Transscleral Delivery of Bioactive Protein to the Choroid and Retina

Abstract

purpose. To investigate the feasibility of transscleral drug delivery to the choroid and retina.

methods. An osmotic pump was used to deliver IgG across the sclera of pigmented rabbits, and levels were measured in the choroid, retina, vitreous humor, aqueous humor, orbit, and plasma over 28 days. This method was then used to deliver an anti–intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (mAb), and its effect on inhibiting vascular endothelial growth factor (VEGF)–induced leukostasis in the choroid and retina was determined by measuring tissue myeloperoxidase (MPO) activity.

results. Levels of retinal and choroidal IgG were significantly higher than baseline at all points up to 28 days (P ≤ 0.01). IgG levels in the orbit, vitreous humor, aqueous humor, and plasma were negligible (P > 0.05). MPO activity in the choroid of eyes treated with anti–ICAM-1 mAb was 80% less (P = 0.01) than in eyes receiving an equal rate of delivery of an isotype control antibody. Inhibition of MPO activity in the retina was 70% (P = 0.01). The plasma concentration of anti–ICAM-1 mAb was 31,000-fold less than the concentration in the osmotic pump.

conclusions. Minimally invasive transscleral delivery can be used to deliver therapeutic levels of bioactive drugs to the choroid and retina with negligible systemic absorption. This method of ocular drug delivery may be used in the treatment of a variety of chorioretinal disorders.

Severe vision loss in age-related macular degeneration and diabetic retinopathy is due to pathologic ocular neovascularization, ¹ whereas glaucoma and retinitis pigmentosa are characterized by the degeneration of retinal ganglion cells and photoreceptors, respectively. Together, these diseases are the principal causes of irreversible blindness worldwide. ^{2 3 4} Although inhibitors of angiogenic factors can inhibit ocular neovascularization, ^{5 6} and growth factors can rescue retinal ganglion cells ⁷ and photoreceptors, ⁸ delivering these drugs to the eye remains difficult and potentially dangerous. 

Systemic delivery of these agents in doses capable of achieving therapeutic levels in the eye may alter the physiology of other organs. Repeated long-term intravitreous delivery, as would be required for these chorioretinal disorders, runs the risk of significant local complications such as retinal detachment, endophthalmitis, vitreous hemorrhage, and cataract formation. In addition, linear molecules larger than 40 kDa and globular molecules larger than 70 kDa cannot diffuse across the internal limiting membrane of the retina, ^{9 10 11 12} precluding intravitreous delivery for many of the antiangiogenic therapies under development. Topical eye drops cannot deliver these biologics to the choroid or retina because of long diffusional path length, counter-directional intraocular convection, lacrimation, and corneal impermeability to large molecules. ¹³ Photoactivated liposomes or caged molecules may hold promise for selective delivery ^{14 15} ; however, radiational and thermal damages associated with these modalities, as well as the limited repertoire of drugs that can be enveloped, limit the clinical utility of these approaches at present. 

We hypothesized that transscleral permeation may be a viable alternative because of the sclera’s large and accessible surface area, high degree of hydration rendering it conducive to water-soluble substances, hypocellularity with an attendant paucity of proteolytic enzymes and protein-binding sites, and permeability that does not appreciably decline with age. ^{16 17 18 19} Previous in vitro experiments have demonstrated that the sclera is permeable to molecules as large as 70 kDa, ^{19 20} and we have recently shown that a 150-kDa IgG molecule can diffuse across this tissue. ²¹  

Existing methods of transscleral delivery are either nonselective or destructive. Drugs injected into the subconjunctival space can reach intraocular tissues, ^{22 23 24} but do so in part via systemic absorption. ²⁵ Transscleral iontophoresis can cause unacceptable retinal necrosis and gliosis. ²⁶ We have developed a minimally invasive transscleral drug delivery modality that can deliver therapeutic concentrations of bioactive proteins to the choroid and retina without significant systemic absorption or tissue damage. We tested the efficacy of this delivery strategy by inhibiting growth factor–induced leukostasis with a monoclonal antibody (mAb) directed against a cell adhesion molecule. 

