Measurement of corneal epithelial healing rates and corneal thickness for evaluation of ocular toxicity of chemical substances

Abstract

The epithelial cells bordering a corneal epithelial wound normally slide across the denuded area to close the wound. This reepithelialization generally occurs at a rate of 0.80 to 1.00 mm²/hr in the rabbit. The cornea also swells after wounding and then returns to normal thickness during the healing process. Because this is a predictable process, it is proposed that measurement of corneal healing rate and corneal thickness be used in the evaluation of potential eye irritants. It is shown that several substances, including methylene blue, isopropanol, and benzalkonium chloride, reduce the healing rate of a standardized, 6-mm diameter corneal epithelial wound as compared to a paired, untreated control. Heptanol and isopropanol cause the wounded cornea to swell to a greater thickness and delay the return to normal thickness. Thus, it is clear that toxic substances can inhibit corneal epithelial healing. Measurement of reepithelialization rate and corneal thickness yield quantitative, statistical data that can be used in addition to standard methods in the testing of chemical substances for ocular toxicity.     

Airborne particulate matter impairs corneal epithelial cells migration via disturbing FAK/RhoA signaling pathway and cytoskeleton organization

Background: Cornea is the outmost structure of the eye and exposed directly to the air pollution. However, little is known about the effect of PM2.5 on corneal epithelium, which is critical for maintenance of cornea homeostasis and visual function.

Objective: We investigated the influence of PM2.5 exposure on corneal epithelial migration and the possible mechanisms involved in the process.

Methods: We observed wound healing in mouse model of cornea abrasion, evaluated the migration and mobility of cultured corneal epithelial cells with wound scratch assay and Transwell migration assay, detected the phosphorylation and interaction of FAK/paxillin with immunofluorescence and immunoprecipitation, and determined the RhoA activity and actin reorganization, in response to PM2.5 exposure.

Results: Exposure to PM2.5 remarkably inhibited corneal epithelial cell migration both in mouse model of corneal abrasion and in cell culture model. We found the phosphorylation and interaction of FAK/paxillin, RhoA activity as well as actin reorganization were suppressed by PM2.5 exposure. Moreover, formation of ROS might play a role in the action of PM2.5.

Conclusions: PM2.5 exposure could result in delay of corneal epithelium wound healing by inhibiting cell migration, thus more attention should be paid to the potential risk of corneal infection and effort should be made to protect eyes against impairment induced by PM2.5     

Comparative studies for ciprofloxacin hydrochloride pre-formed gels and thermally triggered (in situ) gels: in vitro and in vivo appraisal using a bacterial keratitis model in rabbits

Abstract

This article reports on comparative in vitro characterization and in vivo evaluation of pre-formed cellulose-based gels, methylcellulose (MC) and carboxymethylcellulose sodium (CMC) and in situ gel-forming Pluronic F127 (PL) for ocular delivery of ciprofloxacin hydrochloride (Cipro) by using a bacterial keratitis model and histological corneal examination. Drug–polymer interactions were studied employing thermal analysis. Further, different concentrations (1–3% w/w or 10–30% w/w) of gels depending on the nature of the polymer used were prepared, characterized for clarity, pH, rheology and in vitro release. Selected gel formulations were evaluated for ocular delivery to Staphylococcus aureus-infected rabbit corneas; and ocular toxicity through histological examination of the cornea. The results demonstrated no Cipro-polymers physicochemical interactions and pseudoplastic flow for all gels used at 35?°C. Both polymer concentrations and drug solubility in the gels are dominantly the rate-determining factors for in vitro drug release. The corneal healing rate for all gel-based formulations was significantly faster (p?<?0.05) than that for Cipro solution-treated rabbits. PL-based gel induced significant swelling/edema of the corneal stroma, compared with MC- and CMC-based gels. In conclusion, cellulose-based polymers have superior ocular tolerability/dramatically less irritant; and superior efficacy with more convenient administration compared with PL and Cipro solution, respectively.     

