**3. Discussion**

It is believed that the antibacterial effect of silver ions is due primarily to the high affinity of the latter to sulfur or phosphorus [27]. Namely, it depends on the silver cations (Ag+), which bind firmly to groups of electron donors in biological molecules containing silver, oxygen or nitrogen. Due to the large number of sulfur-containing proteins on the surface of the bacterial cell, AgNPs can interact with sulfur-containing proteins inside or outside the cell membrane, which affect the viability of the bacterial cell [27]. Silver ions act by replacing other necessary metal ions, such as Ca2+ or Zn+ [28].

Simultaneously, it was suggested that silver ions (especially Ag+) liberated from AgNPs can interact with phosphorus moieties in DNA, leading to inactivation of DNA replication [27] or react with sulfhydryl groups of metabolic enzymes of the chain of bacterial transport of electrons, causing their inactivation [29].

The release of silver ions is only one of the mechanisms of AgNPs action. AgNPs themselves have different physical, chemical and other properties than the solid silver from which they were obtained.

A number of authors sugges<sup>t</sup> that the antibacterial effect of silver nanoparticles is due to the electrostatic interaction between negatively charged bacterial cells and positively charged nanoparticles [30].

Analysis of the electronic state of AgNPs showed that the core of the particles is zero-valent, while the shell is oxidized by interaction with the functional groups of the bandage and the surrounding medium. Earlier, analysis of the O 1s and valence band spectra showed the existence of four states of oxygen atoms on the Ag surface, which reflect the different states of silver atoms as well [31]. The discrimination of these in the Ag 3d spectrum is practically impossible because of insufficient overall energy resolution of electron energy analyzers. The surface atoms of nanoparticles, which, as a rule, are positively charged, should play the main role in interaction with the bacterial membranes. The Ag 3d spectra shoe that a large fraction (0.27) of the Ag atoms is in the Ag+ state, which determines the antimicrobial effect. It is possible that laser radiation in the surface plasmon resonance mode changes the electronic state of AgNPs, which increases the antibacterial activity of the system as a whole.

The wavelength of laser radiation used in the study was chosen by considering the plasmon resonance effect. When the frequency of the external field coincides with the frequency of the localized surface plasmon, a resonance arises, leading to a sharp increase in the field on the surface of the particle and an increase in the absorption cross section [32].

Apparently, the statistically significant effect of enhancing the antibacterial properties of the medical gauze bandage containing AgNPs observed in the present study is caused by laser photothermolysis of bacteria when exposed to laser radiation.

Probably, AgNPs, when irradiated with a laser, absorb energy, which is transformed into heat. The resulting hyperthermia of AgNPs can lead to both local damage of the bacterial cell and intensification of metabolic processes around the heated AgNPs with the subsequent acceleration of silver ionization processes, which in turn leads to the death of the microbial cell.

#### **4. Materials and Methods**

The ordinary medical gauze bandage was used in the study, produced in the Republic of Belarus (State Standard 1172–93) both as a control and for the production of medical gauze bandages containing AgNPs.

The metal vapor technique was used for the preparation of Ag-bearing bandage (Scheme 1).

The silver nanoparticles were obtained by the MVS from pieces of silver (99.99%) using an apparatus described elsewhere [33–36]. In the preparation of silver organosol, isopropanol was used as the organic dispersion medium, which was degassed in the vacuum prior to synthesis by alternating freeze-thaw cycles. Isopropanol (Fluka, 99.8%) was dried over zeolites 4 Å, and distilled under argon.

Silver was evaporated by resistive heating from a tantalum boat. During the synthesis, a vacuum of no more than 10−<sup>2</sup> Pa was maintained in a 5-L quartz glass reactor with using a high-vacuum post. In a typical experiment, 120–150 mL of organic reagen<sup>t</sup> was used in the synthesis and 0.1–0.12 grams of metal were evaporated. The supply of the organic reagen<sup>t</sup> was adjusted with a fine adjustment valve. Before the synthesis, the glass reactor flask was evacuated, immersed in a vessel with liquid nitrogen, and then an organic reagen<sup>t</sup> isopropanol was fed, which was condensed on the cooled walls of the reactor together with the metal vapors for about 1.5 h.

