A Review of the Antibacterial, Fungicidal and Antiviral Properties of Selenium Nanoparticles

The resistance of microorganisms to antimicrobial drugs is an important problem worldwide. To solve this problem, active searches for antimicrobial components, approaches and therapies are being carried out. Selenium nanoparticles have high potential for antimicrobial activity. The relevance of their application is indisputable, which can be noted due to the significant increase in publications on the topic over the past decade. This review of research publications aims to provide the reader with up-to-date information on the antimicrobial properties of selenium nanoparticles, including susceptible microorganisms, the mechanisms of action of nanoparticles on bacteria and the effect of nanoparticle properties on their antimicrobial activity. This review describes the most complete information on the antiviral, antibacterial and antifungal effects of selenium nanoparticles.


Introduction
Despite the high level of medical development, microbial infections remain a significant factor in morbidity and mortality worldwide [1]. Sepsis and septic shock alone account for approximately 30 million clinical cases each year worldwide, of which 6 million are fatal [2]. The development of bacterial resistance to antibiotics is not a new problem and dates back to the 1940s-1960s [3,4]. Resistant microorganisms have a significant impact on human life and economic activity. Antibiotic-resistant bacteria significantly increase the risk of complications and death from bacteremia [1,5].
In addition, antibiotic-resistant bacteria complicate the course of foodborne illness, which accounts for more than a million deaths and 2 billion hospitalizations worldwide over a period of 20 years [6]. Complications of bacterial infections can affect almost all human tissues, organs and systems: gastrointestinal tract (gastritis, stomach ulcer, severe forms of diarrhea), central nervous system (meningitis, encephalitis), kidneys, liver, spleen, musculoskeletal system (reactive arthritis), cardiovascular system (endocarditis) and reproductive system (premature birth, stillbirth) [7][8][9][10][11][12][13]. By 2050, microbial resistance is predicted to cause a decrease in the total population of the Earth by 100-440 million people [14].
Apart from humans, antibiotic-resistant bacteria infect animals and plants, reducing the efficiency of agriculture [15][16][17]. Expected financial losses from antibiotic-resistant bacteria in 2025-2050 could be $85 trillion in GDP and $23 trillion in global trade [18]. The mechanisms of antibiotic resistance include enzymatic modification and inactivation of the antibiotic: hydrolysis, phosphorylation, glycosylation, etc. [19], reduction of cell wall permeability due to lipopolysaccharides or lipid enrichment, and removal of antibiotics from cells using special molecular pumps (multidrug [MDR] efflux pumps) [20].
In addition to bacterial antibiotic resistance, the development of fungal resistance to antifungal drugs is worth noting [21]. More than 300 million severe fungal infections have been registered in the world, of which over a million are fatal [22]. Additionally, the demand for antiviral drugs is increasing, in connection with the resistance of viruses, as well as in connection with the SARS-COV-2 pandemic [23], which affected all countries from 2019 until now. One of the promising ways to solve the problem of microbial resistance is the use of nanotechnology [24][25][26][27]. Antimicrobial nanomaterials operate through disruption of the electron transport chain [28,29], membrane destruction [30], cell division arrest [31], etc., and have garnered considerable attention due to their broad, potent and persistent bactericidal activities.
Selenium nanoparticles are one such type of nanoparticle. The study of their antimicrobial properties is a young branch of science: the vast majority of research papers (>95%) have been published in the last 10 years. According to PubMed NCBI (https://pubmed.ncbi.nlm.nih.gov/ accessed on 15 May 2023), more than 220 papers have been published with the keywords "selenium nanoparticles antimicrobial activity", approximately 150 papers with the keywords "selenium nanoparticles antibacterial activity", approximately 30 papers with the keywords "selenium nanoparticles antifungal activity" and approximately 20 papers with the keywords "selenium nanoparticles antiviral activity" (see Figure 1). It is important to note that every year, an increasing number of researchers pay attention to this problem. Over the past few years, research publication activity worldwide on SeNP antimicrobial properties has been growing by approximately 25-30% per year. Over 100 articles were published in 2022.
Materials 2023, 16, x FOR PEER REVIEW 2 of 48 mechanisms of antibiotic resistance include enzymatic modification and inactivation of the antibiotic: hydrolysis, phosphorylation, glycosylation, etc. [19], reduction of cell wall permeability due to lipopolysaccharides or lipid enrichment, and removal of antibiotics from cells using special molecular pumps (multidrug [MDR] efflux pumps) [20]. In addition to bacterial antibiotic resistance, the development of fungal resistance to antifungal drugs is worth noting [21]. More than 300 million severe fungal infections have been registered in the world, of which over a million are fatal [22]. Additionally, the demand for antiviral drugs is increasing, in connection with the resistance of viruses, as well as in connection with the SARS-COV-2 pandemic [23], which affected all countries from 2019 until now. One of the promising ways to solve the problem of microbial resistance is the use of nanotechnology [24][25][26][27]. Antimicrobial nanomaterials operate through disruption of the electron transport chain [28,29], membrane destruction [30], cell division arrest [31], etc., and have garnered considerable attention due to their broad, potent and persistent bactericidal activities.
Selenium nanoparticles are one such type of nanoparticle. The study of their antimicrobial properties is a young branch of science: the vast majority of research papers (>95%) have been published in the last 10 years. According to PubMed NCBI (https://pubmed.ncbi.nlm.nih.gov/ accessed on 15 May 2023), more than 220 papers have been published with the keywords "selenium nanoparticles antimicrobial activity", approximately 150 papers with the keywords "selenium nanoparticles antibacterial activity", approximately 30 papers with the keywords "selenium nanoparticles antifungal activity" and approximately 20 papers with the keywords "selenium nanoparticles antiviral activity" (see Figure 1). It is important to note that every year, an increasing number of researchers pay attention to this problem. Over the past few years, research publication activity worldwide on SeNP antimicrobial properties has been growing by approximately 25-30% per year. Over 100 articles were published in 2022. The reality of our days is the resistance of microorganisms (bacteria, fungi and viruses) to modern antibiotic, antifungal and antiviral drugs. Today, the scientific community is faced with the task of searching for new potential molecular structures to solve  The reality of our days is the resistance of microorganisms (bacteria, fungi and viruses) to modern antibiotic, antifungal and antiviral drugs. Today, the scientific community is faced with the task of searching for new potential molecular structures to solve the problem of therapy in bacterial, fungal and viral pathogenesis. Selenium nanoparticles act as such antimicrobial agents. These nanoparticles have a fairly wide range of applications in the biomedical industry. For example, they can affect the activity of neutrophils: this immunomodulatory property of selenium nanoparticles can potentially be used in the treatment of cancer and other diseases associated with inflammation [46]. Moreover, selenium nanoparticles have a cytoprotective effect on the cells of the cerebral cortex under conditions of ischemia/reoxygenation [47]. The mechanism of action of this effect is based on the fact that nanoparticles regulate the expression of protective proteins in the cells of the cerebral cortex and reduce the total level of Ca 2+ ions. The combination of selenium nanoparticles with the flavonoid taxifolin makes it possible to increase the neuroprotective effect in the cells of the cerebral cortex under conditions of ischemia/reoxygenation [48]. Beyond the immunomodulatory and neuroprotective effects, selenium nanoparticles also demonstrate anticancer effects. Selenium-sorafenib nanocomplexes showed better anticancer effects on HepG2 hepatic carcinoma cells than pure sorafenib [49] According to patent searches and recent publications, SeNPs may have the several potential commercial applications ( Figure 2). the problem of therapy in bacterial, fungal and viral pathogenesis. Selenium nanoparticles act as such antimicrobial agents. These nanoparticles have a fairly wide range of applications in the biomedical industry. For example, they can affect the activity of neutrophils: this immunomodulatory property of selenium nanoparticles can potentially be used in the treatment of cancer and other diseases associated with inflammation [46]. Moreover, selenium nanoparticles have a cytoprotective effect on the cells of the cerebral cortex under conditions of ischemia/reoxygenation [47]. The mechanism of action of this effect is based on the fact that nanoparticles regulate the expression of protective proteins in the cells of the cerebral cortex and reduce the total level of Ca 2+ ions. The combination of selenium nanoparticles with the flavonoid taxifolin makes it possible to increase the neuroprotective effect in the cells of the cerebral cortex under conditions of ischemia/reoxygenation [48]. Beyond the immunomodulatory and neuroprotective effects, selenium nanoparticles also demonstrate anticancer effects. Selenium-sorafenib nanocomplexes showed better anticancer effects on HepG2 hepatic carcinoma cells than pure sorafenib [49] According to patent searches and recent publications, SeNPs may have the several potential commercial applications (Figure 2,).  The first application is the development of nutritional supplements for humans and veterinary needs [50]. It is noteworthy that SeNPs provide a more dosed supply of Se to the body compared to alternative sources, such as selenium cysteine. In particular, SeNPs, when administered orally to mice, causes less pronounced toxicity (survival is 4.5-5 times higher) and liver failure compared with the same amount of selencysteine also administered inhibited hyperglycemia-induced ROS production in HUVEC by 40-50% [88]. Data on preclinical trials of a drug for the treatment of DM2T based on liposomes containing SeNPs have been published [89] (Table 1). Other popular non-metal nanoparticles are SiNPs. SiNPs have a number of interesting advantages [92,93].
The first advantage is cheap synthesis. SiNPs are synthesized by the microemulsion method in the presence of oils or by the Stobers method, which requires relatively available reagents [94,95]. SiNPs can also be synthesized by a mechanochemical method, while the raw material for the synthesis of SeNPs can be river sand [96].
The second advantage is a wide range of applications, including targeted drug delivery with very high specificity [97][98][99], bioimaging [100][101][102], the creation of sensors for the detection of glucose, narcotic substances, or nucleic acids [103][104][105], the up-conversion of light (improved photosynthesis processes in agricultural plants), the protection of cereals from drought [106], and the creation of materials with antibacterial properties. SiNPs have the ability to significantly modify the surface and composition to create SiNP-based nanocomposites with antimicrobial properties [107][108][109].
The third advantage is the practically absent toxicity against eukaryotic cells. Oral administration of 1000-2000 mg/kg SiNPs did not have toxic effects in in vivo experiments and did not cause damage to internal organs in rats [110,111].
The disadvantages of SiNPs include the complexity/high cost of surface modification or the addition of metal NPs and the difficulty of obtaining SiNPs with uniform characteristics [93]. As a rule, precious-metal NPs and/or modification by several agents at once are required [104,108,109]. Antimicrobial properties have not been described for SiNPs without surface modification or addition of metal NPs [92]. SeNPs have been described as having their own antimicrobial properties [112][113][114]. In addition, modified SeNPs with antimicrobial properties can be obtained by biosynthesis, which significantly reduces the cost of their production [115][116][117].
Recently, a series of reviews has been published on the antibacterial, antifungal, anticancer, antiviral and antiparasitic properties of SeNPs [118][119][120]. It should be noted that in these works, the main emphasis is on a detailed description of the antimicrobial mechanisms of SeNPs and the contribution of conjugates to the properties of SeNPs. However, the numerical dependences of the properties of SeNPs-n particular, their size and method of preparation-are described to a lesser extent. In addition, it remains unknown to what extent the "size-antimicrobial-property" dependences of SeNPs are preserved when moving from one type of microorganism to another.
In this literature review, we aimed to analyze the dependence of the antimicrobial effect of selenium nanoparticles on their size, features of synthesis and microorganism species such as viruses, bacteria and fungi. There is no doubt that the relevance of this topic is great.
in these works, the main emphasis is on a detailed description of the antimicrobial mechanisms of SeNPs and the contribution of conjugates to the properties of SeNPs. However, the numerical dependences of the properties of SeNPs-n particular, their size and method of preparation-are described to a lesser extent. In addition, it remains unknown to what extent the "size-antimicrobial-property" dependences of SeNPs are preserved when moving from one type of microorganism to another.
In this literature review, we aimed to analyze the dependence of the antimicrobial effect of selenium nanoparticles on their size, features of synthesis and microorganism species such as viruses, bacteria and fungi. There is no doubt that the relevance of this topic is great.

