**Inorganic Nanoparticles: Innovative Tools for Antimicrobial Agents**

Mario Kurtjak, Nemanja Aničić and

Marija Vukomanovicć

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67904

#### **Abstract**

Resistance of bacteria to antibiotics is an urgent problem of humanity, which leads to a lack of therapy for serious bacterial infections. Development of new antibiotics has almost ceased in the last decades—even when a new antibiotic is launched, very soon the resistance of bacteria appears. There is a long list of applications where antimicrobial protection is required to achieve effective treatment. However, if we use the sameantibiotics for all these applications, we will remain caught in the "vicious circle" of constant discovery of new synthetic antibiotics and very fast development of their resistantspecies.Therefore,weneedtofindalternativestrategiesthatwillberoutinelyusedforsomespecificconditions(wounds,implants,etc.).Thus,wewillkeeptheactivityofantibioticsandsavethemforacuteconditions(pneumonia,meningitis,etc.).Anoptionfor designing alternative antimicrobial strategies is to go back to the antimicrobials that were used before the discovery of antibiotics, i.e., inorganic antimicrobial agents includ‐ ingions(Ag<sup>+</sup> , Cu+ /Cu2+, Zn2+, Ga3+,etc.)ornanoparticles(Ag/AgO,Cu/Cu<sup>2</sup> O/CuO,ZnO, Ga/Ga2 O3 ,TiO<sup>2</sup> ,MgO,V<sup>2</sup> O3 ,etc.).Herewearegoingtosummarizethemainpropertiesof inorganic antimicrobials as well as advantages, disadvantages and perspectives for their application.

**Keywords:** Ag/AgO, Cu/Cu<sup>2</sup> O/CuO, ZnO, Ga/Ga<sup>2</sup> O3 ,TiO<sup>2</sup> ,MgO,V<sup>2</sup> O3 ,functionalized Au

#### **1. Introduction**

Resistance of bacteria to antibiotics is becoming an increasingly urgent problem of the humanity. The most serious threat comes from vancomycin‐resistant *Enterococcus* (VRE, mainly *E. faecium*),

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

methicilin‐resistant *Staphylococcus aureus* (MRSA), *Klebsiella* (especially *K. pneumoniae*), *Acinetobacter baumanii, Pseudomonas aeruginosa*, *Enterobacter* and *Escherichia coli* (the so‐called "ESKAPE" patho‐ gens,), Gram‐positive *Mycobacterium tuberculosis* and some other Gram‐negative bacteria [1]. Soon there will be no available antibiotics to treat infections with these pathogens. The problem first appeared in hospitals and grew promptly asaconsequence of uncontrolled application of antibiotics not only in the healthcare but also in agriculture, stock breeding, poultry breed‐ ing, etc. However, overuse and misuse are not the only factors that speed up the spread of resistance. Some mechanisms of resistance do not destroy the antibiotic and leave it active in the environment. Thus, bacteria themselves help maintain the antibiotic environment; fur‐ thermore, the drug can be released into other environments and alter them. Many precau‐ tions against drug misuse and overuse led to the reduction of antibiotic application in the last decade. Consequently, the spreading of resistance slowed down, but it did not decrease. We could get rid of the resistant strains with new antibiotics. Unfortunately, development of new antibiotics has almost ceased in the last decades. Investments in research and devel‐ opment of new kinds of antibiotics were minimized due to their unprofitability. And even when a new antibiotic is launched, very soon the resistance of bacteria to the new antibiotic appears.

What can we deduce from all these facts? Instead of focusing only on development of new antibiotics, which will sooner or later create resistance, we should focus on preventing the resistance itself. There is a long list of applications where antimicrobial protection is required in order to achieve effective treatment. However, if we use the same antibiotics for all these applications, we will remain caught in the "vicious circle" of constant discovery of new synthetic antibiotics and very fast development of their resistant species. Therefore, we need to find alternative strategies that will be routinely used for some specific conditions (such as insufficient and slow wound healing, rejection of medical implants during their incorporation into the body due to the presence of bacteria on the surface of the implant, unsuccessful use of autologous, allogeneic or xenografts in tissue engineering because of the development of infection, etc.). Thus, we will keep the activity of the antibiotics and save them for urgent, acute conditions (like pneumonia, meningitis, peritonitis, etc.). One option for designing these alternative antimicrobial strategies is to go back to the antimicrobials that were used before the discovery of antibiotics, i.e., inorganic antimicrobial agents. There are a lot of inorganic substances with the capacity to kill bacteria or to inhibit bacterial growth. They are applicable in the form of antibacterial ions (i.e. Ag<sup>+</sup> , Cu+ /Cu2+, Zn2+, Ga3+, etc.) or antibacterial nanoparticles (i.e. Ag/AgO, Cu/Cu<sup>2</sup> O/CuO, ZnO, Ga/Ga<sup>2</sup> O3 ,TiO<sup>2</sup> ,MgO, V2 O3 ,functionalized Au, etc.). The new knowledge brought especially by the emergence and progress of biomaterials science and nanotechnology might enable: (i) local, targeted action without side effects in the organism, (ii) improved transport towards and eased penetration into the pathogenic species, leading to higher efficiency, (iii) unique opportunity for devel‐ opment of effective medicines.

This chapter provides detailed overview of various inorganic antimicrobial agents, their physicochemical properties and various mechanisms of action on bacterial/mammalian cells.

## **2. Antibacterial ions**

#### **2.1. Silver (I) (Ag+ )**

methicilin‐resistant *Staphylococcus aureus* (MRSA), *Klebsiella* (especially *K. pneumoniae*), *Acinetobacter baumanii, Pseudomonas aeruginosa*, *Enterobacter* and *Escherichia coli* (the so‐called "ESKAPE" patho‐ gens,), Gram‐positive *Mycobacterium tuberculosis* and some other Gram‐negative bacteria [1]. Soon there will be no available antibiotics to treat infections with these pathogens. The problem first appeared in hospitals and grew promptly asaconsequence of uncontrolled application of antibiotics not only in the healthcare but also in agriculture, stock breeding, poultry breed‐ ing, etc. However, overuse and misuse are not the only factors that speed up the spread of resistance. Some mechanisms of resistance do not destroy the antibiotic and leave it active in the environment. Thus, bacteria themselves help maintain the antibiotic environment; fur‐ thermore, the drug can be released into other environments and alter them. Many precau‐ tions against drug misuse and overuse led to the reduction of antibiotic application in the last decade. Consequently, the spreading of resistance slowed down, but it did not decrease. We could get rid of the resistant strains with new antibiotics. Unfortunately, development of new antibiotics has almost ceased in the last decades. Investments in research and devel‐ opment of new kinds of antibiotics were minimized due to their unprofitability. And even when a new antibiotic is launched, very soon the resistance of bacteria to the new antibiotic

What can we deduce from all these facts? Instead of focusing only on development of new antibiotics, which will sooner or later create resistance, we should focus on preventing the resistance itself. There is a long list of applications where antimicrobial protection is required in order to achieve effective treatment. However, if we use the same antibiotics for all these applications, we will remain caught in the "vicious circle" of constant discovery of new synthetic antibiotics and very fast development of their resistant species. Therefore, we need to find alternative strategies that will be routinely used for some specific conditions (such as insufficient and slow wound healing, rejection of medical implants during their incorporation into the body due to the presence of bacteria on the surface of the implant, unsuccessful use of autologous, allogeneic or xenografts in tissue engineering because of the development of infection, etc.). Thus, we will keep the activity of the antibiotics and save them for urgent, acute conditions (like pneumonia, meningitis, peritonitis, etc.). One option for designing these alternative antimicrobial strategies is to go back to the antimicrobials that were used before the discovery of antibiotics, i.e., inorganic antimicrobial agents. There are a lot of inorganic substances with the capacity to kill bacteria or to inhibit bacterial

,functionalized Au, etc.). The new knowledge brought especially by the emergence and progress of biomaterials science and nanotechnology might enable: (i) local, targeted action without side effects in the organism, (ii) improved transport towards and eased penetration into the pathogenic species, leading to higher efficiency, (iii) unique opportunity for devel‐

This chapter provides detailed overview of various inorganic antimicrobial agents, their physicochemical properties and various mechanisms of action on bacterial/mammalian

, Cu+

O3 ,TiO<sup>2</sup>

O/CuO, ZnO, Ga/Ga<sup>2</sup>

/Cu2+, Zn2+, Ga3+,

,MgO,

growth. They are applicable in the form of antibacterial ions (i.e. Ag<sup>+</sup>

etc.) or antibacterial nanoparticles (i.e. Ag/AgO, Cu/Cu<sup>2</sup>

opment of effective medicines.

appears.

40 Antibacterial Agents

V2 O3

cells.

Silvernitrate(AgNO<sup>3</sup> )waswidelyusedfortreatmentofulcers,burnwoundsanddifferentinfections until the discovery of penicillin and sulpha drugs completely drove it out from the market [2]. In 1965, the favourability ofAgNO<sup>3</sup> over antibiotics was shown and burn treatmentprocedurethatinvolvedfrequentwettingofacottongauzedressingwith0.5wt.% AgNO<sup>3</sup> solution was established [2, 3]. In spite of reduced mortality from severe burns and strong action against *Staphylococcus aureus*, haemolytic streptococci, *Pseudomonas aeruginosa* and *Escherichia coli*,the0.5%AgNO<sup>3</sup> method had several disadvantages. It required very clean wounds,sodeepandlargeburnswerepronetoinvasiveinfection(usuallyby*P. aeruginosa*) leadingtosepsisanddeath.0.5%AgNO<sup>3</sup> was hypotonic, sensitive to light, inactive against *Aerobacter*, *Paracolon*, *Klebsiella* and a number of cutaneous saprophytes and could cause methaemoglobinaemia.AlthoughtheprecipitationofAgNO<sup>3</sup> withdifferentanionsresultedinlowabsorptionofsilverintothebodythroughthewounds,itconsequentlycausedNa<sup>+</sup> ,K<sup>+</sup> , Ca2+, Mg2+ and Cl<sup>−</sup> depletion in serum [2–4].Hence,animprovementwastriedbycombining AgNO<sup>3</sup> with a sulpha drug to obtain silver sulphadiazine [2].Many otherionicAg drugsemerged,butAg‐sulfadiazineremainedthemostwidelyused,althoughitdelaysthewoundhealing process [2, 5].Furtherdevelopmentwent tosystems forcontrolleddeliveryofAg<sup>+</sup> ions,Ag‐containingwounddressings,cathetersandantibacterialcoatings[6, 7]whichfloodedthe market recently [8].

TheantibacterialactionofAg<sup>+</sup> ions is currently explained by three mechanisms:


There are two forms ofresistance: complexation ofAg<sup>+</sup> inside cells orreduced permeability toAg<sup>+</sup> combined withanupgradedactive effluxmechanismtopumpAg out ofthe cell[5]. SeveralGram‐ negative and positive bacteria have been reported to beAg‐resistant, including *Pseudomonas aeruginosa*, *Pseudomonas stutzeri*, members of the *Enterobacteriaceae* and *Citrobacter spp*. [6]. It has been recently shown that the toxic values of AgNO<sup>3</sup> and commercial Ag‐containing wound dressings for *P. aeruginosa, E. coli*, *S. aureus* and human fibroblasts are very similar and that thiol‐containing molecules reduce their toxicity towards both prokaryotes and eukaryotes [14].

#### **2.2. Copper (I, II) (Cu+ , Cu2+)**

Cu+/2+hasalsobeenknownasasterilizing,antisepticandantimicrobialagent[15] used to treat avarietyofskindiseases,syphilis,tuberculosisandanaemia,andtofightmildew[10, 16, 17]. Inmodernhealthcare,theantimicrobialeffectofCuisveryeffectivelyusedinhospitalwaterdistribution systems [16, 17]. Recent research has focused on "contact killing" mechanism [17]. In2008,theUSEnvironmentalProtectionAgency(EPA)proclaimedCu‐surfacesasefficientantimicrobials [17].Cuisthefirstmetaltobeawardedsuchastatus[17]. Cu is an essential micronutrientandmorethan30typesofproteinsthatcontainCuionsareknowntoday[18]. Intheseenzymes,Cuservesasanelectrondonor/acceptorbyalternatingbetweentheredoxstatesCu(I)andCu(II)[19].Dietaryintakesof0.9–1.4mgofCuforanadult(a70‐kgperson) and50μgperkgbodyweightperdayininfantsarerecommendedbytheWHO[16, 20]. Cu ions are also toxic to prokaryotes and eukaryotes at higher cellular concentrations, and the involvementofCu(andZn)inphagosomalkillingofbacteriaengulfedbymacrophagesisanimportant defence mechanism [10, 21].

Theantibacterial/toxicityactionofCu(I,II)iscurrentlyexplainedbythefollowingmechanisms:


Bacteria have evolvedarange of mechanisms to protect themselves from the toxic effects of excess Cu ions: exclusion by a permeability barrier; intra‐ and extracellular sequestration of Cu ions by cell envelopes and metallothionein‐like Cu‐scavenging proteins in the cytoplasm and periplasm; active transport membrane efflux pumps; reduction in the sensitivity of cellular targets to Cu ions; extracellular chelation or precipitation by secreted metabolites including Cu; and adaptation and tolerance via up‐regulation of necessary genes in the presence of Cu [16, 19, 25]. Active extrusion of Cu from the cell appears to be the chief mechanism of Cu tolerance in bacteria and has been extensively studied in Gram‐ positive and Gram‐negative bacteria. However, due to the multiple targets and mostly non‐specific mechanisms of damage exerted by Cu, this bacterial tolerance is relatively low, as compared to the resistance to antibiotics (i.e., 10‐fold lower sensitivity to Cu as opposed to 1000‐fold less sensitivity to methicillin, for example, by methicillin‐resistant *S. aureus*).

#### **2.3. Zinc(II) (Zn2+)**

**2.2. Copper (I, II) (Cu+**

42 Antibacterial Agents

**, Cu2+)**

important defence mechanism [10, 21].

*S. aureus*).

Cu+/2+hasalsobeenknownasasterilizing,antisepticandantimicrobialagent[15] used to treat avarietyofskindiseases,syphilis,tuberculosisandanaemia,andtofightmildew[10, 16, 17]. Inmodernhealthcare,theantimicrobialeffectofCuisveryeffectivelyusedinhospitalwaterdistribution systems [16, 17]. Recent research has focused on "contact killing" mechanism [17]. In2008,theUSEnvironmentalProtectionAgency(EPA)proclaimedCu‐surfacesasefficientantimicrobials [17].Cuisthefirstmetaltobeawardedsuchastatus[17]. Cu is an essential micronutrientandmorethan30typesofproteinsthatcontainCuionsareknowntoday[18]. Intheseenzymes,Cuservesasanelectrondonor/acceptorbyalternatingbetweentheredoxstatesCu(I)andCu(II)[19].Dietaryintakesof0.9–1.4mgofCuforanadult(a70‐kgperson) and50μgperkgbodyweightperdayininfantsarerecommendedbytheWHO[16, 20]. Cu ions are also toxic to prokaryotes and eukaryotes at higher cellular concentrations, and the involvementofCu(andZn)inphagosomalkillingofbacteriaengulfedbymacrophagesisan-

Theantibacterial/toxicityactionofCu(I,II)iscurrentlyexplainedbythefollowingmechanisms:

**1.** Direct generation of ROS through Fenton‐type reactions [19, 22]. Radicals can cause oxidative damage to proteins, nucleic acids and lipids, which lead to cell death [23].

**2.** Indirect generation of reactive oxygen species by inactivation of antioxidants and thiol depletion [19, 23].SuchreactionsofCucanleadto theinhibitionofrespiratoryenzyme-

**3.** Competition with other metal ions for important binding sites on proteins [6, 17, 19]. Site‐specificinactivationbyCuionscanalsooccurinFe‐Sdehydratases,thecytoplasmic-

**4.** By cross‐linking within and between strands of DNA, Cu may cause helical structure

Bacteria have evolvedarange of mechanisms to protect themselves from the toxic effects of excess Cu ions: exclusion by a permeability barrier; intra‐ and extracellular sequestration of Cu ions by cell envelopes and metallothionein‐like Cu‐scavenging proteins in the cytoplasm and periplasm; active transport membrane efflux pumps; reduction in the sensitivity of cellular targets to Cu ions; extracellular chelation or precipitation by secreted metabolites including Cu; and adaptation and tolerance via up‐regulation of necessary genes in the presence of Cu [16, 19, 25]. Active extrusion of Cu from the cell appears to be the chief mechanism of Cu tolerance in bacteria and has been extensively studied in Gram‐ positive and Gram‐negative bacteria. However, due to the multiple targets and mostly non‐specific mechanisms of damage exerted by Cu, this bacterial tolerance is relatively low, as compared to the resistance to antibiotics (i.e., 10‐fold lower sensitivity to Cu as opposed to 1000‐fold less sensitivity to methicillin, for example, by methicillin‐resistant

[6].

O2

was required

functionanddisruptionofrespirationleadstoROSasexplainedforAg<sup>+</sup>

disordersandDNAdenaturation[16, 24].SomestudieshaveshownthatH<sup>2</sup>

fortheDNAbreakage,whichquestionstherelevanceofthismechanism[16].

enzymesneededtomakebranched‐chainaminoacids[17, 23].

Zn2+ is also an essential micronutrient for the development, growth and differentiation of all living systems, including bacteria, and exhibits antibacterial action only at higher concentrations when its homeostasis is overcome. The adult human body contains approxi‐ mately 1.5–2.5 g of Zn2+ [22, 26–28] with essential role in cell membrane integrity, development and maintenance of the body's immune system, managing insulin action and blood glucose concentration, bone and teeth mineralization, normal taste and wound healing[22]. Zn is a constituent of more than 300 enzymes that haveacentral role in reconstruction of the wound matrix [26, 29]. Zn in castor oil hasaspecial place in the treatment of nappy (diaper) rash [26].Avast range of zincated bandages, dressings, emollients, shampoos and creams are available commercially. In normal wound healing, body creates a higher amount of Zn2+ in the wound margin at a certain stage—during the formation of granulation tissue, scar tissue and re‐epithelialization. It is believed that the addition of Zn at this stage might accelerate wound healing. Experimental studies have shown that topical ZnO reduced the initial haem‐ orrhagic phase and promoted the regrowth of damaged skin and hair [26]. The antibacterial properties of Zn2+ ions are exploited especially in oral healthcare for prevention of caries, gingivitis and periodontitis. Zn− salts are used in mouthwashes and toothpastes [30]. The effect of Zn2+ ions is most probably only bacteriostatic, so oral‐care products are designed for frequent use, while bactericidal action can be obtained in combinations with fluoride or Triclosan [30–33].

TheantibacterialactionofZn−ionsisaconsequenceofthefollowingmechanisms[6, 30–32]:


Resistance of bacteria to toxic levels of Zn2+ can be due to extracellularaccumulation, sequestration bymetallothioneins,intracellularphysicalsequestration,and/orcanbeeffluxbased[35].Arecent study compared the Cu and Zn resistance of MRSA and methicillin‐susceptible *S. aureus* in a global collection of species [36].WhiletherewasnodifferenceintheirCu−suscep‐ tibility,thereweresignificantlymoreZn‐resistantMRSAstrains,whichalsohadanencoded-Zn resistance [36].SimilarlytoAg,recentprogressofZn−antimicrobialshasgoneinthedirec‐ tion ofZnO nanoparticles andincorporation ofionicZninto zeolites, polymers, bioactiveceramicsandglassestoachievebetterefficiencyandlocalaction[6].

#### **2.4. Gallium(III) (Ga3+)**

Antibacterial properties of Ga3+ were first mentioned in 1931 [37]. Initially, it was mainly investigated for cancer diagnosis and treatment [38, 39]. Intensive research of Ga(III) asan antibacterial agent in the 2000s revealed great efficacy against*M. tuberculosis* [40] and *P. aeruginosa* [41, 42].A recent study has shown that Ga(NO<sup>3</sup> ) 3 at safe therapeutic dosage (10mg/kg)protectsmicefrom*M. tuberculosis* infection [43].APhase‐1clinicalstudyisbeingconductedsince2010,whichtestsGaniteinhumanpatientssufferingfromcysticfibrosis,andchronically infected by *P. aeruginosa* [37, 38, 44]. Current results show that intravenous Ganite infusion for 5 days decreases the amount of *P. aeruginosa* in the lung without any serious adverseeffect [38, 44]. Subcutaneous applicationofGa‐maltolatewaseffectivein reducing-*S. aureus*, *A. baumannii* and *P. aeruginosa*colonizationinburnwoundsof thermallyinjuredmouse model [42]. These data support a potential use of Ga‐maltolate *in vivo*, especially in topical administration for the prevention and treatment of wound infections. Besides Ga(NO<sup>3</sup> )3 andGa‐maltolate,someother formsofGa(III)have alsobeenused,i.e.,chloride [45], citrate [46], desferriox‐amine B and other complexes [46–48].

