October 2011
Volume 52, Issue 11
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Multidisciplinary Ophthalmic Imaging  |   October 2011
ToF-SIMS Analysis of Dexamethasone Distribution in the Isolated Perfused Eye
Author Affiliations & Notes
  • Jenifer Mains
    From the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland.
  • Clive G. Wilson
    From the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland.
  • Andrew Urquhart
    From the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland.
  • Corresponding author: Andrew Urquhart, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, Scotland, G4 0RE; andrew.urquhart@strath.ac.ul
Investigative Ophthalmology & Visual Science October 2011, Vol.52, 8413-8419. doi:10.1167/iovs.11-8199
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      Jenifer Mains, Clive G. Wilson, Andrew Urquhart; ToF-SIMS Analysis of Dexamethasone Distribution in the Isolated Perfused Eye. Invest. Ophthalmol. Vis. Sci. 2011;52(11):8413-8419. doi: 10.1167/iovs.11-8199.

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Abstract

Purpose.: To illustrate the ability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize and demonstrate the spatial distribution of dexamethasone within ocular tissues.

Methods.: Dexamethasone sodium phosphate was administrated to perfused and nonperfused ovine eyes via intravitreal injections. The vitreous humor, the lens, and the retina of the eyes were then removed and divided into front, middle, and back sections. ToF-SIMS analysis was performed on each cross-section of the vitreous humor using Bi3+ cluster source and images of drug distribution within the sections generated.

Results.: In the positive ion spectra, four key drug fragment peaks were identified and in the negative ion spectra, one key drug peak was identified. All five important drug peaks were successfully imaged in each tissue section and their distribution within the section illustrated. The drug was shown in the nonliving eye to move by diffusion alone, whereas in the living eye the drug was shown to distribute faster within the vitreous and penetrate through to the back of the retina and also into the lens.

Conclusions.: The results illustrate the ability of ToF-SIMS to characterize and provide spatial information about drug distribution within ocular tissues. Key differences in drug movement through the vitreous humor, toward both the anterior and the posterior tissues, in the living eye and the nonliving ovine eye were demonstrated, showing that dexamethasone sodium phosphate distribution through the vitreous is not determined by diffusion alone.

Dexamethasone (see Fig. 1 for chemical structure) is a glucocorticoid drug currently used in the treatment of various inflammatory disease states, including inflammatory conditions of the eye such as uveitis and conjunctivitis. Dexamethasone has been administered to the eye using various routes of administration, including topical, systemic, and intravitreal injection (IVI). Using the topical route of administration, dexamethasone has been shown to be of benefit as an inhibitor of inflammation after ocular surgical procedures such as vitrectomy or lensectomy. It has also been used topically in combination with antibiotics to reduce anterior eye inflammation. 1 Although dexamethasone has been shown to be useful in the treatment of anterior eye inflammation, after topical administration dexamethasone experiences poor penetration into vitreous and posterior eye tissues 2 ; as a result of this, topical treatment will have little effect in the treatment of posterior eye inflammatory conditions. The systemic route of dexamethasone administration has also been shown to be no better than the topical route, with only 0.1% of the injected dose penetrating into the eye. 2 To penetrate to the back of the eye and treat posterior eye inflammation, the intravitreal route of administration is a more feasible option. Using the intravitreal route, dexamethasone has been shown to reduce inflammation and lessen the breakdown of the blood–ocular barrier 3,4 and, more recently, a dexamethasone implant has been shown to improve vision in patients with from macular edema. 5 Although dexamethasone administration via the intravitreal route has been shown to benefit patients with from macular edema, it has been associated with numerous adverse events, 6 8 with the two principal side effects reported to be cataract formation 8 and increased intraocular pressure. 6,7  
Figure 1.
 
The chemical structure of dexamethasone. Note the fluorine atom bonded to the ninth carbon atom in the tetracyclic ring, which provides a unique chemical label for the drug.
Figure 1.
 