Synthesis Methods of Selenium Nanoparticles
In this subsection, we discuss various methods for the synthesis of selenium nanoparticles. SeNPs can be synthesized using a wide range of methods: sonochemical, reflux, microwave, hydrothermal, gamma irradiation, pulsed laser ablation, physical evaporation and "green synthesis" (biological reduction) ( Figure 3). The most common method for selenium nanoparticle synthesis is chemical reduction [114]. The most commonly used precursors are SeO2, Na2SeO3, NaHSeO3 and H2SeO3 [121][122][123][124]. Less-common precursors are H2Se, Na2SeO4, SeCl4 or cyclo-octeno-1,2,3-selenadiazole [125][126][127][128]. As stabilizers, substances such as polysaccharides, quercetin, gallic and ascorbic acids and polyvinyl alcohol are used [129]. In addition to the stabilizer, a reducing agent (or reducer) is added to the solution, such as potassium tetrahydroborate (KBH4), ascorbic acid, hydrazine chloride, hydrazine hydrate (N2H4•3H2O) or dimethylsulfoxide (C2H6OS) [130]. Sometimes substances of biological origin, mainly plant extracts, are used as reducing agents. This method of synthesis is called biological reduction [131,132]. The As stabilizers, substances such as polysaccharides, quercetin, gallic and ascorbic acids and polyvinyl alcohol are used [129]. In addition to the stabilizer, a reducing agent (or reducer) is added to the solution, such as potassium tetrahydroborate (KBH 4 ), ascorbic acid, hydrazine chloride, hydrazine hydrate (N 2 H 4 ·3H 2 O) or dimethylsulfoxide (C 2 H 6 OS) [130]. Sometimes substances of biological origin, mainly plant extracts, are used as reducing agents. This method of synthesis is called biological reduction [131,132]. The sonochemical method is a type of chemical reduction method in which the formation of metal NPs is formed by mixing soluble salts that react with each other to form a precipitate. Ultrasound accelerates of sediment formation. For exposure, a Ti tip immersed in a salt solution is used. It is noteworthy that, during precipitation in the presence of organic compounds, it is possible to obtain conjugated NPs [133]. The hydrothermal method is based on the reduction of inorganic precursors of SeNPs (Na 2 SO 3 ) in aqueous solutions in the presence of organic reducing agents (for example, L-ascorbate) at an elevated temperature (~90 • C). An elevated temperature is necessary to accelerate the Se reduction reaction [134]. The reflux method is a variant of the chemical reduction method that occurs when the reaction mixture is boiled for a long time, which makes it very similar to the hydrothermal method. To increase the boiling time, a special unit with a refrigerant is used to condense the solvent vapors and return them to the reaction mixture [135]. The microwave method for the synthesis of nanoparticles is based on the reduction reaction of selenium-containing precursors in a medium with a reducing agent [136]. Microwave radiation provides a multiple acceleration of the reduction reaction due to heating of the reaction. The time of microwave exposure determines the type of crystal lattice of the synthesized Se nanoparticles [128,137]. In the method of gamma irradiation on the reduction of SeO 2 to Se 0 [138], recovery occurs due to processes associated with water radiolysis [139]. In addition, it has been shown that gamma radiation can enhance the biogenic synthesis of SeNPs using fungi [140,141]. Principally, the microwave method and the use of gamma radiation can be attributed to the group of chemical reduction methods, but we singled them out separately, since they additionally need high-energy electromagnetic sources.
Laser ablation in a liquid belongs to the class of physical synthesis methods. It usually consists of obtaining nanoparticles using intense laser radiation from the surface of massive crystalline selenium targets immersed in a liquid. The laser ablation method has been described in detail [142]. During further laser irradiation of a colloid with nanoparticles, it is possible to obtain smaller NPs. This process is called fragmentation [143]. Physical evaporation is a method for synthesizing nanoparticles by treating a metal target with a laser in a vacuum, inert gas or atmospheric air. The method is essentially laser ablation carried out not in a solution, but in a gas and/or vacuum [144,145], so these methods were combined in this work.
In the case of the biological reduction method, biogenic synthesis and bioorganic synthesis are distinguished. In a significant number of works, the synthesis of SeNPs was carried out by biological reduction ("green synthesis"). This approach is also based on the reduction of selenium-containing precursors to Se 0 . The essential difference between the first and the second type is that biogenic synthesis uses cellular structures [115,131,[146][147][148][149] and bioorganic synthesis uses noncellular extracts of plant and microbial origin to synthesize nanoparticles [117,132,[150][151][152][153]. In this case, reducing agents are usually secondary metabolites, such as flavonoids, thiamine and capsaicin, as well as other agents containing amino groups, extracted from various plants: Spirulina platensis, Azadirachta indica, Trigonella foenum-graecum, Allium sativum, etc. [152,[154][155][156][157]. The use of extracts during synthesis provides antibacterial, anticancer, or antioxidant properties of SeNPs [154][155][156][157]. SeNPs can be synthesized by cultivating microorganisms in a medium with an excess of SeNP precursors (sodium selenite (Na 2 SeO 3 ) or SeO 2 ) [158]. For the synthesis of SeNPs, biomass and/or cell-free supernatants of bacterial cultures (Lactobacillus brevis, Lactobacillus casei, Bacillus licheniformis, Pseudomonas alcaliphila, etc.) [158][159][160][161][162], fungi (Aspergillus oryzae, Penicillium citrinum, Mariannaea sp.) [140,141,163] or yeasts (Saccharomyces cerevisiae, Magnusiomyces ingen) [164,165] can be used. Figure 4a shows the percentages of the various methods for the synthesis of selenium nanoparticles found in the literature.     Each symbol means the MIC value taken from a separate publication. Colors correspond to synthesis methods: orange crosses-laser ablation, blue triangles-microwave method, cyan triangles-chemical reduction, green squares-biological reduction, orange triangles-gamma irradiation.

