Development of Functional Composite Cu(II)-Polyoxometalate/PLA with Antimicrobial Properties

Novel composite self-disinfecting films of polylactic acid (PLA) filled with nanosized particles of double sodium–copper(II) paratungstate B Na2Cu3(CuOH)2[W12O40(OH)2]·32H2O (POM) were developed. The solvent casting (POM/PLA film) and solvent-free melt extrusion methods (Extr. POM/PLA film) were applied for film preparation. The copper (II) ion release to water from both types of the films after 10 days at different temperatures demonstrated that the PLA matrix acts as a diffusion barrier, and the resulting concentration of released copper in water at room temperature remained low, at 0.79% for POM/PLA film and 0.51% for Extr. POM/PLA film. The POM-containing films reveals a significant inhibitory effect against E. coli ATCC 25922 in the agar diffusion test. The numbers of CFUs in washes of the films after incubation for 24 h were found to be 3.6 log CFU mL–1 (POM/PLA film) and 4.1 log CFU mL–1 (Extr. POM/PLA film). The films combine the antibacterial properties of POM and a bio-based polymer matrix, which makes them a prospective coating material for applications in hospital indoor environments. Excellent thermal stability of POM gives a technological advantage for industrial manufacturing to allow the processing of novel composite material in the solvent free (molten) state.