Vascular endothelial growth factor (VEGF), a 46-kDa homodimeric globular glycoprotein, induces the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor and retinal vascular endothelium and regulates leukocyte adhesion to endothelial cells. ^{27 28} Inhibition of ICAM-1 also decreases VEGF-induced leukostasis and angiogenesis in the cornea. ²⁹ Because ICAM-1 mediates leukocyte endothelial adhesion and extravasation into surrounding tissue, ³⁰ myeloperoxidase (MPO) activity can be used to quantify the tissue sequestration of leukocytes. ³¹ We therefore investigated whether transscleral delivery of a mouse anti-human ICAM-1 mAb, which inhibits rabbit neutrophil adhesion through cross-reactivity to rabbit ICAM-1, could inhibit VEGF-induced leukostasis in the choroid and retina by measuring MPO activity in these tissues. 

Methods

Osmotic Pump Implantation

The experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and guidelines developed by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Dutch-belted rabbits were anesthetized with intramuscular ketamine (40 mg/kg; Abbott, N. Chicago, IL) and xylazine (10 mg/kg; Bayer, Shawnee Mission, KS). Osmotic pumps (ALZET; ALZA, Palo Alto, CA) were loaded with drug and incubated at 37°C before implantation. The osmotic pump was implanted subcutaneously between the scapulae and connected to a brain infusion kit (ALZA), which was modified so that the tip could be secured to, and face, the orbital surface of the sclera with a single biodegradable polyglactin 910 suture (Ethicon, Somerville, NJ) in the superotemporal quadrant of the eye, 14- to 16-mm posterior to the limbus (near the equator; Fig. 1 ). Care was taken to make a partial thickness pass through the sclera. If uvea, blood, or vitreous was observed during the procedure, the experiment was terminated. 

Collection of Ocular Tissue and Blood

Blood was collected by cardiac puncture before surgical enucleation of the eyes under deep anesthesia. Aqueous humor of each eye was collected using a 30-gauge needle. Vitreous humor, retina, choroid, and orbital tissue of both eyes were dissected and isolated under a microscope. The choroid of the treated eye was separated into two hemispheres: proximal (in which the tip of the pump was centered) and distal to the tip of the pump. Animals were killed with intracardiac pentobarbital (100 mg/kg; Vortech, Dearborn, MI). 

Pharmacokinetics Experiments

ALZET 2ML4 osmotic pumps (4 weeks, 2.5 μl/h) containing fluorescein isothiocyanate (FITC)–conjugated rabbit IgG (15.5 mg/ml; Sigma, St. Louis, MO) were implanted in one eye of each animal. Animals were killed at 3, 5, 13, 20, and 28 days after implantation, and fluorescence was measured in ocular tissues and plasma. Clearance of FITC–IgG was determined by implanting osmotic pumps (model 2001D; ALZET; 24 hours, 8 μl/h) in one eye of each animal for 1 day and measuring fluorescence in ocular tissues at 1, 3, 5, and 9 days after explantation. 

Analysis of Possible Enzymatic Degradation of FITC–IgG

Choroid and retina obtained from eyes in which osmotic pumps containing FITC–IgG were implanted were subjected to protein precipitation with 20% trichloroacetic acid (Sigma), ³² and the supernatants were assayed for residual fluorescence, which would suggest cleavage of FITC from IgG. As a confirmatory test, tissue homogenates were placed in a diffusion chamber separated by fresh virgin sclera ²¹ to determine diffusion kinetics of fluorescent molecules in the tissue, which was compared with diffusion kinetics of FITC–IgG, because significant differences between the diffusion of tissue homogenate fluorescence and the diffusion of native FITC–IgG also would suggest cleavage of FITC from IgG. Briefly, an in vitro transscleral diffusion apparatus was constructed by attaching fresh sclera to 2 spectrophotometry polystyrene cuvettes (Sigma), each with a 5 × 10 window fashioned 2 mm from the bottom, with a small amount of cyanoacrylate tissue adhesive (Ellman International, Hewlett, NY). Transscleral diffusion of fluorescent molecules at 37°C in a 5% CO₂ atmosphere was determined by sampling every 30 minutes over 3 hours. 

Fluorescence Measurements

Fluorescence was measured (excitation, 495 nm; emission, 525 nm) using a MPF 44A fluorescence spectrophotometer (Perkin–Elmer, Newton, MA) in a right-angle geometry with 3-nm bandwidths. Corrections were made for tissue autofluorescence using the values for the nonimplanted eye. 