Influence of the Method of Synthesis of Selenium Nanoparticles on the Resulting Size and Shape of Nanoparticles
Does the method of synthesis of nanoparticles affect their size? The results of our analysis are presented in Figure 4b. Obviously, physical methods of synthesis, such as laser ablation or microwave irradiation, make it possible to achieve a narrow size distribution of nanoparticles ( Figure 4b). Most often, these are spherical particles less than 200 nm. Chemical or biological synthesis methods produce a wide range of particle sizes from 5 to 500 nm ( Figure 4b). Within the framework of one type of synthesis, a preparation of nanoparticles with a wide size distribution of nanoparticles is usually obtained. At the same time, the vast majority of publications have synthesized spherical selenium nanoparticles. In some cases, other shapes, such as elongated cylinders [166], polygons [167] and granules [146], are observed.

Influence of Selenium Nanoparticle Synthesis Method on the Minimum Inhibitory Concentration in Antibacterial Studies
It has been established that the selenium nanoparticle synthesis method affects the value of the minimum inhibitory concentration in antibacterial studies ( Table 1). The results  Figure 4c. It was noted that when using physical methods for the synthesis of selenium nanoparticles, the minimum inhibited concentration for effective antibacterial action did not exceed 100 µg/mL. When using microwave generation of nanoparticles, the MIC is approximately 100-300 µg/mL, which is significantly worse compared with nanoparticles obtained by other methods.
It should be noted that there are few studies that use nanoparticles synthesized using physical methods. Most likely, in the future, we should expect clarification of the data presented. For the methods of chemical and biological synthesis, the distribution of the obtained values of the minimum inhibited concentration is quite wide. In some cases, MICs of less than 1 µg/mL have been reported. At the same time, the average efficiency of nanoparticles obtained by both chemical and biological synthesis does not differ significantly. In general, the average efficiency of nanoparticles obtained by chemical/biological methods and using laser ablation does not differ significantly.

Dependence of the Effective Concentration of Selenium Nanoparticles on Their Size for the Study of Antiviral Activity
We consider it necessary to start presenting the results with a study of antiviral activity. There are very few such research publications [120]. It seems that this is due to the increased complexity of organizing scientific work with viruses. Figure 5 shows the effective concentration of selenium nanoparticles depending on their size in the study of antiviral activity. The dependence is described with the equation y = 0.035·x + 1.08. By the nature of this dependence, it can be concluded that with a decrease in the size of nanoparticles, their effective concentration for antiviral action also decreases. This means that as the size of nanoparticles decreases, on average, they become more effective against viruses. It should be noted that selenium nanoparticle synthesis, which is used in most antiviral studies, is a chemical reduction method. In the majority of research papers, spherical nanoparticles with a diameter of 10 to 200 nanometers were used. Published papers mainly use the H1N1 influenza virus (H1N1 influenza infecting Madin Darby canine It should be noted that selenium nanoparticle synthesis, which is used in most antiviral studies, is a chemical reduction method. In the majority of research papers, spherical nanoparticles with a diameter of 10 to 200 nanometers were used. Published papers mainly use the H1N1 influenza virus (H1N1 influenza infecting Madin Darby canine kidney cell line) [179][180][181][182]. In addition, there are research publications investigating Enterovirus (Enterovirus 71-EV71) [183,184], hepatitis virus (HAV) [81,185,186], Cox-B4 virus (enteroviruses) [81], herpes virus (HSV-2 Herpes simplex II), influenza virus H1N1 [187] and adenovirus (Adenovirus strain 2) [187].
In the published research papers, two problems are investigated. The first task is to study the general antiviral action of selenium nanoparticles. The second task is to increase antiviral activity with the help of selenium nanoparticles in case of resistance of the virus to antiviral drugs. In other words, in the first task, researchers use "pure" selenium nanoparticles, and in the second task, selenium nanoparticles are processed (=functionalized) with the help of antiviral drug molecules. That is, selenium nanoparticles act as substrates for targeted delivery.

Dependence of the Minimum Inhibitory Concentration of Selenium Nanoparticles on Their Size and Shape in the Study of Antibacterial Activity
A large number of studies are published on this topic every year. For convenience, we present Table 2, which contains the analyzed publications of the antibacterial action of selenium nanoparticles. It should be noted that in most published research papers, three types of bacteria are used as objects: Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. A box-and-whisker plot of the values of the minimum inhibitory concentration for these three types of bacteria is shown in Figure 6a. MIC, µg/ml MIC, µg/ml MIC, µg/ml  Table 2 were used in this figure.

BS -
The antibacterial activity of the extract, AsAc, and Na 2 SeO 3 was enhanced by producing the SeNPs, which significantly inhibited the growth of S. marcescens, E. cloacae, and A. faecalis bacterial strains.    BC-bactericidal effect, BS-bacteriostatic effect, FC-fungicidal effect, FS-fungistatic effect.
Se nanospheres are described in a significant portion of the studies [117,[121][122][123]168]. However, other forms of SeNPs are described in a number of works: nanowires, nanorods and nanotubes. The form of SeNPs depends on the method and conditions of synthesis (pH, the presence and nature of the conjugate) [112,222]. Nanowires have bacteriostatic activity, but their MIC is comparable to or~4-16 times lower than that of spherical SeNPs [112,222]. Comparable or higher MICs against bacteria and fungi were also found for Se nanorods compared with spherical SeNPs [112,222]. The literature also describes selenium-containing nanotubular structures with antibacterial activity [221,222]. However, it is difficult to compare the data of these works with the rest, since it is not possible to accurately estimate the final concentration of SeNPs in the obtained composites. The works devoted to the synthesis of "true" Se nanotubes are few and describe their potential application in technology, for example, in the creation of photosensors or solar cells; there are practically no works on antimicrobial applications of Se nanotubes [223].
Using regression analysis, it was found that the smaller the size of the selenium nanoparticles, the lower the necessary concentration of selenium nanoparticles for effective inhibition of bacterial growth (Figure 6b-d). Graphs are described by the following equations: y = 0.35x + 31.75 for the bacterium E. coli; y = 1.8x + 4.55 for the bacterium B. subtilis; and y = 0.19x + 34.65 for the bacterium S. aureus. For the bacterium B. subtilis, the regression coefficient was 1.8 ( Figure 6c); for the bacterium E. coli, it was 0.35 ( Figure 6b); and for the bacterium S. aureus, the lowest value was 0.19 ( Figure 6d). In other words, a more efficient relationship between size and MIC was observed for B. subtilis than for E. coli and S. aureus.
A number of studies have shown that SeNPs have different antibacterial activities against Gram-positive and Gram-negative bacteria. A more pronounced antibacterial effect of SeNPs against Gram-negative bacteria compared to Gram-positive bacteria was shown. This property is of particular interest because, at present, among the bacteria that cause bacteremia, including sepsis, the proportion of Gram-negative bacteria is significantly increasing [224].

