Enhancing antifungal properties of textiles through hydrogel composite layers incorporating copper nanoparticles

Materials

Polypropylene (PP) nonwoven fabrics were purchased from Hangeulbujigpo (Seoul, Republic of Korea). The PP fabric was a melt-blown nonwoven fabric with a basis weight of 40 g/m² and a thickness of 0.38 mm. These specifications were used to ensure reproducibility of the coating and antimicrobial evaluation experiments. Copper nitrate trihydrate (Cu(NO₃)₂·3H₂O), trisodium citrate dihydrate, ascorbic acid, sodium hydroxide, sodium carbonate, ammonium persulfate, N,N′-methylenebisacrylamide (MBAA), N,N,N′,N′-tetramethylethylenediamine (TEMED), sodium alginate, stannous chloride (SnCl₂), palladium chloride (PdCl₂), and other analytical-grade reagents were obtained from Sigma-Aldrich or Daejung Chemicals and used as received. Deionized (DI) water was used throughout all experiments. Luria-Bertani (LB) and Potato dextrose broth were purchased from BD Difco.

Synthesis of copper nanoparticles (CuNPs)

Copper nanoparticles were synthesized using an aqueous reduction method based on ascorbic acid as a reducing agent. ²⁷ Briefly, copper nitrate trihydrate 0.1 mol/L and trisodium citrate 0.2 mol/L were dissolved in DI water to prepare a copper precursor solution (CS). Then, a 1 M ascorbic acid solution (AA) were prepared. The pH of copper precursor solution (CS) and ascorbic acid solution (AA) were adjusted to pH 5, 7, 11 using sodium hydroxide to regulate particle size. Solutions were stirred together at room temperature to initiate reduction.

The reaction was allowed to proceed for 3 h, after which the resulting CuNPs were collected by centrifugation at 4000 rpm for 20 min. The nanoparticles were washed three times with DI water to remove residual reactants and dried at 50 °C. Particle size and morphology were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively.

Surface activation and CuNP coating of polypropylene fabrics

Because pristine PP fabrics possess chemically inert and hydrophobic surfaces, a surface activation process was performed prior to CuNP deposition. Before chemical activation, the PP fabrics were thermally flattened at 110 °C for 5 min to flatten the fabrics and immersed in an alkaline solution containing NaOH (8 g/L) and Na₂CO₃ (4 g/L) at 80°C for 20 min to increase surface wettability. Subsequently, the fabrics were treated sequentially with a sensitizing solution containing SnCl₂ (2 g/L)/HCl (0.65 mol/L) and an activation solution containing PdCl₂ (0.01 mol/L)/HCl (0.32 mol/L) to generate catalytic sites on the fiber surface.

Activated PP fabrics were immersed in the copper precursor solution (CS) for 1 h, followed by the addition of 10 min treatment of ascorbic acid solution (AA) to induce in situ reduction and deposition of CuNPs onto the fabric surface. This coating procedure was repeated up to five times to control surface coverage, and the resulting samples were designated as PP–CuNP–nX, where n indicates the number of coating cycles. After coating, the fabrics were thoroughly rinsed with DI water and dried at room temperature.

Preparation of hydrogel and Hydrogel–CuNP composite

The hydrogel matrix was prepared using acrylamide (121.932 g/L) as the primary monomer, MBAA (0.18 g/L) as a crosslinker, sodium alginate (4.068 g/L) as a secondary polymer, and ammonium persulfate (1.224 g/L) as an initiator. All components were dissolved in DI water under stirring to obtain a homogeneous precursor solution.

For hydrogel–CuNP composite samples, pre-synthesized CuNPs were dispersed into the hydrogel precursor solution at predefined concentrations, followed by sonication to ensure uniform dispersion. Gelation was initiated by ammonium persulfate/TEMED redox activation and allowed to proceed at room temperature until complete polymerization.

