Antimicrobial properties of ZnO nanomaterials: A review

Author’s Accepted Manuscript Antimicrobial Properties of ZnO Nanomaterials: A Review Rajesh Kumar, Ahmad Umar, Girish Kumar, Hari Singh Nalwa www.elsevier.com/locate/ceri

PII: DOI: Reference:

S0272-8842(16)32312-4 http://dx.doi.org/10.1016/j.ceramint.2016.12.062 CERI14372

To appear in: Ceramics International Received date: 14 November 2016 Revised date: 9 December 2016 Accepted date: 10 December 2016 Cite this article as: Rajesh Kumar, Ahmad Umar, Girish Kumar and Hari Singh Nalwa, Antimicrobial Properties of ZnO Nanomaterials: A Review, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2016.12.062 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antimicrobial Properties of ZnO Nanomaterials: A Review Rajesh Kumar1, Ahmad Umar2,3*, Girish Kumar1*, Hari Singh Nalwa4 1

PGDepartment of Chemistry, JCDAV College, Dasuya 144205, Punjab, India Department of Chemistry, Faculty of Arts and Sciences, Najran University, PO Box 1988, Najran 11001, Kingdom of Saudi Arabia 3 Promising Centre for Sensors and Electronic Devices (PCSED), Najran University, PO Box 1988, Najran 11001, Kingdom of Saudi Arabia 4 Advanced Technology Research, 26650 The Old Road, Suite 208, Valencia, California 91381, USA * [email protected](A.U.) 2

*

[email protected](G.K.)

Abstract Waterborne diseases significantly affect the human health and are responsible for high mortality rates worldwide. Traditional methods of the treatment are now insignificant as maximum bacterial strains have developed multiple antibiotic resistance toward commonly used antibiotic drugs. Recently, ZnO nanostructure, due to its biocompatible nature, has attracted the attention of the scientific community to explore and to understand their cytotoxicity, interaction with biomolecules such as proteins, nucleic acids, fats, cell membranes, tissues, biological fluids, etc., and bio-safety for proper utilization in biomedical applications. Herein, we have reviewed the recent developments for the fabrication of ZnO nanomaterials with variable morphologies, factors influencing the growth, morphology and surface defects, and various laboratory methods to evaluate the antibacterial activities toward Gram-positive as well as Gramnegative strains. A comparative study is carried out to evaluate the mechanistic approach of ZnO nanomaterials toward Gram-positive as well as Gram-negative bacterial cells.

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ZnO nanomaterials can interact chemically as well as physically to exhibit antibacterial activities. Chemical interactions of the ZnO nanomaterials with bacterial cells lead to the photo-induced production of reactive oxygenated species (ROS), the formation of H2O2, and release of Zn2+ ions. In contrast, the physical interaction can show biocidal effects through cell envelope rupturing, cellular internalization or mechanical damage. Finally, surface activation through amine functionalization of ZnO nanoparticles for better antibacterial effects and cytotoxicity of ZnO nanoparticles toward cancer cells are also reviewed.

graphical abstract

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Keywords: ZnO Nanomaterial, Antibacterial, Gram-Positive and Gram-Negative Strains Cytotoxicity, Biocompatibility, Amine Functionalization

1.

INTRODUCTION The present day situation of the basic requirement of safe and sufficient drinking

water is at the alarming stage. Waterborne diseases such as typhoid fever, dysentery, cholera, and diarrhea, etc. are the major cause of mass population deaths worldwide and the problem is replicating at an alarming rate. The extensive use of traditional antibacterial drugs led to the bacterial resistance toward these antibacterial drugs, further intensifying the problem[1]. In addition, horizontal gene transfer by conjugation and transduction, etc. are also other reported possible ways for resistance[2]. Antibacterial agents find their potential applications in food, packaging, cosmetics, medical, healthcare industries, and so on. These antimicrobial agents are commonly classified into, organic or inorganic agents. However, inorganic antibacterial agents are reported to be advantageous over organic antibacterial agents due to higher stability and improved safety. Recent advancement in the field of nanotechnology has opened new channels for its application in a variety of fields. One of the explored applications is the utilization of nanomaterials as antimicrobial agents against bacteria, viruses, and other pathogenic microorganisms. Among the various nanomaterials, metal oxides are extensively studied due to their non-toxicity, stability, efficient biological properties, etc. A variety of nanomaterials such as ZnO, Fe2O3[3], TiO2[4], Ag2O[5], CaO[5], MgO[6] and

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CuO[3,7,8] have been used as efficient bactericidal agents for bacteria. Among the variously reported semiconductor nanomaterials, ZnO, an n-type semiconductor material, has been explored extensively as compared to other materials as anantimicrobial agent. It is biocompatible, non-toxic, photochemically stable, etc. Moreover, it has also been listed as a generally recognized as safe (GRAS) material by the U.S. Food and Drug Administration (21CFR182.8991)[9,10].It shows significant bactericidal properties over a broad range of Gram-positive as well as Gram-negative bacteria, including Escherichia coli,S. enteritidis, Streptococcus pyogenes, Aeromonas hydrophila, B. subtillis, S. aureus,L.

monocytogenes,

Klebsiella

pneumonia,

P.

aeruginosa,

Salmonella

typhimurium, E. faecalis, etc.[11]. Number of mechanisms such as metal ion release, generation of reactive oxygen species (ROS)[12], membrane dysfunction, and nanoparticles

penetration

or

internalization[13],interruption

and

blockage

of

transmembrane electron transportation, etc. have been proposed for explaining the interactions between the nanomaterials and disrupting of the bacterial cell wall[14,15]. Nanostructured ZnO with band gap 3.28 eV, has very high exciton binding energy of 60 meV. As a result, free electrons and holes have sufficient lifetime and may induce photogeneration of reactive oxygen species (ROS) on the surface of ZnO from adsorbed oxygen and water molecules[16]. These ROS include highly reactive species such as superoxide free radicals, hydroxyl free radical and H2O2, which damages cellular components like nucleic acid, proteins, enzymes, membrane, etc., causing various degrees of oxidative stress[17,18].Importantly, it has been reported that the size, morphology and specific surface area of the ZnO nanomaterials greatly affect the

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antibacterial properties[19,20]. Therefore, the surface area of the ZnO nanomaterials is related to the antibacterial activities; i.e., increasing the surface area enhances the antibacterial properties while decreasing the surface area leads to the reduction in the antibacterial properties[21]. The morphology of the ZnO nanomaterials can be tuned by controlled uses of the surfactants to prevent the agglomeration and to stabilize them either by steric repulsion or by electrostatic repulsions. In addition, functionalization of ZnO surface with polar groups further enhances the interactions between the ZnO nanomaterials and the bacterial cell wall. In this review, methods of preparation of versatile morphologies of ZnO, methods to evaluate biocidal activities, and mechanistic approach of ZnO nanomaterials towards Gram-positive as well as Gram-negative bacterial cells have been carried out. 2. ZINC OXIDE NANOSTRUCTURE BASED ANTIMICROBIAL AGENTS The antimicrobial activities of the ZnO nanostructures are greatly affected by the particle size and the morphology. Number of methods have been reported in literature for the synthesis and growth of the ZnO nanomaterials of versatile morphologies such as, nanorods[22–25],

nano/micro-flowers[26–30],microspheres[31],nano-powders[32–35],

nanotubes[36], quantum dots[37,38],thin films[38]and nanoparticles[8,39–58]and capped nanoparticles[59]etc. for their potential applications in the field of antimicrobial agents(Table1). ZnO crystal structure belongs to the C3v point group with each O2- ion surrounded by four Zn2+ ions at the corners of a tetrahedron, and vice versa. ZnO crystal possesses a number of defects and interstitial sites on its surface, which are responsible for its

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versatile properties such as mechanical, thermal, electrical and optical. Excess Zn2+ ions on the surface act as donor interstitials resulting in n-type conductivity. In addition, wurtzite ZnO crystal contains unstable polar symmetry oriented around [0001] terminated by Zn-atoms, and another oriented along [000 1 ] terminated by O-atoms on its surfaces. These two orientations are also responsible for the various physic-chemical properties of ZnO nanostructures. Additionally, there are some stable and non-polar faces such as

[01 1 0] , [0 1 10] , [1 1 00] , [ 1 100] , [ 1 010] , [10 1 0] , that favor the ZnO crystals to acquire a number of morphologies(Fig.1)[60].

Fig.1. (a) ZnO unit cell with wurtzite structure.(b) Various crystal planes of ZnO Wurtzite structure.[Reprinted with permission from Ref.[60], Copyright© 2015, Author].

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2.1. Nanorods One-dimensional ZnO nanorods possess high surface-volume ratio, crystallinity and charge confinement ability as compared to other ZnO nanostructures. Methods including wet processing routes such as hydrothermal, solution combustion chemical bath depositions, etc., and physical vapor-phase processing routes including sputtering techniques, thermal evaporation, and vapor-phase transport are reported widely in the literature. Among the various synthetic techniques, a hydrothermal method is a more convenient and reliable method for the synthesis of 1-D ZnO nanostructures. However, the quantity and quality of these 1-D ZnO nanorods dependon the type of capping agent, pH, and concentration and growth time of the reaction solution. The repeating units during the hydrothermal growth attack the crystal structure at aright angle to the c-axis with [0001] and the [000 1 ] polar surfaces. The polar [0001] and the [000 1 ] surfaces are among the most common crystal orientations of ZnO[61][62]. The concentration of the precursor and the pH, i.e., Zn2+:OH− ratio is the key factor for controlling the morphology and aspect ratio of the 1-D ZnO nanorods. The growth unit formed is an anionic Zn(OH ) 4 2 complex, which preferentially is adsorbed on the positive polar [0001] phase of the hexagonal wurtzite ZnO nuclei. Hu et al.[63]demonstrated that at a pH > 7 of the growth solution, free OH− ions are also strongly attached to the unstable polar plane oriented around [0001] containing Zn2+ ions. At higher concentration of OH−ions, linear chains undergo cyclization may be due to the greater magnitude of an intermolecular reaction than intramolecular reaction resulting in the formation of 1-D nanostructures. This results in anisotropy growth along the c-axis

