JBNB  Vol.2 No.5 , December 2011
Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells
Abstract: Lipidic nanovesicles (so called liposomes) were one the earliest forms of nanovectors. One of their limits was our lack of knowledge on the delivery pathway of their content to the target cell cytoplasm. The present communication describes an efficient way to enhance the delivery. Pulsed electric fields (PEF) are known since the early 80’s to mediate a fusogenic state of plasma membranes when applied to a cell suspension or a tissue. Polykaryons are detected when PEF are applied on cells in contact during or after the pulses. Heterofusion can be obtained when a cell mixture is pulsed. When lipidic nanovesicles, either small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), are electrostatically brought in contact with electropermeabilized cells by a salt bridge, their content is delivered into the cytoplasm in electropermeabilized cells. The PEF parameters are selected to affect specifically the cells leaving the vesicles unaffected. It is the electropermeabilized state of the cell membrane that is the trigger of the merging between the plasma membrane and the lipid bilayer. The present investigation shows that the transfer of macromolecules can be obtained; i.e. 20 kD dextrans can be easily transferred while a direct transfer does not take place under the same electrical parameters. Cell viability was not affected by the treatment. As delivery is present only on electropermeabilized cells, a targeting of the effect is obtained in the volume where the PEF parameters are over the critical value for electropermeabilization. A homogeneous cytoplasm labeling is observed under digitised videomicroscopy. The process is a content and “membrane” mixing, following neither a kiss and run or an endocytotic pathway.
Cite this paper: nullPascal, D. , Valérie, R. , Stefan, W. , Remy, O. , Louise, C. , Pauline, H. , Alain, M. and Justin, T. (2011) Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells. Journal of Biomaterials and Nanobiotechnology, 2, 527-532. doi: 10.4236/jbnb.2011.225063.

[1]   E. Neumann, G. Gerisch and K. Optaz, “Cell Fusion Induced by High Electric Impulse Applied to Dictyostelium,” Naturwissenschaften, Vol. 67, 1980, pp. 414-415. doi:10.1007/BF00405493

[2]   J. Teissie, V. P. Knutson, T. Y. Tsong and M. D. Lane, “Electric Pulse-Induced Fusion of 3T3 Cells in Mono- layer Culture,” Science, Vol. 216, 1982, pp. 537-538. doi:10.1126/science.7071601

[3]   H. Mekid and L. M. Mir, “In Vivo Cell Electrofusion,” Biochimica et Biophysica Acta (BBA)—General Subjects, Vol. 1524, No. 2-3, 2000, pp. 118-130. doi:10.1016/S0304-4165(00)00145-8

[4]   J. Teissie and M. P. Rols, “Fusion of Mammalian Cells in Culture Is Obtained by Creating the Contact between Cells after Their Electropermeabilization,” Biochemical and Biophysical Research Communications, Vol. 140, No. 1, 1986, pp. 258-266. doi:10.1016/0006-291X(86)91084-3

[5]   C. Ramos, D. Bonato, M. Winterhalter, T. Stegmann and J. Teissié, “Spontaneous Lipid Vesicle Fusion with Electropermeabilized Cells,” FEBS Letters, Vol. 518, 2002, pp. 135-138. doi:10.1016/S0014-5793(02)02676-5

[6]   J. Heuving, F. Pincet and S. Cribier, “Hemifusion and Fusion of Giant Vesicles Induced by Reduction of Inter-Membrane Distance,” The European Physical Journal E: Soft Matter and Biological Physics, Vol. 14, No. 3, 2004, pp. 269-276. doi:10.1140/epje/i2003-10151-2

[7]   D. Tareste, F. Pincet, E. Perez, S. Rickling, C. Mioskowski and L. Lebeau, “Energy of Hydrogen Bonds Probed by the Adhesion of Functionalized Lipid Layers,” Biophysical Journal, Vol. 83, No. 6, 2002, pp. 3675-3681. doi:10.1016/S0006-3495(02)75367-8

[8]   F. Pincet, L. Lebeau and S. Cribier, “Short-Range Specific Forces Are Able to Induce Hemifusion,” European Biophysics Journal, Vol. 30, No. 2, 2001, pp. 91-97. doi:10.1007/s002490100131

[9]   C. G. Schuette, K. Hatsuzawa, M. Margittai, A. Stein, D. Riedel, P. Küster, M. K?nig, C. Seidel and R. Jahn, “Determinants of Liposome Fusion Mediated by Synaptic SNARE Proteins,” Proceedings of the National Academy of Sciences USA, Vol. 101, No. 9, 2004, pp. 2858-2863. doi:10.1073/pnas.0400044101