Dependence of the Minimum Inhibitory Concentration of Selenium Nanoparticles on Their Size in the Study of Antifungal Activity
Data were analyzed not only on the antiviral and antibacterial but also on the antifungal effect of selenium nanoparticles. Many more research papers have been devoted to the antifungal effect of selenium nanoparticles than to the antiviral effect; however, this effect is less than the antibacterial effect. In most fungal studies, the following species are used: Candida [126,150,202,[209][210][211][212] and Fusarium [210,[213][214][215]. Apart from these, the following fungal species are used: Colletotrichum [214,220], Puccinia [217], Aspergillus [127,216], Cryptococcus [150], Penicillium [218], Rhizoctonia [219], Pyricularia [220] and Alternaria [220]. Data on articles taken for analysis are also presented in Table 2. Figure 7 shows the dependence of the minimum inhibitory concentration on the size of the nanoparticles. Using regression analysis, we can conclude that the smaller the size of the nanoparticles, the lower the value of the minimum inhibitory concentration (dependence equation y = 1.39x − 40.11). The graph shows the methods by which selenium nanoparticle synthesis was obtained. Most of the analyzed results use nanoparticles obtained by biological synthesis. We have presented research papers that use physical synthesis methods (laser ablation, gamma irradiation) to obtain selenium nanoparticles to study their antifungal effect. The nanoparticles used in these articles are usually spherical, and their size varies from 20 to 130 nm. Materials 2023, 16, x FOR PEER REVIEW 28 of 48 Figure 7. Dependence of the minimum inhibitory concentration of selenium nanoparticles on their size in the study of antifungal activity. Preparations of nanoparticles synthesized using gamma irradiation are marked in blue, purple-using the method of laser ablation in a liquid, pink-using biosynthesis.

Mechanisms of Selenium Nanoparticle Antimicrobial Action
We decided to present the mechanisms of the antibacterial action of selenium nanoparticles in the form of a list (see below) and in the form of a diagram (Figure 8a).
(1) Degradation of proteins due to the bactericidal action of selenium nanoparticles [112]. (2) Slow emission of selenium ions from the surface of nanoparticles can lead to their interaction with -SH, -NH or -COOH functional groups of proteins and enzymes and the subsequent loss of their tertiary and quaternary structure and functions [125]. (3) SeNPs contribute to the inactivation of the natural mechanisms of membrane transport of ions and nutrients through the cell walls, which blocks the vital activity of the cell [225]. (4) Hyperproduction of ROS, disturbance of membrane potential, and depletion of internal ATP [199]. (5) Inhibition of the activity of the dehydrogenase enzyme, as well as destruction of the integrity of the cell membrane [125]. (6) Inhibition of the ability of bacteria to attach to the surface and form bacterial films [146]. (7) Photocatalytic action against bacteria [226].
Thus, SeNPs can potentially be candidates for antibacterial substitutes and additives against antibiotic-resistant bacteria. Antimicrobial NPs can damage bacterial cells through multiple pathways. This multimodal antimicrobial behavior makes nanoparticles attractive, as bacteria are expected to have difficulty developing resistance to multiple forms of attack [227]. Researchers have also found that the viability of eukaryotic cells is preserved under the effective antibacterial action of selenium NPs [199].

Mechanisms of Selenium Nanoparticle Antimicrobial Action
We decided to present the mechanisms of the antibacterial action of selenium nanoparticles in the form of a list (see below) and in the form of a diagram (Figure 8a).
(1) Degradation of proteins due to the bactericidal action of selenium nanoparticles [112].
(2) Slow emission of selenium ions from the surface of nanoparticles can lead to their interaction with -SH, -NH or -COOH functional groups of proteins and enzymes and the subsequent loss of their tertiary and quaternary structure and functions [125]. (3) SeNPs contribute to the inactivation of the natural mechanisms of membrane transport of ions and nutrients through the cell walls, which blocks the vital activity of the cell [225]. (4) Hyperproduction of ROS, disturbance of membrane potential, and depletion of internal ATP [199]. (5) Inhibition of the activity of the dehydrogenase enzyme, as well as destruction of the integrity of the cell membrane [125]. (6) Inhibition of the ability of bacteria to attach to the surface and form bacterial films [146]. (7) Photocatalytic action against bacteria [226].
Thus, SeNPs can potentially be candidates for antibacterial substitutes and additives against antibiotic-resistant bacteria. Antimicrobial NPs can damage bacterial cells through multiple pathways. This multimodal antimicrobial behavior makes nanoparticles attractive, as bacteria are expected to have difficulty developing resistance to multiple forms of attack [227]. Researchers have also found that the viability of eukaryotic cells is preserved under the effective antibacterial action of selenium NPs [199].
In the case of the bacteriostatic action of selenium nanoparticles, the activity of the dehydrogenase enzyme is inhibited, and the integrity of the cell membrane is destroyed. This effect was observed when using selenium nanoparticles stabilized with arabinogalactan polysaccharide [125]. Researchers suggest possible mechanisms of the antibacterial action of selenium nanoparticles, which are triggered by the contact of the nanoparticle with a living cell.
In particular, this is the hyperproduction of ROS on the surface of nanoparticles, followed by a cascade of the LPO (lipid peroxidase) reaction, damage to cell membranes and organelles, blocking of the transcriptional gene and activation of apoptosis genes, as well as impaired synthesis of a number of cellular proteins and enzymes. In addition, the adhesion of nanoparticles on the cell surface can be accompanied by depolarization of the cell membrane, destruction of its integrity and, subsequently, cell death [125]. In the case of the combined bacteriostatic and bactericidal action of selenium nanoparticles, the ability of bacteria to attach to the surface and form bacterial films is presumably inhibited. This conclusion was made in the study of selenium nanoparticles with a silver shell [146]. For convenience, some mechanisms are shown schematically in Figure 8a,d.  In the case of the bacteriostatic action of selenium nanoparticles, the activity of the dehydrogenase enzyme is inhibited, and the integrity of the cell membrane is destroyed. This effect was observed when using selenium nanoparticles stabilized with arabinogalactan polysaccharide [125]. Researchers suggest possible mechanisms of the antibacterial action of selenium nanoparticles, which are triggered by the contact of the nanoparticle with a living cell.
In particular, this is the hyperproduction of ROS on the surface of nanoparticles, followed by a cascade of the LPO (lipid peroxidase) reaction, damage to cell membranes and organelles, blocking of the transcriptional gene and activation of apoptosis genes, as well as impaired synthesis of a number of cellular proteins and enzymes. In addition, the adhesion of nanoparticles on the cell surface can be accompanied by depolarization of the cell membrane, destruction of its integrity and, subsequently, cell death [125]. In the case of the combined bacteriostatic and bactericidal action of selenium nanoparticles, the ability of bacteria to attach to the surface and form bacterial films is presumably inhibited. This conclusion was made in the study of selenium nanoparticles with a silver shell [146]. For convenience, some mechanisms are shown schematically in Figure 8a,d.
Antifungal mechanisms includes antibiofilm activity [221,228], ROS generation and oxidative stress (with the addition of antifungal drug ketoconazole) [229] and influence on expression of fungicidal drug resistance genes [230] (Figure 8b).
The antiviral action of SeNPs is realized through several mechanisms: disruption of the functioning of viral capside proteins (in particular, hemagglutinin and neuraminidase activities of influenza virus), blocking of the virus-induced activation of the AKT-p52-Caspase3-dependent proapoptotic pathway, inhibition of viral replication in the host cell and enhancement of the action of antiviral drugs [183,184,187] (Figure 8c). Antifungal mechanisms includes antibiofilm activity [221,228], ROS generation and oxidative stress (with the addition of antifungal drug ketoconazole) [229] and influence on expression of fungicidal drug resistance genes [230] (Figure 8b).

