Purpose: Standard mouthwashes are characterized by a short residence time and not prolonged activity. Mucoadhesive formulations improve a close contact with the mucosal surface with a consequent provision of sustained drug release.
The aim of the present study was to verify the mucoadhesive properties of a new mouthwash formulated to treat oromucosal disease, containing a mucoadhesive polymer and two active ingredients (anti-inflammatory/analgesic agent and antiseptic agent).
To measure the mucoadhesion of the new formulation, different in vitro testing methods were used:
• Rheological synergism (mechanistic assay)
• Inclined plane (dynamic assay)
• Lectin binding displacement on buccal cells (cellular assay)
The tests were performed comparing the new formulation versus the same product without polymer (control).
The efficacy and tissue permeability properties of the formulation were also assessed.
Methods: Rheological Synergism
Rheological synergism method has been developed for studying mucoadhesion activity based on Hassan and Gallo rheological synergism method. This method investigates the changes in the rheological properties of the mucoadhesive product when mixed with mucin. Rheological measurements were performed using a rotational rheometer equipped with a plate-cone (37°C) measuring system. Interaction between formulation and mucin was quantified by calculating the rheological synergistic parameter Δƞ at all sliding gradient tested.
The test measures mucoadhesiveness as a function of the retention of the mucoadhesive material in contact with a mucosal substrate (mucin film). In this study, a thermostatic "inclined plane" with a variable inclination between 30 and 90 ° was used. On the plane, 2.5 ml of 8% porcine mucin suspension (w / w) in buffer phosphate was set and dried obtaining a surface area film of 28 cm2. About 1 g of sample at 37 ° C was placed on the top of the plane and, once the plane is inclined at 45 °, the sample was allowed to slide on it until it fell on the microbalance. The % of product adhered with and without the biological substrate was calculated by normalizing the difference with the quantity of sample loaded.
Lectin Binding Displacement
Mucoadhesive effect was determined in vitro evaluating the displacement in the binding between lectin and glycoprotein on buccal mucosal cells. Buccal mucosal cells (TR-146) were preincubated with or without test product for 15 minutes at 37°C, treated with biotinylated lectin, then streptavidin peroxidase was added, and peroxidase activity was measured using O-Phenylenediamine as substrate. Reaction product was quantified by spectrophotometric reading of absorbance at 450 nm. Lectin displacement induced by product application was expressed as percent of inhibition vs positive control. For resistance time evaluation, cells were placed in donor compartment of Franz cells and incubated with test solution or vehicle under continuous flux of artificial saliva at 36°C for different times. Finally, cells were removed and treated as previously described for lectin displacement evaluation
Anti-inflammatory activity was assessed in an in vitro macrophage-monocyte inflammation model. The basic bactericidal activity of the combination was investigated in a quantitative suspension test, on Staphylococcus aureus and Pseudomonas aeruginosa. The permeability properties of one of the active ingredients was evaluated in vitro study on reconstituted Human Oral Epithelium.
All tests were performed in comparison with a control mouthwash with the same composition except for the mucoadhesive polymer.
Results: Rheological synergism and inclined plane test results showed that the new developed mouthwash is characterized by a mucoadhesive performance superior to the control. Similar results were found by “lectin binding displacement test”. The new formulation displays a high mucoadhesive performance (range from 64% to 51% inhibition lectin binding vs control), also when used in conditions that better mimic in vivo situation (dilution). Moreover, evaluation of resistance of mucoadhesive layer to salivary action showed that it possesses a high retention capacity, mostly after the first hour of salivary solution application and mucoadhesive properties were found after two hours (14.1% decrease vs time 0).
Anti-inflammatory and antiseptic activity of the formulation and tissue penetration of the active ingredient were confirmed .
Conclusion: Different in vitro testing methods confirm the mucoadhesive properties of the mouthwash. The adhesive characteristics of the formulation containing a mucoadhesive polymer were demonstrated in mechanistic (rheological synergism) and dynamic (inclined plane) assays and the results revealed a good consistency with the data obtained in the cellular test (lectin binding displacement).
The three methods adopted to assess the adhesion properties prove to be highly related and could be usefully performed, in the early development phase, as an integrated approach to characterize the adhesiveness of formulations containing mucoadhesive polymers.