Imaging-Based Drug Penetration Profiling in an Excised Sheep Cornea Model

Abstract

Formulations designed to address ocular conditions and diseases are predominantly administered topically. While in vitro test systems have been developed to assess corneal permeation under extended contact conditions, methods focusing on determining the penetration depth and kinetics of a substance within the cornea itself rather than through it, are scarce. This study introduces a method for time-dependent penetration depth analysis (10 and 60 min) by means of a semiquantitative imaging method in comparison with a quantitative corneal depth-cut technique, employing fluorescein sodium at concentrations of 0.2 and 0.4 mg/mL as a small molecule model substance and sheep cornea as a human surrogate. Excised tissues exhibited sustained viability in modified artificial aqueous humor and maintained thickness (746 ± 43 µm) and integrity (electrical resistance 488 ± 218 Ω∙cm²) under the experimental conditions. Both methods effectively demonstrated the expected concentration- and time-dependent depth of penetration of fluorescein sodium, displaying a significantly strong correlation. The traceability of the kinetic processes was validated with polysorbate 80, which acted as a penetration enhancer. Furthermore, the imaging-based method enabled detecting the retention of larger structures, such as hyaluronic acid and nanoemulsions from the commercial eyedrop formulation NEOVIS^® TOTAL multi, inside the lacrimal layer.

Introduction

Treatment of diseases and pathological conditions of the eye is typically conducted by the application of topical formulations, such as eye drops, creams, or ointments. However, the development of effective topical formulations for the eye remains a challenge due to tear flow, naso-lachrymal drainage, and blinking, which thoroughly clear the corneal surface of an active pharmaceutical ingredient (API) before effective doses penetrate and permeate the cornea, as the eye’s outermost barrier, to the designated target tissue [1]. Furthermore, the cornea itself effectively limits transport into the eye due to its multilayered structure [2,3]. It is composed of five distinct layers, namely the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium, which contribute to its structural integrity, transparency, and critical barrier function. The multilayered epithelium of surface cells with tight junctions, wing cells, and basal columnar cells is particularly difficult for substances to overcome [4]. Although the corneal barrier could easily be bypassed by intravitreal injections, this form of application requires administration by health professionals, and thus, topically administered products retained the greatest market share of ophthalmologic products in 2022 [5]. To assess the corneal permeation of APIs from topical formulations in ophthalmic formulation development, various in vitro and ex vivo test systems have been established. For in vitro ocular drug delivery models, primary cell culture [6], immortalized cell lines [7,8], or reconstructed tissue culture [9,10] are being used.

However, 3D culture models do not provide full-thickness tissue layers, which can subsequently lead to a false overestimation of drug penetration [11]. Therefore, ex vivo permeation studies are conducted either as diffusion cell assemblies with the whole cornea as the diffusion membrane or as whole eye setups, both considering all corneal layers and allowing analysis of drug amounts after passage of the entire corneal tissue. Of course, the human cornea represents the gold standard in these studies, but as it is scarce and mainly reserved for transplantation, animal surrogate tissues from different species, such as rabbits or pigs, have been used [12]. Nevertheless, due to the excellent barrier properties, the comparably small corneal area present for diffusion in these animal tissues would either require specialized study setups and highly sensitive devices for quantification of trace amounts of permeant or a prolonged study time. Prolonging the duration of such ex vivo experiments not only leads to a vastly increased contact time of formulation to the tissue compared to in vivo conditions but also comes at the risk of reduced corneal integrity, accompanied by a decrease in viability and corneal swelling that might lead to overestimated permeation results [13]. Additionally, not every API that is topically applied for the treatment of eye diseases has its target site behind the corneal barrier. Instead, diseases like dry eye disease or keratitis are typical impairments of the cornea that rely on drug integration into the lacrimal film or penetration into the cornea rather than permeation through it [14].

In these cases, standard permeation experiments are less suitable since their focus lies on the amount delivered to the basolateral compartment. Accordingly, a detailed analysis of both substance retention on the eye’s surface and its residence time-dependent distribution into the cornea is needed in formulation development, yielding conclusive results in a suitable timeframe under conditions that ensure corneal integrity to better mimic in vivo conditions. To this end, we present an adaptation of the Franz diffusion cell permeation setup, directly analyzing the penetration of substances into the corneal tissue and the distribution therein using an image-based approach. The sheep cornea, which displays all layers found in the human cornea (Figure S1), was used as a surrogate [15,16]. The total corneal thickness of sheep, pig, and rabbit corneas is similar to that of humans, but the relative epithelial thickness is more analogous to the human cornea in sheep and rabbits [11,17,18,19,20]. The larger corneal area of sheep compared to pigs and rabbits offers improved handling and analysis options, making it the most suitable surrogate for the presented study [11]. The experimental conditions were optimized to ensure prolonged tissue viability and integrity over the duration of the experiments. The model was established by analyzing the time- and concentration-dependent penetration kinetics of the small model molecule fluorescein sodium (FS), which has broadly been used in eye research [21,22,23,24,25], as well as the influence of polysorbate 80 (P80) on FS-penetration semi-quantitatively by confocal laser scanning microscopy (CLSM).

The image-derived results were then confirmed by quantitative analysis of extracted FS from corneal depth cuts. Moreover, the applicability of the image-based technique to more complex formulations containing larger molecules was demonstrated by analyzing an eyedrop nanoemulsion formulation inspired by the composition of the lacrimimetic commercial product NEOVIS® TOTAL multi and subsequent assessment of the penetration of its key components or the adhesion to surface cells, i.e., macromolecular hyaluronic acid and the lipid emulsion droplets.

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Materials

Dimethylthiazolyldiphenyl-tetrazolium bromide (MTT) and 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES) were purchased from Carl Roth GmbH + Co.KG (Karlsruhe, Germany). Dimethylsulfoxide (DMSO) was obtained from VWR International (Radnor, PA, USA). Sodium dodecylsulfate (SDS) was purchased from Caesar & Loretz GmbH (Hilden, Germany). Triton^® X-100, Ethylendiamine, N-ethyl-N9-(3-dimethyl-aminopropyl) carbodiimide, Fluorescein-5(6)-isothiocyanate (FITC), and 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) were obtained from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s minimum essential medium (DMEM) and penicillin plus streptomycin were purchased from Biochrom (Berlin, Germany). Fluorescein sodium was supplied by Merck KGaA (Darmstadt, Germany). Montanox™ 80 PPI (polysorbate 80 for injection) was a gift from Seppic (La Garenne Colombes, France). Sodium hyaluronate (HA), Hydroxypropylmethylcellulose (HPMC), Miglyol 812, and Soybean lecithin lipoid p75 were kindly provided by Horus Pharma (Nice, France). All chemicals used to prepare buffers were of analytical grade or higher. Milli-Q^® water (Q-Gard^® Purification Catridge, Type 1 water) was used throughout the experiments. Sheep heads of class A sheep (Regulation (EU) No. 1308/2013), breed: Einola, 4–6 months old, following the routine slaughtering procedure, were purchased from a local commercial slaughterhouse (Regulation (EU) No. 1069/2009, category three material (article 10a) used for scientific purposes (article 16f.).

Viehmeister, K.; Manuelli, A.; Guerin, C.; Kappes, S.; Lamprecht, A. Imaging-Based Drug Penetration Profiling in an Excised Sheep Cornea Model. Pharmaceutics 2024, 16, 1126. https://doi.org/10.3390/pharmaceutics16091126

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