Efficacy Experiments

ALZET 2001D osmotic pumps, one containing mouse anti–ICAM-1 IgG2a mAb (2 mg/ml) from clone BIRR0001 and one containing mouse nonimmune IgG2a mAb (2 mg/ml) (R&D Systems, Minneapolis, MN), were implanted in the superotemporal quadrant of each eye. The surgeon was masked to the identity of the two pumps. Six hours after implantation, animals were anesthetized, and 0.5% proparacaine (Alcon, Ft. Worth, TX) and 0.3% ofloxacin (Allergan, Hormigueros, PR) eye drops were topically applied. After pump placement, 2 μg of human recombinant VEGF (VEGF₁₆₅), diluted in 100 μl of pyrogen-free Dulbecco’s phosphate-buffered saline (PBS; Sigma), was injected into the vitreous body through the inferonasal pars plana of each eye with a 30-gauge needle. To normalize intraocular pressure, 100 μl of aqueous humor was removed with a 30-gauge needle. Animals were killed 24 hours after implantation and MPO activity was measured in ocular tissues. To ensure that the intravitreous injection did not provide an intraocular conduit for the antibodies, 2 animals were implanted with ALZET 2001D osmotic pumps containing FITC–IgG, and a 30-gauge needle was used to perforate the inferonasal sclera. The fluorescence in ocular tissues, 24 hours later, was compared with that in animals without the perforation. 

MPO Assay

Myeloperoxidase was extracted by freezing, thawing, and sonicating tissue in 50 mM potassium phosphate buffer, pH 6.0 (Sigma), containing 0.5% hexadecyltrimethylammonium bromide (Sigma), three times. MPO activity in supernatants was measured by the change in absorbance at 460 nm that resulted from decomposition of 0.0005% hydrogen peroxide in the presence of 0.167 mg/ml O-dianisidine (Sigma) ³¹ and compared with the activity of 1 unit of MPO (Sigma), using a microplate reader (model MR4000; Dynatech, Chantilly, VA). The assay was performed in masked fashion. 

Mouse IgG2a ELISA

A 96-well plate was coated with 10 mg/ml goat anti-mouse IgG2a-affinity purified antibody (Bethyl, Montgomery, TX) in 0.05% sodium carbonate, pH 9.6 (Bethyl), covered with parafilm, and stored overnight at 4°C. The plate was washed twice with washing buffer (50 mM Tris with 0.05% Tween-20, pH 8.0; Sigma), and nonspecific adsorption sites were blocked by incubation in postcoat buffer (50 mM Tris with 1% bovine serum albumin, pH 8.0; Sigma) for 30 minutes at room temperature (25°C). After two washings with washing buffer, mouse IgG2a reference standard (Bethyl), and unknown samples of blood diluted in postcoat buffer containing 0.05% Tween-20 (Bethyl) were added to the wells. The plate was incubated for 1 hour at room temperature before it was washed twice with washing buffer. Horseradish peroxidase–conjugated goat–anti-mouse IgG2a (Bethyl) was diluted (1:20,000) in postcoat solution containing 0.05% Tween-20 and added to the wells. After 1 hour of incubation at room temperature, the plate was washed twice with washing buffer. The 3,3′,5,5′-tetramethyl benzidine peroxidase substrate solution and peroxidase solution B (both from Kirkegaard and Perry, Gaithersburg, MD) were quickly mixed and added to the wells. The reaction was carried out for exactly 10 minutes in the dark at room temperature and then stopped with 2 M H₂SO₄ (Sigma). The absorbance was measured at 450 nm. 

Statistics

Tissue concentrations of FITC–IgG were compared by standard linear ANOVA, and the paired Student’s t-test was used to compare MPO levels between eyes. All probability values were two-tailed. An α level of 0.05 was used as the criterion to reject the null hypothesis of equality of means. 

