A Novel Biodegradable Composite Polymer Material Based on PLGA and Silver Oxide Nanoparticles with Unique Physicochemical Properties and Biocompatibility with Mammalian Cells

A method for obtaining a stable colloidal solution of silver oxide nanoparticles has been developed using laser ablation. The method allows one to obtain nanoparticles with a monomodal size distribution and a concentration of more than 108 nanoparticles per mL. On the basis of the obtained nanoparticles and the PLGA polymer, a nanocomposite material was manufactured. The manufacturing technology allows one to obtain a nanocomposite material without significant defects. Nanoparticles are not evenly distributed in the material and form domains in the composite. Reactive oxygen species (hydrogen peroxide and hydroxyl radical) are intensively generated on the surfaces of the nanocomposite. Additionally, on the surface of the composite material, an intensive formation of protein long-lived active forms is observed. The ELISA method was used to demonstrate the generation of 8-oxoguanine in DNA on the developed nanocomposite material. It was found that the multiplication of microorganisms on the developed nanocomposite material is significantly decreased. At the same time, the nanocomposite does not inhibit proliferation of mammalian cells. The developed nanocomposite material can be used as an affordable and non-toxic nanomaterial to create bacteriostatic coatings that are safe for humans.


Introduction
Bacterial antibiotic resistance is an urgent problem of world health care. In the world, the death of patients due to antibiotic-resistant bacteremia exceeds total death from diabetes mellitus and cancer [1]. One of the ways to overcome antibiotic resistance is to use metal oxides nanoparticles (NPs) [2], in particular, silver oxide NPs [3]. The presumptive mechanisms of the antibacterial action of NPs of metal oxides are ROS generation, violation of the integrity of the bacterial cell wall, inhibition of ATPase activity, genotoxic and photocatalytic actions, violation of bacterial DNA replication processes, and suppression of the expression of numerous genes [4][5][6][7][8][9][10][11]. The silver oxide nanoparticles advantages are high antibacterial efficiency, good biocompatibility with mammalian cells, and low synthesis cost [12][13][14].
Obviously, the range of applications for PLGA-based materials can be significantly expanded by acquiring new exclusive properties. The idea of creating a material based on PLGA and silver is not new. Previously, several materials with silver salts [33], silver nanoparticles [34], and even silver fibers [35] were prepared. Nanocomposites based on PLGA and Ag NPs demonstrate good bio-compatibility to mammalian cells on the one hand, and antibacterial action, on the other [37,38]. In addition, PLGA/Ag NPs nanocomposites are biodegradable, so they can be used in surgery [39]. The synthesis of a polymer in the presence of silver NPs allows one to control the physicochemical nature of the resulting composire's properties. It expands the possible range of their application [40,41].
Silver oxide nanoparticles have one important advantage over metallic silver nanoparticles [37]. Nanoparticles of their silver oxide dissolve better in aqueous solutions and, as a consequence, lead to a more rapid increase in the concentration of silver ions in aqueous solutions. Currently, an active search is under way for ways to improve the antibacterial properties of PLGA/Ag NPs nanocomposites, including a modification of the method for synthesizing nanoparticles [42]. It should be noted that most materials based on metallic silver nanoparticles did not have the best mechanical and physicochemical properties, and the nanoparticles themselves were randomly distributed in the material. In the present study, using laser ablation, we synthesized silver oxide nanoparticles and fabricated a nanocomposite with a gradient concentration of nanoparticles in the bulk.
Laser ablation is a method of synthesizing NPs of metals and their oxides by laser irradiation of a target metal contained in a solvent. This method makes it possible to fairly finely control the size of the resulting NPs due to the synthesis conditions (radiation parameters and the solvent used) [43][44][45][46]. Additional advantages of laser ablation are its high speed and low cost of synthesis [47]. The unusual physicochemical properties of the nanocomposite have been investigated, its bacteriostatic activity has been determined, and its biocompatibility has been studied.

