2.2. Silver nanoparticle suspension

The silver nanoparticle solution named Argovit was kindly donated by Professor Dr. Vasily Burmistrov from the Scientific and Production Center Vector-Vita (Russia). Argovit is a preparation of highly dispersed AgNPs with an overall concentration of 200 mg/mL (20%) of PVP-coated AgNPs in water. The content of metallic silver in Argovit preparation is 12 mg/mL, stabilized with 188 mg/mL of PVP. AgNPs dilutions were calculated according to metallic silver content in Argovit preparation.

Several dissolutions of Argovit were prepared in distilled water to obtain the following concentrations according with the metallic silver content in each preparation: 0.0025, 0.005, 0.025, 0.05, 0.15 and 0.25%.

2.3. Preparation of AgNPs coated leather

Porcine leather was cut off to take pieces of 225 cm². Then, the square pieces were sprinkled with 7 mL of dissolutions of AgNPs to obtain the leather-coated with different concentrations of AgNPs as mentioned above (2.2). Subsequently, the leather was dried for 20 minutes at room temperature.

2.4. Quantification of metallic silver on AgNPs coated-leather

After drying, the leather coated with AgNPs was prepared to assess the concentration of metallic silver on the leather by inductively coupled plasma/atomic emission spectrometry (ICP-AES) (Inductively coupled plasma-atomic emission spectrometry, Model ICP-Iris intrepid according to EPA Method 6010). The leather quadrates were reduced to small pieces between 2 and 4 mm. Subsequently, 1 gr of sample was digested in 10 mL of HNO₃ concentrated at 140 °C for 15 minutes. After 3 hours, the samples were filtered and filled to 100 mL with distilled water, then the ICP-AES analysis was performed.

2.5. Texture analysis of the leather coated with AgNPs

Texture and softness change after the application of AgNPs were evaluated with a TA.XTPlus Texture Analyzer (Texture Technologies Corp., Hamilton, MA). The test was performed with a speed of 40 mm/s and load handling of 4 and 30 Kg, with a minimum speed of 0.01 mm/s, to determine the final smoothness of the leather. Initially, the electrode, which was used as a point of penetration in different areas of the leather, was calibrated, then, five repetitions per sample were performed, and softness was reported as mm of penetration.

2.6. Antibacterial and antifungal analysis

For microbiological assays, the leather was cut off to obtain 1 cm² pieces, then, the antimicrobial activity of AgNPs impregnated on the leather was tested against P. mendocina and P. syringae bacteria and the fungi T. mentagrophytes. These specimens were obtained from the American Type Culture Collection (Manassas, VA).

Microorganisms were cultured in a nutrient agar medium and maintained at 4 °C before their use. Fungi T. mentagrophytes was propagated under aseptic conditions in potato dextrose broth (PDB) growth media for 5 to 7 days (Figure 1A). Then, fungi mycelium was removed from the broth for its weighing (Figure 1B) and following deposition (one gram of mycelium) on the leather coated with AgNPs (Figure 1C) to investigate the spreading of fungus on the leather after 20 days of incubation at 37 °C. Thus, T. mentagrophytes susceptibility was determined through the final mycelium weight (after incubation time) on AgNPs-coated leather. A negative control for T. mentagrophytes (leather without treatment) was considered.

Figure 1 A) T. mentagrophytes mycelium broth; B) fungi mycelium taken from the broth for its weighing for experiment; C) deposition of fungi mycelium on pieces of leather coated with AgNPs. 

The spread of both genres of Pseudomonas was performed in a potato dextrose agar (PDA) medium at 37 °C for 24 hours. The antibacterial activity of leather coated with Argovit AgNPs was evaluated by a zone of inhibition (ZoI) test. Small pieces of leather coated with AgNPs (dried and not dried after coating) were placed on agar plates spread with each bacteria culture in potato dextrose agar (PDA). Next, each Petri plates (containing bacteria inoculum and AgNPs-coated leather) according to method (^{Hudzicki, 2009}) were dried, and the ZoI was measured to assess the bacterial sensitivity to leather coated with AgNPs.

3.1. Preparation of leather coated with AgNPs

In order to develop leather footwear material coated with AgNPs, we tried a simple method based on sprinkling AgNPs directly on the material and let it dried. Thus, the ICP-AES results showed that the metallic silver content in the leather was not proportional to the AgNPs concentration used (Table 1).

