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

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ABSTRACT

Purpose

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

Methods

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.

Results

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.

Conclusions

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.

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Abbreviations

Ab:

antibody

Chol:

cholesterol

cryo-TEM:

cryo-transmission electron microscope

DAC:

dual asymmetric centrifugation

DDAB:

dimethyldioctadecylammonium (bromide salt)

DSPE-PEG2000 :

2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]

FCS:

fetal calf serum

GD2:

disialoganglioside

HN buffer:

HEPES/NaCl buffer

HSPC:

hydrogenated soybean phosphatidylcholine

L:

liposomes

PBS:

phosphate buffered saline

POPC:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

Rhd-PE:

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

siRNA:

short interfering RNA

SPIT:

sterol-based post-insertion technique

TL:

total lipid

VEGF-A:

vascular endothelial growth factor A

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ACKNOWLEDGMENTS

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.

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Authors and Affiliations

  1. Department of Pharmaceutical Technology & Biopharmacy, Albert-Ludwigs University, Sonnenstr. 5, 79104, Freiburg, Germany

    Joanna E. Adrian, Annette Steinbach & Regine Süss

  2. Center of Paediatrics & Adolescent Medicine Division of Paediatric Haematology & Oncology, University Medical Center Freiburg, Mathildenstr. 1, 79106, Freiburg, Germany

    Joanna E. Adrian, Alexander Wolf & Jochen Rössler

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  1. Joanna E. Adrian
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  2. Alexander Wolf
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  3. Annette Steinbach
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  4. Jochen Rössler
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  5. Regine Süss
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Corresponding author

Correspondence to Joanna E. Adrian.

Additional information

Jochen Rössler and Regine Süss contributed equally as senior scientists.

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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). https://doi.org/10.1007/s11095-011-0457-y

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  • DOI: https://doi.org/10.1007/s11095-011-0457-y

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