4.2. Preparation of Usnic Acid-Chitosan Nanoparticles (UA-CS NPs)

The methodology used for the preparation of UA-CS NPs was a single-step ionotropic gelification method as described earlier with slight modifications [22,39]. Briefly, low molecular weight chitosan (1.2% w/v) was initially dissolved in 1% acetic acid in a total volume of 100 mL of deionized water. The chitosan solution was continuously agitated at the room temperature for 12 h and added with 0.3 g Tween 60 to obtain a homogenous mixture. The mixture was stirred for 60 min at 50 ℃. The usnic acid (0.5%, w/v) solution prepared in dimethyl sulfoxide was slowly added and allowed to mix by stirring for 2 h at room temperature. Two different concentrations of TPP, which are 0.5% and 1.0%, were used for the preparation of UA-CS NPs. The solution of TPP was prepared in a total of 40 mL deionized water and was slowly added dropwise with continuous stirring for 2 h. The light yellow color mixture was centrifuged (13,000 rpm for 15 min) at 4 ℃. Similarly, CS NPs (unloaded with UA) was also prepared using 0.5% and 1.0% TPP. The obtained pellet was washed repeatedly for three times with deionized water and kept at −70 ℃ in a refrigerator to solidify. Finally, the frozen samples were freeze-dried using a freeze dryer (FD8518, ilShinBiobase Co. Ltd., Dongducheon, Korea).