Porcine Corneas Research Articles

Acellular Porcine Cornea Produced by Supercritical Carbon Dioxide Extraction: A Potential Substitute for Human Corneal Regeneration.

The aim of this study was to develop a non-cytotoxic, biocompatible innovative acellular porcine cornea (APC) for corneal wound healing and corneal blindness treatment. APC was produced by using supercritical carbon dioxide (SCCO2) to decellularize the porcine cornea. Decellularization of the porcine cornea was examined by hematoxylin and eosin staining and 4,6-diamidino-2-phenylindole, dihydrochloride staining. The residual DNA content of APC was analyzed in comparison with the native porcine cornea. Virus inactivation up to at least 6 log10 was confirmed for the stepwise process of APC for 4 different model viruses. In addition, a series of in vitro and in vivo tests in accordance with ISO-10993 biocompatibility assay and animal performance tests were performed. APC produced by the SCCO2 process revealed complete decellularization, without any residual non-collagenous proteins. The scanning electron microscopy structural features of the decellularized cornea were similar to those of human. APC was found to be nontoxic and exhibited excellent biocompatibility in both in vitro and in vivo studies. The animal performance test proved that APC exerted excellent adaptability on the cornea and no sign of irritation and good compatibility in lamellar corneal transplantation. APC manufactured by SCCO2 technology revealed complete cells and non-collagenous protein removal compared with the Triton-sodium dodecyl sulfate decellularization process. APC showed excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future.

Corneal riboflavin gradients and UV-absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1 to 0.5%

Avoiding damage of the endothelial cells, especially in thin corneas, remains a challenge in corneal collagen crosslinking (CXL). Knowledge of the riboflavin gradients and the UV absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1% to 0.5% could optimize the treatment. In this study, we present a model to calculate the UV-intensity depending on the corneal thickness.Ten groups of de-epithelialized porcine corneas were divided into 2 subgroups. Five groups received an imbibition of 10 min and the other five groups for 30 min. The applied riboflavin concentrations were 0.1%, 0.2%, 0.3%, 0.4% and 0.5% diluted in a 15% dextran solution for each subgroup. After the imbibition process, two-photon fluorescence microscopy was used to determine fluorescence intensity, which was compared to samples after saturation, yielding the absolute riboflavin concentration gradient of the cornea. The extinction coefficient of riboflavin solutions was measured using a spectrophotometer. Combining the obtained riboflavin concentrations and the extinction coefficients, a depth-dependent UV-intensity profile was calculated for each group.With increasing corneal depth, the riboflavin concentration decreased for all imbibition solutions and application times. The diffusion coefficients of 10 min imbibition time were higher than for 30 min. A higher RF concentration and a longer imbibition time resulted in higher UV-absorption and a lower UV-intensity in the depth of the cornea. Calculated UV-transmission was 6 percentage points lower compared to the measured transmission.By increasing the riboflavin concentration of the imbibition solution, a substantially higher UV-absorption inside the cornea is achieved. This offers a simple treatment option to control the depth of crosslinking e.g. in thin corneas, resulting in a lower risk of endothelial damage.

New HPLC-UV analytical method for quantification of metronidazole: Application to ex vivo ocular kinetic assessments following the administration of thermosensitive ocular in situ gel

Metronidazole eye drops have been used to treat Acanthamoeba keratitis. However, ophthalmic preparations also have some limitations, one of which is the rapid elimination of the drug, that reducing the effectiveness of the drug. Accordingly, an alternative delivery approach can be applied to overcome this issue. Additionally, as one of critical steps in the formulation development, analyitical methods that allow the quantification of metronidazole in ex vivo corneal permeation and deposition should also be developed. Here, we report a validated high-performance liquid chromatography method (HPLC-UV) according to ICH guidelines for the measurements of metronidazole concentrations following formulation of thermosensitive ocular in situ gel and its administration in ex vivo porcine corneas. The development of extraction techniques and optimization of HPLC conditions were optimized using analytical quality by design. Xselect™ CSHTM C18 HPLC column (Water, 3.0 × 150 mm, particle size 3.5 m) was used to separate all analytes by isocratic elution with mobile phases of acetate buffer and acetonitrile with LLOQ value of 0.08 μg/mL. The resulting method proved to be selective, precise, and accurate and was succefully applied to determine ocular kinetic profiles of metronidazole from thermosensitive ocular in situ gel in ex vivo porcine corneas, showing that this approach was able to improve the concentration of metronidazole in the corneal tissues. We, therefore, suggested that HPLC-UV approach developed in this study has the potential to be used in drug release evaluation, therapeutic drug control research, ocular kinetics, and toxicological evaluation.