Hydrogel coating on CuNP-Coated PP fabrics

To fabricate hydrogel-coated CuNP fabrics, PP–CuNP samples were first immersed in an aqueous TEMED solution (11.12 µL/25 mL) to promote polymerization at the fabric surface. The samples were then transferred to the hydrogel precursor solution and allowed to react for 1 h, forming a thin hydrogel layer encapsulating the CuNP-coated fibers.

After coating, the fabrics were rinsed thoroughly with DI water to remove unreacted monomers and dried at ambient conditions. The resulting samples are denoted as HG–PP–CuNP.

Preparation of CuNP-Coated and hydrogel-modified PP samples

The antimicrobial effects of the experimental groups, as listed in Table 1, on fungi and bacteria were evaluated. CuNPs were synthesized by varying the pH to control the particle size (Table 1). The antifungal properties of CuNP-0.25s were evaluated according to the following CuNP concentrations: 15 mg/L (CuNP-0.25-PC1), 150 mg/L (CuNP-0.25-PC2), and 1500 mg/L (CuNP-0.25-PC3) (Table 1). CuNPs were coated one to five times on the PP fabric: PP-CuNP-1X to 5X (Table 1). HG-CuNPs were prepared at two CuNP concentrations of 1500 mg/L and 15000 mg/L: HG-CuNP-0.25-PC3 and HG-CuNP-0.25-PC4 (Table 1). Finally, HG-PP-CuNPs were prepared by coating the hydrogel films on PP-CuNP: HG-PP-CuNP-5X (Table 1).

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Table 1.

Experiment details for antimicrobial assay.

Description	Case name	Substrate	Particle size of CuNPs	Particle concentration	Number of coatings
Copper microparticles (CuMP) and nanoparticles (CuNP) with three different particle sizes	CuMP-2.0	None	2.0 μm	-	-
	CuMP-1.5		1.5 μm	-	-
	CuNP-0.25-PC1		0.25 μm	15 mg/L	-
	CuNP-0.25-PC2			150 mg/L
	CuNP-0.25-PC3			1500 mg/L
Polypropylene fabric (PP) coated with CuNP depending on the number of coatings (1X to 5X)	PP-CuNP-1X	Polypropylene fabric	0.25 μm	-	1X
	PP-CuNP-2X		0.25 μm	-	2X
	PP-CuNP-3X		0.25 μm	-	3X
	PP-CuNP-4X		0.25 μm	-	4X
	PP-CuNP-5X		0.25 μm	-	5X
Hydrogel (HG) containing CuNP with four different particle concentrations (PC3 to PC4)	HG-CuNP-PC3	Hydrogel	0.25 μm	1500 mg/L	-
	HG-CuNP-PC4		0.25 μm	15000 mg/L	-
PP-CuNP-5X coated with hydrogel (HG)	HG-PP-CuNP-5X	PP-CuNP-5X	0.25 μm	-	-

Antifungal activity evaluation

Antifungal activity was evaluated using Botryotinia fuckeliana (KCTC 6973) and Cladosporium cladosporioides (KCTC 16680). Escherichia coli (E. coli) (KCTC 1682) was used as a representative Gram-negative model for supplementary antibacterial assessment. Potato dextrose agar (PDA) media were prepared according to standard protocols, and fungal spores were inoculated at the center using cork bore (6.5 mm) or spore suspensions (5 × 10⁵cells/mL) uniformly spread depending on the experimental configuration.

CuNPs, PP–CuNP fabrics, hydrogel–CuNP composites, and hydrogel-coated PP–CuNP fabrics were placed on PDA plates and incubated at 25 °C. Antifungal performance was assessed by measuring inhibition zones and fungal growth areas. All antifungal experiments were performed in triplicate, and results are reported as mean ± standard deviation.

Antibacterial activity evaluation

Antibacterial activity was assessed using Escherichia coli (KCTC 1682). Bacterial cultures were grown in Luria–Bertani (LB) broth, adjusted to a standardized optical density, and spread uniformly onto LB agar plates using cotton swab. Test samples were placed on the agar surface and incubated overnight at 37 °C.

Inhibition zones were measured and calculated using Equation (2) to evaluate antibacterial efficacy. All antibacterial experiments were conducted in triplicate, and results are presented as mean ± standard deviation.

Characterizations

The size and morphology of CuNPs were observed using scanning electron microscopy (SEM). Particle size was evaluated with a particle size analyzer (90plus, Brookhaven) using the dynamic light scattering (DLS) technique. The mean particle size was determined from three measurements.

The antifungal and antibacterial properties were measured in terms of inhibition efficiency % and inhibition zone as shown in Equations (1) and (2), respectively. When CuNP-0.25 was added to potato dextrose agar (PDA) media and both fungi were inoculated in PDA media, the inhibition efficiency % was calculated as follows:

i n h i b i t i o n e f f i c i e n c y % = d c − d t d c × 100

(1)

i n h i b i t i o n z o n e ( c m ) = i n h i b i t i o n l e n g t h − s a m p l e w i d t h

(2)

Physical characteristics of CuNPs

The physical characteristics of CuNPs in three experimental cases, CuMP-2.0, CuMP-1.5, and CuNP-0.25, were evaluated, as shown in Table 1. Nearly spherical particles were observed in the SEM images (Figure 1(a)). The mean particle sizes varied according to the pH of the reactants (Figure 1(b)–(e)). As the pH increased, particle size decreased. The mean particle size was measured three times by DLS. The mean particle sizes were 2092.83 nm at pH 5, 1618.97 nm at pH 7, and 228.52 nm at pH 11 (Figure 1(e)). The sample name was determined based on DLS measurements.OPEN IN VIEWER

Physical characteristics of PP-CuNP and HG-PP-CuNP

The physical characteristics of PP-CuNPs produced in five different experimental cases, PP-CuNP-1X to PP-CuNP-5X, were evaluated, as listed in Table 1. SEM images were obtained by varying the number of coatings (Figure 2). As a result, when the number of coatings increased from 1X to 5X, more CuNPs were densely attached to the surface of the PP fabric, and CuNPs were more agglomerated (Figure 2). In addition, the color of PP-CuNP became darker with increasing number of coatings (Figure 2). The physical characteristics of the HG-PP-CuNPs were also identified (Figure 2). The TEMED and HG solutions were applied to PP-CuNP-5X to form hydrogel films on the PP-CuNP-5X fabric. As a result, the hydrogel film was effectively coated onto the fabric surface without significantly damaging the CuNP coating layers (Figure 2).OPEN IN VIEWER

HG-CuNP antibacterial properties

The antibacterial properties of HG-CuNP-PC4 (Table 1) were evaluated against the gram-negative bacterium E. coli (KCTC 1682). The control represents bacterial growth without HG-CuNP-PC4 (Figure 3(a) and (b)). The inhibition zone was calculated by using Equation (2). The results showed an average inhibition zone of 1.02 cm for HG-CuNP-PC4 (Figure 3(b)), indicating observable antibacterial activity against E. coli under the tested conditions.OPEN IN VIEWER

PP-CuNP and HG-PP-CuNP antibacterial properties

The antibacterial properties of PP-CuNP-5X and HG-PP-CuNP-5X (Table 1) were determined using the gram-negative bacterium E. coli (KCTC 1682). The bacterial solution and control represent bacterial growth without PP fabric and with untreated PP fabric, respectively (Figure 3(c)). The results showed an average inhibition zone of 0.4 cm for HG-PP-CuNP-5X (Figure 3(d)), indicating observable antibacterial activity against E. coli. In contrast, PP-CuNP-5X did not show a comparable level of antibacterial activity.