The chemical structure of dexamethasone. Note the fluorine atom bonded to the ninth carbon atom in the tetracyclic ring, which provides a unique chemical label for the drug.
Determining the extent of dexamethasone drug distribution within ocular tissues has involved the use of various different analytical techniques. Standard analytical methods often used in determining ocular pharmacokinetics, including high-performance liquid chromatography 9,10 and liquid chromatography–mass spectrometry, 4 have been developed and used previously in the rabbit eye. In addition to these methods, tracking the movement of radiolabeled dexamethasone has also been performed. 11 To prepare ocular tissues for analysis via these commonly used methods, each ocular tissue is typically extracted and is then homogenized before performing a method of drug extraction from the tissue. The destructive nature of the standard analytical techniques has led to the desire to investigate alternative routes of measuring drug distribution within ocular structures and has included the development of a fluorescence polarization immunoassay method 12 and also a nuclear magnetic resonance (NMR) imaging method. 13 Using 1H- and 19F-NMR, Midelfart and colleagues 13 were able to demonstrate the presence of dexamethasone and various amino acids in the aqueous humor of the rabbit eye, without the need for lengthy drug extraction techniques required when performing standard analytical techniques on ocular tissue samples. Although this technique was shown to be a useful alternative technique in detecting drug presence within the aqueous humor, drug detection may not be as straightforward in one of the more complicated tissues of the eye such as the lens and the retina. In addition to this, NMR provided no spatial awareness of the drug location within the tissue. Interest in mapping drug distribution within ocular tissues has recently increased, especially when the target site of action is the posterior eye, which is particularly difficult to reach. Posterior eye conditions such as diabetic retinopathy 14,15 and age-related macular degeneration 16,17 have limited effective treatment options available and understanding spatial drug distribution patterns in ocular tissues could potentially improve treatment options in these conditions. 
Time of flight secondary ion mass spectrometry (ToF-SIMS) is an extremely surface sensitive analytical technique that can characterize the chemical composition of a wide variety of systems, including biological tissue, 18,19 drug formulations, 20,21 and polymers. 22,23 ToF-SIMS of samples requires no sample modification, unlike a similar but well-used technique of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF/MS), 24 which requires the sample to be coated with a matrix. In ToF-SIMS measurements a primary ion beam is fired at the surface of the sample of interest. The primary ions then collide with the sample, causing sample fragmentation and the generation of secondary ions. The secondary ions generated are then accelerated to the detector with a known kinetic energy, enabling an accurate ion mass to be determined from the flight time required by the ion. 25 The potential of ToF-SIMS to map drug distribution within ocular tissue, without the need for prior drug extraction, has previously been demonstrated in our group, using amitriptyline as a model basic drug. 26 Using ToF-SIMS and the multivariate analytical technique, principal component analysis (PCA), key physiological differences between ocular subtypes were identified and, in addition to this, the presence of the model drug in drug-treated tissue samples was demonstrated. However, this study focused on the use of PCA to data mine mass spectra from specific ocular tissue types to determine drug distribution, rather than use key mass fragments to build ion maps to determine drug distribution. In this study, we present for the first time how the technique of ToF-SIMS can be successfully used to image as well as spatially determine drug distribution in the lens, vitreous, and retinal ocular tissues, after intravitreal drug administration. Moreover, we also demonstrate key differences in the movement and distribution of dexamethasone within the vitreous humor, the lens, and retina of the living eye and the nonliving eye. 