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direction of the wurtzite ZnO forming 1-D nanostructures such as nanorods at higher pH of the growth solution[64]. Ramani et al.[22]evaluated the effect of concentration of pyridine as base as well as a capping agent for the growth of 1-D ZnO nanostructures. A lower concentration of pyridine resulted in the formation of uniform rod-like particles of 30–35 nm but still higher pyridine concentration leads to elongated rods with diameter 80–100 nm and lengths up to 2 μm. It was concluded that at higher concentration of pyridine, there is the absence of steric stabilization in pyridine-ZnO adduct favoring the Ostwald ripening and subsequent growth in one direction. Jansson et al.[25]synthesized ZnO nanorods through a seeded solution-based hydrothermal growth method. Seeded ZnO nanoparticles, spin-coated onto glass coverslipswere used as a substrate for ZnO nanorod growth using 20 mM zinc nitrate hexahydrate and 20 mM hexamethylenetriamine for 1 h. Jain et al.[23]used Zn(CH3COO)2·2H2O and NaOH aqueous solutions in stoichiometric ratio followed by the hydrothermal treatment for 120 °C for 24 h in a stainless steel vessel with Teflon liner for the synthesis of ZnO nanorods of average diameter ~45 nm and length ~250 nm. The morphology is also found to be dependent on the type of the solvent used during hydrothermal growth. Talebian et al.[24]obtained ZnO structures with rods, spheres, and flower-like morphologies for 1-hexanol, ethylene glycol and water as solvents, respectively, during an un-catalyzed, template and surfactant-free synthesis. The important parameters like nucleation and growth of the nanomaterials are greatly affected by the solubility of the precursor in the solvents and their saturated vapor pressure[65]. Fast growth for ZnO nanostructures has been observed for solvents like 1-hexanol and

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ethylene glycol, with lower saturated vapor pressures. Additionally, as solvent molecule chain length of the solvent molecule affects the interface-solvent interactions and the polarity, the growth of the ZnO nanostructures is further affected[66,67]. 1-Hexanol with lower polarity thus favors branched rod-like morphologies of ZnO crystals. Okyay et al.[68] reported dense growth of ZnO nanorods on the glass surface. Initial, room temperature sonochemical deposition of ZnO seed layer, using 0.005 M zinc acetate dihydrate dissolved in isopropyl alcohol serve as excellent nucleation sites for the guided orientation and growth of the ZnO nanorods in the subsequent step using 0.04 M zinc nitrate tetrahydrate (Zn(NO3)2·4H2O) and 0.04 M hexamethylenetetramine ((CH2)6N4) as precursors. Fig.2 represents the flow diagram for the growth of sonochemically deposited ZnO nanorods.

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Fig.2. Flow chart of sonochemical synthesis of (a) ZnO seed layer (b) ZnO nanorods on the glass slides. [Reprinted with permission from ref [69] CopyrightThe Royal Society of Chemistry].

2.2. Nano/Micro-flowers It is presumed that the various 1-D nanostructures, especially nanorods, rearrange to form some secondary structures such as nano/micro-flowers. Wahab et al.[27] proposed the

initial

formation

of

[Zn(OH)2 ]

and

[Zn(OH)4 ]2



precipitates

though

stoichiometrically not favored, from zinc acetate dihydrate and sodium hydroxide precursors at pH>9. Laudise and Ballman [70] reported that the higher the growth rate along a plane, the more rapidly it disappears. In wurtzite ZnO, the growth rates for ZnO

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in different planes have the sequence [0001] > [011 1 ] > [01 1 0] > [01 1 1] > [000 1 ] . As a result the [0001] plane disappears quickly, giving pointed tips for the nanorods, whereas the plane [000 1 ] with slowest growth rate results in the formation of a flat plane. These pointed tips aggregate to form flower-like morphologies for ZnO. Wahab et al.[27] reported the formation of micro-flowers from nanorod aggregation with a base diameter of 2–3 μm, with an individual nanorod of 150–200 nm diameter and length of 2 μm. The pointed tips may be formed at the either end, thereby resulting in the formation of leaflike structure, which may further arrange to form nano/micro-flowers. The zincate ion growth units, i.e. Zn(OH ) 4 2 , were supposed to form such morphologies by Mohan Kumar et al.[28]. Umar et al.[71] also observed aggregation of triangular shaped petals of different sizes with pointed tips and wider bases. The later were joined together to form an almost spherical assembly of magnificent flowers. Each petal of nanoflower was about 400–500 nm in length, with basal diameters of 200–300 nm. The average width of each nanoflower was about 1.0–1.5 μm. Fig. 3 (a) and (b) represents the corresponding low and high magnification images of ZnO nanoflowers, whereas Fig.3 (c)–(e) shows low and high magnification TEM images of as-synthesized ZnO nanoflowers synthesized through a facile solution process. Fig.3 (f) represents an HRTEM image exhibiting well-defined lattice fringes with an interplanar distance of ∼0.52 nm.

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Fig.3. Typical (a) and (b) low and highmagnification FESEM images,(c) and (d) low, (e) high-resolution HRTEM images (f) HRTEM image exhibiting well-defined lattice fringes at a distance of ∼0.52 nm for ZnO nanoflowers prepared by facile solution process. (Inset in Fig.3. (b) shows corresponding EDS spectrum of ZnO nanoflowers). [Reprinted with permission from ref [71], Copyright2013,American Scientific Publishers] Khan et al.[29] reported a marked effect on the morphology and crystalline size of the ZnO nanoflowers synthesized through CTAB assisted growth. With increasing growth temperature, larger, broader, and less dense arrow-like petals were formed. The size of the nanoflowers was also decreased with arise in temperature from 25oC to 75oC. The ZnO nanoparticles of crystallite sizes of 23.73, 82.58, 69.65, 88.82 nm were observed at

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25oC, 35oC, 55oC and 75oC temperatures, respectively. Similar results were reported by Li et al.[32].

2.3. Nanopowders and nanoparticles It is well known now that the particle size and the morphology of the ZnO particles can be conveniently controlled by using suitable surface stabilizing agents or surfactants[34,53,57,58]. Stankovi´c et al.[34] used polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) and poly (α,γ,L-glutamic acid)(PGA) for the synthesis of ZnO nanoparticles through a low-temperature hydrothermal method at a growth temperature of 120oC and pH=11 for 24 h. In the presence of PVP, hexagonal shaped prismatic rods of length 1 μm diameter of about 100 nm were formed (Fig. 4(a)). Nanoparticles of spherical morphology with average diameter of around 30 nm were formed if PVA was used as the surface stabilizing agent (Fig. 4(b)), whereas PGA resulted in the formation of ZnO nanoparticles with ellipsoidal shape having a length of 500–600 nm and diameter of 100 nm (Fig. 4(c)).

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Fig.4. FESEM images of hydrothermally synthesized ZnO nanoparticles in the presence of (a) PVP,(b) PVA, and (c) PGA. [Reprinted with permission from Ref. [34], Copyright 2013,Elsevier B.V.] Sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA), and polyoxyethylene sorbitan monolaurate (Tween 20) were used to give sheet-like ZnO particles, nanorods, and spherical ZnO nanoparticles, respectively by Huang et al. [53]. Some other stabilizing agents such as starch, polyethyleneglycol (PEG), ascorbic acid (AsA), mercaptoacetic acid (MAA),etc. are also reported in the literature[54,57–59]. PEG molecules are supposed to form hydrogen bonding with ZnO nuclei, thereby stabilizing them[72]. Still another important factor which affects the growth of ZnO nanoparticles is the pH of the solution. The particle sizes of the hexagonal shaped ZnO nanoparticles were

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reported 38 ± 2, 25 ± 4 and 15 ± 4 nm at pH values 7.2, 6.0 and5.0, respectively[39].A number of research groups have proposed some green synthetic routes for the synthesis of ZnO nanomaterials. Plant leaf extracts such as Tamarindus indica (L.)[26], Epigallocatechin gallate (EGCG), a tea catechin[35], curryleaf (Murrayakoenigii)[45], Cassiafistula[48], powder extract of dry ginger rhizome (Zingiber officinale), Solanum nigrum leaf extract, Moringa oleifera leaf extract[51], Vitex negundo plant extract[52], and even reproducible bacteria, aeromonashydrophila as an eco-friendly reducing and capping agent[46]. The particle size can also be controlled through calcination temperature, which results in the agglomeration of ZnO nano-grains due to high surface energy and high surface tension[55].The solubility of the Zn2+ ion precursor is also found to be a detrimental factor for controlling the particle size. ZnO particle synthesized using Zn(CH3COO)2.2H2O and ZnSO4were larger in size as compared to the particles obtained from Zn(NO3)2 precursor. It was suggested that the solubilities of these salts in the aqueous medium may have played a vital role. From the observations by Raghupathi et al.[56] it can be concluded that the greater the solubility of the precursor, the smaller is the particle size.

2.4. Nanowires 1D nano/microstructured ZnO nanowires has also been synthesized and utilized for effective antimicrobial activities. ZnO nanowires with high aspect ratio provide sufficiently large surface area for the production of reactive oxygenated species. High

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aspect ratio for ZnO nanowires can be achieved through proper and controlled homogeneous nucleation[73]. For this purpose many approaches have been adopted and reported in the literature. According to Gao et al.[74], the proper concentration of the

NH 4 ions in the growth solution serves the above said purpose. A number of additives such as NH4OH, NH4Cl, (NH4)2CO3 etc. are added to the growth solution. Other additives like polyethyleneimine (PEI) also affect the aspect ratio of the nanowire structures significantly[75,76]. Wang et al. [77] observed ZnO nanowire arrays with different orientations synthesized through vapor phase process using Zn powder as source materials. The orientations of these arrays were studied through Confocal laser scanning microscopy (CLSM) and field-emission scanning electron microscope (FE-SEM) techniques. Other factors like growth time and temperature also affect the dimensions and hence the aspect ratio of the ZnO nanowires [78]. A shorter growth time results in thinner and organized nanowires, whereas a longer growth time favors formation of thicker and random orientations of the nanowires.

