Biochemical Behavior, Influence on Cell DNA Condition, and Microbiological Properties of Wool and Wool–Copper Materials

Abstract

The paper presents the study concerning the preparation and physio-chemical and biological properties of wool–copper (WO-Cu) materials obtained by the sputter deposition of copper onto the wool fibers. The WO-Cu material was subjected to physio-chemical and biological investigations. The physio-chemical investigations included the elemental analysis of materials (C, N, O, S, and Cu), their microscopic analysis, and surface properties analysis (specific surface area and total pore volume). The biological investigations consisted of the antimicrobial activity tests of the WO-Cu materials against colonies of Gram-positive (Staphylococcus aureus) bacteria, Gram-negative (Escherichia coli) bacteria, and fungal mold species (Chaetomium globosum). Biochemical–hematological tests included the evaluation of the activated partial thromboplastin time and pro-thrombin time. The tested wool–copper demonstrated the ability to interact with the DNA in a time-dependent manner. These interactions led to the DNA’s breaking and degradation. The antimicrobial and antifungal activities of the WO-Cu materials suggest a potential application as an antibacterial/antifungal material. Wool–copper materials may be also used as customized materials where the blood coagulation process could be well controlled through the appropriate copper content.

1. Introduction

Bleeding is a major cause of mortality and morbidity following both military and civilian trauma [1]. Since bleeding is simultaneously accompanied by bacteria invasion, hemostatic and antibacterial dressings may offer effective control as a part of the prehospital treatment.

Subsequent wound healing of the skin [2] presents a very complex biological process [3,4,5], and thus, its effective treatment has become one of the most important challenges for healthcare [6,7,8,9,10,11]. Therefore, various wound dressings have been tested [12,13,14,15,16], among them polymer composites exhibiting effective hemostatic [17,18,19] and antibacterial properties [20,21,22,23,24,25,26].

As the continuation of our research program directed towards antibacterial polymer–metal materials [27,28,29,30,31,32], we present here the study concerning the wool–copper (WO-Cu) material consisting of the keratin matrix [33,34,35] and copper—the transition element existing mainly in three oxidation states (0, +1, and +2) [36], with positive redox potentials (Cu⁺/Cu = 0.52 V; Cu²⁺/Cu = 0.38 V; Cu²⁺/Cu⁺ = 0.52 V) [37] and coordination numbers 2–4 [36], exhibiting a rich coordination chemistry [38,39,40] and a broad spectrum of diverse biological activities.

Copper’s therapeutic spectrum comprises by antipathogenic (antibacterial [41,42,43,44,45,46,47,48,49,50], antiviral [51,52,53,54,55,56,57,58,59,60], and antifungal [61,62,63,64,65,66,67,68,69,70] activity), anticancer [71,72,73,74,75,76,77,78,79,80], hemostatic [81,82,83,84,85,86,87,88,89,90], angiogenic [91,92,93,94,95,96,97,98,99,100], and osteogenic [96,101,102,103,104,105,106,107,108,109] applications.

In spite of the great medical potential of both components of the WO-Cu material, only a few reports concerning the biochemical investigations of this combination of materials have been published [110,111].

The tested WO-Cu materials were prepared using DC magnetron sputtering to deposit copper on the wool fabrics. The WO-Cu samples were characterized by a complex of physio-chemical and biological/biochemical tests. The main biological aim of the current study is to test the major hemolysis parameters of the wool–copper materials, namely activated partial thromboplastin time (APTT) and pro-thrombin time (PT) [112]. Plasmid relaxation assay is a common method to determine the potential of the tested probe to directly interact with the DNA. As a result of these interactions, it is possible to induce DNA breaks, resulting in a change in an electrophoretic mobility. Herein, we analyzed the potential of wool fibers and wool–copper materials to directly interact with the plasmid DNA.

2. Materials and Methods

All edited figures (Figures 1–4 and 9) in the manuscript were obtained using the BIOVIA Draw 2017 for Academics program.

2.1. Materials

2.1.1. Materials Used for Fabrication

The following materials were used to fabricate the investigated WO-Cu samples:

• Copper target from Testbourne Ltd. (Basingstoke, UK) with 99.99% purity;
• Woolen-adjacent fabric, manufactured by SDL ATLAS Textile Testing Solutions (Rock Hill, SC, USA). The characteristics of the woolen-adjacent fabric are presented in

  Table 1. Characteristics of the textile material.

