Breton M.,University Paris - Sud |
Breton M.,CNRS Gustave Roussy Institute |
Breton M.,Laboratoire Of Vectorologie Et Therapeutiques Anticancereuses |
Amirkavei M.,University Paris - Sud |
And 5 more authors.
Journal of Membrane Biology | Year: 2015
Giant unilamellar vesicles (GUV) are widely used cell membrane models. GUVs have a cell-like diameter and contain the same phospholipids that constitute cell membranes. The most frequently used protocol to obtain these vesicles is termed electroformation, since key steps of this protocol consist in the application of an electric field to a phospholipid deposit. The potential oxidation of unsaturated phospholipids due to the application of an electric field has not yet been considered even though the presence of oxidized lipids in the membrane of GUVs could impact their permeability and their mechanical properties. Thanks to mass spectrometry analyses, we demonstrated that the electroformation technique can cause the oxidation of polyunsaturated phospholipids constituting the vesicles. Then, using flow cytometry, we showed that the amplitude and the duration of the electric field impact the number and the size of the vesicles. According to our results, the oxidation level of the phospholipids increases with their level of unsaturation as well as with the amplitude and the duration of the electric field. However, when the level of lipid oxidation exceeds 25 %, the diameter of the vesicles is decreased and when the level of lipid oxidation reaches 40 %, the vesicles burst or reorganize and their rate of production is reduced. In conclusion, the classical electroformation method should always be optimized, as a function of the phospholipid used, especially for producing giant liposomes of polyunsaturated phospholipids to be used as a cell membrane model. © 2015, Springer Science+Business Media New York.
Mir L.M.,University Paris - Sud |
Mir L.M.,French National Center for Scientific Research |
Mir L.M.,Laboratoire Of Vectorologie Et Therapeutiques Anticancereuses
Methods in Molecular Biology | Year: 2014
Thirty years after the publication of the first report on gene electrotransfer in cultured cells by the delivery of delivering electric pulses, this technology is starting to be applied to humans. In 2008, at the time of the publication of the first edition of this book, reversible cell electroporation for gene transfer and gene therapy (nucleic acids electrotransfer) was at a cross roads in its development. In 5 years, basic and applied developments have brought gene electrotransfer into a new status. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here, as an introduction to the large range of applications described in this book. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted, as well as the large range of new specialized electrodes, developed also in the frame of the other electroporation-based treatments (electrochemotherapy). Indeed, electric pulses are now routinely applied for localized drug delivery in the treatment of solid tumors by electrochemotherapy. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed: noticeably, the first molecular description of the crossing of a lipid membrane by a nucleic acid was reported in 2012. The progress in the understanding of cell electroporation as well as developments of technological aspects, in silico, in vitro and in vivo, have contributed to bring gene electrotransfer development to the clinical stage. However, spreading of the technology will require not only more clinical trials but also further homogenization of the protocols and the preparation and validation of Standard Operating Procedures. © 2014 Springer Science+Business Media New York.