Natural, chrome-tanned pig leather for lining details, provided by a local manufacturer, without a finish coating, and with a thickness of 0.89 mm, was used. The evaluated samples had dimensions of 50/50 mm and an average weight of 2.3 g.

Gelatin from Merck KGaA (Darmstadt, Germany); Glutaraldehyde (25% aqueous solution) from Sigma-Aldrich (Darmstadt, Germany); Zn(NO₃)₂·6H₂O (CAS:10,196-18-6) and NaOH were purchased from Sigma-Aldrich (Darmstadt, Germany). All solutions were prepared with distilled water.

Figure 1 schematically presents the stages of modification of leather samples.

All experiments were performed in a minimum of triplicate to ensure reproducibility and reliability of the results. Data are expressed as mean values ±standard error (SE).

Figure 2 shows micrographs of pure leather samples with a distinct fibrous structure. Figure 3 presents microscopic images of samples obtained by method 1 (Le_ZnO-1). It is evident that the leather sample is coated with cross-linked gelatin, with Zn-NPs impregnated into the hydrogel structure. The microscopic ivestigations of the these samples reveal that the nanoparticles ZnO have transitioned fom spherical to flower-like structures, indicating that the nucleation of ZnO crystal structures has commenced on the leather surface. Clustering and agglomeration of ZnO particles is observed at specific locations in the leather matrix and these particles change from nano-size (from 50 to 200 nm for single psrticles) to micro-size (above 500 nm and above 1 μm for clusters). This agglomeration of particles is likely to have a more significant effect due to the larger surface area of ZnO. In Figure 4 the modified leather samples Le_ZnO-2 show a different morphological structure. The ZnO particles again form a flower-like structure, they also form agglomerates, but are situated on the hydrogel’s surface, suggesting they display higher activity.

Some authors have proposed an explanation mechanism for the in situ growth of ZnO nanostructures (Souza, D. et al., 2018). Their research shows that at the beginning of the reaction in the sol-chemical solution, few ZnO nuclei and many growth units are present. When the fabrics are immersed in this solution for shorter residence times (0 h and 1 h), the ZnO nuclei immediately adhere to the fiber surface and the large amount of growth units around these nuclei leads to rapid growth of ZnO particles. And in our study, similar phenomena are most likely observed, i.e., for the nucleation and crystal formation processes.

The formation of the observed “flower-like structures” can be explained by heterogeneous nucleation of ZnO nanoparticles on gelatin matrices. Functional groups such as amino and carboxyl act as binding sites for Zn²⁺ions, initiating localized nucleation. Upon addition of NaOH, supersaturation drives crystal growth along specific wurtzite planes, while the organic matrix restricts orientation and promotes radial assembly. This confinement results in the characteristic flower-like morphology. Recent studies confirm similar mechanisms (Nichelson, A., et al., 2025) reported that surfactant-assisted and sonochemical synthesis promoted the self-assembly of ZnO into flower-petal morphologies, highlighting the role of organic matrices and solution conditions in directing growth (Wang, Y. et al., 2016).

Elemental analysis shows the presence of Zn in an amount reported on this area of 1 μm³. A Figure 5 present the elemental analysis of pure leather (Figure 5a), and modified leathers (Figures 5b, c). Elemental analysis of the modified leather samples shows that in sample Le_ZnO-1 (Figure 5b) there is about 11.6 wt. % of ZnO into the gelatin hydrogel, while in sample Le_ZnO-2 (Figure 5c) there is approximately 64.6 wt. % of per μm³ area. In sample Le_ZnO-2, the area is most likely coated with agglomerated ZnO particles, as well as SEM analyses prove the location of the zinc particles on the gelatin surface, showing the highest activity. The results fully correlate with the results for the antimicrobial activity.

On Figure 6 are presented spectrum of analized samples.

