Lin YY, Macaes B, Meneshian A, Hung CF, Wu TC. Prospects of RNA interference therapy for cancer. Gene Ther. 2006;13:464–77.

    Article  PubMed  CAS  Google Scholar 

  12. Gantert M, Lewrick F, Adrian JE, Rossler J, Steenpass T, Schubert R, et al. Receptor-specific targeting with liposomes in vitro based on sterol-PEG(1300) anchors. Pharm Res. 2009;26:529–38.

    Article  PubMed  CAS  Google Scholar 

  13. Zhang S, Cordon-Cardo C, Zhang HS, Reuter VE, Adluri S, Hamilton WB, et al. Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int J Cancer. 1997;73:42–9.

    Article  PubMed  CAS  Google Scholar 

  14. Hettmer S, Ladisch S, Kaucic K. Low complex ganglioside expression characterizes human neuroblastoma cell lines. Cancer Lett. 2005;225:141–9.

    Article  PubMed  CAS  Google Scholar 

  15. Kazarian T, Jabbar AA, Wen FQ, Patel DA, Valentino LA. Gangliosides regulate tumor cell adhesion to collagen. Clin Exp Metastasis. 2003;20:311–9.

    Article  PubMed  CAS  Google Scholar 

  16. Navid F, Armstrong M, Barfield RC. Immune therapies for neuroblastoma. Cancer Biol Ther. 2009;8:874–82.

    Article  PubMed  CAS  Google Scholar 

  17. Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, Harel-Bellan A, et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 2003;63:3919–22.

    PubMed  CAS  Google Scholar 

  18. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234:466–8.

    PubMed  CAS  Google Scholar 

  19. Adrian JE, Morselt HW, Suss R, Barnert S, Kok JW, Asgeirsdottir SA, et al. Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells. J Control Release. 2010;144:341–9.

    Article  PubMed  CAS  Google Scholar 

  20. Adrian JE, Kamps JA, Scherphof GL, Meijer DK, van Loenen-Weemaes AM, Reker-Smit C, et al. A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats. Biochim Biophys Acta. 2007;1768:1430–9.

    Article  PubMed  CAS  Google Scholar 

  21. Tettamanti G. Ganglioside/glycosphingolipid turnover: new concepts. Glycoconj J. 2004;20:301–17.

    Article  PubMed  CAS  Google Scholar 

  22. Kowanetzand M, Ferrara N. Vascular endothelial growth factor signaling pathways: therapeutic perspective. Clin Cancer Res. 2006;12:5018–22.

    Article  Google Scholar 

  23. Jakovljevic G, Culic S, Stepan J, Bonevski A, Seiwerth S. Vascular endothelial growth factor in children with neuroblastoma: a retrospective analysis. J Exp Clin Cancer Res. 2009;28:143.

    Article  PubMed  Google Scholar 

  24. Massing U, Cicko S, Ziroli V. Dual asymmetric centrifugation (DAC)–a new technique for liposome preparation. J Control Release. 2008;125:16–24.

    Article  PubMed  CAS  Google Scholar 

  25. Ivanov AI. Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol Biol. 2008;440:15–33.

    Article  PubMed  CAS  Google Scholar 

Download references


We thank Sabine Barnert for instrumental assistance in the cryo-TEM analysis as well as Ulrich Massing and Vittorio Ziroli for inspiring discussions and help in adapting the DAC method in our laboratory. The study was financially supported by a grant of the Wilhelm-Sander-Stiftung.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Joanna E. Adrian.

Additional information

Jochen Rössler and Regine Süss contributed equally as senior scientists.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Adrian, J.E., Wolf, A., Steinbach, A. et al. Targeted Delivery to Neuroblastoma of Novel siRNA-anti-GD2-liposomes Prepared by Dual Asymmetric Centrifugation and Sterol-Based Post-Insertion Method. Pharm Res 28, 2261–2272 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: