Table 2.

Methods used to evaluate the muco-adhesion properties of the formulations and comparison of their key features.

Techniques	Principle of the Technique	Mechanism	Real Time	Relevant	Feasibility	Cost	References
Indirect
Mucin particle	Determined through variation in size, ζ and turbidity.	+	No	+	+++	+	[149,158]
Microgravimetric	Evaluation of material adsorbed using quartz crystal microbalance based on resonance frequency.	++	Yes	+	++	++	[125,155]
AFM	Probe tip modified with polymer/mucin, measure attractive/repulsive forces exerted at molecular level.	+++	Yes	+	+	+++	[157,159]
Optical techniques	Changes in properties of incident light on surface immobilised mucin on binding with nanoparticle (ellipsometry and surface plasmon resonance).	++	Yes	+	++	+++	[150,160]
Diffusion/particle tracking	Impediment to the unhindered diffusive movement of nanoparticles (multiple particle tracking).	+++	Yes	++	+++	++	[161]
Direct
Cytoadhesion	Adhesion of fluorescent-labelled nanoparticles in cell culture using fluorescent microscopy.	++	Optional	++	++	++	[160]
Ex vivo methods	Retention of labelled nanoparticles in mucosal tissue/based on weight difference of nanoparticles.	+	Optional	++	++	++	[162]
In vivo administration/ ex vivo analysis	Administration of labelled nanoparticles to living animals, evaluation upon sacrifice.	+	No	+++	++	++	[163,164]
In vivo imaging	Natural trafficking of labelled-particles (e.g., barium sulphate, technetium-99 m).	+	Yes	+++	+	+++	[29,165]