Non-contact acoustic micro-tapping optical coherence elastography (AμT-OCE) in the cornea: Mechanical model, ex vivo and in vivo results, and direct comparison with mechanical tests

The cornea is one of the primary determinants of visual performance. Its highly pressurized anisotropic structure of collagen fibrils embedded in a hydrated proteoglycan matrix forms the clear refractive primary lens of the eye. Despite recent success in corneal topography, there are no clinical tools to predict shape changes from vision-correction therapies such as LASIK and CXL due to the absence of non-contact methods that can map corneal elasticity in vivo. Here we review our newest results in quantification of corneal elasticity. Recently, we produced a technology combining non-contact excitation of sub-mm wavelength mechanical waves in the cornea using air-coupled acoustic micro-tapping (AμT), with 1.6 MHz scan-rate phase-sensitive OCT to track their propagation three-dimensionally in real time. However, corneal moduli reconstruction from measured wavefields requires an anisotropic elastic model and cornea’s bounded, non-flat geometry must be taken into account. Thus, a nearly incompressible transversally isotropic (NITI) model of corneal elasticity was developed. We have performed multiple ex vivo and in vivo studies in rabbit, porcine and human corneas and compared AμT-OCE results directly with conventional mechanical tests (tensile extensometry and shear rheometry). These studies demonstrate that in-plane Young’s and out-of-plane shear moduli can be accurately mapped with non-contact, non-invasive AμT-OCE.

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

Statistical analysis of corneal OCT speckle: a non-parametric approach.

In biomedical optics, it is often of interest to statistically model the amplitude of the speckle using some distributional approximations with their parameters acting as biomarkers. In this paper, a paradigm shift is being advocated in which non-parametric approaches are used. Specifically, a range of distances, evaluated in different domains, between an empirical non-parametric distribution of the normalized speckle amplitude sample and the benchmark Rayleigh distribution, is considered. Using OCT images from phantoms, two ex-vivo experiments with porcine corneas and an in-vivo experiment with human corneas, an evidence is provided that the non-parametric approach, despite its simplicity, could lead to equivalent or better results than the parametric approaches with distributional approximations. Concluding, in practice, the non-parametric approach should be considered as the first choice to speckle modeling before a particular distributional approximation is utilized.

Biomechanical Response After Corneal Cross-linking With Riboflavin Dissolved in Dextran Solution Versus Hydroxypropyl Methylcellulose.

To evaluate corneal stiffening in porcine eyes induced by corneal cross-linking (CXL) using riboflavin dissolved in either aqueous dextran or hydroxypropyl methylcellulose (HPMC) solution. Fifty-one porcine corneas were divided into three groups of 17 each. After deepithelialization, the first (Dresden) group was treated for 30 minutes with 0.1% riboflavin (riboflavin-5-monophosphate in 0.9% NaCl) dissolved in hypertonic 20% dextran and the second (HPMC) group for 30 minutes with isotonic solution containing 0.1% riboflavin and 1.1% HPMC. Thereafter, corneas of both groups were irradiated using 5.4 J/cm2 (irradiance of 9 mW/cm2 for 10 minutes; 10*9). After CXL, all corneas were kept in an isotonic 16% dextran bath for 2 hours to obtain an equal hydration state. The third group served as the control group. Stress-strain measurements were performed on 5-mm-wide strips. Corneal thickness was monitored throughout the entire course of the experiments. The required stress for a 10% strain was increased by 83% in the Dresden group and 35% in the HPMC group compared to the control group. Resultant Young's modulus (at 10% strain) was 2.53 ± 0.73, 1.87 ± 0.50, and 1.47 ± 0.44 Pa for the Dresden, HPMC, and control groups, respectively. The differences between the Dresden and HPMC groups (P = .006), the Dresden and control groups (P < .001), and the HPMC and control groups (P = .014) were statistically significant. Pachymetry measurements showed a significantly increased corneal thickness after application of HPMC compared with the Dresden group (P = .002) and control group (P = .041). The biomechanical stiffening of the cornea by CXL can be achieved using dextran- and HPMC-based riboflavin solutions in porcine corneas with an application time of 30 minutes. Dextran-based riboflavin solutions seem to induce a slightly stronger biomechanical response in this setting. HPMC solutions induce less thinning than dextran solutions. [J Refract Surg. 2021;37(9):631-635.].

