Nanoscale, Electric Field-Driven Water Bridges in Vacuum Gaps and Lipid BilayersHo, Ming-Chak; Levine, Zachary; Vernier, P.
doi: 10.1007/s00232-013-9549-4pmid: 23644990
Formation of a water bridge across the lipid bilayer is the first stage of pore formation in molecular dynamic (MD) simulations of electroporation, suggesting that the intrusion of individual water molecules into the membrane interior is the initiation event in a sequence that leads to the formation of a conductive membrane pore. To delineate more clearly the role of water in membrane permeabilization, we conducted extensive MD simulations of water bridge formation, stabilization, and collapse in palmitoyloleoylphosphatidylcholine bilayers and in water–vacuum–water systems, in which two groups of water molecules are separated by a 2.8 nm vacuum gap, a simple analog of a phospholipid bilayer. Certain features, such as the exponential decrease in water bridge initiation time with increased external electric field, are similar in both systems. Other features, such as the relationship between water bridge lifetime and the diameter of the water bridge, are quite different between the two systems. Data such as these contribute to a better and more quantitative understanding of the relative roles of water and lipid in membrane electropore creation and annihilation, facilitating a mechanism-driven development of electroporation protocols. These methods can be extended to more complex, heterogeneous systems that include membrane proteins and intracellular and extracellular membrane attachments, leading to more accurate models of living cells in electric fields.
Biological Properties of Melanoma and Endothelial Cells after Plasmid AMEP Gene Electrotransfer Depend on Integrin Quantity on CellsBosnjak, Masa; Prosen, Lara; Dolinsek, Tanja; Blagus, Tanja; Markelc, Bostjan; Cemazar, Maja; Bouquet, Celine; Sersa, Gregor
doi: 10.1007/s00232-013-9550-ypmid: 23649038
The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Antiangiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antiangiogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plasmid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on MatrigelTM or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.
Planning of Electroporation-Based Treatments Using Web-Based Treatment-Planning SoftwarePavliha, Denis; Kos, Bor; Marčan, Marija; Županič, Anže; Serša, Gregor; Miklavčič, Damijan
doi: 10.1007/s00232-013-9567-2pmid: 23780414
Electroporation-based treatment combining high-voltage electric pulses and poorly permanent cytotoxic drugs, i.e., electrochemotherapy (ECT), is currently used for treating superficial tumor nodules by following standard operating procedures. Besides ECT, another electroporation-based treatment, nonthermal irreversible electroporation (N-TIRE), is also efficient at ablating deep-seated tumors. To perform ECT or N-TIRE of deep-seated tumors, following standard operating procedures is not sufficient and patient-specific treatment planning is required for successful treatment. Treatment planning is required because of the use of individual long-needle electrodes and the diverse shape, size and location of deep-seated tumors. Many institutions that already perform ECT of superficial metastases could benefit from treatment-planning software that would enable the preparation of patient-specific treatment plans. To this end, we have developed a Web-based treatment-planning software for planning electroporation-based treatments that does not require prior engineering knowledge from the user (e.g., the clinician). The software includes algorithms for automatic tissue segmentation and, after segmentation, generation of a 3D model of the tissue. The procedure allows the user to define how the electrodes will be inserted. Finally, electric field distribution is computed, the position of electrodes and the voltage to be applied are optimized using the 3D model and a downloadable treatment plan is made available to the user.
On the Electroporation Thresholds of Lipid Bilayers: Molecular Dynamics Simulation InvestigationsPolak, Andraž; Bonhenry, Daniel; Dehez, François; Kramar, Peter; Miklavčič, Damijan; Tarek, Mounir
doi: 10.1007/s00232-013-9570-7pmid: 23780415
Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the “electrical” properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.
Nanosecond Electric Pulse Effects on Gene ExpressionChopinet, Louise; Batista-Napotnik, Tina; Montigny, Audrey; Rebersek, Matej; Teissié, Justin; Rols, Marie-Pierre; Miklavčič, Damijan
doi: 10.1007/s00232-013-9579-ypmid: 23831956
Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.
Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coliHaberl, Saša; Jarc, Marko; Štrancar, Aleš; Peterka, Matjaž; Hodžić, Duša; Miklavčič, Damijan
doi: 10.1007/s00232-013-9580-5pmid: 23831957
The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.