Introduction
Copper is an inexpensive, earth-abundant metal with moderate toxicity and extremely broad use as a green and sustainable alternative for numerous metal-catalyzed organic reactions [1,2]. Copper is well recognized as having antimicrobial activity, but it has not been applied widely to the clinical setting [3], including health care-associated infections, which pose a real danger nowadays. These infections occur while receiving medical or surgical care, or in any other conditions related to treatment. Despite the efforts of environmental and infection control specialists, infections acquired during hospitalization are still one of the leading causes of morbidity and mortality in hospitals around the world. The development of antimicrobial surfaces or coatings offers an opportunity to reduce the risk of infection associated with poor hygiene and ineffective room cleaning [4]. Self-cleaning or self-disinfecting antimicrobial surfaces were developed and put into use only 50 years ago to complement existing infection control methods. The self-disinfecting surfaces proposed in 1964 by Kingston and Noble were developed based on the hypothesis that self-disinfecting solutions reduce the spread of germs as a result of the suppression of bacteria growth during its deposition on the surface in the anthropogenic environment [5]. All the above mentioned self-disinfecting materials can be divided into two main categories, namely anti-adhesive/anti-fouling (polyethylene glycol; polyethylene terephthalate films, modified through the addition of polyethylene oxide) and antimicrobial, either by inhibiting the growth of bacteria or by directly killing the microorganisms (triclosan, silver and copper surfaces) [4]. Thus, the creation of new antibacterial coatings based on soluble and thermoplastic polymeric materials is an urgent task. One of the promising sustainable plastics is polylactic acid (PLA), an inexpensive and bio-based material [6][7][8][9][10][11][12][13]. Table 1 shows data on the improvement of the antibacterial properties of PLA composites by introducing additives with antibacterial properties. Designing new polymeric material with antibacterial properties and the ability to form thin films is a challenging but prospective issue for various biomedical applications. Poly lactic-b-4vinylpyridine (PDLA-b-P4VP); Agnanoparticles arbitrary PLLA/PDLA 255 -P4VP 46 against E. coli, 83.2% reduction; PLLA/PDLA 255 -P4VP 46, 75, 98 /Ag against E. coli, 100% reduction [10] Cunanoparticles 0.3 wt.% Activity against mix of P. fluorescens and P. putida: 6.0 log CFU·mL -1 (CuNPs-C-PLA), 7.4 log CFU·mL -1 (blank PLA) [11] Electrospinning method Fibrillar particles of Ag-sepiolite and Cu-sepiolite 5 wt.% 35% (P. putida), 85% (S. cerevisiae) reduction [12] Copper salt ion-exchange zeolite nanoparticles (nZH-Cu) 1, 2, and 3 wt.% PLA 1%, nZH-Cu 55.3% (S. aureus), and 62.7% (S. Typhi) reduction; PLA ≥2%, nZH-Cu 100% (S. aureus and S. Typhi) reduction [13] Special attention should be paid to enhancing the antibacterial properties of PLA with inorganic molecules, e.g., copper(II)-containing polyxometalates. These compounds compare favorably to other antibacterial agents in terms of thermal stability, low solubility in water, and insolubility in organic solvents, as well as ease of preparation from aqueous solutions. Polyoxometalates (POM) is a large class of polyoxoanions formed by the metals of V and VI groups of the Periodic Table with broad application in medicine [14][15][16]. Thus, these compounds can exhibit antitumor activity [17], but their antibacterial properties also deserve great attention. The antibacterial activity of pure inorganic POMs can be divided into two main types, synergistic and direct. Many inorganic POMs did not show significant antibacterial activity at pharmacologically acceptable concentrations but showed synergistic activity with conventional antibiotics. For example, polyoxotungstates had a synergistic effect in combination with β-lactam antibiotics on MR/VR S. aureus (+) [18][19][20][21][22]; MR S. epidermis (+) , MR S. auricularis (+) , and MS S. haemolyticus (+) [23]; and MZR H. pylori (−) and CLR H. pylori (−) [22]. Only a few POMs had direct antibacterial activity [16,18,19,[24][25][26][27] tested against various strains of S. aureus to show significant antibacterial activity, demonstrating a minimum inhibitory concentration (MIC) < 100 mg mL −1 , while the effective antibiotics had MIC values of 0.001-10 mg mL -1 . In the review of Bijelic et al. [16], there was only one copper(II)-containing polyoxotungstate K 12 [Cu 3 (PW 9 O 34 ) 2 ] mentioned as having MIC values of 400 mg mL -1 and 200 mg mL -1 against MRSA strains SR3605 and ATCC43300, respectively. In combination with oxacillin, this POM was reported to have FIC values of 0.141 and 0.070 towards the MRSA strains SR3605 and ATCC43300, respectively.
In this study, we report PLA composites with double sodium-copper(II) paratungstate B possessing antibacterial properties and characterized by low copper (II) ion release. The films were prepared by two methods, namely the solvent casting method and the melt extrusion method. The comprehensive characterization of the obtained composite films included determination of physical properties as well as release of Cu(II) ions in water. The surface morphology of the films was studied by scanning electron microscopy (SEM) to demonstrate encapsulation of POM particles inside the polymer matrix, which prevents active leaching of metals upon contact with aqueous environments.
The antibacterial activity of Cu(II)-containing paratungstates B was studied against E. coli. The double sodium-copper(II) paratungstate B Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O revealed stability, and its structure was reliably determined and retained during the change of the number of water molecules, some of which were uncoordinated and located in the structure channels [28,29]. Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·nH 2 O (n = 32, 34) nanoparticles can be prepared by an inexpensive low energy consuming method. Its synthesis occurs either as a result of self-assembly in an acidified aqueous solution of Cu 2+ and WO 4 2− [28,29], or as a result of the hydrolytic transformation of ammonium metatungstate (NH 4 ) 6
Microscopic analysis showed that the POM grain surface has fuzzy blurred edges. The grain size of the sample was within the range of 200-400 nm ( Figure 1). Uniform surface contrast in BEC mode pointed to the single-phase of the obtained salt ( Figure 2). On the micrographs of POM powder in characteristic X-ray emission, there were no regions with different surface morphologies, and there was an even distribution of Na, Cu, W, and O without segregation and liquation (apparent heterogeneities are explained by different relief of the sample surface); see Figure S1. These clearly indicate the formation of a single-phase sample. The grain size of the sample was within the range of 200-400 nm ( Figure 1). Uniform surface contrast in BEC mode pointed to the single-phase of the obtained salt ( Figure 2). On the micrographs of POM powder in characteristic X-ray emission, there were no regions with different surface morphologies, and there was an even distribution of Na, Cu, W, and O without segregation and liquation (apparent heterogeneities are explained by different relief of the sample surface); see Figure S1. These clearly indicate the formation of a single-phase sample.  X-ray spectral microanalysis was carried out in various areas of the powder surface. The results of elemental analysis presented in Figure S2 show an identical theoretical molar ratio of elements Na:Cu:W = 2.00:5.00:12.00.
The FTIR spectra of POM is shown in Figure S3a. The grain size of the sample was within the range of 200-400 nm ( Figure 1). Uniform surface contrast in BEC mode pointed to the single-phase of the obtained salt ( Figure 2). On the micrographs of POM powder in characteristic X-ray emission, there were no regions with different surface morphologies, and there was an even distribution of Na, Cu, W, and O without segregation and liquation (apparent heterogeneities are explained by different relief of the sample surface); see Figure S1. These clearly indicate the formation of a single-phase sample.  X-ray spectral microanalysis was carried out in various areas of the powder surface. The results of elemental analysis presented in Figure S2 show an identical theoretical molar ratio of elements Na:Cu:W = 2.00:5.00:12.00.
The FTIR spectra of POM is shown in Figure S3a. X-ray spectral microanalysis was carried out in various areas of the powder surface. The results of elemental analysis presented in Figure S2 show an identical theoretical molar ratio of elements Na:Cu:W = 2.00:5.00:12.00.
The FTIR spectra of POM is shown in Figure S3a.

Preparation and Characterization of PLA Composite Films
Two types of composite films were successfully obtained by (i) solvent casting (POM/PLA film) and (ii) melt extrusion (Extr. POM/PLA film) methods. Figure 3 represents evolution of torque during compounding. As could be seen, the addition of POM particles to PLA reduced the viscosity of the compound after 3 min of blending. This time was assumed to be a homogenization time, taking into account that significantly longer time may cause thermal degradation of the polymer matrix. After 5 min of compounding, the material extruded out as a round-shaped filament and was collected. For further studies, the filaments were molded into 1 mm sheets by hot-pressing between steel plates with Teflon spacers. Specimens of Extr. PLA appeared transparent with a slightly yellowish color, and Extr. POM/PLA compounds were opaque and yellowish.
particles to PLA reduced the viscosity of the compound after 3 min of blending. This time was assumed to be a homogenization time, taking into account that significantly longer time may cause thermal degradation of the polymer matrix. After 5 min of compounding, the material extruded out as a round-shaped filament and was collected. For further studies, the filaments were molded into 1 mm sheets by hot-pressing between steel plates with Teflon spacers. Specimens of Extr. PLA appeared transparent with a slightly yellowish color, and Extr. POM/PLA compounds were opaque and yellowish. The morphology of the films was studied by SEM. The micrograph of POM/PLA film (see Figure 4a) shows many white spots that are evenly distributed over the surface of the PLA material, and there are no large-scale agglomerations. The sizes of the spot ranged from 0.45 to 2.68 μm (Table 2), which allowed us to identify them as POM particles, as shown in Figure 1. Any similar white spots on the micrograph of blank PLA film (see Figure 4b) were detected. POM particles on the surface of Extr. POM/PLA film (see Figure  5a) were different from the particles on the POM/PLA film, as shown in Figure 4a, by smaller size (0.18-1.98 μm, Table 2), as well as a lesser quantity. It can be assumed that extrusion compounding breaks the agglomerates of the POM particles. A comparison of Figures 4a,b and 5a,b shows that the film obtained by the extrusion method is smoother than the film from the solvent casting method. Additionally, it can be seen that POM particles sedimented from solution during film drying, and thus in the bottom side of the film, the amount of POM was larger than in the top side. Both sides of extruded films had very similar amounts of particles because the rapid solidification of the melt prevented particle sedimentation. The morphology of the films was studied by SEM. The micrograph of POM/PLA film (see Figure 4a) shows many white spots that are evenly distributed over the surface of the PLA material, and there are no large-scale agglomerations. The sizes of the spot ranged from 0.45 to 2.68 µm (Table 2), which allowed us to identify them as POM particles, as shown in Figure 1. Any similar white spots on the micrograph of blank PLA film (see Figure 4b) were detected. POM particles on the surface of Extr. POM/PLA film (see Figure 5a) were different from the particles on the POM/PLA film, as shown in Figure 4a, by smaller size (0.18-1.98 µm, Table 2), as well as a lesser quantity. It can be assumed that extrusion compounding breaks the agglomerates of the POM particles. A comparison of Figures 4a,b and 5a,b shows that the film obtained by the extrusion method is smoother than the film from the solvent casting method. Additionally, it can be seen that POM particles sedimented from solution during film drying, and thus in the bottom side of the film, the amount of POM was larger than in the top side. Both sides of extruded films had very similar amounts of particles because the rapid solidification of the melt prevented particle sedimentation.      The particle distribution in the surface of the films was analyzed and is represented in Figure 6. As can be seen, particles up to 9 μm in size were present in solvent casted film, whereas particles in extruded film were limited to 2 μm. Even more, there was a strong tendency for narrowing of the particle size distribution and shifting the majority of particle sizes around 530 nm in extruded films. Solvent casted films had very broad size distribution, with the majority of particles around 750 nm. The particle distribution in the surface of the films was analyzed and is represented in Figure 6. As can be seen, particles up to 9 µm in size were present in solvent casted film, whereas particles in extruded film were limited to 2 µm. Even more, there was a strong tendency for narrowing of the particle size distribution and shifting the majority of particle sizes around 530 nm in extruded films. Solvent casted films had very broad size distribution, with the majority of particles around 750 nm. Several characteristics of composite films are summarized in Table 2.  Several characteristics of composite films are summarized in Table 2. ; and 957 cm -1 (ν(W = O t ))) agree well with those determined previously for the salts with the paratungstate B anion [28][29][30]. The band attributed to stretching vibrations of C = O is located at 1748-1749 cm -1 for all types of PLA film ( Figure S3b-e), which is slightly shifted [10]. The superimposition of the ATR-FTIR spectra are shown on Figure S4. Differences between the spectra of blank PLA film and POM/PLA-containing film can also be seen in the 1050-1250 cm -1 range, attributed to C-O and C-O-C stretching vibrations.

