Do Iron Oxide Nanoparticles Have Significant Antibacterial Properties?

The use of metal oxide nanoparticles is one of the promising ways for overcoming antibiotic resistance in bacteria. Iron oxide nanoparticles (IONPs) have found wide applications in different fields of biomedicine. Several studies have suggested using the antimicrobial potential of IONPs. Iron is one of the key microelements and plays an important role in the function of living systems of different hierarchies. Iron abundance and its physiological functions bring into question the ability of iron compounds at the same concentrations, on the one hand, to inhibit the microbial growth and, on the other hand, to positively affect mammalian cells. At present, multiple studies have been published that show the antimicrobial effect of IONPs against Gram-negative and Gram-positive bacteria and fungi. Several studies have established that IONPs have a low toxicity to eukaryotic cells. It gives hope that IONPs can be considered potential antimicrobial agents of the new generation that combine antimicrobial action and high biocompatibility with the human body. This review is intended to inform readers about the available data on the antimicrobial properties of IONPs, a range of susceptible bacteria, mechanisms of the antibacterial action, dependence of the antibacterial action of IONPs on the method for synthesis, and the biocompatibility of IONPs with eukaryotic cells and tissues.


Introduction
Nowadays, the application of nanotechnological solutions, such as the use of nanoparticles, is one of the promising ways to overcome antibiotic resistance in bacteria [1][2][3][4][5].
Iron is one of the most abundant elements on Earth and the fourth-most abundant element in the Earth's crust. Iron makes up more than 85% of the mass of the Earth's core and about 5% of the mass of the Earth's crust [15,16]. In living systems, iron is one of the key microelements. It has several important functions: it is a cofactor of several enzymes (catalase) and transport proteins (hemoglobin), ETC proteins (cytochromes and FeS proteins), and is necessary for DNA repair [17][18][19][20]. Iron is also found in the regulatory proteins of enterobacteria Salmonella enterica, including Fur, Fnr, NorR, SoxR, IscR, and NsrR [21][22][23][24][25][26]. Several bacteria can accumulate iron oxides in special organelles called magnetosomes-for example, Magnetospirillum magneticum [27]. It is assumed that they provide bacteria with the constant magnetic dipole, presumably for navigation purposes [28]. It was shown in Magnetospirillum magneticum wild-type and DmagA1-/-that magnetosomes plays a key role in magneto-aerotaxis. Magneto-aerotaxis is the direct motion of bacteria downward in microaerobic environments favorable to growth [29].

Susceptible Microorganisms
A list of microorganisms susceptible to the toxic action of IONPs is presented in Table 1. A minimum of 10 species of Gram-negative and 11 species of Gram-positive bacteria, as well as three fungal species susceptible to IONPs, have been mentioned in the  (Table 1). The majority of the indicated microorganisms have epidemiological significance [65]. A range of the bacteriostatic concentrations for IONPs is quite wide and makes up 25-2000 µg/mL.  [90] IONPs have antimicrobial activity against both Gram-positive (including Staphylococcus aureus) and Gram-negative (including Escherichia coli) bacteria [33]. The data about the dependence of the antibacterial action of IONPs on the bacterial group (Gram-positive or Gram-negative) are ambiguous. On the one hand, there are data about the comparable effects of IONPs against Gram-negative and Gram-positive bacteria [91], similar to CuO [92], which distinguishes IONPs from ZnO NPs [93]. On the other hand, there are data about the more pronounced bacteriostatic action of Fe 3 O 4 against Gram-negative bacteria compared to Gram-positive [66]. The authors linked the indicated differences with the peculiarities of the cell wall structure and metabolism of Gram-positive and Gram-negative bacteria [66].

The Mechanisms of Antibacterial IONP Activity
One of the main mechanisms of IONP toxicity is ROS generation [5,94], including in photocatalysis, Fenton reactions, or similar ones [88]. ROS, in turn, have a genotoxic action, damaging DNA molecules ( Figure 1) [94]. An increase in ROS concentration can be caused by a decrease in the activity of antioxidant system enzymes (SOD, catalase, and glutathione reductase) [67]. Metal ions are able to bind mecapto (-SH), amino (-NH), and carboxyl (-COOH) groups of proteins, including enzymes, which leads to inactivation or partial inhibition [95]. Additionally, IONPs damage the bacterial cell wall integrity, as shown in reference [94]. The direct binding of IONPs with the cell wall of Staphylococcus aureus was shown by scanning electron microscopy [96]. IONPs can cause a decrease in the expression of antibiotic resistance genes (ARGs) in antibiotic-resistant bacteria found in operating rooms [5]. IONPs are able to disturb the function of F 0 /F 1 -ATPase and reduce the rate of H + flow through the membrane and the redox potential [66]. The mechanisms of the antimicrobial action for IONPs have been suggested in several studies based on their size and are common for other types of metal oxide nanoparticles [95,97]. An ability to inhibit DNA replication by the inactivation of topoisomerase is described for nanoparticles with small sizes [98]. It was shown by the method of electron microscopy that Fe 2 O 3 NPs can bind directly with the cell wall of E. coli. IONPs can also penetrate into the cytoplasm, concentrate in it, and cause vacuole formation and cell wall disruption [84,99].
One of the main mechanisms of IONP toxicity is ROS generation [5,94], including in photocatalysis, Fenton reactions, or similar ones [88]. ROS, in turn, have a genotoxic action, damaging DNA molecules ( Figure 1) [94]. An increase in ROS concentration can be caused by a decrease in the activity of antioxidant system enzymes (SOD, catalase, and glutathione reductase) [67]. Metal ions are able to bind mecapto (-SH), amino (-NH), and carboxyl (-COOH) groups of proteins, including enzymes, which leads to inactivation or partial inhibition [95]. Additionally, IONPs damage the bacterial cell wall integrity, as shown in reference [94]. The direct binding of IONPs with the cell wall of Staphylococcus aureus was shown by scanning electron microscopy [96]. IONPs can cause a decrease in the expression of antibiotic resistance genes (ARGs) in antibiotic-resistant bacteria found in operating rooms [5]. IONPs are able to disturb the function of F0/F1-ATPase and reduce the rate of Н + flow through the membrane and the redox potential [66]. The mechanisms of the antimicrobial action for IONPs have been suggested in several studies based on their size and are common for other types of metal oxide nanoparticles [95,97]. An ability to inhibit DNA replication by the inactivation of topoisomerase is described for nanoparticles with small sizes [98]. It was shown by the method of electron microscopy that Fe2 O3 NPs can bind directly with the cell wall of E. coli. IONPs can also penetrate into the cytoplasm, concentrate in it, and cause vacuole formation and cell wall disruption [84,99]. Fe3O4 IONPs can concentrate between the outer and inner membranes of the cell wall in Gram-negative bacteria due to binding with the FHL complex in the inner membrane. Therefore, Fe3O4 IONPs have more pronounced antimicrobial actions against Gram-negative bacteria [66].
Bactericidal and antibiofilm activities were shown in Fe3O4 IONPs. Positively charged and neutral IONPs promoted a higher reduction of Streptococcus mutans biofilms compared with negatively charged IONPs [89]. IONPs coated with oleic acid can prevent biofilm formation by S. aureus and P. aeruginosa [85]. IOPNs have the ability to adsorb and penetrate into bacterial biofilms due to their physicochemical characteristics, such as a surface charge, hydrophobicity, and high surface area ratio by volume [100,101].
