*3.5. Evaluation of the Bio-Compatibility of CuI@AG*

Due to the membrane-damaging effects of CuI@AG composites in pathogenic fungi, we studied the outcomes of the interaction of composites and human red blood cells (RBCs). An *in vitro* toxicity assay to evaluate the hemolytic activity of the composites at different

doses was conducted (Figure 11A). As shown in Figure 11B,C, the composites exhibited little hemolytic activity at doses where the effective antifungal activity occurs. Side effects in host cells (RBCs) result in their exposition to composite materials at 12.5 µg/mL. We can also observe a decreased hemolytic activity at higher doses of the materials. This behavior might indicate the aggregation of the composite materials in the exposure media. The homo-aggregation of the materials decreases its surface area, thus its reactivity toward surrounding RBCs.

**Figure 11.** Evaluation of the hemolitic activity of CuI@AG. (**A**) Schematic representation of the interaction of red blood cells and CuI@AG. (**B**,**C**) Qualitative and quantitative evaluation of the hemolitic activity of CuI@AG at different doses.

It is important to remark that the interactions of NMs and living cells are complex and depend on the surface properties of the NMs and the functional groups present in the different cell membranes [40,41]. Exposure media also influence the surface properties of NMs, thus also their bio-activity. For example, Ag NPs can inhibit the growth of some, but not all, fungal strains. Another example indicates that the preparation of ketoconazoleloaded chitosan–gellan gum nanoparticles are more effective against *Aspergillus niger* than unmodified NPs and ketoconazole alone [42,43]. Current findings are not contradictory: the differences arise from the lack of uniformity in the experimental conditions among research groups.

Also, numerous reports indicate increased biocompatibility of metallic NPs after polymer functionalization. In the present study, we use Arabic gum and chitosan as CuI coatings. It is important to point up that Arabic gum is negatively charged under experimental conditions, whereas chitosan has a positive charge. RBCs are also nega-

tively charged, avoiding electrostatic interactions with Arabic gum coated CuI materials. The AFM study of the interactions of fungal cells and CuI@polymers shows the adsorption of the composites to the fungal cells. After adsorption, the NMs enter the fungal cell, causing its death. Fungal cells contain a cell wall that can serve as a reservoir for CuI accumulation. RBS lack a cell wall. Numerous reports indicate that lixiviation of ions is favorable after a direct interaction with biological entities. CuI@AG does not interact closely due to the electrostatic repulsion of negatively charged surfaces. Penetration of NMs into the cells is not favorable under such conditions.

Although in this manuscript we present the most relevant findings related to the antifungal activity of CuI materials, previous studies show that CuI@Ch become unstable in the exposure media (aggregation), decreasing their antifungal activity. We also observe that, due to their positive charge under physiological conditions, these materials exert hemolytic activity. Our results show the same trend during the antifungal or bio-compatibility evaluation. The activity of CuI@Ch NMs is dose and time-dependent due to their instability in the exposure media. Current studies of our research group aim to improve CuI@Ch stability.

Finally, in a previous work, Pd@Ag nanosheets were studied as potential alternatives to develop efficient antifungal agents. The materials exhibit enhanced antifungal activity and biocompatibility at low doses [35]. The composites (CuI@AG) under study in the present manuscript also exhibits enhanced antifungal activity and biocompatibility in human red blood cells. CuI@AG composite materials can be a better option for large scale applications due to their lower cost and simple fabrication.

#### **4. Conclusions**

In work work, CuI@AG and CuI@Ch composites were synthesized using a facile and reproducible technique. The materials exhibit a 3D shell ordered structure that might suit them for numerous applications. We demonstrated antifungal activity at low doses and short exposure times. We also demonstrate the enhanced activity of CuI by comparing it with Cu NPs antifungal activity. The CuI NMs are bio-compatible with RBCs at the doses required to exert antifungal activity. The materials display excellent antifungal activity against biofilm-forming pathogenic fungi. The materials are also efficient to inhibit the growth of filamentous fungi (*Fusarium oxysporum*). To our knowledge, this is one of the first studies that demonstrate the efficacy of nano-antifungals to inhibit *S. schenckii* growth. Future studies will investigate the activity of the materials using *S. schenkii* yeasts. Commercially available fungal therapies show limited effectiveness due to their reduced fungal cell penetration and the development of drug-resistant strains. In this work, we demonstrate the multistep antifungal mechanism of CuI composites. Multiple studies have shown the strong antimicrobial effects of single metallic NPs on several species of microorganisms. However, some NPs are effective against certain species but have little or no effect on others. Aiming to formulate a broad-spectrum antifungal, Cu, I, and chitosan integrate the present formulation. The combination of different antifungal agents renders enhanced activity at low doses. This favors its biocompatibility with host cells and reduce the capacity of the pathogen for resistance development. It is important to remark that the synthesis of CuI@AG develops within minutes under ambient conditions. The materials are efficient, bio-compatible, broad-spectrum fungicidal agents, and of low cost. Due to all the above considerations, CuI@bio-polymer composites turn out to be potent candidates to develop efficient antifungal agents for biomedical or environmental applications.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2309-608 X/7/2/158/s1, Figure S1. SEM-EDX analysis of CuI nanostructured materials. (A) SEM micrograph of powdered CuI NMs. (B) EDX analysis highlighting copper content. (C) EDX analysis highlighting iodide content. The mass (wt) and atomic (at) percentages of the elements confirm the formation of CuI materials; Figure S2. SEM micrographs illustrating the behavior of powdered CuI nanostructured materials at different doses in phosphate buffered medium in the presence (A\*–C\*) and absence (A–C) of *S. schenckii*; Figure S3. UV-Vis characterization of Cu@AG colloidal suspension.The spectra