Thermal Properties
The thermal behavior of the different POM/PLA composite films was studied by DSC to evaluate how the introduction of POM particles can affect the glass transition (T g ) and melting (T m ) temperatures and crystallization parameters of PLA. The addition of the POM filler to PLA resulted in decreasing melting point (T m ), as shown in Table 3 for both solvent cast and extruded films: by 4 • C for PLA/POM, and by 7 • C for Extr. POM/PLA. The fact of T m depression is the evidence of better dispersion of POM particles within the PLA matrix during melt compounding, as compared to dispersion in solution. Additionally, in the case of the film developed by the solvent casting method (PLA/POM), the T g value increased with addition of POM, and in the case of the Extr. POM/PLA film, the T g value decreased with the addition of POM; see Table 3. Additionally, melting points of extruded films were higher than the solvent cast, which was due to differences in the degree of crystallinity. PLA usually consists of α and α' crystalline phase mixture, but the casting of the film from a melt at 120 • C transferred all α phase to α' [31]. Relatively low total crystallinity and low T g allowed uniform thin films to be produced by hot-press or roll-mills. The DSC thermogram is shown in Figure S5.  2 ]·32H 2 O sample was investigated first, using 0.020 mg weights in Milli-Q water (10 cm 3 ) at room temperature and 40 ± 1 • C. The particles were initially 1.12 ± 0.20 µm in diameter, and after 10 days in Milli-Q water, the size did not change significantly (1.20 ± 0.34 µm). Released total Cu(II) ion concentrations in Milli-Q water were low, amounting to 42.49 ± 0.53 µM and 47.21 ± 0.27 µM at room temperature and 40 ± 1 • C, respectively. This indicates that 1.59 ± 0.02% and 1.79 ± 0.01% by weight of the original POM Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O were released into solution.

Copper(II) Ion Release from PLA Composite Films
The mixture of PLA (0.3311 g) and POM (0.0102 g, 3% w/w) was used for the film preparation by the solvent casting method. The copper content in POM was 8.24% w/w, which corresponds to 0.8 mg. Therefore, the films contained 0.25 w% of copper (to the total weight of the mixture). The copper content in the film obtained by extrusion was also 0.25% w/w in the case of 3% w/w of the POM, which was used to preparation of compound. The copper ion release value was sown on the basis of the POM weight (8.24% w/w) for convenience. Over 10 days of copper release from two types of PLA composite films in Milli-Q water at room temperature and 40 ± 1 • C, the film size, weight, and thickness did not change significantly (Tables 2 and 4). In the case of Extr. PLA composite film, released total Cu(II) ion concentrations in Milli-Q water were lower than in Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O itself, namely 4.09 ± 0.27 µM and 4.56 ± 0.22 µM at room temperature and 40 ± 1 • C, respectively. This amounts to 0.51 ± 0.03% and 0.57 ± 0.03% of the total Cu(II) content. In its turn, released total Cu(II) ion concentrations in Milli-Q water for PLA composite film was found to be 1.73 ± 0.15 µM (0.79 ± 0.07%) and 1.89 ± 0.20 µM (0.86 ± 0.09%) at room temperature and 40 ± 1 • C, respectively. Therefore, the novel POM/PLA composite is characterized by remarkably low release of copper ions to the environment.

