Ag2O Nanoparticles as a Candidate for Antimicrobial Compounds of the New Generation

Antibiotic resistance in microorganisms is an important problem of modern medicine which can be solved by searching for antimicrobial preparations of the new generation. Nanoparticles (NPs) of metals and their oxides are the most promising candidates for the role of such preparations. In the last few years, the number of studies devoted to the antimicrobial properties of silver oxide NPs have been actively growing. Although the total number of such studies is still not very high, it is quickly increasing. Advantages of silver oxide NPs are the relative easiness of production, low cost, high antibacterial and antifungal activities and low cytotoxicity to eukaryotic cells. This review intends to provide readers with the latest information about the antimicrobial properties of silver oxide NPs: sensitive organisms, mechanisms of action on microorganisms and further prospects for improving the antimicrobial properties.


Introduction
Since the moment of their discovery, antibiotics have been the "golden standard" in the treatment of many bacterial infections [1,2]. Unfortunately, the uncontrolled use of overthe-counter (OTC) antibiotics available without prescription has led to the emergence of new antibiotic-resistant bacterial strains. Diseases caused by such bacteria are not amenable to treatment. This phenomenon is called antibiotic resistance [3][4][5]. The development of antibiotic resistance in bacteria led to a new wave of growth in the number of infectious diseases and the necessity to search for new antimicrobial agents [6]. One of the ways to overcome antibiotic resistance in bacteria is the use of metal and metal oxide nanoparticles (NPs) [7]. Fungal diseases are a multi-national problem. More than 150 million people in the world have severe fungal diseases. More than 1.5 million cases of fungal diseases have a lethal outcome [8]. The problem is exacerbated by the development of fungal resistance to antifungal drugs [9]. There are reports about the antifungal properties of metal oxide NPs [10,11]. Since the beginning of the COVID-19 pandemic, special attention has been given to the search for inexpensive and effective antiviral agents [12,13].
The antimicrobial properties of silver and its compounds have been known since ancient times. The first references to the use of silver are dated back to 3500-1000 B.C. In particular, silver was used for dishware production and water storage; later on, there were attempts to use silver powder to treat various diseases [14][15][16]. It has been shown many times in the literature that nanoparticles (NPs) of silver and its compounds have significant bactericidal, fungicidal and antiviral activities [17][18][19]. Ag 2 O NPs have attracted particular attention of researchers in the field of nanomaterials because of their unique properties that ensure multiple functions and a wide field of application. The most significant applications      The antimicrobial effect against Gram-positive bacteria is more pronounced than against Gram-negative ones. [73] 45 Ag 2 O NPs with polyhedral shape 400-700 Escherichia coli Bactericidal 10 µg/mL The antimicrobial effect of cubic NPs is two times higher than that of octahedral NPs.  [87] *-concentration is not directly indicated in article in µg/mL and is calculated based on description in Materials and Method sections. Original data are shown in brackets.
When assessing a ratio of reports about the bactericidal and bacteriostatic activity of Ag 2 O NPs (Table 1), we found that bacteriostatic activity was described in about 75% of studies and bactericidal activity in 25% of studies. It is worth noting that the ratio of reports about the bactericidal and bacteriostatic activity of Ag 2 O NPs (equal to 1:3) is comparable to other widely used metal oxide NPs with antimicrobial activities, for example, iron oxides or ZnO NPs [7,88]. Iron oxides or ZnO NPs demonstrated high cytotoxicity in contradistinction to Ag 2 O NPs [89][90][91]. Having the same antimicrobial activity with other metal oxide NPs and low cytotoxicity makes Ag 2 O NPs an interesting candidate for the role of new generation antiseptic. For antifungal activities, the ratio shifted towards a reduction of the fungicidal activity. Only 15% of studies indicate the presence of the fungicidal effect and 85% contain data about the fungistatic effect. Therefore, fungi have higher resistance to Ag 2 O NPs compared to bacteria. This effect can be explained by the higher resistance of eukaryotic cells to the genotoxic effect of metal ions compared to prokaryotes, in particular, due to differences in the structure of the genetic apparatus and function of the reparation systems [92][93][94].

