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1From the Department of Pharmaceutics and Pharmaceutical Chemistry and the 2Utah Center for Advanced Imaging Research (Radiology), University of Utah, Salt Lake City, Utah; and 3Aciont Inc., Salt Lake City, Utah.
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METHODS. Thirty-minute 2-mA anodal constant current transscleral and transcorneal iontophoresis (current density, 10 mA/cm2) was performed on three New Zealand White rabbits in vivo. Intravitreal injection and passive delivery were the controls. Transscleral and transcorneal iontophoresis experiments were conducted with the electrode device placed in the superior cul-de-sac away from the limbus and on the cornea adjacent to the limbus, respectively. During iontophoresis, the current delivered into the eye was monitored using a probe ion (Mn2+) with MRI. The distributions of the ion in the aqueous and vitreous humor after iontophoresis, passive delivery, and intravitreal injection were also determined by MRI.
RESULTS. With the short application time, passive diffusion did not deliver a significant amount of the ion into the eye. Whereas transscleral iontophoresis delivered the ion into the vitreous, transcorneal iontophoresis delivered the ion into the anterior chamber. The current pathways during iontophoresis were mainly from the electrode into the eye, perpendicular to the electrode–eye interface beneath the electrode. Electric current along the surface of the globe was relatively minimal. With the present transscleral iontophoresis protocol, the ion penetrated the sclera and traveled as far as 1.5 mm from the electrode–conjunctiva interface into the vitreous. For transcorneal iontophoresis, the ion penetrated the cornea and filled the entire anterior chamber.
CONCLUSIONS. MRI can be a useful technique in the study of the penetration of probe compounds in the eye during and after iontophoresis, such as in iontophoresis protocol and device testing. Ocular pharmacokinetic studies using MRI are noninvasive and provide real-time data without perturbation and compound redistribution that can occur during dissection and assay in traditional pharmacokinetic studies. With MRI, it was shown that transscleral iontophoresis, transcorneal iontophoresis, and intravitreal injection deliver ions to different parts of the eye.
Manganese (Mn) is a common contrast agent used in MRI. For example, it has been used in the imaging of the liver13 and in neuroaxonal tracing of the optic nerve.14 Manganese-enhanced magnetic resonance imaging (ME-MRI) works under the principle that Mn2+ ion enhances the relaxivity of the water protons, reducing the proton spin-lattice relaxation times (T1). As a result, T1-weighted MR images show enhanced signal intensities at the locations of the Mn2+ ions. The higher the concentration of Mn2+ ion in the vicinity, the more the signal is enhanced. ME-MRI is a noninvasive method of studying ocular iontophoresis. The approach not only reduces the number of animals required in pharmacokinetic studies, but it also allows the determination of the concentration profiles in the eye (e.g., in the vitreous) in real time.
In the present study, the target sites of ion delivery and the electric current pathways during transscleral and transcorneal iontophoresis were to be determined and compared using MRI in a rabbit model in vivo. Intravitreal injection and passive delivery were the controls. The distributions of the probe ion after iontophoresis, intravitreal injection, and passive delivery were also to be monitored with MRI. These results should improve our understanding of ocular iontophoresis and help in the optimization of ocular iontophoresis for site-specific drug delivery into the eye.
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Materials and Methods |
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Iontophoresis Electrode Device
Flexible tubing (3/16 in. inner diameter x 5/16 in. outer diameter x 1/4 in. length; Silastic; VWR Scientific) was the outer shell of the electrode device (Fig. 1A) . One end of the tubing was sealed by silicone elastomers (NuSil Technology, Carpinteria, CA), and a piece of silver foil was inserted at the back of the shell adjacent to the seal as the conductive element. A layer of liquid silver paint (
0.05 cm thick) was put on the silver surface to increase the effective surface area of the Ag electrode. The electrode chamber (donor compartment) was then filled with approximately 2% agarose gel in normal saline (0.9%) with 0.4 mM MnCl2. The final volume of the agarose-filled chamber was between 0.04 and 0.07 mL. The silver surface outside the electrode was covered with a nonconductive paint. The Ag/AgCl return electrode was prepared by electrochemically plating a silver sheet in saturated KCl solution.
