Mechanistic Studies Of Electroporation

The degree of permeabilization of the muscle cells is dependent on the electric field intensity, length of pulses, shape and type of electrodes, and cell size.

A. Electric Field Intensity

The influence of electrode configuration on the electric field distribution has been shown by measuring 51Cr-EDTA uptake in vivo (19,20). These studies indicated two uptake phases in which the membrane permeabilization increases, followed by irreversible membrane damage. Both plate electrodes (18,21) or a pair of wire electrodes (22) have been shown to be effective. The calculated electric field distribution was more homogenous for plate electrodes than for needle electrodes. Also, the needle diameter determines 3 parameters: in rabbit liver, the decrease of the electric field intensity near the electrodes is steeper with smaller electrode diameter; the area covered with a given electric field is smaller with smaller electrodes (23); and there is a more homogenous electric field with larger electrode diameter.

Recently, square-wave electric pulses of different field strength and duration have been evaluated. Data for highvoltage pulses of short duration or low-voltage pulses of long duration are found in Table 1. Despite the large variation in conditions used for the target tissue or organ, the expression achieved in all these cases represents efficacious levels of gene products, some of which are within the range that is necessary to treat human diseases.

Theoretical and practical data also suggest that the cell size in the region perpendicular to the electric field plays a crucial role in determining the permeabilization parameters (24). The larger the ''functional'' size of the cell, the lower is the field strength necessary (25). For example, tissues containing cells

Figure 1 Schematic depiction of the electroporation process in the skeletal muscle using penetrating electrodes. (A) Electroporation system is placed next to the target muscle; (B) the injection needle and the electrodes are inserted into the muscle; (C) plasmid is injected into the target muscle; (D) at a selected time interval after plasmid injection, typically 2 min, a square-wave electric pulse is generated; (E) effect of the electric field at the muscle fiber level; (F) plasmid injection followed by electroporation increases uptake by 10- to 1000-fold into the target muscle; notice the muscle fiber nuclei that contain numerous copies of the injected plasmid; and (G) direct plasmid injection into the skeletal muscle, without electroporation results in a low plasmid copy number uptaken and expressed by the muscle fiber. See the color insert for a color version of this figure.

Figure 1 Schematic depiction of the electroporation process in the skeletal muscle using penetrating electrodes. (A) Electroporation system is placed next to the target muscle; (B) the injection needle and the electrodes are inserted into the muscle; (C) plasmid is injected into the target muscle; (D) at a selected time interval after plasmid injection, typically 2 min, a square-wave electric pulse is generated; (E) effect of the electric field at the muscle fiber level; (F) plasmid injection followed by electroporation increases uptake by 10- to 1000-fold into the target muscle; notice the muscle fiber nuclei that contain numerous copies of the injected plasmid; and (G) direct plasmid injection into the skeletal muscle, without electroporation results in a low plasmid copy number uptaken and expressed by the muscle fiber. See the color insert for a color version of this figure.

that communicate through tight gap junctions amplify transmembrane potential changes. Thus, the skeletal muscle can be electroporated at lower field intensities (26) with decreased tissue damage, compared with other tissues.

In studies of the electric field-mediated enhancement of gene and drug delivery, different types of electrodes have been used. These include clamp or caliper, tweezers, paddles, and needle arrays. In large mammals such as pigs, dogs, or humans, the increased resistance of the skin, the thickness of the subcutaneous fat tissue, and the concern for tissue damage that occurs with the proportionally increased intensity of the electric field makes use of some percutaneous electrodes inefficient and impractical. Thus, the electrode design must be adapted to the application, organ, and animal species. For cutaneous and subcutaneous tumors in animals and humans, calipers or needles (27-29) are used. For liver, tweezers, or occasionally needles (23,30-32); for skin, calipers, meander electrode, plate-and-fork electrodes, or small needles

(29.33.34); and for the skeletal muscle, calipers or needles

B. Association with Carrier Molecules

Despite the recent advances in the technology of plasmid transfer into tissues, additional improvements in electropora-tion techniques and plasmid formulations are needed. For example, the entire electroporation procedure in theory can be completed without causing permanent damage to the cell. However, under some conditions, electroporation procedures inflict fatal stress on some skeletal muscle cells (16,36) and

Table 1 Examples of Square-wave Pulses Electric Field Intensity and Pulse Length

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