Absorption peaks (cm^-1) of the FTIR spectrum: 3308, 2,924, 2,852, 1,647, 1,558, 1,456, 1,234, 1,033 cm^-1 were assigned to–NH, –CH₃, =CH₂, –C=O, –NH, –C=O (in amide III) and C–N (in amine) groups. The collagen macromolecules in the leather and in the gelatin hydrogel contain polar groups in the side chains of their constituent amino acid residues, which are amino or amide groups and carboxyl groups, and can bind to metal atoms. The presence of Zn particles with the positive charge of zinc ions (Zn⁺) on the leather surface can interact electrostatically with the negative charge of the carboxylate residue (RCOO−) or with lone pair electrons of N atoms of amino acids (Sirelkhatim et al., 2015).

The peak at 852 cm^-1 confirms the formation of tetrahedral coordination of ZnO (Jain et al., 2020). This peak is present in our spectra and these peaks are most clearly expressed in the composite obtained by Method 3 (Le_ZnO-3).

The UV-VIS absorption spectral analysis of the modified leather material is presented on Figure 7. The bands confirm the conclusions drawn from FTIR analysis about the collagen structure of the gelatin hydrogel and the leather, about the bonds with the crosslinking agent. But most indicative is the appearance of a low peak at 363 nm, which is an evident of the formation of nano ZnO.

The antimicrobial activity of the tested leather samples was evaluated using the Kirby–Bauer disc diffusion method. The test bacterial strains used in this study included Pseudomonas aeruginosa (−), Bacillus subtilis (+), Escherichia coli (−) and Erysipelothrix rhysiopatiae (Gram+). Sterile Mueller-Hinton agar plates were inoculated with the standardized bacterial suspensions. The antimicrobial activity was determined after 24 h of incubation by measuring the diameter (mm) of the inhibition zones around each disc, which appeared as clear, bacteria-free areas.

As could be seen in Table 2 and Figure 8 the samples Le_ZnO-3 exhibits the highest bactericidal effect on Gram-positive spore-forming strain (B. subtillis).

After 24 h of aerobic cultivation of test microorganisms in termostate, presense or absence of inhibitory zones were observed. Performed experiments showed that Gram-positive (B. subtillis) bacteria is more sensitive to the samples with ZnO particles than tested Gram-negative bacteria (E. coli and P.aeruginosa). Our findings align with those reported by previous researchers, demonstrating similar trends in antibacterial performance. For example, in a study conducted by Emami-Karvani and Chehrazi (2011), it was shown that Gram-negative bacteria (E. coli) are more resistant to ZnO nanoparticles than Gram-positive bacteria (S. aureus). Similar results were also previously published by Reddy et al. (2007). Some scientists suggest that the higher sensitivity of Gram-positive bacteria may be related to differences in cell wall structure, cellular physiology, or metabolism. Additionally, variations in surface charge and cell membrane composition could influence the extent to which nanoparticles interact with cells (Girma et al., 2024).

Nanoparticles can damage cells in two ways: indirectly by generating reactive oxygen species (ROS), and directly by disrupting membrane integrity, penetrating the cytoplasm or interfering with essential processes such as proton pump activity.

The relatively low antibacterial activity observed in this study may be attributed to the limited diffusion of active ZnO particles from the impregnated leather samples, which restricts their availability at the site of bacterial interaction (Sukhodub et al., 2024; Zhang et al., 2008). Despite this, zinc oxide nanoparticles (ZnO-NPs) are well known for their strong antimicrobial properties, primarily due to their ability to generate (ROS) such as hydrogen peroxide, superoxide anions, and hydroxyl radicals, which cause oxidative stress and damage to bacterial cells. Additionally, the release of Zn²⁺ ions from ZnO-NPs interferes with bacterial metabolism and enzyme function, while their small size allows them to effectively penetrate bacterial membranes (Bouttier-Figueroa, et al., 2024). They can also interact with intracellular components, leading to structural or functional damage, including to DNA. The effect of nanoparticles depends on their physicochemical characteristics, such as size, shape, surface charge and hydrophobicity. This phenomenon provides a practical way to modulate and optimise their antimicrobial activity against different bacterial groups (Rahman et al., 2024).

The antifungal activity of the tested compounds was evaluated using the disc diffusion method as well. The test fungal strains used in this study included Aspergillus niger, Aspergillus fumigatus, and Candida albicans. Fungal strains were grown on Potato Dextrose Agar at 28 °C for 7 days.