Hybrid thermosensitive-mucoadhesive in situ forming gels for enhanced corneal wound healing effect of L-carnosine

PurposeThermosensitive in situ gels have been around for decades but only a few have been translated into ophthalmic pharmaceuticals. The aim of this study was to combine the thermo-gelling polymer poloxamer 407 and mucoadhesive polymers chitosan (CS) and methyl cellulose (MC) for developing effective and long-acting ophthalmic delivery systems for L-carnosine (a natural dipeptide drug) for corneal wound healing.MethodsThe effect of different polymer combinations on parameters like gelation time and temperature, rheological properties, texture, spreading coefficients, mucoadhesion, conjunctival irritation potential, in vitro release, and ex vivo permeation were studied. Healing of corneal epithelium ulcers was investigated in a rabbit’s eye model.ResultsBoth gelation time and temperature were significantly dependent on the concentrations of poloxamer 407 and additive polymers (chitosan and methyl cellulose), where it ranged from <10 s to several minutes. Mechanical properties investigated through texture analysis (hardness, adhesiveness, and cohesiveness) were dependent on composition. Promising spreading-ability, mucoadhesion, transcorneal permeation of L-carnosine, high ocular tolerability, and enhanced corneal epithelium wound healing were recorded for poloxamer 407/chitosan systems.ConclusionIn situ gelling systems comprising combinations of poloxamer-chitosan exhibited superior gelation time and temperature, mucoadhesion, and rheological characteristics suitable for effective long-acting drug delivery systems for corneal wounds.     

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line

Abstract

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.

Methods: A human corneal epithelial cell culture was treated with travoprost (0.4?mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10?μM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4?mg/ml) and/or PD168393 (10?μM). After being cultured for 24?h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.

Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell–cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.

Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell–cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.     

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman’s membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.     

Toxicity of ingredients in artificial tears and ophthalmic drugs in a cell attachment and spreading test†

Abstract

During re-epithelialization of corneal erosions, the epithelial cells are attached and migrate on a fibronectin-containing substratum. In earlier studies we found that topical application of the plasmin inhibitor aprotinin promoted healing of corneal epithelial defects, presumably inhibiting proteolytic degradation of fibronectin and related adhesive proteins. The present study investigated, using an in vitro cell culture test, the action of various ophthalmic preparations on cell attachment and spreading on a fibronectin-coated substratum. The cell culture test was designed to simulate the situation in the wounded corneal epithelium in vivo. Analysis of 16 different ophthalmic preparations indicated that the toxicity of some of them in the test could be assigned to specific ingredients (benzalkonium chloride, thimerosal sodium, Tween 80) used as additives. In contrast, chlorobutanol, used widely as a preservative, was nontoxic in the test, in concentrations used in ophthalmic preparations. It should be noted that the wounded and inflamed cornea may be more vulnerable to the toxicity of topical mediations than an intact corneal epithelium. hoper tests of the vehicle are therefore recommended before clinical trials are undertaken.     

Epithelial healing in experimental corneal alkali wounds with nondiluted autologous serum eye drops

Purpose: To evaluate the effects of the topical application of nondiluted autologous serum on epithelial healing as an adjuvant therapy in rabbit corneal alkali wounds.

Methods: Central corneal alkali wounds were produced on the right eye of 16 rabbits, by a 60-second application of a 6-mm round filter paper soaked in 1 N sodium hydroxide (NaOH). One group of rabbits (n=8) was treated with non-diluted autologous serum, the other control group (n=8) was treated with physiologic saline, topically 4 times a day. The epithelial healing of the wounds was followed on a slit lamp with and without fluorescein staining daily for 1 week. On day 7, both corneas of each rabbit were excised and assessed histopathologically.

Results: A significant increase in the epithelial healing rate was observed in the autologous serum–treated corneas compared with the controls. Epithelial hyperplasia was noticed in specimens with thickened surface cells with multilayers, revealing disorganization in both groups. The autologous serum–treated epithelium showed a relatively well-organized appearance.

Conclusion: Nondiluted autologous serum eye drops could be used as an adjuvant therapy for promoting the epithelial healing process during the repair stage of corneal alkali wounds.     

Nanoparticles laden in situ gel of levofloxacin for enhanced ocular retention

Abstract

Availability of proper concentration of medicament on to the corneal surface is a challenging task. Many novel formulations, i.e. hydrogels, nanoparticles, ocuserts, etc. had been tested to improve ocular bioavailability, out of which our group found, in situ gel and polymeric nanoparticle are the most interesting approach to achieve ocular retention. We found that in situ gel stay only for 12?h and poly(lactic-co-glycolic acid (PLGA) nanoparticles are non mucoadhesive in nature so we try to combine both these formulations and termed it as “Nanoparticle laden in situ gel”. Here we prepare nanoparticle laden in situ gel containing levofloxacin encapsulated PLGA nanoparticle, incorporated in chitosan in situ gel and evaluated its ocular retention by gamma scintigraphy in rabbits. The observations of acquired gamma camera images showed good retention over the entire precorneal area. From static and dynamic gamma scintigraphy evaluation, we can be interpret that developed nanoparticle laden in situ gel formulation cleared at a very slow rate and remained at corneal surface for longer duration than marketed formulation, in situ gel and nanosuspension alone.     