2.5. Other morphologies ZnO

nanomaterials

of

some

other

morphology

including

microspheres[31],

nanotubes[36], quantum dots[37], thin films[38] etc., synthesized through surfactant-free, microwave assisted, hydrothermal, surfactant CTAB (cetyltrimethylammonium bromide) assisted solution method and DC reactive magnetron sputtering, respectively, are also

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reported in the literature. Shinde et al.[31]reported that the particle size and morphology of ZnO nanostructures can be controlled by changing the reaction time and the microwave power. For irradiation time of 1–3 min, ZnO microspheres with an average diameter of 1.5 μm are formed. Each microsphere was further composed of thin nanoplates of typical thickness 20 nm, whose density was further increased with longer microwave irradiation for 5 min (Fig. 5(a)). Aal et al.[36] hydrothermally synthesized nanotubes with a hexagonal inner as well as outer wall, having a wall thickness of less than 2 nm. The average and length of the as-synthesized nanotubes were 17 nm and 2 μm, respectively (Fig. 5(b)).Quantum dots with smooth and spherical surfaces of size about 5-6 nm were successfully synthesized by Wahab et al.[37] at very high pH of 12.4 at 95oC refluxing for 2 h. Fig.5(c) justifies the above details and also shows that assynthesized QD are grown in very high yield. Carvalho et al.[38] deposited ZnO thin films of 50 to 600 nm thickness through theDC Reactive Magnetron Sputtering technique. As the deposition time was increased, the thickness of the ZnO thin films was also increased. Thin films of thickness 50 and 100 nm were more compact with smaller particle sizes than thicker films.

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Fig.5.(a) High FESEM image of ZnO microspheres synthesized through microwave irradiation; [Reprinted with permission from Ref. [31], Copyright © 2012 Scientific & Academic Publishing], (b) HRTEM image of hexagonal ZnO nanotubes; [Reprinted with permission from Ref. [36], Copyright © 2015 Elsevier B.V.], (c) Low magnification TEM image of ZnO QD; [Reprinted with permission from Ref. [37], Copyright © 2014 Elsevier B.V.], and (d) SEM image of ZnO thin film with thickness 600 nm; [Reprinted with permission from Ref. [38], Copyright © 2014 Elsevier B.V.].

3. METHODS FOR STUDYING ANTIMICROBIAL ACTIVITIES OF ZnO In order to evaluate the antimicrobial activities, a number of experimental techniques are reported in the literature and given in Table-1. Depending upon the type of the bacterial species,i.e., Gram-positive or Gram-negative, and the type of data to be extracted one or more techniques are applied so as to get accurate and significant results. In all the

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methods of bacterial assays are incubated at 37oC for a particular time in the growth medium. The growth medium may contain trypticase soy broth, Luria–Bertani broth, nutrient agar and tryptic soyagar (TSA) etc. as culture media. Depending upon the technique used the parameters like minimum inhibition concentration (MIC), Zone of inhibition (ZOI), colony count, or optical densities of the bacterial culture are evaluated. 3.1. Disk-Diffusion Method It is also referred to as Kirby–Bauer antibiotic testing (KB testing). In this method, Mueller-Hinton agar as media is taken in a Petri dish. The pH of the media is generally maintained around physiological pH of 7.4. A bacterial suspension containing a known concentration

is

applied

on

the

Mueller-Hinton

agar

media[8,22,23,26,31,45,47,51,54,79].The assay is allowed to dry. A definite amount of the ZnO nanomaterials is soaked on the filter paper disks using a particular solvent under aseptic conditions. The disks are allowed to dry and subsequently mounted over the media in the Petri dish carefully. A disk soaked only with solvent is used as a control. These plates are incubated for 24 h at 37oC so as to provide proper growth conditions for the bacteria growth. As a result of bactericidal activities of the ZnO nanomaterials, no bacterial growth is observed around the disk up to a particular distance depending upon the concentration of the ZnO nanomaterials. The minimum concentration at which ZnO exhibits antimicrobial activities is called minimum inhibition concentration (MIC) and the zone around the disk where no bacterial growth is observed is referred to as the zone of inhibition. Lower the value of MIC and greater the diameter of the inhibition zone, higher is the antimicrobial activity[50,53].

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3.2. Well diffusion method The technique for the formation of Mueller-Hinton agar media and culture formation is same as that of the disk-diffusion method. In contrast to the disk-diffusion method, a well is bored in the media plate instead of mounting filter paper disks. The well is filled with suspensions of the ZnO nanoparticles of varying concentrations. The MIC and ZOI are calculated

in

a

similar

fashion

like

that

of

the

disk-diffusion

method[8,35,39,43,46,48,49,52,55,80].Aseptic conditions are also mandatory to maintain a contamination-free environment. In Fig.6 (a)–(d) zone of inhibitions for Klebsiella aerogenes, Escherichia coli, Staphylococcus aureus and Pseudomonas desmolyticum, respectively, are shown after the incubation at 37oC for 36 h of the culture against ZnO Nps having 500and1000 μg concentrations. A positive standard (Ciprofloxacin) is used to study the viability of the ZnO NPs as antibacterial agents[48].

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Fig.6 (a)–(d) ZI for K. aerogenes, E. coli, S. aureus and P. desmolyticum, respectively, treated through agar well diffusion method. [Reprinted with permission from Ref.[48], Copyright © 2015 Elsevier Ltd.]. 3.3. Optical density measurement in liquid broth medium During the incubation period, due to the growth of bacteria the turbidity of the growth solution is increased. This increase in turbidity and hence cell proliferation can be measured through optical density measurements after regular time spans. No reagents and specific

processing

are

required

in

this

technique[22,23,25,30,32,33,36–

38,41,42,56,59,81]. Umar et al.[30]carried out growth inhibition experiments on E. coli strains using ZnO nanoflowers as antibacterial agents.50 μl E. coli strain was inoculated

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with 5 ml of a freshly prepared nutrient broth medium in cultured glass tubes which were then incubated at 37oC for 12 hrs. After this 25, 50, 75, 100, and 150 μg/ml of ZnO nanoflower was introduced into the bacterial suspension and the corresponding bacterial growth was monitored at regular intervals by observing the absorbance at 600 nm in a spectrophotometer. From Fig.7, the optical density of the broth solution was found to decrease regularly with increasing concentration of the ZnO nanoflowers as antimicrobial agents. The bacterial growth in liquid broth involves four phases; viz., lag phase, log phase, stationary phase, and death phase. Lag phase involves the adaptations of the bacterial culture towards growth conditions, log phase also known as logarithmic phase or exponential phase is a cell division or growth phase. Stationary phase is an equilibrium phase in which the growth and the death rates of the cells are almost same. The final death phase also called decline phase marks the last phase, which may result due to consumption of the essential nutrients and formation of some growth inhibitors in the medium[23,30,36,56,82].

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Fig.7. Bacterial growth curves of E. coli with increasing concentration of ZnO nanoflowers. [Reprinted with permission from Ref. [30], Copyright © 2013, American Scientific Publishers]. From Fig.7 it can be clearly demonstrated that with increasing concentration of the ZnO nanoflowers, lag phase is shortened. For still higher concentrations as studied by Jain et al.[23], no lag phase is observed for Gram-positive (S. aureus and B. subtilis) as well as Gram-negative (E. coli and A. aerogenes) bacteria. ZnO nanorods act differently for Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, a concentration of ZnO nanorods (50μg/mL) is capable of extending the lag phase and delaying the onset of log phase, whereas for Gram-negative bacteria, at higher concentrations of 100, 150, and 200 μg/mL, the growth curves show the absence of all four phases. In contrast, a higher concentration of 500 μg/mL of ZnO nanorod gives the same results for Gram-negative bacterial strains.

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3.4. Colony-forming unit Measurements The technique is also generally known as spread-plate technique and is applied to count the number of viable bacteria cells. It involves the spreading of the bacterial strains on the agar layer taken in a Petri dish. ZnO nanomaterials are either introduced into the agar plate or are spread as suspensions in a particular liquid medium. Bacterial strains are mounted on the agar plate and are subjected to incubation for a particular time at 37oC temperature. Colony-forming units are counted using a suitable colony count method. The comparison is made by counting the CFUs for bacterial strains incubated in the presence and absence of ZnO nanoparticles (taken as control)[24,32,34,40,42,44,53,58]. Stankovi´c et al.[34] calculated the percentage of bacterial cell reduction (R %) using the Eq. (1), R% 

CFU control  CFUSample CFUSample

…………………(1)

where CFUcontrol = numbers of CFUs per milliliter for the negative control, and CFUsample=CFUs per milliliter in the presence of ZnO dispersion.

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Fig.8.Comparison of CFUs for Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria strains in the presence of ZnO nanorods (Sample-A), ZnO spheres (Sample-B),

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ZnO flowers (Sample-C) and control (Blank) under different conditions. [Reprinted with permission from Ref. [24], Copyright © 2013 Elsevier B.V.]. Fig.8. represents the viable growth of Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria strains in the presence of ZnO nanorods, nanospheres micro-flowers and Blank control in the presence of UV irradiation under dark conditions as studied by Talebian et al.[24].It was concluded that ZnO nanostructures with flower-like morphology exhibited better photocatalytic inactivation as compared to nanorod and nanospheres. Further, the inactivation efficiencies for E. coli and S. aureus under UV light irradiation were better than dark conditions[24]. 3.5. Microtiter plate method A microtiter plate also called microplate consists of flat plate with variable number of small test tubes or wells. Prior to the dispersion of bacterial strains and ZnO suspensions, an indicator solution which may comprise resazurin[57], 2,3,5-triphenyltetrazolium chloride

(TTC),

crystal

violet[80]

and

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT)[83]etc. is placed into the wells. After that, bacterial strains of known and different concentrations are dispersed into the wells of the microtiter plate. Known concentrations of ZnO nanomaterials diluted in sterile broth medium are then dispersed into the wells. The palate is then subjected to incubation at 37°C for a definite time interval. The change in the absorbance of the indicator helps to determine the bacterial cell viability. Table1:Synthesis, morphologies, and dimensions of ZnO nanostructuresfor antibacterial activities against various Gram-positive and Gram-negative bacterial strains.

