Statens Seruminstitut Rabbit Cornea Research Articles

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

Fluocinolone Acetonide Loaded Chitosan Nanofiber Scaffolds for Treatment of Ocular Disorders: In Vitro Characterization, Ex-Vivo Corneal and Ex-Vivo Scleral Evaluation

Purpose Drugs administered in the ocular region need to overcome ocular barriers without permanently damaging the ocular tissues. Moreover, ocular disorders of the posterior segment are more difficult to treat due to invasive procedures required to reach the posterior segment. Hence, to treat posterior disorders of the eye an attempt was made to develop nanofiber (NF) scaffolds for effective management of chronic posterior uveitis. Nanofibers (NFs) were formulated using the electrospinning technique. Methods NF scaffolds were formulated using the electrospinning technique. The effect of different concentrations of chitosan on NF production was studied by considering different ratios of chitosan (CS) and polyvinyl alcohol (PVA). Physicochemical characterization of NFs was performed to evaluate developed NFs. Results The optimized NF scaffold had a diameter of 129 ± 3 nm. NF scaffolds were found to have a tensile strength of 0.2882 ± 0.078 N/m2, thickness of 0.16 ± 0.05 mm, and drug entrapment of 95 ± 2.0%. The bioadhesive strength of the NF was found to be 257.3 ± 0.04 g/cm2 indicating high bioadhesion of NFs to the ocular tissues. The in-vitro, ex-vivo corneal and ex-vivo scleral drug release after 12 h was found to be 78.4 ± 1.0%, 65.33 ± 0.2% and 78.41 ± 1.0%, respectively. Ex-vivo whole eye model experiment indicated a concentration of about 40 ± 1.75% of drug permeated from corneal layer to the vitreous humor after 12 h. The Hen’s egg test-chorioallantoic membrane study (HET-CAM) study and in-vitro cytotoxicity study on Statens Seruminstitut Rabbit Cornea (SIRC) cell lines indicated that the developed drug-loaded NF scaffolds were found to be non-toxic as compared to pure drug, thus suggesting cytocompatibility. Conclusion Results of HET-CAM, sterility and ex-vivo studies indicate that the developed formulation is non-toxic, sterile, and effective for the ocular delivery of fluocinolone acetonide to the posterior segment of eye.

A promising synthetic citric crosslinked β-cyclodextrin derivative for antifungal drugs: Solubilization, cytotoxicity, and antifungal activity

Effective antifungal therapy for the treatment of fungal keratitis requires a high drug concentration at the corneal surface. However, the use of natural β-cyclodextrin (βCD) in the preparation of aqueous eye drop formulations for treating fungal keratitis is limited by its low aqueous solubility. Here, we synthesized water-soluble anionic βCD derivatives capable of forming water-soluble complexes and evaluated the solubility, cytotoxicity, and antifungal efficacy of drug prepared using the βCD derivative. To achieve this, a citric acid crosslinked βCD (polyCTR-βCD) was successfully synthesized, and the aqueous solubilities of selected antifungal drugs, including voriconazole, miconazole (MCZ), itraconazole, and amphotericin B, in polyCTR-βCD and analogous βCD solutions were evaluated. Among the drugs tested, complexation of MCZ with polyCTR-βCD (MCZ/polyCTR-βCD) increased MCZ aqueous solubility by 95-fold compared with that of MCZ/βCD. The inclusion complex formation of MCZ/βCD and MCZ/polyCTR-βCD was confirmed by spectroscopic techniques. Additionally, the nanoaggregates of saturated MCZ/polyCTR-βCD and MCZ/βCD solutions were observed using dynamic light scattering and transmission electron microscopy. Moreover, MCZ/polyCTR-βCD solution exhibited good mucoadhesion, sustained drug release, and high drug permeation of porcine cornea ex vivo. Hen’s Egg test-chorioallantoic membrane assay and cell viability study using Statens Seruminstitut Rabbit Cornea cell line showed that both MCZ/polyCTR-βCD and MCZ/βCD exhibited no sign of irritation and non-toxic to cell line. Additionally, antifungal activity evaluation demonstrated that all isolated fungi, including Candida albicans, Aspergillus flavus, and Fusarium solani, were susceptible to MCZ/polyCTR-βCD. Overall, the results showed that polyCTR-βCD could be a promising nanocarrier for the ocular delivery of MCZ.

Nutrient Supplementation Improves Contact Lens-Induced Corneal Cell Damage Based on a SIRC Cellular Model.

The long-term use of contact lenses may damage the structure of the ocular surface and cause metabolic disorders in corneal cells. Vitamins and amino acids help maintain the physiological function of the eye. In the present study, the effects of nutrient (vitamin and amino acid) supplementation on corneal cell repair after contact lens-induced damage was investigated. High-performance liquid chromatography was used to quantify the nutrient contents of minimum essential medium, and the MTT assay was used to measure the viability of corneal cells. A Statens Seruminstitut rabbit cornea cellular model was established to simulate contact lens-induced keratopathy and investigate the effects of vitamin and amino acid supplementations on corneal cell repair. The high water content lens group (78%) has a cell viability as high as 83.3%, whereas the cell viability of the low water content lens group (38%) is only 51.6%. The 32.0% difference between the two groups confirms the correlation between water content of lens and corneal viability. Vitamin B2, vitamin B12, asparagine, and taurine supplementation may help improve contact lens-induced damage.

Thermogels containing sulfated hyaluronan as novel topical therapeutics for treatment of ocular surface inflammation.

The development of long lasting therapeutic agents is critically important for efficient treatment of chronic diseases. We herein report a rational strategy to develop a therapeutic thermogel featured with prolonged anti-inflammatory and corneal-protective effects. Specifically, a hyaluronic acid with different sulfation degrees and an amine-terminated poly(N-isopropylacrylamide) are conjugated to achieve the thermogels. In vitro studies reveal that the thermogels are highly biocompatible to statens seruminstitut rabbit cornea cells and their anti-inflammatory properties are strongly dependent on the sulfation degree. In a rabbit model of ocular inflammation, single-dose topical administration of a thermogel formulation could repair defects in corneal epithelium (∼99% thickness restored), prevent corneal cell apoptosis (∼68.3% cells recovered), and suppress ocular surface inflammation (∼4-fold decrease) for a follow-up period of 7 days. This high treatment efficacy of the thermogel can be attributed to its potent inhibition in selectin-mediated leukocyte infiltration as well as effective corneal protection. These findings show a great promise for topical treatment of ocular inflammation and advancement of ophthalmic formulations using the bioactive thermogel as a therapeutic component that is not rapidly cleared from the eye and thus considerably reduces administration times.

Sorafenib Repurposing for Ophthalmic Delivery by Lipid Nanoparticles: A Preliminary Study

Uveal melanoma is the second most common melanoma and the most common intraocular malignant tumour of the eye. Among various treatments currently studied, Sorafenib was also proposed as a promising drug, often administered with other compounds in order to avoid resistance mechanisms. Despite its promising cellular activities, the use of Sorafenib by oral administration is limited by its severe side effects and the difficulty to reach the target. The encapsulation into drug delivery systems represents an interesting strategy to overcome these limits. In this study, different lipid nanoparticulate formulations were prepared and compared in order to select the most suitable for the encapsulation of Sorafenib. In particular, two solid lipids (Softisan or Suppocire) at different concentrations were used to produce solid lipid nanoparticles, demonstrating that higher amounts were able to achieve smaller particle sizes, higher homogeneity, and longer physical stability. The selected formulations, which demonstrated to be biocompatible on Statens Seruminstitut Rabbit Cornea cells, were modified to improve their mucoadhesion, evaluating the effect of two monovalent cationic lipids with two lipophilic chains. Sorafenib encapsulation allowed obtaining a sustained and prolonged drug release, thus confirming the potential use of the developed strategy to topically administer Sorafenib in the treatment of uveal melanoma.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

Fibrosis as a result of polyhexamethylene guanide exposure in cultured Statens Seruminstitut Rabbit Cornea (SIRC) cells.

Previous research studies on the toxicity of polyhexamethylene guanidine (PHMG) as a humidifier disinfectant majorly focused on lung fibrosis. Considering that disinfectants in humidifiers are released in aerosol form, the eyes are directly exposed and highly vulnerable to the detrimental effects of the PHMG. Therefore, in the present study we investigated the adverse effects of PHMG on the eyes; considering fibrosis as a manifestation of PHMG toxicity in the eye, we evaluated fibrosis-related biomarkers in cultured Statens Seruminstitut Rabbit Cornea (SIRC) cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, fibrosis-related biomarkers were evaluated through polymerase chain reaction (PCR) and immunoblotting, and oxidative stress was evaluated using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA). Polyhexamethylene guanidine showed cytotoxicity in a time and concentration-dependent manner. Fibrosis related biomarkers including transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA), matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP) and hemeoxygenase-1 (HO-1) increased in both gene and protein levels. Oxidative stress also increased in the PHMG-treated cultured cells. The findings of the present study suggest that PHMG could cause toxicity in the eye as manifested by fibrosis.