3.2. Effect of the AgNPs on the texture of leather

Textural changes on the leather were evaluated, after the application of AgNPs, through a texturometer. The data represent the penetration distance in the leather. Table 2, shows a decrease of leather softness when coated with AgNPs, and it was inversely proportional to the concentration of AgNPs.

After the analysis of the content of metallic AgNPs, it was evident that the sprinkling strategy used does not allow a homogenous impregnation of AgNPs in the leather. This method must be optimized to use a sharp sprinkler system. The textural properties were also affected by the application of AgNPs. However, it is possible that, after homogenous impregnation of AgNPs in the leather, its use for manufacturing footwear with suitable softness, not so different from the raw material, is possible.

3.3. Antibacterial and antifungal activity of AgNPs impregnated in leather

Leather sprinkled with AgNPs was used in the ZoI assays to investigate their antimicrobial properties against major bacterium and fungi present in footwear. The results from the ZoI assays showed that the antibacterial effect of AgNPs-coated leather against P. mendocina and P. syringae was evident when the content of silver metallic nanoparticles was 0.05%, higher concentrations of AgNPs did not increase the ZoI (Table 3).

The diameter of the ZoI was also measured for antibacterial activities against P. mendocina and P. syringae bacterium (Table 4). Horizontal and vertical diameters of the ZoI revealed that the inhibition of bacterial growth was evident at 0.05% of metallic silver content; also, a larger inhibition zone was detected when bacteria were exposed to leather coated with 0.25% of metallic AgNPs.

The antibacterial effect of AgNPs-coated leather could be affected by the difussion of AgNPs in a solid surface like the fibers of the leather, which could reduce their availability, and therefore, their activity as a microbicide. However, this was not a limitiation since both results of ZoI tests against P. mendocina and P. syringae revealed a clear inhibition of bacterial growth. The diameter of ZoI reached up to 2.6 cm after 24 hours of bacterial exposure to AgNPs-coated leather. When both bacterial genres were compared, P. syringae resulted to be more sensitive to the antimicrobial effect of AgNPs than P. mendocina, as demonstrated by the diameter size obtained from the ZoI test (Table 4).

In the case of the antifungal activity of the AgNPs-coated leather against T. mentagrophytes, it was observed that a silver metallic content of 0.5% decreased fungal growth; nevertheless, this amount of metallic silver was did not eliminate fungal growth, as it is shown by mycelium weight (Table 5).

The antifungal property of AgNPs against T. mentagrophytes was not demonstrated using the metallic AgNPs content herein tested. This can be explained by the diffusion limitation of silver ions through the fungal cell wall. However, after mycelium weighting of control and treated groups, it was evident that the higher concentrations of metallic AgNPs (0.05 and 0.25%) produced in an amount similar to the fungal dry weight. This may suggest that the activity of AgNPs-coated leather is fungistatic rather than fungicidal. The action mechanisms of silver ions with fungi have been reported by the interaction of Ag+ with the NH⁺ groups or with the positive-charged groups present in lysine residues in the mycelium. This may explain the electrostatic forces interaction favoring the fungistatic activity of Ag⁺ (^{Thakkar et al., 2010}). It has been reported that besides AgNPs, other metals like Au, Cd, and magnetite had an intracellular interaction with bacterial proteins producing bacterial growth inhibition. Nevertheless, in the case of fungi, this interaction is extracellular, limiting the ability of AgNPs to interact with vital intracellular mechanisms (^{Thakkar et al., 2010}).

Due to the complexity of fungal and bacterial infections in extreme cases of dermatoses, it is necessary to combine dermal, oral, and even laser treatment (^{Gupta & Paquet, 2014}). This proposal becomes a treatment complement to inhibit the growth and spread of dermatophytes, especially on the sole and forefoot. This work presents a technology that can be used for covering the lining of the footwear and act as added value to prevent and control bacterial infections in high-risk users.

To extend the study of the possibility of using AgNPs in the shoe industry, some further experiments are necessary: repetitive application of fungi on the same pieces of leather; influence of temperature, humidity and time of leather contact with fungi and comparison with spore germination under the same conditions, among others.