Antimicrobial Properties of POM and PLA Composite Films
After incubation, the antibacterial activity of POMs (Table 5) was evaluated by measurements of the inhibition zone width, see Figure 7. The POM Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O (T9) showed the largest inhibition zone diameter (see Table 5) and was chosen as antimicrobial filler for PLA films. Along with this, Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O in suspension showed activity against E. coli, with a MIC of 250 µg mL −1 . The Cu 2+ release at room temperature from POM determined in Section 2.3.1. was 1.59 ± 0.02%. Thus, the calculated MIC(Cu 2+ ) = 3.9 µg mL −1 . Along with this, the antimicrobial effect of copper(II) sulfate against E. coli was studied in [32]; MIC (CuSO 4 ·5H 2 O) = 18.8 µg mL −1 , where the value MIC(Cu 2+ ) = 3.0 µg mL −1 was calculated, which is almost the same as the value MIC(Cu 2+ ) reported in this study. These results allowed us to assume that POM antimicrobial action occurs due to the presence of copper(II) ions. Additionally the MBC value of 750 µg·mL −1 was determined ( Figure S6), which was not much higher than the MBCs of nanoparticulate metals, and metal oxides against E. coli (500 µg·mL −1 of Cu 2 O; 250 µg·mL −1 of CuO; 250 µg·mL −1 of Cu), mentioned in [33], could be the evidence of antimicrobial activity. The POM Na2Cu3(CuOH)2[W12O40(OH)2]·32H2O (T9) showed the largest inhibition zone diameter (see Table 5) and was chosen as antimicrobial filler for PLA films. Along with this, Na2Cu3(CuOH)2[W12O40(OH)2]·32H2O in suspension showed activity against E. coli, with a MIC of 250 μg mL −1 . The Cu 2+ release at room temperature from POM determined in Section 2.3.1. was 1.59 ± 0.02%. Thus, the calculated MIC(Cu 2+ ) = 3.9 μg mL −1 . Along with this, the antimicrobial effect of copper(II) sulfate against E. coli was studied in [32]; MIC (CuSO4·5H2O) = 18.8 μg mL −1 , where the value MIC(Cu 2+ ) = 3.0 μg mL −1 was calculated, which is almost the same as the value MIC(Cu 2+ ) reported in this study. These results allowed us to assume that POM antimicrobial action occurs due to the presence of copper(II) ions. Additionally the MBC value of 750 μg·mL −1 was determined ( Figure S6), which was not much higher than the MBCs of nanoparticulate metals, and metal oxides  The agar diffusion method was reported as a useful method in testing antimicrobial properties of specific polymer materials with antimicrobial adding aimed at creating antimicrobial coatings [9,34]. Figure 8 shows the agar diffusion tests after 24 h against E. coli for POM/PLA composite films, where blank PLA films served as a control. Control films did not induce any inhibition zone in either bacterial strain, while the POM/PLA composite films formed an inhibition zone of about 1-2 mm. The inhibition zone was not uniform due to distribution of Cu(II) paratungstate B microbeads within the films and, thus, nonuniform copper release.  [33], could be the evidence of antimicrobial activity. The agar diffusion method was reported as a useful method in testing antimicrobial properties of specific polymer materials with antimicrobial adding aimed at creating antimicrobial coatings [9,34]. Figure 8 shows the agar diffusion tests after 24 h against E. coli for POM/PLA composite films, where blank PLA films served as a control. Control films did not induce any inhibition zone in either bacterial strain, while the POM/PLA composite films formed an inhibition zone of about 1-2 mm. The inhibition zone was not uniform due to distribution of Cu(II) paratungstate B microbeads within the films and, thus, nonuniform copper release. Polymer composites showed the presence of an inhibition zone, so the number of CFU·mL −1 was counted after the inoculation of the films. The antibacterial activity of POM/PLA films was compared with a control polymer matrix [9]. The number of CFUs within the resulting suspensions was counted after 24 h of incubation; see Table 6. Results demonstrated high activity of POM-containing PLA films compared to the control polymer matrix. In particular, a cell loads equal to 4.1 log CFU·mL −1 was measured at the end of the test period in the Extr. POM/PLA sample ( Figure S7a); this value was lower than that measured in the blank Extr. PLA sample (5.4 log CFU·mL −1 ; Figure S7b). In the case of films made by the solvent casting method, the POM/PLA sample ( Figure S8a) was found to have a cell load equal to 3.6 log CFU·mL −1 ; this value was lower than that measured in blank PLA sample (5.8 log CFU·mL −1 ; Figure S8b). This experimental result confirms the Polymer composites showed the presence of an inhibition zone, so the number of CFU·mL −1 was counted after the inoculation of the films. The antibacterial activity of POM/PLA films was compared with a control polymer matrix [9]. The number of CFUs within the resulting suspensions was counted after 24 h of incubation; see Table 6. Results demonstrated high activity of POM-containing PLA films compared to the control polymer matrix. In particular, a cell loads equal to 4.1 log CFU·mL −1 was measured at the end of the test period in the Extr. POM/PLA sample ( Figure S7a); this value was lower than that measured in the blank Extr. PLA sample (5.4 log CFU·mL −1 ; Figure S7b). In the case of films made by the solvent casting method, the POM/PLA sample ( Figure S8a) was found to have a cell load equal to 3.6 log CFU·mL −1 ; this value was lower than that measured in blank PLA sample (5.8 log CFU·mL −1 ; Figure S8b). This experimental result confirms the efficiency of PLA films filled with Cu(II)-containing POMs to prevent the E. coli proliferation in novel smart coating. SEM analysis of the biomass on the films (Figure 9a,b) allowed us to observe the morphological changes of the E. coli cells during a 24 h period. Healthy cells from blank PLA film had smoother surfaces (Figure 9a), while E. coli cells from PLA-POM film had a coarser surface structure, and cell destruction was noticeable; see Figure 9b [35,36]. SEM analysis of the biomass on the films (Figure 9a,b) allowed us to observe the morphological changes of the E. coli cells during a 24 h period. Healthy cells from blank PLA film had smoother surfaces (Figure 9a), while E. coli cells from PLA-POM film had a coarser surface structure, and cell destruction was noticeable; see Figure 9b [35,36]. It can be assumed that the toxic antimicrobial effect of POM/PLA composite films is completely due to the action of POM. Recent studies indicate that the release of metal ions is the driving force of the antimicrobial properties of, in essence, nanoparticles [37][38][39]. A similar approach can be applied for understanding antimicrobial activity of POMs, from which Cu(II) ions can be released. The Cu(II) ion release from the POM/PLA composite films in water is quite low; moreover, at room temperature, its value is 2 (POM/PLA film) and 3 (Extr. POM/PLA film) times less than that from POM itself. At the same time, POM/PLA composite films showed a high value of antibacterial activity. According to the literature data [39,40], the following mechanism may occur in the presence of bacteria: (1) adsorption of bacteria on the polymer surface triggers the diffusion of water through the polymer matrix due to the medium surrounded the bacteria; (2) water with dissolved oxygen reaches the surface of embedded POM particles, allowing dissolution or corrosion processes and in this way Cu(II) ions are realized; (3) Cu(II) ions reach the composite surface, damaging the bacteria membrane; (4) afterwards, Cu(II) ions can diffuse into the bacteria. It can be assumed that the toxic antimicrobial effect of POM/PLA composite films is completely due to the action of POM. Recent studies indicate that the release of metal ions is the driving force of the antimicrobial properties of, in essence, nanoparticles [37][38][39]. A similar approach can be applied for understanding antimicrobial activity of POMs, from which Cu(II) ions can be released. The Cu(II) ion release from the POM/PLA composite films in water is quite low; moreover, at room temperature, its value is 2 (POM/PLA film) and 3 (Extr. POM/PLA film) times less than that from POM itself. At the same time, POM/PLA composite films showed a high value of antibacterial activity. According to the literature data [39,40], the following mechanism may occur in the presence of bacteria: (1) adsorption of bacteria on the polymer surface triggers the diffusion of water through the polymer matrix due to the medium surrounded the bacteria; (2) water with dissolved oxygen reaches the surface of embedded POM particles, allowing dissolution or corrosion processes and in this way Cu(II) ions are realized; (3) Cu(II) ions reach the composite surface, damaging the bacteria membrane; (4) afterwards, Cu(II) ions can diffuse into the bacteria.

