Skip to main content
SpringerLink
Log in

Magnetic and Acoustically Active Microbubbles Loaded with Nucleic Acids for Gene Delivery

  • Protocol
  • First Online:
Nanotechnology for Nucleic Acid Delivery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 948))

Abstract

Targeted gene or drug delivery aims to locally accumulate the active agent and achieve the maximum local therapeutic effect at the target-site while reducing unwanted effects at nontarget sites. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected magnetic nanoparticles (MNPs), phospholipids, and nucleic acid are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres or microbubbles. This manuscript describes the protocols for preparation of magnetic lipospheres and microbubbles for nucleic acid delivery, and it also describes the procedures for labeling the components of the bubbles (lipids, MNPs, and nucleic acids) for the visualization of the vectors and their characterization, such as magnetic responsiveness and ultrasound contrast effects. Protocols are given for the transfection procedure in adherent cells, evaluation of the association of the magnetic vectors with the cells, reporter gene expression analysis, and cell viability assessment.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mykhaylyk O, Antequera YS, Vlaskou D, Plank C (2007) Generation of magnetic nonviral gene transfer agents and magnetofection in vitro. Nat Protoc 2:2391–2411

    Article  CAS  Google Scholar 

  2. Plank C, Anton M, Rudolph C, Rosenecker J, Krotz F (2003) Enhancing and targeting nucleic acid delivery by magnetic force. Expert Opin Biol Ther 3:745–758

    Article  CAS  Google Scholar 

  3. Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG (1987) Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 84:8463–8467

    Article  CAS  Google Scholar 

  4. Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME (1996) Ultrasound-mediated transfection of mammalian cells. Hum Gene Ther 7:1339–1346

    Article  CAS  Google Scholar 

  5. Bao SP, Thrall BD, Miller DL (1997) Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 23:953–959

    Article  CAS  Google Scholar 

  6. Newman CM, Lawrie A, Brisken AF, Cumberland DC (2001) Ultrasound gene therapy: on the road from concept to reality. Echocardiography 18:339–347

    Article  CAS  Google Scholar 

  7. Tata DB, Dunn F, Tindall DJ (1997) Selective clinical ultrasound signals mediate differential gene transfer and expression in two human prostate cancer cell lines: LnCap and PC-3. Biochem Biophys Res Commun 234:64–67

    Article  CAS  Google Scholar 

  8. Lawrie A, Brisken AF, Francis SE, Tayler DI, Chamberlain J, Crossman DC, Cumberland DC, Newman CM (1999) Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro. Circulation 99:2617–2620

    Article  CAS  Google Scholar 

  9. Hernot S, Klibanov AL (2008) Microbubbles in ultrasound-triggered drug and gene delivery. Adv Drug Deliv Rev 60:1153–1166

    Article  CAS  Google Scholar 

  10. Ward M, Wu J, Chiu JF (1999) Ultrasound-induced cell lysis and sonoporation enhanced by contrast agents. J Acoust Soc Am 105:2951–2957

    Article  CAS  Google Scholar 

  11. Wu J, Ross JP, Chiu JF (2002) Reparable sonoporation generated by microstreaming. J Acoust Soc Am 111:1460–1464

    Article  Google Scholar 

  12. Tachibana K, Uchida T, Ogawa K, Yamashita N, Tamura K (1999) Induction of cell-membrane porosity by ultrasound. Lancet 353:1409

    Article  CAS  Google Scholar 

  13. Basta G, Venneri L, Lazzerini G, Pasanisi E, Pianelli M, Vesentini N, Del Turco S, Kusmic C, Picano E (2003) In vitro modulation of intracellular oxidative stress of endothelial cells by diagnostic cardiac ultrasound. Cardiovasc Res 58:156–161

    Article  CAS  Google Scholar 

  14. Dijkmans PA, Juffermans LJ, Musters RJ, van Wamel A, ten Cate FJ, van Gilst W, Visser CA, de Jong N, Kamp O (2004) Microbubbles and ultrasound: from diagnosis to therapy. Eur J Echocardiogr 5:245–256

    Article  CAS  Google Scholar 

  15. Pitt WG, Husseini GA, Staples BJ (2004) Ultrasonic drug delivery–a general review. Expert Opin Drug Deliv 1:37–56

    Article  CAS  Google Scholar 

  16. Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R (2002) Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:372–380

