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16.2.2 Impact of Genetics
In view of recent advances in medical genetics of AMD and related diseases by
genome-wide association studies, it is advantageous to consider other drugs and
other routes. About 70% of the risk of developing AMD is accounted for by alleles
in several complement cascade genes and the LOC387715/HTRA1 locus, and imply
a role of the innate immune system in AMD etiology. A more thorough understanding of the workings of the innate immune system in the eye and at the blood-retina
barrier is important. Meta-analyses predict additional risk alleles. Other risk factors
were discovered in epidemiological studies and include diet, smoking, light exposure, blue irides, drinking, and others. It remains to be determined what initiates the
disease process in AMD, and it is possible that any number of putative causative
events start the disease. We need to know more about the precipitating events in
AMD, and how the body normally protects itself from these insults. Such information might generate better drugs.
Better knowledge of the VEGF-mediated pathways that cause neovascularization may provide alternative potential therapies. New drugs may prove useful that
block: (1) the interaction of VEGF with its receptor, (2) the formation of VEGF or
promote its inactivation or breakdown, (3) the formation of its receptor, and (4) the
action of the receptor’s signaling pathway.
16.3 Better Tools for Delivery and Treatment
Given this current state of knowledge, nonetheless, it is equally clear that we need
better tools and approaches to deliver a drug to its correct target. We recognize the
need for effective, convenient, safe, and inexpensive drug delivery. No matter how
potent a new drug might be, its delivery is a concern. Delivery to an inappropriate
target can be life threatening. Ultimately it comes down to simple arithmetic: What
is the balance between minimizing side-effects of a drug and maximizing the duration of time for which the correct dose of drug is delivered to the target cell?
16.3.1 Barriers to Success
Regardless of the type of drug, each takes a perilous journey to its subcellular target.
These include but are not limited to – (1) physical barriers such as fascia, membranes, linings, or blood vessel walls, (2) voluminous gaps or interstitial and intracellular spaces that result in dilution, (3) convection, pressure, and flow barriers such
as solvent flow including blood, aqueous, and lymph flow, which can force drugs
away from the target cell, (4) binding of the drug to extracellular matrices such that
charge-charge or hydrophobic interactions bind or entangle the drug, (5) enzymatic
activities that metabolize the drug, (6) compartmentalization, sequestration, and
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entrapment that keep a drug from its target, (7) transport processes such as those at
the nuclear pore or cell membrane that actively transport drug out of a target subcellular compartment, and (8) failure to activate the drug or agent intracellularly.
16.3.2 Physics-Based Approaches
As we have so few tools in the armamentarium of drug delivery to the eye (Geroski
and Edelhauser 2000), it is wise to fully consider all those available and then carefully develop new ones to supplement existing technology.
Physics in medical therapy is often found in radiation oncology: Ionizing radiation (such as X-rays, gamma-rays, and particles) is used to kill cancerous tumors.
Laser photocoagulation, cryotherapy, and TTT are the standard of treatment for
several ophthalmological diseases, and all are straightforward applications of physics. Cautery, cutting, etc., in surgery are but too common to recognize the underlying physical principles. Given the broad use of physical principles in medicine is
seems useful, if not wise, to consider physical approaches to deliver drugs.
16.3.2.1 Physical Methods to Deliver Drugs to a Target Cell
in the Posterior Segment
Pressure changes: (1) hydrodynamic pressure is very effective in liver (Liu et al.
1999) but it seems unlikely to be applicable to the eye, as the sclera is tough, pre-
venting stretching of retinal cells, without excess pressure damaging the ONH. An
exception may be bleb formation that stretches cells, such as, subretinal blebs, (2)
Stretching the plasma membrane by sonoporation is effective in vitro. A laser beam
focused to one micron size can open pores transiently if the laser is focused selectively on the cell membrane in vitro (Nikolskaya et al. 2006). Direct microinjection
into the target cell or into its nucleus by ballistic or jet injection: This route seems
unlikely given the toughness and thickness of the sclera. The application of electric
fields in therapy is well known. Defibrillators are commonplace. Tiny current densities are effective in neurostimulation. Examples include the cochlear implant for
sensory stimulation and the cardiac pacemaker, widely used and highly successful
for low or irregular heart rhythms. Low-voltage iontophoresis has been used since
the early 1900's to deliver charged drugs into skin and the eye.
16.3.2.2
History of Electrical Fields in Medicine
There is a long history, back to Roman times, for the use of electric fields in medicine.
Quoting from Wikipedia http://en.wikipedia.org/wiki/Cranial_electrotherapy_
stimulation, accessed on July 28, 2010:
“Electrotherapy” has been in use for at least 2000 years, as shown in the (clinical) literature
of the early Roman physician, Scribonius Largus. (He wrote) in the Compositiones Medicae
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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
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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 sol-
vent, 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 compensation 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
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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 com-
pared 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.