Materials
In the study, Na 2 WO 4 2H 2 O, HNO 3 , and Cu(NO 3 ) 2 were used as purchased from Sigma-Aldrich without further purification; aqueous solutions were prepared using Milli-Q demineralized water.
Polylactic acid (PLA 3052 D) with a specific gravity of 1.24 g mL −1 , molecular weight of ca.

Preparation of PLA Composite Films
Prior to film preparation, POM particles were suspended in ethanol and sonicated to break the agglomerates of particles and then dried at room temperature.

The Solvent Casting Method
Two types of films were prepared, namely blank PLA (0.33 g) and the mixture of PLA (0.33 g) and POM (0.01 g); thus, the content of POM in PLA-POM film was 3 wt.%. For this purpose, required amounts of PLA and a mixture of PLA with POM were dissolved in 3 mL of chloroform (10 wt.%) at vigorous stirring. After stirring for 30 min, 0.30 mL of DMSO was added. Addition of DMSO extends the time of solvents evaporation and prevents film from shrinkage during solvent casting. The resulting solution was poured onto a clean glass surface. The resulting films were dried for 12 h at room temperature and then 1 h in a vacuum oven at 50 mbar and 105 • C, which is sufficient to remove the DMSO completely.

The Melt Extrusion Method
PLA granules were milled into a powder by a laboratory knife mill Retsch SM100 with a 1 mm sieve. Next, POM powder was added to PLA and mechanically pre-mixed in a beaker. The POM load was set as 3 wt.% of the compound. Five grams of the premixed blend was loaded into a Haake MiniCTW micro-conical counter-rotating twin-screw extruder at 205 • C, which allowed the thermal stability of PLA to be maintained, and blended for 5 min at 150 rpm via a backflow channel. The compound of pure PLA was prepared under the same conditions. Extruded filament was hot-pressed into a thin film between stainless steel plates at 120 • C and pressure of 70 bar. The low glass transition point of PLA of around 60 • C allowed manufacturing of thin films by hot pressing with no difficulties.

Copper(II) Ion Release Studies from POM
For studying Cu(II), ion release from an air-dried POM sample, 20 ± 1 mg of POM, was added to 10 mL of Milli-Q water. The experiment was performed in a glass vial at room temperature and at 40 ± 1 • C under atmospheric conditions. POM samples were kept in water for 10 days. All experimental points were performed in triplicate. The supernatant was analyzed for Cu(II) content using the ICP-MS method.

Copper(II) Ion Release Studies from Composite PLA Films
Rectangular composite films (2 cm × 3 cm) were immersed in closed glass tubes with 10 mL of Milli-Q water. Total Cu(II) ion release from the films after 10 days at room temperature and 40 ± 1 • C was studied under standard atmospheric conditions. All experimental points were performed in triplicate [9].

Scanning Electron Microscopy (SEM)
Field-emission scanning electron microscopy (FE-SEM) was used to analyze cross sections of PLA composite films and to prove the presence of POM particles in the films. The PLA composite films were cross-sectioned, and FE-SEM analyses were performed by using a MIRA 3 XMU field emission scanning electron microscope (Tescan USA Inc., Cranberry Township, PA, USA).
The films' morphology was studied by means of a tabletop scanning electron microscope TM-1000 Hitachi. The Zeiss EVO MA15 (Carl Zeiss Microscopy GmbH, Jena, Germany) in the secondary electron scattering mode was used for E. coli imaging. Particle size distribution was analyzed by Image J freeware (developed by National Institutes of Health USA) and AMC Statistics add-in for MS Excel (developed by the Royal Society of Chemistry).

Fourier Transform Infrared Spectroscopy (FT-IR) of PLA Composite Films
Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the chemical changes in the PLA matrix due to its potential interactions with POM after thermal processing. Spectra were obtained from two types of blank PLA films and two PLA-POM-containing films using an IRTracer-100 FTIR spectrophotometer (Shimadzu, Kyoto, Japan). This study was conducted in the 400-4000 cm −1 range with a 2 cm −1 resolution, and number of scans of 40.

Thermal Characterization of PLA Composite Films
Differential scanning calorimetry (DSC) tests were carried out using a Q200 calorimeter (TA Instruments Mod., New Castle, DE, USA). Tests were performed in the temperature range of −25-210 • C at a heating rate of 10 • C min −1 for PLA and PLA-POM composite films. The glass transition (T g ), melting point (T m ), and enthalpy of fusion (∆H m ) were measured. Degree of crystallinity was calculated according to the following equation: where ∆H m is the enthalpy of melting, ∆H c is cold crystallization enthalpy, ω is the weight fraction of PLA, and ∆H 0 m is melting enthalpy of 100% crystalline PLA (107 J/g).