Silver Oxide Nanoparticles Preparation and Characterization
Silver oxide nanoparticles were synthesized by laser ablation in liquid with pulsed ytterbium-doped fiber laser (Pokkels, Moscow, Russia). We used commercially available Ag ≥ 99.9% silver (SigmaAldrich, Saint Louis, MO, USA) as a target metal. The target metal was placed in a container with 10 mL of deionized Milli-Q water (with a resistivity of at least 18 MΩ/cm) so that the upper surface of a piece of metal was immersed in water by~1 mm. The target metal was irradiated with a laser beam directed from top to bottom. The tank was continuously cooled using a water-cooling system. The laser beam was focused using an F-theta objective (F = 10 cm). The calculated diameter of the laser beam at the waist area was~50 µm. To ensure uniform irradiation of the target metal surface, the position of the laser beam was constantly changed using a galvo-mirror system with a scanning speed of 500 mm/s, and the area of the irradiation zone was~1 cm 2 . The time and parameters of the irradiation were selected in preliminary experiments [48]. Parameters of laser pulses were λ = 1064 nm, τ = 4-200 ns, energy 2 mJ, average power ≤ 20 W, and repetition frequency 20 kHz. The irradiation time was 10 min. The schematic diagram of the laser ablation setup is shown in Figure 1. irradiation were selected in preliminary experiments [48]. Parameters of laser pulses were λ = 1064 nm, τ = 4-200 ns, energy 2 mJ, average power ≤ 20 W, and repetition frequency 20 kHz. The irradiation time was 10 min. The schematic diagram of the laser ablation setup is shown in Figure 1. To obtain nanoparticles uniform in size, the resulting colloid of nanoparticles was centrifuged in an analytical centrifuge. The speed and time of centrifugation were selected in such a way as to precipitate NPs with a size of more than 60 nm for their subsequent removal. The distribution of Ag NPs on size and zeta potential was evaluated by dynamic light scattering method with Zetasizer Ultra Red Label (Malvern Panalytical, Malvern, UK). The measuring features were described earlier [49]. The size, shape, surface structure, and element composition of nanoparticles were studied with Libra 200 FE HR transmission electron microscope (Carl Zeiss, Jena, Germany). Atomic force microscopy was performed using an SII Nanopics 2100 atomic force scanning microscope (KLA-Tencor, Milpitas, CA, USA), in the dynamic force microscopy (DFM) mode. The NPs aqueous colloids spectra were obtained with Cintra 4040 spectrophotmeter (GBC Scientific Equipment, Braeside, Australia). The features of the optical spectrum have been described in detail earlier [50].

Composite Fabrication, Production of Plates from Composite Material, and Rheological Properties
The PLGA-based composite with silver oxide NPS was synthesized by the low-temperature technology developed by us earlier [47]. Production of plates from composite material. The PLGA-based material was heated to 40 °C and rolling up to 1000 μm thickness film. Further films were cut into 20 mm × 25 mm (area 10 cm 2 ) rectangle piece. Each film piece was placed in 20 mL water. Nanoparticles were added to the material at the stage of polymerization at concentrations of 0.001, 0.01, or 0.1% of the weight of the polymer matrix [51].

Thermal Characteristics Assay
Thermal characteristics assay was carried out by differential scanning calorimetry with DSC 3 Excellence (Mettler Toledo, Columbus, OH, USA) [52]. Thermograms in the heating and cooling modes were constructed to assess thermal characteristics. The temperatures of glass transition (Tg) and heat capacity change (ΔCp) were also evaluated at different dopant conentrations. To obtain nanoparticles uniform in size, the resulting colloid of nanoparticles was centrifuged in an analytical centrifuge. The speed and time of centrifugation were selected in such a way as to precipitate NPs with a size of more than 60 nm for their subsequent removal. The distribution of Ag NPs on size and zeta potential was evaluated by dynamic light scattering method with Zetasizer Ultra Red Label (Malvern Panalytical, Malvern, UK). The measuring features were described earlier [49]. The size, shape, surface structure, and element composition of nanoparticles were studied with Libra 200 FE HR transmission electron microscope (Carl Zeiss, Jena, Germany). Atomic force microscopy was performed using an SII Nanopics 2100 atomic force scanning microscope (KLA-Tencor, Milpitas, CA, USA), in the dynamic force microscopy (DFM) mode. The NPs aqueous colloids spectra were obtained with Cintra 4040 spectrophotmeter (GBC Scientific Equipment, Braeside, Australia). The features of the optical spectrum have been described in detail earlier [50].

Composite Fabrication, Production of Plates from Composite Material, and Rheological Properties
The PLGA-based composite with silver oxide NPS was synthesized by the lowtemperature technology developed by us earlier [47]. Production of plates from composite material. The PLGA-based material was heated to 40 • C and rolling up to 1000 µm thickness film. Further films were cut into 20 mm × 25 mm (area 10 cm 2 ) rectangle piece. Each film piece was placed in 20 mL water. Nanoparticles were added to the material at the stage of polymerization at concentrations of 0.001, 0.01, or 0.1% of the weight of the polymer matrix [51].