The effect of gatifloxacin 0.3% or moxifloxacin 0.5% on corneal healing,ocular tolerability and toxicity following pterygium surgery

Purpose: To compare the rate of corneal epithelial healing and ocular tolerability following pterygium surgery between gatifloxacin and moxifloxacin.

Methods: In this double masked, prospective, controlled study 40 patients were randomized to receive prophylactic topical gatifloxacin 0.3% or moxifloxacin 0.5% following pterygium surgery. Patients were examined on days 1, 3, 7 and 21 post-operatively or until complete corneal epithelial healing. The primary outcome measure was the area of corneal epithelial defect during the post-operative period. Patients graded post-operative ocular pain, foreign body sensation, tearing, general burning sensation and burning sensation post-antibiotic drops instillation on a scale of 1–5. Conjunctival hyperemia and superficial punctate keratopathy (SPK) were measured on a scale of 0–3.

Results: No significant differences between groups were found in terms of corneal epithelial defect percentage over time (p?=?0.989) and there was no significant difference between groups on each of the post-operative days. No significant differences were noted in terms of post-operative ocular pain, foreign body sensation, tearing, general burning sensation, burning sensation post-antibiotic drops instillation, conjunctival hyperemia and SPK.

Conclusions: Gatifloxacin and moxifloxacin showed equivalent results in terms of corneal epithelial healing and ocular tolerability following pterygium surgery. This study suggests that there was no apparent added epithelial toxicity due to the presence of benzalkonium chloride in the gatifloxacin preparation when compared to moxifloxacin.     

An in vitro evaluation for corneal damages after instillation of eye drops using rat debrided corneal epithelium: changes in corneal damage of benzalkonium chloride by addition of thickening agent

Benzalkonium chloride (BAC) is known to cause corneal epithelial damage. In this study we investigated the effect of a BAC solution containing a thickening agent, which enhanced residence time in the eyes, on corneal wound healing using in vivo rat model debrided corneal epithelium. 0.5% or 1.0% methylcellulose (MC), carboxymethylcellulose (CMC) and hydroxypropyl-methylcellulose (HPMC) were used as the thickening agent. The levels of corneal wound healing of rat eyes injected with saline were alone approximately 45.0% at 12 h and 93.6% at 24 h after corneal epithelial abrasion, and healing was almost complete at 36 h. The healing rate in the rat eye treated just with MC, CMC and HPMC was higher than that in those injected with saline. In contrast to the treatment result using only this thickening agent, the healing rate in the eye treated with BAC was lower than that in those injected with saline: the corneal wounds in the BAC-treated eye showed approximately 20% healing at 12 h after abrasion. The injection of 0.02% BAC solution containing MC, CMC and HPMC more significantly delayed the healing than did the injection of 0.02% BAC alone. The results show that the in vivo evaluation method for corneal damage using rat debrided corneal epithelium reflects a toxic change depending upon residence time. These findings provide valuable safety and efficacy information for use in the design of eye drops.     

Toxic Effects of Drugs on the Corneal Epithelium: A Review

Abstract

The corneal epithelium being an exposed tissue can be easily injured by chemical agents. Because of its accessibility it can be readily examined by clinical methods. Clinical reports on the adverse drug effects on the cornea as well as data from in vivo and in vitro corneal experiments are gradually increasing our knowledge of the toxic effects of drugs on the corneal epithelium. In this paper the adverse effects of drugs given directly to the eye and administered systemically are reviewed. It is obvious that a large number of drugs can produce cytotoxic effects on the corneal epithelium.     

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust

Objective: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair.

Materials/methods: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH₁ transgenic) mice and measured wound repair using the electric cell impedance sensing system.

Results: Wound closure in epithelial cells from WT mice occurred within 24?h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH₁ transgenic mice, control wounds were repaired up to 8?h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24?h. Likewise, cells from DDAH₁ transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC.

Discussion/conclusion: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.     

