Fig. 16.4  Subretinal injection followed by electroporation at different voltages. In all panels 1 mL 2 mg/mL pVAX-tdTomato in water were injected and three blebs were raised. Five different voltage settings (0, 25, 40, 70, and 100 V) were used. Electroporation followed with a constant 1 ms pulse, 1 s interval, and 5 pulses on (a, c, e, g, and i, and 10 pulses with b, d, f, h, and j). In (a, b) the samples received 0 V, in (c, d), 25 V, in (e, f), 40 V, in (g, h), 70 V, and in (i, j), 100 V. Each panel represents a flatmount of the eye excluding the lens. The tips of the florets correspond to the center of the cornea, and the central area from the midpoint or greatest bulge of each petal inwards corresponds to the RPE sheet. The red fluorescence is punctate; each dot corresponds to a single RPE cell. When no voltage was applied, there was no evidence of tdTomato expression (red). When 25 V was applied, there was no expression of tdTomato; however, when 40–100 V were applied, there was an accumulation of tdTomato fluorescence. The scale bar represents 1 mm. This caption is quoted from and the figure images are reproduced with permission from Johnson et al. (2008)

Weaknesses – Caution must be observed as a maximum safe voltage is reached precipitously with further increases resulting in cell death. Loss of intracellular ATP by leakage through electroporetic pores in the cell membrane is a concern. Some transfection media are designed to match intracellular concentrations of ions and ATP so that the opening of pores does not deplete the cell of essential constituents. In some cases, these media help to reduce cell death in cell lines. Transfection efficiency seems low compared to several viral delivery systems. The size, shape, and volume of a subretinal bleb may interfere with electrical field strength, and the kinetics of bleb resorption have not been factored into electroporation strategies.

16.4  Outstanding Issues in Electric Fields for the Delivery of Drugs

(1) Electrodes must be configured, designed, and placed considering the anatomy and physiology of the region near the eye: Current must not pass along or through the cranial nerves, optic nerve, or extraocular muscles. (2) Electrode set-up alone may be insufficient to obtain an optimum electric field. To electroporate irregularly shaped objects (the retina, RPE, choroid, or sclera), protection of other nearby tissues must be done. Shielding must be placed appropriately. Insulating materials

Fig. 16.5  Expression of tdTomato in mouse RPE cells following subretinal injection and electroporation. An adult C57BL/6J mouse was subretinally injected and electroporated as described (Johnson et al. 2008). Three days later the eye was excised and the RPE sheet was flatmounted. The RPE were stained for f-actin with phalloidin and imaged with a confocal microscope. The image highlights the expression and accumulation of tdTomato fluorescence in RPE cells. The cells were established to be RPE based on several quantitative criteria. These included: the presence of the actin ring bounding each cell, polygonality and number of neighboring cells, size of each cell, regularity of each cell, and number of binculeate cells. Also it was apparent that these were RPE cells based on the location and face adjacent to the removed neural retina (removed during dissection and preparation of the flatmount), and RPE pigment that blocked show-through of the underlying choroid. The expression pattern, including a significant fraction of RPE cells displaying tdTomato, indicates a high transfection efficiency of the combined subretinal injection and electroporation approach

may be placed or injected near or into adjacent sensitive tissues. Insulation placed on electrodes may help to prevent collateral damage. (3) Electric fields may damage plasma membrane proteins even in an electric field that does not cause thermal denaturation. (4) Histology alone may not be sensitive enough to study subtle changes in the tissue barriers after application of an electric field. Other special tests are needed, c.f., ERGs. (5) Long-term safety and efficacy have not been assessed except with a few select drugs. (6) Large animal studies have not been conducted.

(7) Electroporation likely is the basis of electro-shock therapy, and shielding the brain from an electrical-based therapy in the eye is critical. (8) Time: These treatments take time, much longer than a simple intravitreal injection.

16.5  Summary

In this review, we critiqued the current state of electrical fields as means of delivery of therapeutic agents intracellularly in the posterior segment of the eye. Such physical approaches are used in conjunction with the formulation of drugs and other physical techniques such as the subretinal or suprachoroidal injection of agents in a location immediately abutting the relevant target cell, for example, the RPE cell or the photoreceptor cell. Electric field–tissue interactions are complex and poorly understood, and the application of an electric field to a tissue requires optimization for each particular ocular target. The use of electric fields should be given consideration when other simpler delivery approaches fail or when a simple route needs augmentation. Electric fields offer several unique advantages that other approaches lack. No other approach aids in transiently and reversibly breaching any membrane of the cell. Numerous examples abound in the use of electroporation in vitro. In vitro electroporation is by far the most efficient way to transfect DNA, RNA, or protein into a cell without the use of viruses. Even viral transfection is fraught with potentially lethal hazards. Provided that great care is taken to minimize cellular damage by the electric field, iontophoresis and electroporation are delivery approaches worthy of further consideration in vivo.

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