Novel Formulation of Eye Drops Containing Choline Salicylate and Hyaluronic Acid: Stability, Permeability, and Cytotoxicity Studies Using Alternative Ex Vivo and In Vitro Models.

This work investigated the potential of a novel formulation of eye drops containing a non-steroidal anti-inflammatory drug—choline salicylate (CS)—and hyaluronic acid (HA). Thus, these drops may exert both anti-inflammatory and regenerative activity. The experiment was conducted through the careful characterization of physicochemical properties, stability, and quality of eye drops. Moreover, microbiological analysis, as well as penetration and cytotoxic studies, were performed. The UV, HPLC-UV, and HPLC-MS/MS methods were used to determine the purity and stability of CS. The penetration rate of CS was assessed using a hydrophilic membrane and ex vivo porcine cornea model. Additionally, the cytotoxic effects were evaluated using the SIRC cell line. The interaction between HA and CS was tested using size-exclusion chromatography and IR spectrophotometry. As a result, HA increased the viscosity of the drops, which prolonged their contact with the ocular surface, thus ensuring more effective penetration of CS into the corneal structure. After long-term storage, an interaction in the pharmaceutical phase between CS and HA was observed. However, this interaction did not affect the viability of rabbit corneal cells. Our findings showed that eye drops with CS and HA, stored at 2–8 °C in light-protected conditions, met the criteria of stability and safety.

Hydrogels derived from acellular porcine corneal stroma enhance corneal wound healing

Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. Statement of significanceExtracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Establishment of a Robust and Simple Corneal Organ Culture Model to Monitor Wound Healing.

The use of in vitro systems to investigate the process of corneal wound healing offers the opportunity to reduce animal pain inflicted during in vivo experimentation. This study aimed to establish an easy-to-handle ex vivo organ culture model with porcine corneas for the evaluation and modulation of epithelial wound healing. Cultured free-floating cornea disks with a punch defect were observed by stereomicroscopic photo documentation. We analysed the effects of different cell culture media and investigated the impact of different wound sizes as well as the role of the limbus. Modulation of the wound healing process was carried out with the cytostatic agent Mitomycin C. The wound area calculation revealed that after three days over 90% of the lesion was healed. As analysed with TUNEL and lactate dehydrogenase assay, the culture conditions were cell protecting and preserved the viability of the corneal tissue. Wound healing rates differ dependent on the culture medium used. Mitomycin C hampered wound healing in a concentration-dependent manner. The porcine cornea ex vivo culture ideally mimics the in vivo situation and allows investigations of cellular behaviour in the course of wound healing. The effect of substances can be studied, as we have documented for a mitosis inhibitor. This model might aid in toxicological studies as well as in the evaluation of drug efficacy and could offer a platform for therapeutic approaches based on regenerative medicine.

Full-field strain mapping for characterization of structure-related variation in corneal biomechanical properties using digital image correlation (DIC) technology

Characterization of structure-related variation in corneal biomechanical properties is important for the design of corneal implants with a controllable degradation. In the current study, we conducted an in-vitro inflation experiment on contact and structural-damaged porcine corneas and mapped the strain distribution during using digital image correlation (DIC) algorithm. The inflation experiment was performed with a pressure loading device, a steady-state pressure transducer and DIC system. Corneal samples with uniformly-distributed paint particles were fixed on the loading device, and then were subjected increasing pressure load through injection with a constant velocity (0.134 ​ml/min). Three-dimensional (3D) DIC algorithm was performed to present a map of the full-field strain distribution on the corneal surface. The results showed a significant difference in the strain distribution between the intact and damaged corneas. The time-strain history also exhibited differently when the two types of cornea samples were subjected to inflation. It indicated that the DIC technology is validated to characterize structure-related variation in corneal biomechanical properties.

Clearance of Gram-Negative Bacterial Pathogens from the Ocular Surface by Predatory Bacteria

It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addition, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular surface to clear the tested Gram-negative bacterial pathogens from the ocular surface.

Dexamethasone-Loaded Nanostructured Lipid Carriers for the Treatment of Dry Eye Disease.

Dry eye disease (DED) or keratoconjunctivitis sicca is a chronic multifactorial disorder of the ocular surface caused by tear film dysfunction. Symptoms include dryness, irritation, discomfort and visual disturbance, and standard treatment includes the use of lubricants and topical steroids. Secondary inflammation plays a prominent role in the development and propagation of this debilitating condition. To address this we have investigated the pilot scale development of an innovative drug delivery system using a dexamethasone-encapsulated cholesterol-Labrafac™ lipophile nanostructured lipid carrier (NLC)-based ophthalmic formulation, which could be developed as an eye drop to treat DED and any associated acute exacerbations. After rapid screening of a range of laboratory scale pre-formulations, the chosen formulation was prepared at pilot scale with a particle size of 19.51 ± 0.5 nm, an encapsulation efficiency of 99.6 ± 0.5%, a PDI of 0.08, and an extended stability of 6 months at 4 °C. This potential ophthalmic formulation was observed to have high tolerability and internalization capacity for human corneal epithelial cells, with similar behavior demonstrated on ex vivo porcine cornea studies, suggesting suitable distribution on the ocular surface. Further, ELISA was used to study the impact of the pilot scale formulation on a range of inflammatory biomarkers. The most successful dexamethasone-loaded NLC showed a 5-fold reduction of TNF-α production over dexamethasone solution alone, with comparable results for MMP-9 and IL-6. The ease of formulation, scalability, performance and biomarker assays suggest that this NLC formulation could be a viable option for the topical treatment of DED.

Corneal Lamb wave imaging for quantitative assessment of collagen cross-linking treatment based on comb-push ultrasound shear elastography

Keratoconus, a serious corneal disorder, often causes highly irregular astigmatism and different degrees of visual impairment. Riboflavin/UVA corneal collagen cross-linking(CXL) is currently approved for effective treatment of keratoconus by enhancing the mechanical strength of collagen fibers in the cornea. However, few methods are capable of quantitatively and non-destructively assessing the mechanical properties of the cornea before and after CXL treatments. This study developed a corneal viscoelasticity imaging method based on comb-push ultrasound shear elastography (CUSE) and implemented this method on a Verasonics™ Vantage 256 ultrasound open system with a high-frequency linear array ultrasound transducer. Push beams were generated by three teeth each consisting of 10 elements (working frequency = 10.41 MHz) for inducing Lamb wave propagation in the cornea, and then the system immediately switched to the plane wave imaging mode using 60 elements in the middle (working frequency = 18 MHz). This method can provide a high-resolution 2D Lamb wave velocity image overlapping with a B-mode image as well as quantitative viscoelasticity estimation according to experimentally obtained phase velocity dispersion of Lamb waves. The validation experiments were performed on ex vivo porcine corneas, and the accuracy of elasticity estimation was verified by a tensile test. The results showed that the shear elasticity increased and the viscosity decreased after CXL treatment. The shear elasticity results (reported as mean ± standard deviation) of one control group with no CXL treatment and three CXL-treated groups named as 10 min, 30 min, and 60 min groups according to UV irradiation time were 14.62 ± 3.38 kPa, 49.47 ± 3.63 kPa, 116.54 ± 23.99 kPa, and 197.89 ± 39.64 kPa, respectively, which was in agreement with the results of tensile tests. The ultrasound safety measurement indicated that this method could have acceptable safety, but further to ocular tissue and vision function. The study demonstrated the possibility of using a commercial ultrasound system to obtain high-resolution images of corneal mechanical properties as well as the ability to quantify changes induced by CXL treatment. Therefore, the proposed method could serve as a helpful tool in the studies related in corneal biomechanics.

The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

Asymmetry in Drug Permeability through the Cornea.