After the synthesis was completed, the cooling was stopped; the reactor was cut off from the vacuum post with a slide gate. Argon was fed to the reactor; the co-condensate of metal and organics was heated to the melting point. The obtained colloidal silver solution in isopropanol was impregnated with a bandage, which was before modification in a vacuum flask. The excess organosol was removed by drying in a vacuum of 1 Pa at a temperature of 80 ◦C.

Four strains of Gram-negative microbes (*Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Escherichia coli*, *Moraxella* spp.) and two strains of Gram-positive bacteria (*Staphylococcus aureus*, *Staphylococcus* *haemolyticus*) were used. The strains were sowed from purulent wounds of patients of surgical departments in Grodno (Belarus).

The sampling for microbiological testing was performed in patients with purulent wounds using standard disposable sterile tampons by Heinz Herenz company, within an hour the material was delivered to a microbiological laboratory where a pure microbial culture was isolated and identified with a BioMerieux Vitek apparatus, antibiotic susceptibility of each microorganism was defined.

The bacteria sensitivity to the six most commonly used antibiotics in surgical hospitals in Belarus (amoxicillin (ACC), cephalexin (CFL), gentamicin (GEN), ciprofloxacin (CIP), cefazolin (CZ), erythromycin (ERI)) is presented in Table 8. Definition of antibacterial sensitivity was performed by diffusion into agar using discs.


**Table 8.** Antibacterial sensitivity of the strains of microbes used in the study.

S = the microorganism is sensitive to this antibiotic, I = moderate resistance of the microorganism to the antibiotic presented, R = the microorganism is resistant to this antibiotic. ACC = amoxicillin, CFL = cephalexin, GEN = gentamicin, CIP = ciprofloxacin, CZ = cefazolin, ERI = erythromycin.

Then, the isolated culture of the microbe was inoculated on sloping beef-extract agar, after 24 h cultivation, a sterile 0.85% solution of NaCl (5 mL) was flushed and diluted to the desired concentration with the same solution by successive inoculation into Petri dishes with agar of different concentrations of the microorganism. The desired concentration corresponded to the formation, after seeding, with a pipette, of 0.1 mL of the microbe suspension and placing the Petri dish in a 24 h thermostat for about 100 CFU. The following concentrations were used in the study: 0.5 × 10−<sup>6</sup> for *Staphylococcus aureus*, 0.5 × 10−<sup>5</sup> for *Staphylococcus haemolyticus*, *Klebsiella pneumoniae* and *Escherichia coli*, for *Pseudomonas aeruginosa* 1 × 10−<sup>7</sup> and for *Moraxella* spp. 1 × 10−6.

The obtained suspension of microorganisms (0.1 mL) was seeded on a Petri dish with beef-extract agar. Then, two bands of medical gauze bandage were placed on each cup, measuring 1.5 × 4 cm. A standard medical gauze bandage was used as a control, medical gauze bandage containing AgNPs was used in the experimental groups. After that, all Petri dishes were placed in a thermostat at a temperature of 37.0 ◦C on 1 day for cultivation. After 24 h, CFU were counted on both sides of the edge of the bandage at a distance from the edge equal to the diameter of one colony ad oculus and using a binocular magnifier glass.

In addition, the percentage decrease in the number of CFU was calculated by the Formula (1):

$$\text{Percentage reduction of CFU (\%)}=100 \times (\text{A}-\text{B})/\text{A} \tag{1}$$

where A is the average value of the number of CFU along the edge of the bandage in the control groups; and B is the average value of the number of CFU in the groups with medical gauze bandage containing AgNPs.

