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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    ABSTRACT: The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.
    Biophysical Journal 02/2014; 106(4):801-12. · 3.83 Impact Factor
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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). · 2.28 Impact Factor

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