Synthesis Methods
Methods for the synthesis of Ag 2 O nanoparticles can be divided into physical, chemical and biological, otherwise referred to as "green synthesis" [95].
Chemical methods include various types of precipitation. The simplest method is realized when mixing AgNO 3 with NaOH at high temperatures [13,58,75,96].
In this case, NP synthesis occurs in two stages described by the reaction equations: Modifications of the method are possible: the addition of strong oxidizers, for example, K 2 S 2 O 4 , and KOH as a base [19,50]. Sometimes AgNO 3 is obtained directly at the moment of synthesis upon the oxidation of silver foil with nitric acid; then, precipitation with alkali described above is performed [77]. To prevent the premature aggregation of synthesized Ag 2 O NPs, a surfactant-for example, citrate, polyethylene glycol, triethylene glycol, chitosan, urea and other compounds-can be added to the reaction mixture [40,82,[96][97][98][99]. Another method for Ag 2 O NP production is the reduction of AgNO 3 using organic acids citrate, acetate and oleic acid [45,53,56]. In the literature, this method is sometimes called the sol-gel method [100]. A method of Ag 2 O production upon the reduction of complex compounds, for example, ammoniate [Ag(NH 3 ) 2 ] x , is described [59,101]. To obtain NPs with a complex chemical composition, the drying of metal oxide NPs in the AgNO 3 solution is used, as in the case of TiO 2 /Ag 2 O NPs [47].
Chemical and physical methods used today for NP synthesis can be expensive, require high temperatures and pressure or lead to the generation of waste that is hazardous for the environment [103]. Therefore, biological methods for the synthesis of nanomaterials, the so-called "green synthesis", are preferable [26,104]. Moreover, silver oxide NPs obtained using biological methods have several advantages: low cost of synthesis, high antimicrobial activity, low cytotoxicity to mammalian cells and the possibility to use in pharmacology and biomedicine, like for NPs obtained by classical methods [105]. Similar to Ag NPs, "green" synthesis using extracts of medicinal plants is one of the methods for improving the antimicrobial properties of Ag 2 O NPs [106].
"Green synthesis" of Ag 2 O NPs consists of, as a rule, the reduction of water-soluble salt AgNO 3 in an extract of medicinal plants or cultural liquid of non-pathogenic/weakly pathogenic microorganisms [107][108][109].
However, cases of real biosynthesis of Ag 2 O NPs are described, for example, synthesis by bacteria isolated from seeds of agricultural crops and cultivated in medium with the addition of AgNO 3 [110,111] and soil bacteria Nitrobacter sp. [61]. In addition, methods for synthesis of Ag/Ag 2 O NPs by silver reduction in the medium of Fusarium oxysporum mycelium or dead biomass of yeasts [56,80] were described.

Methods for Studying Ag 2 O NPs
Dozens of methods have been applied to describe the parameters of Ag 2 O NPs. These methods are commonly used to study other Me/Me x O y NPs [26]. To determine the size and shape of Ag 2 O NPs, various microscopic methods are used: atomic force microscopy (AFM) [112], scanning tunneling microscopy (STM) [113], scanning electron microscopy (SEM) [114] and transmission electron microscopy (TEM). The indicated methods allow us to image dry NPs and assess their size, shape, distribution on the surface of composite materials. To assess the elementary composition, proportion of organic impurities and conjugates, the following methods are used: UV-vis spectroscopy [115], Fourier transform infrared spectroscopy (FT-IR) [116,117], energy dispersive spectroscopy (EDX) [118], X-ray photoelectron spectroscopy (XPS) [119] and thermal gravimetric analysis (TGA) [120].
To determine the crystalline structure of NPs, the X-ray diffraction (XRD) method is applied [121,122]. To assess the hydrodynamic radius of NPs and stability of NP colloids in solvents, the dynamic light scattering (DLS) method and measurement of zeta potential, respectively, are used [123]. Assessment of the NP surface area and rheological properties of obtained nanomaterials is carried out by differential scanning calorimetry (DSC) and the Brunauer-Emmett-Teller (BET) method, respectively [124,125]. In the case of NP embedding into a polymeric material, it is possible to assess NP spatial distribution inside a polymeric matrix using modulation interference microscopy (MIM) [126].