Materials and Methods
Chemicals
Dexamethasone phosphate disodium salt (purity ≥ 98%) was purchased from Sigma-Aldrich (Dorset, UK). Isopropyl alcohol used to clean silicon wafers before sample mounting was obtained from Fischer Scientific (Loughborough, UK). 
Ocular Tissue Preparation
Preparation of ocular tissues for intravitreal dexamethasone administration was based on a method detailed previously. 26 Briefly, a batch of ovine eyes was collected from a local abattoir within 1 hour of slaughter and then cannulated using one of the long posterior ciliary arteries that typically wrap around the optic nerve. One long posterior ciliary artery was cannulated because, within the eye, the lateral and medial ciliary arteries merge. It has previously been reported that no difference in ocular blood flow occurred when both arteries were perfused, compared with the perfusion of only one artery. 27 In the ovine eye, the long posterior arteries supply the iris, ciliary body, and anterior choroid and also branch out to form the short posterior arteries, which supply the posterior choroid. 28 The long posterior ciliary arteries also provide a reinforcing branch to the choroidoretinal artery and this vessel supplies the choroid and the retina via the retinal arterioles, which branch out from the choroidoretinal artery. 29 In previous ocular perfusion studies, perfusion of one of the ciliary arteries has been shown to maintain the viability of the eye over a prolonged time period, with slow deterioration of Bruch's membrane 30,31 and maintenance of oxygen and glucose consumption, indicating retinal tissue perfusion. To establish if flow systems remained operational within the eye, a small volume of perfusion fluid was slowly pumped through the eye and the vortex veins were inspected for fluid exit. On successful initiation of perfusion fluid flow, the eye was introduced into the perfusion system and perfused with physiologic media, based on a method previously used by Koeberle et al. 32  
Throughout the experiment, the perfused eye was maintained at 37°C to mimic typical in vivo body temperature. Details of the composition of the perfusion fluid administered into the eyes via the ciliary artery are presented in Table 1; all components were purchased from Sigma-Aldrich. A solution containing dexamethasone at a concentration of 100 mg/mL was prepared in deionized water. Once arterial perfusion pressure was maintained, to investigate the distribution of dexamethasone within the ovine eye 100 μL of the solution was injected into the vitreous of the isolated perfused eye. The injection was administered 5 mm into the vitreous, approximately 3 to 4 mm from the limbus, using a 1-mL syringe and 23-gauge needle. After injection the needle was drawn very slowly from the eye to minimize leakage from the injection site. An IVI was also administered to nonperfused eyes as a control, using the same method used for the perfused eyes. One hour after the IVI, for the isolated perfused ovine eyes, the perfusion procedure was terminated and the perfused eyes were removed from the perfusion kit. Using liquid nitrogen, both the perfused eyes and the nonperfused eyes were then immediately snap frozen before the tissue dissection procedure was performed. In the first instance, the lens was removed by cutting away the anterior eye in a circular manner. From the posterior section, the vitreous was removed by slicing the eye at four positions from the anterior to the posterior section. The sclera was then pressed back to enable cryosectioning of the retina and all tissues were stored at −80°C before sectioning. Using embedding matrix (Shandon M-1; Thermo Fisher Scientific, Cheshire, UK), the lens, vitreous, and retina were mounted onto a cryostat chuck. Each tissue was then cut through the center of the tissue to produce 20-μm-thick sections using a cryostat (Leica CM1850; Leica Microsystems, Milton Keynes, UK). Tissue sections were mounted directly onto 1 × 1 cm silicon wafers that were previously cleaned with isopropyl alcohol. 
Table 1.
 
