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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: 2.23). 02/2008; 233(1):94-105. DOI: 10.3181/0704-RM-113
Source: PubMed

ABSTRACT 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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    • "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.
    Journal of Nanoparticle Research 04/2013; 15(4). DOI:10.1007/s11051-013-1511-y · 2.28 Impact Factor
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    • "However, diffusion is only effective for small molecules because bigger molecules like plasmid DNA diffuse slower and transmembrane transport is limited by steric hindrances. Convective transport, which occurs when a constituent of a fluid is carried along with the fluid, has also been proposed as a mechanism involved in the cellular uptake of uncharged molecules (Zaharoff et al., 2008). This would be possible due to electric field-induced cell-movement, cell swelling and electrodeformation. "
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    ABSTRACT: Electroporation is a widely used technique for enhancing the efficiency of DNA delivery into cells. Application of electric pulses after local injection of DNA temporarily opens cell membranes and facilitates DNA uptake. Delivery of plasmid DNA by electroporation to alter gene expression in tissue has also been explored in vivo. This approach may constitute an alternative to viral gene transfer, or to transgenic or knock-out animals. Among the most frequently electroporated target tissues are skin, muscle, eye, and tumors. Moreover, different regions in the central nervous system (CNS), including the developing neural tube and the spinal cord, as well as prenatal and postnatal brain have been successfully electroporated. Here, we present a comprehensive review of the literature describing electroporation of the CNS with a focus on the adult brain. In addition, the mechanism of electroporation, different ways of delivering the electric pulses, and the risk of damaging the target tissue are highlighted. Electroporation has been successfully used in humans to enhance gene transfer in vaccination or cancer therapy with several clinical trials currently ongoing. Improving the knowledge about in vivo electroporation will pave the way for electroporation-enhanced gene therapy to treat brain carcinomas, as well as CNS disorders such as Alzheimer's disease, Parkinson's disease, and depression.
    Progress in Neurobiology 10/2010; 92(3):227-44. DOI:10.1016/j.pneurobio.2010.10.001 · 10.30 Impact Factor
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    ABSTRACT: Biological membranes form transient, conductive pores in response to elevated transmembrane voltage, a phenomenon termed electroporation. These pores facilitate electrical and molecular transport across cell membranes that are normally impermeable. By applying pulsed electric fields to cells, electroporation can be used to deliver nucleic acids, drugs, and other molecules into cells, making it a powerful research tool. Because of its widely demonstrated utility for in vitro applications, researchers are increasingly investigating related in vivo clinical applications of electroporation, such as gene delivery, drug delivery, and tissue ablation. In this thesis, we describe a quantitative, mechanistic model of electroporation and concomitant molecular transport that can be used for guiding and interpreting electroporation experiments and applications. The model comprises coupled mathematical descriptions of electrical transport, electrodiffusive molecular transport, and pore dynamics. Where possible, each of these components is independently validated against experimental results in the literature. We determine the response of a discretized cell system to an applied electric pulse by assembling the discretized transport relations into a large system of nonlinear differential equations that is efficiently solved and analyzed with MATLAB. We validate the model by replicating in silico two sets of experiments in the literature that measure electroporation-mediated transport of fluorescent probes. The model predictions of molecular uptake are in excellent agreement with these experimental measurements, for which the applied electric pulses collectively span nearly three orders of magnitude in pulse duration (50 ts -20 ms) and an order of magnitude in pulse magnitude (0.3 -3 kV/cm). The advantages of our theoretical approach are the ability to (1) analyze in silico the same quantities that are measured by experimental studies in vitro, (2) simulate electroporation dynamics that are difficult to assess experimentally, and (3) quickly screen a wide array of electric pulse waveforms for particular applications. We believe that our approach will contribute to a greater understanding of the mechanisms of electroporation and provide an in silico platform for guiding new experiments and applications.
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