Thermal Characteristics Assay
Thermal characteristics assay was carried out by differential scanning calorimetry with DSC 3 Excellence (Mettler Toledo, Columbus, OH, USA) [52]. Thermograms in the heating and cooling modes were constructed to assess thermal characteristics. The temperatures of glass transition (Tg) and heat capacity change (∆C p ) were also evaluated at different dopant conentrations.

Hydrogen Peroxide Concentration Measurement
The measurement of hydrogen peroxide concentration was carried out by highly sensitive modified chemiluminescence with Biotox-7A-USE ultrasensitive chemiluminometer (ANO Engineering Center-Ecology, Moscow, Russia). This method is based on luminol oxidation by horseradish peroxidase in presence of p-iodophenol [48]. The measurement and calibration procedure was described earlier for different cases [53][54][55]. The samples were placed in 1 mL of a "counting solution" (1 µM Tris-HCl buffer with pH 8.5, 50 µM p-iodophenol, 50 µM luminol, 10 nM horseradish peroxidase). The "counting solution" was prepared immediately before the measurement. The minimum H 2 O 2 concentration evaluated by modified chemiluminescence method is <1 nM [55].

Mesurement of Long-Lived Reactive Protein Species Concentrations
Concentrations of long-lived reactive protein species (LRPS) were measured by chemiluminescence of X-irradiated protein solutions [58,59]. In this method, free radicals amount evaluated by light quanta emission during free-radical interactions. The experiments were carried out at room temperature, in the dark, and in plastic polypropylene vials for liquid scintillation counting (Beckman, Brea, CA, USA). We used large volumes (20 mL) in comparison with standard volume (0.1 mL) to improve (in ≤200 times) the sensitivity of method [60]. All samples were irradiated by X-rays and incubated 30 min after X-rays exposure, and chemiluminescence intensities were measured with Biotox-7A-USE chemiluminometer (ANO Engineering Center-Ecology, Moscow, Russia). The non-treated by X-radiation proteins were used as controls. The detailed description of method may be found in [61].

Mesurement of 8-Oxoguanine Concentration
Concentrations of 8-oxoguanine in samples were measured by a non-competitive enzyme-linked immunosorbent assay (ELISA) with anti-8-oxoguanin monoclonal antibodies [62]. Before measuring, samples of DNA (350 µg/mL) were boiled 5 min in water bath for and cooled 3-4 min in ice to induce DNA denaturation. Each sample (42 µL) was added into well of 98-well plate. Wells with samples were dried by incubation 3 h at 80 • C to adsorb DNA on plate bottom surface. Blocking of nonspecific binding was carried out by incubation of samples with 300 µL of 1% skimmed milk (in 0.15 M Tris-HCl buffer, pH 8.7, supplemented by 0.15 M NaCl) overnight and at room temperature. Wells were washed twice (300 µL/well) with 50 mM Tris-HCl buffer (pH 8.7) supplemented by 0.15 M NaCl and 0.1% Triton X-100. Further, 100 µL/well of anti-8-OG antibodies in appropriate dilution was added in each well. Wells were washed twice as described previously after 20 min incubation. Further samples were incubated in 80 µL/well solution of horseradish peroxidase-conjugated with secondary antibodies (1:1000 dulition) in blocking buffer and were washed 3 times as described previously. Subsequently, 100 µL of chromogenic substrate (18.2 mM ABTS and hydrogen peroxide (2.6 mM) in 75 mM citrate buffer, pH 4.2) was added to each well [63]. After 15 min incubation, 50 µL/well of 1.5 mM NaN 3 was added to 0.1 M citrate buffer (pH 4.3) to stop the reaction [63]. The optical density of samples at 405 nm was evaluated with photometer (Titertek Multiscan, Vantaa, Finland). The method was described in more detail earlier.

Assay of Bacteriostatic Activity
Bacteriostatic activity was evaluated by experiments with Gram-negative Escherichia coli bacteria (LenReaktiv, St. Petersburg, Russia). E. coli were cultured and subcultured in LB medium in sterile solution by standard protocol [58]. Bacterial cells concentrations in liquid Materials 2021, 14, 6915 5 of 18 medium were evaluated by spectrophotometry with drop UV5Nano Excellence (Mettler Toledo, Columbus, OH, USA). Equal amount of BL medium with same concentration of E. coli was inflicted on previously sterilized with 70% ethanol nanocomposite film (size 10-15 mm and 1000 µm thickness) in sterile hoop and sealed by glass slide. Bacteria were cultures 24 h, at 37 • C, approximately 150 rpm in shaker incubator ES-20 (Biosan). After incubation, concentration of bacteria was measured by spectrophotometry as described above [64].

Assay of Biocompatibility with Mammalial Cells
Studies of biocompatibility were carried out on SH-SY5Y cell line of human neuroblastoma. The SH-SY5Y is a standard cell model. SH-SY5Y cell line is a subclone from the SK-N-SH cells, which were isolated from the four-year-old female patient with neuroblastoma. The differentiation is the process of implementing a genetically determined program for the formation of a specialized cell phenotype, reflecting their ability regarding certain profile functions. SH-SY5Y cells are also interesting in that they can grow not only in monolayers but also form cell aggregates, which also take root on substrates SH-SY5Y usually used in the study of cells proliferation and differentiation in different conditions. For example, SH-SY5Y cells can spontaneously divide into one of two phenotypes, similar to "neuroblast" or "epithelial" phenotypes [65]. The SH-SY5Y cells were cultured by standard protocol in DMEM (Biolot, Moscow, Russia) supplemented with 10% fetal bovine serum (Gibco, USA) and 30 µg/mL gentamicin in a CO 2 incubator (Binder, Tuttlingen, Germany). SH-SY5Y cell (10 4 cells/cm 2 ) were seeded on material samples (20 mm × 20 mm) in 35 mm Petri dishes (1 sample/dish) in 3 mL cell culture medium and cultured for 3 days in CO 2 incubator. Cells viability assay were carried out by fluorescent microscopy. Cells were stained for 10 min at 37 • C by 2 µg/mL Hoechst 33342 (Sigma, Saint Louis, MO, USA) to indicate all cells and 2 µg/mL propidium iodide (Sigma, USA) to indicate death cells ( Figure 1). Stained cells were washed by phosphate buffer and analyzed with confocal microscope Leica DMI6000 (Leica, Munich, Germany). At least 500 cells were analyzed in each sample [66].
Proliferation assay was carried out on cells in logarithmic growth phase (3 days of growth on nanocomposite). We used a number of cells in a state of mitosis (mitotic index) to estimate cell proliferation. Cells were stained with the Hoechst 33342 fluorescent dye at 15 min and further analyzed by fluorescence microscopy. Depending on distribution of chromatin in nuclei, cells were categorized into mitosis stage: prophase (P), metaphase (M), anaphase (A), and telophase (T). On each sample surface, at least 500 cells were analyzed. The mitotic index (MI) was calculated by the formula MI = (P + M + A + T)/N × 100%, where P, M, A, and T are the numbers of cells at the stage of prophase, metaphase, anaphase, and telophase, respectively, and N is the total number of analyzed cells [67]. A typical stained cell culture specimen is shown in Figure 2.

Statistic
The all data are presented as means ± SE. Data from at least three independent experiments were used in each experimental condition. Significance of differences between samples means was evaluated with two sample two-tailed t-tests. GraphPad Prims (Graph-Pad Software, San Diego, CA, USA), Origin (OriginLab Corporation, Northampton, MA, USA), and SigmaPlot (Systat Software Inc., San Jose, CA, USA) software were used to data statistical processing.

Statistic
The all data are presented as means ± SE. Data from at least three independent experiments were used in each experimental condition. Significance of differences between samples means was evaluated with two sample two-tailed t-tests. GraphPad Prims (GraphPad Software, San Diego, CA, USA), Origin (OriginLab Corporation, Northampton, MA, USA), and SigmaPlot (Systat Software Inc., San Jose, CA, USA) software were used to data statistical processing.

Results
Ag2O NPs were successfully synthesized by laser ablation in water. The concentration of nanoparticles in the working fluid of the ablation reactor was determined using the DLS Malvern Ultra Red Label ( Figure 3a) and was almost 350 million particles per mL. The nanoparticle size distribution is monomodal and rather narrow (Figure 3a). The hydrodynamic diameter of nanoparticles is ~35 nm, and the half-width of the distribution is 25-45 nm. The distribution of nanoparticles by zeta potential values is also monomodal (Figure 3b). The average ζ-potential of nanoparticles is −25 mV (from −8 to −40 mV). The absorption spectrum of an Ag2O NPs colloid is demonstrated in Figure 3c. It is shown that the absorption spectrum of nanoparticles corresponds to the absorption spectrum of silver oxide nanoparticles. The size of the nanoparticles was independently verified by TEM (Figure 3d). It is shown that the bulk of the nanoparticles in the photographs have a size from 20 to 40 nm.