Composition of Perfusion Fluid Administered to Isolated Perfused Eye
Table 1.
 
Composition of Perfusion Fluid Administered to Isolated Perfused Eye
Chemical Concentration
Tissue culture medium 1000 mL
Sodium bicarbonate 2.2 g/L
Atropine sulfate 0.005 g/L
EDTA 0.2922 g/L
Penicillin G 100 kU/L
Streptomycin 75.6 kU/L
Gentamycin 0.08 g/L
Insulin bovine 50 U/L
Bovine holo-transferrin 0.0025 g/L
Sodium selenite 2.4 μg/L
ToF-SIMS Analysis
A ToF-SIMS instrument (Ion-TOF IV; ION-TOF GmbH, Münster, Germany) using a Bi3+ cluster source and a single-stage reflectron analyzer was used to perform time of flight–secondary ion mass spectrometry. Spectra were acquired in both positive and negative modes by rastering a primary ion energy of 25 kV, along with a pulsed target current of approximately 1 pA and postacceleration energy of 10 kV across the sample surface. Analysis was performed on a sample area of 500 × 500 μm of each tissue. The primary ion dose density was maintained at <1012 ions/cm2 throughout to ensure static conditions. A common insulating effect of biological surfaces is positive primary ion beam induced surface charging. To account for this, low-energy electrons (20 eV) were delivered to the sample surface throughout the analysis. Data processing was performed using imaging software (SurfaceLab 6 Image; ION-TOF GmbH), for spectroscopy and image analysis. 
Results
To analyze lens, retina, and vitreous samples by ToF-SIMS, both positive and negative spectra were obtained for all samples. For each of the tissue subtypes and tissue samples, perfused eye samples, and nonperfused eye samples, analysis was performed on three replicates of each sample. ToF-SIMS analysis was also performed on a sample of dexamethasone alone. In ToF-SIMS analysis, the ion beam is rastered across the sample surface and a complete mass spectrum is obtained for each point on the sample hit. From the mass spectra produced, key ions of interest can then be selected and an image of the distribution pattern of the ion in the sample generated. Mass spectra obtained for each sample contained numerous peaks with mass-to-charge ratio (m/z) ranging from 1 to 870. Peaks at the lower end of the mass spectra dominated, with greater peak intensities achieved in the m/z range of 1 to 200, a common occurrence in ToF-SIMS. 22 A peak list was generated for both the positive and negative spectra, using a method detailed previously by Urquhart et al. 22 Peak lists were initially generated using the pure drug substance sample. Using the positive spectra obtained for the drug and tissue samples, 172 ion peaks were selected and added to form the peak list. In the negative pure drug spectra, 292 ion peaks were selected and added to form the peak list. The positive and negative ion peak lists generated were then applied to each of the tissue samples. Positive spectra obtained from a perfused eye lens sample and a nonperfused lens sample were then directly compared with the positive spectra obtained for the pure drug sample. Using all three spectra, key drug peaks present in the lens samples were identified. This process was repeated for both the retina and the vitreous tissue samples and used to generate a list of fundamental drug ion fragments to be imaged across the samples. Across the samples in the positive spectra, four significant dexamethasone fragments were identified, attributed to their presence in the spectra obtained for the drug, the lens, the vitreous, and the retina samples (m/z = 63, 109, 187, 289). 
Images of the distribution of the significant positive ion drug peaks in the nonperfused eye treated with dexamethasone are shown in Figure 2. A high signal intensity was obtained for drug peak m/z = 63 in the vitreous samples. Similar signal intensities were achieved at the front and the middle of the vitreous, with the greatest signal intensity recorded in the back section of the vitreous toward the retina. A high signal intensity was also seen at the front of the retina, with intensity decreasing in the middle of the retina and becoming extremely low at the back of the retina. In the lens samples, the presence of the molecular ion m/z = 63 was negligible. The remaining three chosen drug peaks (m/z = 109, 187, 289) showed similar intensities in the nonperfused eye. Signals were detectable in the vitreous samples with low intensity at the front and in the middle of the vitreous but increasing in intensity at the back of the vitreous. In the retina, although signal intensity was low, the intensity decreased moving through the retina, with the highest signal intensity detected at the front of the retina, next to the vitreous, and the lowest at the back. Again in the lens samples, the presence of all three drug peaks was insignificant. 