  Parameter
  Surface mass	125 (±5) g/m2—determined in accordance with the standard ISO 3801:1977 [113], Textiles. Woven fabrics. Determination of mass per unit length and mass per unit area. International Organization for Standardization: Geneva, Switzerland, 1977.
  Textile material structure	Plain weave fabric.
  The pH of the aqueous extract	7.5 (±0.5)—determined in accordance with the method described in the standard ISO 3071:2020 [114], Textiles. Determination of the pH of aqueous extracts. International Organization for Standardization: Geneva, Switzerland, 2020.
  The residue after dissolution in methane dichloride	0.5 (±0.1)%—determined in accordance with the method described in the standard ISO 3074:2014 [115], Wool. Determination of dichloromethane-soluble matter in combed sliver. International Organization for Standardization: Geneva, Switzerland, 2014.
  Thickness	0.40 (±0.5) mm—determined in accordance with the method described in the standard ISO 5084:1996 [116], Textiles. Determination of thickness of textiles and textile products. International Organization for Standardization: Geneva, Switzerland, 1996.

  .

2.1.2. Microbiological Strains Used for Antimicrobial Activity Assesment

The following bacterial and fungal strains were purchased from Microbiologics (St. Cloud, MN, USA):

• Escherichia coli (ATCC 25922);
• Staphylococcus aureus (ATCC 6538);
• Chaetomium globosum (ATCC 6205).

2.2. Methods

2.2.1. Magnetron Sputtering Modification

The wool underwent copper coating using the magnetron sputtering method. The sputtering procedure parameters are presented in

Table 2. The sputtering procedure.

Method	Magnetron Sputtering
Equipment	DC magnetron sputtering system by P.P.H. Jolex s.c. (Czestochowa, Poland)
Target	Copper target of 99.99% purity from Testbourne Ltd. (Basingstoke, UK)
Distance between the target and substrate	15 cm
Deposition time	5, 10, and 15 min
Working atmosphere	Argon
Working pressure	2.4 × 10−3 mbar
Power discharge	0.5 kW
Power density	0.7 W/cm2

.

2.2.2. Wool—Copper Material Physio-Chemical Characterization

Atomic Absorption Spectrometry with Flame Excitation (FAAS)

The copper content of WO-Cu materials was assessed by prior sample mineralization (Figure 1), using single-module Magnum II microwave mineralizer from Ertec (Wroclaw, Poland) and subsequent FAAS determination, in a similar way as described earlier [27,28,29,30,31,32].

The FAAS determinations were performed using flame excitation with a Thermo Scientific Thermo Solar M6 (LabWrench, Midland, ON, Canada). The spectrometer was equipped with a 100 mm titanium burner, coded lamps with a single-element hollow cathode, background correction: D2 deuterium lamp.

The following Equation (1) [117] was used in order to calculate the total copper content (bulk copper content) in the WO-Cu material:

$$M=\frac{C\times V}{m}$$

(1)

where

• M—total copper content (bulk copper content) (mg/kg);
• C—metal concentration in the tested solution (mg/L);
• V—volume of the sample solution (mL);
• m—mass of the mineralized sample (g).

In order to evaluate the durability/washing fastness of the copper coating, the cooper concentration in WO-Cu materials after washing was determined using the FAAS method and compared with the copper concentration in the same sample before the washing procedure. The washing of the functionalized wool fabrics was performed in accordance with the EN ISO 105-C06:2010 standard [118]. For that purpose, a standard detergent was applied with a concentration equal to 4 g/L. The washing was carried out for 30 min at 40 °C.

Microscopic Analysis

The assessment of the surface morphology of the tested samples was carried out using the optical and scanning electron microscopy.

Optical microscopy investigations were performed with the use of a DM6 M microscope (Leica, Wetzlar, Germany). The applied magnification levels were equal to 150× and 2000×.

The SEM (scanning electron microscope) analysis of the WO-Cu materials was carried out with the Phenom ProX G6 scanning electron microscope (Thermo Fisher Scientific, Waltham, MA, USA). The SEM microscopic analysis was performed under low vacuum (60 Pa), and the energy of the probe beam was equal to 15 ekV. The back-scattered electron detector was used. The applied magnifications were 1000× and 8000×.

The performance of an EDS system was evaluated by measuring the resolution of a known set of elemental standards (Oxford Instruments, Abingdon, UK) in line with the ISO 15632:2012 [119].

Specific Surface Area and Total Pore Volume Analysis

In order to assess the specific surface area and total pore volume, the Brunauer–Emmet–Teller method (BET) was applied. The Autosorb-1 apparatus (Quantachrome Instruments, Boynton Beach, FL, USA) was used for the measurements with nitrogen (−195.8 °C) applied as a sorption agent. Before the analysis, the samples were dried for 24 h at 105 °C. After that, the samples were degassed at room temperature. For each measurement, ~2 g of a given sample was weighed and used.