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It should be pointed out that the effects of the strong magnetic fields on the applied iontophoretic current and Mn2+ ion are negligible. The equivalent electric field on the ions due to the Lorentz force is insignificant compared with the electric field, E, provided by iontophoresis. Briefly, Fc = (ev) x B, where Fc is force, e is charge, B is magnetic field, and v is charge velocity. When the magnetic field is perpendicular to the current (i.e., maximum effect), Fc = evB. The force on an ion during iontophoresis is related to the electric field, Fc = eE. Combining these two equations, E = vB. When B = 1.5 T and v 
6 x 10-6 m/s (ion velocity normally encountered during iontophoresis: 6 x 10-4 cm2/s per V at 1 V/cm), E = 9 x 10-6 V/m, which is negligible compared with the applied electric fields in the present iontophoresis study. Furthermore, the distribution of Mn2+ ion in aqueous solution is homogeneous in the bore magnet, suggesting that the effects of the magnetic force on the ion are insignificant relative to Brownian motion.
MRI Calibration
The signal equation of the spin-echo imaging is
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Experiments were conducted to create a calibration curve between manganese-enhanced signal intensity and Mn2+ concentration. MnCl2 standard solutions of 0.01 to 4 mM Mn2+ were prepared in saline and in deionized water. The solutions (5 mL in capped glass vials) were imaged with spin-echo pulse sequences: TR was 50, 100, 200, 400, 800, 1600, and 2400 ms, with TE fixed at 9 ms for T1 measurements, and TE was 15, 30, 45, and 60 ms, with TR fixed at 2400 ms for T2 measurements. The relaxation times T1 and T2 of these standards were determined by curve fitting of the signal intensity of a region-of-interest (ROI) with respect to the time variables TE and TR, using equation 2 . This experiment allowed us to determine the concentrations of Mn2+ by direct comparison and correct for T2 effects at higher concentrations of the contrast agent.
Calibration experiments to identify possible Mn2+ binding in the eye were conducted with enucleated eyes and extracted vitreous humor in vitro. In the equilibration experiments with enucleated rabbit eyes, the eyes were equilibrated in 100 mL MnCl2 solutions of 0.04 to 1 mM Mn2+ in saline for 48 hours. The eyes were then removed from the solutions and scanned. In the experiments with rabbit vitreous humor, the vitreous humor was extracted from freshly excised eyes. Known amounts of concentrated Mn2+ solutions were then added to the vitreous humor in vials. The T1 of Mn2+ in vitreous humor and in saline was compared.
In Vivo Experiments
Transscleral and transcorneal iontophoresis experiments were conducted on three rabbits. After the rabbits were anesthetized with 1 mg/kg diazepam intraperitoneally (IP), 0.1 mg/kg glycopyrrolate intramuscularly (IM), 25 to 50 mg/kg ketamine IM, and 5 to 10 mg/kg xylazine IM, an electrode device with MnCl2 was placed on the conjunctiva–sclera in the superior cul-de-sac (under the upper eyelid) away from the limbus or on the cornea adjacent to (or touching) the limbus for transscleral and transcorneal iontophoresis, respectively. The position of the electrode for transscleral iontophoresis in the superior cul-de-sac was maintained naturally by the pressure from the eyelid. The silver foil sticking out from the electrode (Fig. 1a) was then fixed by medical adhesive tape, and the electrode in the cul-de-sac was further secured by adhesive tape over and on top of the upper and lower eyelids. The upper conjunctiva was chosen because of the larger space available for the electrode device in the superior cul-de-sac than that in the inferior cul-de-sac of rabbits. For transcorneal iontophoresis, the position of the electrode on the cornea was maintained by the pressure from the eyelid and secured by medical adhesive tape. During iontophoresis, the position of the electrode was checked by comparing the relative electrode position with the anatomic structures of the eye in the MR images. Both the left and right eyes were used, but they were not used concurrently. The return electrode (approximately 4 cm2 surface area) was placed on a piece of gauze pad (approximately 40 cm2 surface area) wetted in saline and clamped to the ear. A 2-mA anodal constant current DC (10 mA/cm2) was applied to the eye for 30 minutes with a constant-current iontophoretic device (Phoresor II Auto, model PM850; Iomed, Inc., Salt Lake City, UT). The delivery and distribution of Mn2+ ion in the eye were monitored by MRI. At the end of the iontophoresis experiment, the pH of the electrode was checked and was found to be between pH 5 and 7. Between each experiment, the animals were allowed an approximately 1-month washout period.