Iron oxide nanoparticles have both magnetic and paramagnetic properties [68,87,102]. Fe3 O4 NPs with high paramagnetic activity are also named superparamagnetic iron oxide nanoparticles (SPIONs) [103,104]. SPIONs in the presence of the alternating magnetic fields cause cell death and biofilm destruction due to the vibration damage, local hyperthermia, and ROS generation. All of the above-mentioned factors lead to the dissociation of bacteria from a biofilm, damage of the bacterial cell wall, membrane rupture, the fusion of different cells with each other, and death [69]. Fe 3 O 4 IONPs can concentrate between the outer and inner membranes of the cell wall in Gram-negative bacteria due to binding with the FHL complex in the inner membrane. Therefore, Fe 3 O 4 IONPs have more pronounced antimicrobial actions against Gram-negative bacteria [66].
Bactericidal and antibiofilm activities were shown in Fe 3 O 4 IONPs. Positively charged and neutral IONPs promoted a higher reduction of Streptococcus mutans biofilms compared with negatively charged IONPs [89]. IONPs coated with oleic acid can prevent biofilm formation by S. aureus and P. aeruginosa [85]. IOPNs have the ability to adsorb and penetrate into bacterial biofilms due to their physicochemical characteristics, such as a surface charge, hydrophobicity, and high surface area ratio by volume [100,101].
Iron oxide nanoparticles have both magnetic and paramagnetic properties [68,87,102]. Fe 3 O 4 NPs with high paramagnetic activity are also named superparamagnetic iron oxide nanoparticles (SPIONs) [103,104]. SPIONs in the presence of the alternating magnetic fields cause cell death and biofilm destruction due to the vibration damage, local hyperthermia, and ROS generation. All of the above-mentioned factors lead to the dissociation of bacteria from a biofilm, damage of the bacterial cell wall, membrane rupture, the fusion of different cells with each other, and death [69].
In 80% of studies, IONPs show only bacteriostatic action. The bactericidal action of IONPs is described in the literature in 20% of cases.
Aqueous coprecipitation is the most widely used chemical method of IONP synthesis [105,109]. In this method, IOPNs are synthesized by the simultaneous precipitation of Fe 2+ and Fe 3+ salts (molar ratio 1:2) in a basic solution at room temperature or under heat [105,109,110]. The advantage of the coprecipitation method is the low cost of IOPNs synthesis. It is important in cases of large-scale production [27]. The disadvantages of the method are the large size distribution of produced IONPs, aggregation, poor crystallinity, a high possibility of oxidation, and poor magnetic property [111]. The change of pH in the solution can improve the properties of IONPs synthesized by coprecipitation [112].
The thermal decomposition is a nonaqueous synthesis in which organometallic compounds such as Fe(Acac) 3 , Fe(C 2 O 4 ) × 2 H 2 O, Fe(CH 3 COO) 2 , or ferrocene suffer decay at high temperatures in organic solvents (high boiled) or via being solvent-free in the presence of stabilizing surfactants like aliphatic amine and fatty acids [113]. This method may generate high-quality IONPs with close distributions of particle sizes and a high magnetism and degree of crystallinity [113]. Addition advantages of this method are the high yield and absence of IONP aggregation [114]. The main disadvantage of this method is the insolubility of produced IONPs in water. Therefore, further steps are required to make their surfaces hydrophilic and use IONPs in biological solutions [115].
The sol-gel method (wet-chemical method) is a sum of reactions of condensation and hydrolysis between iron alkoxides and salts (e.g., chlorides, nitrates, and acetates) [116]. The main advantage of this method is a good homogeneity and size and high purity and quantity of IONPs [116]. The disadvantages of the method are the requirements for compliance with exact values of the pH, temperature, and concentration of the reagents during a synthesis; high cost of precursors; and low wear resistance of synthesized IONPs [117].
Laser ablation synthesis in a solution is a synthesis that is triggered by the immersion of pulsed laser beams on the target material in a liquid solution [118]. Laser ablation synthesis allows to work with a wide range of materials and solvents. The size and clustering of IONPs are difficult to control [118]. Laser ablation allows the synthesis of FeOx crystal to a few atom clusters in the following modification: phosphonates as an aqueous solution and bulk iron as a target [72].
