Mechanistic Analysis of Electroporation-Induced Cellular Uptake of Macromolecules

Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Durham, North Carolina 27708, USA.
Experimental Biology and Medicine (Impact Factor: 2.17). 02/2008; 233(1):94-105. DOI: 10.3181/0704-RM-113
Source: PubMed


Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.

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Available from: Fan Yuan, Aug 04, 2014
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    • "We believe that the expression of PS on the exterior of the membrane occurs as a function of pulse settings, (pulse duration, τ, and electric field strength, ED) therefore, the number and size of induced membrane pores. Dosimetric trends of cells exposed to electrical pulses have been reported [21], [26], [27], [28], [29], but this is the first account of a quantifiable non-linear trend in PS externalization from an nsPEF in live-cells. Additionally, we have shown that rapid increases in intracellular calcium occur at doses far below that required for PS externalization. "
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    ABSTRACT: High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to nsPEF. A series of experiments were performed to determine the dosimetric trends of PS expression caused by nsPEF as a function of pulse duration, τ, delivered field strength, ED, and pulse number, n. To accurately estimate dose thresholds for cellular response, data were reduced to a set of binary responses and ED50s were estimated using Probit analysis. Probit analysis results revealed that PS externalization followed the non-linear trend of (τ*ED (2))(-1) for high amplitudes, but failed to predict low amplitude responses. A second set of experiments was performed to determine the nsPEF parameters necessary to cause observable calcium uptake, using cells preloaded with calcium green (CaGr), and membrane permeability, using FM1-43 dye. Calcium influx and FM1-43 uptake were found to always be observed at lower nsPEF exposure parameters compared to PS externalization. These findings suggest that multiple, higher amplitude and longer pulse exposures may generate pores of larger diameter enabling lateral diffusion of PS; whereas, smaller pores induced by fewer, lower amplitude and short pulse width exposures may only allow extracellular calcium and FM1-43 uptake.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "Some theoretical studies were also accomplished to explain underlying concepts of the cell electroporation (Li and Lin 2011; DeBruin and Krassowska 1999a, b; Talele et al. 2010; Bilska et al. 2000; Movahed and Li 2013; Zhao et al. 2010; Miklavcic and Towhidi 2010). So far, the published studies on the single-cell studies and nanofluidics have explained many aspects of the reversible cell electroporation, such as cell membrane permeabilization (Li and Lin 2011; DeBruin and Krassowska 1999a, b; Talele et al. 2010; Bilska et al. 2000; Zhao et al. 2010; Miklavcic and Towhidi 2010), the electrokinetics in nanochannels (Movahed and Li 2011a, b; Zangle et al. 2010), uptakes of fluid, ions, and macromolecules by the cell during the electroporation (Li and Lin 2011; Movahed and Li 2012a, b; Zaharoff et al. 2008; Granot and Rubinsky 2008), and the electrokinetic motion of nanoparticles in nanochannels. However, up to date, it is not clear yet how the nanoparticles (nanoscale bio-samples such as QDots) from the surrounding liquid will be transported into the opening of the nanopores during electroporation, what forces move these nanoparticles toward the nanopores, and how close the nanoparticle should be in order to be attracted into the opening of the nanopores created on the cell membrane. "
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    ABSTRACT: Nanoparticle transport to the opening of the single nanopore created on the cell membrane during the electroporation is studied. First, the permeabilization of a single cell located in a microchannel is investigated. When the nanopores are created, the transport of the nanoparticles from the surrounding liquid to the opening of one of the created nanopores is examined. It was found that the negatively charged nanoparticles preferably move into the nanopores from the side of the cell membrane that faces the negative electrode. Opposite to the electro-osmotic flow effect, the electrophoretic force tends to draw the negatively charged nanoparticles into the opening of the nanopores. The effect of the Brownian force is negligible in comparison with the electro-osmosis and the electrophoresis. Smaller nanoparticles with stronger surface charge transport more easily to the opening of the nanopores. Positively charged nanoparticles preferably enter the nanopores from the side of the cell membrane that faces the positive electrode. On this side, both the electrophoretic and the electro-osmotic forces are in the same directions and contribute to bring the positively charged particles into the nanopores.
    Full-text · Article · Apr 2013 · Journal of Nanoparticle Research
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    • "The mechanism of electroporation is not well understood. The current thought is that electroporation creates small pores in the plasma membrane, allowing large molecules to enter the cytosol through simple diffusion [17,18]. Originally, polymers in solution were believed to bind to DNA and assist in transport across the membrane [19]. "
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    ABSTRACT: Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use. We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells. Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
    Full-text · Article · Apr 2012 · Biological Procedures Online
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