16.3.2  Physics-Based Approaches

of 46 AD that his patients should stand on a live black torpedo fish for the relief of a variety of medical conditions, including gout and headaches. Claudius Galen (131–201 AD) also recommended using the shocks from the electrical fish for medical therapies.

16.3.2.3  Safety Concerns with Electric Fields

For an electric field-based treatment to be effective, exposures that exceed suggested occupational and public limits might be required. Standards for occupational and public exposure limits to electrical and magnetic fields can be found at http:// www.who.int/peh-emf/publications/elf_ehc/en/index.html.

Exposure of the head to sufficient electric or magnetic fluxes induce phosphenes (Taki et al. 2003). The effect is direct and experimentally repeatable. The exact mechanisms by which phosphenes are generated is not known, whether a physiological property of the retina or central visual pathways. Nonetheless, these phosphenes suggest an effect of electromagnetism on physiological processes and a route that may be exploited for treatment purposes. These potential treatment avenues must be carefully balanced by the serious risks of exposures that cause severe damage by excessive heating.

Safety precautions should be taken in any procedure employing electric fields, electronics, or electrical equipment. The reader’s environment health and safety office is a resource for appropriate information. It is obvious that voltages and currents that are too high will result in massive cell death, and in extreme circumstances tissue will vaporize and burn, leading to electrocution and death. Less appreciated is that vasoconstriction occurs during application of low currents. Only a short duration of vasoconstriction is needed to result the pooling of blood and thrombus formation. However, as with any therapeutic approach, given proper deference, the electric field can be a useful tool in the delivery of drugs to a specific target cell or tissue. Here we review evidence that electrical fields can be used to deliver drugs to specific targets in the cell and subcellular compartments without damage to surrounding tissues in living animals.

16.3.2.4  Definitions of Electric Field Methods

An understanding of electric fields in drug delivery requires definitions of the methods by which particles are acted upon:

(a)Electrophoresis is the technique to move charged particles in an electric field over macroscopic distances in realistic amounts of time. In the laboratory, it is routine to move double-stranded DNAs of 1–10 kilobase lengths in an electric field of a few volts per cm, over distances of 1–15 cm in about an hour in an agarose gel in an aqueous medium. The velocity of electrophoretic movement is directly proportional to the strength of the electric field, the dielectric constant

of the media, the zeta potential (surface charge of the particle), and inversely proportional to the viscosity of the media and the log of the molecular weight of the DNA.

(b)Iontophoresis is a noninvasive method of moving large concentrations of a charged substance by using direct electrical current. A chamber, reservoir, or a patch containing a charged drug is connected to an electrode of the same sign, repelling the drug and moving it into tissue. Typical settings are 1–10 mA/cm2 for 1–10 min. Iontophoresis may increase the effective permeability of a drug by

(a) electrophoresis of the drug through extracellular spaces among cells, (b) the electric field may increase the number and size of microscopic pores through tissues by unclogging them, and (c) concomitant electroendosmosis, as discussed in the next paragraph.

(c)Electroendosmosis is the movement of an uncharged solute with a polar solvent, carrying the drug with the solvent into a tissue. Tissues themselves have charged groups on the surfaces of membranes and in extracellular matrices, which in the case of skin or sclera carry net negative charges at neutral pH. These stationary groups remain ionized as long as the solution is neutral or basic. These fixed negative groups in an electric field are attracted by the anode. As they are immobile in the tissue, they cannot migrate. This results in compen-

sation by the counterflow of H3O+ ions toward the cathode. As the H+ ions are shared across numerous H2O molecules there is a net solvent migration toward the cathode. Transscleral electroendosmosis enhances the flux of positively charged drugs and retards negative ones. For small drugs, the effects of electroendosmosis are relatively small compared to electrophoresis, but the contribution of electroendosmosis to transport increases with molecular size of the drug. For macromolecules and nanoparticles, the effect of electroendosmosis is expected to be dominant.

(d)Electrostimulation is mediated through a direct effect of an electric field on voltage-gated ion channels, opening or closing the channel.

(e)Electroporation, also known as electropermeabilization, is a large increase in permeability of the plasma membrane caused by an externally applied electrical field. The increased permeability of the cell membrane is attributed to formation of small holes or pores when the voltage across the plasma membrane exceeds its dielectric coefficient. Typically, a potential difference of 0.1–1 V across the plasma membrane, which is about 7.5 nm in thickness, is sufficient to transiently open pores with a diameter in the range of 1–10 nm on the surface of the plasma membrane. During or shortly after the application of the field, small pores may fuse with others to form large pores. Numerous commercial instruments that provide accurate pulses and pulse trains of appropriate voltages are readily available. These are used routinely in the research laboratory to transfect bacteria and eukaryotic cells grown in culture. Upwards of 50–90% of eukaryotic cells are transfected and survive under controlled conditions in vitro. The application of electroporation to living animals has been tested successfully, though at reduced and quite variable transfection efficiencies. Morphology, ongoing physiologic processes, and distribution/distortion of electric fields are more difficult to control in vivo, which lead to compromises and a generally less

efficacious outcome in living animals. Even so, remarkable results have been obtained recently.