The following is currently known about the mechanism of antibacterial action of Ga3+ ions:


Considerable progress has been recently made in the development of Ga delivery systems using phosphate‐based glasses [52–54], cellulose [55],scaffolds[56], phosphosilicates [57, 58] and titanium implants [59].BecausebacteriacannotdiscriminatebetweenFe(III)andGa(III), they will not sense an increase of Ga3+ concentration and a decrease of Fe3+.However,since-Ga(III)entersmicrobialcellsbyexploitingspecificFe(III)‐uptakemechanisms,mutationsinthese pathways could block Ga from reaching its cellular targets, ultimately making bacte‐ ria less susceptible to Ga's inhibitory activity, as it has been observed in laboratory studies of Ga(III) antibacterial mechanism, in which resistant strains were created by genetic modificationof*P. aeruginosa* [60, 61].Nevertheless,suchmutationscouldnevercompletelyprevent Ga3+ entranceinto bacterial cells and only 2–4 times higher Ga(III) concentrationswerealreadyeffectiveagainsttheresistantstrains.

## **3. Antibacterial nanoparticles**

#### **3.1. Ag nanoparticles**

**2.4. Gallium(III) (Ga3+)**

44 Antibacterial Agents

Ga(NO<sup>3</sup> )3

**4.** IncreasedproductionofH<sup>2</sup>

Antibacterial properties of Ga3+ were first mentioned in 1931 [37]. Initially, it was mainly investigated for cancer diagnosis and treatment [38, 39]. Intensive research of Ga(III) asan antibacterial agent in the 2000s revealed great efficacy against*M. tuberculosis* [40] and

(10mg/kg)protectsmicefrom*M. tuberculosis* infection [43].APhase‐1clinicalstudyisbeingconductedsince2010,whichtestsGaniteinhumanpatientssufferingfromcysticfibrosis,andchronically infected by *P. aeruginosa* [37, 38, 44]. Current results show that intravenous Ganite infusion for 5 days decreases the amount of *P. aeruginosa* in the lung without any serious adverseeffect [38, 44]. Subcutaneous applicationofGa‐maltolatewaseffectivein reducing-*S. aureus*, *A. baumannii* and *P. aeruginosa*colonizationinburnwoundsof thermallyinjuredmouse model [42]. These data support a potential use of Ga‐maltolate *in vivo*, especially in topical administration for the prevention and treatment of wound infections. Besides

andGa‐maltolate,someother formsofGa(III)have alsobeenused,i.e.,chloride

The following is currently known about the mechanism of antibacterial action of Ga3+ ions:

biological interactions for Ga3+ that would not be possible for Fe3+ [49, 50].

the main reason or only a consequence of the Ga3+ antibacterial action.

ferric‐binding protein and non‐ribosomal peptide microbial siderophores [49]).

**1.** Ga3+ follows uptake and transport pathways for Fe3+;unlikeFe(III),itcannotbereducedto the oxidation state (+2); small amounts of non‐bound Ga can exist in solution at physiological conditions, versus insignificant amounts of non‐bound Fe3+, permitting

**2.** Most bacteria require Fe for growth [37]. If bacteria use Ga instead of Fe, it will prevent theirmultiplication,whichiscrucial forharming theorganism (asobservedinbacterial-

**3.** Ga(III) can affect the synthesis of siderophores by regulation of gene expression [68] leadingtoshortageofFeinsidecellandinhibitionofmanyFe‐requiringenzymes.

Ga3+ quenches the superoxide ion signal [51],anditisnotyetclearwhethertheROSsare-

Considerable progress has been recently made in the development of Ga delivery systems using phosphate‐based glasses [52–54], cellulose [55],scaffolds[56], phosphosilicates [57, 58] and titanium implants [59].BecausebacteriacannotdiscriminatebetweenFe(III)andGa(III), they will not sense an increase of Ga3+ concentration and a decrease of Fe3+.However,since-Ga(III)entersmicrobialcellsbyexploitingspecificFe(III)‐uptakemechanisms,mutationsinthese pathways could block Ga from reaching its cellular targets, ultimately making bacte‐ ria less susceptible to Ga's inhibitory activity, as it has been observed in laboratory studies of Ga(III) antibacterial mechanism, in which resistant strains were created by genetic modificationof*P. aeruginosa* [60, 61].Nevertheless,suchmutationscouldnevercompletelyprevent Ga3+ entranceinto bacterial cells and only 2–4 times higher Ga(III) concentrations-

)3

was noticed due to Ga3+ antibacterial action [51].However,

at safe therapeutic dosage

*P. aeruginosa* [41, 42].A recent study has shown that Ga(NO<sup>3</sup>

[45], citrate [46], desferriox‐amine B and other complexes [46–48].

O2

werealreadyeffectiveagainsttheresistantstrains.

Ag nanoparticles show bactericidal action in both Gram‐positive and Gram‐negative bacteria, with higher efficiency induced by smaller particles[12, 32, 62] and quite intriguing dependence of the efficiency on the shape, falling in the order: triangular nanoplates, nano‐ spheres, nanowires [63]. In Ag nanoparticles, there are three sources of bactericidal activity: Ag, Ag ions and nanosize. As the three sources are interlacing, it is difficult to determine what effect comes from each of them. Ag nanoparticles come into contact with bacterial cells. Positively charged Ag nanoparticles attach to bacterial membrane by electrostatic interac‐ tions, while negatively charged ones attach due to high affinity of Ag (soft acid) for P‐ and S‐containing molecules (soft bases) [63–65]. Then, Ag ions are released into the cell and inhibit respiratory enzymes, which facilitates the generation of ROS and consequently dam‐ ages the cell membrane [66]. The uptake of Ag can be recognized by irregular pits. They can be dissolved by oxygen orH<sup>2</sup> O2 .AFenton‐like reaction was suggested to account for the observed generation of OH• radicals at pH below 7.4 [67]. Then, S‐containing proteins in the membrane or inside the cells and P‐containing elements like DNA are likely to be the preferential sites for Ag nanoparticle binding. Disruption of membrane morphology may causeasignificant increase in permeability, leading to leaking of the internal components resulting in cell death [63].

ExposureofmurinemacrophagestoAgNPsshowedmitochondrialdamage,apoptosisandcelldeathabrogatedinthepresenceofAgion‐reactive,thiol‐containingcompoundssuggest‐ ingthecentralroleofAgionsinAgNPtoxicity[68].FurtherresearchshowedthatAg<sup>+</sup> ions weretheonlyactivepartoftheAgNPs[69].Testingunderanaerobicconditions(ionreleasewasnegligible)showedthatAgNPswereineffectiveagainst*E. coli*K12.IftheAgNPswereexposed to air prior to the antibacterial test under anaerobic conditions, their antibacterial properties were enhanced and bacterial survivability depended on released ions.