Mechanisms of the Antimicrobial Activity
Antimicrobial properties of NPs are conditioned, first of all, by the antimicrobial properties of elements being their constituents. Silver ions show high toxicity to microorganisms. For example, Ag + causes the death of Aspergillus niger spores at a concentration of 5.5 × 10 −5 M (0.00006% w/w) and higher [127]. Ag NPs exert a significant antibacterial effect beginning from a concentration of 20 µg/mL [128,129]. It is shown that silver can be accumulated in microorganisms as Ag 0 , Ag 2 O or Ag + [130]. Five mechanisms (as a minimum) of the antibacterial activity are described for these forms (Figure 2) [131]. The first mechanism is binding to the bacterial cell wall and disruption of the cell wall integrity, resulting in direct damage of the cell envelope and cytoplasmic components [96,97,100]. It is assumed that after Ag 2 O NP penetration into a bacterial cell, the release of Ag 0 and/or Ag + having the bactericidal activity according to the mechanisms described below takes place [132,133].
The second mechanisms of toxicity is binding to SH-groups of proteins with the subsequent disorder of their function [134]. Silver-induced inactivation of bacterial enzymes, in particular, dehydrogenases of the respiratory chain, is described [110]. This, in turn, inhibits ATP synthesis, disturbs the energy balance in cells, enhances an intracellular ROS production and causes oxidative stress [110,135]. Moreover, Ag 2 O NPs are able to release O 2 , which can also exert antibacterial activity [96].
The third mechanism is the oxidative stress described above. ROS cause protein modifications and exert a genotoxic effect [136][137][138]. An increase in ROS generation leads to the destruction of the cell wall and biofilms of both Gram-positive and Gram-negative bacteria [123].
The fourth mechanism of the antibacterial activity of Ag 2 O NPs is the genotoxic activity of Ag compounds, which after penetration inside a bacterial cell interact not only with proteins but also with phosphoric acid residues in DNA molecules [59,139].
It is assumed that silver compounds from Ag 2 O NPs and Ag NPs are also capable of binding to the N7 atom of guanine in DNA, therefore disturbing the process of its replication, inhibiting cell division [139].
The fifth mechanism is photocatalytic activity. The addition of Ag 2 O NPs can enhance the photocatalytic properties of other metal NPs. In particular, composites of Ag 2 O/TiO 2 NPs and Ag 2 O/ZnO NPs demonstrate enhanced photocatalytic activity compared to TiO 2 or ZnO NPs [140][141][142]. Furthermore, photocatalytic activity of Ag 2 O NPs was demonstrated. It is interesting that the photocatalytic activity of Ag 2 O NPs enhanced after the conjugation of Ag 2 O NPs with certain pharmaceutical agents, for example, moxifloxacin [48,62].
It is notable that Ag 2 O NPs possess high toxicity to pathogenic microorganisms and low toxicity to soil microorganisms. In particular, soil Nitrobacter sp., Bacillus sp. and Pseudomonas strains are able to synthesize Ag 2 O NPs from AgNO 3 in amounts sufficient for the growth inhibition of pathogenic microorganisms of the human oral cavity [49,54,61,78,143]. Specific Ag 2 O NP cytotoxicity to pathogenic microorganisms is an attractive feature for the creation of eco-friendly antimicrobial materials and preparations.

Methods for Improving Antimicrobial Properties
In meta-analysis, we found a dependence of the bacteriostatic activity (expressed in MIC) on NP size (Figure 1b). When a NP's size decreases, an increase in its toxicity to microbes is observed. This dependence corresponds to the literature data about NPs of other metal oxides [7,144], and can be explained by a growth in the release of Ag + , Ag 0 and Ag 2 O from NPs into the surrounding solution due to an increase in the area to volume ratio.
Antimicrobial properties of Ag 2 O NPs can be improved at the initial stage of NP synthesis: precipitation of Ag 2 O NPs. For example, precipitation of Ag 2 O NPs in medium with low (10 mM) or high (100 mM) concentration of AgNO 3 lead to obtaining cubic or octahedral Ag 2 O NPs, respectively [74]. Cubic Ag 2 O NPs showed more pronounced bacteriostatic effects compared to octahedral [74].
The most common other modifications of Ag 2 O NP synthesis are NP coating with polymers, Ag 2 O NP inclusion into other nanocomposites or fusion with NPs of oxides of other elements and NP synthesis in the medium of a substrate of the biological origin-most often an extract of plant leaves (Figure 1c) [34,47,118].
Coatings can be conditionally divided into two large groups. The first group includes organic polymers: chitosan, polyethersulfone, cellulose acetate, polyvinyl alcohol, polyethylene terephthalate and starch [41][42][43]57,96]. This modification commonly had bacteriostatic and fungistatic activity [39,43]. Pharmaceutical preparations, in particular, aspirin and moxifloxacin, can be assigned to the second group [43,62]. For example, Ag 2 O NP coating with aspirin increased their bacteriostatic and fungistatic activity by 50% compared to non-conjugated NPs. In the case of Ag 2 O NP conjugation with moxifloxacin, a more pronounced increase in the bacteriostatic and fungistatic activity of Ag 2 O NPs (by 2-3 times) was shown [62]. Ag 2 O NP coating with chitosan allows practically 100% inhibition of the bacterial growth to be achieved irrespective of their Gram stain group [40]. An opportunity to use conjugates chitosan/Ag 2 O NPs for the creation of fabrics and cloths with the bacteriostatic properties is shown [40,41].
Examples of nanocomposites with Ag 2 O NPs are relatively rare. Among them, composites with ZrO 2, TiO 2 NPs, H 2 Ti 3 O 7 ·2H 2 O and graphene oxide can be highlighted [60,122,123]. The addition of graphene oxide resulted in a dose-dependent increase in the antibacterial properties of Ag 2 O NPs. It is notable that in the case of graphene oxide, an enhancement of the bacteriostatic properties against Gram-negative bacteria was more pronounced [46].
It is worth noting that all modifications of Ag 2 O NP synthesis enhance their antimicrobial properties compared to the chemical synthesis methods, in particular, precipitation ( Figure 1c). Therefore, the selection of the conditions of Ag 2 O NP synthesis can make it possible to obtain NPs with high antimicrobial activity against antibiotic resistance bacteria. There are data that show that a synergetic effect is possible due to the use of several methods to improve the bacteriostatic activity of Ag 2 O NPs [75], for example, the synthesis of complex composites Cu·PES/CA/Ag 2 O NPs. This composite had more pronounced bacteriostatic properties compared to PES/CA/Ag 2 O NPs [42].
A growth in the studies devoted to the creation of various composites with the addition of Ag/Ag 2 O NPs (Table 1) allows us to suggest that the development of new composite materials with Ag 2 O NP introduction and, as a consequence, the extension of application fields for Ag 2 O NP-based nanomaterials will be promising investigations in this field [60,118,122,123].

