Skip to main content

Advertisement

SpringerLink
Log in

Optimization of Magnetic Nanoparticle-Assisted Lentiviral Gene Transfer

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Targeting of specific cells and tissues is of great interest for clinical relevant gene- and cell-based therapies. We use magnetic nanoparticles (MNPs) with a ferrimagnetic core (Fe3O4) with different coatings to optimize MNP-assisted lentiviral gene transfer with focus on different endothelial cell lines.

Methods

Lentiviral vector (LV)/MNP binding was characterized for various MNPs by different methods (e.g. magnetic responsiveness measurement). Transduced cells were analyzed by flow cytometry, fluorescence microscopy and iron recovery. Cell transduction and cell positioning under physiological flow conditions were performed using different in vitro and ex vivo systems.

Results

Analysis of diverse MNPs with different coatings resulted in identification of nanoparticles with improved LV association and enhanced transduction properties of complexes in several endothelial cell lines. The magnetic moments of LV/MNP complexes are high enough to achieve local gene targeting of perfused endothelial cells. Perfusion of a mouse aorta with LV/MNP transduced cells under clinically relevant flow conditions led to local cell attachment at the intima of the vessel.

Conclusion

MNP-guided lentiviral transduction of endothelial cells can be significantly enhanced and localized by using optimized MNPs.

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

Access this article

Log in via an institution

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

Similar content being viewed by others

Abbreviations

bPAEC:

bovine pulmonary arterial endothelial cell

CMV:

cytomegalovirus

DMEM:

Dulbecco’s modified eagle’s medium

DMF:

dimethylformamide

DMSO:

dimethyl sulfoxide

eGFP:

enhanced green fluorescent protein

EPC:

endothelial progenitor cell

FCS:

fetal calf serum

FSA:

lithium 3-[2-(perfluoroalkyl)ethylthio]propionate

HBSS:

Hank’s balanced salt solution

HBSS++:

Hank’s balanced salt solution + MgCl2 and CaCl2

hlEPC:

human late endothelial progenitor cell

HUVEC:

human umbilical vein endothelial cell

IP:

infectious particles

LDH:

lactate dehydrogenase

LV:

lentiviral vector

meEPC:

murine embryonal endothelial progenitor cell

MNP:

magnetic nanoparticle

MOI:

multiplicity of infection

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromid

PALD:

palmitoyldextrane

PB:

polybrene

PBS:

phosphate buffered saline

PEI:

polyethylenimine

PFA:

paraformaldehyde

RT:

reverse transcriptase

SDS:

sodium dodecyl sulfate

SO:

silicon oxide

SiOx/Phosphonate:

silicon oxide layer with surface phosphonate groups

V’30:

LV transduction without MNPs for 30 min

V’ON:

LV transduction without MNPs overnight

VP:

viral particles

VSV.G:

glycoprotein of vesicular stomatitis virus

References

  1. Pfeifer A, Verma IM. Virus vectors and their application. In: Howley PM, Knipe DM, Griffin D, Lamb RA, Martin A, Roizman B, Straus SE, editors. Fields Virology. Philadelphia: Lippincott-Raven Publishers; 2001. p. 469–91.

    Google Scholar 

  2. Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol. 1998;9(5):457–63.

    Article  PubMed  CAS  Google Scholar 

  3. Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent. Gene Ther. 2000;7(1):20–3.

    Article  PubMed  CAS  Google Scholar 

  4. Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annu Rev Biochem. 2005;74:711–38.

    Article  PubMed  CAS  Google Scholar 

  5. Goff SP. Retroviridae: the viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors. Fields virology, vol 2. 4th ed. Philadelphia: Lippincott-Raven Publishers; 2001. p. 1871–939.

    Google Scholar 

  6. Desrosiers RC. Nonhuman lentiviruses. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, Straus SE, editors. Fields Virology, vol 2. 4th ed. Philadelphia: Lippincott-Raven Publishers; 2001. p. 2095–121.

    Google Scholar 

  7. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature. 2010;467(7313):318–22.

    Article  PubMed  CAS  Google Scholar 

  8. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009;326(5954):818–23.