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M or ph olo gy

Method of preparation

Dim ensi ons

Bac teri a

Met hods appl ied

Un ca pp ed Zn O S ma ll Na no ro ds Lo ng Na no ro ds

Aqueous solution route

70– 80 nm 30– 35 nm D=8 0– 100 nm, L=2 μm

Sta phy loc occ us aur eus

Liqu id brot h meth od Disk diffu sion meth ods

Na no ro ds

Hydrothermal method

D=4 5nm , L=2 50 nm

#

Sal mo nell a typ him uri um * Pro teus vul gar is* Kle bsie lla pne um oni ae* Sta phy loc occ us aur eus #

Bac illu s

Liqu id brot h meth od, Disk diffu sion meth

Prop osed mode of actio n ROS produ ction

ROS produ ction

Important results

R e f .

ZOI diameter increased with concentrations of ZnO nanorods (5–45 μg mL−1) for all strains except for Proteus vulgaris. Toxicity order: uncapped-ZnO < Long nanorods < Small nanorods. Gram-positive Staphylococcus aureus bacterium, due to a thicker peptidoglycan layer in cell wall offered a higher resistanceto ROS.

[ 2 2 ]

MIC = ~64 and ~256 μg mL-1 respectively, for Gram-positive (S. aureus and B. subtilis) and Gramnegative (E. coli and A. aerogenes) bacterial strain. Gram-positive bacterial cells exhibited no growth at alower concentration of 100 μg mL-1. On the other hand, for Gramnegative cells, the inhibition concentration was recorded to be 500 μg mL-1 ; i.e., antibacterial activity of ZnO nanorods is higher towards Gram-positive bacterium than Gram-negative bacterium

[ 2 3 ]

27 | P a g e

sub tilis

ods

#

Ent ero bac ter aer oge nes #

Na no ro ds Na no sp her es Na no flo we rs Na no ro ds

Hydrothermal method

76 nm 65 nm 45 nm

Esc heri chi a coli * Aer oba cter aer oge nes * Esc heri chi a coli * Sta phy loc occ us aur eus

Colo ny coun ts meth od

ROS produ ction

Liqu id Brot h meth od

Zn2+ releas e

ZnO flower-like showed significantly higher photocatalytic inactivation than ZnO rod- and sphere-like against E. coli compared with S. aureus

[ 2 4 ]

#

Hydrothermal method

D=5 0 nm, L500 nm

Pse udo mo nas eru gin osa *

[ 2 5 ]

28 | P a g e

Ca uli flo we r lik e

Green synthesis using leaf extract of Tamarindus indica (L.)

19– 37 nm

Sta phy loc occ us epi der mid is# Sta phy loc occ us aur eus # Pse udo mo nas aer ugi nos a* Bac illu s sub tilis

Disk diffu sion meth od

Mech anical effect , ROS produ ction and dissol ution of ZnO

ZOI = 13.1 ± 0.28 mm, MIC= 25 mg mL-1 ZOI = 11.3 ± 0.57 mm, MIC= 50 mg mL-1 ZOI = 12.6 ± 0.76 mm, MIC= 25 mg mL-1 ZOI = 12.0 ± 0.50 mm, MIC= 50 mg mL-1 Gram-positive bacteria more susceptible for antimicrobial activity against ZnO NPs than Gram-negative bacteria

[ 2 6 ]

Liqu id brot h meth od

Nano partic le intern alizati on, cell wall dama ge

MIC = 5 μg mL-1 for all the strains.

[ 2 7 ]

#

Mi cro Fl ow ers

Aqueous solution route

Bas e dia met er ~2– 3 μm, indi vidu

Pro teus mir abil is* Sta phy loc occ us aur eus #

Esc heri

29 | P a g e

al nan orod dia met er ~15 0– 200 nm

Na no flo we rs

Simple solution method

Len gth of each peta l~ 800 nm

chi a coli * Sal mo nell a typ him uri um * Kle bsie lla pne um oni ae* Esc heri chi a coli * Sta phy loc occ us aur eus

Well diffu sion meth od

----

Disk diffu sion meth od

Mech anical dama ge, ROS produ ction and dissol ution of ZnO

MIC = 25 mg L-1 for all the strains.

[ 2 8 ]

ZOI = 20 mm# ZOI = 21 mm* # ZOI = 21 mm ZOI = 18 mm* ZOI = 23 mm# ZOI = 19 mm* ZOI = 19 mm# ZOI = 17 mm* -1 (All values for 0.5 mg mL of ZnO nanoflower concentrations)

[ 2 9 ]

#

Na no flo we rs

CTAB assisted solution method

23.7 nm 82.5 nm 69.6 nm 88.8 nm

Esc heri chi a coli * Sta phy loc occ us aur

30 | P a g e

eus #

Na no flo we rs

Simple solution method

Mi cro sp her es

Microwave process

Typi cal peta l leng th 400 – 500 nm, base dia met ers 200 – 300 nm, flow er dia met er 1.0– 1.5 μm. Ultr athi n nan opla tes of typi cal thic knes s of ∼20 nm,

Esc heri chi a coli

Liqu id brot h meth od

Mech anical dama ge, ROS produ ction and dissol ution of ZnO

MIC = 25 μg mL-1.

[ 3 0 ]

Esc heri chi a coli * Sta phy loc occ us aur eus

Liqu id brot h meth od Disk diffu sion meth od

Mech anical dama ge and ROS produ ction

ZOI = 25.7 mm ZOI = 17.6 mm, (MIC = 25 mg L-1 for both the strains.) Inhibitory efficacy for Gram-negative bacteria E. coli is more compared to Gram-positive bacteriaS. aureus

[ 3 1 ]

#

31 | P a g e

Na no po wd ers

Commercial

Na no po wd ers

Commercial

sphe re dia met er of 1.5 μm 70 ± 15 nm

----

Esc heri chi a coli *

Bac illu s sub tilu s# Bac illu s me gat eriu m# Sta phy loc occ us aur eus

Liqu id brot h meth od Colo ny coun t meth od Liqu id brot h meth od Disk diffu sion meth ods

Leaka ge of intrac ellula r conte nts and mem brane disru ption ROS produ ction

MIC = 3 mmol l-1

[ 3 2 ]

ZOI = 24 mm, MIC= >5 μg mL-1 ZOI = 20 mm, MIC= >5 μg mL-1 ZOI = 22 mm, MIC= >1 μg mL-1 ZOI = 18 mm, MIC= >5 μg mL-1 ZOI = 24 mm, MIC= >0.5 μg mL-1 ZOI = 22 mm, MIC= >0.5 μg mL-1 ZOI = 16 mm, MIC= >5 μg mL-1 ZOI = 14 mm, MIC= >5 μg mL-1 Gram-negative bacteria more susceptible for antimicrobial activity against ZnO NPs than Gram-positive bacteria

[ 3 3 ]

#

Sar cin a lute a# Esc

32 | P a g e

Pri sm ati c na no ro ds Na no sp her es Na no elli ps oid

Hydrothermal

D=1 00 nm, L-1 μm 30 nm 100 nm

heri chi a coli * Pse udo mo nas aer ugi nos a* Kle bsie lla pne um oni ae* Pro teus vul gar is* Esc heri chi a coli * Sta phy loc occ us aur eus

Colo ny coun ts meth od

ZnO nanospherical particles with average diameter of 30 nm showed highest antibacterial activity for both E. coli and S. aureus bacterial strains.

[ 3 4 ]

#

33 | P a g e

Na no po wd ers

Green synthesis through EGCG assisted

9– 19 nm

Pse udo mo nas aer ugi nos a* Kle bsie lla aer oge nes * Esc heri chi a coli * Sta phy loc occ us aur eus

Well diffu sion meth od

----

Liqu id brot h meth od

ROS gener ation and Zn2+ ion releas e

ZOI = 4.33±0.33 mm ZOI = 13.33±0.33 mm ZOI = 4.33±0.33 mm ZOI = 3.67±0.33 mm

[ 3 5 ]

ZOI= 11.3 ± 0. 62 mm MIC=0.55 ± 0.04 mg mL-1 ZOI= 14.8 ± 0.38 mm MIC=0.45 ± 0.04 mg mL-1 ZOI= 9.8 ± 0.30 mm MIC=0.55 ± 0.04 mg mL-1 ZOI= 12.2 ± 1.61 mm MIC=0.45 ± 0.04 mg -1 mL ZOI= 10.8 ± 0.56 mm MIC=0.55 ± 0.04 mg mL-1 ZOI= 11.8 ± 4.59 mm MIC=0.65 ± 0.04 mg mL-1 ZOI= 13.2 ± 0.54 mm MIC=0.65 ± 0.04 mg mL-1 ZOI= 11.0 ± 0.82 mm MIC=0.75 ± 0.04 mg mL-1 ZOI= 10.0 ± 2.56 mm MIC=0.75 ± 0.04 mg -1 mL

[ 3 6 ]

#

Na not ub es

Hydrothermal

Wal l thic knes s is 2nm and D= 17 nm, L=2 μm

Aci net oba cter bau ma nnii * Esc heri chi a coli * Kle bsie lla

34 | P a g e

pne um oni a* Pro teus mir abil is* Pse udo mo nas aer ugi nos a* Sal mo nell a typ hi* Bac illu s sub tilis

-mL-1 ZOI= 12.7 ± 1.57 mm mL-1 -mL-1

MIC=0.75 ± 0.04 mg MIC=0.60 ± 0.04 mg MIC=0.60 ± 0.04 mg

#

Mic roc occ us lute us# Sta phy loc occ us aur eus #

MR SA# Sta

35 | P a g e

Qu ant um dot s Na no par ticl es Th in fil ms

Solution methods

5–6 nm 10– 15 nm

Reactive DC magnetron sputtering

Na no par ticl es

Chemical precipitation method

50– 100 nm 200 – 600 nm (Fil m thic knes ses) 15 ± 4 nm

phy loc occ us epi der mid is# Stre pto coc cus pne um oni a# Esc heri chi a coli *

Liqu id brot h meth od

Esc heri chi a coli *

Liqu id brot h meth od

Sta phy loc occ us epi der mid

Well diffu sion meth od

Nano partic le intern alizati on, cell wall intera ctions ROS gener ation and Zn2+ ion releas e

MIC = 25 μg mL-1.

[ 3 7 ]

Thin films of 50 and 100 nm thickness exhibited less pronounced antibacterial effect than the thicker films (200–600 nm).