However, the amount of released ions from the Ag NPs at their MIC was always lower than the MIC of Ag<sup>+</sup> ions [70–72]. Recent research [73, 74] showed much higher intracellular dis‐ solution of Ag NPs compared to extracellular ones. The ion release from the Ag NPs is size‐ specific and surface‐dependent. The toxicity of 20–80‐nmAg NPs follows this size dependence and is mainly assigned to the released ions. However, the 10‐nm Ag NPs are much more toxic. Importantly, immobilized Ag NPs were more efficient than Ag<sup>+</sup> ion‐releasing substrates, even though they released much lower amount of ions and the immobilized Ag NPs were not internalized[72, 75]. Ag NPs can change the lipid composition of the membrane, anchor and incorporate into the outer membrane, and it is currently believed that the outer membrane damage is mainly "nano‐specific" [72]. Ag NPs enhance the transport of Ag<sup>+</sup> ions into the cell and could avoid bacterial resistance that involves efflux systems. However, *E. coli* easily develop resistance to Ag NPs as well as Ag<sup>+</sup> after 100–200 generations of exposure to Ag NPs [76]and several studies have shown low efficiency of Ag NPs against Ag‐resistant bacteria [71, 72].

#### **3.2. Cu/CuO nanoparticles**

In Cu nanoparticles, there is a coincidence of antibacterial effect of ions and nano‐sized particles.TheefficiencyofCuwasimprovedbydecreasingthedimensions,butitwashigherfor Gram‐positive bacteria [32].Cunanoparticleshavegreataffinityforaminesandcarboxylgroups, so they bind to the ones on the surface of bacteria and release the ions inside. These ionscantheninteractwithDNAmoleculesandintercalatewithnucleicacidstrands[77]. It isbelievedthathere,theroleofROSismuchlargerthaninAgnanoparticles,sincetheycanbegeneratedbyCuO aswell as the releasedCu<sup>+</sup> /Cu2+ ions by their dissolution [78]. Somescientists,ontheotherhand,emphasizetheroleofthereleasedionsmore[32]. Both Cu and CuO antibacterialnanoparticlescauselipidperoxidation,cellwall andmembranedamageandoxidativedamagetoDNA.TheygenerateROSintheabsenceofanycells,inextracellularaswellasintracellularenvironment.CuOnanoparticlesaremuchmoretoxictomammaliancells than Cu2+ionsandalsomuchmorecytotoxicthanZnOandTiO<sup>2</sup> NPs[79]. In general, it has been shown that trends in bactericidal activity were similar to trends in cytotoxicity, i.e. more powerful bactericidal agents [80] were more toxic towards human cells [81].

#### **3.3. ZnO nanoparticles**

ZnO has so far been found to be the most effective metal oxide antimicrobial, with efficiency comparable to Ag[32]. If ZnO nanoparticles are shined with UV light, their antibacterial effect can become strongly bactericidal as a consequence of photocatalysis. ZnO is a semiconduc‐ tor with adirect 3.3‐eV band gap [82]. Absorption of light with energy greater than 3.3 eV induces the electron transfer from the valence to the conduction band and separation of charge, generatingahole (h<sup>+</sup> )in the valence band and an electron (e−)in the conduction band[82, 83]. At the surface of the excited ZnO particle, the valence band holes abstract electrons from water and/or hydroxide ions, generating hydroxyl radicals (OH•). Electrons can reduceO<sup>2</sup> to pro‐ duce the superoxide anion O−•. The obtained OH• and O−• can induce lipid peroxidation in membranes, DNA damage due to strand breakage or oxidized nucleotides and oxidation of amino acids and protein catalytic centres [83]. Negative charge of OH• and O−• prevents these species from passing through the membrane into the cell, so they can exert only outside damage. They can also combine withH<sup>+</sup> to createH<sup>2</sup> O2 , which can pass into the cell and create internal damage leading to cell death [82]. ZnO nanoparticles show bactericidal properties as well as ROS generation also in complete absence of light. This effect has been tried to be explained by surface defects and the oxidative role of oxygen or halogens adsorbed on their surfaces [31, 82]. Suchamechanism would be enhanced in an aerobic environment and it was observed that oxy‐ gen annealing and formation of nanoholes on the surface, which both stimulated a high amount of adsorbed oxygen atoms on the ZnO surface, increased the ROS production and enhanced the antibacterial properties [31, 82]. ZnO nanorods are stronger antimicrobials than nanospheres, and flower‐shaped nanoparticles with exposed polar are even stronger[82].

#### **3.4. TiO2 nanoparticles**

TiO<sup>2</sup> nanoparticlescanaccountfortheirantibacterialeffectasaconsequenceofproductionof-OH•radicals.TheydonotpossessanyantibacterialpropertiesintheabsenceofUVlightdueto weak interaction with bacterial surface because of negative charge. In contrast, recent stud‐ ieshaveshownthatTiO<sup>2</sup> particles exhibited high tendency to bond to the *E. coli* membrane viaVanderWallsandreceptor‐ligandinteractions.Theextentoftheseinteractionswasmorepronounced thanin thecaseofZnOnanoparticles.Asaresult,whenilluminatedwithUVlight,TiO<sup>2</sup> wasmorepowerfulthanZnO.AsopposedtoTiO<sup>2</sup> , it did not show up‐regulation ofROS‐relatedproteinsbutrathercausedmembranedamageviadirecttransferofROSmol‐ ecules from particle surface towards the bacterial membrane [84].Ofinterestismetaldoping (e.g.withAg)ofTiO<sup>2</sup> ,whichcanimproveitsantibacterialpropertiessignificantlyandenablevisible‐light‐induced photocatalytic activity [85, 86].

#### **3.5. Functionalized Au nanoparticles**

**3.2. Cu/CuO nanoparticles**

46 Antibacterial Agents

**3.3. ZnO nanoparticles**

generatingahole (h<sup>+</sup>

**3.4. TiO2**

TiO<sup>2</sup>

They can also combine withH<sup>+</sup> to createH<sup>2</sup>

 **nanoparticles**

begeneratedbyCuO aswell as the releasedCu<sup>+</sup>

cells than Cu2+ionsandalsomuchmorecytotoxicthanZnOandTiO<sup>2</sup>

In Cu nanoparticles, there is a coincidence of antibacterial effect of ions and nano‐sized particles.TheefficiencyofCuwasimprovedbydecreasingthedimensions,butitwashigherfor Gram‐positive bacteria [32].Cunanoparticleshavegreataffinityforaminesandcarboxylgroups, so they bind to the ones on the surface of bacteria and release the ions inside. These ionscantheninteractwithDNAmoleculesandintercalatewithnucleicacidstrands[77]. It isbelievedthathere,theroleofROSismuchlargerthaninAgnanoparticles,sincetheycan-

scientists,ontheotherhand,emphasizetheroleofthereleasedionsmore[32]. Both Cu and CuO antibacterialnanoparticlescauselipidperoxidation,cellwall andmembranedamageandoxidativedamagetoDNA.TheygenerateROSintheabsenceofanycells,inextracellularaswellasintracellularenvironment.CuOnanoparticlesaremuchmoretoxictomammalian-

has been shown that trends in bactericidal activity were similar to trends in cytotoxicity, i.e.