Results
Ag 2 O NPs were successfully synthesized by laser ablation in water. The concentration of nanoparticles in the working fluid of the ablation reactor was determined using the DLS Malvern Ultra Red Label ( Figure 3a) and was almost 350 million particles per mL. The nanoparticle size distribution is monomodal and rather narrow (Figure 3a). The hydrodynamic diameter of nanoparticles is~35 nm, and the half-width of the distribution is 25-45 nm. The distribution of nanoparticles by zeta potential values is also monomodal (Figure 3b). The average ζ-potential of nanoparticles is −25 mV (from −8 to −40 mV). The absorption spectrum of an Ag 2 O NPs colloid is demonstrated in Figure 3c. It is shown that the absorption spectrum of nanoparticles corresponds to the absorption spectrum of silver oxide nanoparticles. The size of the nanoparticles was independently verified by TEM (Figure 3d). It is shown that the bulk of the nanoparticles in the photographs have a size from 20 to 40 nm.
Silver is known to form several different oxides. The elemental composition was evaluated by energy dispersive X-ray spectrometry ( Figure 4). It was found that the nanoparticles obtained by us consist of two chemical elements: silver and oxygen. The ratio of atoms Ag/O = 1.94; thus, it can be assumed that the nanoparticles are predominantly composed of Ag 2 O.
The resulting NPs were transferred into PLGA polymer using a previously developed low-temperature technology [47]. The composite PLGA/Ag 2 O NPs had visually smooth surfaces. The surface relief was studied using atomic force microscopy (Figure 5a). It is shown that the composite material has no significant flaws, cavities, holes, cracks, or breaks. Both the analytical slow scan and the high-speed scan did not reveal any defects. The roughness of the test samples was ≤0.633 nm (Figure 5b).
Atomic force microscopy provides information on the surface topology but does not answer the question of the relative position of both polymer and nanoparticles in the composite material. It is known that PLGA and silver oxide differ significantly in their optical properties from each other. In this regard, we applied modulation interference microscopy, which allows us to isolate patterns in materials that differ in refractive index and other optical properties. The refractive index was determined at the wavelength of the laser microscope.
( Figure 3b). The average ζ-potential of nanoparticles is −25 mV (from −8 to −40 mV). The absorption spectrum of an Ag2O NPs colloid is demonstrated in Figure 3c. It is shown that the absorption spectrum of nanoparticles corresponds to the absorption spectrum of silver oxide nanoparticles. The size of the nanoparticles was independently verified by TEM (Figure 3d). It is shown that the bulk of the nanoparticles in the photographs have a size from 20 to 40 nm.  Silver is known to form several different oxides. The elemental composition was evaluated by energy dispersive X-ray spectrometry ( Figure 4). It was found that the nanoparticles obtained by us consist of two chemical elements: silver and oxygen. The ratio of atoms Ag/O = 1.94; thus, it can be assumed that the nanoparticles are predominantly composed of Ag2O.  The refractive index of the unmodified PLGA is 1.47 at 405 nm, and the refractive index of the silver oxide is 1.19 at 405 nm. Thus, the refractive index of PLGA and silver oxide nanoparticles differs by almost 0.3 units. It has been shown that PLGA without nanoparticles does not have any pronounced surface structure (Figure 6a). When silver oxide nanoparticles are added to the polymer at a concentration of 0.001%, the composite surface breaks down into domains that differ significantly in the change in the phase of the laser radiation (Figure 6b). It should be noted that domains are rather large, on average 0.5 µm × 0.5 µm, and domains of much larger sizes are encountered. The domains begin to merge with each other, forming elongated structures several micrometers long, during increasing of dopant concentration in the polymer to 0.01 or 0.1% (Figure 6c,d). The data obtained make it possible to assert that the nanoparticles in the polymer are not evenly distributed. It can be assumed that domains with a large phase incursion are the centers of concentration of nanoparticles in the polymer.
Thermograms of the composite material obtained in the heating and cooling mode are shown in Figure 7a. The glass transition of the material is clearly visible 320-325 K, which is observed for all samples. Based on the results of differential scanning calorimetry, the glass transition temperatures Tg and the change in heat capacity ∆C P of the samples under study were determined, the concentration dependences of which are shown in Figure 7b,c, respectively. The glass transition temperature is in the range 319-321 K and corresponds to the literature data for pure PLGA [52]. The ∆C P values vary within 7% of 0.5 J/(g × K), which is comparable to the measurement error.
Silver is known to form several different oxides. The elemental composition was evaluated by energy dispersive X-ray spectrometry (Figure 4). It was found that the nanoparticles obtained by us consist of two chemical elements: silver and oxygen. The ratio of atoms Ag/O = 1.94; thus, it can be assumed that the nanoparticles are predominantly composed of Ag2O.   