*2.3. Characterization of CuI Nanostructured Materials*

CuI nanostructured materials were characterized by scanning electron microscopy (SEM-EDX) (Carl Zeiss, Sigma-HDVP and EDX detector QUANTAX Brucker) and atomic force microscopy. Thin films of CuI@AG and CuI@Ch were deposited on a glass substrate using doctor blade technique. The surface roughness and morphology at microand nanometer scale were measured with a ScanAsyst atomic force microscope (Bruker, Dimension Edge with Scan Assist). The samples were analyzed in tapping mode in air with OTESPA-R3 tips (silicon; f0: 300 kHz; k: 26 N/m). Data was acquired on square frames of 10 × 10, 5 × 5 and 2.5 × 2.5 µm. Images were recorded using height, amplitude and phase channels. Amplitude mode was used to evaluate the topography of the films; height mode images were used to obtain quantitative measurements, and phase images to evaluate changes in composition. A resolution of 512 × 512 pixels was used. Measurements were made by triplicate on different zones of each sample.

#### *2.4. Evaluation of the Antifungal Activity of CuI Nanostructured Materials*

**Fungal strains and culture conditions.** A general scheme for the antifungal evaluation of CuI@AG and/or CuI@Ch is illustrated in Figure 1. The antifungal activity of Cu@AG against *C. albicans* was used as a reference to demonstrate the enhanced activity of CuI@AG. The concentration of copper in the Cu NPs is the same as the concentration used for CuI NMs.

**Figure 1.** Schematic representation of the methodological approach for the evaluation of the antifungal activity of CuI@biopolymer against pathogenic fungi (*C. albicans*, *S. schenckii*).

*Sporothrix schenckii***.** The *S. schenckii* sensu stricto wild-type strain UAA-307 was obtained from a human lymphocutaneous sporotrichosis case. The fungus identification was previously performed by biochemical, morphological, and molecular biology techniques (polymerase chain reaction sequencing of the calmodulin gene, accession number KJ921740 in GenBank) [19]. Conidial suspensions were prepared as previously reported [20]. Briefly, fungal mycelium was incubated at 28 ◦C for three days in sabouraud dextrose broth under orbital agitation at 150 rpm (Shaker Lumistell, IR0-60, Temperature Control). The fungal suspension was filtered on sterile filter paper (pore size 0.45 mm) and harvested by centrifugation at 750× *g*, for 20 min at 4 ◦C. The fungal cells were washed then with sterilized phosphate buffer solution (PBS) and counted in a Neubauer chamber before all experiments. The bioavailability of the fungi was determined by trypan blue staining. In all the experiments, a stock fungal suspension of 1 × 10 7 conidia/mL in distilled water was used.

*Candida albicans***.** *C. albicans* strain used in this study was a clinical isolate, and isolates were cultured in Sabouraud Dextrose Agar (SDA), then incubated at 37 ◦C for 48 h. Fungal biomass was collected afterwards by a sterile loop from the surface of SDA medium and resuspended in 1 mL of PBS. An inoculum of *C. albicans* was placed in 3 mL of Potato Dextrose broth and left to grow in a shaking water bath (60 rpm) at 35 ◦C for 48 h (resulting in 1 × 10 <sup>7</sup> CFU/mL according to the growth phase). The fungal suspension was centrifuged next at 3500 rpm for 5 min, decanted, and suspended in distilled water (stock fungal suspension).

*Fusarium oxysporum***.** *Fusarium oxysporum* strain was isolated from samples that present fungal infection. The infected samples were placed in a PDA medium to allow fungal growth. After a small portion of the growing mycelium is reseeded in a box with fresh PDA medium and allowed to grow at 28 ◦C or room temperature for 5 to 7 days. Analysis of conidia production is conducted by optical microscopy. Conidia were concentrated in phosphate buffer. Fungal concentration was determined by UV-Vis spectroscopy (1 × 10 <sup>7</sup> CFU/mL).

**Drip dilution test.** For the drip dilution test, six decimal dilutions were made in sterile distilled water (by duplicate) to evaluate the fungal growth in each dilution and to demonstrate fungal bioavailability under the experimental conditions. The initial concentration of fungal cells was 1 × 10<sup>7</sup> CFU/mL. From each dilution, an aliquot of 10 µL was laid on a Petri dish with SDA. The drops were allowed to dry to avoid movement displacement. They were left in an incubator at 28 ◦C for 48 h (*C. albicans*) or 72 h (*S. schenckii*).

**Interaction test.** 100 µL are taken from the stock fungal suspension, placed in Eppendorf tubes and diluted to 1 mL. The dilution of the fungal suspension is 1 × 10<sup>6</sup> CFU/mL. The interaction of fungi with NMs at different concentrations was performed then according to Table 1. Each experimental condition was evaluated by duplicate. The interaction was carried out for 5 h in a water bath at 37 ◦C. The tubes were removed next from the water bath, then 100 µL were taken from each Eppendorf tube and placed on SDA (two boxes per tube), dispersed with boiling pearls and left in an incubator for 48 h (*C. albicans*) and 72 h (*S. scenckii*) for their subsequent analysis or counting at 28 ◦C. The fungicidal activity of the materials was checked by determining the MIC (minimal inhibitory concentration) and MFC (minimal fungicidal concentration). The MIC was defined as the lowest concentration of the composite that significantly (visually) inhibited fungal growth with respect to CFU. The MFC was defined as the lowest concentration of the composite that completely inhibited fungal growth.

**Table 1.** Determination of the MIC and MFC of CuI@polymer composites. The exposure of pathogenic fungi was evaluated at different composites concentrations.


**Evaluation of the interaction of** *S. schenckii* **and** *C. albicans* **with CuI by Atomic Force Microscopy (AFM).** The analysis of the interactions of CuI@AG and CuI@Ch with the fungi was carried out by Atomic force microscopy. A 10 µL sample of the different treatments specified in the paragraph *Interaction test* was withdrawn after the exposure time was finished. The sample was deposited on a mica substrate and fixed with absolute ethanol. The samples were analyzed using a Dimension edge with a scan assyst microscope (Bruker) on tapping mode. Topography, phase, and amplitude images were collected using tapping mode in the air at room temperature. OTESPA-R3 probes (K: 26 N/m, f0: 300 kHz) were used for the analysis.

**Evaluation of the biocompatibility of CuI colloidal materials.** In vitro toxicity studies were carried out using human whole blood. Heparin-stabilized human blood was freshly collected. Physiological saline solution (PSS) was added to test tubes (10 mL of PSS in each tube). Different amounts of CuI@AG were added to each tube according to Table 2. A 100 µL sample of whole blood was added to each tube. All samples were prepared in triplicate, and the suspension was briefly vortexed before putting the samples in a water bath (37 ◦C) for 5 h. The mixtures were centrifuged next at 3500 rpm for 5 min. The amount of hemoglobin was determined by UV–Vis spectroscopy (Thermo Scientific, Helios Omega UV-Vis) and measured with the reference wavelength of 525 nm.


**Table 2.** Determination of the biocompatibility of CuI@polymer composites. The exposure of RBCs was evaluated at different composites concentrations.