The permeability through the cornea determines the ability of a drug or any topically applied compound to cross the tissue and reach the intraocular area. Most of the permeability values found in the literature are obtained considering topical drug formulations, and therefore, refer to the drug permeability inward the eye. However, due to the asymmetry of the corneal tissue, outward drug permeability constitutes a more meaningful parameter when dealing with intraocular drug-delivery systems (i.e., drug-loaded intraocular lenses, intraocular implants or injections). Herein, the permeability coefficients of two commonly administered anti-inflammatory drugs (i.e., bromfenac sodium and dexamethasone sodium) were determined ex vivo using Franz diffusion cells and porcine corneas in both inward and outward configurations. A significantly higher drug accumulation in the cornea was detected in the outward direction, which is consistent with the different characteristics of the corneal layers. Coherently, a higher permeability coefficient was obtained for bromfenac sodium in the outward direction, but no differences were detected for dexamethasone sodium in the two directions. Drug accumulation in the cornea can prolong the therapeutic effect of intraocular drug-release systems.

Partitioning and Spatial Distribution of Drugs in Ocular Surface Tissues.

Ocular drug absorption after eye drop instillation has been widely studied, but partitioning phenomena and spatial drug distribution are poorly understood. We investigated partitioning of seven beta-blocking drugs in corneal epithelium, corneal stroma, including endothelium and conjunctiva, using isolated porcine tissues and cultured human corneal epithelial cells. The chosen beta-blocking drugs had a wide range (−1.76–0.79) of n-octanol/buffer solution distribution coefficients at pH 7.4 (Log D7.4). In addition, the ocular surface distribution of three beta-blocking drugs was determined by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) after their simultaneous application in an eye drop to the rabbits in vivo. Studies with isolated porcine corneas revealed that the distribution coefficient (Kp) between the corneal epithelium and donor solution showed a positive relationship and good correlation with Log D7.4 and about a 50-fold range of Kp values (0.1–5). On the contrary, Kp between corneal stroma and epithelium showed an inverse (negative) relationship and correlation with Log D7.4 based on a seven-fold range of Kp values. In vitro corneal cell uptake showed a high correlation with the ex vivo corneal epithelium/donor Kp values. Partitioning of the drugs into the porcine conjunctiva also showed a positive relationship with lipophilicity, but the range of Kp values was less than with the corneal epithelium. MALDI-IMS allowed simultaneous detection of three compounds in the cornea, showed data in line with other experiments, and revealed uneven spatial drug distribution in the cornea. Our data indicate the importance of lipophilicity in defining the corneal pharmacokinetics and the Kp values are a useful building block in the kinetic simulation models for topical ocular drug administration.

Solubilized ubiquinol for preserving corneal function

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol – the reduced and active form of coenzyme Q10 and a powerful antioxidant – significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or β-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus.

PurposeWe aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus.MethodsThe study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase–mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment.ResultsWe demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment.ConclusionsThis chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment.Translational RelevanceThe cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.

Atorvastatin-Eluting Contact Lenses: Effects of Molecular Imprinting and Sterilization on Drug Loading and Release.

Statins are receiving increasing attention in the ophthalmic field. Their activity as 3-hydroxy-3-methylglutaryl–CoA (HMG–CoA) reductase inhibitors is clinically used to regulate cholesterol levels and leads to pleiotropic effects, which may help in the management of diabetes-related ocular pathologies. This work aims to design bioinspired contact lenses (CLs) with an affinity for atorvastatin by mimicking the active site of HMG–CoA reductase. Sets of imprinted and nonimprinted 2-hydroxyethyl methacrylate (HEMA) hydrogels were synthesized, varying the contents in functional monomers that bear chemical groups that resemble those present in HMG–CoA reductase, namely, ethylene glycol phenyl ether methacrylate (EGPEM), 2-aminoethyl methacrylate hydrochloride (AEMA), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). The hydrogels were characterized in terms of suitability as CLs (solvent uptake, light transmission, mechanical properties, and biocompatibility) and capability to load and release atorvastatin. Three sterilization protocols (steam heat, gamma radiation, and high hydrostatic pressure) were implemented and their effects on hydrogel properties were evaluated. Copolymerization of AEMA and, particularly, APMA endowed the hydrogels with a high affinity for atorvastatin (up to 11 mg/g; KN/W > 200). Only high hydrostatic pressure sterilization preserved atorvastatin stability and hydrogel performance. Permeability studies through the porcine cornea and sclera tissues revealed that the amount of atorvastatin accumulated in the cornea and sclera could be effective to treat ocular surface diseases.