    Article  CAS  Google Scholar 

  17. Bekeredjian R, Chen SY, Frenkel PA, Grayburn PA, Shohet RV (2003) Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart. Circulation 108:1022–1026

    Article  Google Scholar 

  18. Unger EC, Porter T, Culp W, Labell R, Matsunaga T, Zutshi R (2004) Therapeutic applications of lipid-coated microbubbles. Adv Drug Deliv Rev 56:1291–1314

    Article  CAS  Google Scholar 

  19. Tsutsui JM, Xie F, Porter RT (2004) The use of microbubbles to target drug delivery. Cardiovasc Ultrasound 2:23

    Article  Google Scholar 

  20. Vlaskou D, Mykhaylyk O, Krötz F, Hellwig N, Renner R, Schillinger U, Gleich B, Heidsieck A, Schmitz G, Hensel K, Plank C (2010) Magnetic and acoustically active lipospheres for magnetically targeted nucleic acid delivery. Adv Funct Mater 20:3881–3894

    Article  CAS  Google Scholar 

  21. Vlaskou D, Pradhan P, Bergemann C, Klibanov AL, Hensel K, Schmitz G, Plank C, Mykhaylyk O (2010) Magnetic microbubbles: magnetically targeted and ultrasound-triggered vectors for gene delivery in vitro. AIP Conference Proceedings 1311:485–494

    Article  CAS  Google Scholar 

  22. Plank C, Vlaskou D, Schillinger U, Mykhaylyk O, Brill T, Rudolph C, Huth S, Krötz F, Hirschberger J, Bergemann C (2005) Localized nucleic acid delivery using magnetic nanoparticles, Eur. Cells Mater 10(Suppl 5):8

    Google Scholar 

  23. Hellwig N, Plank C, Vlaskou D, Bridell H, Sohn HY, Pohl U, Krotz F (2005) Ultrasound-enhanced microbubble-magnetofection: a new approach for targeted delivery of nucleotides in vivo. J Vasc Res 42:86–87

    Google Scholar 

  24. Vlaskou D, Mykhaylyk O, Giunta R, Neshkova I, Hellwig N, Kroetz F, Bergemann C, Plank C (2006) Magnetic microbubbles: new carriers for localized gene and drug delivery. Mol Ther 13:S290–S290

    Article  Google Scholar 

  25. del Pino P, Munoz-Javier A, Vlaskou D, Rivera Gil P, Plank C, Parak WJ (2010) Gene silencing mediated by magnetic lipospheres tagged with small interfering RNA. Nano Lett 10:3914–3921

    Article  Google Scholar 

  26. Holzbach T, Vlaskou D, Neshkova I, Konerding MA, Wortler K, Mykhaylyk O, Gansbacher B, Machens HG, Plank C, Giunta RE (2010) Non-viral VEGF(165) gene therapy - magnetofection of acoustically active magnetic lipospheres (‘magnetobubbles’) increases tissue survival in an oversized skin flap model. J Cell Mol Med 14:587–599

    CAS  Google Scholar 

  27. Unger EC, McCreery TP, Sweitzer RH, Caldwell VE, Wu Y (1998) Acoustically active lipospheres containing paclitaxel: a new therapeutic ultrasound contrast agent. Invest Radiol 33:886–892

    Article  CAS  Google Scholar 

  28. Stride E, Porter C, Prieto AG, Pankhurst Q (2009) Enhancement of microbubble mediated gene delivery by simultaneous exposure to ultrasonic and magnetic fields. Ultrasound Med Biol 35:861–868

    Article  Google Scholar 

  29. Lentacker I, De Smedt S, Demeester J, Van Marck V, Bracke M, Sanders N (2007) Lipoplex-loaded microbubbles for gene ­delivery: a Trojan horse controlled by ultrasound. Hum Gene Ther 18:1046–1046

    Google Scholar 

  30. Mykhaylyk O, Vlaskou D, Tresilwised N, Pithayanukul P, Möller W, Plank C (2007) Magnetic nanoparticle formulations for DNA and siRNA delivery. J Magn Magn Mat 311:275–281

    Article  CAS  Google Scholar 

  31. Sanchez-Antequera Y, Mykhaylyk O, Thalhammer S, Plank C (2010) Gene delivery to Jurkat T cells using non-viral vectors associated with magnetic nanoparticles. Int J Biomedical Nanoscience and Nanotechnology 1:202–229