Methods for Studying the Characteristics of Selenium Nanoparticles
The antiviral action of SeNPs is realized through several mechanisms: disruption of the functioning of viral capside proteins (in particular, hemagglutinin and neuraminidase activities of influenza virus), blocking of the virus-induced activation of the AKT-p52-Caspase3-dependent proapoptotic pathway, inhibition of viral replication in the host cell and enhancement of the action of antiviral drugs [183,184,187] (Figure 8c).

Methods for Studying the Characteristics of Selenium Nanoparticles
To describe the physicochemical characteristics of selenium NPs, a number of methods are usually used in the analyzed literature. Basically, in all articles, data on morphology, size and elemental composition are given.
Various microscopic methods are used to characterize the morphology of NPs. The most common is transmission electron microscopy (TEM) [231], and rarely atomic force microscopy (AFM) [232], scanning tunnelling microscopy (STM) [233] or scanning electron microscopy (SEM).
To characterize the sizes of NPs, the dynamic light scattering (DLS) method is most often used [234]. The method makes it possible to measure the hydrodynamic radius of nanoparticles, that is, the size of the NPs themselves and their solvate shell. The lesscommonly used CPS Disc Centrifuge method allows estimation of the size of nanoparticles; however, with sizes less than 7 nm, the procedure can take several hours. Often, the size distribution of NPs is calculated using photographs or reconstructions obtained using microscopy. Differential centrifugal sedimentation (DCS) [235], particle size mobility scanning (SMPS) [236] and ion occlusion scanning (SIOS) are relatively inexpensive methods for determining particle size based on the Coulter counting principle [237]. The rarely used nanoparticle tracking analysis (NTA) method provides information on the diffusion of nanoparticles and their size [238].
A large number of methods are used to characterize the elemental composition of NPs. It should be noted that in biological applications, there are no NPs consisting of selenium oxide, and this greatly simplifies the task, since for most nanoparticles from other elements, it is necessary to prove the absence or presence of oxides [239]. Selenium oxide is soluble in water. When the surface of a selenium nanoparticle is oxidized, selenium oxide goes into solution, and the surface again consists of selenium atoms. The process continues until the complete dissolution of the selenium NP. To characterize the chemical composition, energy dispersive spectroscopy (EDX) [240] is usually used; this is very convenient since this method is usually integrated into modern transmission electron microscopes. X-ray photoelectron spectroscopy (XPS) is also often used [241]. In addition, the crystal structure of nanoparticles is often studied using the X-ray diffraction (XRD) method [242]. Selenium nanoparticles with impurities and conjugates are usually characterized with absorption spectroscopy in the UV-visible region of the spectrum [243] and Fourier transform IR spectroscopy (FTIR) [244,245].
Sometimes, differential scanning calorimetry and the Brunauer-Emmett-Teller (BET) method [246] are used to characterize NPs; these methods are used to study the surface area and rheological properties of NPs. Modulation interference microscopy (MIM) is used to study the spatial distribution of nanoparticles inside a polymer matrix [247]. The stability of NP colloids in a solvent is studied by measuring the zeta potential [227].