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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 high-
lights 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 tech-
nique 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 rela-
tionship 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 tech-
nologies 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 anti-
apoptotic 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,
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modification of extracellular space, pro-inflammatory and immune response,
blood flow modification, alterations to morphology of retinal layers, hemorrhage,
necrosis/apoptosis/paraptosis of tissue, fibrosis, and RPE hyperplasia all have
been reported in conjunction with too much current or voltage, or too long a
duration of application (Butterwick et al. 2007).
6. Pain: Electrostimulation of pain receptors occurs when the current is applied
rapidly. For iontophoresis, slowly changing the current up or down prevents this
sensation.
16.3.2.7
Possible Strategies to Improve Electric Field-Mediated
Drug Delivery
1. Pretreatment of the targeted tissues with hyaluronidase increases the ratio of
electrotransferred cells while reducing the need for high electric voltage, hence
decreasing the risk of tissue damage.
2. In general, bigger electrodes offer a lower current density: There is a need for
novel electrodes for ocular treatment depending on cell to be treated.
16.3.3 Experiences with Iontophoresis
Animal studies demonstrated how current and ions flow through the eye. An illuminating set of studies was the use of MRI to monitor iontophoresis in real time
(Li et al. 2004, 2008; Molokhia et al. 2009). Manganese ions are detected by nuclear
magnetic resonance, showing where the current is flowing within the eye in an electric field, and this analysis can be performed on live animals in real time while
iontophoresis is in progress. In transscleral iontophoresis, manganese ions moved
macroscopic distances within the eye of a living anesthetized rabbit. These ions
penetrated the sclera. Via a transcorneal route, the manganese ions became fully
distributed in the anterior chamber.
16.3.3.1 Examples of Iontophoresis
Iontophoresis to deliver drugs into the cornea has been used extensively in Europe
(Hughes and Maurice 1984). Examples include: Iodide iontophoresis was used
to treat dry eye symptoms in patients with ocular surface disease. Numerous
classes of drugs have been delivered including antibiotics, antifungals, antivirals, antiinflammatories, and analgesics (Eljarrat-Binstock and Domb 2006). A 0.2 mA current
level or 1.6 mA/cm
1984). No unpleasant sensation was described with a 0.2-mA current applied to the
anesthetized human cornea. However, currents over 2 mA cause pain (Hughes and
2
density does not appear to cause damage (Hughes and Maurice
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Maurice 1984). These current levels caused muscular contractions in rabbits that
were under general anesthesia. Also, switching a current of about 1 mA on and off
caused a shock. Current should be applied and reduced gradually. Parel and
colleagues (Behar-Cohen et al. 1997, 2002) developed a “coulomb-controlled”
device that adjusts voltage and current if tissue resistance charges during iontophoresis. With this device, iontophoresis is safe up to 50 mA/cm2 for 5 min. Tissue
properties change during and after iontophoresis. Present hypotheses are that barriers within or surrounding tissues are altered by exposure to the electric field. These
alterations may increase permeability of the tissue to the flow or diffusion of a drug
after iontophoresis.
In addition to current flow, proper electrode placement is important in transscleral iontophoresis. Placement of one electrode at the pars plana provides maximal
delivery of a drug into the vitreous. Ongoing experiments will test whether the par
plana exhibits the lowest electrical resistance or whether the pars plana is most vulnerable to barrier breakdown during iontophoresis (Molokhia et
al. 2008).
Li and colleagues discovered that iontophoresis could be used to create a drug
depot in the sclera that subsequently undergoes sustained drug release. This strategy
reduces the number and frequency of iontophoretic treatments. Triamcinolone acetonide phosphate was delivered into the eye from one electrode, and calcium ions
were supplied from the other electrode simultaneously. Calcium ions and the phosphate moiety on the triamcinolone acetonide analog precipitate when they come
into contact, forming a reservoir of drug in the sclera. The precipitate dissolved
slowly, providing a slow-release formulation of drug that could be used to treat
uveitis in an animal model. The slow release formulation was effective over long
periods of time and prevented symptoms (Higuchi et al. 2007).
16.3.3.2 Summary of the Strengths and Weaknesses of Iontophoresis
Strengths – Over short distances, high concentrations of drugs can be delivered in a
short period of time. These treatments are clinically useful and are in common practice in Europe. Iontophoresis is a method of choice for charged drugs, which pose
difficulties in crossing membranes and hydrophobic barriers. Iontophoretic pretreatment increases delivery into the eye in vivo, suggesting that electric fields increase
the permeability of biological matrices or membranes.
Weaknesses – It is impractical to transport drugs macroscopic distances (~1–2 cm)
from the anterior surface of the cornea to the posterior segment of the eye by iontophoresis due to the weak electric field applied across the eye, the low mobility of
drugs, and short duration of treatment. Anterior segment structures may be sensitive
to electric field strength, and it appears necessary and advantageous to avoid current
flow through or near these structures. For delivery to the posterior segment, the
transscleral, not transcorneal, route appears to be the better approach. As with all
new drug delivery approaches, careful consideration and evaluation of collateral
tissue damage, both intraocular and extraocular, is needed.
