Inorganic Nanoparticles: Innovative Tools for Antimicrobial Agents

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 same­ antibiotics for all these applications, we will remain caught in the “vicious circle” of constant discovery of new synthetic antibiotics and very fast of their for 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 + , Cu + /Cu 2+ , Zn 2+ , Ga 3+ ,­etc.)­or­nanoparticles­(Ag/AgO,­Cu/Cu 2 O/CuO,­ZnO,­ Ga/Ga 2 O 3 ,­TiO 2 ,­MgO,­V 2 O 3 ,­etc.).­Here­we­are­going­to­summarize­the­main­properties­ of inorganic antimicrobials as well as advantages, disadvantages and perspectives for their application.


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,mainlyE. faecium), methicilin-resistant Staphylococcus aureus(MRSA),Klebsiella(especiallyK. pneumoniae),Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter and Escherichia coli(theso-called"ESKAPE"pathogens,), Gram-positive Mycobacterium tuberculosis and some other Gram-negative bacteria [1]. Soontherewillbenoavailableantibioticstotreatinfectionswiththesepathogens.Theproblem firstappearedinhospitalsandgrewpromptlyasaconsequenceofuncontrolledapplication of antibiotics not only in the healthcare but also in agriculture, stock breeding, poultry breeding,etc.However,overuseandmisusearenottheonlyfactorsthatspeedupthespreadof resistance.Somemechanismsofresistancedonotdestroytheantibioticandleaveitactivein the environment. Thus, bacteria themselves help maintain the antibiotic environment; furthermore, the drug can be released into other environments and alter them. Many precautions 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 developmentofnewkindsofantibioticswereminimizedduetotheirunprofitability.Andeven 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 requiredinordertoachieveeffectivetreatment.However,ifweusethesameantibioticsfor 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 needtofindalternativestrategiesthatwillberoutinelyusedforsomespecificconditions (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 developmentofinfection,etc.).Thus,wewillkeeptheactivityoftheantibioticsandsave themforurgent,acuteconditions(likepneumonia,meningitis,peritonitis,etc.).Oneoption 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.Theyareapplicableintheformofantibacterialions(i.e.Ag + , Cu + /Cu 2+ , Zn 2+ , Ga 3+ , etc.)orantibacterialnanoparticles(i.e.Ag/AgO,Cu/Cu 2 O/CuO,ZnO,Ga/Ga 2 O 3 ,TiO 2 ,MgO, V 2 O 3 ,functionalizedAu,etc.).Thenewknowledgebroughtespeciallybytheemergenceand progressofbiomaterialsscienceandnanotechnologymightenable:(i)local,targetedaction withoutsideeffectsintheorganism,(ii)improvedtransporttowardsandeasedpenetration intothepathogenicspecies,leadingtohigherefficiency,(iii)uniqueopportunityfordevelopmentofeffectivemedicines.
This chapter provides detailed overview of various inorganic antimicrobial agents, their physicochemical properties and various mechanisms of action on bacterial/mammalian cells.
TheantibacterialactionofAg + ions is currently explained by three mechanisms: 1. Ag + ions react with thiol groups of the respiratory and transport proteins in the cell membrane [6,9] so that cellular respiration and electron transfer are blocked [6,9], membrane potential and permeability are disrupted, leading to cell death [10].

Increasedproductionofreactiveoxygenspecies(ROS).Disruptionofcellularrespiration
and inactivation of intracellular thiol-based antioxidants increases the oxidative stress caused by reactive radicals that are generated by the Fenton reactions [9,13].
Bacteriahaveevolvedarangeofmechanismstoprotectthemselvesfromthetoxiceffectsof 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].ActiveextrusionofCufromthecellappearstobe the chief mechanism of Cu tolerance in bacteria and has been extensively studied in Grampositive 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).

Zinc(II) (Zn 2+ )
Zn 2+ 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 approximately 1.5-2.5 g of Zn 2+ [22,[26][27][28] with essential role in cell membrane integrity, development and maintenance of the body's immune system, managing insulin action and blood glucose concentration,boneandteethmineralization,normaltasteandwoundhealing [22]. Zn is a constituentofmorethan300enzymesthathaveacentralroleinreconstructionofthewound matrix [26,29].Znincastoroilhasaspecialplaceinthetreatmentofnappy(diaper)rash [26]. A vast range of zincated bandages, dressings, emollients, shampoos and creams are available commercially. In normal wound healing, body creates a higher amount of Zn 2+ in the wound margin at a certain stage-during the formation of granulation tissue, scar tissue andre-epithelialization.ItisbelievedthattheadditionofZnatthisstagemightaccelerate woundhealing.ExperimentalstudieshaveshownthattopicalZnOreducedtheinitialhaemorrhagic phase and promoted the regrowth of damaged skin and hair [26]. The antibacterial properties of Zn 2+ ions are exploited especially in oral healthcare for prevention of caries, gingivitis and periodontitis. Zn− salts are used in mouthwashes and toothpastes [30]. The effectofZn 2+ ions is most probably only bacteriostatic, so oral-care products are designed forfrequentuse,whilebactericidalactioncanbeobtainedincombinationswithfluorideor Triclosan [30][31][32][33].