Preparation of Ophthalmic Microemulsions Containing Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation

Royal jelly is a natural substance secreted by worker honeybees that possesses antioxidant, anti-inflammatory, and other biological activities. The purpose of this study was to formulate microemulsions with incorporated Lithuanian royal jelly for possible ophthalmic delivery and to evaluate the quality of the microemulsions in vitro. The oil in water type microemulsions were prepared by the oil titration method, incorporating royal jelly, surfactant, co-surfactant, oil, and water. Physicochemical characteristics of the microemulsions and the quantity of 10-hydroxy-2-decenoic acid released in vitro were assessed. The in vitro assessment of prepared microemulsions formulations was performed with the Statens Seruminstitut rabbit cornea (SIRC) cell culture model. The results revealed that the droplet size of all microemulsion formulations was 67.88–124.2 nm and the polydispersity index was lower than 0.180. In the in vitro release study, the release of 10-hydroxy-2-decenoic acid depended on the amount of royal jelly incorporated and on the ratio of surfactant and co-surfactant in formulations. The in vitro tests with the SIRC cell culture line have shown that all formulations were found non-irritating.

Comparative behaviour of electrospun nanofibers fabricated from acid and alkaline hydrolysed gelatin: towards corneal tissue engineering

Gelatin is a hydrolysed form of collagen which is categorised as type A (acid hydrolysed) or type B (base hydrolysed), based on method of hydrolysis. In this work, we have studied the comparative properties of electrospun nanofibers of gelatin A (GA) and gelatin B (GB) prepared using a ternary solvent (glacial acetic acid/ethyl acetate/water). In addition, silk fibroin was used to improve the integrity and stability of gelatin nanofibrous sheets. Silk fibroin is known to serve as a potential implantable biomaterial because of its optimal mechanical stability, transparency, cell proliferation and tunable degradability. GA scaffold and silk permeated gelatin A nanofibrous scaffold (SFG) showed 50-70% of transparency comparable to that of adult rat and goat cornea. ATR-FTIR analysis showed the presence of native functional groups and bonds. Scanning electron microscope (SEM) micrographs revealed the porous nature of the scaffolds i.e., 48.08± 3.35 and 51.38 ± 3.72 for GA and GB, respectively. GB nanofibrous scaffolds got dissolved instantly in phosphate buffer solution (PBS), while GA scaffolds remained stable up to ∼ 11 h at 25˚C, however got degraded within 12 h when incubated at 37˚C. Furthermore, SFG scaffolds physically cross-linked with ethanol vapour displayed relatively improved stability when examined for at least 6 days at 37°C, minor weight loss when incubated in a lysozyme solution and facilitated the cellular proliferation significantly. Liquid retention capacity of the electrospun GA nanofibrous scaffolds was found to be nearly 800% from its original weight within 48 h. The electrospun nanofibrous scaffolds i.e., SFG facilitated ~72% of cellular proliferation compared to the control for SIRC [Statens Seruminstitut Rabbit Cornea] fibroblasts cells by 5th day of cell culture. Comparative outcomes reveal that the electrospun GA nanofibers possess significantly better integrity and noted stability in an aqueous environment compared to GB nanofibrous scaffold. Moreover, owing to the enhanced integrity and better stability the GA nanofibrous scaffold in combination with silk fibroin could be exploited in corneal tissue engineering applications where transparency plays an indispensable characteristic.

Protective effect of lipidure on ocular surface and soft contact lens against desiccation damage

AbstractPurposeThe insertion of a contact lens (CL) disturbs the homeostasis of the tear film, favouring its evaporation and producing friction between the lens and the eyelids and the lens and the ocular surface. These events may induce or aggravate a pre‐existing condition of eye dryness. Lipidure is a biocompatible polymer of 2‐methacryloyloxi‐ethyl‐phosphorylcholine, showing a structure similar to the phosphatidylcholine polar bases; it is endowed with excellent antifouling and moisture retaining abilities. Aim of this study has been to address the protective effects of lipidure, with or without either hyaluronic acid (HA) or hydroxyl‐propyl‐methyl‐cellulose (HPMC), on desiccation of Statens Seruminstitut Rabbit Cornea (SIRC) cells and explanted porcine corneas. Moreover, the ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated.MethodsThe protective effect of lipidure was evaluated measuring viability on SIRC cells and explanted porcine corneas leaving them dried at 37°C, 5%CO2 and 95% humidity for 1 h. The ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated leaving them air‐dried at 26°C and 46% humidity.ResultsThe protective effect of 0.1% lipidure±HA (0.15%) or HPMC (0.3%), was compared to trehalose (3%), a sugar known in nature to protect cells from dessiccation damage. Results obtained indicate that lipidure either alone or in association with HA or HPMC is able to improve corneal epithelial cells survival under desiccation conditions, both in vitro and ex vivo, similar or even better than threalose alone. Moreover, lipidure in association with HPMC can better preserve morphology and transparency of CL from dehydration.ConclusionsIn conclusion, these studies support the use of lipidure both in artificial eye drops to protect the ocular surface form dryness, and as wetting agents for CL improve their tolerance over the eye surface.

In Vitro Cytotoxicity of Zinc Oxide Nanoparticles in Cultured Statens Seruminstitut Rabbit Cornea Cells

The possibility of eye exposure for workers participating in manufacturing of nanoparticles or consumers using products containing nanoparticles has been reported, but toxicity studies on the eye are scarce. In this study, cytotoxicity of five nanoparticles including silver, ceria, silica, titanium and zinc were tested using Statens Seruminstitut Rabbit Cornea (SIRC) cells. When cells were treated with nanoparticles with concentrations of 1–100 μg/mL for 24 hr, zinc oxide nanoparticles showed higher toxicity to cornea cells. LC50 of zinc oxide nanoparticles was less than 25 μg/mL but those of other nanoparticles could not be calculated in this test, which means more than 100 μg/mL. Generation of reactive oxygen species was observed, and expression of apoptosis related biomarkers including Bax and Bcl-2 were changed after treatment of zinc oxide nanoparticles, while no other significant toxicity-related changes were observed in cornea cells treated with Ag, CeO2, SiO2 and TiO2 nanoparticles.

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models.

Many chemicals have been reported to induce phototoxicity. The absorbance of light energy within the sunlight range is a common characteristic of phototoxicity. The 3T3 NRU phototoxicity test (PT) in 3T3 mouse skin fibroblasts has been used to identify the phototoxic potential induced by excited chemicals after exposure to ultra violet (UV). However, as phototoxicity may occur in ocular cells, it is necessary to develop a more suitable test for cornea-derived cells. In this study, we attempted to establish a new in vitro PT method in rabbit corneal cell lines (SIRC). We evaluated five ophthalmic agents, ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline, for their cytotoxic potential and in vitro phototoxicity. The results obtained using 3D human corneal models revealed that the UV-induced eye tissue toxicity by the test substances showed good correlation with those obtained using the in vitro phototoxicity test. However, the results from the 3D PT for ciprofloxacin, norfloxacin, and tetracycline in the 3D human cornea model were only partially comparable. Therefore, we suggest the SIRC cell line as a new phototoxicity test model; however, a sequential testing strategy, such as 3D PT, was also proposed to obtain relevant information for topical eye agents.

Phenotypic characterization of the SIRC (Statens Seruminstitut Rabbit Cornea) cell line reveals a mixed epithelial and fibroblastic nature

The aim of the present study was to investigate, in the Statens Seruminstitut Rabbit Cornea (SIRC) cell line, the presence of epithelial and fibroblastic markers, comparing their levels with those of the human Retinal Pigmented Epithelial (ARPE-19) cell line, and the Human Keratocyte (HK) cell line, respectively. SIRC cells, often described as of epithelial origin, are used as a corneal epithelial barrier model to study the permeability of ophthalmic drugs. However, they show a morphology that is more consistent with a fibroblastic cell phenotype, similar to corneal keratocytes. Our comparative analyses of cell type specific markers demonstrated that SIRC do not express cytokeratins 19 and 16 (typical of ARPE-19) and cytokeratin 9 (typical of HK); they do express cytokeratins 3 and 18 common to all three cell lines, and cytokeratin 12 typical of ARPE-19. Tight junction proteins were absent in HK, and lower in SIRC than in ARPE-19. All cell lines expressed the markers lumican and vimentin, with SIRC expressing intermediate levels between HK and ARPE-19; alpha-SMA was highly expressed in all lines. These markers, considered typical of fibroblasts, can be, however, expressed by epithelial cells during wound healing. These results might suggest that long-term in vitro cultivation of cell lines leads to a derangement of their specific phenotype, most likely due to genetic and epigenetic factors. This could be the reason why SIRC cells came to exhibit a hybrid nature between epithelial and fibroblastic cells.