ZnO has so far been found to be the most effective metal oxide antimicrobial, with efficiency comparable to Ag[32]. If ZnO nanoparticles are shined with UV light, their antibacterial effect can become strongly bactericidal as a consequence of photocatalysis. ZnO is a semiconduc‐ tor with adirect 3.3‐eV band gap [82]. Absorption of light with energy greater than 3.3 eV induces the electron transfer from the valence to the conduction band and separation of charge,

At the surface of the excited ZnO particle, the valence band holes abstract electrons from water

duce the superoxide anion O−•. The obtained OH• and O−• can induce lipid peroxidation in membranes, DNA damage due to strand breakage or oxidized nucleotides and oxidation of amino acids and protein catalytic centres [83]. Negative charge of OH• and O−• prevents these species from passing through the membrane into the cell, so they can exert only outside damage.

and/or hydroxide ions, generating hydroxyl radicals (OH•). Electrons can reduceO<sup>2</sup>

O2

and flower‐shaped nanoparticles with exposed polar are even stronger[82].

damage leading to cell death [82]. ZnO nanoparticles show bactericidal properties as well as ROS generation also in complete absence of light. This effect has been tried to be explained by surface defects and the oxidative role of oxygen or halogens adsorbed on their surfaces [31, 82]. Suchamechanism would be enhanced in an aerobic environment and it was observed that oxy‐ gen annealing and formation of nanoholes on the surface, which both stimulated a high amount of adsorbed oxygen atoms on the ZnO surface, increased the ROS production and enhanced the antibacterial properties [31, 82]. ZnO nanorods are stronger antimicrobials than nanospheres,

nanoparticlescanaccountfortheirantibacterialeffectasaconsequenceofproductionof-OH•radicals.TheydonotpossessanyantibacterialpropertiesintheabsenceofUVlightdue-

)in the valence band and an electron (e−)in the conduction band[82, 83].

more powerful bactericidal agents [80] were more toxic towards human cells [81].

/Cu2+ ions by their dissolution [78]. Some-

, which can pass into the cell and create internal

NPs[79]. In general, it

to pro‐

Au nanoparticles alone are considered biocompatible and bioinert [87–89]. OnlyAu NPswithsizebelow3nmarecytotoxicduetotheirirreversiblebindingtokeybiopolymers[90]. InternalizationofAuNPsintoacellissize‐,shape‐andcharge‐dependant.Thefastestuptakewasobserved for40–50nmsize;itwashigher fornanospheresvsnanorodsandpositivelychargedNPspenetratemoreeasily[91–93].AuNPscanbeusedasantibacterialagentsonlyiftheyareirradiatedwithNIRlight(photothermaltreatment)orifsomeantibacterialcompo‐ nent is added to them [77, 85, 89]. Interestingly, some studies have also shown antibacterial activityofAunanoparticleswithnon‐antibacterialcomponentsaddedtothem,likeC/Aucoreshell [94]orfunctionalizedAunanoparticles[95–97].Aunanoparticlesascarriersenableentryof the addedmoleculesinto bacterial cells,where they can directly affect someimportantmolecules, otherwise protected by the cell wall and membrane. Concentration of otherwise inactivemoleculesonthesurfaceofAunanoparticleenables(orincreases)someinteractionsthat lead to bacterial death [95]. In this way, 4,6‐diamino‐2‐pyrimidinethiol was able to chelate Mg2+ionswhenattachedtotheAunanoparticle[95] and induced damage of the outer mem‐ brane,leadingtoincreasedpermeabilityofthecellularmembrane.Nanoparticlesenteredthecell, where chelation of Mg2+andinteractionoftheparticleswithDNAresultedininhibitionof protein synthesis. Cell death followed as a consequence of leakage of intracellular contents [95]. The antibacterial action of Au/4,6‐Diamino‐2‐pyrimidinethiol NPs involves changingthemembranepotentialandinhibitionofATPsynthaseactivitiestodecreasetheATPlevelandinhibitionoftheribosomesubunitfortRNAbinding,indicatingacollapseofbiologicalprocess [98].Alternatively,inaminoacid‐functionalizedAuNPs,astructuresimilartoanti‐ microbial peptides was created and enabled strong electrostatic interactions between cationic functionalizationatAuNPsandbacterialmembraneresultingindamageofthemembranecompactness and structure which provided antibacterial action in *E. coli* and *S. aureus* [97].

#### **3.6. Gallium‐containing nanoparticles**

Investigation of Ga‐based antibacterial nanoparticles has begun only very recently and it started with Ga2 O3 nanoparticles (100nm)showing theiranti‐biofoulingpropertiesagainst-*E. coli* and *S. aureus* [99].However, concentrationsup to 25mg/L (133μM)exhibited onlyveryweak(towards*S. aureus*)ornoinhibition(towards*E. coli*)ofplanktonicgrowth.Furtherinvestigation showed antibacterial action of Ga2 O3 nanorods (50×200 nm) which createdan inhibition zone in *E. coli* already at 25 mg/L concentration, whereas at least 50 mg/L- concentrationwasneeded foraninhibitionzonein*S. aureus* [100]. By contrast, bulk Ga2 O3 did not create anyinhibition zone. They also presented good photocatalytic properties ofthissemiconductorwithabandgapof4.9eV,butphotocatalysiswasnotresponsiblefortheobserved antibacterial action, since the test was performed in dark and Ga2 O3 nanoparticles cancreatereactiveoxygenspecies(onlyOH•radicals)onlyunderdirectvisiblelightillumi‐ nation [101].AntibacterialactivitywasshownalsoforGaNnanoparticles(50nm)[102] and anti‐biofouling activity was observed towards *S. aureus*, *E. coli*, *P. aeruginosa* and *Pseudomonas putida*.AnotherstudyaboutanewGa(III)deliverysystemintheformofKGa[Fe(CN)<sup>6</sup> ]/PVP-NPs(averagesizeof15nm)waspublished,whichdemonstratedtheirverygoodbiocompat‐ ibilitywithHeLacellsuntilatleast1.1mMconcentration, theirmostprobableendocytoticpenetration into the cells and untargeted distribution in the cytoplasm, and *in vitro* exchange ofGa(III)byFe(III)fromFe(II)suggestingtheirabilitytosequesterFe(II)andconsequentlyreleaseGa(III)[103].AveryrecentstudyoneutecticGaInalloynanoparticles(averagesizearound100nm)hasalsoshowntheirlow*in vitro*cytotoxicityagainstHeLacellsforatleast-21mg/L(0.2mMGa)concentrationandendocytosis,fusionanddegradationoftheeutectic-GaIn nanoparticles with release of Ga3+ionsinsideHeLacells[104]. The *in vivo*injectionofthese nanoparticles into mice caused no tissue damage, no allergic reaction, exhibited very low acutetoxicity(maximumtolerateddoseof700mg/kg),whileGaandInwereexcretedwithboth faeces and urine [104].However,theantibacterialpropertiesofKGa[Fe(CN)6]/PVPandeutecticGaInalloynanoparticleshavenotbeenevaluated.Ontheotherhand,Narayanasamyetal.incorporatedGa(III)‐tetraphenylporphyrinintopolymernanoparticles(averagesizeof-300nm)anddemonstratedtheirefficiencyagainst*Mycobacterium smegmatis* as well as against HIVinmacrophages, and did not show any sign of cytotoxicity formacrophages even at-2mM concentrations despiteinternalization of the nanoparticlesinto all compartments ofthe cells [105].Anotherway for thelocaldelivery ofGa(III)‐tetraphenylporphyrinwasbyitsconjugationtoPtnanoparticles(averagesizearound30nm)[106]. Bactericidal properties against *S. aureus*weredemonstratedundervisuallightillumination.However,thenon‐conju‐ gatedPtnanoparticleswerenottested,soitisnotclearhowlargetheircontributionwasandtowhatextenttheywereonlydeliverersofGa(III)‐tetraphenylporphyrin.Firstinvestigationon antibacterial performances of elemental Ga nanoparticles [107]confirmedactivityagainst-*P. aeruginosa*withMICat0.1mg/ml,lowtoxicityatthisconcentrationandwidetherapeuticwindow, which gives a good promise to this material for further investigations and design for biomedical applications.

