*3.8. Antibacterial Test*

The antibacterial activity of the PEEK films coated with various concentrations of silver nanoparticles by a green method using the Tollens reagent and a monosaccharide were tested against two Gram-negative bacteria: *Escherichia coli* and *Serratia marcescens* and one Gram-positive bacterium: *Bacillus licheniformis*, evaluating the zone of inhibition by a contact method direct with medium agar as shown in Figures 12–14. The results of the zone of inhibition measured with the ImageJ software are shown in Table 3.

**Figure 12.** Inhibition zone of the PEEK (1), PEEK/Ag0.04 (2), PEEK/Ag0.08 (3) and PEEK/Ag0.12 (4) coated with one layer (**a**) and two layers (**b**) of silver with *Escherichia coli*.

**Figure 13.** Inhibition zone of the PEEK (1), PEEK/Ag0.04 (2), PEEK/Ag0.08 (3) and PEEK/Ag0.12 (4) coated with one layer (**a**) and two layers (**b**) of silver with *Serratia marcescens*.

**Figure 14.** Inhibition zone of the PEEK (1), PEEK/Ag0.04 (2), PEEK/Ag0.08 (3) and PEEK/Ag0.12 (4) coated with one layer (**a**) and two layers (**b**) of silver with *Bacillus licheniformis*.


**Table 3.** Antibacterial activity of silver nanoparticles deposited in PEEK films.

SD: standard deviation.

For the antimicrobial test, an uncoated PEEK film was used as a control in the six plates, which did not present antimicrobial activity. The amount of silver deposited on the PEEK/Ag0.04 sample with a single layer was not enough to prevent the proliferation of the Gram-negative bacteria shown in Figures 12 and 13, stimulating bacterial growth in the silver-free sites that are shown in the SEM images, similar to that reported by Seuss et al. [30]. While the polymer coated with a layer AgNPs in a concentration of 0.08 mol/L presented inhibition against *E. coli* but not against *S. marcescens*. The PEEK/Ag0.12 system with a single layer and all the samples coated with two layers of AgNPs had antibacterial properties, which increased the zone of inhibition with the amount of Ag<sup>+</sup> ions deposited in the substrate, favoring the bactericidal effect.

The antibacterial efficiency of PEEK films coated with AgNPs shown in Table 3 was higher for *E. coli* compared to *S. marcescens*, and this was due to the presence of an envelope of two membranes which have different proteins and phospholipids that prevent the passage of silver nanoparticles inside the cells. [16,30,58]. Although the *S. marcescens* bacterium is resistant to traditional antibiotics, the AgNPs synthesized by a green method and deposited on a polymeric PEEK substrate had antibacterial efficiency against this microorganism, because the nanoparticles easily crossed the cytoplasmic membrane due to its small size, causing damage to the organelles of the cell and leading to the death of the microorganism, similar to that described by Baghayeri et al. [59] and Mathew et al. [4].

Figure 14 shows that the uncoated Polyetheretherketone films had no antimicrobial effect on the Gram-positive bacterium *Bacillus licheniformis* similar to a Gram-negative bacterium. Figure 14a,b show that the polyetheretherketone films coated with a single layer of silver nanoparticles in concentrations of 0.04 and 0.08 mol/L did not exhibit antibacterial activity in *B. licheniformis*, while the PEEK/Ag0.12 system and all polymeric films coated with two layers of metallic silver counteracted the growth of Gram-negative bacteria, by releasing silver ions in humid conditions. In addition, the amount of nanoparticles in the culture medium increased the zone of inhibition of bacterium B. licheniformis as shown in Table 3 [60,61]. The growth of the Gram-positive bacterium was higher in comparison with

the Gram-negative bacteria, because the bacterium *Bacillus licheniformis* has a thicker peptidoglycan layer in its membrane, which regulates and prevents the path of AgNPs in low concentrations to the cell as indicated by Mathew T. et al. [4]. Similar results with a difference in antibacterial activity were observed by Sikder et al. [62,63] when exploring antibacterial surfaces on PEEK and Ti6Al4V, and such studies were performed in the case of Gram-negative (*E. coli*) and gram-positive (*S. aureus*) bacteria. Apart from the difference in the diameter of inhibition zone, their research also presented SEM images which prove the variance in interactions of Ag+ ions with negative and positive strains of bacteria. And therefore, these results show a similar trend to the present work. Likewise, Mosselhy D.A. et al. and Ur Rehman et al. [16,64] described a similar effect to the one reported in this study, where the antibacterial properties increased with the increase of the silver nanoparticle ratio and the humid environment in which the samples were installed, such studies demonstrated the effectiveness of the AgNPs coatings in solid state as an antibacterial system.