Results

Transscleral Diffusion of Intact Immunoglobulins

FITC–IgG was delivered to the superotemporal scleral surface at a rate of 2.5 μl/h for 28 days via an osmotic pump. Levels of retinal and choroidal fluorescence, a quantitative marker of IgG concentration, were significantly higher than baseline at all times (Fig. 2 ; n = 4 per time, P ≤ 0.01 for each time). Levels in the orbit, vitreous humor, and aqueous humor were negligible (Fig. 3 ; n = 4 per time, P > 0.05 for each time). No fluorescence was detected in the plasma at any time. The concentration of IgG in the choroid in the hemisphere proximal to the pump, which reached a plateau of 6% of the concentration in the osmotic pump, was roughly 50% greater than in the distal hemisphere and 50% greater than the overall retinal concentration. The elimination of fluorescence from the choroid and retina followed first-order kinetics, with half-lives of approximately 3 days (Fig. 4 ; n = 4 per time). 

To confirm the continued linkage of FITC to IgG, protein precipitation of tissue homogenates at various times was performed. Virtually all fluorescence was protein-bound (99.6% in retina and 99.8% in choroid, at 28 days; n = 3), indicating that the IgG molecule crossed the sclera intact and did not undergo significant cleavage over the time studied. Additionally, the in vitro transscleral diffusion of fluorescence from retinal tissue homogenates (mean permeability coefficient = 6.2 × 10⁻⁶ cm/sec) and choroidal homogenates (mean permeability coefficient = 5.6 × 10⁻⁶ cm/sec) was not significantly different (P > 0.05 for both comparisons) from that of FITC–IgG (mean permeability coefficient = 4.6 × 10⁻⁶ cm/sec), ²¹ indicating that the tissue fluorescence emanated from intact FITC–IgG. Iatrogenic perforation of the sclera at the injection site did not result in increased intraocular delivery (Table 1 , indicating that lateral surface diffusion did not play a significant role in transscleral entry. 

Bioactivity of Transsclerally Delivered Protein

VEGF-induced leukostasis in the retina and choroid, as measured by MPO activity, was markedly inhibited by the delivery of anti–ICAM-1 mAb (Fig. 5) . Myeloperoxidase activity in the choroid of the eye treated with anti–ICAM-1 mAb (2 mg/ml delivered at 8 μl/h) was 80% less (P = 0.01) than in the eye receiving an equal rate of delivery of an isotype control antibody (n = 5). Inhibition of MPO activity in the retina was 70% (P = 0.01; n = 5). As expected, the diffusion of MPO, whose molecular weight is 70 kDa, into the vitreous humor was minimal in both groups of eyes. The plasma concentration of anti–ICAM-1 mAb, 64.5 ± 73.4 ng/ml, was 31,000-fold less than the concentration in the osmotic pump. 

Discussion

We have demonstrated that antibodies can be delivered across the sclera into the choroid and retina without significant proteolytic degradation, loss of bioactivity, or systemic absorption. The target selectivity (choroid/retina to plasma) was on the order of 700:1. The low levels of fluorescence in the nonimplanted eye and the spatial concentration gradient observed in the choroid of the implanted eye also indicate no significant systemic absorption. We confirmed that the fluorescence detected in the choroid and retina of the implanted eye was not due to fluorescein that may have been cleaved from the FITC–IgG complex by subjecting the tissue extracts to protein precipitation and by analyzing the diffusion kinetics of the fluorescent molecules in the tissues. The extremely low levels of fluorescence in the vitreous humor also suggest that the antibody was not cleaved from FITC, which would have readily diffused across the retina. ¹¹ The retained bioactivity of the anti–ICAM-1 mAb also confirmed intact transscleral delivery of the protein. 

Transscleral delivery of anti–ICAM-1 mAb resulted in significant suppression of VEGF-induced leukostasis. The site through which VEGF was injected into the vitreous is unlikely to have served as a conduit for the mAb because experiments with FITC–IgG revealed no significant increase in intraocular concentration of fluorescence resulting from the creation of a scleral perforation at the pars plana, 20-mm distant from the pump tip. Furthermore, even if the perforation resulted in increased vitreous levels of mAb, it is unlikely to have had any impact on the retinal or choroidal vasculature, owing to the diffusion barrier of the internal limiting membrane of the retina. ^{9 10 11 12}  

We speculate that the high ocular selectivity is due to the sponge-like nature of the sclera. Although bolus injections of drugs into the subconjunctival space may overwhelm the absorptive capacity of the sclera, leading to systemic absorption, ²⁵ the gradual delivery used in these experiments may permit more complete scleral absorption because the drug-sclera contact time is greater than the lag time to steady state flux. ³³ A long-term transscleral delivery device may be clinically feasible because the human eye is remarkably tolerant of foreign bodies overlying the sclera, such as scleral buckles used in treating retinal detachment, even for years. ³⁴ Moreover, human sclera is hypocellular ¹⁶ and has a large surface area (16.3 cm²), ¹⁸ both of which would facilitate diffusion. 