    Article  PubMed  CAS  Google Scholar 

  9. Pariente N, Morizono K, Virk MS, Petrigliano FA, Reiter RE, Lieberman JR, et al. A novel dual-targeted lentiviral vector leads to specific transduction of prostate cancer bone metastases in vivo after systemic administration. Mol Ther. 2007;15(11):1973–81.

    Article  PubMed  CAS  Google Scholar 

  10. Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Kruger A, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 2002;9(2):102–9.

    Article  PubMed  CAS  Google Scholar 

  11. Hofmann A, Wenzel D, Becher UM, Freitag DF, Klein AM, Eberbeck D, et al. Combined targeting of lentiviral vectors and positioning of transduced cells by magnetic nanoparticles. Proc Natl Acad Sci U S A. 2009;106(1):44–9.

    Article  PubMed  CAS  Google Scholar 

  12. Alexiou C, Arnold W, Klein RJ, Parak FG, Hulin P, Bergemann C, et al. Locoregional cancer treatment with magnetic drug targeting. Cancer Res. 2000;60(23):6641–8.

    PubMed  CAS  Google Scholar 

  13. Strebhardt K, Ullrich A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat Rev Cancer. 2008;8(6):473–80.

    Article  PubMed  CAS  Google Scholar 

  14. Chorny M, Fishbein I, Yellen BB, Alferiev IS, Bakay M, Ganta S, et al. Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields. Proc Natl Acad Sci U S A. 2010;107(18):8346–51.

    Article  PubMed  CAS  Google Scholar 

  15. Polyak B, Fishbein I, Chorny M, Alferiev I, Williams D, Yellen B, et al. High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents. Proc Natl Acad Sci U S A. 2008;105(2):698–703.

    Article  PubMed  CAS  Google Scholar 

  16. Hatzopoulos AK, Folkman J, Vasile E, Eiselen GK, Rosenberg RD. Isolation and characterization of endothelial progenitor cells from mouse embryos. Development. 1998;125(8):1457–68.

    PubMed  CAS  Google Scholar 

  17. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet. 2000;25(2):217–22.

    Article  PubMed  CAS  Google Scholar 

  18. Pfeifer A, Hofmann A. Lentiviral transgenesis. Methods Mol Biol. 2009;530:391–405.

    Article  PubMed  CAS  Google Scholar 

  19. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998;72(11):8463–71.

    PubMed  CAS  Google Scholar 

  20. Mykhaylyk O, Antequera YS, Vlaskou D, Plank C. Generation of magnetic nonviral gene transfer agents and magnetofection in vitro. Nat Protoc. 2007;2(10):2391–411.

    Article  PubMed  CAS  Google Scholar 

  21. Mykhaylyk O, Sanchez-Antequera Y, Vlaskou D, Hammerschmid E, Anton M, Zelphati O, et al. Liposomal magnetofection. Methods Mol Biol. 2010;605:487–525.

    Article  PubMed  CAS  Google Scholar 

  22. Mykhaylyk O, Vlaskou D, Tresilwised N, Pithayanukul P, Möller W, Plank C. Magnetic nanoparticle formulations for DNA and siRNA delivery. J Magn Magn Mat. 2007;311(1):275–81.

    Article  CAS  Google Scholar 

  23. Sanchez-Antequera Y, Mykhaylyk O, Thalhammer S, Plank C. Gene delivery to Jurkat T cells using non-viral vectors associated with magnetic nanoparticles. Int J Biomedical Nanoscience and Nanotechnology. 2010;1(2/3/4):202–29.

    Article  CAS  Google Scholar 

  24. Mykhaylyk O, Zelphati O, Hammerschmid E, Anton M, Rosenecker J, Plank C. Recent advances in magnetofection and its potential to deliver siRNAs in vitro. Methods Mol Biol. 2009;487:111–46.

    PubMed  CAS  Google Scholar 

  25. Kowalski JB, Tallentire A. Substantiation of 25 kGy as a sterilization dose: a rational approach to establishing verification dose. Radiat Phys Chem. 1999;54(1):55–64.

    Article  CAS  Google Scholar 

  26. Krill CE, Birringer R. Estimating grain-size distributions in nanocrystalline materials from X-ray diffraction profile analysis. Philos Mag A. 1998;77(3):621–40.

    Article  CAS  Google Scholar 

  27. Mykhaylyk O, Zelphati O, Rosenecker J, Plank C. siRNA delivery by magnetofection. Curr Opin Mol Ther. 2008;10(5):493–505.