[ 3 8 ]

ROS gener ation and Zn2+ ion releas e

ZOI = 17.00 ± 1.00 mm ZOI = 12.00 ± 1.00 mm ZOI = 6.67 ± 0.58 mm ZOI = 6.67 ± 0.58 mm ZOI = 7.67 ± 0.58 mm ZOI = 5.67 ± 0.58 mm

[ 3 9 ]

36 | P a g e

is# Bac illu s sub tilis #

Sta phy loc occ us aur eus #

Ent ero bac ter aer oge nes #

Sal mo nell a par aty phi * Kle bsie lla pne um oni a*

37 | P a g e

Na no par ticl es

Commercial

Na no par ticl es

Commercial

20 nm 90– 200 nm 20 nm <10 nm 14 nm 25 nm 35– 45 nm 10– 30 nm 230 – 241 7 nm

Esc heri chi a coli *

Colo ny coun ts meth od

ROS gener ation and Zn2+ ion releas e

5 9 7 1 92 7 3 10 (No of bacterial colonies formed after 4 h of ambient illumination) No correlation was observed between Zn2+ ion release, ROS production, zeta potential and antibacterial activity.

[ 4 0 ]

Esc heri chi a coli * Sal mo nell a typ him uri um * Bac illu s sub tilis

Liqu id brot h meth od

ROS gener ation and intera ction betwe en ZnO nanop articl es and bacter ia mem brane wall

The antibacterial activity increases with increasing particle concentration and decreasing particle size.

[ 4 1 ]

#

Sta phy loc occ us aur

38 | P a g e

eus #

Na no par ticl es

Commercial

200 nm

Na no par ticl es

Commercial

----

Esc heri chi a coli * List eria mo noc yto gen es# Sta phy loc occ us aur eus

Liqu id brot h meth od CFU

H2O2 produ ction and mem brane disru ption

Photoactivation of ZnO nanoparticles exhibited better antibacterial activity against Listeria monocytogenes and E. coli bacteria as compared to non-photoactivation.

ZOI = 1.5 cm

Well diffu sion meth od

MIC =39 μg ml -1

[ 4 2 ]

[ 4 3 ]

#

Na no par ticl es

Commercial

20 nm 60 nm

Sta phy loc occ us aur eus

Colo ny coun t meth od

ROS and H2O2 produ ction

ZnO nanoparticles of 60 nm diameter were less effective than a particle of 20 nm diameter. Upon ultrasonication, the antibacterial activity of 60 nm particles was pronounced than 20 nm ZnO nanoparticles.

[ 4 4 ]

#

Pse udo mo nas aer ugi nos a*

39 | P a g e

Na no par ticl es

Green Biofabrication of zinc oxide nanoparticles using leaf extract of curryleaf

12 nm

Sta phy loc occ us aur eus

Disk diffu sion meth od

----

ZOI= 14.07 ± 0.50mm ZOI= 13.87 ± 0.76mm

MIC =25 μg ml -1 MIC =100 μg ml 1 (ZOI for 200 μg mL-1for both the strains)

[ 4 5 ]

Well diffu sion meth od

----

MIC=2.9 ± 0.01 μg mL-1 ZOI= 22 ± 1.8 mm MIC=1.2 ± 0.02 μg mL-1 MIC=2.4 ± 0.08 μg mL-1 MIC=1.2 ± 0.04 μg mL-1 MIC=1.5 ± 0.01 μg mL-1 MIC= 2.4 ± 0.05 μg mL-1

[ 4 6 ]

#

Bac illu s sub tilis #

Na no par ticl es

Green reproducible bacteria, Aeromonas hydrophila as eco-friendly reducing and capping agent

57.7 2 nm

Pse udo mo nas aer ugi nos a* Esc heri chi a coli * Sta phy loc occ us aur eus #

Aer om ona s hyd rop hila * Ent ero

40 | P a g e

Na no par ticl es

Green synthesis using Cassiafistula plant extract

5– 15 nm

coc cus fae cali s Stre pto coc cus pyo gen es# Kle bsie lla Aer oge nes * Esc heri chi a coli * Sta phy loc cus aur eus

Well diffu sion meth od

----

Well diffu sion meth od

H2O2 produ ction

ZOI= 9.67 ± 0.33 mm ZOI= 4.67 ± 0.33 mm ZOI= 4.67 ± 0.33 mm ZOI= 4.00 ± 0.00 mm (ZOI for 1000 μg per well for all the strains)

[ 4 8 ]

ZOI= 11 mm ZOI= 10 mm (ZOI values for 1000 μg mL-1for all the strains)

[ 4 9 ]

#

Na no par ticl es

Green synthesis of ZnO nanoparticles using ginger

23– 26 nm

Pse udo mo nas des mol ytic um * Kle bsie lla pne um

41 | P a g e

rhizome extract

oni a* Sta phy loc occ us aur eus #

Na no par ticl es

Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract

20– 30 nm

Sta phy loc occ us aur eus

Disk diffu sion meth od

ZOI= 18 mm ZOI= 17 mm ZOI= 11 mm ZOI= 7 mm

[ 5 0 ]

ZOI= 23.8 ± 0.76 mm ZOI= 23.3 ± 0.57 mm ZOI= 22.8 ± 0.76 mm ZOI= 19.6 ± 0.76 mm ZOI= 23.0 ± 0.50 mm (ZOI for 200 μg mL-1 for all the strains)

[ 5 1 ]

#

Na no par ticl es

Green synthesis of ZnO nanoparticles using leaf extract of Moringa oleifera

16– 20 nm

Sal mo nell a par aty phi * Vib rio cho lera e* Esc heri chi a coli * Sta phy loc occ us aur eus #

Bac

Disk diffu sion meth ods

Disru ption of the cell mem brane and gener ation

42 | P a g e

illu s sub tilis

of surfac e oxyge n speci es

#

Na no par ticl es

Green synthesis of ZnO nanoparticles using Vitex negundo extract

75– 80 nm

Pse udo mo nas aer ugi nos a* Pro teus mir abil is* Esc heri chi a coli * Esc heri chi a coli * Sta phy loc occ us aur eus

Well diffu sion meth od

ROS gener ation and Zn2+ ion releas e

Diff usio n disk meth od, CFU

Cellul ar intern alizati on

ZOI= 16 mm ZOI= 19 mm

[ 5 2 ]

#

Na no par ticl es of var iab

Precipitation method

60– 150 nm

Stre pto coc cus aga lact iae# Sta

Low concentrations of ZnO nanoparticles were ineffective for cellular damage. PVA-coated ZnO nanoparticles at higher concentrations (0.016M) in the EG aqueous medium exhibited excellent antibacterial activities.

[ 5 3 ]

43 | P a g e

le mo rp hol ogi es

phy loc occ us aur eus #

Na no par ticl es

Precipitation method

Esc heri chi a coli * Kle bsie lla pne um oni ae* Shi gell a dys ent eria e* Sal mo nell a typ hi* Pse udo mo nas aer ugi nos a* Bac illu s sub tilis

Diff usio n disk meth od

ROS produ ction

----

[ 5 4 ]

44 | P a g e

#

Sta phy loc occ us aur eus #

Na no par ticl es

Precipitation method

Bac illu s sub tilis

Well diffu sion meth od

H2O2 produ ction

ZOI = 16 ± 1.3 mm, MIC= 10 mM ZOI = 15 ± 0.5 mm, MIC= 8 mM ZOI = 10 ± 0.7 mm, MIC= 16 mM ZOI = 12 ± 1.2 mm, MIC= 21 mM The antibacterial efficiency was found to be directly proportional to the ZnO nanoparticle concentration and inversely proportional to their particle size. Gram-positive bacteria were more susceptible for antibacterial attacks than Gram-negative bacteria.

[ 5 5 ]

Liqu id brot h meth od

ROS and H2O2 produ ction

ZnO nanoparticles having 4-7 mM concentration inhibited 95% of growth for most of the microorganisms except Salmonella typhimurium.

[ 5 6 ]

#

Sta phy loc occ us aur eus #

Na no par ticl es

Solvothermal synthesis

12 nm

Stre pto coc cus pyo gen es# Esc heri chi a coli * Sta phy loc occ us aur eus #

Sta phy

45 | P a g e

loc occ us epi der mid is# Stre pto coc cus pyo gen es# Ent ero coc cus fae cali s# Bac illu s sub tilis #

Esc heri chi a coli * Pro teus vul gar is* Sal mo nell a typ him uri um

46 | P a g e

*

Na no par ticl es

Wet chemical method

10– 30 nm

Esc heri chi a coli * Pse udo mo nas aer ugi nos a* Sta phy loc occ us aur eus

Micr otite r plate – base d meth od

ROS produ ction

MIC=500 μg mL-1 MIC=500 μg mL-1 MIC=125 μg mL-1 Antimicrobial activities were more pronounced for Gram-positive bacterial strains than the Gram-negative bacterial strains.

[ 5 7 ]

Colo ny coun t meth od

Mem brane dysfu nctio n broug ht about by the intera ction of ZnO with

Antimicrobial activities for PEG-coated ZnO nanoparticles were enhanced as the size of the particles was reduced from microscale to nanoscale. Gram-negative bacteria were more susceptible to antibacterial attacks than positive bacteria.

[ 5 8 ]

#

Na no par ticl es

Wet chemical method

40 nm– 1.2 μm

Esc heri chi a coli * Sta phy loc occ us aur eus #

47 | P a g e

the cell mem brane . PE GCo ate d As A co ate d M A A co ate d pol ys or bat e 80 (T80 ) co ate d na no par ticl es Na no par ticl es

Precipitation method

7–9 nm

Esc heri chi a coli *

Liqu id brot h meth od

ROS produ ction

4–6% growth inhibition 4–6% growth inhibition 13 % growth inhibition 43% growth inhibition (As compared to control )

[ 5 9 ]

Wet chemical method

10– 15 nm

Pse udo mo nas aer ugi nos

Well diffu sion meth od

ROS produ ction

ZOI = 16 mm for 100 μg ml-1 of ZnO nanoparticles

[ 8 0 ]

48 | P a g e

a* Na no par ticl es

Green synthesis using extract of Caltropis procera fruit or leaves

90– 100 nm

Na no par ticl es

Precipitation method

12 nm 45 nm 2.0 μm

Vib rio cho lera e* Esc heri chi a coli * Esc heri chi a coli *

Liqu id brot h meth od

----

Liqu id brot h meth od Disk diffu sion meth ods

ROS gener ation and Zn2+ ion releas e

MIC = 5 × 105particles/ml MIC = 1× 107 particles/ml

[ 8 4 ]

ZOI = 31 mm ZOI = 27 mm ZOI = 22 mm

[ 8 5 ]

* Gram-negative bacteria #Gram-positive bacteria ZOI = Zone of inhibition MIC = Minimum inhibition concentration MBC = Minimum bacterial concentration D = Diameter L = Length

4. PROPOSED

MECHANISMS

FOR

ANTIMICROBIAL

ACTIVITIES

AGAINST GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA In order to establish an efficient and effective mechanism of antimicrobial activities of ZnO nanomaterials, the detailed explanation about the compositional and structural features of the bacterial cell envelope and cell wall is compulsory. However, the exact mechanism is still debatable.

