*3.3. Evaluation of the Antifungal Activity of CuI*

Following our methodology, different variables were evaluated to optimize the interaction of CuI materials and pathogenic fungi. Our first attempt involved the use of CuI powdered material at different doses (0.5, 2.5, and 12.5 mg/mL). Although still a common practice, the use of powdered nanomaterials for biomedical applications is cumbersome, since stable colloidal suspensions in exposition media are difficult to be obtained (Figure S2). The evaluation of the antifungal activity of CuI against *S. schenckii* was hampered by the aggregation of CuI materials in phosphate buffer, decreasing the interactions of NPs and fungi and limiting the reproducibility of observed results.

Because of the above results, stable CuI colloidal suspensions were prepared by using biopolymers as surfactants. Their antifungal activity was evaluated next at different concentrations of CuI colloids and times of exposition of pathogenic fungi. To avoid the aggregation of the NMs, their interactions with fungi were evaluated in distilled water. Initially, the following concentrations (75, 100, and 125 µg/mL) were evaluated at short exposition times (2 and 3 h). We observed good antifungal activity of the materials at all tested concentrations (Figure 6A). For instance, CuI@Ch shows antifungal activity with very high inhibition growth percentages (>90%). In addition, CuI@AG shows moderate activity after 2 h of exposition with an inhibition of 28% for the highest tested concentration. However, after 3 h of exposition, the activity of CuI@AG is very similar to CuI@Ch with inhibitions near 100%. Figure 6B shows that the fungi are viable (under the experimental conditions) in the growth controls at different serial decimal dilutions. Figure 6C illustrates the antifungal activity of the materials at different doses evaluated by the drop dilution method. Fungal growth is only registered for control groups. Figure 6D–G and Figure S4 show that longer exposition time (5 h) of fungi to these materials concentrations, result in complete growth inhibition. Figure S4 illustrates a slightly decreased antifungal activity of non-freshly prepared CuI@Ch colloidal suspension.

Freshly prepared CuI@Ch shows an enhanced antifungal activity in comparison to CuI@AG due to the synergic action of CuI and the polymer. However, its improved antifungal activity varies with time (Figure S4). A recent report remarks the importance to study the stability of biopolymers in different exposure media [22]. Accordingly, under experimental conditions, CuI@Ch tend to form aggregates, decreasing antifungal activity. Lixiviation of copper ions is one of the proposed mechanisms of CuI antifungal activity. For CuI@Ch composite, complex formation (Cu-NH2) results in decreased bioavailability of Cu ions or amino groups, the chemical species responsible for antifungal activity [23,24]. In addition, decreased solubility in the exposure media lowers the stability of CuI@Ch colloidal suspension. Earlier reports point out the role of the physicochemical properties of chitosan and its antifungal activity. For example, the high molecular weight chitosan shows a better antifungal activity. Moreover, the susceptibility of the fungi specie is an important consideration [25].

Figure S5 shows the results of the antifungal activity of Cu NPs. Figure S5A illustrates the presence of fungi after their interaction with Cu NPs treatment. In contrast, CuI materials inhibit the growth of the fungi under the same experimental conditions. Figure S6 illustrates the viability of the fungal cells under experimental conditions (top row Figure S6A). It is evident the effect of Cu NPs treatment at different doses. CuI composites achieve more than 90% growth inhibition. For Cu NPs, lower efficiencies are attained under the same experimental conditions (Figure S6B). Accordingly, a recent study discusses the effect of Cu and CuO NPs to inhibit *Colletotrichum gloeoesporioides*. Cu and CuO NPs affect fungal growth at high doses (500 mg/mL) and long exposure times [26]. Cu NPs exert morphology-dependent antifungal activity. For example, the sharp edges of marigold-like petal nanostructures injure the cellular wall and membrane, and cause the death of the yeast (*C. albicans*) [27].

