Our paper focuses on analysis of mechanisms of gene electrotransfer, which has in last years emerged as the most promising non-viral method for delivery of plasmid DNA, oligonucleotides and short RNA molecules. We present new experimental and theoretical results on different steps involved in gene electrotransfer of plated cells and cells in a suspension combined with theoretical analysis of the underlying biophysical phenomena. In our in vitro study we addressed opened questions of this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane, visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency, and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We demonstrate that the crucial step is DNA insertion into the electropermeabilized membrane which is governed by electrophoretic force. The inserted DNA is then slowly transferred into the cytosol, where also nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that number of DNA molecules interacting with the electropermeabilized cell membrane increases with higher DNA concentration or addition of electrophoretic LV pulses to HV, while transfection reach saturation suggesting that there is a maximal number of DNA molecule which can be successfully transferred. We also explain the observed differences between transfection of cell suspensions and plated cells due to more homogeneous size, shape and movement of suspended cells. From the presented results we propose that DNA is most probably translocated through the stable electropores or alternatively through electro-stimulated endocytosis, possibly dependent on pulse parameters. Understanding the relation between permeabilization, electrophoresis, interaction, viability and transfection efficiency can aid to faster optimization of optimal electric protocol for specific application. Objaveljno v reviji Scientific Reports NPG: A'', IF=5.08 http://www.nature.com/scientificreports
COBISS.SI-ID: 10952788
We have showed experimentally and theoretically the importance of interaction of DNA with the cell membrane, which was analyzed in collaboration with group of dr. M.P. Rols, CNRS, Tolouse, with TOTO dye for different pulses and magnesium concentrations. Furthermore, we for the first time systematically compared different electric pulses used of EGT: long duration, short duration and combinations of electropermeabilizing short HV and electrophoretic long LV pulses, all pulses were analyzed in terms od transfection efficiency, viability and interaction with the cell membrane. Our results show that for translation of a given gene electrotransfer protocol to clinical practice it is advisable to use long-duration millisecond pulsing protocols or combination of HV+LV pulses. On the other hand, for certain biotechnological applications where total yield of transfected cells and/or preserved viability is crucial, short duration protocols could be more optimal.
COBISS.SI-ID: 9897812
The mechanisms of DNA entry into cytoplasm during gene electrotransfer are so far not clear. One of hypothesis is electro-stimulated endocytotic uptake. We have developed a protocol which enables us visualization of endocytosis during gene electrotransfer. We have shown that we can observe temperature dependent endocytosis and increased vesiculation after exposure to stress. However, our results do not show increased endocytosis after pulse delivery suggesting that the hypothesis of DNA translocation through hydrophilic pores in the lipid bilayer is more plausible.
COBISS.SI-ID: 8747092
We have developed a first multiscale three-dimensional model of tissue electroporation consisting of random distributed core-shell spheres representing cells with given size distribution. So far different models existed, either describing single cell electroporation or 3D models of "bulk " tissue electroporation, while PI had previosly developed a static solution for connecion of a single cell electroporation with a multicell model. However no model yet had coupled dynamic single-cell solution with a multicellular enviroment, which is presented in this paper. We investigated the temporal evolution of the electric conductivity of such cell system during application of an applied electric field. We compute the volume fraction of electroporated cells, showing a hyperbolic tangent dependence of electric field. The collective physical processes causing the transient permeability of the cell membranes can be understood by analogy with the physics of a two-state system with an external field. The model enables incorporation of cells of different shapes, spacial organisation and sizes.
COBISS.SI-ID: 9058388
Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools, however its application is limited also due to low efficiency of gene-transfer techniques. Therefore two non-viral transfection methods: lipofection and gene electrotransfer were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electrotransfection. Transfection efficiency and cell viability were inversely proportional for both approaches. The importance of optimisation of electric pulse parameters for gene electrotransfer to achieve both maximal transfection efficiency and maximal cell viability was shown in the experiments, where we obtained that only narrow window of possible electric parameters lead to high transfection yield. Too short pulses (8×200 µs) gave almost no transfection, while too long pulses (8 ×10 ms) drastically reduced cell viability. Optimisation was therefore performed for the 8 × 2 ms and 8×5 ms pulsing protocols, and the best results were obtained using 8 × 2 ms with 0.8 kV/cm pulses (44 % transfection, with 74 % viability). High transfection efficiencies were obtained also in several other media (iso-KPB, SMEM, RPMI),while low transfection in DMEM and MEM was attributed to the relatively high calcium concentration (1.8 mM) which reduces viability and consequently electrotransfection. The total yield of transfected cells (% of transfected viable cells) for the optimal lipofection and optimal electrotransfection protocols were similar (32% vs. 32.5%, respectively). Both of these methods are therefore effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in-vivo application to skeletal muscle.
COBISS.SI-ID: 28284967