2.2.3. Wool–Copper Material Biological Characterization

Antimicrobial Properties

The antibacterial and antifungal activity of WO-Cu materials was evaluated according to the EN ISO 20645:2006 [120] and EN 14119: 2003 [121] standards, respectively. The test were performed against E. coli, S. aureus (tryptic soya agar (TSA) was used), and C. globosum (complete mineral salt agar with glucose was used), analogously to our previous works [27,28,29,30,31,32]. The following concentrations of inoculum were used: E. coli = 1.3 × 10⁸ CFU/mL; S. aureus = 1.9 × 10⁸ CFU/mL; C. globosum = 2.5 × 10⁶ CFU/mL. The samples were 30 mm in diameter and were incubated for 24 h at 37 °C in the case of the E. coli and S. aureus and for 14 days at 29 °C in the case of the C. globosum (in accordance with the above-mentioned ISO standards).

Biochemical Properties

• Plasmid relaxation assay

The plasmid relaxation assay was performed similarly to the procedure of Juszczak et al. [122]. The pUC19 plasmid was isolated from the DH5α E. coli cells with Isolate II Plasmid Mini Kit (Meridian Bioscience, OH, USA) according to the manufacturer’s instruction. The isolated plasmid quantity and quality were determined by the A260/A280 ratio and gel electrophoresis, respectively. The native form of the pUC19 exists mainly in the supercoiled form (CCC), which is characterized by a relatively high electrophoretic mobility. The plasmid was digested with the restrictase PstI (New England Biolabs, Ipswich, MA, USA) to induce linear (L) form. Topological differences between the CCC and L forms of the plasmid account for their different electrophoretic mobility. The plasmid at 50 ng μL⁻¹ was incubated for 2 h and 24 h with WO and WO-Cu samples. Then, the samples were subjected to 1% agarose gel electrophoresis with ethidium bromide staining, visualization under the UV light (302 nm), scanning by a CCD camera, and analysis with the GeneTools 4.3.9.0 by Syngene (Cambridge, UK) software. During the electrophoresis, we also separated 4 μL of 1 kb DNA ladder (GeneRuler 1 kb DNA Ladder, Thermo Scientific, Waltham, MA, USA).

2.

Activated Partial Thromboplastin Time (aPTT) and Pro-thrombin Time (PT)

The standard human blood plasma lyophilizates, i.e., Dia-CONT I (Diagon Kft, Budapest, Hungary), were dissolved in 1 mL of a deionized water. A square piece of each sample (1 mg) was added to 200 µL of plasma, vortexed, and incubated for 15 min at 37 °C. For the aPTT measurements, the Dia-PTT (kaolin and cephalin) reagent (Diagon Kft, Budapest, Hungary) was resolved and 0.025 M CaCl₂ solution reagent (Diagon Kft, Budapest, Hungary) prepared according to the manufacturer’s instruction. The aPTT measurements were performed using a K-3002 OPTIC coagulometer (KSELMED^®, Grudziadz, Poland). For each sample, 50 µL of plasma sample and 50 µL of suspension of Dia-PTT were introduced into a measuring cuvette and placed in the thermostat of the coagulometer at 37 °C. The mixture was left for 3 min; then, the measurement was started by adding 50 µL of 0.025 M CaCl₂ solution to the cuvette.

For the PT assessment, cuvettes with 100 µL of plasma sample were incubated at 37 °C in the thermostat of the coagulometer for 2 min. Next, 100 µL of Dia-PT (Diagon Kft, Budapest, Hungary) was added, and the measurement was started. Dia-PT contained tissue thromboplastin from rabbit brain, calcium ions, and preservative and was shaken each time before adding in order to obtain a homogeneous suspension.

3. Results and Discussion

3.1. Magnetron Sputtering Modification

The wool samples were modified by the surface deposition of metallic copper using a direct current (DC) magnetron sputtering system. Wool fibers consisted of 95–98% proteins (about 80–85% keratin), lipids (0.1%), and minerals (0.5%) [34,123,124,125]. The structure of the keratin chain is given in Figure 2.

The average amino acidic content in wool is presented in

Table 3. Average amino acid composition of wool [121,122].