**Figure 6.** Evaluation of the antifungal activity of CuI@AG, CuI@Ch and chitosan to inhibit *C. albicans* growth. (**A**) Log reduction on the CFU/mL of *C. albicans* exposed to different amounts of CuI colloids or chitosan. (**B**) Viability of control group (decimal dilutions) under experimental conditions. (**C**) Evaluation of the fungicidal activity of CuI@AG against *C. albicans* (drop dilution test). (**D**–**G**) Enumeration of the CFU/mL of *C. albicans* exposed for 5 h to different amounts (0, 75, 100 and 125 µg/mL) of CuI NMs (spread plate procedure).

> Our results indicate that the conidia of *S. schenckii* are more susceptible than the yeast of *C. albicans* to CuI treatment. For example, after 5 h of the exposition of these fungi to CuI@Ch, total growth inhibition is observed at all evaluated concentrations. Also, if the time of exposition is reduced (2 h), then the fungal growth inhibition is achieved at a

concentration of 75 µg/mL of CuI@Ch. CuI@AG shows similar behavior for the treatment of *S. schenckii*. For example, after 5 h of the exposition of *S. schenckii* to CuI@AG composite, the minimum inhibitory concentration (MIC) is 12.5 µg/mL, whereas the minimum fungicidal concentration (MFC) is 25 µg/mL. At shorter exposition times (2 h), 125 µg/mL of CuI@AG composite must be used to inhibit the fungal growth (Figures S7 and S8). To our knowledge, there are not many reports regarding the development of effective and specific compounds for the treatment of *S. schenckii* infections. Potassium iodide, azoles, and amphotericin B are among the very few options for fungal infection treatment. However, these treatments result in side deleterious effects. Except for KI, the cost of the treatment is elevated, a barrier for general application in developing countries [28]. In this work, we demonstrate the effective antifungal activity of CuI materials at low doses. Their affordable cost and biocompatibility suit them as strong candidates for the development of new broad-spectrum antifungal agents.

As previously discussed, *C. albicans* is more resistant to CuI treatment. For example, after the exposition of these pathogenic fungi to CuI@AG for 5 h, fungal growth is observed at the lower evaluated doses (12.5 and 25 µg/mL). However, good inhibition activity is reached, since the fungal growth in the control group is abundant, impeding the total (CFU), direct count. On the other hand, after treatment with 12.5 µg/mL, only 50 CFU are observed. Also, treatment with 25 µg/mL decreases the growth to 5 CFU. CuI@Ch colloids exhibit the same behavior discussed for *S. schenckii*. The materials inhibit the growth of *C. albicans* at all evaluated concentrations after 5 h of exposition. However, variable effectiveness is also observed. The variability might also be a consequence of the instability of the polymer at the exposure media (pH and ionic strength).

Figure S9 shows the preliminary results on the fungicidal activity of CuI@AG against *Fusarium oxysporum*. Our results indicate fungal growth inhibition at high doses (75 µg/mL) and 20 h exposure. Future studies aim to optimize the different interaction variables to increase antifungal efficiency. Previous studies discuss the advantages of the use of NMs for the control of fungal plant diseases. Carbon, silver, silica, non-metal oxides, polymer composites and aluminosilicates show efficient activity to promote plant growth and to inhibit plant pathogens [29]. CuI@AG is a potent candidate for the development of fungicidal agents for crop protection. Also, NMs are efficient for the treatment of superficial fungal and yeast infections. Among the therapeutic agents, Ag, CuO, polymers and ZnO NPs have been investigated. NPs can also be used as antifungal carriers for the treatment of superficial fungal infections (liposomes, nanofibrous) [30] (Chapters 5, 6). From all the above, it is evident that nanotechnology offers numerous strategies to overcome the challenges that currently face the health sector.

In summary, CuI@AG shows the best antifungal activity among the different materials evaluated in this study (Cu@AG, Chitosan, CuI@Ch). Figure 7 summarizes the effective doses of the materials to inhibit the growth of pathogenic fungi. Figure 7B shows the efficiency of CuI@Ch as fungicidal at low doses. However, Figure S4 shows the variable behavior of this material. This behavior is due to its instability in the exposure medium. Figure 7C shows the enhanced antifungal activity of CuI@AG in comparison to chitosan. Although numerous reports remark the antifungal activity of this polymer, CuI@AG is more effective. Figure 7D resumes the MIC and MFC for the antifungals under study.


