An in vitro technique for measuring contact angles on the corneal surface and its application to evaluate corneal wetting properties of water soluble polymers

Abstract

The development of an in vitro technique capable of measuring the extent and duration of corneal wetting by demulcent polymers is reported. The technique required modification of a contact angle goniometer to facilitate the measurement of contact angles of demulcent polymer solutions on freshly enucleated rabbit eyes. Contact angles of the test solutions on the corneal surface was measured under physiologically relevant conditions. Contact angle measurements were performed under conditions to ensure that the tissue was not dehydrated and the native surface characteristics of the tissue were unchanged. Additionally, experimental procedures for contact angle measurements were developed in order to provide a partial simulation of in vivo fluid dynamics that is typically observed upon topical instillation of a drop of a test polymer solution into the eye of a patient. This multistep experimental procedure was initiated by briefly dipping a freshly enucleated rabbit eye in the test polymer solution. The treated eye was then immersed upside-down in an oxygenated, lactated Ringer's solution and placed in the chamber of the goniometer. This medium was used to maintain ocular tissue viability and corneal surface integrity over the duration of the experiment. For measurement of contact angles, a well defined air bubble was slowly introduced into the chamber of the goniometer at close proximity to the enucleated eye. The air bubble was then entrapped on the corneal surface. Angle of contact of the air bubble against the corneal epithelial surface was visually measured with the goniometer. A new air bubble was entrapped at intervals of 5 min and the contact angle was measured as a function of time. The degree and duration of reduction of contact angle was observed to depend on the type and concentration of the water soluble polymer used. Most demulcents were found to rapidly desorb from the corneal surface. For these demulcent polymers the contact angle returned to pre-treatment values within 25 min of the start of the experiment. Hyaluronic acid showed the most sustained wetting of the cornea. The in vitro kinetic measurement of contact angle demonstrated the time dependent, marginally functional mucomimetic properties of demulcent polymers. The kinetics of desorption was considered to partially simulate the fluid dynamics in the eye. The validity of the inverted air bubble as well as this new technique for the kinetic measurement of contact angles was established on synthetic (polyethylene) surface prior to its utilization on freshly enucleated ocular surface. The measured contact angle and estimates of critical surface tension for polyethylene surface was in close agreement with values reported in the literature and with values obtained from conventional techniques of measurement of contact angle. The contact angle of lactated Ringer's solution on the rabbit corneal surface range from 45 to 55°. Removal of mucin from the corneal surface increased the contact angle of the cornea in the range of 55–65°. This increase in contact angle of the corneal epithelium devoid of mucin indicated decreased wetting and demonstrated its greater hydrophobicity than the mucin coated epithelium. Adsorption of demulcent polymers solutions decreased the contact angles to as low as 30° indicating enhanced wetting of the cornea. The measurement of contact angles of various demulcent polymer solutions possessing different surface tensions made it possible to estimate the critical surface tension of the rabbit corneal epithelium. The critical surface tension, representing the minimal surface tension of a formulation necessary to completely wet and spread over the corneal epithelium was estimated at 39 dyn/cm. Therefore, the reported air bubble trapping along with the kinetic technique for measurement of contact angles may be useful as an in vitro screening tool for the evaluation of new demulcent polymers. The technique may also be used for the identification of superior tear substitutes for the treatment of dry eyes.