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Monitoring the Degradation of Reduction-Sensitive Gene Carriers with Fluorescence Spectroscopy and Flow Cytometry

  • Constantin Hozsa
  • Miriam Breunig
  • Achim Göpferich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 991)

Abstract

Polycations like poly(ethylene imine) (PEI) or poly(l-lysine) (pLL) form nanometer-sized complexes with nucleic acids (polyplexes) which can be used for gene delivery. It is known that the properties of these ­carriers can be greatly improved by introducing disulfide bridges on the polymers, thus making them reduction sensitive. However, little is known about how such modified carriers behave intracellularly.

Here, we describe a method that uses the reduction-sensitive fluorescent dye BODIPY FL l-cystine to label PEI and pLL. Our probe is activated under reductive conditions leading to strongly increased fluorescence intensity. Subsequently, we show how the intracellular route of polyplexes made from these labeled polymers can be monitored by flow cytometry.

Key words

Poly(ethylene imine) Poly(l-lysine) Redox-sensitive gene carrier Disulfides Flow cytometry BODIPY FL l-cystine Polyplexes 

References

  1. 1.
    De Laporte L et al (2006) Design of modular non-viral gene therapy vectors. Biomaterials 27:947–954CrossRefGoogle Scholar
  2. 2.
    Wagner E, Kloeckner J (2006) Gene delivery using polymer therapeutics. In: Satchi-Fainaro, Ronit and Duncan, Ruth (eds) Advances in polymer science: polymer therapeutics I. Springer, BerlinGoogle Scholar
  3. 3.
    Won YY et al (2009) Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes. J Control Release 139:88–93CrossRefGoogle Scholar
  4. 4.
    Luten J et al (2008) Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J Control Release 126:97–110CrossRefGoogle Scholar
  5. 5.
    Yadava P et al (2007) Evaluation of two cationic delivery systems for siRNA. Oligonucleotides 17:213–222CrossRefGoogle Scholar
  6. 6.
    Miyata K et al (2004) Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression. J Am Chem Soc 126: 2355–2361CrossRefGoogle Scholar
  7. 7.
    Bauhuber S et al (2009) Delivery of nucleic acids via disulfide-based carrier systems. Adv Mater 21:3286–3306CrossRefGoogle Scholar
  8. 8.
    Dhananjay J et al (2009) Bioreducible polymers for efficient gene and siRNA delivery. Biomed Mater 4:025020CrossRefGoogle Scholar
  9. 9.
    Oupicky D et al (2001) Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo. Gene Ther 8:713–724CrossRefGoogle Scholar
  10. 10.
    Felgner JH et al (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269:2550–2561Google Scholar
  11. 11.
    Lin C, Engbersen JF (2009) The role of the disulfide group in disulfide-based polymeric gene carriers. Expert Opin Drug Deliv 6:421–439CrossRefGoogle Scholar
  12. 12.
    Da Poian AT et al (1998) Kinetics of intracellular viral disassembly and processing probed by Bodipy fluorescence dequenching. J Virol Methods 70:45–58CrossRefGoogle Scholar
  13. 13.
    Lee Y et al (2007) Visualization of the degradation of a disulfide polymer, linear poly ­(ethylenimine sulfide), for gene delivery. Bioconjug Chem 18:13–18CrossRefGoogle Scholar
  14. 14.
    Kunishima M et al (1999) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride: an efficient condensing agent leading to the formation of amides and esters. Tetrahedron 55:13159–13170CrossRefGoogle Scholar
  15. 15.
    Breunig M et al (2008) Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo. J Control Release 130:57–63CrossRefGoogle Scholar
  16. 16.
    Lungwitz U et al (2005) Polyethylenimine-based non-viral gene delivery systems. Eur J Pharm Biopharm 60:247–266CrossRefGoogle Scholar
  17. 17.
    Brissault B et al (2003) Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjug Chem 14:581–587CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Constantin Hozsa
    • 1
  • Miriam Breunig
    • 1
  • Achim Göpferich
    • 1
  1. 1.Lehrstuhl für Pharmazeutische TechnologieUniversität RegensburgRegensburgGermany

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