A control experiment was also conducted to examine possible MR image artifacts due to the applied electric current during iontophoresis (2 mA for 30 minutes) without the presence of Mn2+ ion. Another control experiment involved passive delivery, using the electrode device with MnCl2 as the donor but without application of electric current. The electrode device was placed on the limbus, covering parts of both the cornea and sclera. The same rabbits in the iontophoresis experiments were involved in these control experiments.
Intravitreal injections (0.1 mL) of 0.4 mM and 1.0 mM MnCl2 in saline were performed with the rabbits under anesthesia. A 30-gauge 0.5-in. needle was used in this procedure. The needle was inserted at the center of the sclera surface under the upper eyelid through the sclera (conjunctiva, sclera, choroid, and retina) into the vitreous. The distribution of the ion after the injections was monitored by MRI.
Theories and Equations for Iontophoresis and Diffusion
For iontophoretic transport within and from an ocular electrode device, the conventional Nernst-Planck equation can be used. The flux, J, of an ion across a planar surface during iontophoresis is expressed as16
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is the combined porosity and tortuosity factor; 
is the electrical potential at position x; and vf is the average velocity of the convective solvent flow resulting from pressure and electroosmosis. The flux of an ion is also related to the electric current carried by the ion as
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/x is the electric field applied and u (/x) is the migration velocity. The distance of ion migration, h, can be expressed as
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In the analysis of the intravitreal injection data, a simple model of passive diffusion from a spherical source can be used. The concentration at radial position r at time t, Cr, from the center of an initially uniformly distributed spherical source of concentration, C0, and radius a is calculated by17
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Results |
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4 seconds). There is no significant difference between the T1 and T2 of Mn2+ in deionized water and those in saline. Figure 2B presents the SI/S0 versus Mn2+ concentrations in saline and in water. As can be seen in the figure, SI/S0 reaches a maximum of approximately 0.5 at about 0.6 to 1.0 mM Mn2+ and is relatively linear with respect to Mn2+ concentration below 0.4 mM. The concentration of MnCl2 used in the present study was therefore chosen to be 0.4 mM in saline, unless otherwise specified.
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Passive Delivery
Representative MR images during passive delivery through the electrode device are shown in Figure 3 . The eyelids, anterior chamber, lens, vitreous, and other structures of the eye were visible in the MR images. Mn2+ in the donor compartment of the electrode device in the superior cul-de-sac appears as a high-signal-intensity rectangular shape. The outer shell of the device (background signal with an inverse U-shape) enclosing the Mn2+ compartment can also be seen in the superior cul-de-sac between the donor compartment and the upper eyelid. The weaker signal near the top of the Mn2+ compartment is due to signal interference from the Ag metal electrode. Although no external pressure was applied to the electrode device on the eye, the pressure due to the contact with the eyelid was significant and deformed the eye surface. As can be seen in Figure 3 , there were no significant changes in the signal intensity of the tissues (±15%) in the eye under and around the site of the electrode application over the duration of the application. There were slightly higher signal intensities than the background control on the surface of the eye, but they were within the noise. The amount of Mn2+ delivered into the eye was below the detection limit of the present MRI technique (<0.02 mM).
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0.2 cm in diameter) of the Mn2+ spot in the vitreous (i.e., the ROI) was determined at different time points after the injection (0–35 minutes). Data points beyond 35 minutes were not used, because the free-diffusion assumption of equation 7 would not be valid when the Mn2+ concentration boundary reached the retinal barrier. The signal intensity was then converted to the Mn2+ concentration by using the SI/S0 versus concentration data in Figure 2B . Five times higher signal intensity at this location relative to the background (vitreous without Mn2+) was observed immediately after the injection. This is consistent with the initial concentration of Mn2+. Using the present method, the diffusion coefficient of Mn2+ in the vitreous was estimated to be 1.3 x 10-5 cm2/s (uncertainty: ±0.4 x 10-5 cm2/s). This value is consistent with free diffusion of Mn2+ in an aqueous medium and suggests that the vitreous behaves like a free aqueous diffusion medium, as stated in the literature.19 The demonstration of free diffusion of the ion in the vitreous provides the basis of the analysis of ion distribution after iontophoresis in the present study.
