A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport of genes.
ABSTRACT 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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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 · 3.62 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 · 3.23 Impact Factor
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ABSTRACT: Advances in biotechnology have led to an accelerated discovery of macromolecular therapeutics such as peptides, proteins and polynucleotides. These macromolecules can be targeted against a variety of diseases, each requiring delivery to a well-defined compartment of the body. As such therapeutics are often prone to degradation before reaching their target site, or do not reach their target site at all, they often require a special formulation. This review focuses on several types of materials that are currently under investigation for the delivery of nucleic acid therapeutics and aims to pinpoint the limitations of these materials with the ultimate goal to identify the material challenges which, in our opinion, will constitute a new generation of ‘intelligent’ materials for nucleic acid delivery. Such ‘intelligent’ materials should be able to sense and respond to environmental changes. The generated response to these environmental changes should give the material new properties that favor the intracellular delivery of their payload. Besides dealing with material properties, we especially aim to focus on the biological barriers such intelligent materials will have to deal with when used for the delivery of nucleic acids. Furthermore, we briefly discuss the advanced light microscopy techniques that are often used to visualize and quantify the steps of the delivery process of nucleic acids.Materials Science and Engineering R Reports 11/2007; DOI:10.1016/j.mser.2007.06.001 · 11.79 Impact Factor