Unraveling the mechanism of antifungal action of encapsulated eugenol/chitosan nanoparticles against Aspergillus fumigatus

Abstract

Aspergillus fumigatus can cause an invasive life-threatening infection in people who have weakened immune systems, underlying diseases or respiratory infections such as COVID-19. Azole-resistant A. fumigatus infections are difficult to treat and have high mortality. This study aimed to investigate the efficacy of encapsulated eugenol in chitosan biopolymer nanoparticles (NPs) to increase its antifungal activity against A. fumigatus. The physical properties of eugenol-encapsulated chitosan nanoparticles (EECNPs), size, polydispersity and the release profile of eugenol were examined by DLS, XRD and FTRI techniques. The antifungal activity of EECNPs against A. fumigatus was examined using CLSI-M38 microbroth dilution assay, time killing assay, reactive oxygen species (ROS) activity, destruction of cell membrane integrity/permeability, and cell wall morphological damage by electron microscopy. The eugenol/chitosan ratio of 1.00:1.00 was considered the optimal ratio with the highest encapsulation efficiency (43.1%) and loading capacity (30.8%). EECNPs were characterized by a hydrodynamic radius of 330.9 nm, a polydispersity index (PDI) of 0.298 and the zeta potential of 18.9 mv. The average size of the EECNPs was 300 nm according to TEM micrographs. The antifungal activities were determined at 150–300 μg/mL as MIC concentrations. The time killing assay showed that the nanoparticles could inhibit the fungal growth after 12 h. The toxicity assay revealed more than 60% survival of human embryonic kidney (HEK293) cell line in 2 × MIC concentration. The release of reactive oxygen species (ROS), and extracellular potassium in A. fumigatus caused more damaging on cell membrane integrity, through increasing 600 μg/mL concentration of nanoparticles. Our results revealed that the encapsulation of eugenol in chitosan biopolymer to produce EECNPs not only increased the half-life of eugenol but also improved its antifungal activity against A. fumigatus by a combined mechanism of damaging cell surfaces, cell membrane integrity, and the induction of ROS activity. This study is the first to report that EECNPs has the potency to use as a natural antimicrobial agent due to embracing the destructive properties on cell membrane integrity in A. fumigatus.