AA Abbr. (a,b/)	a/	Trp	His, Met	Ala, Ile, Lys, Phe, Tyr	Pro, Thr, Val	Gly	Asp c/, Leu, Ser	Arg, Cys d/	Glu e/
	b/	W	H, M	A, I, K, F, Y	P, T, V	G	D, L, S	C, R	E
AA cont. [%]		0–0.5 f/	0.6–0.9	3.5–5	5.8–6.5	4–8	7–8	7–10	13–16

Amino acids (AA) abbreviations: ^a,b/ tri- or one-letter codes. AA cont. (%), amino acid percentage content in wool. ^c/ Includes asparagine. ^d/ Calculated as cysteine, cystine, and cysteic aid. ^e/ Includes glutamine. ^f/ Dependent on applied conditions of keratin degradation.

[124,125,126].

The copper electron configuration of 1s2 2s2 2p6 3s2 3p6 3d10 4s1 4p0p 0p 0 enables its electro-donor as well electro-acceptor reactivity [36]. These reactivities of metallic copper were summarized and illustrated recently [32]. Physio-chemical investigations on the chemisorption of amino acids and peptide on copper phases have been the subject of numerous reports [127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165]. Their summed information is listed in Supplemental Information Table S1 and presented schematically in Figure 3.

The subsequent deposition of further copper layers on the wool surface–copper monolayer resulted in the supposed formation of Cu-Cu bonds [166,167,168,169,170,171,172,173] and is presented in Figure 4.

3.2. Physico-Chemical Characteristic of WO-Cu Materials

3.2.1. Atomic Absorption Spectrometry with Flame Excitation (FAAS)

The evaluation of the copper bulk concentration in wool–copper materials was performed with the use of the FAAS method and is presented in

Table 4. Results of the determination of copper bulk content in WO-Cu samples.

Sample	Sp.T. a/ (min.)	Copper Bulk Concentration		Sample Name e/ WO-Cu(SpT) (MBC)	Copper Bulk Concentration after Washing
		(mg/kg) b,c/	MBC d/ (mol/kg)		(mg/kg) b,c/	MBC d/ (mol/kg)
WO	-	-	-	WO	-	-
WO-Cu(5)	5	3510	0.055	WO-Cu(5)(0.06)	2990	0.05
WO-Cu(10)	10	9020	0.14	WO-Cu(10)(0.14)	7960	0.13
WO-Cu(15)	15	24,270	0.38	WO-Cu(15)(0.38)	20,820	0.33

^a/ Sp.T., sputtering deposition time (min). ^b/ The results calculated from Equation (1). ^c/ The results were measured in triplicate and are presented as a mean value with ± deviation equal to approximately 2%. ^d/ MBC, copper molal bulk concentration (M_Cu = 63 550 mg/mol). ^e/ WO-Cu^(SpT)( MBC), wool fabric/copper material after corresponding sputtering deposition time (Sp.T.) with copper molal bulk concentration (MBC).

.

The copper bulk content in the WO-Cu samples depends on the applied sputtering deposition time, and the correlation bewteen the copper bulk content and the deposition time is almost linear: 5 min process—3510 mg/kg (WO-Cu⁽⁵⁾(0.06)); 10 min process—9020 mg/kg (WO-Cu⁽¹⁰⁾(0.14)); 15 min process—24,270 mg/kg (WO-Cu⁽¹⁵⁾(0.38)). In addition, the distribution of copper in a WO-Cu material’s bulk after the magnetron sputtering process is uniform.

The copper bulk concentration in the investigated samples after the performed washing (in accordance with the EN ISO 105-C06:2010 standard [118]) is lower; however, the cooper coatings are still present on the surface of the wool fabric. For the WO-Cu⁽⁵⁾(0.06) sample, the copper bulk content lowered to ~85% of the original value, for the WO-Cu⁽¹⁰⁾(0.14) to ~88% of the initial bulk concentration, and for the sample WO-Cu⁽¹⁵⁾(0.38) to ~86% of the copper bulk content prior to the washing. Therefore, it may be concluded that the functionalized samples exhibit a satisfactory washing fastness.

3.2.2. Microscopic Analysis

Figure 5 presents the selected images of the surface morphology before (a,b) and after (c,d) the modification process, obtained using optical microscopy under different magnification levels. It can be clearly observed that the sample was successfully coated by copper (change in the sample color). The copper coating is present not only on the upper fibers of the fabric (placed on the surface) but also on the lower fibers (placed deeper within the fabric). Moreover, the coating is uniform; however, some small spot defects are visible under higher magnification. This may be caused by the presence of some impurities on the surface of the fibers.

Figure 6 shows the SEM images of the surface of the selected samples before (a,b) and after (c,d) the deposition of copper under different magnifications. It can be observed that both the upper and lower wool fibers are uniformly coated with copper. However, after the magnetron sputtering process, some spot defects are present within the coating (visible as black spots on the surface of the fibers). This is consistent with the results from the optical microscopy.

Both the optical microscopy (Figure 5b) and SEM (Figure 6a,b) observations revealed that the surface of the wool fibers before the magnetron sputtering process consisted of a network of overlapping scales. The untreated wool fibers exhibited a coronal-reticulate pattern of scales [174]. Scales are not only responsible for the characteristic morphological structure of the wool fibers but also play an important role by influencing their properties and by protecting them from damage [175]. From the performed microscopic observation, it may be concluded that the scales remained clearly visible after the magnetron sputter deposition of copper, and their pattern was not altered (Figure 5d and Figure 6c,d). However, the SEM images (Figure 6b,d) indicate that the surface of the scales is smoother after the copper deposition.

Figure 7 presents the EDS spectra acquired for all of the tested samples. The results of EDS analysis of the chemical composition of the WO sample and WO-Cu samples are listed in

Table 5. EDS analysis of the chemical composition of the WO sample and WO-Cu samples.

Sample Name	Element Symbol	Element Name	Atomic Conc. (%)	Weight Conc. (%)
WO	C	Carbon	44.583	39.000
	N	Nitrogen	35.083	35.800
	O	Oxygen	19.049	22.200
	S	Sulfur	1.284	3.000
	Cu
WO-Cu(5)(0.06)	C	Carbon	44.496	32.368
	N	Nitrogen	26.842	22.777
	O	Oxygen	21.029	20.380
	S	Sulfur	2.571	4.995
	Cu	Copper	5.062	19.481
WO-Cu(10)(0.14)	C	Carbon	53.169	36.300
	N	Nitrogen	13.937	11.100
	O	Oxygen	22.758	20.700
	S	Sulfur	2.633	4.800
	Cu	Copper	7.503	27.100
WO-Cu(15)(0.38)	C	Carbon	37.122	18.382
	N	Nitrogen	22.829	13.187
	O	Oxygen	17.264	11.389
	S	Sulfur	2.040	2.697
	Cu	Copper	20.745	54.346

Atomic Conc. (%), % as a function of the number of atoms; Weight Conc. (%), % as a function of weight of atoms.

.

In the case of the unmodified sample (WO), the elemental analysis showed the presence of carbon, nitrogen, oxygen, and sulfur, which are the main natural constituents of wool [176]. For the samples modified by the sputter deposition of copper, i.e., WO-Cu⁽⁵⁾(0.06), WO-Cu⁽¹⁰⁾(0.14), and WO-Cu⁽¹⁵⁾(0.38), an additional peak corresponding to copper was present.

The relative content of copper increases with the increasing sputter deposition time, which is consistent with the results obtained from the atomic absorption spectrometry. The EDS results confirmed the presence of copper coating on the surface of the wool fabric, and the growing relative content of copper is probably due to the higher thickness of the coating, which is in turn caused by the longer deposition time.

3.2.3. Specific Surface Area and Total Pore Volume Analysis

Table 6. The results of the BET analysis of the investigated samples.

Sample Name	Specific Surface Area (SSA)	Total Pore Volume (TPV)
	m2/g	cm3/g
WO	0.2653	7.527 × 10−4
WO-Cu(5)(0.06)	0.1929	7.394 × 10−4
WO-Cu(10)(0.14)	0.1854	7.254 × 10−4
WO-Cu(15)(0.38)	0.1783	7.103 × 10−4

The results were measured in duplicate and are presented as a mean value with ± deviation equal to approximately 2%.

presents the results of the BET analysis of the untreated wool fabric (sample WO) and wool–copper (WO-Cu) materials (WO-Cu^(SpT) (MBC)). It was revealed that the specific surface area of the wool fabric was lowered significantly from 0.2653 m²/g to 0.1783–0.1929 m²/g as a result of the magnetron sputtering deposition of copper. Similarly, the total pore volume also dropped from 7.527 × 10⁻⁴ cm³/g (for the unmodified wool) to 7.103–7.394 × 10⁻⁴ cm³/g (for the samples coated with copper). The observed effect is probably due to the decrease in the porosity of the wool fibers, which results from the fact that the deposited copper filled in the pores present on the fibers’ surface. This is also in agreement with the SEM observations, which showed that the surface of the copper-coated wool fibers is smother than the surface of the untreated wool fibers (Figure 5). The higher deposition time, i.e., the thicker the copper coating, the lower the specific surface area and the lower the total pore volume.

The N₂ adsorption–desorption isotherms acquired for the unmodified WO sample and WO-Cu^(SpT) (MBC) WO-Cu materials are presented in Figure 8. As it may be observed, the adsorption branch of the isotherm resembles the type II isotherm, which is S-shaped/sigmoid-shaped, according to the International Union of Pure and Applied Chemistry (IUPAC) classification [177,178,179]. This type of isotherms occurs in the case of the physisorption of gases on non- or mesoporous adsorbents and is associated with the monolayer formation followed by the multilayer adsorption [177,178,179]. Since for all of the samples, the observed point B, i.e., the so-called “knee” defined as the beginning of the middle almost linear section, is not so distinctive (a more gradual curvature occurs), it may be concluded that the monolayer formation is overlapped by the multilayer adsorption [178]. It may be observed that with increasing relative pressure, the multilayer adsorption takes place, resulting in an increasing isotherm slope [177]. For all samples, the appearance of the H3 hysteresis loop was observed. The occurrence of the hysteresis loop is a result of the capillary condensation associated with the presence of mesopores [178,179], and the H3 hysteresis loop is related to the slit-shaped pores [178,180].

3.3. Microbiological Properties

The antimicrobial properties of WO-Cu^(t)(MBC) materials were investigated by the disk diffusion method, using Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria and the representative fungus species (C. globosum) according to the EN ISO 20645:2006 [120] and EN 14119:2003 [121] standards. Microbiological test results are presented in

Table 7. Results of antimicrobial activity tests of WO-Cu^(SpT)(MBC) materials.

POLYM-Cu(MBc) Material	Average Inhibition Zone (mm)				LIT.
	Bacteria		Fungi
	E. coli	S. aureus.	C. globosum	A. niger
WO	0 a/				[111]
WO	0	0	0		This work b/
WO-Cu(0.06)	1	1	1
WO-Cu(0.14)	1	1	1
WO-Cu(0.38)	3	2	1
PET	0	0	0		[27]
PET-Cu(0.11)	1	1	3
PET-Cu(0.22)	2	1	3
PLA	0	0	0		[28]
PLA-Cu(0.16)	2	1	1
PLA-Cu(0.43)	2	1	3
PLA	0	0	0	0	[29]
PLA-ALG	0	0	0	0
PLA-ALG-Cu(+2)(0.21)	3	2	3	3
PLA-ALG-Cu(+2)(1.16)	3	4	3	3

^a/ The wool fabric showed antibacterial efficacy towards SA if interpreted according to the agar diffusion test as “no growth” under the textile sample [111]. ^b/ Concentration of inoculum (CFU/mL): E. coli, 1.9 × 10⁸; S. aureus, 1.9 × 10⁸; C. globosum, 2.1 × 10⁶. Polymers: WO, wool fiber; PET, poly(ethylene terephthalate); PLA, polylactic acid; ALG, alginate; Cu(MBc), copper molal bulk concentration; PLA-ALG-Cu⁽⁺²⁾, PLA-ALG complex with copper sulfate.

, and the images are shown in Figure 9.

The unmodified material (control/WO) exhibited strong growth of bacterial and fungal colonies, covering the entire surface of the samples placed on the Petri dishes (Figure 10a,c–e; Table 7). WO-Cu^(SpTt)(MBC) materials showed a good inhibitory effect against the E. coli and S. aureus bacteria and fungus species (C. globosum), expressed by the zones of inhibition (from 1 to 3 mm) and no visible growth on/under the samples (Figure 10b,d,f; Table 7). This assessment was made based on the criteria of the antimicrobial effect according to the EN ISO 20645:2006 [120]. The results obtained in accordance with the EN ISO 20645:2006 and EN 14119:2003 standards [120,121] confirmed the antimicrobial protection ability of the wool–copper (WO-Cu) materials (WO-Cu^(SpT)(MBC)) against various, representative types of microorganisms.

The average ZOI of the investigated copper-plated polymers (WO-Cu (this work), PET-Cu [27], and PLA-Cu [28]) (Table 7) are in the range of 1–4 mm due to the low solubility of copper in water [36] and PLA-ALG-Cu⁽⁺²⁾ due to formation of strong complexation of cupper ions by alginate [29].

The antibacterial activity of WO-Cu can be caused by released copper ions formed during the surface copper corrosion (Figure 9, path (1)) or copper-contact kill—the interaction of copper’s surface with bacteria membrane (Figure 9, path (2)).

Table 8. The literature’s in vitro antibacterial activity data of some human pathogenic bacteria by disc diffusion assay (ZOI, mm) of copper succinate (Cu(Succ)₂) and copper nanoparticles (CuNPS).

CuNPS Synth. a–i/	Pathogens								Ref.
	E. coli		S. aureus		B. subtilis		C. albicans
	ZOI	Conc./Amount	ZOI	Conc./Amount	ZOI	Conc./Amount	ZOI	Conc./Amount
CuSO4 → CuNPS a/	11	25 μg/mL	9	25 μg/mL					[185]
Cu(OAc)2 → CuNPS b/	10	25 μg/mL	10	25 μg/mL					[184]
			23	100 μg/mL
Cu(OAc)2 → CuNPS c/	2.6	0.21 mg/cm2	5.6	0.21 mg/cm2	2.8	0.21 mg/cm2	0	0.21 mg/cm2	[186]
Cu(Succ.)2 → CuNPS d/	14	70 μL	10	70 μL					[187]
Cu(Succ.)2	30	70 μL	34	70 μL
Cu(OAc)2 → CuNPS e/	25	50 μL	21	50 μL			23	50 μL	[188]
WO→
CuSO4 → CuO NPS f/	4	0.28 M	4.2	0.28					[189]
	5	0.56 M	5.5	0.56 M
CuCl2 → CuO NPS g/	26	50 μL	21	50 μL			23	50 μL	[190]
WO → WO-CuSO4 → WO-CuNPS h,i/ + WO-Cu2O NPS h,i/			21	50 μL					[191]
			18	50 μL

Copper derivatives: CuNPS, copper nanoparticles; CuO NPS, copper(II)oxide nanoparticles; Cu(OAc)₂, copper acetate; Cu(Succ)₂, copper succinate. CuNPS Syntheses: ^a/ Bio-reduction using Dryopteris manniana extract (terpenoids, phenolics, alkaloids, flavonoids, steroids, saponins, and tannins present in the D. manniana leaf extracts serve as effective reducing agents); ^b/ bio-reduction using Piper nigrum fruit extract (alkaloids, polyphenolic compounds, and terpenoids present in the Piper nigrum fruit extract serve as effective reducing agents and as stabilizing and capping agents); ^c/ reduction by ascorbic acid; ^d/ reduction by NaBH₄; ^e/ the reduction of copper acetate hydrate by modified polyol method; ^f/ biosynthesis of copper (II) oxide nanoparticles (CuO NPs) using CuSO₄, glucose, and Halomonas elongate; ^g/ biosynthesis of copper (II) oxide nanoparticles (CuO NPs) using CuCl₂ and apple peel extract under microwave (MW) irradiation; ^h,i/ two-stage process consisting of (1) chelation of CuSO₄ (0.5 M or 0.05 M) on wool and (2) subsequent reduction of CuSO₄ complexed by means of ascorbic acid (Cu²⁺:Ascorbic acid = 0.5:0.6) ^h/ or sodium borohydride (0.05 M:0.15 M) ^i/.

contains the comparison of the representative literature data on diffusion disc assays (ZOI; mm) of copper derivatives and copper nanoparticles.

These are higher ZOI data compared to those of POLYM-Cu due to the substantially greater solubility of copper ions, derivatives, and nanoparticles in aqueous media (e.g., [36,184,192]).

3.4. Biochemical Properties

3.4.1. Plasmid Relaxation Assay

We investigated the possibility for direct interaction of wool fabric and wool–copper (WO-Cu) materials with the DNA. For this purpose, we used the plasmid relaxation assay. The results obtained from the electrophoretic mobility shift analysis (EMSA) showed that the pUC19 plasmid, which we isolated from the DH5α E. coli cells, is presented in the supercoiled form (CCC). Overnight treatment at 37 °C with the restrictase PstI led to a linear form (L) of the plasmid. Incubation of the plasmid with the WO-Cu⁽⁵⁾(0.06), WO-Cu⁽¹⁰⁾(0.14), and WO-Cu⁽¹⁵⁾(0.38) samples showed a possibility of DNA adducts or breaks, which affected topological changes of the plasmid and led to the appearance of the OC form, whereas incubation with the WO sample did not affect the plasmid conformation, and the observed results were similar to the control (Figure 11A). This result demonstrates the possibility of the induction of the DNA single-strand breaks by the wool threads in vitro. After longer incubation (24 h), the L form of the plasmid also appeared for the WO sample. In the case of the WO-Cu⁽⁵⁾(0.06), WO-Cu⁽¹⁰⁾(0.14), and WO-Cu⁽¹⁵⁾(0.38) samples, we observed smears indicating significant DNA degradation (Figure 11B).

Copper was found as one of the first antimicrobial agents in history. In ancient Egypt, copper solutions were used to clean wounds and to purify water [193]. Studies indicate that copper, especially Cu²⁺, has the ability to interact with DNA [194,195]. Moreover, copper ions can induce Fenton-like reactions and geneate reactive oxygen species (ROS), especially highly reactive hydroxyl radicals [196]. Our results suggest the potential of wool–copper (WO-Cu) materials to directly interact with DNA. These findings could be associated with the antimocrobial activity against the tested species. However, it should be emphasized that the ability to interact with DNA does not imply genotoxic activity for humans. Even if copper from the WO-Cu materials had the opportunity to interact with the genomic DNA, humans have effective DNA repair systems, such as the base excision repair (BER), which remove DNA damage and maintain genome stability [197]. Additionally, the antioxidant systems present in cells protect them from reactive oxygen species [198].

3.4.2. Activated Partial Thromboplastin Time (aPTT) and Pro-Thrombin Time (PTT)

When a foreign body comes into contact with blood, plasma proteins are adsorbed on its surface, which leads to the initiation of the coagulation cascade through the activation of the coagulation factors and the adhesion and activation of platelets. As a result, a fibrin network is formed [199,200,201,202].

Over the last decades, the carboxyl group (-COOH) and the hydroxyl group (-OH) have found a wide application in the design and production of materials characterized by a high compatibility with blood. Carboxyl groups have the ability to bind calcium ions (Ca²⁺) present in the blood, contributing to the improvement of their antithrombotic properties. However, the presence of hydroxyl groups on the materials’ surface promotes their excellent hydrophilicity and leads to antibiofilm properties, which may contribute to the inhibition of the thrombogenesis process by limiting the formation of thrombi [203,204].

The blood compatibility of wool materials containing keratin is crucial, and therefore, the assessment of the blood coagulation properties is very important in the context of their use in the medical sector. Activation partial thromboplastin time (aPTT) and pro-thrombin time (PT) tests are commonly used to assess the antithrombogenicity of the biomaterials in vitro [205,206]. The results obtained for the investigated wool–copper materials are presented in Figure 12 (aPTT) and Figure 13 (PT).

The aPTT results, which are the measure of the intrinsic, i.e., contact, coagulation mechanism of blood plasma through thrombin formation and fibrin clot polymerization, show that the clotting times of the modified samples were longer compared to the unmodified sample and the control sample. Moreover, as the amount of the metallic copper on the wool surface increased, the aPTT also increased. In the study presented by Kang et al., the researchers observed that the activation partial thromboplastin time (aPTT) slightly increased with the increase in the number of the grafted carboxyl groups on the surface of the polyurethane (PU) and poly(ethylene terephthalate) (PET) membranes. This result suggests a possible relationship between the presence of the carboxyl groups and the blood coagulation process [207,208]. Therefore, the prolonged aPTT time can potentially also be attributed to the presence of carboxyl groups that constitute the wool structure.

At the same time, it was observed that the presence of amino groups (-NH₂) and carboxyl groups (-COOH) in wool did not have a significant impact on the PT (pro-thrombin time). Similarly, the surface concentration of metallic copper had no significant effect on the PT time. Therefore, it can be concluded that there was no disruption of the external factors of the blood coagulation pathway.

4. Conclusions

This paper investigated the biological potential of the new wool–copper materials (WO-Cu^(SpT)(MBC)) obtained by the sputter deposition of copper on the fabric made from wool fibers. The following conclusions were obtained:

• The wool samples (WO) were successful modified by the surface deposition of metallic copper using a direct current (DC) magnetron sputtering system;
• The obtained results confirmed the antimicrobial protection of wool–copper materials (WO-Cu^(SpT)(MBC)) against representative type of bacterial and fungal microorganisms according to EN ISO 20645:2006 and EN 14119:2003 standards [116,117];
• The investigated wool–copper materials have the ability to interact with bacterial DNA, resulting in breaks and changes in the conformation of the plasmid;
• The activated partial thromboplastin time (aPTT) of the modified samples (WO-Cu^(t)(MBC)) was longer compared to the unmodified wool sample (WO). As the amount of the metallic copper on the wool surface increased, the aPTT also increased. No change was observed in the case of the pro-thrombin time;
• The good antimicrobial and antifungal effect of the wool–copper materials (WO-Cu^(SpT)(MBC)) suggests a potential application as an antibacterial/antifungal material. Moreover, wool–copper (WO-Cu) materials may be applied as new customized materials, where the blood coagulation process could be well controlled by the copper concentration.