Mass Spectrometry
An inductively coupled plasma mass spectrometer (ICP-MS) equipped with a hexapole collision cell (PQExCell, Thermo Electron, Winsford, UK) was used to determine the Cu(II) ion content released from POM-containing composite films (see Section 3.4). (T9)) was tested by the agar diffusion method against the E. coli ATCC 25922 strain. The inoculum of bacterial strain was prepared from fresh overnight culture in nutrient broth (NB) (5 g L −1 peptone, 3 g L −1 meat extract) that were incubated at 37 • C. The agar diffusion test was performed in nutrient agar (NA) (10 g L −1 peptone from meat, 10 g L −1 meat extract, 5 g L −1 NaCl, 18 g L −1 agar) and 1% of the bacterial culture in order to obtain 10 5 CFU·mL −1 . The diffusion was carried out by pouring 0.1 mL of the media into Petri dishes. The dishes were left for 15 min to dry in air, and the weight of a corresponding POM (~0.05 g) mentioned in Table 5 was placed on the sterile filter-disks on the agar surface and incubated for 24 h at 37 • C. Antibacterial activity was evaluated by measurements of width of the inhibition zone.

Antimicrobial Studies
The amounts of 2000, 1000, 750, 500, 250, 200, 150, 100, 50, and 20 µg·mL −1 of suspended POM were used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) required to prevent the growth of bacteria after transferring them onto media. Approximately 2.7 × 10 7 CFU·mL −1 of E. coli were added to the POM suspension at a dilution of 1 in 100. Incubation was then carried out in a shaking incubator (200 rpm at 37 • C in air) for 24 h. Inoculated POM-free broth was used as negative controls. Bacteria growth was then assessed by plating each POM/bacterial suspension onto NA plates, followed by the incubation at 37 • C for 24 h [33].

Antimicrobial Properties of PLA Composite Films
Antimicrobial effects of PLA composite films and pure PLA films as a control were investigated by the agar diffusion method. PLA films were prepared as described in Section 3.3 and kept under vacuum for 3 h in order to reach complete vaporization of the traces of chloroform. The resulted films were sterilized for 2 h by means of UV light. The inoculum of bacterial strains was prepared from the fresh overnight culture in NB followed by incubation at 37 • C. The agar diffusion test was performed in 1% of the bacterial culture in order to obtain 10 5 CFU mL −1 . The diffusion technique was conducted by pouring 0.2 mL −1 of the media into Petri dishes. The dishes were then left for 15 min to dry in air, and film samples were placed on the agar surface to incubate at 37 • C for 24 h. Antibacterial activity was evaluated by measurements of the inhibition zone width [9].
The antibacterial activity of the PLA composite films against E. coli was also tested according to Japan Industrial Standard JIS Z 2801:2000 [41,42]. The inoculum of bacterial strains was prepared from fresh overnight culture in NB as described above. Then the strains were centrifuged for 30 min at 5000 rpm and diluted by saline solution (50 mL; 0.9 g of sodium chloride dissolved in 1 L of Milli-Q water). The final concentration of 27 × 10 6 CFU· mL −1 was determined by the serial dilution method. An aliquot of the resulting cell suspension (1 mL) was then placed onto both untreated surfaces and nanostructured films (3 × 3 cm 2 ) that were then incubated at 25 • C for 16 h in sterile Petri dishes. After incubation, 5 mL of saline solution were added in each Petri dish. Before sampling, the dishes were shaken for 180 s. The wash was serially diluted, and aliquots of each dilution were spread on NA. The medium was incubated at 37 • C for 24 h. The number of CFUs within the resulting suspensions was enumerated. Data from JIS Z 2801:2000 are usually expressed as an antibacterial value calculated from the difference between the log CFU on the treated surface and that measured on the untreated surface, although in some cases, the actual data as CFU cm −2 are presented. A number of checks are imposed by JIS Z 2801 to validate the results.

Conclusions
Novel composite POM/PLA films show strong potential as coatings with antimicrobial activity, convenient for application in hospital indoor environments. POMs have strong advantages over conventional antibiotics due to excellent thermal stability, which makes possible the application of melt extrusion solvent-free methods for the development of the coatings based on thermoplastic polymers. One could conclude that melt extrusion is an efficient and technological approach for antimicrobial surface preparation with uniform distribution of bioactive filler. The antimicrobial activity of Cu(II)-containing polyoxometalates was confirmed by the agar diffusion test. The MIC = 250 µg·mL −1 and MBC = 750 µg· mL −1 values were found for Na 2 Cu 3 (CuOH) 2 [W 12 O 40 (OH) 2 ]·32H 2 O against E. coli. The agar diffusion method showed 1-2 mm inhibition zone diameters for both types of films. The values of the antimicrobial activity of films against E. coli were determined to be higher for films obtained by the solvent casting method (3.6 log CFU·mL −1 ) than for films obtained by the extrusion method (4.1 log CFU·mL −1 ).