    Article  CAS  Google Scholar 

  32. Suzuki M, Mikami T, Matsumoto T, Suzuki S (1977) Preparation and antitumor activity of o-palmitoyldextran phosphates, o-palmitoyldextrans, and dextran phosphate. Carbohydr Res 53:223–229

    Article  CAS  Google Scholar 

  33. Snyder F, Stephens N (1959) A simplified spectrophotometric determination of ester groups in lipids. Biochim Biophys Acta 34:244–245

    Article  CAS  Google Scholar 

  34. Schneider M (1999) Characteristics of SonoVuetrade mark. Echocardiography 16:743–746

    Article  Google Scholar 

  35. Sanchez-Antequera Y, Mykhaylyk O, van Til NP, Cengizeroglu A, de Jong JH, Huston MW, Anton M, Johnston IC, Pojda Z, Wagemaker G, Plank C (2011) Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells. Blood 117:e171–e181

    Article  CAS  Google Scholar 

  36. Mykhaylyk O, Zelphati O, Hammerschmid E, Anton M, Rosenecker J, Plank C (2009) Recent advances in magnetofection and its potential to deliver siRNAs in vitro. Methods Mol Biol 487:111–146

    CAS  Google Scholar 

  37. Mykhaylyk O, Zelphati O, Rosenecker J, Plank C (2008) siRNA delivery by magnetofection. Curr Opin Mol Ther 10:493–505

    CAS  Google Scholar 

  38. Mykhaylyk O, Sanchez-Antequera Y, Vlaskou D, Hammerschmid E, Anton M, Zelphati O, Plank C (2010) Liposomal magnetofection. Methods Mol Biol 605:487–525

    Article  CAS  Google Scholar 

  39. Wilhelm C, Gazeau F, Bacri JC (2002) Magnetophoresis and ferromagnetic resonance of magnetically labeled cells. Eur Biophys J 31:118–125

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Dr. Christian Bergemann (chemicell GmbH, Berlin) for providing a large selection of MNPs for the preparation of the MAALs. The authors greatly appreciate the contribution of Prof. Alexander L. Klibanov from the Department of Biomedical Engineering, Division of Cardiovascular Medicine, University of Virginia, as well as the contributions of Prof. Georg Schmitz and Mrs Karin Hensel from the Institute of Medical Engineering, Department for Electrical Engineering and Information Sciences, Ruhr-University Bochum, for their input on the evaluation of ultrasound responsiveness of MAALs/MMBs. The ultrasound images in phantom gel were obtained with the kind contribution of Priv. Doz. Dr. Klaus Woertler from the Institute of Radiology, Klinikum rechts der Isar, TU Munich. The authors would also like to thank Priv. Doz. Dr. Axel Walch, Dr. Shingi Takenaka, and Mrs. Luise Jennen of the Helmholtz Zentrum München, German Research Center for Environmental Health, for the TEM images of the MAALs and MMBs.

This work was supported by the Nanobiotechnology program (www. nanobio.de) of the German Federal Ministry of Education and Research, Nanobiotechnology grants 13N8186 and 13N8538. Financial support of the German Research Foundation DFG Research Unit FOR917 (Project PL 281/3-1), BMBF project ELA 10/002, and German Excellence Initiative via the “Nanosystems Initiative Munich (NIM)” is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

    Dialechti Vlaskou, Christian Plank & Olga Mykhaylyk

Authors
  1. Dialechti Vlaskou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Plank
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olga Mykhaylyk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dialechti Vlaskou .

Editor information

Editors and Affiliations

  1. , Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandstraße 5-13, Munich, 81377, Germany

    Manfred Ogris

  2. , Department of Pharmaceutical Sciences, Wayne State University, Mack Avenue 259, Detroit, 48201, USA

    David Oupicky

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Vlaskou, D., Plank, C., Mykhaylyk, O. (2013). Magnetic and Acoustically Active Microbubbles Loaded with Nucleic Acids for Gene Delivery. In: Ogris, M., Oupicky, D. (eds) Nanotechnology for Nucleic Acid Delivery. Methods in Molecular Biology, vol 948. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-140-0_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-140-0_15

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-139-4

  • Online ISBN: 978-1-62703-140-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation