The CONPs were synthesized using a modified ionotropic gelation.37 (link) First, 1 mg/mL chitosan solutions were prepared by dissolving the medium molecular weight CS (50,000–190,000 Da, 75–85% deacetylated, Aladdin Biochemical Technology Co. Ltd, USA) in a solution of hydrochloric acid to 0.04% (v/v). The CS solution was stirred for 1 h and adjusted to pH = 5.5 by NaOH. The oleuropein solutions were prepared by dissolving 0.72 mg oleuropein (Jiangsu Caiwei Biology Science and Technology Co. Ltd, China) in 1mL water. Next, add 1 mL of oleuropein solution to 5 mL of CS solution, stir for a few minutes, and adjust the pH to 5.5. 2 mg/mL tripolyphosphate solution was obtained by dissolving tripolyphosphate (Sigma Aldrich, USA) in distilled water and added dropwise to the CS solution with a volume ratio of 1:5. Finally, the suspensions were stirred for 30 min at room temperature. The concentration of the oleuropein solution used for preparing CONPs was 100 mg/L. Five drops of the suspension were prepared by allowing a single drop of nanoparticle suspension to dry overnight at room temperature on a 200-mesh copper grid. Transmission electron microscope (TEM, HT7700, Hitachi, Japan) was used to observe the morphology of the synthesized nanoparticles (Figure 1).

Transmission electron microscopy (TEM) image of CONPs (scale bar = 100 nm).