**Figure 7.** Summary of the antifungal activity of CuI NMs (5 h of exposure). (**A**) CuI@AG against *S. schenckii*. (**B**) CuI@Ch against *S. schenckii*. (**C**) CuI@AG and chitosan against *C. albicans*. (**D**) MIC and MFC values.

> The materials presented in this study offer numerous advantages against some previously reported antifungal compounds (Table 3). For instance: (a) the compounds can be synthesized using a green route under ambient conditions; (b) the materials show excellent antifungal activity at low doses and short exposure times; (c) the materials exhibit different mechanisms to inhibit the growth of different pathogenic fungi; (d) the materials show biocompatibility at the doses that exhibit antifungal activity; (e) Low cost.


**Table 3.** Comparison of the properties of non-conventional antifungal agents.

### *3.4. Evaluation of the Interaction of Fungi and CuI by AFM*

Because of their physicochemical properties, NMs can display different mechanisms to destroy pathogenic microorganisms. Thus, numerous analytical techniques are applied to elucidate the antimicrobial activity of NMs. In this study, we use AFM to investigate the capacity of CuI NMs to inhibit the growth of pathogenic fungi. As previously discussed in this work, CuI NMs exhibit antifungal activity at very low doses. This capacity is not only due to their composition but also to the different and individual antifungal mechanisms shown by the elements present in the composite.

The analysis of the interaction of CuI and *Candida* by optical microscopy (Figure S10) shows the capacity of the materials to inhibit biofilm formation. The colony-forming ability of *C. albicans* decreases as the amount of CuI NMs increases. The inhibition of biofilm formation is a result of the high affinity between fungi cells and NMs. Our observations are supported by previous studies that discuss the effectiveness of iodine-containing polymers to inhibit the growth of biofilm-forming microorganisms. The materials avoid the adhesion of bacteria (*S. aureus*) or fungi (*C. albicans*) to surfaces, limiting biofilms formation [32].

AFM allows a closer examination of the interactions of CuI colloids and pathogenic fungi. There are no differences in the affinity exhibited by the colloids to adhere and penetrate the fungal cell (Figures 8–10). Short interaction times (1 h) demonstrate the adherence of the materials to fungi cell membranes. We can also observe differences in the stability of the colloids in the exposition media. For example, CuI@AG materials do not agglomerate, facilitating their interaction with fungal cells. This close interaction (CuI@AG-*C. albicans*) results in cell damage at all evaluated concentrations, as represented in Figure 8. Contrarily, CuI-Ch materials form big aggregates at the surface of the *C. albicans* membrane (Figure 9). Their increased size due to agglomeration decreases their antifungal activity. As previously discussed, the low stability of the materials renders poor reproducibility. This observation supports our results of the antifungal activity of these materials evaluated by classical microbiology methods.

**Figure 8.** Evaluation of the exposition of *C. albicans* to different doses of CuI@AG for 1 h. Height, amplitude and phase images are presented for: Control group (**A**–**C**), and *C. albicans* exposed to different doses of CuI@AG: 75 µg/mL (**D**–**F**); 100 µg/mL (**G**–**I**); 125 µg/mL (**J**–**L**). Blue dotted circles indicate the presence of NMs disrupting fungal cells.

**Figure 9.** Evaluation of the exposition of *C. albicans* to different doses of CuI@Ch for 1 h. Height, amplitude and phase images are presented for: Control group (**A**–**C**), and *C. albicans* exposed to different doses of CuI@Ch: 75 µg/mL (**D**–**F**); 100 µg/mL (**G**–**I**); 125 µg/mL (**J**–**L**). Blue dotted circles indicate the presence of aggregated NMs on the surface (**D**–**F**) or penetrating the cells (**G**–**L**). Figures E and F denote the presence of nanomaterials on the surface of the fungi. In particular, face change in F corroborates the presence of the nanomaterials on the surface. On the other hand, K and L illustrate the penetration of nanomaterials in fungal cells.

**Figure 10.** Evaluation of the exposition of *S. schenckii* to different doses of CuI@Ch for 1 h. Height, amplitude and phase images are presented for: Control group (**A**–**C**), and *S. schenckii* exposed to different doses of CuI@Ch: 75 µg/mL (**D**–**F**); 100 µg/mL (**G**–**I**); 125 µg/mL (**J**–**L**). Blue dotted circles indicate the penetration of the material on the fungal cell and exposition of its components. White dotted circles indicate morphological changes in *S. schenckii* due to the almost complete fungal cell coverage of the composites.

The AFM analysis of the morphological changes experimented by fungi after interacting with CuI materials indicates the penetration and disruption of the cells (Figure 8). Previous studies reported that the adherence of NMs to the membranes of microorganisms increases the lag stage of the bacterial growth period, extending the reproduction time of the microorganisms [37,38]. In accordance, in this study, the growth of pathogenic fungi was slower as a result of their exposure to CuI composites.

Kim et al. [31] reported that as a result of the interactions of *C. albicans* and nano-Ag, the fungi membranes exhibit significant changes which were manifested by the formation of "pits" on their surfaces, followed by pore development and cell death [31]. Our results indicate similar findings, the formation of a pit on the surface of *C. albicans* due to CuI penetration (Figures 8 and S11). Following the adherence of NMs to the membranes, different mechanisms of antifungal activity become active. For instance, the adhesion of CuI@Ch to fungal cell results in increased permeability of fungal membrane, then leakage of cellular contents, followed of cell death [25].

To our knowledge, there are not many reports regarding the antifungal activity of iodine based materials [32,39]. For example, the preparation of PVP-Iodine liposome hydrogels and their capacity to inhibit *C. albicans* growth was reported [33]. Human keratinocytes infected with *C. albicans* underwent antifungal treatment (PVP-iodine liposome hydrogels and commercially available Betaisodona). The iodine-based treatment results in epithelial alterations of the keratinocytes. Additionally, PVP-iodine liposome hydrogels show a multi-step antifungal mechanism. First, the adherence of the material to the cell wall of *C. albicans*, followed by membrane damage and adsorption of the active agent into the fungal cell. Our results illustrate similar findings. However, lower doses and exposure times are required.

The alteration on the morphology of *S. schenckii* due to interactions with CuI were evaluated by AFM. The results are presented in Figures 10 and S12. The high affinity of CuI colloidal materials to the *S. schenckii* cell surface results in the total coverage of fungal cells at all evaluated concentrations. AFM images show the typical morphological characteristics of these fungi by observation of the control group, demonstrating its bioavailability under test conditions (Figure 10A–C). CuI@Ch firmly adheres to fungal cells, causing damage at short exposition times (Figure 10G–L). As previously discussed by *C. albicans*, CuI@Ch forms bigger aggregates in the exposition media. Treatment of *S. schenckii* with doses of 100 and 125 µg/mL (for one hour) results in damage to fungal cells.

Figure 10G,H illustrate the formation of a cavity in *S. schenckii* (dotted blue circles). This asseveration is supported by tapping phase analysis (Figure 10I), which highlights the location of interfaces within the sample. Phase contrast is evident in the circled region, indicating the interface between NMs (light zones) and fungi (dark zone). The penetration of the materials disrupts the membrane exposing fungi cellular components. Figure 10J,K illustrate the changes in the morphology (dotted white circles) of *S. schenckii* due to interaction with the highest dose of composites. Figure 10I confirms the presence of morphologically modified fungi due to the almost complete coverage of fungi surface.

By AFM studies, we demonstrate that the highest susceptibility of *S. schenckii* to CuI materials is due to closer interactions of the materials and fungal cells. There are evident differences in the size of the fungal cells evaluated in this study (conidia of *S. schenckii* vs. *C. albicans* yeast), a variable that might influence the reported results. There are no significant differences in the activity of CuI@Ch or CuI@AG. For practical applications, CuI@AG is more suitable. Because of its abundance, low-cost, high hydrophilicity, and low toxicity. These properties suit it as an excellent candidate for the development of composites for biomedical and environmental applications.