#### **3.7. Nanostructured MgO**

MgO exhibits a broad range of antimicrobial activities against both Gram‐positive and Gram‐negative bacteria comparable to ZnO [107]. The molecular mechanism of MgO's antibacterial activity is still unclear. Some reports show dominant role of ROS which cause lipid peroxidation and diffuse inside the cell and cause cell death[108–110]. Other reports show non‐ROS mechanism and suggest polar interaction with components of the cell wall (e.g. lipopolysaccharides), which cause membrane disintegration and bacterial death [111] similar to antimicrobial peptides [112]. In both mechanisms' descriptions, the surface defect sites were related to the production of ROS. In the latter case, generation of ROS species was attributed to defects in general [111]. In the former case, oxygen could be reduced at the surface oxygen vacancy [109]. This is consistent with the mechanism of ROS species generation at the surface of MgO. However, for this to happen, energy is required to support electron transfer from vacancy towards the molecular oxygen [113]. Other mechanism of ROS generation was completely ignored in explanation of MgO antibacterial activity [113]. Till date, the following properties of MgO are known: (i) MgO exhibits contact‐based antibacte‐ rial action [108]; (ii) increasing the pH in bacterial suspension due to MgO hydration did not contribute to its antibacterial activity [108]; (iii) dissolved Mg2+ were not causing harm to bac‐ teria [108]; (iv) AFM and SEM morphology studies confirmed deterioration of bacterial mem‐ brane, which indicated membrane leakage [111, 114]; (v) TEM study showed lack of MgO particle internalization in bacteria, which indicated that MgO particles are "doing the dam‐ age" outside of the bacteria [111]. It has been shown that the bactericidal potential of MgO is proportional to its specific surface area. The MgO with the highest specific surface area (BET) exhibited the most effective antibacterial activity[115]. Improvement in the effectiveness of the bacteria/surface contact is also achieved by Li‐doping which enhanced the creation of oxygen vacancies and improved antibacterial activity [116]. The strength of the nanopar‐ ticle interaction is inversely proportional to the size, i.e. smaller particles exhibited stronger agglomeration [117]. The agglomeration could strongly influence the further processing of MgO nanoparticles[118]. MgO particles containing microrods exhibited moderate antibac‐ terial activity, while nano‐textured microrods showed strongly improved antibacterial activ‐ ity. As‐prepared particles exhibited reduced agglomeration, lower specific surface area and improved bactericidal potential when compared to the commercial MgO nanoparticles. We attributed the difference in antibacterial activity toareduced concentration of non‐emissive defects at the surface of nano‐textured MgO microrods[118]. Magnesium is the second most abundant intracellular cation in the human body [119] essential in many physiological pro‐ cesses like enzyme activity, membrane processes, functioning of muscle and neural tissue, and so on. [119]. The clinical study showed the ability of MgO to reduce hypertension (1g for 21 days)[120]. Although *in vitro* studies pointed out toxic effect of MgO on human cells [121], at aconcentration of 0.2 mg/ml in suspension MgO particles were able to eliminate bacteria while at the same time showed potential to exhibit bioactive properties on the cells. In this context, there isapossibility to exploit multifunctional properties of MgO to design medicine‐relevant devices, which exhibit both bioactive and antimicrobial properties.

#### **3.8. Nanostructured V2 O5**

concentrationwasneeded foraninhibitionzonein*S. aureus* [100]. By contrast, bulk Ga2

observed antibacterial action, since the test was performed in dark and Ga2

for biomedical applications.

48 Antibacterial Agents

**3.7. Nanostructured MgO**

did not create anyinhibition zone. They also presented good photocatalytic properties ofthissemiconductorwithabandgapof4.9eV,butphotocatalysiswasnotresponsibleforthe-

cancreatereactiveoxygenspecies(onlyOH•radicals)onlyunderdirectvisiblelightillumi‐ nation [101].AntibacterialactivitywasshownalsoforGaNnanoparticles(50nm)[102] and anti‐biofouling activity was observed towards *S. aureus*, *E. coli*, *P. aeruginosa* and *Pseudomonas putida*.AnotherstudyaboutanewGa(III)deliverysystemintheformofKGa[Fe(CN)<sup>6</sup>

NPs(averagesizeof15nm)waspublished,whichdemonstratedtheirverygoodbiocompat‐ ibilitywithHeLacellsuntilatleast1.1mMconcentration, theirmostprobableendocytoticpenetration into the cells and untargeted distribution in the cytoplasm, and *in vitro* exchange ofGa(III)byFe(III)fromFe(II)suggestingtheirabilitytosequesterFe(II)andconsequentlyreleaseGa(III)[103].AveryrecentstudyoneutecticGaInalloynanoparticles(averagesizearound100nm)hasalsoshowntheirlow*in vitro*cytotoxicityagainstHeLacellsforatleast-21mg/L(0.2mMGa)concentrationandendocytosis,fusionanddegradationoftheeutectic-GaIn nanoparticles with release of Ga3+ionsinsideHeLacells[104]. The *in vivo*injectionofthese nanoparticles into mice caused no tissue damage, no allergic reaction, exhibited very low acutetoxicity(maximumtolerateddoseof700mg/kg),whileGaandInwereexcretedwithboth faeces and urine [104].However,theantibacterialpropertiesofKGa[Fe(CN)6]/PVPandeutecticGaInalloynanoparticleshavenotbeenevaluated.Ontheotherhand,Narayanasamyetal.incorporatedGa(III)‐tetraphenylporphyrinintopolymernanoparticles(averagesizeof-300nm)anddemonstratedtheirefficiencyagainst*Mycobacterium smegmatis* as well as against HIVinmacrophages, and did not show any sign of cytotoxicity formacrophages even at-2mM concentrations despiteinternalization of the nanoparticlesinto all compartments ofthe cells [105].Anotherway for thelocaldelivery ofGa(III)‐tetraphenylporphyrinwasbyitsconjugationtoPtnanoparticles(averagesizearound30nm)[106]. Bactericidal properties against *S. aureus*weredemonstratedundervisuallightillumination.However,thenon‐conju‐ gatedPtnanoparticleswerenottested,soitisnotclearhowlargetheircontributionwasandtowhatextenttheywereonlydeliverersofGa(III)‐tetraphenylporphyrin.Firstinvestigationon antibacterial performances of elemental Ga nanoparticles [107]confirmedactivityagainst-*P. aeruginosa*withMICat0.1mg/ml,lowtoxicityatthisconcentrationandwidetherapeuticwindow, which gives a good promise to this material for further investigations and design