Cytotoxicity to Human Cells
Data on Ag 2 O NP cytotoxicity are ambiguous and constantly being enriched. There are data about the toxicity of Ag 2 O NPs/Aspergillus terreus to Dalton's lymphoma ascites (DLA) cells, which enables the use of Ag 2 O NPs in the therapy of oncological diseases [36]. High cytotoxicity of Ag 2 O/Ag NPs reported against breast cancer cell line MCF-7 and lung cancer cell line A549. Mechanisms of toxicity are genotoxic effects and ROS overproduction and membrane disruption [146]. Cytotoxicity of Ag NPs and consequently Ag 2 O NPs against eukaryotic cells is actively studied. Induction of apoptosis and necrosis by Ag 2 O/Ag NPs was shown on lung cells lines A549, MRC-5, bronchial cells BEAS-2B and NIH3T3, 3Dcultures of human primary small airway epithelial cell, etc. [147][148][149][150][151]. The ways to increase the cytotoxicity of Ag NPs against cancer and decrease against normal cells have been researched [152]. An interesting approach is using different coating agents; for example, Ag NP cytotoxicity increases in range "PVP > citrate > plant extracts > without coating", but in the case of PVP and citrate, increased predominantly anticancer activity [153].
However, many studies report the low cytotoxicity of Ag 2 O NPs to eukaryotic cells. For example, Ag 2 O NPs did not affect the survival and migration of 3T3 fibroblast cells [63]. It was shown for Ag/Ag 2 O NPs/R. mucilaginosa that the cytotoxic action against eukaryotic cells was realized at concentrations 4-10 times higher than the cytotoxic action against bacteria and fungi [80]. For nanocomposites based on borosiloxane and PLGA and Ag 2 O NPs, the high bactericidal activity was found at Ag 2 O NP concentrations from 1 µg/ml; with that, the survival and the proliferation rate of eukaryotic cells on the above mentioned composites was comparable to these parameters obtained on the culture plastic [52,53]. Low cytotoxicity allows Ag 2 O NPs to be used for wound healing [37].
We assume that the cause of high biocompatibility with eukaryotic cells in the majority of studies is the use of Ag 2 O NP conjugates and composites instead of "pure" Ag 2 O NPs. We also proposed that Ag 2 O is more biologically invert compared to pure Ag.
Metal oxide NPs were potential drug delivery systems. The moderate/low cytotoxicity of Ag 2 O/Ag NPs makes them a perfect candidate for drug delivery systems [154][155][156]. Ag 2 O/Ag NPs can be used in anticancer and antiviral therapy [157][158][159]. Ag 2 O/Ag NPs can also be used as a photoactivated drug delivery unit, for example, in the localized induction of bone regeneration [160].

Conclusions
A search for antimicrobial agents of the new generation that allow us to overcome bacterial antibiotic resistance is an important task for world public health. Candidates for such agents are Ag 2 O NPs. Over the last three years, the interest of researchers in Ag 2 O NPs has increased manifold. The reason for this is the high toxicity to Gram-positive and Gram-negative bacteria, including antibiotic resistance, as well as fungi having epidemiological significance. Moreover, Ag 2 O NPs are inexpensive and easy to produce, and the field of their possible application includes regenerative medicine, prosthetics, therapy of oncological diseases, as well as the development of a wide spectrum of materials with antimicrobial properties (textile and construction). Ag 2 O NP cytotoxicity to eukaryotic cells and nonpathogenic microorganisms is significantly lower than against human pathogens, which makes Ag 2 O NPs an attractive candidate for the role of an antimicrobial agent safe for humans and the environment. Extension of the list of composite materials with the addition of Ag 2 O NPs and, as a consequence, an increase in the number of application fields for Ag 2 O NP-based nanomaterials can be considered the expected outcomes of investigations in this field.