Self-assembled lignin nanoparticles produced from elephant grass leaves enable selective inactivation of Gram-positive microorganisms

In this study, we added value to lignocellulosic biomass-derived lignin by converting it into antimicrobial nanoparticles using a simple self-assembling method in solution. Transmission electron microscopy (TEM) and zeta potential analyses showed that the self-assembled lignin nanoparticles (SA-LNPs) had a spherical-like morphology, 80 nm average size, and a surface charge of −29 ± 4 mV. Previous studies have shown that LNPs are toxic to bacteria, though the potential mechanisms of action leading to antimicrobial properties of LNPs are lacking in the literature. Therefore, we conducted a thorough investigation of the antibacterial activity of SA-LNPs using four bacteria strains: Escherichia coli and Pseudomonas aeruginosa (Gram-negative) and Bacillus subtilis and Lactobacillus fermentum (Gram-positive). The antimicrobial assays performed in saline media revealed that SA-LNPs were selectively toxic to Gram-positive bacteria, and no significant antimicrobial effects were found against the Gram-negative strains. Time-kill experiments showed that 25 μg mL−1 SA-LNPs were able to inactivate more than 90% of the Gram-positive bacteria after 30 min exposure. We conducted in vitro and in vivo assays to evaluate the production of reactive oxidative species (ROS), such as glutathione and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFA). These assays indicated that oxidative stress was not the underlying mechanism involved in the antimicrobial activity of SA-LNPs. This finding corroborates that SA-LNPs could scavenge radicals of 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazin-1-yl (DPPH), confirming their strong antioxidant property. Although direct oxidative stress was ruled out as the probable mechanism of action, we still cannot dismiss an indirect pro-oxidant effect resulting from the SA-LNPs-containing adsorbed ROS coming into direct contact with the cell wall.