49 | P a g e

The outermost covering of the bacterial cell is a cell envelope, which consists of thecell wall (absent in eukaryotic cells) and plasma membrane or cytoplasmic membrane (similar to that of eukaryotic cells)[86]. The main functions of the bacterial cell wall are to provide strength, rigidity, maintain shape, and to act as a cushion to prevent any mechanical damage, and finally to protect the cell from rupturing due to turgor pressure developed inside the cell as a result of endosmosis[87].Unlike other organisms, the bacterial cell wall has a layer of peptidoglycan also called murein, a heteropolymer composed polysaccharide backbone of equal amounts of alternating N-Acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues cross-linked through short peptide linkages[88]and is located outside the plasma membrane[89]. This peptidoglycan porous layer maintains the rigidity of the cell wall and is also responsible for the determination of bacterial cell morphology. On the basis of composition, structure of bacterial cell wall and Gram staining characteristics, bacterial cells are classified into two main categories;viz.,Gram-positive (+) and Gram-negative (-) bacteria[90]. Compared to a Gram-positive cell wall, a Gram-negative cell wall is more complex both structurally and chemically. These structural properties of the cell wall are very important in influencing the effect of ZnO and other nanomaterials bacteria. The basic differences between the structural and compositional properties of the cell walls of Gram positive and Gramnegative bacteria are presented diagrammatically in Fig.9. Usually, antibacterial drugs such as penicillin, etc., deactivate the penicillinbinding proteins or transpeptidases enzyme responsible for the production of peptidoglycan. Reproduction of the bacterial cells through binary fission requires a

50 | P a g e

million of peptidoglycan subunits (NAM-NAG+oligopeptide) attached to existing subunits[91].

Fig.9. Cell wall structures of (a) Gram-positive bacteria and (b) Gram-negative bacteria.

In a broader classification, different antibacterial mechanisms can be categorized into (a) chemical and (b) physical interactions of ZnO nanomaterials and their corresponding secondary species with the bacterial cells and their cell components[15].

4.1. Chemical interactions of ZnO nanomaterials with the bacterial cells 4.1.1. Generation of reactive oxygenated species (ROS)

51 | P a g e

This is the most commonly studied and most widely accepted mechanism for the antimicrobial activities by different nanomaterials. The ROS are highly reactive ionic species, radical anions, and molecules of oxygen such as O•-2, HO•, H2O2, which damage the cells and various cellular components through oxidative stress[92,93]. In biological systems, ROS are generated through aerobic pathways in electron transport chains during bio-redox reactions. In biological systems there are a number of counter mechanisms forthese ROS by producing a number of reducing equivalents[94– 98]. The imbalance between the generated ROS and their detoxification by the cellular systems is termed as oxidative stress. This oxidative stress as a result of generated ROS, is supposed to induce a variety of nonpathogenic disorders including cancer, cell or tissue inflammations, and myocardial disorders. It is important to regulate the balance between the generated ROS and the antioxidation of excess of ROS. Biomolecules, enzymes, and vitamins are the naturally occurring antioxidants present in the biological systems to control the negative effects of reactive free radicals species[99]. ZnO is an n-type semiconductor material and can undergo a number of photoinduced molecular transformations in the presence of UV-visible light. ZnO has alarge exciton binding energy (60 meV) and thus it is possible to generate an excitons,i.e., bound electron–hole pairs when irradiated with incident photon having λ < 390 nm[22,23].When ZnO nanomaterials are exposed to UV or visible light, redox processes  ) in the occur resulting in the generation of electron/hole pairs with free electrons (e CB

empty conduction band and positive holes (h VB ) in the valence band (Fig. 10). Electronhole pairs are diffused out to the surface of ZnO particles thereby initiating a series of

52 | P a g e

oxidation-reduction reactions on the surface of the photocatalyst. The holes (h VB ) in the valence band ionize and oxidize water to generate HO free radicals. The conduction  ) with oxygen undergoes a series of reactions to produce intermediates band electron (e CB

like O -2 , HO2 , H 2 O 2 and HO in the reduction process on the surface of the ZnO nanoparticles[41,42,44,54,57,59,61,81,100].It is important to mention that, negatively charged superoxides and hydroxide radicals anions can’t penetrate the cytoplasmic membranes; it is only H2O2 which penetrates the cells and performs biocidal activities[101]. The concentration of these ROS increases with the concentration of the ZnO nanomaterials, irrespective of the method used for determining the antibacterial activities[23,61,102]. Limited-time UV exposure can enhance the rate of formation of these ROS[24,26,45,47,51]. Raghupathi et al.[56] reported the reduction of viable cell count for S. aureus upon 15 min UV irradiation of ZnO nanoparticles, however, a longer time exposure for 30 min resulted in complete removal of viable-cell as compared to the control. The results confirm the formation of ROS. This enhancement in antibacterial activity in the presence of UV light confirms the generation of the reactive oxygen species by the ZnO nanomaterials[85]. However, the results can be challenged as UV irradiations themselves exhibit biocidal effects.

53 | P a g e

Fig.10. Photo-induced ROS production from ZnO nanomaterials. Many comparative studies have shown that ZnO nanomaterials exhibit better bactericidal or bacteriostatic activities against Gram-positive bacteria as compared to Gram-negative bacteria. Azam et al.[7,8] reported larger inhibition zones for Grampositive bacteria such as B. subtilis and S. aureus as compared to Gram-negative bacteria such as E. coli and P. aeruginosa using ZnO nanoparticles. Jain et al.[23] analyzed MIC of ~64 and ~256 μg/mL respectively, for ZnO nanorods against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and A. aerogenes) bacterial strains. Better antibacterial activities for ZnO nanomaterials against Gram-positive bacteria than Gram-negative bacteria may be attributed the structural and compositional differences. As shown in Fig.11, Gram-negative bacteria have an extra outer plasma membrane with a thick lipopolysaccharide layer on it. This layer is much thicker than the

54 | P a g e

peptidoglycan layer present in Gram-positive bacteria. These two are the important structural features of a Gram-negative bacterial cell, which oppose the lipid peroxidation in the presence of ROS generated through ZnO nanomaterials[23,103,104].

Fig.11.Gram-negative bacteria cell damage through the production of ROS. According to Tsuneda et al.[105], surface negative charges arose from the ionization of carboxylic, phosphate and amino groups on the bacterial cells also play a major role in the association of the ZnO nanoparticles to the bacterial surface and in bacterial aggregation. Due to this surface negative charge, ZnO nanoparticles with a positively charged hole (h+) are trapped well in the peptidoglycan layer of Gram-positive bacteria as compared to the thin peptidoglycan layer of Gram-negative bacteria[36]. Trapped ZnO nanoparticles produce sufficient ROS resulting in the damage to bacterial cell structure (Fig.12).

55 | P a g e

Fig.12.Gram-positive bacteria cell damage through the production of ROS. The Zeta potential, which is the electric potential difference between the medium and the stationary layer of the medium adhered to the bacterial cell, is directly affected by the concentration of

the ROS

and hence the

concentration of the

ZnO

nanomaterials[106]. Zeta potential showed more negativity with more pronounced effect in Gram-positive bacterial strains than in Gram-negative strains. Fig. 13 represents the zeta potential value of some of the Gram positive and Gram negative bacteria. Gram negative bacteria were found to exhibit higher negative zeta potential value as compared to Gram-positive bacteria as the former possess an additional layer of negatively charged lipopolysaccharide[107].

56 | P a g e

Fig.13. Zeta potentials of Gram positive and Gram negative bacteria. Reproduce with permission from Ref. [107], Scientific Reports 2015;5: 9578.

For Gram-positive bacteria, a stagnation in Zeta potential was observed for ZnO nanorod concentration of150 and 200 μg/mL, whereas the same effect was seen at400 and 500 μg/mL for Gram-negative bacteria[23]. In contrast, Ng et al.[40] reported no correlation between the ROS production, Zn2+ release, zeta potential and the antibacterial activity. In contrast,

Shinde et al.[31] reported ZnO microspheres to be better

antibacterial agents against Gram-negative bacteria as compared to Gram-positive bacteria. The results were explained on the basis of a thinner peptidoglycan layer (5-10 nm) and the presence of a lipopolysaccharide layer having functionalities such as amide,

57 | P a g e

phosphate, and carboxyl, which reacts with nanomaterials and disrupts the cell membranes in Gram-negative bacteria through the formation of ROS. Although the bactericidal activities of ZnO nanomaterials are well explained through the formation of ROS, a number of contradictions are also reported. The production of ROS is well explained in the presence of UV or visible light and oxygen, but there are reports which indicate the antimicrobial activities for ZnO even in the dark, definitely either through another way of ROS generation, or due to some other mechanism[108– 110]. The generation of ROS in dark conditions can be attributed to the defects present on the surface of ZnO crystal. Wu et al.[111] reported mainly four types of defects in ZnO, three types of oxygen vacancies with neutral ( VO0 ), singly ionized ( VO ) and doubly ionized ( VO ) charge states and the fourth one corresponds to interstitial Zn2+ ions (Zni). As ZnO is n-type of semiconductor, these defects are responsible for the trapping of the majority carriers i.e., electrons. These electrons are released to reduce oxygen to generate ROS under dark conditions through the different steps, as represented in Eqs. 2-5.   O 2  e (From defects)  O 2 ……………….……….(2)

O2  H 2 O  HO2  HO …………………….(3) HO2  HO2  H 2 O 2  O 2 …………………….(4) H 2 O 2  O2  HO  HO  O 2 …………..….(5) Prassana et al.[112], in order to investigate and confirm the generation of ROS even in the dark, carried out fluorescence studies of the hydroxyl terephthalic acid

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generated in the presence of ZnO nanoparticles from terephthalic acid. Significant increase in the fluorescence intensity due to the production of hydroxyl terephthalic acid even after 5 min clearly indicates the generation of hydroxyl radicals from aqueous suspensions of ZnO nanoparticles which further increases with the time (Fig.14 a). However, the concentration of the generated hydroxyl radicals is much smaller in dark than in the presence of visible light (Fig.14 b).