Figure 2.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z (mass-to-charge ratio) in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for nonperfused eye tissue sections.
Figure 2.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z (mass-to-charge ratio) in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for nonperfused eye tissue sections.
Distributions of the significant positive ion drug peaks in the perfused eye samples treated with dexamethasone are shown in Figure 3. For drug peak m/z = 63 in the vitreous samples a high signal intensity, similar to that seen in the back of the vitreous of the nonperfused eye samples, was seen across the front, middle, and back sections of the vitreous. Signal intensities in the retina of the perfused eye samples were higher than those in the nonperfused eye samples. High signal intensities were seen at the front and in the middle sections of the retina, with intensity decreasing at the back of the retina. In the lens samples of the perfused eye, a low intensity of mass peak m/z = 63 was detected at the back of the lens (in contact with the vitreous); however, at the front and in the middle of the lens signal intensities were still insignificant. For drug peak m/z = 109, significant and relatively equal signal intensity was seen in the front vitreous, middle vitreous, front retina, and middle retina, with seemingly even distribution across the samples. At the back of the vitreous and the back of the retina, the signal intensity was slightly reduced. In the lens samples, again a low intensity was detected at the back of the lens, with negligible drug detection at the front and in the middle of the vitreous. A similar pattern of intensity was shown for drug peak m/z = 187 for the retina and vitreous samples, with the exception for the lens tissue where for all samples front to back, signal intensities were negligible. For drug mass peak m/z = 289, a similar pattern of intensity to drug peak m/z = 187 was noted across all samples with reduced signal intensity. 
Figure 3.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for perfused eye tissue sections.
Figure 3.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for perfused eye tissue sections.
As with the positive spectra, key drug peaks present in the perfused eye and nonperfused eye samples were identified by directly comparing the negative spectra of samples with the negative spectra obtained for the pure drug samples. After this, the process was again repeated for both the retina and the vitreous to generate a complete list of crucial drug ion fragments to be imaged across the sample. Across the samples in the negative spectra, just one critical dexamethasone fragment (m/z = 19) was identified due to its presence in the spectra obtained for the drug, the lens, the vitreous, and the retina samples. Distribution of the m/z = 19 drug fragment in the nonperfused eye samples treated is shown in Figure 4A. In the nonperfused eye, fluorine signal intensity was low at the front of the vitreous but high in the middle and in the back vitreous samples. Interestingly, a spot of very high signal intensity was seen in the middle vitreous and also in the front of the retina. At the front of the retina, significant peak intensity was recorded and, again, a small area of larger signal intensity was seen. In the remaining retina samples, lower intensities were seen in the middle and at the back of the retina. Peak intensity in the lens samples was marginal. Distribution of the m/z = 19 drug fragment in the perfused eye samples treated is shown in Figure 4B. In the perfused eye greater dexamethasone distribution was apparent. Across the vitreous samples, high, evenly distributed signal intensities were seen from front to back, with slightly greater intensity seen at the back of the vitreous. In the retina samples, m/z = 19 peak intensity was much greater in the perfused eye compared with that in the nonperfused eye, with slightly reduced signal intensity at the back of the retina compared with that at the front and the middle retina samples. Again, in the lens samples signal intensities were marginal, with a slightly greater signal intensity noted at the back of the lens (see Fig. 5 for increased clarity). 
Figure 4.
 