CuNP antifungal property

After CuNP-0.25 was mixed with PDA media at concentrations of 15 mg/L, 150 mg/L, and 1500 mg/L (Table 1), the antifungal properties against B. fuckeliana (KCTC 6973) and C. cladosporioides (KCTC 16680) were evaluated (Figure 4). The growth patterns of B. fuckeliana and C. cladosporioides were different. B. fuckeliana grew radially from the center starting from the center of the fungal inoculation, but C. cladosporioides grew irregularly as spores spread (Figure 4(c) and (f)). Because the growth patterns of the two fungi were different, the diameter of the fungal growth was measured for B. fuckeliana and the growth area was measured using ImageJ for C. cladosporioides. The inhibition efficiency % was calculated using Equation (1).OPEN IN VIEWER

Figure 4 shows the antifungal activity against B. fuckeliana and C. cladosporioides at different concentrations of CuNP (CuNP-0.25-PC1, CuNP-0.25-PC2, and CuNP-0.25-PC3 in Table 1). CuNP-0.25-PC3 effectively inhibited both B. fuckeliana and C. cladosporioides, with 65% inhibition efficacy against B. fuckeliana (Figure 4(b)) and nearly 100% inhibition efficacy against C. cladosporioides (Figure 4(e)). C. cladosporioides did not grow after inoculation with CuNP-0.25-PC3, whereas B. fuckeliana did grow; however, the growth morphology differed from that of the control (Figure 4(c) and (f)). The results indicate that CuNPs had a stronger antifungal effect against C. cladosporioides than against B. fuckeliana.

Additional antifungal experiments were conducted to confirm the antifungal properties of CuNP-0.25 not dispersed in the PDA media. CuNP-0.25 was deposited directly on the center of the PDA medium where the fungal suspensions were spread. As a result, the abnormal growth of the two fungi around CuNP-0.25, which turned yellow for B. fuckeliana and white for C. cladosporioides (Figure 5(a) and (b)), indicated that CuNPs have antifungal properties but are ineffective, unlike CuNPs dispersed in PDA media. This finding suggests that the dispersing media can enhance the antifungal effects of CuNPs.OPEN IN VIEWER

HG-CuNP antifungal properties

The antifungal properties of HG-CuNPs were evaluated using two concentrations of CuNPs inside the HG: HG-CuNP-PC3 and HG-CuNP-PC4 (Figure 5(c) and (d), Figure 6, and Table 1). As a result, HG-CuNP-PC3 did not show significant antifungal properties against either B. fuckeliana or C. cladosporioides (Figure 5(c) and (d)), although C. cladosporioides (Figure 5(d)) showed slight inhibition around HG-CuNP-PC3 on the second day (Figure 5(d)). Because C. cladosporioides is more sensitive to CuNPs than B. fuckeliana, the antifungal effects of HG-CuNP-PC3 (Figure 5(d)) and CuNP-0.25-PC3 (Figure 4) were similar. HG-CuNP-PC4 showed significant antifungal effects against both fungi (Figure 6(a) and (b)). For B. fuckeliana, the color of the mycelium changed from white to yellow (Figure 6(a)), indicating antifungal properties. However, for C. cladosporioides, an inhibition zone was observed (Figure 6(b)). Therefore, we measured the diameter of the inhibition zone of HG-CuNP-PC4. The average inhibition zone was 2.31 cm on the second day and 0.92 cm on the seventh day (Figure 6(b) and (c)). Although the diameter of the inhibition zone decreased as the fungi grew, we found that HG-CuNP-PC4 showed effective antifungal properties against both B. fuckeliana and C. cladosporioides (Figure 6(a) and (b)).OPEN IN VIEWER

PP-CuNP and HG-PP-CuNP antifungal properties

To evaluate the antifungal properties of PP-CuNP-5X and HG-PP-CuNP-5X (Table 1), the fabrics were placed on PDA media where B. fuckeliana and C. cladosporioides spore suspensions were spread evenly. The fungal spores and control in Figure 7(a) and (b) represent the fungal growth results without PP fabric and with untreated PP fabric, respectively. Notably, outstanding antifungal performance was obtained with HG-PP-CuNP-5X, which produced an average inhibition zone of 2.15 cm against C. cladosporioides on the third day (Figure 7(b) and (c)). Furthermore, PP-CuNP-5X showed fungal growth in the contact area with B. fuckeliana; however, in HG-PP-CuNP-5X, the fungal mycelium color changed from white to yellow (Figure 7). These results demonstrate that the antifungal properties against both fungi were significantly improved by the hydrogel film layer (Figure 7).OPEN IN VIEWER