Microbial strains were cultivated on the Pronadisa beef-extract agar manufactured by Laboratorios Conda, S.A., which was prepared and sterilized according to the instructions of the manufacturer.

Sterilization of the experimental and control samples of the bandage was carried out by autoclaving at 121 ◦C during 16 min with a Cliniklav-25 vacuum autoclave.

Laser irradiation was performed by a Rodnik-1 therapeutic laser apparatus, a wavelength of 470 ± 30 nm (blue region of the spectrum), with a power of 5 mW, for 5 min. Laser irradiation was performed 2 and 4 h after inoculating the Petri dish and placing the dressing on it.

The schematic view of the irradiation experiment is shown on the Figure 12.

Figure 13 shows a Petri dish with agar, and two pieces of bandage placed on it irradiated with a laser.

**Figure 13.** The scheme of the laser experiment; 1—laser and 2—two pieces of the bandage.

The frequency selected in the study is determined by the frequency of plasmon resonance of AgNPs, the power and time of the exposure are determined experimentally.

Statistical processing of the results was carried out using the program Statistica 10.0. The difference between the groups was estimated using the nonparametric Mann-Whitney U-test at a given 5% significance level.

To assess the degree of increase in the antibacterial effect, the difference between the percentage decrease in the number of CFUs in the group where a AgNPs-containing bandage without laser irradiation was used and the percentage reduction in the group with laser irradiation was calculated 4 h after seeding the Petri dish and placing the bandage on it using the Formula (2):

Difference in the percentage reduction of CFU (%) = 100 × (A − B)/A = 100 × (A − C)/A (2)

where A is the average value of the number of CFU along the edge of the bandage in the control group (ordinary medical gauze bandage without laser irradiation); B is the average value of the CFU in the band with medical gauze bandage containing AgNPs with laser irradiation four hours after seeding the Petri dish and placing the bandage on it; and C is the average value of the number of CFUs in the group with medical gauze bandage containing AgNPs without laser irradiation.

Micrographs of the Ag-cotton sample were obtained by transmission electron microscopy (TEM) using a JEOL JEM 2100F/UHR instrument with a resolution of 0.1 nm. Prior to the test, 0.1 g of the sample was placed in 30 mL of isopropanol and sonicated for 300 s. A drop of the resulting mixture was placed on a carbon-coated copper TEM grid and dried for 1 h. The size of the Ag-containing particles was determined as the maximum linear dimension. To construct a histogram of the particle size distribution, the TEM data on 192 particles were processed. Identification of the chemical composition of the particles and the surface of the samples was carried out using an energy dispersive analysis (EDA) on a JED-2300 instrument. The interplanar distances in structures visible in high resolution TEM photographs were calculated using the electron diffraction patterns obtained with the fast Fourier transform in the ImajeJ 1.4 code. To assign the electron diffraction patterns to the crystallographic faces of the silver compounds, a crystallographic JCPDS database was used.

The X-ray photoelectron spectra were recorded using a XSAM-800 spectrometer (Kratos, Manchester, UK) with Mg Kα radiation at an operating power of 90 W of the X-ray tube in the

fixed analyzer transmission mode at ~20 ◦C and a pressure in the analytical chamber of ~10−<sup>8</sup> Pa. Survey and high-resolution spectra of appropriate core levels were recorded with step sizes of 1 and 0.1 eV, respectively. The photoelectron spectra were approximated by Gauss function or the sum of Gauss functions, and the background caused by secondary electrons and photoelectrons that lost energy, was approximated by the straight line. The energy scale of spectrometer was calibrated according to the standard procedure, while considering the following binding energies: 932.7, 368.3, and 84.0 eV for Cu2p3/2, Ag3d5/2, and Au4f7/2, respectively. Quantification was performed using atomic sensitivity factors included in the software of the spectrometer. The samples were fixed to the sample holder by double-sided conductive adhesive tape. Sample charging was corrected by referencing to the C-C/C-H state deconvoluted in the C ls spectrum (284.8 eV).