Large-scale synthesis is a modification of the coprecipitation method with controlled heating and addition polyacrilic acid salts or sodium oleate as the surfactant [103] The hydrothermal method is a synthesis of IONPs from iron precursors at high pressure and temperature conditions in an aqueous medium [103,104]. Aqueous synthesis methods generate particles with low crystallization [122]. Replacing water with other organic solvents allows the formation of IONPs with high crystallinity and controlled shapes. This method is named solvothermal synthesis [113]. The disadvantage of this method is the long time it takes for synthesis (hours to days) [123].

Dependence of the Antimicrobial Action of IONPs on the Size and Type of Iron Oxides
The majority of studied IONPs have a spherical shape (Table 2), which excludes a contribution of the shape into the antimicrobial action. Therefore, we assessed the sizes and compositions of IONPs. Based on the analyzed literature data, we did not reveal an association between the IONPs' size and the minimum bacteriostatic concentrations (Figure 2a). Several IONP types are distinguished depending on the oxide on which basis they are synthesized: NPs based on hematite (α-Fe 2 O 3 ) [5,68] [124][125][126], and Fe 3 O 4 [83,127]. We found that Fe 2 O 3 NPs show more pronounced bacteriostatic actions compared to Fe 3 O 4 NPs (Figure 2b). For more detailed analyses, we assessed the contribution of a method for IONP synthesis of their antimicrobial properties. method is the long time it takes for synthesis (hours to days) [123].

Dependence of the Antimicrobial Action of IONPs on the Size and Type of Iron Oxides
The majority of studied IONPs have a spherical shape (Table 2), which excludes a contribution of the shape into the antimicrobial action. Therefore, we assessed the sizes and compositions of IONPs. Based on the analyzed literature data, we did not reveal an association between the IONPs' size and the minimum bacteriostatic concentrations (Figure 2a). Several IONP types are distinguished depending on the oxide on which basis they are synthesized: NPs based on hematite (α-Fe2O3) [5,68], β-Fe2O3, γ-Fe2O3, ε-Fe2O3 [124][125][126], and Fe3O4 [83,127]. We found that Fe2 O3 NPs show more pronounced bacteriostatic actions compared to Fe3O4 NPs (Figure 2b). For more detailed analyses, we assessed the contribution of a method for IONP synthesis of their antimicrobial properties.  Table 2. In (a), the grey color shows the values for the IONPs obtained without "green synthesis", and the green color shows the values for the IONPs obtained by "green synthesis". *-p < 0.05 by the Mann-Whitney U test.

Dependence of the Antimicrobial Action of IONPs on a Synthesis Method
The methods for IONPs synthesis are multiple and include coprecipitation [105], thermal decomposition [70], low-temperature synthesis [71], the sol-gel method [106], hydrothermal method [69], electrochemical method [83], laser ablation [91,107], sonochemical, microwave, microemulsion methods, matrix-mediated method using PVA, and many others [32,84,108]. IONPs synthesized by the low-temperature method from iron sulfate showed an antimicrobial effect against E. coli, P. aeruginosa, Serratia marcescens, and Listeria monocytogenes, exerting bacteriostatic action and inhibiting biofilm formation [71]. NPs obtained by laser ablation had comparable bacteriostatic effects against Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens) and Gram-positive (Staphylococcus aureus) bacteria. The bacteriostatic actions of IONPs do not depend on a solvent (SDS or DMF) or bacterial group (Gram-positive or Gram-negative) [91].  Table  2. In (a), the grey color shows the values for the IONPs obtained without "green synthesis", and the green color shows the values for the IONPs obtained by "green synthesis". *-p < 0.05 by the Mann-Whitney U test.

Coprecipitation Method
The most common method for IONP synthesis when studying the antimicrobial properties is the coprecipitation of salts Fe 3+ /Fe 2+ [56,73,75,86,[127][128][129]. This synthesis method is the most available. Modifications of the method are possible. For instance, the addition of oleic acid for the generation of conjugated IONPs [127,129], as well as the coprecipitation of different metal salts, allow us to obtain composite NPs-for example, based on FeSO 4 × 7 H 2 O and Co(NO 3 ) 2 × 6 H 2 O [76]. One of the methods for improving the antimicrobial properties of IONPs is the use of composites-for example, α-Fe 2 O 3 /Co 3 O 4 [105]. Composite NPs have more pronounced antimicrobial actions against B. subtilis, S. aureus, E.coli, and S. typhimirium. The synergistic effect of Fe 2 O 3 and Co 3 O 4 was observed compared to oxides used individually. However, upon the strong bacteriostatic action (practically a full inhibition of the bacterial growth at a concentration of 1200 mg/mL), the bactericidal action was almost absent [76]. α-Fe 2 O 3 /ZnO NPs show more pronounced bacteriostatic actions against Gram-positive Bacillus subtilis and Staphylococcus aureus and Gram-negative Escherichia coli and Salmonella typhi than IONPs and ZnO NPs; with that, the size of the inhibition zone increases when the ZnO concentration in the composite is increased [76], (Table 2).
Compared to Fe 3 O 4 NPs, the composite Fe 3 O 4 /SiO 2 NPs has a more pronounced photocatalytic bactericidal action against Escherichia coli and Staphylococcus aureus; with that, the effect was higher against Gram-positive bacteria [130]. The use of the combined method for IONPs synthesis allows achieving a significant bacteriostatic effect against Staphylococcus aureus, Xanthomonas, Escherichia coli, and Proteus vulgaris [83].

"Green Synthesis"
The so-called "green synthesis" has aroused considerable interest. It is a modification of synthesis methods (as a rule, coprecipitation) with the application of plant extracts used as a reducing agent [83,[106][107][108][109][110].
IONPs synthetized by the "green" method show comparable antimicrobial effects against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria [68]. However, the antimicrobial effect of 50-100 µg/µL of IONPs is about three times lower than that of 20 µg/mL of streptomycin. IONPs synthesized in the presence of Punica granatum peel extract exert a bacteriostatic effect on Pseudomonas aeruginosa; with that, these IONPs do not have hemolytic activity against erythrocytes [70]. IONPs in complex with Cynometra ramiflora extract have more pronounced bacteriostatic effects against Gram-positive S. epidermalis compared to Gram-negative E. coli [88]. NPs synthesized in the medium of the Zea mays extract did not have their own antimicrobial and antifungal properties but significantly enhanced the bacteriostatic action of kanamycin and rifampicin against Grampositive Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus and Gram-negative Escherichia coli and Salmonella typhimurium, as well as the antifungal activity these antibiotics against six strains of Candida [87]. In addition to the antimicrobial properties, IONPs obtained as a result of "green" synthesis had antioxidant properties and inhibited their proteasome activity, which allowed us to regard IONPs as possible candidates for cancer therapy [87].
Compared to Fe 3 O 4 NPs, Fe 3 O 4 /Malva sylvestris NPs had more pronounced bacteriostatic and bactericidal effects against Staphylococcus aureus, Corynebacterium sp., Pseudomonas aeruginosa, and Klebsiella pneumoniae and exerted cytotoxic action against the Hep-G2 and MCF-7 cell lines [82].
IONPs synthesized in the Argemone mexicana extract had more pronounced bacteriostatic activity against E. coli, P. mirabilis, and B. subtilis than pure IONPs, which was comparable with the effects of streptomycin [73].       Fe 3 O 4 NPs synthesized with a Couroupita guianensis extract inhibited the growth of E. coli, S. typhimurium, K. pneumoniae, and S. aureus and induced the apoptosis of the hepatocellular carcinoma (HepG2) cell line [81]. IONPs synthesized in a Ruellia tuberosa extract inhibited the growth of E. coli, K. pneumoniae, and S. aureus in a dose-dependent manner. The IONP effectiveness turned to be higher than that of streptomycin. The mechanism of antimicrobial action is the photocatalytic generation of ROS [74]. Fe 2 O 3 /Citrus sinensis NPs exerted a comparable bacteriostatic action against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria. The inhibitory effect of Fe 2 O 3 /Citrus sinensis NPs was comparable with chlorhexidine, hexachlorophene, benzalkonium chloride, and phenol taken in equal concentrations [121]. α-Fe 2 O 3 NPs, in combination with a Sida cordifolia extract, had comparable bacteriostatic activity against E. coli, K. pneumoniae, B. subtilis, and S. aureus. The bacteriostatic effect against Gram-positive bacteria was more strongly pronounced and was comparable with the effect of neomycin [119]. Unfortunately, for several extracts-for example, Couroupita guianensis-"green synthesis" leads to an enhancement of IONP cytotoxicity [81]. In another study, the antioxidant properties were described for Fe 3 O 4 NPs synthesized by the "green method" [87]. In a meta-analysis, we found that IONPs generated by the "green synthesis" method had three times more pronounced bacteriostatic activity than IONPs generated by the coprecipitation method ( Figure 2c).

Additional Methods for Increasing the Antimicrobial Activity of IONPs
Iron oxide nanoparticles have both magnetic and paramagnetic properties [68,87,[102][103][104]. The use of an alternating magnetic field allows additional increases in the bactericidal action of Fe 3 O 4 NPs against E. coli and S. aureus, causing cell death and biofilm destruction due to the photocatalytic generation of ROS, and local hyperthermia and vibration damage occurred under the action of the magnetic field. All of the above-mentioned factors lead to the dissociation of bacteria from the biofilm, damage of the bacterial cell wall, membrane rupture, the fusion of different cells with each other, and death [69].
Fe 2 O 4 composite NPs with the addition of different ratios of Co and Mn have magnetic properties due to Fe 2 O 4 and inhibit the growth of E. coli and cause damage to E. coli and B. subtilis in a dose-dependent manner [102].
IONP conjugation with carbon nanotubes allows achieving a bactericidal effect against Gram-negative (E. coli and K. pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria; with that, the CFU were reduced by two and more times compared to the control [77]. Carbon nanotubes/IONPs accelerated wound healing in mice in a wound-healing test by 25% and 50% compared to IONPs or carbon nanotubes taken individually. It is worth noting that, in this study, the size of the inhibition zone increased insignificantly upon a considerable decrease in the CFU; therefore, the antimicrobial effect of IONPs assessed by a size of the inhibition zone in the majority of studies can be underestimated. In contrast to other IONPs types, Fe 3 O 4 IONPs coated with oleic acid exert a different effect on the growth and viability of Gram-positive (Enterococcus hirae) and Gram-negative (E. coli) bacteria. More pronounced antimicrobial action was observed against Gram-negative bacteria [127]. The authors linked this phenomenon with differences in the cell wall structure; in particular, with the ability of Fe 3 O 4 IONPs to concentrate between the outer and inner membranes of the cell wall in Gram-negative bacteria and the presence of the FHL complex in the inner membrane of E. coli, which is an additional target for IONP Fe 3 O 4 . Fe 3 O 4 NPs covered with oleic acid cause a reduction in the growth of kanamycin-and ampicillin-resistant E. coli strains due to retardation of the logarithmic growth phase, lag phase extension, reduction of the H + flow through the membrane, and redox potential [85,131]. IONPs coated with oleic acid not only inhibit the growth of S. aureus and P. aeruginosa but also prevent biofilm formation [131].
Surface modification is also a key way to improve IONP the antibacterial properties [78] The conjugation of IONPs with chitosan enhanced the bactericidal action of IONPs against Bacillus subtilis and Escherichia coli due to ROS generation [78]. Fe 3 O 4 NPs covered with polyethylene glycol (PEG) exert a dose-dependent bactericidal action against the E. coli and S. aureus and antibiotic-resistant Micrococcus luteus strain. The mechanism of toxicity resides in a decrease in the activity of the antioxidant system enzymes (SOD, catalase, and glutathione reductase) and, as a consequence, enhancement of ROS generation and lipid oxidation [67].
Fe 3 O 4 NPs conjugated with chitosan have bactericidal and fungicidal actions against Candida albicans, Aspergillus niger, and Fusarium solani [90]. Coating with alginate or tobramycin did not have a significant effect on the bacteriostatic activity of Fe 3 O 4 NPs against P. aeruginosa [84].
Conjugation with polyethylene glycol (PEG) and chitosan allows not only improving the antimicrobial properties of IONPs but also reducing the undesirable adsorption of IONPs on liver macrophages [133,134].
One of the methods for improving the antimicrobial properties is the use of a combination of "green synthesis" and a change in the NP compositions-for example, the addition of gold. The bacteriostatic effect of the mixture Urtica/α-Fe 2 O 3 •Ag NPs against S. aureus, Bacillus sp., Klebsiella sp., and E. coli was higher compared to Urtica/α-Fe 2 O 3 NPs. An increase in the inhibition zone was proportional to the silver concentration in the composite. Both Urtica/α-Fe 2 O 3 •Ag NPs and Urtica/α-Fe 2 O 3 NPs had more pronounced effects on the growth of the Gram-negative strains [36]. Some of the mechanisms of action of Fe 3 O 4 and Ag NPs are membrane damage, a decrease in the redox potential, and H + fluxes, which lead to the inhibition of the activity of bacterial F o /F 1 -ATPase [131].
The combined use of NPs from iron oxides and gold does not reduce the growth of the bacterial biomass of the E. coli culture but prevents bacterial cell division [75]; as a consequence, E. coli alters their morphology from rods to filaments with a length of several micrometers. The mixture of Fe 3 O 4 and Au NPs inhibits the growth of the kanamycinresistant Escherichia coli and Salmonella typhimurium strains more effectively than Fe 2 O 3 NPs [75].
An approach to an improvement in the antimicrobial properties of IONPs by their conjugation with antibiotics is described by the example of gentamicin [79]. With that, a more pronounced bacteriostatic effect was achieved against Gram-positive B. subtilis and S. aureus than Gram-negative E. coli and P. areuginosa. A conjugation with gentamicin reduced the minimum inhibitory concentration against all indicated strains by more than ten times [79]. In several cases, the conjugation of IONPs with antibiotics can give an opposite result. IONPs conjugated with amoxicillin enhanced the growth of Pseudomonas aeruginosa and Staphylococcus aureus [86]. The presence of organic acids (humic acid) additionally accelerates bacterial growth. In general, it is possible to significantly influence the antimicrobial activity of IONPs by additives, coatings, and conjugates, which, undoubtedly, can be promising in the development of this direction.

Biocompatibility of IONPs
It is shown that IONPs have good biocompatibility and biodegradability. In particular, the intravenous injection of 0.8 mg/kg of γ-Fe 2 O 3 NPs did not influence the weight gain in rats or cause the activation of apoptosis in HUVEC cells [61]. After intravenous injection, NPs were found in rat lungs, liver, and kidneys but not in the brain or heart. A significant proportion of NPs was eliminated with urine after 72 h [61]. In general, IONPs show an absence or low cytotoxic effects on cell cultures. For example, no adverse effect of IONPs coated with polyethyleneimine, dimercaptosuccinate, or citrate on primary rat cerebellar cortex astrocytes and cultured murine astrocytes was observed [62,135]. IONPs conjugated with PEG-phospholipids (WFION) did not influence the viability of the B16 F10 cell line at doses up to 0.75-mg Fe/mL [136]. Fe 3 O 4 NPs show a bacteriostatic effect and, at the same time, do not exert a hemolytic action [66,70]. In several cases, IONPs enhance Casp3-dependent apoptosis in HUVEC cells, cause ROS generation, membrane damage, changes in the cytoskeleton, and so on [137]. In general, the cytotoxic properties of IONPs are manifested at much higher concentrations than the antimicrobial properties.

Disadvantages of IONPs
The disadvantages of IONPs include relatively weak antimicrobial action against several strains and insufficient biocompatibility with eukaryotic cells. For example, Fe 2 O 3 NPs inhibit the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis less effectively than ZnO and CuO NPs. The inhibitory action of Fe 2 O 3 NPs on Escherichia coli growth was lower than that of ZnO and CuO NPs [138]. This effect may be explained by differences in the antibacterial properties of considered metals. Iron Fe 2+ is necessary for the proliferation of bacteria [31]. Fe 3+ inhibited E. coli growth in concentrations above 0.25-1 mM, but Fe 3+ had only a bacteriostatic effect without bactericidal action [139]. Zn 2+ and Cu + decrease the viability of Staphylococcus aureus and Escherichia coli in concentrations of 2.41 and 0.46 mM, respectively [140]. Fe NPs exert more pronounced bacteriostatic actions against Pseudomonas aeruginosa than Fe 3  Unfortunately, IONPs has not only bacteriostatic and bactericidal activities but toxicity for some eukaryotic cell lines [108]. The main mechanism of IONP toxicity is the production of ROS, which leads to increasing the level of lipid peroxidation, decreasing the antioxidant enzymes, and protein aggregation [141][142][143][144]. IONPs can lead to cell iron overload. Iron overload causes serious deleterious and leads to cell death [142,143]. In addition, a high dose of IONPs increases the lipid metabolism, the breakage of iron homeostasis, and exacerbates the loss of murine liver functions in vivo [145].
The IONP applications in biomedicine are limited due to a lack of control and prediction of the final IONP properties, such as IONP interactions with cells [146]. An important aspect of IONPs in biomedical applications is their surface chemistry [147]. The coating of IONPs by PEG reduces protein adsorption, increases stability to the IONPs, decreases the IONP uptake by culture cells in and by entire organisms in vivo, and increases IONP retention times in the blood flow [148][149][150]. Unfortunately, PEG can be oxidized by host enzymes, which leads to a loss of some PEG-IONP proteins [148]. Proteins are commonly the first biomolecules that IONPs encounter when they interact with biological systems in vitro or in vivo [146]. IONPs may be coated by bovine serum albumin (BSA) or fetal bovine serum [151]. BSA forms a protective layer on the NPs to improve the biocompatibility and transport of the IONPs. BSA-coated IONPs allow to accumulate the drug in the tumor due to an enhanced permeability and retention and to reduce the risk of hypersensitivity reactions [152]. Drugs released from BSA-coated IONPs can be triggered by protease digestion in target tissues, and finally, the unfolding BSA protein on the IONPs can facilitate their clearance by phagocytes after drug delivery [153]. Additionally, BSA coating supports the colloidal stability of the IONPs in cell culture experiments [151]. Multiple specialized characterization methods are widely used to characterize IONP surfaces: TEM, UV-visualization, MD simulation, isothermal titration calorimetry, ζ-potential measuring, etc. [151]. The antibacterial properties of BSA-IONPs remain unclear.

Conclusions
IONPs have found wide applications in different fields of biomedicine. The antibacterial activities of IONPs are of special interest. However, the situation with the antimicrobial activities of IONPs is ambiguous. On the one hand, the antibacterial activities of IONPs depend, to a significant extent, on the microbial strain, and the inhibitory actions of IONPs are often less pronounced than that of NPs of other metal oxides (CuO or ZnO). On the other hand, IONPs show less-pronounced cytotoxic properties and better biocompatibility in vivo compared to CuO or ZnO NPs. We assume that, in the near future, IONPs will allow achieving a balance between antimicrobial actions and biocompatibility in vivo. In this case, IONPs can be considered potential antimicrobial agents of the new generation. Based on the analyzed data, we believe that the most promising method for increasing the antimicrobial properties of IONPs and improving biocompatibility is "green synthesis" and other variants of the additive or composite generation of nanoparticles.

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.