The resulting NPs were transferred into PLGA polymer using a previously developed low-temperature technology [47]. The composite PLGA/Ag2O NPs had visually smooth surfaces. The surface relief was studied using atomic force microscopy ( Figure  5a). It is shown that the composite material has no significant flaws, cavities, holes, cracks, or breaks. Both the analytical slow scan and the high-speed scan did not reveal any defects. The roughness of the test samples was ≤0.633 nm (Figure 5b). Atomic force microscopy provides information on the surface topology but does not answer the question of the relative position of both polymer and nanoparticles in the composite material. It is known that PLGA and silver oxide differ significantly in their optical properties from each other. In this regard, we applied modulation interference microscopy, which allows us to isolate patterns in materials that differ in refractive index and Thermograms of the composite material obtained in the heating and cooling mode are shown in Figure 7a. The glass transition of the material is clearly visible 320-325 K, which is observed for all samples. Based on the results of differential scanning calorimetry, the glass transition temperatures Tg and the change in heat capacity ΔCP of the samples under study were determined, the concentration dependences of which are shown in Figure 7b,c, respectively. The glass transition temperature is in the range 319-321 K and corresponds to the literature data for pure PLGA [52]. The ΔCP values vary within 7% of 0.5 J/(g × K), which is comparable to the measurement error.   Addition of Ag2O NPs at concentration of 0.001 in composite increases rate of H2O2 generation in comparison with the control by almost two times. The rate of H2O2 generation increases more than three times at Ag2O NPs concentration of 0.01% and almost five times at concentration 0.1%. The effect of the composite material on the generation of hydroxyl radicals has been investigated. PLGA polymer did not influence the generation of OH-radicals. In this case, composite PLGA/Ag2O NPs significantly increase OH-radicals generation.
Elevated generation of ROS often leads to biomacromolecule modification or destruction. The effect of composite PLGA/Ag2O NPs on the formation of 8-oxoguanine in DNA was evaluated (Figure 9a). PLGA did not change the rate of 8-oxoguanine generation. Ad- dition of Ag2O NPs to the polymer significantly increased the rate of 8-oxoguanine generation. At a concentration Ag2O NPs of 0.001%, the rate of 8-oxoguanine generation increases by 60% compared to the control, at a concentration of 0.01% by 130%, and at a concentration of 0.1%, by almost three times. The effect of composite PLGA/Ag2O NPs on the formation of LRPS was studied (Figure 9b). It was shown that PLGA containing no silver oxide nanoparticles did not influence on LRPS generation or LRPS decay rate. It was found that when Ag2O NPs appear in the polymer, the rate of LRPS generation increases significantly. The increasing in the rate The effect of composite PLGA/Ag 2 O NPs on the formation of LRPS was studied (Figure 9b). It was shown that PLGA containing no silver oxide nanoparticles did not influence on LRPS generation or LRPS decay rate. It was found that when Ag 2 O NPs appear in the polymer, the rate of LRPS generation increases significantly. The increasing in the rate relative to the control by about 40% is observed at a concentration of silver oxide nanoparticles of 0.001%. With an increase in the concentration of Ag 2 O NPs to 0.01%, the rate of LRPS generation increases by almost two times, at a concentration of 0.1%-more than 2.5 times. At the same time, silver oxide nanoparticles have almost no effect on the average half-life of active forms of proteins. In all groups, the half-life is about 4-5 h.
The effect of composite PLGA/Ag 2 O NPs on E. coli growth was studied ( Figure 10). It was shown that PLGA containing no silver oxide nanoparticles did not influence on E. coli bacteria growth. The addition of Ag 2 O NPs in the polymer at a concentration of 0.001% decreased the density of bacterial cultures grown by more than half compared to the control, at a Ag 2 O NPs concentration of 0.01% (by almost 95%), and at a Ag 2 O NPs concentration of 0.1% (by more than by 97%). The effect of composite PLGA/Ag 2 O NPs on the viability of eukaryotic cells was investigated (Figure 11a). The almost of non-viable cells on control substrates (culture plastic) was no more 4%. Approximately the equal almost of death cells was observed on PLGA without silver oxide nanoparticles or 0.001% silver oxide nanoparticles. At the same time, when using the TiNbTaZr medical alloy as a substrate, an almost one and a half greater number of nonviable cells was determined (almost 6%). Number of nonviable cells were about 5.5% of the cells in case of PLGA and silver oxide nanoparticles (0.01%) and 6.5% of the cells in case of PLGA and silver oxide nanoparticles (0.1%).
The influence of the composite material on the mitotic index (percentage of cells in a dividing state) was investigated (Figure 11b). The number of proliferated cells was determined by fluorescence microscopy. The mitotic index for cells cultured on the culture plastic is 1.2%. In the case of medical alloy TiNbTaZr, the mitotic index is almost 2%. When cells were grown on composite PLGA/Ag 2 O NPs, the mitotic index was 1.0-1.4%, which did not statistically differ from the control values.
Density of the cell culture growth on plastic is on average 1000 cells/mm 2 of surface ( Figure 11c). The cell culture density reaches almost 1500 cells/mm 2 in case of grown on TiNbTaZr medical alloy. When cells were grown on composite PLGA/Ag 2 O NPs, the density of the cell culture was 800-1100 cells/mm 2 , which did not statistically differ from the control values.  The surfaces of composite PLGA/Ag 2 O NPs have been shown to be suitable for cell life, growth, and proliferation ( Figure 11d). Moreover, the degree of suitability is comparable to that of control. At the same time, the degree of suitability of the composites is somewhat lower than that of the TiNbTaZr medical alloy. With the duration of the experiment for 72 h of growth, a completely confluent monolayer of cells is not formed on all materials. Only individual elements of the monolayer formed. Maximum confluence of cells on all studied material was about 70-75% of the surface area.

