Dye encapsulating experiments were conducted in a series of 250 ml flasks by mixing chitosan with aqueous Congo Red (CR) solutions. In all experiments, the dye solution (50 ml) is prepared at a desired initial concentration by diluting the stock dye solutions with distilled water and transferring them into an Erlenmeyer flask with a magnetic stirrer. The chitosan solution (1 ml, 1% w/v) was then added to the dye solution and stirred magnetically at 300 rpm. Dye encapsulated chitosan nanoparticles are obtained by adding the TPP solution at different volumes (1% w/v) to this solution. Dye encapsulated nanoparticles are magnetically stirred until stable, and then the nanoparticles are removed by filtration.
The amount of residual dye was determined at 495 nm, which is the maximum wavelength absorption of CR, by means of a UV–vis spectrophotometer (Genesys 10S, ThermoFisher Scientific, USA). In all experiments, the encapsulation efficiency is calculated by the following equation: