Preparation of Cotton–Zinc Composites by Magnetron Sputtering Metallization and Evaluation of their Antimicrobial Properties and Cytotoxicity

The aim of this investigation was to evaluate the biological properties of cotton–zinc composites. A coating of zinc (Zn) on a cotton fabric was successfully obtained by a DC magnetron sputtering system using a metallic Zn target (99.9%). The new composite was characterized using scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), UV/Vis transmittance, and atomic absorption spectrometry with flame excitation (FAAS). The composite was tested for microbial activity against colonies of Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and antifungal activity against Aspergillus niger and Chaetomium globosum fungal mold species as model microorganisms. Cytotoxicity screening of the tested modified material was carried out on BALB/3T3 clone mouse fibroblasts. The SEM/EDS and FAAS tests showed good uniformity of zinc content on a large surface of the composite. The conducted research showed the possibility of using the magnetron sputtering technique as a zero-waste method for producing antimicrobial textile composites.


Introduction
Cotton is a popular fabric based on cellulose [1,2]-the most widespread biopolymer on Earth [3][4][5]. Consequently, the physicochemical properties of a cotton fabric follow from the cellulose polymer and depend on its chain length, topology, and surface condition [6][7][8][9][10]. One of the many applications of cellulose is as medical textiles, mainly wound dressings with various functions and purposes. However, the hydrophilic nature of cotton and its products, due to their large specific surface area and hydrophilicity, provide an excellent environment for the development of pathogenic microorganisms [11][12][13]. Therefore, antibacterial pre-functionalization of cotton intended for medical use is a standard finishing step, usually based on equipping the cotton matrix with organic/inorganic antimicrobials [12,[14][15][16].

No
Preparation Antibacterial Activity Ref.
The antibacterial efficiency of COT-ZnO (Ec 67% and Sa 100% after 1 h incubation) resisted the intensive laundry regimes used in hospitals. [65]

No Preparation
Antibacterial Activity Ref.
[ The modification of textiles by means of magnetron sputtering does not require the use of any chemicals, can be achieved in one process cycle in a single industrial installation, and does not involve the emission of toxic substances to the environment or the production of pollutants [53,[74][75][76][77][78][79][80]. Therefore, this method can be considered eco-friendly and zero-waste.
Only a few studies have been devoted to other zinc compounds such as zinc sulfide (ZnS) deposited on polyethylene terephthalate (PET) [102] and gallium-doped zinc oxide (GZO) deposited on a transparent flexible substrate based on cellulose derivatives [103]. Only in two studies was metallic zinc used for surface functionalization of polymer nanofibers, namely, for polyamide [104], and for polyethylene (PE) and polytetrafluoroethylene (PTFE) [105].
As part of our research program dedicated to biologically active functionalized phosphonates [106,107] and biofunctionalization of textile materials [108][109][110][111][112][113][114], we present the preparation and physicochemical and biological properties of the COT-Zn polymer hybrid. The aim of this work was to modify the surface of cotton fabric with zinc using the DC (direct current) magnetron sputtering method to produce a new antimicrobial, multifunctional composite material.

Magnetron Sputtering
The medical cotton fabric (COT) was modified using a DC (Direct Current) magnetron sputtering system produced by P.P.H. Jolex s.c. (Czestochowa, Poland) and a zinc target. The distance between the target and the substrate was 15 cm. The deposition of coatings was carried out in the atmosphere of argon. The following parameters were used for the modification: discharge power 700 W, with the resulting power density 0.72 W/cm 2 and working pressure 2.0 × 10 −3 mbar. In order to differentiate the zinc content (Zn (0) + Zn (2+) ) in the composites COT-Zn, three different deposition variants were used, i.e., 5 min one-sided (sample name: COT-Zn-5(1 s)), 10 min one-sided (COT-Zn-10(1 s)), and 10 min two-sided (COT-Zn-10(2 s)).

SEM/EDS-Scanning Electron Microscopy/Energy-Dispersive X-ray Spectroscopy
The microscopic structure was examined using a HITACHI S-4700 scanning electron microscope (Tokyo, Japan) equipped with a Thermo NORAN EDS X-ray microanalyzer (Waltham, MA, USA). The topography analysis of the tested samples was carried out in low vacuum at a beam energy of 10 kV and magnifications of 400×, 1600×, and 3000×. The study was conducted under low vacuum in the presence of steam. Water vapor dissipates excess charge, making it possible to image nonconductive materials without coating the surface with gold.

Atomic Absorption Spectrometry with Flame Excitation-FAAS
Zinc content in the composite samples was determined using the Thermo Scientific Thermo Solar M6 atomic absorption spectrometer (Waltham, MA, USA) equipped with a 100 mm titanium burner, coded lamps with a single-element hollow cathode, and a D2 deuterium lamp for background correction. The sample was prepared using a singlemodule Magnum II microwave mineralizer from Ertec (Wroclaw, Poland).
The total zinc content of the sample M (mg/kg) was calculated according to the following formula [115]: where C is the Zn concentration in the tested solution (mg/L), m is the mass of the mineralized sample (g), and V is the volume of the sample solution (mL).

Biological Experiments Antibacterial Activity
The antibacterial activity of COT-Zn composites was tested by the agar (Mueller Hinton medium) diffusion method (PN-EN ISO 20,645:2006), on colonies of Gram-negative (E. coli; ATCC 25,922) and Gram-positive (S. aureus; ATCC 6538) bacteria [116]. The test was initiated by pouring each agar into sterilized Petri dishes and allowing it to solidify. The surfaces of agar media were inoculated with the overnight broth cultures of bacteria (ATCC 25,922: 1.5 × 10 8 CFU/mL, ATCC 6538: 2.5 × 10 8 CFU/mL). Samples of sterile COT-Zn discs and a control, unmodified sample (10 mm) were placed on the inoculated agar and incubated at 38 • C for 24 h. The diameter of a clear zone around the sample was measured as an indication of inhibition of the microbial species. All tests were carried out in duplicate.

Antifungal Activity
The antifungal activity of the COT-Zn composites was tested according to PN-EN 14,119:2005 against A. niger (ATCC 6275) and C. globosum (ATCC 6205) [117]. Specimens of the tested material were placed on agar plates; the samples of sterile modified COT-Zn discs (20 mm) and the control, unmodified sample were placed on inoculated agar (pH:6.2) and incubated at 30 • C for 14 days. The agar was inoculated with the selected fungus (ATCC 6275: CFU/mL = 3.5 × 10 6 , ATCC 6205: CFU/mL = 3.0 × 10 6 ). The level of antifungal activity was assessed by examining the extent of fungal growth: in the contact zone between the agar and the specimen, on the surface of specimens, and, if present, the extent of the inhibition zone around the specimen. All tests were carried out in duplicate.

Extract Preparation and Cell Treatment
Extracts of test materials (COT and COT-Zn composites) were prepared according to EN ISO 10993-12-2012 [118] using the exposure medium, i.e., cDMEM with a lower (5%) concentration of FBS, in order to avoid the masking of toxicity by the protective effect of the serum. Test materials were autoclaved (120 • C, 20 min) and left at 70 • C for 5 h to dry. Next, the materials were immersed in the exposure medium using the extraction ratio of 0.1 g of the material to 1 mL of the exposure medium, i.e., the predetermined additional volume of the exposure medium needed for the maximum soaking of the test material. Extraction was performed in shaken vials that were incubated at 37 • C for 24 h. The pH values of extracts (ca. 8) were adjusted to 7.4 using 1 N HCl (POCH, Gliwice, Polska). Extracts, the preparations, and sample abbreviations are summarized in Table 2.

COT COT-Zn /b E-COT (100%) E-COT-Zn (100%) Extraction /c Dilution
0.25 mL 0.75 mL a/ cDMEM with a lower (5%) concentration of FBS; b/ COT-Zn-10 (1 s) was used for cytotoxicity assays; c/ 1 mL of the exposure medium + the predetermined additional volume of the exposure medium needed for the maximum soaking of the test material (ni). Cells were exposed to the extracts at selected concentrations (100% and 50% for unmodified cotton; 100%, 50%, and 25% for modified cotton) for 24 h, i.e., after 24 h adherence of cells, the supernatant above the cells was aspirated and replaced with 100 µL of an appropriately concentrated extract or control solution (negative control, NC-exposure medium treated in the same way as extracts; positive control-SDS (Sigma-Aldrich, Saint Louis, MO, USA) in the concentration range 0-150 µg/mL). During the experiment cells were examined with the Olympus IX70 (Tokyo, Japan) inverted microscope. Each sample was tested in triplicate per experiment, and three independent experiments were performed.

The Neutral Red Uptake (NRU) Assay
After the 24 h incubation of BALB/3T3 clone A31 cells with the extracts, the medium was gently aspirated, and the wells were washed with 150 µL of Dulbecco's phosphatebuffered saline (PBS) with Ca 2+ and Mg 2+ ions (BI, Kibbutz Beit-Haemek, Israel). Then, 100 µL of NR (Sigma-Aldrich, Saint Louis, MO, USA) solution (50 µg/mL, prepared in culture medium) was added to each well, and the plates were incubated further for 3 h (37 • C; 5% CO 2 ). Afterward, the NR solution was removed, the wells were washed with 150 µL of PBS, and 150 µL of desorbing solution [1% glacial acetic acid (POCH, Gliwice, Polska), 50% ethanol (POCH, Gliwice, Polska), and 49% deionized water] was added to each well. Absorbance was read at 540 nm using a Multiskan™ GO spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Results were expressed as the percentage cell survival (OD of exposed vs. OD of control unexposed cells).

SEM/EDS-Scanning Electron Microscopy/Energy-Dispersive X-ray Spectroscopy
The analysis of changes in the morphological structure of fibers in cotton fabric under zinc modification was carried out using scanning electron microscopy. Figure 1 shows SEM images of the samples before and after magnetron sputtering, at magnifications of 400×, 1600×, and 3000×.
According to the image analysis, it can be noted that the fibers in cotton fabric were characterized by a rather smooth surface with characteristic parallel ridges and grooves. The fibers in cotton fabric with a magnetron-sputtered zinc layer were characterized by a rougher surface with visible ridges and grooves in the fibers. The zinc coating exhibited a regular distribution of the applied modifier particles.
In order to confirm and verify the uniformity of the zinc coating of the fibers, an energy-dispersive X-ray spectroscopy (EDS) study was performed, which provided a chemical analysis of the tested fabric and its elemental composition. The EDS surface analysis ( Figure 2) in the form of individual "mapping" and the quantitative spot analysis showed the content of characteristic elements in the cotton fiber surface.
The study indicated a uniform distribution of zinc on the surface-modified fibers. The black areas visible in the images were the spaces between the fibers in the yarn (threedimensional structure). Due to the dense and uniform zinc coating of the fibers, carbon became less visible, which was confirmed by the quantitative analysis of the content of elements on the fiber surface (Table 3) and EDS punctate analysis diagram ( Figure 2).  According to the image analysis, it can be noted that the fibers in cotton fabric were characterized by a rather smooth surface with characteristic parallel ridges and grooves. The fibers in cotton fabric with a magnetron-sputtered zinc layer were characterized by a rougher surface with visible ridges and grooves in the fibers. The zinc coating exhibited a regular distribution of the applied modifier particles.
In order to confirm and verify the uniformity of the zinc coating of the fibers, an energy-dispersive X-ray spectroscopy (EDS) study was performed, which provided a chemical analysis of the tested fabric and its elemental composition. The EDS surface analysis (Figure 2) in the form of individual "mapping" and the quantitative spot analysis showed the content of characteristic elements in the cotton fiber surface. According to the image analysis, it can be noted that the fibers in cotton fabric were characterized by a rather smooth surface with characteristic parallel ridges and grooves. The fibers in cotton fabric with a magnetron-sputtered zinc layer were characterized by a rougher surface with visible ridges and grooves in the fibers. The zinc coating exhibited a regular distribution of the applied modifier particles.
In order to confirm and verify the uniformity of the zinc coating of the fibers, an energy-dispersive X-ray spectroscopy (EDS) study was performed, which provided a chemical analysis of the tested fabric and its elemental composition. The EDS surface analysis ( Figure 2) in the form of individual "mapping" and the quantitative spot analysis showed the content of characteristic elements in the cotton fiber surface. The study indicated a uniform distribution of zinc on the surface-modified fibers. The black areas visible in the images were the spaces between the fibers in the yarn (threedimensional structure). Due to the dense and uniform zinc coating of the fibers, carbon became less visible, which was confirmed by the quantitative analysis of the content of elements on the fiber surface (Table 3) and EDS punctate analysis diagram ( Figure 2). The nature of the so-called zinc-cellulose interface matter (Zn-cell-OH) still remains unclear, since the zinc atom is reactive, and cellulose possesses reactive hydroxyl functions. It is well documented that zinc reacts with alcohols with the temporary formation of zinc alcoholate intermediates, which, in an aqueous environment, rearrange into zinc oxide [119][120][121][122].
The reactions of zinc with alcohols presumably proceeded in accordance with Figure  3. The nature of the so-called zinc-cellulose interface matter (Zn-cell-OH) still remains unclear, since the zinc atom is reactive, and cellulose possesses reactive hydroxyl functions. It is well documented that zinc reacts with alcohols with the temporary formation of zinc alcoholate intermediates, which, in an aqueous environment, rearrange into zinc oxide [119][120][121][122].
The reactions of zinc with alcohols presumably proceeded in accordance with Figure 3.  Subsequently, zinc atoms of a metallic monolayer deposited on the cellulose surface reacted during deposition with cellulose, forming corresponding alcoholates (Figure 4), which could subsequent hydrolyze to cellulose and ZnO. The following layers of deposited zinc atoms were attached to the lower zinc layer, forming appropriate zinc multilayers. The zinc atoms of the upper layer were oxidized to Subsequently, zinc atoms of a metallic monolayer deposited on the cellulose surface reacted during deposition with cellulose, forming corresponding alcoholates (Figure 4), which could subsequent hydrolyze to cellulose and ZnO.  Subsequently, zinc atoms of a metallic monolayer deposited on the cellulose surface reacted during deposition with cellulose, forming corresponding alcoholates (Figure 4), which could subsequent hydrolyze to cellulose and ZnO. The following layers of deposited zinc atoms were attached to the lower zinc layer, forming appropriate zinc multilayers. The zinc atoms of the upper layer were oxidized to ZnO, by reaction of zinc atoms with oxygen or water. This mechanism was partly con- The following layers of deposited zinc atoms were attached to the lower zinc layer, forming appropriate zinc multilayers. The zinc atoms of the upper layer were oxidized to ZnO, by reaction of zinc atoms with oxygen or water. This mechanism was partly confirmed by EDS test (Table 3). The results for COT were as follows: C (40.0%), O (60.0%); the results for COT-Zn-10 (1 s) were as follows: C (3.2%), O (17.7%), Zn (79.7%). This suggests that carbons of the cellulose skeleton were covered by O-Zn moieties. Cellulosederived oxygens were masked by zinc atoms; hence, the oxygen revealed in the EDS test was presumably derived from the passivated layer, i.e., from ZnO. The ratio of Zn:O ≈ 4.5:1 (Zn 1.2 O 1.1 ) suggests a nearly quantitative character of passivation. It is worth adding that the formation of ZnO during sputtering with pure zinc has been described in a few papers using pure Zn target in an argon-oxygen [123][124][125] or oxygen atmosphere [126]. Another example of the formation of a metal-polymer interface during Zn sputtering onto PE, PTFE, and PI surfaces was described by Pertsin and Volkov [105].

Atomic Absorption Spectrometry with Flame Excitation-FAAS
The determination of zinc content in COT-Zn composite samples was assessed by the FAAS method, and the results are presented in Table 4.

Antibacterial Activity
The COT-Zn composites were tested in vitro for antimicrobial activity against Grampositive S. aureus and Gram-negative E. coli. Results of the tests are illustrated in Figures  5 and 6.   A comparison of these results (ZID) with representative data from the literature is given in Table 5.  A comparison of these results (ZID) with representative data from the literature is given in Table 5.  The literature data related to the antibacterial activity of zinc-based composites cannot be used as direct comparative data, due to the difference in the applied test methods. The presented results (Table 5) indicate that, independently of the type of base material used for modification with Zn and zinc compounds, and irrespective of the applied test method to evaluate antimicrobial activity, the expected result was achieved. Results of these tests demonstrate the antimicrobial protection against various bacterial microorganisms of COT-Zn-10 (1 s/2 s) composites for E. coli and S. aureus (Table 5), expressed by the visible zones of inhibition of bacterial growth on the Petri dishes (Figures 5b and 6b).

Antifungal Activity
The results of the antifungal activity tests (ZID) in accordance with PN-EN 14119:2005 against colonies of A. niger (ATCC 6275) and C. globosum (ATCC 6205) for the cotton sample and COT-Zn composites are illustrated in Figures 7 and 8 and presented in Table 6 (comparison with representative data from the literature).
these tests demonstrate the antimicrobial protection against various bacterial microorganisms of COT-Zn-10 (1 s/2 s) composites for E. coli and S. aureus (Table 5), expressed by the visible zones of inhibition of bacterial growth on the Petri dishes (Figures 5b and 6b).

Antifungal Activity
The results of the antifungal activity tests (ZID) in accordance with PN-EN 14119:2005 against colonies of A. niger (ATCC 6275) and C. globosum (ATCC 6205) for the cotton sample and COT-Zn composites are illustrated in Figures 7 and 8 and presented in Table 6 (comparison with representative data from the literature).       [117]. /e Dependent on the green method applied. /f Dependent on the green method applied. f Cotton patch (5 × 5 cm 2 ) saturated for 5 min in 10% aqueous solution of ZnO and/or MnO 2 /ZnO. /g Concentration of inoculum: A. niger: CFU/mL = 3.5 × 10 6 , C. globusum: CFU/mL = 3.0 × 10 6 . /h Visible growth on sample surface.
As anticipated, the unmodified sample (100% cotton) did not inhibit the growth of A. niger or C. globosum, as expressed by the strong visible fungal growth covering the entire surface of the COT samples (Figures 7a and 8a). Antifungal activity and protection against A. niger and C. globosum were demonstrated by COT-Zn-10 (1 s/2 s) samples modified by magnetron sputtering metallization. The results revealed visible zones of fungal growth inhibition in Petri dishes (Figures 7b and 8b), with no fungal growth on the surface of the composites.
The zinc oxide impact on bacteria or fungi depends on its morphology (particle size and shape), concentration, exposure time, pH, etc. [148]. This is illustrated by the corresponding ZID values summarized in Tables 5 and 6. Generally, ZnO NPs had ZID values over 10 mm, revealing the dependence of antimicrobial activity on zinc concentration (ZID = f(ZnX)). In a few cases, the ZnO NP ZIDs were comparable with the ZIDs of precursory zinc salts (ZnCl 2 , ZnSO 4 , or Zn(OAc) 2 ). Since zinc metal presents lower solubility than ZnO (1 µg/L vs. 3.6 µg/L) [149] and much lower solubility than ZnO NPs [150][151][152][153], the metallic zinc in COT-Zn composites presented lower solubility in inoculum media than ZnO NPs and, consequently, a lower ZID. Therefore, the process of releasing antimicrobial zinc ions from COT-Zn (COT-Zn→COT-Zn(OH) 2 →COT + Zn 2+ ) is much longer, and these composites should preserve their antimicrobial nature/characteristics for much longer.

Cytotoxicity
Cytotoxicity assays [154][155][156] are crucial in biomaterial science with respect to the therapeutic potential of nanocomposites and nanostructures, as well as the legal and normative requirements for medical devices and biomaterials [157][158][159][160][161][162][163][164]. These include an array of methods, mostly fluorescent and colorimetric, providing quantitative estimations of the number of viable cells in a culture [154,155]. The neutral red uptake (NRU) assay is one of the most used cytotoxicity tests in biomedical and environmental applications [165] and is based on the natural tendency of neutral red dye to incorporate to living cell lysozymes.
As cells begin to die (a loss of cell viability), their ability to bind neutral red diminishes (decrease in neutral red uptake), corresponding to colorimetric changes.

Cytotoxicity Experiments
Macroscopic observations of the extracts showed no changes in transparency for unmodified cotton, while the modified cotton had a visibly darker color, brightening after sedimentation, and leaving a black residue (Figure 9A.). Microscopic analysis of the exposed cells revealed fibers present in all extract samples and small fragments present only in the modified cotton extracts ( Figure 9B). . Light microscopy images of BALB/3T3 clone A31 cells exposed for 24 h to negative control (a,d), 100% unmodified cotton extract (b,e), and 100% extract from COT-Zn sample (c,f), before and after incubation with NR, respectively.
The results of the NRU assay showed no decrease in viability of fibroblasts exposed to the unmodified cotton extracts at both tested concentrations. COT-Zn extracts significantly reduced cell viability, causing almost 100% cell death, irrespective of the extract concentration ( Figure 10). Treatment of cells with SDS resulted in a concentration-dependent decrease in cell viability ( Figure 11). . Light microscopy images of BALB/3T3 clone A31 cells exposed for 24 h to negative control (a,d), 100% unmodified cotton extract (b,e), and 100% extract from COT-Zn sample (c,f), before and after incubation with NR, respectively.
The results of the NRU assay showed no decrease in viability of fibroblasts exposed to the unmodified cotton extracts at both tested concentrations. COT-Zn extracts significantly reduced cell viability, causing almost 100% cell death, irrespective of the extract concentration ( Figure 10). Treatment of cells with SDS resulted in a concentration-dependent decrease in cell viability ( Figure 11).  . Viability of BALB/3T3 clone A31 cells exposed for 24 h to SDS (positive control), assayed with NRU test. Viability presented as a percentage of the negative control (NC; culture medium with vehicle, i.e., 2% H2O in culture medium).
Zinc compounds also reveal strong anticancer activity (e.g. [136,[179][180][181]) reflected additionally by nearly 2400 document results on Anticancer Zinc [182] and 1-700 documents results on Anticancer Activity of Zinc abstracted in the Scopus Base [183]. Therefore the COT-Zn composites with cytotoxic activity against BALB/3T3 clone A31 cells should also reveal anticancer character. These investigations will be continued.   . Viability of BALB/3T3 clone A31 cells exposed for 24 h to SDS (positive control), assayed with NRU test. Viability presented as a percentage of the negative control (NC; culture medium with vehicle, i.e., 2% H2O in culture medium).
Zinc compounds also reveal strong anticancer activity (e.g. [136,[179][180][181]) reflected additionally by nearly 2400 document results on Anticancer Zinc [182] and 1-700 documents results on Anticancer Activity of Zinc abstracted in the Scopus Base [183]. Therefore the COT-Zn composites with cytotoxic activity against BALB/3T3 clone A31 cells should also reveal anticancer character. These investigations will be continued. SDS concentration (µl/mL) Viability (% of negative control) Figure 11. Viability of BALB/3T3 clone A31 cells exposed for 24 h to SDS (positive control), assayed with NRU test. Viability presented as a percentage of the negative control (NC; culture medium with vehicle, i.e., 2% H 2 O in culture medium).
Zinc compounds also reveal strong anticancer activity (e.g. [136,[179][180][181]) reflected additionally by nearly 2400 document results on Anticancer Zinc [182] and 1-700 documents results on Anticancer Activity of Zinc abstracted in the Scopus Base [183]. Therefore the COT-Zn composites with cytotoxic activity against BALB/3T3 clone A31 cells should also reveal anticancer character. These investigations will be continued.

Conclusions
In summary, COT-Zn composites with different Zn content were prepared by DC magnetron sputtering technology using a zinc metal target. The composite samples were characterized by SEM, EDS, and FAAS. The biological properties of the materials were verified by cytotoxicity screening and antimicrobial activity tests against colonies of E. coli and S. aureus bacteria and A. niger and C. globosum fungi. The in vitro determined antimicrobial properties of COT-Zn composites revealed the antibacterial and antifungal activities.
In vitro studies showed also that COT-Zn composites containing merely 9 g/kg Zn were cytotoxic. The ability to adapt clean and zero-waste magnetron sputtering methods to an industrial scale provides the possibility to obtain sustainable materials for use in a variety of applications. The prepared fiber composites have great application potential as an antimicrobial material in the field of biomedical engineering (e.g., rehabilitation, medical devices); however, due to their cytotoxicity they have limited possibilities of use as a material interacting with cells of the human body.