Recent developments in novel drug delivery systems for wound healing

Introduction: Complete regeneration and restoration of the skin’s structure and function with no or minimal scarring remains the goal of wound healing research. Novel pharmaceutical carriers have the potential to deliver wound healing drugs such as antibiotics, antimicrobials, human EGFs, and so on. Thus, offering a potential platform to overcome the limitations of conventional wound dressings.

Areas covered: This review will describe various techniques such as microspheres, nanoparticles, liposomes, solid lipid nanoparticles, nano and microemulsions, sponges and wafers, and so on, that are successfully applied as carriers for wound healing drugs. Results of various studies including in vitro and in vivo experiments are also discussed.

Expert opinion: Controlled and localized delivery of wound healing drugs to the wounds is more convenient than systemic administration as higher concentrations of the medication are delivered directly to the desired area in a sustained manner. They are also capable of providing optimum environmental conditions to facilitate wound healing while eliminating the need for frequent changes of dressings. As the number of people suffering from chronic wounds is increasing around the world, controlled delivery of wound healing agents have enormous potential for patient-friendly wound management.     

Thermosensitive in situ nanogel as ophthalmic delivery system of curcumin: development,characterization, in vitro permeation and in vivo pharmacokinetic studies

Context: The nanogel combining cationic nanostructured lipid carriers (CNLC) and thermosensitive gelling agent could enhance preocular retention and ocular permeation capacity of curcumin (CUR).

Objective: The purpose of the study was to develop and characterize a thermosensitive ophthalmic in situ nanogel of CUR-CNLC (CUR-CNLC-GEL) and evaluate in vitro and in vivo properties of the formulations.

Materials and methods: The physicochemical properties, in vitro release and corneal permeation, were evaluated. Ocular irritation and preocular retention capacity were also conducted. Finally, pharmacokinetic study in the aqueous humor was investigated by microdialysis technique.

Results: The solution–gel transition temperature of the optimized formulation diluted by simulated tear fluid was 34?±?1.0?°C. The CUR-CNLC-GEL displayed zero-order release kinetics. The apparent permeability coefficient (P_app) and the area under the curve (AUC_0→∞) of CUR-CNLC-GEL were 1.56-fold and 9.24-fold, respectively, than those of curcumin solution (CUR-SOL, p?<?0.01). The maximal concentration (C_max) was significantly improved (p?<?0.01). The prolonged mean residence time (p?<?0.01) indicated that CUR-CNLC-GEL is a controlled release formulation.

Discussion and conclusion: Those results demonstrated that CUR-CNLC-GEL could become a potential formulation for increasing the bioavailability of CUR in the aqueous humor by enhancing corneal permeation and retention capacity.     

Novel nanostructured lipid carrier-based inserts for controlled ocular drug delivery: evaluation of corneal bioavailability and treatment efficacy in bacterial keratitis

Objectives: The aim of the present study was to develop novel ofloxacin (OFX)-loaded nanostructured lipid carrier (NLC)-based inserts for ocular application for treatment of bacterial keratitis.

Methods: NLC loaded with 0.3% OFX was prepared by means of high shear homogenization and 0.75% chitosan oligosaccharide lactate (COL) was added. Glycerin or PEG 400 at the range of 1 – 15% was added to NLCs as plasticizers and inserts were developed by solvent casting evaporation. Characterization, in vitro release, microbiological, ex vivo and in vivo studies were performed.

Results: The inserts developed with the addition of glycerin (Ins3_OFX) was found as optimal. The kinetic studies revealed that the release of Ins3_OFX was a combination of diffusion and swelling. Ins3_OFX was more bioadhesive in texture profile analysis studies. In the in vivo studies performed with rabbits, the pre-ocular retention time was enhanced up to 24 h and C_max was increased almost six times in comparison with commercial. The rabbits were infected with Staphylococcus aureus and keratitis was confirmed. This group was treated with Ins3_OFX and they recovered in 7 days with no significant sign of conjunctival redness and corneal opacity.

Conclusion: NLC-based inserts developed with COL and glycerin may be offered as appropriate vehicles for ocular delivery.     