(A) F ion distribution in the nonperfused eye (500 × 500 μm images). (B) F ion distribution in the perfused eye (500 × 500 μm images).
Figure 4.
 
(A) F ion distribution in the nonperfused eye (500 × 500 μm images). (B) F ion distribution in the perfused eye (500 × 500 μm images).
Figure 5.
 
Enhanced-contrast ToF-SIMS images, from the perfused eye back of the lens sample, altering saturated pixel percentage from 0.4% to 2.5% as an aid for clarity. (A) m/z = 19, (B) m/z = 63, and (C) m/z = 109. All images are 500 × 500 μm.
Figure 5.
 
Enhanced-contrast ToF-SIMS images, from the perfused eye back of the lens sample, altering saturated pixel percentage from 0.4% to 2.5% as an aid for clarity. (A) m/z = 19, (B) m/z = 63, and (C) m/z = 109. All images are 500 × 500 μm.
Discussion
Dexamethasone phosphate was selected for investigation in this work because of its anti-inflammatory effect in both the anterior and the posterior eye and also its structural chemistry. Dexamethasone sodium phosphate is a relatively large, slightly lipophilic drug molecule, with a molecular weight of 516.45 and a log P of 0.56 (Fig. 1). The presence of the fluorine group makes it an ideal candidate for studying distribution using ToF-SIMS, in that fluorine-based anions tend to dominate ToF-SIMS negative ion spectra due to the stability of the fluorine anion. 33,34  
The four drug peaks identified in the positive ion spectra represent molecular fragments of dexamethasone sodium phosphate. The peak at m/z = 63 represents C5H3 +, a commonly encountered hydrocarbon species in positive ToF-SIMS spectra of molecules containing aromatic ring structures. 35 It is possible that m/z = 63 could be a fragment of an amino acid, such as phenylalanine, tryptophan, or tyrosine; however, due to the uneven distribution within the vitreous of the nonperfused eye, m/z = 63 represents a single-ring fragment of the drug. Mass peak m/z = 109 represents C7H9O+, which represents another single-ring–based structure, arising typically in mass spectrometry of steroid drug structures. 36 The peak at m/z = 187 remains unassigned, although the similarity in distribution of this ion peak to that of the other drug peaks suggests it is likely to represent a bicyclic ring fragment of the drug. The final drug peak identified in the positive spectra at m/z = 289 represents C18H25O3 +, a tricyclic ring fragment generated from dexamethasone. 37 In the positive spectra, there is a potential chance that a proportion of the ion intensity measured for each molecular ion fragment could be influenced by the signal generated by molecular ions emitted from biological molecules released from the tissue surface. In the case of negative spectra, the ion intensity signal of the characteristic fluorine ion of the drug was high and is highly unlikely to be influenced by the secondary ions generated from the tissue sample. For this reason, m/z = 19 (F) was also selected to monitor drug distribution through the eye and to validate the patterns of drug distribution recorded for key drug peaks obtained in the positive spectrum. 
All the positive and negative drug fragments selected to monitor drug distribution in ocular tissue cross-sections followed very similar patterns of drug distribution within each of the eye tissue sections; however, it is noted that the signal intensities obtained for each individual ion are not the same. Because the individual ion fragments are generated from the same parent drug compound, in a simplistic view it would be anticipated that each ion fragment would generate the same signal intensity, given that the concentration of the drug contained in the tissue cross-section from which the ion is generated is the same. In ToF-SIMS this is not the case because the yield of secondary ions generated is not a simple process and can depend on several factors. Two important factors, which experience variability and influence secondary ion generation and therefore signal intensity, are the yield of secondary ions generated per impact of the primary ion beam and also the probability that a given particle will be released from the sample surface as a certain ion. 38 In addition, there is also the influence of matrix-effect phenomena, where the yield of secondary ions emitted can vary depending on the environment in which they are generated. The sample environment can change during analysis and this again can influence the secondary ion yield, although by using the spectra preprocessing steps discussed previously, the impact of this effect can be limited. 39  
The nonperfused eye was selected as an initial means of determining the feasibility of imaging drug location within the ocular tissue cross-sections. In the nonperfused eye, flow and circulation within the isolated eye are redundant and, as a result, drug movement will occur by diffusion alone. There are also no clearance systems operating within the nonperfused eye; therefore, no drug would have been removed from the eye during the course of the experiment and drug concentrations would have remained high in the vitreous. In the vitreous of the nonperfused eye, drug fragment distribution was successfully imaged and drug diffusion from the front section of the vitreous to the middle and back sections was evident. In the nonperfused eye a differential concentration gradient is present in the vitreous, providing a motive force, which drives drug movement through the vitreous to the retina, and this is shown by the presence of drug in the positive spectrum in the front section of the retina only (Fig. 2) This is confirmed by the generation of a much greater signal intensity of F in the front section of the retina, compared with the middle and back sections, in the negative spectrum (Fig. 4A). Because the drug solution was injected into the front of the vitreous, the lack of uniformity in drug distribution across the vitreous (from front to back) is likely due to the vitreal structure. The vitreous is a fluid-based structure composed of approximately 99% water. However, although composed of predominantly water, the vitreous has a gel-like structure attributed to its viscoelastic properties to collagen, hyaluronic acid, and proteoglycans that are contained within. 40 It has previously been shown that dexamethasone diffusion through the vitreous is four- to fivefold slower than its diffusion in water. 41 In the nonperfused eye lens images, the drug was not shown to be present due to the lack of flow operating in the dead eye and, thus, the drug did not penetrate into the ocular lens. In addition we also see a potential element of drug clumping in the vitreous and the retina of the nonperfused eye; this could be explained by drug localization on structures contained within these tissues when flow systems are not in operation within the eye. 
