Interaction of hyaluronic acid with mucin, evaluated by gel permeation chromatography

Hyaluronic acid (HA) is known to increase the ocular bioavailability of oph thalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require consi derable amounts of mucin (M) that are difficult to obtain from ocular surfa ces. Thus, we developed an alternative method, based on gel permeation liqu id chromatography, to examine the interaction of HA with microgram quantiti es of mucin. HA (from human umbilical cord or rooster comb) were fractionat ed using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay b ased on the histological dye Stains-all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bou nd, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoad hesion of HA could be obtained. This method could be useful to study the in teraction between IIA and microgram quantities of ocular M (mucin), obtaine d from individual patients or normal subjects.