Antimicrobial mechanisms of CuNPs

The antifungal activity of copper nanoparticles (CuNPs) has generally been attributed to ion-mediated mechanisms, particularly the release of Cu²⁺ ions and the induction of oxidative stress in fungal cells.^13,14 The present findings extend this understanding by demonstrating that antifungal efficacy is not determined solely by intrinsic nanoparticle properties such as size, composition, or surface density, but is strongly influenced by the surrounding physicochemical environment that regulates ion mobility and bioavailability.^20,22

When CuNPs were dispersed in aqueous PDA media, pronounced antifungal activity was observed against Botryotinia fuckeliana and Cladosporium cladosporioides. In contrast, identical CuNPs deposited on agar surfaces or immobilized on hydrophobic polypropylene (PP) fabrics exhibited markedly reduced antifungal effects. Because nanoparticle composition, size, and loading were maintained constant, these differences are best explained by variations in moisture availability and ion transport rather than intrinsic nanoparticle characteristics.^18,21

Role of hydrogel matrix in enhancing moisture-mediated ion transport

The introduction of hydrogel matrices substantially altered this behavior. By providing a moisture-rich microenvironment and continuous diffusion pathways, hydrogels facilitate ion mobility and prolong the local availability of biologically active copper species.^25,26 Both hydrogel-dispersed CuNPs and hydrogel-coated CuNP fabrics exhibited significantly enhanced antifungal performance compared with their dry counterparts, despite unchanged nanoparticle chemistry. These results indicate that environmental regulation functions as a primary activating factor in this system.

Although direct quantification of Cu²⁺ release was not performed in the present study, the enhanced antifungal activity observed under moisture-rich hydrogel-assisted conditions is consistent with previously reported ion-mediated antimicrobial mechanisms of copper-based nanoparticles.^13,15,22 Collectively, the findings demonstrate that antifungal functionality can be activated or suppressed through environmental engineering, without modifying nanoparticle composition or increasing loading.^24,27 Therefore, the present results should be interpreted as biological evidence that the hydrogel environment facilitates moisture-mediated antifungal activity, rather than as direct quantitative proof of Cu²⁺ release kinetics. Because the antibacterial assay was performed only against E. coli, the antibacterial result should not be interpreted as evidence of broad-spectrum antibacterial activity. Future studies will include Gram-positive bacteria, such as Staphylococcus aureus, to evaluate the broader antibacterial applicability of the hydrogel-coated CuNP textile system.

Comprehensive physicochemical characterization, including detailed ion-release kinetics and crystallographic analysis such as X-ray diffraction (XRD), would further strengthen the mechanistic interpretation. However, the primary objective of the present work was to elucidate the environmental activation of antifungal function rather than to optimize nanoparticle synthesis. Such analyses will be considered in future studies aimed at correlating structural parameters with moisture-mediated activation behavior.

Environmental engineering as a design strategy for antifungal textiles

From an application perspective, the present work should be regarded as a mechanistic proof-of-concept for antifungal textile design. While standardized washing durability, long-term stability, and regulatory safety assessments remain essential for practical deployment, the current findings demonstrate that antifungal performance of CuNP-coated textiles can be significantly enhanced through environmental regulation without increasing nanoparticle loading.^16,24 These results provide a conceptual framework for the development of antifungal textile systems in which moisture-mediated ion availability is treated as a primary design parameter in medical and agricultural applications. By distinguishing mechanistic clarification from subsequent durability and safety optimization, this approach enables a more systematic and rational development of functional antimicrobial textiles guided by a clearly defined governing mechanism.