The perfused eye system has been developed as an effective method of investigating properties of the eye, ex vivo, while maintaining arterial flow and nutritional status within the eye. 42 Advantages of the isolated eye model compared with performing a full animal study include no anesthetic requirements or concern over animal comfort, control over the physiologic environment, reduced animal usage, controlled drug administration, and control of exposure to substances from systemic circulation into the ocular system, 43 making it an ideal model in which to study drug distribution. In the vitreous samples of the perfused eye, a more even distribution of the drug across the front, the middle, and the back of the vitreous was evident, when compared with that of the nonperfused eye. Because flow systems remain operational within the perfused eye, drug movement in the vitreous occurs by both diffusion and convection. 44,45 There has been some debate over the role of convection in drug distribution in the vitreous, with some suggestion that convection plays a role, although the impact is less than that of diffusion, 44,46 whereas other work has suggested that convection will become important only for larger sized molecules. 47,48 Although dexamethasone sodium phosphate is regarded as a small drug molecule, it is clear from the images that drug movement within the nonperfused and perfused eye differ, with drug movement in the perfused eye occurring not only in the posterior direction but also in the anterior direction, where it was also detected in the back section of the lens. Therefore, in this case drug movement is not caused solely by diffusion but also by circulation systems operating within the eye. In the perfused eye, the signal intensity in the retina images was much larger than the signal obtained in the nonperfused eye retina images for both the positive drug fragments and the negative drug fragment F. The drug was evident in all retinal cross-sections, demonstrating that dexamethasone sodium phosphate can penetrate from the vitreous completely through the retina to the posterior retina, which is extremely desirable in the treatment of posterior eye disease. The transfer of materials at the retinal pigment epithelium and therefore drug movement from the vitreous to the retina and the choroid is not only controlled by flow systems operating within the eye but is also thought to involve drug transporter systems. 40,41 Previously, the involvement of transporter systems in ocular drug delivery, such as p-glycoprotein, organic cation transports, and organic anion transporters, has been demonstrated. 42 44 The involvement of drug transporter systems in dexamethasone movement into the retina was outside the scope of this study; however, the potential impact of drug transporter systems on dexamethasone distribution in ocular tissues should be considered when interpreting the results. Although the signal intensity of drug fragments in the lens is extremely low, the presence of drug within the back section of the lens is apparent, suggesting that dexamethasone will also move toward the anterior eye after intravitreal administration. However, the lack of drug presence in the middle and the front lens sections could suggest that the drug will not move any further forward through the lens and will potentially move back into the vitreous. A similar effect on movement was noted by Tan et al. 49 with intravitreally injected sodium fluorescein in the rabbit eye. Using ocular fluorophotometry, it was shown that sodium fluorescein will penetrate into the ocular lens 2 hours after dosing; however, the substance did not continue a forward diffusion into the anterior chamber but unloaded the absorbed fluorescein back into the vitreous humor 3 hours later. However, due to the short time course of the experiment it is difficult to determine whether the drug would penetrate further into the lens and through to the anterior chamber. It would be interesting in the future to extend the perfusion time and image the drug moving through and out of the lens tissue. 
The results presented in this work illustrate the ability of ToF-SIMS to characterize and provide spatial information about drug distribution within ocular tissues. The capability to characterize drug positioning within an ocular tissue is extremely desirable, such as when determining ocular drug distribution, ocular tissues are homogenized and the drug is then extracted from the tissue. Therefore, no specific information with regard to the spatial positioning of the drug within the tissue can be determined. The ability to map and image drug positioning within individual ocular tissues holds significant value in adding to the understanding of how we can improve drug treatment options in posterior eye disease. Understanding spatial drug distribution within the target site of action, the retina, is especially useful because drug penetration through the retina is key in treating disease states such as diabetic retinopathy and age-related macular degeneration. In addition, here we have also demonstrated key differences in drug movement through the vitreous humor, toward both the anterior and the posterior tissues, in the living eye and the nonliving ovine eye, demonstrating that dexamethasone sodium phosphate distribution through the vitreous is not determined by diffusion alone and will involve the circulatory flow systems operating within the eye and drug transporter systems. 
Footnotes
 Supported by a studentship from AstraZeneca and the University of Strathclyde (JM).
Footnotes
 Disclosure: J. Mains, None; C.G. Wilson, None; A. Urquhart, None
The authors thank David Scurr from Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham for assistance with ToF-SIMS measurements. 
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Figure 1.
 