Angiogenesis plays an integral role in the pathogenesis of a variety of posterior segment disorders. Because laser photocoagulation, the only proven therapy for intraocular neovascularization, ^{35 36} is quite destructive, there has been a rapid growth in the development of drugs targeting VEGF receptors, integrins, matrix metalloproteinases, and other molecules. Although these potentially effective therapies are under development, there is a parallel need to develop drug delivery systems to the choroid and retina that are safe, effective, and feasible for long-term use. We have described a nondestructive and minimally invasive method for the targeted delivery of high molecular weight compounds to the posterior segment of the eye. This drug delivery method exhibits linear kinetics of absorption and elimination, with the potential to deliver constant doses of medication. It is robust because it is not limited to delivering antiangiogenic drugs, but can be extended to neuroprotective agents or vectors for gene transfer, which hold promise in the treatment of glaucoma ^{7 37} and other chorioretinal degenerations. ^{8 38}  

 Supported by the Heed Ophthalmic Foundation (JA); the American Ophthalmological Society–Knapp Testimonial Fund (JA); a Foundation Fighting Blindness Career Development Award (JA); the Massachusetts Lions Eye Research Fund (APA); and the Roberta Siegel Research Fund (APA). E. S. Gragoudas is a Research to Prevent Blindness Scientific Investigator.

 Submitted for publication August 5, 1999; revised October 15, 1999; accepted October 26, 1999.

 Commercial relationships policy: N.

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

 

Figure 1.

 

Schematic of osmotic pump placement.

Figure 2.

 

Concentration of FITC–IgG (1 mg/ml delivered at 2.5 μl/h) in the choroid (proximal hemisphere [▪] and distal hemisphere [▴]) and the retina (•). *P < 0.01, #P < 0.005, †P < 0.001 versus day 0. n = 4 for all times.

Figure 3.

 

Concentration of FITC–IgG (1 mg/ml delivered at 2.5 μl/h) in the orbit (▪), vitreous humor (▴), and aqueous humor (•). P > 0.05 for all tissues at all times versus orbital tissue of fellow eye (⋄), which had the highest fluorescence of any tissue in and around that eye. n = 4 for all times.

Figure 4.

 

Clearance of FITC–IgG (1 mg/ml delivered at 8 μl/h from day 0 to day 1) in the choroid (proximal hemisphere [▪], t _1/2 = 2.89 days; and distal hemisphere [▴], t _1/2 = 3.14 days) and the retina (•, t _1/2 = 3.36 day). n = 4 for all times.

Table 1.

 

Concentration of FITC–IgG (Delivered for 24 Hours at 8 μl/h) in Tissues as a Percentage of Its Concentration in Osmotic Pump, with and without the Presence of a Scleral Perforation in the Inferonasal Pars Plana

Tissue	Without Scleral Perforation	With Scleral Perforation	P
Choroid, proximal hemisphere	1.84 ± 0.51%	2.06 ± 0.36%	0.67
Choroid, distal hemisphere	0.88 ± 0.20%	0.99 ± 0.14%	0.58
Retina	0.66 ± 0.22%	0.55 ± 0.08%	0.60
Vitreous humor	0.04 ± 0.06%	0.12 ± 0.04%	0.19

Figure 5.

 

MPO activity in vitreous humor, choroid, and retina after intravitreous injection of 2 μg VEGF₁₆₅ in eye treated with anti–ICAM-1 mAb (unshaded bars) and isotype control mAb (shaded bars). n = 5.

The authors thank Robert Rothlein at Boehringer Ingelheim (Ridgefield, CT) and Napoleone Ferrara at Genentech (San Francisco, CA) for their generous gifts of anti–ICAM-1 mAb and VEGF, respectively; Tiansen Li at the Massachusetts Eye and Ear Infirmary for the use of a sonicator; and Ambati M. Rao and Balamurali K. Ambati for critical review of this article.