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16.3.4 Experiences with Electroporation
Electroporation is a highly successful strategy in transfecting naked DNAs, RNAs,
and nucleic acid analogs into eukaryotic and bacterial cells in laboratory experiments. In vitro transfection efficiencies range from 50 to 90%, depending on the cell
line in eukaryotic cells. Bacterial transformation of plasmids by electroporation
usually results in about 1010 transformants per mg of plasmid DNA. Given routinely
high success rates, it is intriguing to test electroporation for nucleic acid and drug
delivery in vivo in living animals that model the human condition. Currently there
are huge collections of mouse mutations that are orthologous with eye-disease causing mutations in human patients, making the testing of therapeutic agents first in
mouse models an exceptionally productive approach to translational medicine.
Along these lines, highly relevant mouse mutations have been found and others
have been deliberately constructed to study disease etiology and normal biology.
Causative gene lesions and alleles associated with increased risks of disease are
now proven for many retinal diseases as the result of gene engineering approaches,
including electroporation, in the laboratory. Several tools and reagents, including
but not limited to viruses, plasmids, ribozymes, siRNA, and oligonucleotides, can
be delivered into affected cells in animal models of retinal diseases. Unfortunately,
some of these techniques intermittently or variably work in experimental systems.
These reliability issues cause great concern and consternation as we try to translate
this technology into clinical practice. There are numerous well-known risks and
probably many more unknown risks in implementing these new technologies. That
said, there is nothing more exciting in the field of biology than gene therapy.
16.3.4.1 Examples of Electroporation in Living Animals
1. Following delivery by intravitreal injection of naked plasmid and electropora-
tion, a reporter gene was successfully expressed in RGCs (Dezawa et al. 2002).
2. Subretinal injection of a naked plasmid into neonatal rat and mouse eyes, fol-
lowed by electroporation, results in expression in daughter terminally differenti-
ated retinal cells (Matsuda and Cepko 2004, 2007).
3. Multiple electroporation pulse trains administered with a 90° rotation between
sets of pulses is more effective than without rotation of the field. The interpreta-
tion is that the rotation increases the surface area of cell membrane that is exposed
to the electric field (Heller et al. 2007).
4. In our laboratories we sought to deliver plasmid DNAs and smaller nucleic acids
by electroporation following a subretinal injection in juvenile to adult mice. We
demonstrated a modified subretinal injection protocol that avoided passage of the
injection needle through the choroid, and we found this to be highly advantageous
in avoiding blood in the vitreous, which in our hands was impossible to manage
in the mouse eye. We modified the approach (Timmers et al. 2001) by an oblique
transcorneal entry illustrated in Fig. 16.1 (reproduced from (Johnson et al. 2008)).
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Fig. 16.1 The subretinal injection technique. (a) Position of the 34-gauge beveled needle is shown
nearly tangential just before lancing the cornea. (b) This schematic illustrates the position of the
35-gauge blunt needle after puncturing the neural retina and partially inflating the interphotoreceptor space (the subretinal space) to produce subretinal blebs. (c) Presented is a still image from a
video illustrating penetration of the cornea. (d) This panel shows the positioning of the 35-gauge
blunt needle in the center of the anterior chamber. (e) The 35-gauge needle penetrates through the
retina into the subretinal space. (f) The 35-gauge needle is removed from the vitreous after subretinal injection of quantum dots. A small number of quantum dots are evident in the vitreous that
generate a reddish-orange color. (g) Illustrated is a fundus before subretinal injection. The retinal
vessels can be readily detected in the fundus image. A ruddy red background color can be observed
before injection. (h) Shown is the fundus immediately after subretinal injection. The positions of
three blebs surrounding the optic nerve head are located at clock face positions 4, 8, and 11. Each
bleb appears puffy and gray in color with red vessels between the blebs. The optic nerve head is
nearly centered in the image of the fundus. The imaged mouse eyes are about 3 mm in diameter.
This caption is quoted from and the figure images are reproduced with permission from Johnson
et al. (2008)
Next, we optimized the electroporation step by simplifying the electrode design,
based on loops of platinum wire (Fig. 16.2). We adjusted pulse trains, voltage,
duration of pulses for the adult mouse eye, finding that 50 V, 1–5 ms pulse dura-
tion 1 s intervals and two trains of ten pulses were optimal (see Figs. 16.3 and
16.4, reproduced from (Johnson et al. 2008)). Figure 16.5 illustrates that the cells
that were transfected were bounded by actin rings and were often binucleate,
suggesting the identity as RPE cells.
5. In typical electroporation, joule heating is small. But under other conditions
called electron avalanche transfection, the change in temperature is so sharp and
great that the medium vaporizes to form short-lived microbubbles. The rapid col-
lapse of a microbubble can be sufficient to initiate a shockwave. If the bubble
forms and collapses adjacent to a cell membrane, then the shockwave will disrupt
the plasma membrane locally causing a small hole to form transiently. Plasmid
sizes may preclude or limit diffusion of DNA across the plasma membrane pore.
However, the violent microbubble formation and collapse may contribute to a
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