The chemical structure of dexamethasone. Note the fluorine atom bonded to the ninth carbon atom in the tetracyclic ring, which provides a unique chemical label for the drug.
Figure 1.
 
The chemical structure of dexamethasone. Note the fluorine atom bonded to the ninth carbon atom in the tetracyclic ring, which provides a unique chemical label for the drug.
Figure 2.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z (mass-to-charge ratio) in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for nonperfused eye tissue sections.
Figure 2.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z (mass-to-charge ratio) in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for nonperfused eye tissue sections.
Figure 3.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for perfused eye tissue sections.
Figure 3.
 
ToF-SIMS images showing the spatial distribution of drug-specific dexamethasone sodium phosphate drug fragments detailed by the drug fragments' m/z in the front, middle, and back sections of the lens, the vitreous humor, and the retina. The 500 × 500 μm images were obtained from measurements of positive secondary ions and show images obtained for perfused eye tissue sections.
Figure 4.
 
(A) F ion distribution in the nonperfused eye (500 × 500 μm images). (B) F ion distribution in the perfused eye (500 × 500 μm images).
Figure 4.
 
(A) F ion distribution in the nonperfused eye (500 × 500 μm images). (B) F ion distribution in the perfused eye (500 × 500 μm images).
Figure 5.
 
Enhanced-contrast ToF-SIMS images, from the perfused eye back of the lens sample, altering saturated pixel percentage from 0.4% to 2.5% as an aid for clarity. (A) m/z = 19, (B) m/z = 63, and (C) m/z = 109. All images are 500 × 500 μm.
Figure 5.
 
Enhanced-contrast ToF-SIMS images, from the perfused eye back of the lens sample, altering saturated pixel percentage from 0.4% to 2.5% as an aid for clarity. (A) m/z = 19, (B) m/z = 63, and (C) m/z = 109. All images are 500 × 500 μm.
Table 1.
 
Composition of Perfusion Fluid Administered to Isolated Perfused Eye
Table 1.
 
Composition of Perfusion Fluid Administered to Isolated Perfused Eye
Chemical Concentration
Tissue culture medium 1000 mL
Sodium bicarbonate 2.2 g/L
Atropine sulfate 0.005 g/L
EDTA 0.2922 g/L
Penicillin G 100 kU/L
Streptomycin 75.6 kU/L
Gentamycin 0.08 g/L
Insulin bovine 50 U/L
Bovine holo-transferrin 0.0025 g/L
Sodium selenite 2.4 μg/L
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