Modified leather samples show excellent protective effects against fungal strains Aspergillus niger and Candida albicans compared to untreated leather samples, which did not exhibit any antifungal activity (Table 3; Figure 9).

Our results show that the three modified variants inhibit the growth of Candida albicans for 24 h, after which the fungal strain resumes its growth. In Aspergillus niger, inhibition of fungal growth is again observed for 24 h, followed by suppression of sporulation for up to 72 h (Table 3; Figure 9).

Some authors reported that chemically synthesized ZnO nanoparticles exhibit significant antifungal activity against Colletotrichum sp., Erythricium salmonicolor, and Cercospora coffeicola. Although their mechanism of action is not yet fully understood, it is suggested to involve both physical and chemical processes. The physical mechanisms depend on the size and surface properties of the nanoparticles and may disrupt ion and electron transport, membrane integrity, and protein function. The chemical mechanisms include the generation of reactive oxygen species (ROS), ion release through efflux, and direct interaction with the cell membrane, leading to oxidative stress and impaired permeability (Subba et al., 2024).

Upon treatment with ZnO-NPs, compact vacuole-like structures were observed within the hyphae, accompanied by a reduction in cytoplasmic volume. A layer of electron-dense particles formed around the cell wall, likely reflecting the adsorption of ZnO-NPs - a process frequently reported in nanoparticle–cell interactions. Alterations in the physicochemical properties of nanoparticles can lead to disrupted enzyme activity, damage to the cell membrane, impaired nutrient uptake, and genotoxic effects (Mosquera-Sanchez and Arciniegas-Grijalba, 2020).

The reduced antimicrobial activity is likely attributable to the limited diffusion of zinc ions resulting from the crosslinking of gelatin.

Zinc oxide nanoparticles (ZnO-NPs) show strong antibacterial potential against both Gram-positive and Gram-negative bacteria. The antibacterial activity of ZnO-NPs is still under investigation, but research suggests several possible mechanisms. These include direct interaction with bacterial cell surfaces, leading to membrane disruption; the generation of reactive oxygen species (ROS) on the ZnO-NPs surface; and the dissolution of particles, releasing free Zn²⁺ ions. The latter process disturbs cellular zinc ion balance, increasing ROS levels and ultimately causing cell damage and necrosis. It is likely that the antibacterial effects result from a combination of these pathways (Raha and Ahmaruzzaman, 2022). The antifungal activity of ZnO nanoparticles synthesized from E. crassipes extract was established against fungal strains isolated from building materials, including Aspergillus sp., Stachybotrys sp., Fusarium sp. and Phoma sp. Higher concentrations exhibited a stronger antifungal effect, probably due to the larger polydispersity index (PDI) and crystallite sizes.

Given the potential interaction of zinc nanoparticle coatings with human skin, it is essential to evaluate the toxicological implications of zinc ion release and comprehensively assess the safety profile of the proposed technology. Zinc oxide nanoparticles (ZnO-NPs) have been widely investigated due to their broad applicability and are generally considered safe at low concentrations, with GRAS (Generally Recognized As Safe) status granted for certain uses (Hou, J., et al., 2018). Nevertheless, their nanoscale dimensions and enhanced surface reactivity warrant careful toxicological and regulatory evaluation. While most studies to date report limited dermal penetration and low acute toxicity in humans, the long-term effects of chronic exposure, remain insufficiently understood. Since the early 2000s, the safety of nanomaterials has been a central focus of scientific and regulatory discourse, underscoring the need for comprehensive risk assessments that integrate (Fernández-Bertólez, et al., 2024; Casiano-Muñiz and Ortiz-Román, 2024). Nanomaterials fall under the scope of EU regulatory mechanisms (REACH- Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency) (https://echa.europa.eu/regulations/reach). In this context, future research should aim to bridge existing knowledge gaps by combining in vitro, in vivo, and computational approaches, thereby ensuring that the benefits of ZnO nanotechnology are realized without compromising human health or environmental safety.