Fig. 14. Fluorescence spectra of hydroxyl terephthalic acid in aqueous suspensions of ZnO (a) dark (b) visible. [Reprinted with permission from Prasanna et al , 2015 [112]Copyright © 2015ACS Paragon Plus Environment]

4.1.2. Release of Zn2+ ions A detailed study carried out by Li et al.[113] supports the release of Zn2+ ions responsible for ecotoxicity from ZnO nanomaterials in different media. ZnO nanoparticles having a concentration of 5 mg/L (Equivalent to solubility) was taken in

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ultra-pure water. For the comparison of the cytotoxic effects, Zn2+ ions solution, also prepared in ultra-pure water with a concentration lower than 0.1 mg/Lwas used. The TEM images of the treated Gram-negative E. coli strains are shown in Fig. 15. Clearly, the E. coli morphology was deformed on treatment with ZnO nanoparticles or Zn2+ ions solution. The leakage of the intracellular fluid resulted due to Zn2+ionsand osmotic stress can be seen in Fig.15 (b) and (c). The cytotoxic effects are comparable for the E. coli when treated with either ZnO nanoparticles or Zn2+ ion solution. Thus, Zn2+ ions have an important effect on the cytotoxicity of ZnO nanoparticles. The similar toxicity tests conducted by Song et al.[114]showed that dissolved Zn2+ ions can severely inhibit the cell viability to 50% at an equilibrium concentration of 10μg/ml and equivalent to that of ZnCl2.

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Fig.15. TEM images of (a) untreated E. coli cells (b) treated with ZnO nanoparticles and (c) treated withZn2+ ions solution in ultra-pure water.[Reprinted with permission from [113]Copyright© 2011, American Chemical Society]. As reported, Gram-positive bacteria with a thicker peptidoglycan layer, are less susceptible to releaseZn2+ions. This may be due to the fact that, the peptidoglycan layer is negatively charged and Zn2+ ions are trapped in this layer. Hence, very limited toxic activities, as compared to Gram-negative bacteria with a thinner peptidoglycan layer, are observed (Fig.16)[36,115].These Zn2+ions prolong the lag phase of growth interfere with the biochemical processes such as glycolysis, active and passive proton transportation across the membrane, and acid tolerance, etc., and are reported to show bacteriostatic rather than bacteriocidal effects[116].

Fig.16. Cell damage of Gram-positive bacteria through the production of Zn2+ ions. A comparative study carried out by Reddy et al.[117]reported ≥3.4 mM and ≥1 mM ZnO nanoparticle concentrations for complete growth inhibition of Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, respectively. Increased number of CFUs

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was observed, interestingly for E. coli, when treated with ZnO nanoparticles in doses of0.01–1 mM, in contrast to the control. The fact was due to the preference of E. coli bacteria for low concentrations of Zn2+ in the growth medium, which may act as nutrients for it[85,117]. Thus, the important conclusion can be drawn that a very low concentration of ZnO nanoparticles favors the growth, whereas relatively higher concentration doses show growth inhibition effects. Additionally, the number of Zn2+ions in ZnO nanoparticles solution can be increased by maintaining a pH below 6 or above asthe solubility of ZnO increases drastically in these pH values. Hence, the ecotoxic effects of ZnO nanoparticles as an antibacterial agent at these pH values will be more suitable if the Zn2+ions are responsible for the antibacterial activities[118]. Other factors which affect the release of Zn2+ ions from ZnO nanomaterials are; concentration, morphology, surface defects, the type of method applied, and medium of the ZnO sample taken for the evaluation of the antimicrobial activities. Pasquet et al.[119]studied the antimicrobial efficacy of ZnO nanomaterials for some Gram-negative bacterial strains and found the results comparable to those of Zinc gluconate as a source of Zn2+ ions through Agar diskdiffusion method and liquid broth medium. It was suggested that ZnO nanoparticles can’t diffuse from the disks and thus, the antimicrobial activities surely are due to the released Zn2+ ions. Further, two to three times higher dissolution was observed in Mueller-Hinton broth than in pure water. This was attributed to the complexation of released Zn2+ ions by the components of the Mueller-Hinton[119].

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4.1.3. Photoinduced production of H2O2 In addition to the light-induced ROS generated from the ZnO nanoparticles, many reports consider H2O2 as the main species for the antibacterial activities against Grampositive as well as Gram-negative bacteria[12,41,119]. As stated earlier in section 4.1.1, negatively charged ROS can’t penetrate the bacterial cell wall, but H2O2can easily do the same. Sawai et al.[120]considered H2O2generated from ZnO slurry as a main cause for the biocidal mechanism. It can also be assumed that after the membrane disruption by H2O2or HO•, ROS can penetrate into the intracellular space, leading to enhanced biocidal effects (Fig 17).

Fig.17.Schematic representation of Gram-positive bacterial cell damage through the productionof H2O2from ZnO nanomaterials. Zhang et al.[12] estimated the amounts of H2O2 produced from ZnO nanomaterials suspensions. 1μM H2O2 was reported being produced from 0.2 gL-1of ZnO with particle

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size 400 nm (Sample A), whereas no H2O2production was from ZnO nanomaterials with particle size 93 nm (Sample E). However, fluorescence studies showed strong interaction between E. coli lipid vesicles and Sample E nanoparticles. It was thus concluded that ZnO nanomaterials with large-sized particle or aggregates exhibit antibacterial activities through the production of H2O2. In contrast, the interactions between small-sized ZnO particles and lipid vesicles are responsible for the toxic activities.

Fig.18 (a) Concentrations of H2O2 for ZnO suspensions; ZnO Sample-A (Particle size 400 nm and Sample-E (Particle size 93 nm). (b) Bacteria colonies formed in the presence of 1 g/l of different ZnO samples for incubation for24 h at 37oC.[Reproduced with permission from Ref. [12]Crown copyright © 2013 Published by Elsevier Ltd.].

Moreover, according to Jing et al.[121], the smaller the size of the nanomaterial particles the higher is the magnitude of surface oxygen vacancies and defects. These vacancies and defects are responsible for the inhibition of the photo-induced electronsholes recombination and O2 adsorption favoring the formation of ROS. Fig.18 (a) represents the concentrations of H2O2 produced from different concentrations of ZnO suspensions. It can be seen that there is a direct correlation between the

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concentration of the H2O2 produced and the amount of ZnO taken for Sample A. From Fig.18(b), it can be observed that Sample E has maximum ecotoxic activity as compared to other samples, as well as control. The role of H2O2 as a cytotoxic agent for Sample A was further confirmed by the fact that the antimicrobial activities were drastically reduced in the presence of catalase, an H2O2 quencher. However for Sample E, a little change in the cytotoxic activity was observed, confirming no role of H2O2 for its antimicrobial activity. Yamamoto et al.[122] analyzed the H2O2 amount formed from ZnO samples annealed at different temperatures. Interestingly, a good correlation was reported between the amount of H2O2 generated and the c0 value of the hexagonal ZnO crystal lattice for different samples. According to Storz et al.[123] H2O2 undergoes either strong interactions or diffuses through the cell envelope of the bacteria. In the intracellular spaceH2O2oxidizes protein cysteinyl residues, thiols and deactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase. It can also react with Fe2+ ions released from Fe-S protein oxidation to form HO•, which drastically damage the DNA[124]. 4.2. Physical interactions of ZnO nanomaterials with the bacterial cells 4.2.1. Plasma membrane disruption through ZnO interactions According to Stoimenov et al.[125] and Tsuneda et al.[105] at biological pH, the bacterial cell surface is negatively charged due to dissociation of carboxylic and other functional groups. The reported Zeta potential value of positively charged ZnO nanomaterials is +24 mV[41].The surface negative charges of reported to be -41.3,-7.20 and -32.3 mV for B. subtilis, E. coli and P. fluorescensas observed in 1 gL-1 NaCl at pH

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of6.5[126].These opposite charge of the bacterial cells and ZnO nanoparticles are responsible for the strong electrostatic attractions between them (Fig.19). Based upon this mechanistic approach, antibacterial effects of ZnO nanomaterials are studied mostly on E. coli and S. aureus bacteria. A very little is reported for ZnO nanomaterial interactions with other types of bacterial strains. It has been formulated that nanoparticles having a size greater than 10 nm are accumulated on the outer surface of the plasma membranes due to these strong electrostatic interactions, and neutralize the surface potential of the bacterial membrane resulting in increased surface tension and membrane depolarization. This leads to change in membrane textures, rupture, membrane blebs, membrane disruption, morphology alteration, induction of oxidative stress, increase in membrane permeability, particle internalization, leakage of intracellular fluid and components causing bacterial cell death[107,127,128].As interactions play a major role in bactericidal effects, the surface modification in terms of capping agents and templates, of the ZnO nanomaterials can lead to the enhanced interactions with the bacterial cell walls. However, it is stated that these interactions are not merely responsible for antimicrobial activities of the ZnO nanomaterials[41,129]. 4.2.2. Cellular internalization of ZnO nanoparticles In another significant mechanism, it has also been proposed that nanostructures with a size smaller than 10 nm can pass through the cytoplasmic membrane, referred to as particle internalization, and accumulate inside the bacterial cell where they damage the intracellular components including nucleic acids[130,131]. Additionally, it has also been proposed that the ZnO – bacterial cell interactions can also enhance the cell permeability

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(Fig.20)[127,128]. Brayner et al.[129]reported biocidal effects on E. coli bacterial cells through thecellular internalization of ZnO nanoparticles synthesized in diethylene glycol medium.

Fig.19. Various types of physical interactions of ZnO nanomaterials with the bacterial cells leading to the biocidal effects.

