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Lipid-Based Nanoparticles as Nonviral Gene Delivery Vectors

  • Daniele Pezzoli
  • Anna Kajaste-Rudnitski
  • Roberto Chiesa
  • Gabriele Candiani
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1025)

Abstract

Efficient delivery of nucleic acids into cells is a promising technique to modulate cellular gene expression for therapeutic and research applications. Cationic lipid-based liposomes represent one of the most intensively studied and employed nonviral vectors. They are positively charged at physiological pH and spontaneously self-assemble with polyanionic nucleic acids forming nanoscaled complexes named lipoplexes. Here, we draft a simple protocol for the development, characterization, optimization, and screening of liposomal formulations for in vitro gene delivery. In particular, we report as a practical example a quick method to formulate and extrude nanometer-sized unilamellar cationic vesicles composed of DOTAP as cationic lipid and DOPE as zwitterionic helper lipid at 1:1 molar ratio. The physico-chemical characterization of liposomes and lipoplexes involves the measurement of mean diameter and overall surface charge using Dynamic Light Scattering (DLS) and Laser Doppler Microelectrophoresis. The outlined transfection procedure takes into account several experimental parameters affecting the in vitro performance of gene delivery systems, paying special attention to the charge ratio (CR). Gene delivery effectiveness is evaluated both in terms of transfection efficiency and cytotoxicity of the vector to find the optimal transfection conditions. Importantly, the proposed protocol can be easily shifted to different types of nonviral vectors.

Key words

Gene delivery Cationic liposomes Extrusion Charge ratio Transfection efficiency Cytotoxicity DLS Firefly luciferase 

Notes

Acknowledgments

This work was partly supported by the Politecnico di Milano, 5xmille Junior, “SURGES” Project and by the Italian Ministry for Education, University and Research (MIUR)—FIRB Futuro in Ricerca 2008, Grant RBFR08XH0H (to Dr. Candiani, both), as well as by the Italian Ministry of Health Grant Giovani Ricercatori 2009, GR-2009-1471693 (to Dr. Kajaste-Rudnitski).

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Daniele Pezzoli
    • 1
  • Anna Kajaste-Rudnitski
    • 2
  • Roberto Chiesa
    • 3
  • Gabriele Candiani
    • 1
    • 3
  1. 1.INSTM (National Interuniversity Consortium of Materials Science and Technology)Research Unit Milano PolitecnicoMilanItaly
  2. 2.Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene TherapySan Raffaele Scientific InstituteMilanItaly
  3. 3.Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”Politecnico di MilanoMilanItaly

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