shows the characteristic plasmon of Cu NPs; Figure S4. Evaluation of the antifungal activity (*C. albicans*) of CuI@AG (A), chitosan (B) and CuI@Ch (C) at different doses; Figure S5. Evaluation of the antifungal activity (*C. albicans*) of Cu@AG (A), growth from dilution (10−<sup>3</sup> ) (B) at different doses; Figure S6. Evaluation of the antifungal activity of Cu@AG to inhibit C.albicans growth. (A) Viability of control group (decimal dilutions) under experimental conditions (Top row). Evaluation of the fungicidal activity of Cu@AG against *C. albicans* (drop dilution test) at differen NPs concentrations. (B) Log reduction on the CFU/mL of *C. albicans* exposed to different amounts of Cu@AG; Figure S7. Evaluation of the antifungal activity (*S. schenckii*) of CuI@AG at different concentrations and exposition of 5 h. (A) Fungal cell are viable in the growth controls at different serial decimal dilutions under experimental conditions. (B) Evaluation of the antifungal activity of CuI@AG by the drop dilution method. Fungal growth is only observed for growth control. (C–F) Evaluation of the antifungal activity of CuI@AG by the pour plate method. Fungal growth is only observed for growth control (C); Figure S8. Evaluation of the antifungal activity (*S. schenckii*) of CuI@Ch at different concentrations and exposition of 5 h. (A) Fungal cell are viable in the growth controls at different serial decimal dilutions under experimental conditions. (B) Evaluation of the antifungal activity of CuI@Ch by the drop dilution method. Fungal growth is only observed for growth control. (C–F) Evaluation of the antifungal activity of CuI@Ch by the pour plate method. Fungal growth is only observed for growth control (C); Figure S9. Evaluation of the antifungal activity (*F. oxysporumi*) of CuI@AG at different concentrations and exposition of 20 h. (A) Fungal cell are viable in the growth controls at different serial decimal dilutions under experimental conditions. (B–D) Evaluation of the antifungal activity of CuI@AG at different doses by the drop dilution method. Fungal growth is only observed for the fungi exposed to 75 µg/mL of NMs; Figure S10. Optical microscopy examination of CuI-C. albicans interaction at diferent CuI doses. (A) Big aggregates of *C. albicans* are observed for control group. (B–D) The aggregates decrease as the concentration of NMs increases (50, 75 and 100 µg/mL respectively); Figure S11. AFM analysis of the morphological changes in *C. albicans* due to exposure to CuI@AG (50 µg/mL × 1 h). Formation of a pit in fungal cell is observed due to NMs penetration (red dotted circles). A,B,C are the low-magnification images, whereas D,E,F are the high magnification images; Figure S12. AFM analysis of the morphological changes in S. schenckii due to exposure to CuI@Ch (50 µg/mL × 5 h). (A–C) Control group. The fungi maintain their typical morphology under test conditions, demonstrating the bioavailability of the MOs at the end of the experiment. (D–I) The NMs interact closely with fungal cells resulting in their destruction.

**Author Contributions:** Conceptualization, I.E.M.-R.; methodology, I.E.M.-R.; software, I.E.M.-R. and J.E.M.-D.; validation, I.E.M.-R.; formal analysis, J.H.M.-M., I.E.M.-R., Y.R.-L. and A.G.-G.; investigation, J.H.M.-M., I.E.M.-R. and Y.R.-L.; resources, I.E.M.-R. and J.E.M.-D.; data curation, J.H.M.-M., I.E.M.-R., Y.R.-L. and A.G.-G.; writing—original draft preparation, I.E.M.-R. and J.E.M.-D.; writing—review and editing, I.E.M.-R. and J.E.M.-D.; visualization, J.H.M.-M., I.E.M.-R. and Y.R.-L.; supervision, I.E.M.-R.; project administration, I.E.M.-R.; funding acquisition, I.E.M.-R. and J.E.M.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** One of the authors (I.E.M.-R.) was financially supported by the National Council for Science and Technology of Mexico (CONACyT) through the award No. 299078. Meanwhile, J.E.M.-D. was supported by CONACYT via grant A1-S-45928.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in insert article or supplementary material here.

**Acknowledgments:** This work is a submission to the special issue of Journal of Fungi on *Fungal Nanotechnology*. Beforehand, the authors wish to thank Kamel A. Abd-Elsalam for handling this submission. The authors also wish to thank the anonymous reviewers and the associate editor in charge of handlight this paper for their comments and suggestions, which helped to improve the quality of this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of all the compounds are available from the corresponding author.