    PubMed  CAS  Google Scholar 

  28. Mykhaylyk O, Steingotter A, Perea H, Aigner J, Botnar R, Plank C. Nucleic acid delivery to magnetically-labeled cells in a 2D array and at the luminal surface of cell culture tube and their detection by MRI. J Biomed Nanotechnol. 2009;5(6):692–706.

    Article  PubMed  CAS  Google Scholar 

  29. Haim H, Steiner I, Panet A. Synchronized infection of cell cultures by magnetically controlled virus. J Virol. 2005;79(1):622–5.

    Article  PubMed  CAS  Google Scholar 

  30. Benninghoff A, Drenckhahn D. Anatomie, Makroskopische Anatomie, Embryologie und Histologie des Menschen. 16 ed. Drenckhahn D, editor. München: Elsevier; 2004.

  31. Dimitrov DS. Virus entry: molecular mechanisms and biomedical applications. Nat Rev Microbiol. 2004;2(2):109–22.

    Article  PubMed  Google Scholar 

  32. Tempaku A. Random Brownian motion regulates the quantity of human immunodeficiency virus type-1 (HIV-1) attachment and infection to target cell. Journal of Health Science. 2005;51(2):237–41.

    Article  CAS  Google Scholar 

  33. Chomoucka J, Drbohlavova J, Huska D, Adam V, Kizek R, Hubalek J. Magnetic nanoparticles and targeted drug delivering. Pharmacological Research. 2010;62(2):144–9.

    Article  PubMed  CAS  Google Scholar 

  34. Shubayev VI, Pisanic Ii TR, Jin S. Magnetic nanoparticles for theragnostics. Adv Drug Deliver Rev. 2009;61(6):467–77.

    Article  CAS  Google Scholar 

  35. Al-Deen FN, Ho J, Selomulya C, Ma C, Coppel R. Superparamagnetic nanoparticles for effective delivery of malaria DNA vaccine. Langmuir. 2011;27(7):3703–12.

    Article  PubMed  CAS  Google Scholar 

  36. Chorny M, Fishbein I, Alferiev I, Levy RJ. Magnetically responsive biodegradable nanoparticles enhance adenoviral gene transfer in cultured smooth muscle and endothelial cells. Mol Pharm. 2009;6(5):1380–7.

    Article  PubMed  CAS  Google Scholar 

  37. Mykhaylyk O, Sanchez-Antequera Y, Tresilwised N, Doblinger M, Thalhammer S, Holm PS, et al. Engineering magnetic nanoparticles and formulations for gene delivery. J Control Release. 2011;148(1):e63–4.

    Article  Google Scholar 

  38. Plank C, Scherer F, Schillinger U, Bergemann C, Anton M. Magnetofection: enhancing and targeting gene delivery with superparamagnetic nanoparticles and magnetic fields. J Liposome Res. 2003;13(1):29–32.

    Article  PubMed  CAS  Google Scholar 

  39. Anton M, Wolf A, Mykhaylyk O, Koch C, Gansbacher B, Plank C. Optimizing Adenoviral transduction of endothelial cells under flow conditions. Pharm Res. 2011. doi:10.1007/s11095-011-0631-2.

  40. Sanchez-Antequera Y, Mykhaylyk O, van Til NP, Cengizeroglu A, de Jong JH, Huston MW, et al. Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells. Blood. 2011;117(16):e171–81.

    Article  PubMed  CAS  Google Scholar 

  41. Tresilwised N, Pithayanukul P, Holm PS, Schillinger U, Plank C, Mykhaylyk O. Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes. Biomaterials. 2011;33:256–69.

    Article  PubMed  Google Scholar 

  42. Agopian K, Wei BL, Garcia JV, Gabuzda D. A hydrophobic binding surface on the human immunodeficiency virus type 1 Nef core is critical for association with p21-activated kinase 2. J Virol. 2006;80(6):3050–61.

    Article  PubMed  CAS  Google Scholar 

  43. Shin S, Shea LD. Lentivirus immobilization to nanoparticles for enhanced and localized delivery from hydrogels. Mol Ther. Apr;18(4):700-6.

  44. Rathel T, Mannell H, Pircher J, Gleich B, Pohl U, Krötz F. Magnetic stents retain nanoparticle-bound antirestenotic drugs transported by lipid microbubbles. Pharm Res. 2011. doi:10.1007/s11095-011-0643-y.

Download references

Acknowledgements

For excellent technical assistance we are thankful to Christina Stichnote, Institute of Pharmacology and Toxicology, University of Bonn, Germany and Anja Wolf, Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der TU München. For providing human and murine EPCs we thank Ulrich Becher and Katharina Peske, Institute of Internal Medicine II, University of Bonn, Germany and Christian Kupatt, Institute of Internal Medicine I, University of Munich, Germany.

This work was supported by the German Research Foundation within the DFG Research Unit FOR917, by the North Rhine-Westphalia (NRW) International Graduate Research School BIOTECH-PHARMA and by the Ministry of Innovation, Science, Research and Technology of the State of NRW within the junior research group of Daniela Wenzel (“Magnetic nanoparticles (MNPs) - endothelial cell replacement in injured vessels”).

Author information

Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, Biomedical Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany

    Christina Trueck, Katrin Zimmermann & Alexander Pfeifer

  2. NRW International Graduate Research School BIOTECH-PHARMA, Bonn, Germany

    Christina Trueck, Katrin Zimmermann, Bernd K. Fleischmann & Alexander Pfeifer

  3. Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, 81675, Munich, Germany

    Olga Mykhaylyk & Martina Anton

  4. Institute of Physiology I, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany

    Sarah Vosen, Daniela Wenzel & Bernd K. Fleischmann

  5. PharmaCenter Bonn, University of Bonn, Bonn, Germany

    Bernd K. Fleischmann & Alexander Pfeifer

Authors
  1. Christina Trueck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katrin Zimmermann
    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

  4. Martina Anton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sarah Vosen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daniela Wenzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bernd K. Fleischmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alexander Pfeifer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander Pfeifer.

Additional information

Christina Trueck and Katrin Zimmermann contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure S1

Transduction efficiency of various LV/MNP complexes. HUVECs were transduced with LV/MNP (300 fg Fe per VP, MOI 5) with different MNPs and percentage of eGFP positive cells was determined via flow cytometry 48 h after transduction. Controls are: buffer without LVs and MNPs (HBSS++), buffer with LV and without MNPs (LV), LV without MNP but transduction overnight at 37°C in medium (V’ON). n > 3, mean+SEM. (JPEG 15 kb)

High resolution image (TIFF 2145 kb)

Supplementary Figure S2

Dose–response curve of LV/MNP complexes in different solvents. LVs and different concentrations of MNPs (1 to 1,000 fg Fe per VP) were incubated in HBSS++, FCS (serum) or 0.9% (w/v) NaCl (0.15 M) or incubated in HBSS++ with subsequent addition of 1:1 FCS (HBSS++/serum) and transferred to 24well plate in a magnetic gradient field. The supernatant (=uncomplexed virus) was analyzed using p24 ELISA. As positive control LV without MNPs was used and the percentage of complexed virus was calculated. MNPs used for LV complexation were either the positively charged PEI-Mag2 (a) or the negatively charged PALD2-Mag1 (b). n ≥ 3, mean±SEM. (JPEG 16 kb)

High resolution image (TIFF 5.26 MB)

Supplementary Figure S3

Analysis of transduction time of LV/MNP complexes in a magnetic gradient field. HUVECs were transduced with LV/MNP (300 fg Fe per VP, MOI 5) for different time spans (5 to 60 min) and percentage of eGFP positive cells was determined via flow cytometry 48 h after transduction. Controls are: buffer without LVs and MNPs (HBSS++), buffer with LV and without MNPs (LV), LV without MNP but transduction overnight at 37°C in medium (V’ON). n = 3, mean±SEM. (JPEG 9 kb)

High resolution image (TIFF 2313 kb)

Supplementary Table S1

Characteristics of the MNPs (DOCX 30 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Trueck, C., Zimmermann, K., Mykhaylyk, O. et al. Optimization of Magnetic Nanoparticle-Assisted Lentiviral Gene Transfer. Pharm Res 29, 1255–1269 (2012). https://doi.org/10.1007/s11095-011-0660-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-011-0660-x

Key Words

Search

Navigation