4.2.3. Mechanical damage of the cell envelope The presence of surface defects, uneven surface texture, and rough edges and corners on the surface of ZnO nanomaterials results in effective abrasiveness in comparison to the bulk ZnO materials. This contributes to excessive mechanical damage of the bacterial cell membrane[125]. Padmavathy et al.[85] observed enhanced antibacterial activities of ZnO nanoparticles against E. coli for particles with 12 nm diameter, as compared to particles with 2 μm and 45 nm. It was attributed to the mechanical damage of the

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bacterial cell envelope as a result of increased surface area to volume ratio of the smaller ZnO nanoparticles. The orientation of the ZnO nanomaterials is still another important factor affecting the biocidal behavior. Wang et al.[77]reported that randomly oriented ZnO nanowires exhibit better antibacterial activities as compared to regularly oriented ZnO nanowires. Tam et al.[104] observed the toxic effects using ZnO nanomaterials of different morphologies; viz., powders, nanorods, and nanomaterials as antibacterial agents for B. atrophaeus (Fig.20 (a) to (d)).As reported by the author, cellular internalization has been observed in very few cells. For B. atrophaeus, different ZnO morphologies resulted in similar types of cell damage with multiple breaches. Leakage out of the cellular contents has also been observed.

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Fig.20. TEM images of B. atrophaeus(a) control, (b) ZnO powders, (c) ZnOnanorods and (d) ZnO nanoparticles.[Reprinted with permission from Ref. [104], Copyright © 2008, Elsevier B.V.].

5. SURFACE ACTIVATION THROUGH AMINE FUNCTIONALIZATION OF ZnO NANOPARTICLES Amine functionalization of the ZnO nanomaterials can lead to better conjugation with the surface biomolecules of the bacterial cell. Different amines such as isopropylamine (IPA), diethylamine (DEA) and triethylamine (TEA) are used as surface acting agents for amine functionalization of ZnO nanostructures, which have been studied for the evaluation of oxidative stress in the bacterial system[132]. Additionally, amines also exhibit antioxidant effects by counteracting with generated ROS and thus have many commercial applications[133,134]. These amines regulate the ZnO nanoparticles morphology, shape, and size, which are the key parameters for antibacterial activities. Kumar and Ansari et al. [132]investigated the antioxidant properties of amine functionalized ZnO nanoparticles for Gram-negative E. coli and Gram-positive S. aureus bacterial strains. As stated earlier, a low concentration of ZnO favors the bacterial growth; IPA functionalized ZnO exhibited lower growth and a lower reduction in oxidative stress for S. aureus as compared to for E. coli cells with a thin peptidoglycan layer at 0.5 mM concentration. On the other hand, DEA and TEA functionalized ZnO nanomaterials at 0.5 mM, 1 mM, and 1.5 mM concentrations showed better bacterial growth than control. At higher concentrations, however, these functionalized nanoparticles effectively served as

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bacteriostatic as well as bactericidal agents for E. coli. MIC values for IPA, DEA and TEA functionalized ZnO nanoparticles were recorded as 6mM, 9mM, and 12mM, respectively. It was proposed that at higher concentrations the cytotoxic effects of these functionalized ZnO were due to their penetration through the bacterial cell wall, leading to bactericidal effects[59,135].Almost the same results were observed for Gram-positive S. aureus bacteria with a thicker peptidoglycan layer in the cell envelope. However, the MIC values were calculated to be 14 mM, 15 mM and 18 mM for IPA, DEA and TEA functionalized ZnO nanoparticles (Fig.21)[132]. In amine functionalized ZnO nanoparticles, ZnO crystals are hydrogen bonded, leaving smaller alkyl chains to bind with the biomolecules of the bacterial cell wall and penetrate through it, damaging the enzyme systems, proteins, and nucleic acid, and thus inhibiting the cell growth[136,137].

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Fig.21.The growth curve for S. aureus in the presence of IPA, DEA and TEA functionalized ZnO nanoparticles. [Reprinted with permission from Ref. [132]Copyright© 2014, American Scientific Publishers].

6. CYTOTOXICITY OF ZnO NANOPARTICLES TOWARD CANCER CELLS Cancer or malignant tumor is a complex disease leading to abnormal cell division and growth due to cellular malfunctioning. This cellular malfunctioning can occur due to mutations induced bycarcinogens, gene amplification, or chromosomal translocation, or due to apoptosis due to genetic mutations[138,139]. Uncontrolled growth of the cancer cells takes place only when cellular malfunctioning and apoptosis operate simultaneously in the same cell. There are three reported stages of the transformation of normal cells to a

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cancerous cell,i.e., initiation, promotion, and progression[140]. The usual cancer treatments such as cancer therapy, including surgery, radiation, photodynamic therapy and conventional chemotherapy, etc., can pose severe damage to normal body cells also[141]. Multidrug resistance (MDR) have led to the potential abortion of chemotherapy for the treatment of the malignant tumor. Thus, the need of the hour is to develop anti-MDR agents for effective drug delivery for cancer treatment. Recent advancements in the field of nano-biotechnology have triggered research worldwide to explore significant treatment strategies for cancer patients. Materials at nanoscale possess size-related unique morphological, structural, physicochemical, electronic, and magnetic properties significant for many potential biological and medical applications. Among the various nanomaterials, ZnO nanomaterials have been explored extensively owing to their biocompatible nature in several biological applications including antibacterial, antifungal agents. An isoelectric point for ZnO nanoparticles from 9–10 provides the surface a strong positive charge[142]. In contrast, the cancer cells on their outer membrane bear a high concentration of phospholipids with negatively charged functional groups. This provides high membrane potentials[143]. The electrostatic interactions between cancerous cells and ZnO nanoparticles promote ZnO internalization leading to cytotoxicity. Sharma et al.[144]reported that ZnO nanomaterials result in ROS-triggered oxidative damage of DNA and mitochondria-mediated apoptosis in human liver cells. Recently, Wahab et al.[145] reported that ZnO nanoparticles induced oxidative stress in Cloudman S91 melanoma cancer cells. Park et al.[146]compared the cytotoxic process of ZnO nanostructures through various physicochemical characterizations and ROS

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properties. Ostrovsky et al.[147]explained the cytotoxic effects of ZnO nanomaterials with particle size 60 nm toward human glioma cell lines U87, A172, U251, LNZ 308, LN18, LN 229, human normal astrocytes, and on breast and prostate cancer cells. It has been reported that cytotoxic activities of the ZnO nanomaterials are due to interactions with rapidly dividing cancer cells in a specific and proliferation-dependent manner, whereas the quiescent or non-cancerous cells remains unaffected[148]. It has been reported that the tumor suppressor gene, p53 is the master guardian of the cell which activates

the

cellcycle,

DNA

repair,

and

apoptosis

to

maintain

genomic

stability[149,150]. Guo et al.[151] found a UV light-inducedsynergic effect in cytotoxicity suppression of daunorubicin drugs in the presence of ZnO nanoparticles of 20, 60 and 100 nm sizes on leukemia K562 and drug resistance K562/A02cancer cells using MTT assay. The half maximal inhibitory concentration (IC50) values of ZP5 (20 nm), ZP6 (60 nm) and ZP7 (100 nm) nanoparticles on K562 and K562/A02 cell lines were reported to be 13.44, 13.99 and 11.84 μg/mL and 9.11, 8.57 and 8.22 μg/mL, respectively. It can be observed that ZnO nanoparticles also exhibit cytotoxicity toward drug-resistant K562/A02 leukemia cells, implying that these nanoparticles inhibit the functioning of P-glycoprotein, a cell membrane protein, and alter composition properties of the cell membrane of the drug-resistant cells. These changes enhance the drug uptake by the drug-resistant K562/A02 leukemia cells. These studies have shown that ZnO nanoparticles have a great potential in biomedical applications.

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7. CONCLUSION AND FUTURE SCOPE

In conclusion, ZnO nanomaterials with versatile morphologies can be efficiently utilized as antibacterial agents against various Gram-positive as well as Gram-negative bacterial strains through chemical and physical interactions with the cell envelope of the bacterial cells. The ZnO morphology, the presence of surface defects, the concentration of the ZnO nanomaterials, types of the bacterial strain, the method applied for exploring the antibacterial activities, and the features of the cell wall envelope are the main factors affecting the biocidal activities. The cell envelope damage and hence biocidal activities are supposed to be triggered through collective actions of the chemical as well as physical interactions. However, chemical interactions resulting in the production of ROS and H2O2 and release of Zn2+ ion from ZnO solubility are reported to be major causes for the said activities.ROS include species like O -2 , HO2 and HO , which damage the cell envelopes and the cellular components causing various degrees of oxidative stress. Gramnegative bacteria have an extra outer plasma membrane with a thick lipopolysaccharide layer, which is much thicker than the peptidoglycan layer present in Gram-positive bacteria. These structural features of the Gram-negative bacterial cell oppose the lipid peroxidation in the presence of ROS generated through ZnO nanomaterials and make them less susceptible to ZnO attack. Strong electrostatic interactions between the ZnO nanomaterials and bacterial cell surfaces result in increased surface tension and membrane depolarization leading to change in membrane textures, rupture, membrane blebs, membrane disruption, morphology alteration, induction of oxidative stress,

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increase in membrane permeability, particle internalization, leakage of intracellular fluid, and components causing bacterial cell death. These electrostatic interactions can be enhanced through amine functionalization of the ZnO nanomaterials, which resulted in better conjugation with the surface biomolecules of the bacterial cell. Further for better conjugation, and hence antimicrobial activities, ZnO nanoparticles can be engineered through doping with transient as well as representative metals. Importantly, a low concentration of Zn2+ nanoparticle is reported to be favorable for the bacterial growth in some case such as for E. coli, as Zn2+ ions released from ZnO act as nutrients for it. Hence, the future study should focus on the low-level toxicity studies of ZnO nanoparticles. A detailed research is required to bridge the knowledge gaps for the safe biomedical applications of ZnO nanomaterials for the treatment of several infectious diseases and environmental and ecological aspects. The use of nanomaterial-based antibiotics will be a remarkable achievement for the future of the pharmaceutics industry. Acknowledgement

Rajesh Kumar and Girish Kumar want to thank the University Grants Commission, New Delhi for financial assistance in the form of minor research projects.

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