2.
Zn 2+ ionsinhibittheutilizationofthebacterialcarbonsource.Theycandisruptthemetabolism of sugars as well as the amino acid metabolism.

4.
Zn(II) binds to the membranes and slows down the growth of organisms [6], inhibits protease-induced adhesion [34] and reduces the net negative charge on the cell surface and, hence, increases co-aggregation [34].
The following is currently known about the mechanism of antibacterial action of Ga 3+ ions: 1. Ga 3+ follows uptake and transport pathways for Fe 3+ ;unlikeFe(III),itcannotbe reduced to the oxidation state (+2); small amounts of non-bound Ga can exist in solution at physiological conditions, versus insignificant amounts of non-bound Fe 3+ , permitting biological interactions for Ga 3+ that would not be possible for Fe 3+ [49,50].

Cu/CuO nanoparticles
In Cu nanoparticles, there is a coincidence of antibacterial effect of ions and nano-sized particles.TheefficiencyofCuwasimprovedbydecreasingthedimensions,butitwashigher for Gram-positive bacteria [32].Cunanoparticleshavegreataffinityforaminesandcarboxyl groups, so they bind to the ones on the surface of bacteria and release the ions inside. These ionscantheninteractwithDNAmoleculesandintercalatewithnucleicacidstrands [77]. It isbelievedthathere,theroleofROSismuchlargerthaninAgnanoparticles,sincetheycan be generated by CuO as well as the released Cu + /Cu 2+ ions by their dissolution [78]. Some scientists,ontheotherhand,emphasizetheroleofthereleasedionsmore [32]. Both Cu and CuO antibacterial nanoparticles cause lipid peroxidation, cell wall and membrane damage andoxidativedamagetoDNA.TheygenerateROSintheabsenceofanycells,inextracellular aswellasintracellularenvironment.CuOnanoparticlesaremuchmoretoxictomammalian cells than Cu 2+ ionsandalsomuchmorecytotoxicthanZnOandTiO 2 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].

Nanostructured V 2 O 5
Recent studies have highlighted the ability of nanostructured V 2 O 5 to mimic the myeloperoxidase activity [122,123]. The activity is a characteristic of enzyme in human neutrophils, which eliminate bacteria via the catalysis of the hydrogen-peroxide-to-hypochlorite transformation in the presence of chloride ions [124].ThisbiomimeticpropertyofV 2 O 5 was effectively utilizedfortheprocessingofananti-biofoulingship-hullcoatingusingseawater as a source of hydrogen peroxide (100 nM) [123]. However, it has been shown that V 2 O 5 generatesROSonitsown [125], which indicated the possibility to perform a unique mode of antibacterial activity with a two-step mechanism: (i) generation of ROS and (ii) transformation of the generated ROS to antibacterially more potent hypochlorite ions. The use of V 2 O 5 in medicine is limited by its relatively high solubility in aqueous media (>1 g/L). So-formed,highconcentrationsofvanadateionsaretoxictohumancells [126,127]. In vitro studies also showed their bi-phasic nature, as these ions stimulate proliferation of various types of mammaliancells at low concentrations (up to 10 μM) [128,129]. They exhibit an insulin-mimicking action via the inhibition of tyrosine phosphatase [130].Orallyadministeredvanadatesinratmodels stimulatedtheorientationofthefibroblastsinparallelarrays early in the tissue-repair process, i.e., vanadate ions can accelerate tissue repair [131][132][133].

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 thoughCu-andAg-resistancegeneshavebeenfoundassociatedwithantibioticresistance genesinafewcases.Ontheotherhand,themajorproblemofnanoparticlesistheirnonselectivityandconsequenttoxicityforeukaryoticcells.Forthisreason,currentfindings 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 forantibioticresistance.But,theproblemisthatmanyofthetargetsarenotspecificfor bacteria,incontrasttoantibiotics.Particularly,theproductionoffreeradicalsandreactive oxygen species in the absence of any cells needs to be avoided. Designing a wide therapeuticwindow(antibacterialactivityatlowconcentrationsandcytotoxicityathigh concentrationsofinorganicagent)isoneofthegreatestchallengesfortheapplicationof 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.