Discussion
The average size of the synthesized NPs was 25-50 nm (Figure 3a,d). In our work, the average NP size is somewhat smaller than in most other works [16][17][18]22,68]. This tendency can be explained by the differences in the method of synthesizing silver oxide NPs: in these studies, the coprecipitation method and polymer matrices were used that were different from PLGA. The average value of the ζ-potential of the NPs synthesized by us is -30 mV (Figure 3b), which corresponds to stable NPs [69]. The absorption spectra with a peak at 450 nm correspond to silver oxide NPs [20,22]. According to TEM data with elemental analysis, we obtained an Ag:O ratio of~2:1 (Figure 4), which indicates that the nanoparticles we obtained mainly consist of silver (I) Ag 2 O oxide. The modulation interference microscopy method, in contrast to atomic force microscopy ( Figure 5), allows one to assess the uneven distribution of nano-and micro-sized particles [70]. Using modulation interference microscopy, we found that NPs were unevenly distributed over the polymer volume ( Figure 6).
The addition of nanoparticles to PLGA alters the behavior of the polymer during heating and cooling, increases the temperature of glass transition (Tg), and decreases the composite heat capacity compared to the polymer without a dopant (Figure 7). An increase in the temperature of glass transition, and heat-capacity decrease, may indicate a decrease in the internal viscosity of the polymer [52]; thus, the addition of Ag 2 O NPs modifies the physical properties of the nanocomposite. The largest drop in the glass transition temperature and an increase in heat capacity were observed at a dopant concentration of 0.01%. The results show that the Tg can be adjusted by changing the volume fraction of the nanoparticles. With an increase in the volume fraction of NPs, a decrease in Tg is observed first and then an increase in Tg. The nonlinear dependence of Tg and thermal conductivity of the nanocomposite on the dopant concentration was found by us in previous studies and is described in the literature [71,72]. The nonlinear character of the dependence of the Tg of the nanocomposite on the dopant concentration was also found in mathematical modeling [73]. According to the literature, the main mechanism of the complex change in Tg with increasing NPs concentration is competition between the attraction of nanoparticles to polymer chains and rapid diffusion of nanoparticles at the initial stage of polymer composite synthesis [73]. The aggregation of NPs with an increase in their concentration in the polymer matrix can also be one of the mechanisms for changing the properties of the polymer matrix. Aggregation of Ag 2 O NPs is noted during the synthesis by the coprecipitation method [74]. In addition, we detected the formation of Ag 2 O NPs clusters in the nanocomposite by the MIM method, which indicates the role of NP aggregation in the polymer in the regulation of the physical properties of the nanocomposite ( Figure 6).
ROS generation is a general mechanism of nanomaterial toxicity. Excess ROS generation lead oxidative cell stress and disturbance of redox functions and metabolism [75,76]. However, ROS may play physiological role in cell signal transduction and regulation of cell responses to mitogens [77]. PLGA-based material does not influence H 2 O 2 and OH-radical generation ( Figure 8). This fact differs PLGA from some other polymer material, which can increase ROS generation rate [78,79]. Therefore, PLGA is more suitable polymer matrix to development of biocompatible nanocomposites. Development of oxidative stress includes oxidative protein modification, protein radical generation [80], and lipid peroxidation [81].