The goal of electroporation is to open pores transiently, allow diffusion of the drug or agent across the plasma membrane, and provide for the closing or resealing of the pores. The profile of pore closing has three phases: a rapid (microsecond), medium (millisecond), and slow (1–10 s) phase. The latter stage may cause too much equilibration of substances on either side of the membrane, which could be lethal. Obviously, pores that remain open permanently will result in cell death.

Interest in electroporation has been kindled because of the dramatic successes and deadly failures of viral-mediated gene therapies. Immunogenicity of viruses is a major concern in gene therapy; this is mitigated in several ways. Reducing the amount or size of the virus, use of reduced antigenicity viruses, or simply eliminating the virus altogether. Selecting the best virus has led to AAV. Subretinal delivery of genetic material using recombinant AAV has led to promising results in animals and the initiation of clinical trials in LCA2 patients with a defect in RPE65. This route of delivery creates a localized retinal detachment, and there are some concerns about the hazards of the delivery technique and unknown potential risks. This leads to consideration of other potential ways of treating gene defects that cause ophthalmic diseases. Instead of subretinal injection, intravitreal injections have been attempted in animal models of LCA2. Intravitreal injection of naked plasmid DNA is not efficient due to rapid digestion of the plasmid by nucleases, dilution, and nonspecific binding to the vitreous. Also, the plasmid is not delivered to the putative target cells of the retina because of limited diffusion of these very large molecules (MW ~2 million Daltons) within the comparatively large space of the vitreous.

16.3.2.5  Advantages of Electric Fields for DNA Transfection vs. Viral Mediated DNA Delivery

1. Lower immunogenicity of naked DNA compared to high antigenicity of virus proteins.

2. Ease and lower costs of preparation of large quantities of plasmid DNA compared to virus production.

3. Easier preparation of endotoxin-free plasmid with fewer contaminants than are found in viral preparations.

16.3.2.6  Problems of In Vivo Electric Field Applications

1. Size differential matters: Large cells may receive too much current resulting in damage, while small cells receive too little current to allow transfection.

2. Irregular patterns and arrangement of cells can distort the electric field.

3. The voltage at the cell membrane depends on the shape and orientation of the cell, resulting in too many pores in some cells in one orientation and to few pores in differently oriented cells of the same type.

4. Blood, lymph, and aqueous clear out drugs rapidly regardless of delivery method if the drug enters into one of those flow streams. Fluorescence angiography highlights the wash-out process. Also, fluorescein angiography indicates where it is possible to deliver drugs in the eye via circulation. The comparatively short time that fluorescein remains detectable in the eye tells us the short time that ordinary drugs will remain at a target cell in the eye if delivered through the circulation. Coordinated angiography and electroporation pulse delivery could allow a real time choice for maximum drug in the target tissue. Drugs need to be designed to latch on to their target cell in the eye. Comparing the same drug delivery technique in living and postmortem animals underscores the impact of flow and clearance mechanisms on drug delivery. Many approaches appear effective in postmortem animals, but few work as well in the living animal.

5. Damage to cells may take place by any of several routes:

Vasoconstriction by an electric field leads to pooled blood and clot-formation (Palanker et al. 2008). This is distinct from burn damage. There is a clear relationship of current density and pulse duration that defines a threshold above which vasoconstriction occurs. It is important to operate below this threshold to minimize tissue damage, as clots will adversely affect treatment outcomes. If it is necessary to use conditions above the threshold, anticlotting agents may be required.

 Electrochemical reactions at the surface interface between metal and liquids or tissues result in the production of toxic substances. Oxygen and hydrogen gas bubbles are generated by electrolysis at the electrodes, which can interrupt current flow and alter reservoir contents. The pH of reservoir solutions changes during current flow because of electrolysis. The pH change can be great, resulting in damage to biologic membranes and tissues, in essence a chemical burn. One way to prevent pH changes in reservoirs is to continuously replace reservoir solutions and another is to buffer them. Also, toxic agents or pH changes can be minimized by increasing the distance from the metal–liquid interface location (at the cathode and anode) to the contact point with the tissue. Membrane technologies have improved drug delivery and safety by blocking movement of the electrochemical products generated at the electrode. Monitoring the pH near the electrodes or at the site of contact with a tissue may be wise during any electric field treatment.

 Cells may die by any of several routes as caused by an electric field. It may become necessary to reduce or postpone cell death by pretreatment with antiapoptotic or antinecrotic drugs, such as TUDCA (Boatright et al. 2006).

Extent of heating by electric current–Joule heating: The amount of energy delivered to a tissue can result in thermal damage. Temperature rise during an electrical pulse can be estimated and is a function of current density, pulse duration, resistivity of the tissue and medium, tissue density, and the tissue’s heat capacity.

 Damage needs to be monitored during and after electric field application. Edema, inflammation, hypoxia, ischemia, anoxia, glucopenia, reperfusion injury,