The antibacterial mechanism of the silver nanoparticle coatings is possible because of the Ag<sup>+</sup> ions generated by the conversion of metallic silver into the physiological environment where the antimicrobial evaluation occurs [16]. The silver nanoparticles in cationic form penetrate the cell, deforming the cell membrane, and interacting with some proteins; the silver nanoparticles also interact with the sulfur and phosphorus bases contained in the DNA, causing an interruption in DNA replication and subsequent cell death [36,37]. Likewise, Ag<sup>+</sup> ions form free radicals that attack respiratory enzymes which are essential for cell replication [45,59].

The zone of inhibition of the three bacteria analyzed was similar in the PEEK/Ag0.12 samples with a single layer and PEEK/Ag0.04 with two layers. This was possible from the homogeneously distributed silver nanoparticles deposited once on the PEEK substrate when AgNO3 was used in a concentration of 0.12 mol/L. Such particles had a high surface area with particle sizes below 25 nm according to the SEM and AFM analyzes. Finally, the sample with the maximum zone of inhibition against the growth of *E. coli*, *S. marcescens* and *B. licheniformis* was PEEK/Ag0.12 with two coating layers of AgNPs, corroborating the efficacy of the synthesis method by chemical reduction of ammoniacal silver complexes with glucose in obtaining coatings of metallic silver nanoparticles with high surface area [65]. In addition, the increase in the proportion of nanoparticles in the polymeric substrate favors the antibacterial effectiveness by the high liberation of silver ions in the wet conditions of the culture medium as reported by Logeswari et al and Gao et al. [66,67].

#### **4. Conclusions**

A method of coating by a chemical route with ammoniacal silver complexes was used to impregnate polyetheretherketone films with silver nanoparticles to inhibit bacterial growth. The characteristic diffraction peaks of PEEK were kept constant by completely coating the surface of the polymer. The intensity of the silver signals in the diffractograms increased with the amount of silver nanoparticles deposited. The average size of the crystalline domains of AgNPs synthesized by a simple chemical reduction method was less than 30 nm using the Debye–Scherrer formula, corroborating the results by statistical analysis with transmission electron microscopy images. The electrostatic interactions between the polymer and the deposited AgNPs were evidenced by FTIR and TEM. The thermograms showed the proportion of silver adhered to the polymeric substrate. The proportional deposition of silver on the surface of the PEEK was evaluated by scanning electron microscopy and atomic force microscopy, revealing the excellent distribution of the particles when they are synthesized with glucose. In addition, it is evident that the second deposition process leaves the polymer with a thickness around 300 nm for all samples. The action mechanism of AgNPs against the bacterial growth of pathogenic microorganisms is influenced by the conversion of metallic silver to Ag+. The sample that shows the best antibacterial activity in Gram-positive and Gram-negative bacteria for a possible application in the design of air purification equipment is PEEK/Ag0.12 with two layers, since the excellent distribution of the coating improves contact with bacteria, inhibits the replication process, and favors cell death

**Author Contributions:** Conceptualization, A.F.C.-P., J.A.G.C. and J.J.M.Z.; Data Curation, A.F.C.-P., D.T.M.-C., J.A.G.C. and J.J.M.Z.; Formal Analysis, A.F.C.-P., D.T.M.-C. and J.A.G.C.; Funding Acquisition, L.P.-M., C.A.P.V., J.J.M.Z. and C.A.P.G.; Investigation, A.F.C.-P., D.T.M.-C., J.A.G.C. and L.P.-M.; Methodology, J.A.G.C.; Project Administration, C.A.P.G.; Resources, C.A.P.V., J.J.M.Z. and C.A.P.G.; Supervision, J.A.G.C., L.P.-M., C.A.P.V., J.J.M.Z. and C.A.P.G.; Validation, L.P.-M. and C.A.P.G.; Writing—Original Draft, A.F.C.-P.; Writing—Review & Editing, A.F.C.-P., J.A.G.C., L.P.-M. and C.A.P.G.

**Funding:** This work was supported by Universidad Antonio Nariño under the project number 2016216–PI/ UAN-2018-635GFM and by Vicerrectoría de Investigación y Extensión de la Universidad Pedagógica y Tecnológica de Colombia.

**Conflicts of Interest:** The authors declare no conflict of interest.