Corneal epithelial testing strategies for safety evaluation of ophthalmic formulations

The toxicity of an ophthalmic formulation was tested both in vivo and in vitro. Initial tests on transformed human corneal epithelial (HCE-T) cells in monolayer cultures resulted in adverse effects on cell morphology. The adverse effects were unexpected since the formulation caused no damage to the cornea in vivo. These results suggested HCE-T monolayers do not adequately model the intact corneal epithelium. Therefore, further in vitro studies were conducted to investigate reversibility of morphologic changes, proliferation, cell viability, and effects on corneal epithelial barrier function. These tests showed that the formulation had no adverse effects on cell viability and proliferation. Multilayered cultures of HCE-T cells at an air interface provide a morphologic and physiologic model more relevant to the in vivo cornea. This study demonstrates the importance of selecting appropriate models when conducting in vitro toxicity studies so that potentially effective ophthalmic formulations are not rejected based on false positive in vitro endpoints.     

The Use of Cell Lysis as an Index of Ocular Irritation Potential

Abstract

We report an in vitro method for assessing ocular irritation by measuring cell lysis of mouse connective tissue (strain L, Clone 929) and rabbit corneal cell lines (SIRC). Lysis of corneal epithelial cells in vivo leads to swelling and subsequent opacification of the underlying corneal stroma. In vitro lysis was determined by the measurement of changes in absorbance (360 nm) of cell suspensions over a 10-min exposure to test materials. These data were expressed as numbers of cells/ml using linear regression. The regression line obtained was linear and similar for both cell types. Loss of viability and spontaneous cell lysis were minimal over the assay interval. In a separate study, rabbit corneal cells were used to assess alteration of cell membrane integrity following 30-min exposures to test materials by measuring trypan blue dye exclusion in these cells. Activities of test materials were ranked according to the lowest concentration capable of producing statistically significant cell lysis as follows: triethanolamine lauryl sulfate (TLS) < triethanolamine (TEA) < propylene glycol (PG) (0.003, 0.1, and 10%, respectively, p < 0.01). The order was similar when dye exclusion ability was the index of activity except that benzalkonium chloride (BAK) was more active than the other materials. BAK and materials with cationic properties were not easily tested in the proposed suspension assay because of increased absorbance due to protein denaturation and subsequent masking of dissolution, if present. Six commercial formulations coded A through F were tested in the suspension assay as 1:3,000,1:1,000, and 1:300 dilutions and compared to similarly diluted standards to assess cell dissolution ability. A shampoo concentrate acted similarly to TLS and an amphoteric-surfactant-based shampoo acted similarly to TEA at dilutions of 1:3,000. Effects of other formulations were intermediate between these extremes (p < 0.05), but similar to one another. At 1:1,000 dilutions, the effects converged, with all formulations having the same activity, which was significantly less than that of TLS, but significantly greater than that of TEA (p < 0.05). Over the next interval, the effects paralleled that of TLS but were significantly less except for that of the concentrate (p < 0.05). Another formulation showed a plateau-like effect while the remaining one showed a decreased effect owing to its inherent cationic nature. Results were in general agreement with in vivo and in vitro observations as well as with known chemical activities of the materials. A method for estimating the relative ability of materials and formulations to cause cell lysis is proposed which may serve as a model for the assessment of a form of corneal opacity initiated by the loss of integrity of limiting corneal layers.     

Enhancing and sustaining the topical ocular delivery of fluconazole using chitosan solution and poloxamer/chitosan in situ forming gel

Fungal keratitis is a serious disease that can lead to loss of vision. Unfortunately, current therapeutic options often result in poor bioavailability of antifungal agents due to protective mechanisms of the eye. The aim of this work was to evaluate the potential of a chitosan solution as well as an in situ gel-forming system comprised of poloxamer/chitosan as vehicles for enhanced corneal permeation and sustained release of fluconazole (FLU). For this, in vitro release and ex vivo corneal permeation experiments were carried out as a function of chitosan concentration from formulation containing the chitosan alone and combined with the thermosensitive polymer, poloxamer. Microdialysis was employed in a rabbit model to evaluate the in vivo performance of the formulations. The in vitro release studies showed the sustained release of FLU from the poloxamer/chitosan formulation. Ex vivo permeation studies across porcine cornea demonstrated that the formulations studied have a permeation-enhancing effect that is independent of chitosan concentration in the range from 0.5 to 1.5% w/w. The chitosan solutions alone showed the greatest ex vivo drug permeation; however, the poloxamer/chitosan formulation presented similar in vivo performance than the chitosan solution at 1.0%; both formulations showed sustained release and about 3.5-fold greater total amount of FLU permeated when compared to simple aqueous solutions of the drug. In conclusion, it was demonstrated that both the in situ gelling formulation evaluated and the chitosan solution are viable alternatives to enhance ocular bioavailability in the treatment of fungal keratitis.