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%–100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures.

Antioxidant and Osmoprotecting Activity of Taurine in Dry Eye Models.

To assess the protective effects of ophthalmic formulations based on taurine (TAU) and sodium hyaluronate (SH) in ocular surface. Rabbit corneal epithelial cells [Statens Seruminstitut Rabbit Cornea (SIRC)] were subjected to oxidative stress (1 mM H2O2) and treated with the following formulations: 0.2% SH, 0.4% SH, 0.4% SH +0.5% TAU. Reactive oxygen species (ROS) were evaluated by commercial kit. Dry eye was induced by atropine sulfate and topical treatment was carried out with the following formulations: 0.2% SH, 0.4% SH, 0.4% SH +0.5% TAU. Schirmer's test, tear breakup time (TBUT), and tear osmolarity were evaluated. Furthermore, tear matrix metalloproteinase 9 (MMP-9) expression was assessed by Western blot. TAU significantly (P < 0.05) decreased ROS production in SIRC after oxidative stress. Topical administration of atropine in the rabbit eye significantly (P < 0.05) reduced tear volume and TBUT. Tear osmolarity was also significantly (P < 0.05) modified by atropine treatment. All the altered parameters were significantly (P < 0.05) reversed by 0.5% TAU +0.4% SH treatment; furthermore, this formulation was more effective than SH alone. Moreover, tear levels of MMP-9 were significantly (P < 0.05) lower in the group treated with 0.5% TAU +0.4% SH. Altogether these data suggest that TAU has a relevant antioxidant effect in corneal epithelial cells and prevents the ocular surface damage elicited by atropine. Therefore, our findings suggest that TAU in combination with SH may be useful in clinical practice to manage ocular surface diseases.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation.

A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with D-isomers (D-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., L-valine-L-valine-prednisolone (LLP) > L-valine-D-valine-prednisolone (LDP) > D-valine-L-valine-prednisolone (DLP) > D-valine-D-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration.

Effect of hydrophilic polymers on the wettability, static and dynamic, of solid substrate covered by confluent monolayer of air-damaged SIRC cells

The aim of this study was to evaluate the possible implementation of hydrophilic polymers as recovery agents in air-damaged corneal cells. The sessile bubble technique was implemented to measure the wetting properties of four selected polymers: hydroxyethyl cellulose (HEC), sodium chondroitin sulphate (SCS), hydroxypropyl-methylcellulose (HPMC) and poloxamer F127 (PO12), at equilibrium conditions and in the case of advancing and receding contact angle. For testing the wetting properties of the polymers, glass slides covered with a confluent monolayer of Statens Seruminstitut rabbit cornea (SIRC) cells were used. HEC showed best properties for a broad concentration range, as the polymer showed capability to maintain low values of the static (equilibrium) contact angle (average static contact angle – 36.07˚, compared to average static compact angles of HPMC – 38.44˚, PO12 – 38.92˚ and SCS – 37.85˚), i.e. better wettability. Sessile bubble technique provides quick, relatively simple and reliable approach for testing surface properties of the listed polymers. The nature of the surface damage produced by the exposition of SIRC cells was used as a plausible model of evaporative dry eye syndrome, and thus the results may have clinical implementation.

Rubella virus perturbs autophagy.

Autophagy is a cellular catabolic process implicated in numerous physiological processes and pathological conditions, including infections. Viruses have evolved different strategies to modulate the autophagic process. Since the effects of rubella virus (RV) on autophagy have not yet been reported, we evaluated the autophagic activity in the Statens Seruminstitut Rabbit Cornea cell line infected with the To336 strain of RV. Our results showed that RV lowered the levels of microtubule-associated protein 1 light chain 3 B-II (LC3B-II) and the autophagy-related gene 12-autophagy-related gene 5 conjugate, inhibited the autophagic flux, suppressed the intracellular redistribution of LC3B, decreased both the average number and the size of autophagosomes per cell and impeded the formation of acidic vesicular organelles. Induction of autophagy by using rapamycin decreased both the viral yields and the apoptotic rates of infected cultures. Besides its cytoprotective effects, autophagy furnishes an important antiviral mechanism, inhibition of which may reorchestrate intracellular environment so as to better serve the unique requirements of RV replication. Together, our observations suggest that RV utilizes a totally different strategy to cope with autophagy than that evolved by other positive-stranded RNA viruses, and there is considerable heterogeneity among the members of the Togaviridae family in terms of their effects on the cellular autophagic cascade.