Hyaluronic Acid In The Dry Eye Disease Treatment: Correlation Between Molecular Weight And Mucoadhesive Properties.

Tear substitutes, named also artificial tears, are used to treat dry eye disease (DED), either in cases of aqueous deficiency or increased tear evaporation. Substitute mucoadhesion is a process related to the ocular residence time and therefore can be used to measure the effectiveness of eye drops. In this work, the correlation between mucoadhesive performances of different artificial tears based on hyaluronic acid (HA) have been evaluated, also simulating the pathological condition of the tear film during DED..

The mucoadhesive properties of HA and other gelling agents have been evaluated in an in vitro ocular model, where the interaction between HA and MUC2 was measured with three different approaches: rheological assessment, fluorescence analysis, and surface plasmon resonance (SPR). The DED was simulated decreasing the mucin or the polar lipid fraction concentration. Finally, a series of marketed artificial tears were analyzed and compared in terms of HA molecular weight and mucoadhesiveness.

MUC2 was purified by dialysis and characterized by GPC-TDA. Rheological analysis: MUC2 was mixed 1:1 with the HA formulated at different concentrations and analyzed by rheometer at 35°C. The mucoadhesive index is given by (ǀƞ*ǀ of mixture: Sample+Mucine) - (ǀƞ*ǀ of mixture: Sample+Buffer) - (ǀƞ*ǀ of mixture: Buffer+Mucine), where ǀƞ*ǀ was sampled at 1Hz. Fluorescence analyses: Each HA formulation (from 0.5 to 4.5mg/mL) was mixed with MUC2 (final conc. 5mg/mL) and Nile Red (final conc. 0.004 mg/mL) and analyzed with Microplate reader (Ex/Em = 510/600nm). SPR measurements were carried out on a dual flow cell Biacore-X100 instrument. MUC2 was immobilized (255RU) on a CM5 chip. HA analogs (0-1mg/mL) were injected at 10 mL/min (180s) in PBS. 

The rheological analysis shows that unmodified high MW HA has a higher mucoadhesive index than cross-linked HA and other polymers, except for xanthan gum. Reducing the mucin concentration (from 40 to 20mg/mL), as often happens in a moderate DED, high MW HA maintain its mucoadhesive property. At lower Oleic Acid concentrations (a model of DED associated with MGD), the mucoadhesive effect of high MW HA is also well maintained (opposed to low MW HA). The fluorescence and the SPR analysis confirm that the interaction between MUC2 and high MW HA is more stable in comparison to low MW HA or cross-linked HA. Linear correlation has been reported between the MW of the HA formulated in the eye drops on the market and the mucoadhesive index.

 The mucoadhesive performance of unmodified HA was linearly correlated with the MW, while cross-linked and other gelling agents commonly formulated in artificial tears do not show the same mucoadhesive properties, with the exception of xanthan gum. The mucoadhesive performance of the high MW HA was also demonstrated simulating the pathological condition of the tear film during DED by decreasing the MUC2 or the oleic acid concentration. The physical-chemical analysis of a series of marketed artificial tears on the market confirmed the same correlation. In conclusion, tear substitutes have specific properties and  a careful choice should be done when treating patients with DED.