Cytotoxicity to Eukaryotic Cells
In addition to effective antimicrobial activity, it is also important to determine the safety of selenium nanoparticles for eukaryotic cells. This is a fundamental point that will allow the use of selenium nanoparticles in clinical practice. Zeraatkar et al. proved that selenium nanoparticles do not exhibit cytotoxicity to mouse fibroblasts (3T3 cell line) up to a concentration of 64 µg/mL, while the minimum inhibitory concentration is 4-8 µg/mL for Pseudomonas aeruginosa and Acinetobacter baumannii bacteria [117]. In another work by Jason Hou et al., it was shown that at concentrations from 2 to 16 µg/mL, no cytotoxicity was observed for osteoblast precursor cells (MC3T3-E1 osteoblast precursor cell line), which is important, while at concentrations of 4 µg/mL and more, the growth of the bacterium Porphyromonas gingivalis was inhibited [114].
In a published article [191], using selenium nanoparticles synthesized by a biogenic method, it was found that the inhibitory concentrations (IC50 is the concentration sufficient to inhibit the viability of 50% of cells) of SeNPs were 233.08 and 849.21 µg/mL for normal kidney cells and liver cells, respectively. Thus, normal liver cells showed greater viability to selenium nanoparticles compared with kidney cells. Importantly, the minimum inhibitory concentration of such nanoparticles against bacteria of the genera Salmonella, Klebsiella and Escherichia is 25-200 µg/mL, which is much lower than the concentration of cytotoxicity for the studied normal cell lines.
Importantly, the potential application of selenium nanoparticles lies precisely in their selective cytotoxicity; that is, nanoparticles show effective cytotoxicity for cancer cells and are safe for normal cells. For example, an article [131] was published in which the effective concentration for the antiproliferative activity of HeLa cells is 5.5 µg/mL, while the MIC for E. coli and S. aureus bacteria is 50 µg/mL. Thus, we can speak of effective inhibition of both the growth of cancerous and bacterial cells.
Moreover, a study was conducted and published in our laboratory that showed that selenium nanoparticles can have a cytoprotective effect on neuroglial cells of the cerebral cortex during ischemia/reoxygenation [48]. At concentrations as low as 3 µg/mL, selenium nanoparticles inhibit the hyperproduction of ROS in cells. The experiments also used a complex of selenium NPs with taxifolin (a flavonoid that lacks the "high dosage effect" that occurs in selenium NPs), while at a concentration of 10 µg/mL, the Se-TAX complex inhibits ROS hyperproduction and does not have toxicity on brain cells [48].
SeNPs are less toxic in vivo than other organic and inorganic (selenite) sources of Se [248]. This fact makes SeNPs attractive for biomedical applications. However, a number of experiments have shown that concentrations of SeNPs above 2 mg/kg can cause Se toxicity in mammals. [249]. Manifestations of toxicity at concentrations below 5-30 mg/kg according to the WOS standard require the substance to be classified as Toxicity class I-II. In addition, SeNPs (5 µM) exhibit acute toxicity to marine unicellular algae, which makes products based on SeNPs potentially hazardous to the environment [250]. SeNPs have comparable or even greater toxicity against microorganisms than metal NPs, for example, CuNPs [251]. The average MIC/MBC CuNPs against different strains of Escherichia coli and Staphylococcus aureus are~210/235 and~140/160 µg/mL, respectively [252]. For SeNPs, these values can reach <10 µg/mL and <20 µg/mL for MIC and MBC, respectively [114]. The inhibitory concentration for fungi of CuNPs is 13-22 µg/mL [253], which is comparable to or higher than that of SeNPs [150,209,212]. The toxic concentration of CuNPs for animals is 200 µg/kg, which is significantly higher than that of SiNPs, but lower than that of SiNPs [110,111,249]. CuNPs accumulate in the liver, inhibit CYP450 enzymes, and cause activation of pro-inflammatory reactions through the signaling pathway of NF-κB, MAPK, and STAT5 [254]. Thus, SeNPs have more pronounced antimicrobial properties than CuNPs, but may be more toxic against eukaryotes. These facts require caution in further biomedical developments using SeNPs.

Conclusions
Today, the problem of antimicrobial resistance is acute. A potential approach that can solve this problem is the use of selenium nanoparticles for antimicrobial activity against viruses, bacteria and fungi. That is why, in recent years, the interest of the scientific community in this topic has grown significantly. Selenium nanoparticles are especially attractive because of their relatively simple and inexpensive synthesis, as well as their low cytotoxicity for eukaryotic cells. Unfortunately, the chronic cytotoxicity of SeNPs has been little studied; therefore, in the future, it is necessary to carefully and thoroughly investigate this issue. It is expected that the development of scientific research in this area will effectively solve the problem of antimicrobial resistance in the near future. We consider that, in practical terms, it will be especially interesting to develop nanocoatings and nanocomposites with antibacterial properties based on SeNPs. It is probably worth focusing especially on antiviral research due to its potentially high threat. Recently, the number of publications on antiviral properties of SeNPs has been growing.

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

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