## 5 Discussion
flavus_ conidia contain surface-exposed polysaccharides (e.g., \(\beta\)-1, 3-glucan, \(\alpha\)-1, 3-glucan, chitin, and galactomannan) and contain more fibrillary polysaccharides in the cell wall than _A. fumigatus_ conidia [61]. The cell walls \(\beta\)-1, 3-glucan content was comparable in the conidia of the two _A.pergillus_ species; however, the \(\beta\)-1, 3-glucan of _A. flavus_ was more highly branched than that of _A. fumigatus_ (as indicated by the higher MIC value). These architectural variations and compositional differences may be associated with the heterogeneity in the killing dynamics of PEI-AuNP@Van against the conidia of _A. fumigatus_ and _A. flavus_. Furthermore, _A. flavus_ exhibited higher levels of chitin in the cell wall and ergosterol in the cell membrane. It also shows increased peroxidase activity, leading to lower peroxidation of the membrane lipids. Differences in the composition and organization of the conidial cell walls of _A. fumigatus_ and _A. flavus_ may affect their interactions with PEI-AuNP@Van. Therefore, studies were undertaken to understand how PEI-AuNP@Van interacts with the conidial surface of _A. fumigatus_ and _A. flavus_ and the macromolecules responsible for this interaction. Because the conidial cell wall is the first to interact with the PEI-AuNP@Van system, its composition and surface organization may play an important role in the killing kinetics. PEI-functionalized AgNPs have been reported to interact with _C. albicans_ surface proteins and sporangiospores on the _R. arrhizus_ via electrostatic interactions [36; 57]. In this study, the cationic nature of PEI may have enabled binding to the anionic character of the conidia along with vancomycin, leading to vancomycin remaining close to the target macromolecule. As such, the functionalization of vancomycin served as a potent antifungal agent against _A. fumigatus_ conidia (free vancomycin did not show any activity per Figure 3a,b). _A. flavus_ was less susceptible to PEI-AuNP@Van, as indicated in the MIC study. This result indicates that the structural differences and selective surface binding of PEI-AuNP@Van lead to different results for the two species. Furthermore, PEI-AuNP@Van inhibited the germination of both _Asepergillus_ species, and variable dynamics were observed (Figure 4). The conidial susceptibility to PEI-AuNP@Van of _A. fumigatus_ (93% germination inhibition) was much higher than that of _A. flavus_ (35% inhibition). However, voriconazole only inhibited germination by 56 and 42%, respectively, indicating the germination inhibition potency of PEI-AuNP@Van. Similarly, PI staining revealed the differences in killing dynamics of conidia at 8 h of exposure: 38.4% of _A. fumigatus_ conidia were calculated as dead; in contrast, only 28% of conidia of _A. flavus_ were calculated as dead (as shown in Figures 5 and 6). These findings support the structural differences between the conidia of the two _Asepergillus_ species. To answer this question, we investigated the mechanism underlying the antifungal activity of PEI-AuNP@Van by using Raman spectroscopy, TEM analysis, and an endogenous ROS generation assay. ROS are mainly produced in the mitochondria during metabolic redox reactions and are formed as a series of intermediate products during oxygen metabolism. Excessive ROSaccumulation degrades intracellular biomolecules (e.g., proteins, lipids, and DNA), leading to irreversible oxidative damage. In this study, we monitored PEI-AuNP@Van-induced ROS production in the conidia of _A. fumigatus_ and _A. flavus_. The PEI-AuNP@Van-treated conidia of _A. fumigatus_ and _A. flavus_ were stained with H2 DCFDA dye, and FACS analysis was performed. We used 2\({}^{\prime}\), 7\({}^{\prime}\)-dichlorodifluor fluorescein diacetate (H2 DCFDA), a probe that is commonly utilized to measure cellular H2O2. Cell-permeable H2 DCFDA diffuses into cells and is deacetylated by cellular esterase to form 2\({}^{\prime}\), 7\({}^{\prime}\)-dichlorodifluororescein (H2 DCF).