A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport of genes

Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Durham, NC 27708, USA.
Bioelectrochemistry (Impact Factor: 4.17). 11/2006; 69(2):248-53. DOI: 10.1016/j.bioelechem.2006.03.006
Source: PubMed


The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.

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    • "The study revealed that the mannitol solution could significantly increase the extent of tissue penetration of the dextran molecule with molecular weight of 2,000,000 (2M), and that the penetration depth was correlated to the change in the available volume fraction (KAV) of dextran 2M. The present study was designed to investigate whether the pretreatment of 4T1 and B16.F10 tumors with the hyperosmotic solution of mannitol would similarly increase the tumor interstitial space, and therefore the extent of pDNA transport in vivo since previous studies have shown that the rate of interstitial transport depends strongly on the pore sizes in the extracellular matrix 29, 30. In the study, a series of in vitro and ex vivo experiments were first performed to determine the kinetics of tumor cell or tissue volume reduction following the mannitol treatment. "
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    ABSTRACT: Pulsed electric fields can enhance interstitial transport of plasmid DNA (pDNA) in solid tumors. However, the extent of enhancement is still limited. To this end, the effects of cellular resistance to electric field-mediated gene delivery were investigated. The investigation used two tumor cell lines (4T1 (a murine mammary carcinoma) and B16.F10 (a metastatic subline of B16 murine melanoma)) either in suspensions or implanted in two in vivo models (dorsal skin-fold chamber (DSC) and hind leg). The volume fraction of cells was altered by pretreatment with a hyperosmotic mannitol solution (1 M). It was observed that the pretreatment reduced the volumes of 4T1 and B16.F10 cells, suspended in an agarose gel, by 50 and 46%, respectively, over a 20-min period, but did not cause significant changes ex vivo in volumes of hind-leg tumor tissues grown from the same cells in mice. The mannitol pretreatment in vivo improved electric field-mediated gene delivery in the hind-leg tumor models, in terms of reporter gene expression, but resulted in minimal enhancement in pDNA electrophoresis over a few microns distance in the DSC tumor models. These data demonstrated that hyperosmotic mannitol solution could effectively improve electric field-mediated gene delivery around individual cells in vivo by increasing the extracellular space.
    Cancer gene therapy 01/2011; 18(1):26-33. DOI:10.1038/cgt.2010.51 · 2.42 Impact Factor
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    • "These structural features are instrumental for the analysis of the various data obtained with HV plus LV pulse combination modes and the effects of a time lag between the pulses. The electric pulses play two roles: (1) permeabilization of the target cells and (2) enforcement of the electrophoretic transport of DNA toward (Henshaw et al., 2006), as well as across, the permeabilized membrane (Bureau et al., 2000). The electroporator used in the present study was developed to deliver LV pulse(s) after a defined period of time after the HV pulse(s). "
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    ABSTRACT: Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.
    Human gene therapy 11/2008; 19(11):1261-71. DOI:10.1089/hgt.2008.060 · 3.76 Impact Factor
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    ABSTRACT: One of the key issues in electric field-mediated molecular delivery into cells is how the intracellular field is altered by electroporation. Therefore, we simulated the electric field in both the extracellular and intracellular domains of spherical cells during electroporation. The electroporated membrane was modeled macroscopically by assuming that its electric resistivity was smaller than that of the intact membrane. The size of the electroporated region on the membrane varied from zero to the entire surface of the cell. We observed that for a range of values of model constants, the intracellular current could vary several orders of magnitude whereas the maximum variations in the extracellular and total currents were less than 8% and 4%, respectively. A similar difference in the variations was observed when comparing the electric fields near the center of the cell and across the permeabilized membrane, respectively. Electroporation also caused redirection of the extracellular field that was significant only within a small volume in the vicinity of the permeabilized regions, suggesting that the electric field can only facilitate passive cellular uptake of charged molecules near the pores. Within the cell, the field was directed radially from the permeabilized regions, which may be important for improving intracellular distribution of charged molecules.
    Annals of Biomedical Engineering 08/2007; 35(7):1264-75. DOI:10.1007/s10439-007-9282-1 · 3.20 Impact Factor
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