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Discussion |
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In contrast to passive delivery through the electrode device, significant penetration of the probe ion was observed during iontophoresis. According to the MR images in Figures 5 and 6 and the concentration data in Figures 7 and 8 , both transscleral and transcorneal iontophoresis took approximately 12 to 18 minutes to unload the entire content of Mn2+ in the electrode device in the present study. The time to unload the entire content to the eye was independent of the sites of electrode placement. This observation is consistent with the electromobility of Mn2+, the dimension of the electrode device, and the current applied. Under the electric current of 2 mA and with the electrical resistance of the donor compartment in the electrode device being approximately 0.2 k
, the electric field across the electrode chamber was approximately 1 V/cm. According to equation 6 and Mn2+ aqueous electromobility in saline of 4 x 10-4 cm2/s/V, an Mn2+ ion traveled 0.4 cm (approximately the dimension of the electrode device) in 17 minutes at 2 mA, which is consistent with the time observed for Mn2+ to be completely unloaded from the electrode in the MR images.
The main distinctive feature between transscleral and transcorneal delivery was the different target sites of delivery (i.e., different current pathways). For transscleral iontophoresis, the probe ion was predominantly transported directly into the vitreous under the electrode. During the 30-minute iontophoresis transport, the ion penetrated approximately 0.1 cm into the vitreous (Fig. 7) . There was no observable Mn2+-enhanced signal deeper in the vitreous (above the 0.02-mM detection limit) over the duration of the experiments. This is again consistent with electrotransport theory. When the Mn2+ ion left the electrode chamber, the ion was driven by a weaker electric field than that within the electrode chamber, due to the distribution of the current outward in the eye from the electrode–conjunctiva interface (i.e., from the conjunctiva to the sclera and then to the vitreous). As the cross-sectional area of the current passage increased, the electric field decreased (equation 5) , and the migration velocity of the ions decreased. If the electric field in the vitreous had remained the same as that in the electrode chamber, the ion front would have traveled approximately 0.6 cm into the vitreous from the device–conjunctiva interface (instead of the observed 0.1–0.2 cm) after 30 minutes of the iontophoresis application (equation 6) . Some distributions of the ion along the sclera structure outward from the area beneath the electrode were also observed near the end of the iontophoresis application. The MR images also clearly showed a depot of the probe ion loaded in the conjunctiva-sclera-retina tissues under and near the site of iontophoresis (Fig. 5) . The depot was beneath the surface and was not removed by tears and a saline rinse applied to the eye surface. Ion distribution by diffusion after iontophoresis occurred mainly near these tissues and was at a slower rate toward the center of the vitreous than the diffusion rate observed in the intravitreal injection experiments (Fig. 4) . This is consistent with ion clearance through the retina.
In contrast to transscleral iontophoresis, the anterior chamber was the site of ion delivery in transcorneal iontophoresis. The probe ion was transported across the cornea into the anterior chamber and distributed in the aqueous humor (Figs. 6 8) . No ion was observed to be distributed into the vitreous from the anterior chamber through the posterior chamber over the duration of the experiment. It should be pointed out that ion distribution in the anterior chamber during and after transcorneal iontophoresis appeared to occur faster than that in the vitreous during transscleral iontophoresis. One explanation is the difference between the volume of the anterior chamber and that of the vitreous. It is also speculated that this may be a result of deeper penetration of the ion during iontophoresis because of the lesser divergence of the electric field, and/or ion movement due to clearance through the canal of Schlemm in the anterior chamber. Another interesting observation in the MR images during and after transcorneal iontophoresis is the pattern of Mn2+ distribution in the anterior chamber. A lack of probe ion in the middle of the anterior chamber was observed. This distribution pattern is not consistent with simple passive diffusion or simple current driven transport in the eye and may be related to the presence of other structures in the anterior part of the eye (e.g., iris) and/or ion movement due to convection in the anterior chamber. Resolving this question requires further investigation.
With the assumption that Mn2+ ion follows the electric current (composed of all ions available in the electric current pathway), the distribution of the probe ion during iontophoresis can be used to assess the current paths during iontophoresis. Although the present electrode design did not have a seal surrounding the electrode–eye contact surface (Fig. 1A) , only a small amount of Mn2+ was distributed to the surface of the eye outside the area underneath the electrode (total amount of Mn2+ outside on the eye surface is estimated to be less than 10% of the initial amount in the electrode device). Most ions were delivered into the eye during iontophoresis. The electric current pathways during iontophoresis were mainly from the electrode into the eye, perpendicular to the electrode–eye interface. There was some current distributed laterally along the sclera structure after penetration. Although the electric current passed through the globe between the donor electrode device and the return electrode, migration of the ion was slow and did not allow sufficient delivery of the ion (below the detection limit) to the back of the eye over the duration of the present experiments. The results in the present anodal iontophoresis study should also hold for cathodal iontophoresis, provided that the electric current distribution across the eye follows Ohm’s law. However, different electrical polarities may induce different membrane and tissue structural changes. Future studies will examine this question when an anionic contrast agent becomes available.