MgO exhibits a broad range of antimicrobial activities against both Gram‐positive and Gram‐negative bacteria comparable to ZnO [107]. The molecular mechanism of MgO's antibacterial activity is still unclear. Some reports show dominant role of ROS which cause lipid peroxidation and diffuse inside the cell and cause cell death[108–110]. Other reports show non‐ROS mechanism and suggest polar interaction with components of the cell wall (e.g. lipopolysaccharides), which cause membrane disintegration and bacterial death [111] similar to antimicrobial peptides [112]. In both mechanisms' descriptions, the surface defect

O3

]/PVP-

nanoparticles

O3

Recent studies have highlighted the ability of nanostructured V<sup>2</sup> O5 to mimic the myelo‐ peroxidase activity [122, 123]. The activity isacharacteristic of enzyme in human neutro‐ phils, which eliminate bacteria via the catalysis of the hydrogen‐peroxide‐to‐hypochlorite transformation in the presence of chloride ions [124]. This biomimetic property ofV<sup>2</sup> O5 was effectively utilized for the processing of an anti‐biofouling ship‐hull coating using sea water as a source of hydrogen peroxide (100 nM) [123]. However, it has been shown that V<sup>2</sup> O5 generates ROS on its own[125], which indicated the possibility to perform a unique mode of antibacterial activity with atwo‐step mechanism: (i) generation of ROS and (ii) trans‐ formation of the generated ROS to antibacterially more potent hypochlorite ions. The use of V<sup>2</sup> O<sup>5</sup> in medicine is limited by its relatively high solubility in aqueous media (>1 g/L). So‐formed, high concentrations of vanadate ions are toxic to human cells[126, 127]. *In vitro* studies also showed their bi‐phasic nature, as these ions stimulate proliferation of various types of mammalian cells at low concentrations (up to 10 μM) [128, 129]. They exhibit an insulin‐mimicking action via the inhibition of tyrosine phosphatase [130]. Orally adminis‐ tered vanadates in rat models stimulated the orientation of the fibroblasts in parallel arrays early in the tissue‐repair process, i.e., vanadate ions can accelerate tissue repair [131–133]. Vanadates improved the bone‐formation rate, mechanical strength and mineralization[134], while the pro‐oxidant potential of vanadates was not revealed in erythrocytes [135]. These studies confirmed the bioactive potential of vanadate ions when they are properly delivered, which might be effectively applied when designing the antibacterial drug‐delivery system to enable controlled delivery of vanadate ions.

#### **4. Concluding remarks**

Antibacterial ions are prone to similar problems as antibiotics, i.e., biodistribution and bacterial resistance. Nevertheless, they offer new options, especially for local delivery, and the antibiotic resistant bacteria are not always resistant also to antibacterial ions, even though Cu‐ and Ag‐resistance genes have been found associated with antibiotic resistance genes inafew cases. On the other hand, the major problem of nanoparticles is their non‐ selectivity and consequent toxicity for eukaryotic cells. For this reason, current findings are still far from a good substitution of antibiotics. It is very good that nanomaterials have many targets as opposed to antibiotics. This implies that they could be the solution for antibiotic resistance. But, the problem is that many of the targets are not specific for bacteria, in contrast to antibiotics. Particularly, the production of free radicals and reac‐ tive oxygen species in the absence of any cells needs to be avoided. Designing a wide therapeutic window (antibacterial activity at low concentrations and cytotoxicity at high concentrations of inorganic agent) is one of the greatest challenges for the application of inorganic antimicrobial agents. The possibility to modulate therapeutic window has the decision‐making role in the perspective of inorganic antimicrobial agents as an alternative antimicrobial strategy.

#### **Author details**

MarioKurtjak\*,NemanjaAničićandMarijaVukomanovicć \*Addressallcorrespondenceto:marija.vukomanovic@ijs.si AdvancedMaterialsDepartment,JozefStefanInstitute,Ljubljana,Slovenia

#### **References**

of antibacterial activity with atwo‐step mechanism: (i) generation of ROS and (ii) trans‐ formation of the generated ROS to antibacterially more potent hypochlorite ions. The use

Antibacterial ions are prone to similar problems as antibiotics, i.e., biodistribution and bacterial resistance. Nevertheless, they offer new options, especially for local delivery, and the antibiotic resistant bacteria are not always resistant also to antibacterial ions, even though Cu‐ and Ag‐resistance genes have been found associated with antibiotic resistance genes inafew cases. On the other hand, the major problem of nanoparticles is their non‐ selectivity and consequent toxicity for eukaryotic cells. For this reason, current findings are still far from a good substitution of antibiotics. It is very good that nanomaterials have many targets as opposed to antibiotics. This implies that they could be the solution for antibiotic resistance. But, the problem is that many of the targets are not specific for bacteria, in contrast to antibiotics. Particularly, the production of free radicals and reac‐ tive oxygen species in the absence of any cells needs to be avoided. Designing a wide therapeutic window (antibacterial activity at low concentrations and cytotoxicity at high concentrations of inorganic agent) is one of the greatest challenges for the application of inorganic antimicrobial agents. The possibility to modulate therapeutic window has the decision‐making role in the perspective of inorganic antimicrobial agents as an alternative

O<sup>5</sup> in medicine is limited by its relatively high solubility in aqueous media (>1 g/L). So‐formed, high concentrations of vanadate ions are toxic to human cells[126, 127]. *In vitro* studies also showed their bi‐phasic nature, as these ions stimulate proliferation of various types of mammalian cells at low concentrations (up to 10 μM) [128, 129]. They exhibit an insulin‐mimicking action via the inhibition of tyrosine phosphatase [130]. Orally adminis‐ tered vanadates in rat models stimulated the orientation of the fibroblasts in parallel arrays early in the tissue‐repair process, i.e., vanadate ions can accelerate tissue repair [131–133]. Vanadates improved the bone‐formation rate, mechanical strength and mineralization[134], while the pro‐oxidant potential of vanadates was not revealed in erythrocytes [135]. These studies confirmed the bioactive potential of vanadate ions when they are properly delivered, which might be effectively applied when designing the antibacterial drug‐delivery system to

of V<sup>2</sup>

50 Antibacterial Agents

enable controlled delivery of vanadate ions.

**4. Concluding remarks**

antimicrobial strategy.

MarioKurtjak\*,NemanjaAničićandMarijaVukomanovicć \*Addressallcorrespondenceto:marija.vukomanovic@ijs.si

AdvancedMaterialsDepartment,JozefStefanInstitute,Ljubljana,Slovenia

**Author details**


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