[10]   A. Cypionka, A. Stein, J. M. Hernandez, H. Hippchen, R. Jahn and P. J. Walla, “Discrimination between Docking and Fusion of Liposomes Reconstituted with Neuronal SNARE-Proteins Using FCS,” Proceedings of the National Academy of Sciences USA, Vol. 106, No. 44, 2009, pp. 18575-18580. doi:10.1073/pnas.0906677106

[11]   W. Xu and F. Pincet, “Quantification of Phase Transitions of Lipid Mixtures from Bilayer to Non-Bilayer Structures: Model, Experimental Validation and Implication on Membrane Fusion,” Chemistry and Physics of Lipids, Vol. 163, No. 3, 2010, pp. 280-285. doi:10.1016/j.chemphyslip.2009.12.002

[12]   S. Martens, M. M. Kozlov and H. T. McMahon, “How Synaptotagmin Promotes Membrane Fusion,” Science, Vol. 316, No. 5828, 2007, pp. 1205-1208. doi:10.1126/science.1142614

[13]   K. Rosenheck, “Evaluation of the Electrostatic Field Strength at the Site of Exocytosis in Adrenal Chromaffin Cells,” Biophysical Journal, Vol. 75, 1998, pp. 1237- 4123. doi:10.1016/S0006-3495(98)74043-3

[14]   P. Luitel, D. F. Schroeter and J. W. Powell, “Self-Electroporation as a Model for Fusion Pore Formation,” Journal of Biomolecular Structure & Dynamics, Vol. 24, No. 5, 2007, pp. 495-503.

[15]   M. P. Rols and J. Teissie, “Electropermeabilization of Mammalian Cells to Macromolecules: Control by Pulse Duration,” Biophysical Journal, Vol. 75, No. 3, 1998, pp. 1415-1423. doi:10.1016/S0006-3495(98)74060-3

[16]   E. R. Travis and R. M. Wightman, “Spatio-Temporal Resolution of Exocytosis from Individual Cells,” Annual Review of Biophysics and Biomolecular Structure, Vol. 27, 1998, pp. 77-103. doi:10.1146/annurev.biophys.27.1.77

[17]   E. Alés, L. Tabares, J. M. Poyato, V. Valero, M. Lindau and G. Alvarez de Toledo, “High Calcium Concentrations Shift the Mode of Exocytosis to the Kiss-and-Run Mechanism,” Nature Cell Biology, Vol. 1, No. 1, 1999, pp. 40-44. doi:10.1038/9012

[18]   S. O. Rizzoli and R. Jahn, “Kiss-and-Run, Collapse and ‘Readily Retrievable’ Vesicles,” Traffic, Vol. 8, No. 9, 2007, pp. 1137-1144. doi:10.1111/j.1600-0854.2007.00614.x

[19]   J. Teissie and C. Ramos, “Correlation between Electric Field Pulse Induced Long-Lived Permeabilization and Fusogenicity in Cell Membranes,” Biophysical Journal, Vol. 74, 1998, pp. 1889-1898. doi:10.1016/S0006-3495(98)77898-1

[20]   L. V. Chernomordik, D. Papahadjopoulos and T. Y. Tsong, “Increased Binding of Liposomes to Cells by Electric Treatment,” Biochimica et Biophysica Acta (BBA)—General Subjects, Vol. 1070, No. 1, 1991, pp. 193-197. doi:10.1016/0005-2736(91)90163-3

[21]   J. Teissie, M. Golzio and M. P. Rols, “Mechanisms of Cell Membrane Electropermeabilization: A Minireview of Our Present (Lack of ?) Knowledge,” Biochimica et Biophysica Acta (BBA)—General Subjects, Vol. 1724, No. 3, 2005, pp. 270-280. doi:10.1016/j.bbagen.2005.05.006

[22]   M. Miteva, M. Andersson, A. Karshikoff and G. Otting, “Molecular Electroporation: A Unifying Concept for the Description of Membrane Pore Formation by Antibacterial Peptides, Exemplified with NK-lysin,” FEBS Letters, Vol. 462, No. 1-2, 1999, pp. 155-158. doi:10.1016/S0014-5793(99)01520-3

[23]   T. Hampton, “Electric Pulses Help with Chemotherapy may Open New Paths for Other Agents,” Journal of the American Medical Association (JAMA), Vol. 305, No. 6, 2011, pp. 549-551. doi:10.1001/jama.2011.92