Safety of Iontophoresis
Iontophoresis can enhance the penetration of a compound by altering the structure of a biological membrane.6 If membrane alterations are reversible and do not cause any significant side effects, iontophoresis can be considered safe. The issue of safety related to ocular iontophoresis is beyond the scope of the present study as several recent studies have already discussed this topic.9 10 11 12 22 Nevertheless, it deserves some attention. Both transscleral and transcorneal iontophoresis is relatively safe, provided the electric current density is low.12 23 Although no histology was performed in the present study, the present transscleral iontophoresis parameters (total current, current density, and current duration) were chosen based on these previous studies. For example, Parkinson et al.22 have shown that human subjects can tolerate (with no significant side effect) the electric current and current dose similar to those in the present study. Similar total current levels were also shown not to cause any observable side effects in the eye in other studies.11 12 23 Whereas the transscleral iontophoresis parameters in the present study were justified, the parameters of transcorneal iontophoresis were chosen to be the same as those of transscleral iontophoresis to allow direct comparison between these two methods. As a result, the duration of the transcorneal iontophoresis (30 minutes) was longer than those typically used in previous studies (
10 minutes),9 10 23 24 and such applications could lead to corneal damage. With the electrode design and iontophoresis protocol in the present study, both transscleral and transcorneal iontophoresis also caused minor silver burn at the sites of iontophoresis (electric current-driven silver ions precipitating at the eye surface). The silver burn disappeared within several days.
Pharmacokinetic Studies with MRI
The lack of understanding of the mechanisms of ocular drug delivery, pharmacokinetics, distribution, and elimination is partly due to the complicated anatomy of the eye.25 It is also a result of the lack of data due to the invasive approaches taken to study drug distributions in the eye. These invasive approaches are expensive and inconvenient, severely perturb the eye during sampling, and require large numbers of animals in each study. For example, traditional pharmacokinetic studies involve killing animals at different time points after drug administration and assaying different sections of the eye for the drug. A noninvasive approach to the study of ocular pharmacokinetics and drug delivery not only can reduce the number of animals required in ocular drug delivery and pharmacokinetic research, but also allows the determination of the concentration profiles in the vitreous on a real-time basis without perturbation. Sampling of the eye by dissection in traditional pharmacokinetic studies may also result in redistribution of the compound of interest in the eye during assay. MRI can be used to determine the distribution and route of elimination of ions and compounds during and after ocular drug delivery such as iontophoresis, intravitreal injection, and periocular injection without these drawbacks. It is a promising noninvasive complementary technique to the traditional methods.
Although MRI has been used in ocular research previously,14 26 the present study is the first to use MRI as a noninvasive method to retrieve real-time information on the pharmacokinetics and elimination of an ion in the eye. Although the present study has only demonstrated the use of a probe ion in ocular noninvasive pharmacokinetic studies, MRI can be used to monitor the pharmacokinetics and elimination of compounds labeled with a chelating moiety bound to magnetic ions such as diethylene triamine pentaacetic acid (DTPA). The MRI technique can also be used to monitor liposomal and micellar ocular drug delivery27 when the liposomes and micelles are loaded or labeled with Mn or gadolinium (Gd) probes. However, quantitative MRI analyses at this stage are limited to the vitreous and aqueous humor. Additional calibration studies and further fine tuning of the technique are necessary before accurate measurements of the concentration of contrast agents in eye tissues can be obtained.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 1, 2003; revised October 4 and December 12, 2003; accepted December 17, 2003.
Disclosure: S.K. Li, Aciont Inc. (F, C); E.-K. Jeong, None; M.S. Hastings, Aciont Inc. (F, E)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: S. Kevin Li, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112; kevin.li{at}m.cc.utah.edu.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) T1 in saline, (
) T1 in water, (
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T2 in saline, and (x) T2 in water. (B) Relationship between signal intensity ratio SI/S0 and Mn concentration. (
) in saline and (x) in water.
) 1.0 mm (ROI: 
) at the center of the vitreous; and (
) at the center of the anterior chamber. Dotted line: limit of quantitative assessment in the current MRI method.