The presented Ag 2 O NPs in composite increase the rate of H 2 O 2 generation by almost 2 and 5 times at a percentage of nanoparticles 0.001-0.1% (Figure 8a). This behavior of silver oxide nanoparticles in redox reactions leading to ROS generation is in agreement with the works [82,83]. PLGA does not influence OH-radical generation (Figure 8b). Addition of Ag 2 O NPs to PLGA increases rate of OH-radical generation by 1.5-3 times, which differs from literature data [84,85]. 8-oxoguanine is an important marker of DNA oxidative damage; therefore, 8-oxoguanine generation in DNA is of great interest in medicine [86]. 8-oxoguanine can lead to the formation of mismatched nucleotides with adenine and thus lead to GC-TA transversion, which increases risk of mutations. At least four reparation pathways are realized in mammals for deleting 8-oxoguanine from DNA and for preventing its introduction into DNA. The duplicating of protection mechanisms indicates that 8-oxoguanine is an extremely serious danger for cells and that it must be quickly eliminated [87]. (Figure 9a) PLGA did not influence 8-oxoguanine generation in DNA, which differed from literature data [88,89]. The addition of silver oxide nanoparticles in PLGA significantly increases the rate of 8-oxoguanine generation. The rate of 8-oxoguanine generation increases by 1.4, 2.2, and 3 times at a concentration of Ag 2 O NPs of 0.01, 0.01, and 0.1%, respectively. LRPS are not only an important marker of oxidative stress but can also generate secondary radicals [90,91]. PLGA without nanoparticles did not increase the generation of LRPS (Figure 9b). The addition of Ag 2 O NPs increased the generation of LRPS by 25 (for 0.001% NPs), 98 (for 0.01% NPs), and 152% (for 0.01% NPs), compared to control. The generation of hydrogen peroxide, hydroxyl radical, 8-oxoguanine, and LRPS may be the mechanisms of the antimicrobial effects of the PLGA/Ag 2 O NPs nanocomposite [92].
In some cases, biomaterial may be substrate to microorganisms and infection source itself [89,92]. For example, bacteria from air and skin can contaminate medical devices and induce hospital-acquired infections [93].
Microorganisms can grow, forming biofilms on surface of some biomaterials [94]. We observed that ( Figure 10) PLGA, without silver oxide nanoparticles, did not affect E. coli growth. Bacterial growth decreased on polymer with silver oxide nanoparticles. Bacterial cultures density over the composite decreased by 65,94, and 98% at a silver oxide nanoparticles concentration of 0.001, 0.01, and 0.1%, respectively. In our study, we proposed that 0.001% of silver oxide nanoparticles is similar to minimum inhibitory concentration (MIC) 10 µg/mL. MIC, in case of silver oxide nanoparticles coated other by polyethersulfone, cellulose, and hydrogel, is above 2-8000 µg/mL (Table 1) [17][18][19]95,96]. Moreover, the nanocomposite is suitable for anchoring and spreading mammalian cells ( Figure 11). The nanocomposite has comparable biocompatibility (number of living cells, values of mitotic index, and density of cell culture and free surface) with the medical alloy TiNbTaZr or special plastic utensils for growing cells. The PLGA/Ag 2 O NPs nanocomposite obtained by us is a promising candidate for the development of materials with high antibacterial activity and good biocompatibility with human cells. Such materials can find application in surgery, in particular, in prosthetics.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.