2.2.1. Preparation of Surfactin-Loaded CO-CNF Nanoparticles

The detailed procedures for particle preparation and characterization were reported in our previously reported work [13]. Briefly, 100 mL of CO (1 g in 100 mL) solution was mixed with 100 mL of CNF solution (30 mg/ ml) and stirred at 80 °C for 10 min. Later, potassium chloride (KCl) was added as a cross-linker and stirred at 80 °C. After 3 h, the particles were collected by centrifugation, and then sonicated (HOYU Ultrasonic 250, Taipei, Taiwan), and freeze-dried. The surfactin-loaded CO-CNF nanoparticles were prepared by the emulsion method. Three types of nanoparticles were prepared in this study such as 50SNPs (50 mg surfactin-loaded CO-CNF nanoparticles), 100SNPs (100 mg surfactin-loaded CO-CNF nanoparticles), and 200SNPs (200 mg surfactin-loaded CO-CNF nanoparticles) based on the concentration of surfactin. 200 µL of Tween 80 was added to 50 mL of CO-CNF solution and stirred for 1h at 60 °C. Afterwards, surfactin (50/100/200 mg in 2 mL methanol) solution was taken and CO-CNF solution was added to it. As a stabilizer, 8 mL of N,N′-methylenebisacrylamide (MBAA) (1.87 mg/mL) was added. After 2 h of reaction, the obtained particles were sonicated and free-dried. A schematic representation of surfactin loaded nanoparticles preparation was given in Figure 1.

Schematic representation of surfactin-loaded nanoparticles preparation.