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ZnO Applications and Challenges

Ovidiu Oprea1, Ecaterina Andronescu1, Denisa Ficai1, Anton Ficai1, Faik N. Oktar2-4* and Mehmet Yetmez5

1Politehnica University of Bucharest, Faculty of Applied Chemistry and Material Science; 1-7 Polizu St., 011061 Bucharest,

Romania; 2Bioengineering Dept., Faculty of Engineering, Marmara University, Istanbul, Turkey;

3Medical Imaging Techniques

Dept., School of Health Related Professions, Marmara University, Istanbul, Turkey; 4Nanotechnology and Biomaterials Research and

Application Center, Marmara University, Istanbul, Turkey; 5Mechanical Engineering Dept., Faculty of Engineering, Bulent Ecevit

University, Zonguldak, Turkey

Abstract: Metal oxide nanoparticles represent a new class of important materials that are increasingly being developed for use in re-

search and health-related applications. Although the in vitro antibacterial activity of zinc oxides and some other zinc compounds has been

known for quite some time, only in the last few years nanoparticles of ZnO have been investigated for their antibacterial activity, the

knowledge about it remaining deficient. The antimicrobial activity of ZnO nanoparticles is strongly influenced by some factors such as

size and the presence of light. The potential applications include, but are not limited to, topic drugs, cosmetics or component for agents

that control the spread of bacterial strains (antibacterial paint in hospitals, antibacterial coatings for fabrics, antibacterial packaging for

food, etc). The current review aims to present the level of knowledge accumulated on the antibacterial and antifungal activity of ZnO.

Keywords: ZnO nanoparticles, antibacterial activity, antifungal activity.

INTRODUCTION

In recent years, nanoparticles have received increasing attention due to their unique physical and chemical properties. Currently, metal oxide nanomaterials are among the most highly produced nanomaterials; their available applications include catalysis, sen-sors, environmental remediation, medicine, varistors, solar cells, rubber, concrete, foods, cosmetics and personal care products [1-11].

One promising approach is the use of metal oxides nanoparti-cles in health-related applications (hyperthermia, drug delivery, coated nanoparticles) [12-31]. The metal oxides such as titanium oxide, magnesium oxide, zinc oxide and copper oxide are better than nanosilver because of their low cost.

ZnO nanoparticles have been prepared by various methods such as thermal decomposition [32, 33], spray pyrolysis [34, 35], sol-vothermal reaction [36, 37], forced hydrolysis [13], reactive elec-tron beam evaporation technique [38], sol-gel method [39, 40], chemical vapor deposition [41], and hydrothermal method [42, 43]. They can also be produced into a variety of morphologies [44] such as nanowires [45, 46], nanorods [47-49], nanocombs [50, 51], tetra-legs [52], nanoflowers and nanosheet [53, 54].

ZnO has found its way in many applications in daily life such as in drug delivery, cosmetics, medical devices [55-62], dentistry and orthopedics [63-66]. Zinc is an essential trace element, which is found in the human body and has a stimulatory effect on bone for-mation. Human bone contains 0.0120 – 0.0250 wt% Zn [67]. The Zn content of various human tissues is shown in (Table 1) [68]. It can also be used in new bioglass formulations. Rui Lin Du et al. (2006) had shown that a certain concentration of the ionic products from Zn-containing bioactive glass could stimulate osteoblast pro-liferation [67] (Table 2).

*Address correspondence to this author at the Bioengineering Dept., Faculty of Engi-neering, Marmara University, Istanbul, Turkey; Tel: +90 216 348 0292/732; Fax: +90 216 348 0293; E-mail: foktar@marmara.edu.tr

The use of ZnO in cosmetics is not limited to the sunscreens [69] where (together with TiO2) the nanoparticles formulations have an improved performance as they scatter better the ultraviolet light than their respective bulk formulations [70] (Table 2).

In dentistry, ZnO alone or together with eugenol is used as a fill material due to its ability to block microbial leakage [71-74] (Table 2). For this reason, ZnO usually is included in the temporary ce-mentation material for cementation of temporary crowns and bridges. Most of those cements are based with eugenol and they are called as zinc oxide-eugenol (ZOE) cements. Beside temporary cementation, they are also very suitable for permanent cementation and as a cavity liner. They are also commonly used in the tempo-rary filling of teeth, and as a cavity basement in deep cavity prepa-rations. These ZnO containing products are non-toxic to oral tissues when they come into direct contact with freshly cut dentine tissue and the oral mucosa [65]. According to a recent study on ZnO, when it was used in nano-hydroxyapatite (HA)/chitosan compos-ites, it was proven to shorten the setting time, enhance the mechani-cal strength and improve the bioactivity and antibacterial activity of those composites [75]. Otsuka et al. (2004) have used Zn-tri cal-cium phosphate (TCP), as carrier, for controlled zinc release. The results showed that long-term sustained Zn release from Zn-TCP may improve bone mineral density in Zn-deficient osteoporotic rats [76]. Other researchers have discovered that HA-based system holds wide potential for application in clinic as an antibacterial biomaterial, as well as for dental filing and repair [77]. The struc-tural behavior of Zn in synthetic HA provides valuable information for future studies of Zn storage and transport involving calcified hard tissues and therefore the regulation of Zn metabolism in the body [78].

Zn holds also an inhibitory effect on osteoclastic bone resorp-tion. Several researchers have attempted to doper materials with Zn+2 at low concentrations for increasing the bioactivity of bone cells and decreasing and regulating the inflammatory reaction [79].

Besides being an antiseptic material, ZnO is also used as rein-forcing oxide for orthopedic various calcium phosphate based bio-

2 Current Organic Chemistry, 2014, Vol. 18, No. 2 Oprea et al.

ceramics (i.e. hydroxyapatite - HA). But there are limited studies in the literature on the reinforcement of HA structures with Zn or ZnO. Usually HA materials have poor mechanical properties. They cannot be used as heavy-loaded implants [80]. For improving me-chanical reliability of HA metallic materials, ceramic oxides, whiskers, or fibers are used [81]. ZnO seems be a good reinforcing material for HA [63, 64, 82, 83].

D. Norhidayu et al. (2008), have also proved that zinc plays a very important role in proliferative effects on osteoblastic cells apart from inhibiting osteoclastic resorption. Because of this zinc-substituted calcium phosphate has received a considerable attention [83].

Zn substitution in HA structures has been focused to the par-ticular interest in human body. Zn is present in all biological tissues and plays diverse roles in many different biological functions (i.e. enzyme activity, nucleic acid metabolism, maintenance of mem-brane structure and function, hormonal activity, biomineralization, potential pathological calcification etc.). The uptake and release of Zn material in the body are strongly mediated by the bone reservoir, even though it only contains a small portion of the total body Zn. For example, teeth contain a significant amount of Zn, (144.5 mi-crograms Zn/ g tooth substance). Zn concentration has been used also as an indicator of environmental exposure [84]. Kawamura et

al. (2000) found that the optimum Zn content in calcium phosphate ceramics is 0.316 wt % for promoting bone formation [85].

ZnO effect on epithelialization of wounds as well as ZnO bacte-riostatic property promote it as a topical wound dressing. It can be used in treatment of various dermatitis, diaper rashes, diaper wipes, blisters, and open skin sores [59, 86-88] (Table 2).

ZnO has for decades been used in medicine as a mild topical as-tringent (and possible antibacterial agent) in eczema and slight ex-coriations, in wounds, and for hemorrhoids [89]. Although the in

vitro antibacterial activity and efficacy of zinc oxides and some other zinc compounds have been investigated, only in the last few years nanoparticles of ZnO have been investigated for their antibac-terial activity [90-92]. Studies have indicated that ZnO nanoparti-cles at a concentration of between 3 and 10 mM caused 100% inhi-

bition of bacterial growth as a result of the intracellular accumula-tion of nanoparticles [93].

The antibacterial activity of ZnO may be dependent on the size and the presence of UV or visible light. The data suggest that ZnO nanoparticles have a potential application as a bacteriostatic agent in visible light and may have future applications in the development of derivative agents to control the spread and infection of a variety of bacterial strains.

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF ZnO

ZnO is one of five zinc compounds that are currently listed as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (21CFR182.8991) [102, 103].

Many studies have focused on the in vitro antibacterial activity on various bacterial strains (Staphyloccocus aureus, Staphylococcus epidermidis 1487, Streptococcus pyogenes, E. coli, E. coli O157:H7, Listeria monocytogenes, Bacillus subtilis, Bacillus atro-phaeus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Sal-monella enteritidis, Salmonella typhimurium, Enterococcus fae-calis, Enterobacter cloacae, Lactobacillus helveticus) [103-108] and indicate that ZnO nanoparticles are more efficient as antibacterial agent than bulk powder and generally Gram-positive bacteria are more sensitive to ZnO than Gram negatives. This could be simply explained as smaller particles normally have a larger surface to volume ratio which provides a more efficient mean for antibacterial activity [109, 110]. Alternatively, small particles exhibit stronger cellular internalization, higher surface defect concentration, gener-ating larger amount of hydroxyl radicals [110]. ZnO nanoparticles also have antibacterial activity against spores that are high-temperature and high-pressure resistant [111-114].

Jones et al. (2008) demonstrated the influence of the particle size, by choosing three different values: 1 m, 50-70nm and 8nm. While ZnO particles ~1 m reduced growth rates (c. 50%) of Staphylococcus aureus, ZnO nanoparticles, with smaller particle size, were able to reduce c. 99% of growth in colloidal suspension with concentration of 2mM. While 8nm diameter ZnO nanoparti-

Table 1. Summary of Literature Concerning Zinc Concentration in Human Tissues [68]

Bone Muscle Liver Pancreas Kidney Author Date Method Specimen Source

g/ g dry weight

Eggleton 1940 Dithizone Hospital autopsies

163a

(9)b

121-307c

245

(20)

116-562

135

(16)

44-216

186

(20)

68-314

Butt 1954 Emission spectrography Hospital autopsies 219±109d

(241)

190±100

(244)

Tipton 1963 Emission spectrography Hospital autopsies 206

(137)

141±18

(150)

79

(138)

230±19

(145)

Netsky 1969 Atomic absorption

spectrophotometry Hospital autopsies

207±103

(32)

184±75

(34)

Atomic absorption

spectrophotometry Hospital autopsies

163±30

(16)

187-368

203±67

(14)

149-287

258±138

(20)

72-532

104±52

(20)

48-249

205±104

(20)

84-444 McBean 1971

Atomic absorption

spectrophotometry Accidental death

218±94

(9)

135-461

197±48

(10)

94-264

179±66

(10)

91-323

115±28

(10)

86-175

194±49

(10)

101-274

a Mean. b Numbers in parentheses indicate number of samples. c Range. d Standard deviation

ZnO Applications and Challenges Current Organic Chemistry, 2014, Vol. 18, No. 2 3

cles showed more than 95% growth inhibition at 1mM concentra-tion (0.008%), ZnO nanoparticles with relatively larger particle sizes (e.g. 50-70 nm) showed only 40-50% growth inhibition at 5mM [106]. The minimum inhibitory concentration (MIC) for ZnO nanoparticles with smaller size was calculated to be 1mM (0.08 mg mL-1) for Staphylococcus aureus, whereas that for larger particles of ZnO was calculated to be 15mM (1.2 mg mL-1). Increasing the contact time from 10h to 24h leads to no significant variation of the bacterial growth inhibition.

Another approach is represented by the studies which measure the antibacterial activity of ZnO nanoparticles in the presence of light [106].

The study made by Jones et al. (2008) clearly suggests that re-gardless of ZnO nanoparticles size the antibacterial activity of ZnO nanoparticles in the dark is less than that in ambient laboratory

conditions. In the case of the nanoparticles with 8nm diameter, only 30-50% of the antibacterial activity is retained at 5mM concentra-tion up to 10h. By photoactivation of ZnO nanoparticles suspen-sions, performed by exposure with UV light at 254nm for 30 min, the original antibacterial activity can be obtained [106].

By using the light factor in measuring the antibacterial activity, researchers take advantage of the well known photocatalytic activ-ity of ZnO nanoparticles. This photocatalytic activity can act syner-gic with the intrinsic antibacterial effect of ZnO, and promotes it as a good candidate for future topic drugs, cosmetics or simply as a component for agents that control the spread of bacterial strains (antibacterial paint in hospitals [115-119], antibacterial coatings for fabrics [120-125], etc). The overall growth analyses suggest that the ambient laboratory conditions are sufficient for the optimal biocidal activity of the ZnO nanoparticles.

Table 2. Some Applications of ZnO-based Materials and their Characteristics

Applications Material Characteristics Ref.

Biotinylated

silica-coated ZnO-

quantum dots

New biotinylated silica-coated ZnO quantum dots (QDs) were developed for improved bioimaging special due to the

low cytotoxicity, high luminescence, high stability in aqueous solutions and high sensitivity. These materials could

be used as advanced fluorescence probes for biological cell-labeling applications.

[94]

ZnO/Eugenol ZnO/eugenol based sealers are extensively used in dentistry because of its antiseptic role. [71-74]

Co-doped ZnO

particles in silk-fibroin

(SF) peptides

Co-doped ZnO NPs with the diameter of 50 nm can be obtained via a biomineralization technique in presence of silk

as templates. SF-coated Co-ZnO NPs remarkably stimulate the growth of cells when NP concentrations are less than

0.25mg/mL stimulating and the adhesion and proliferation of cell due to the SF peptide coating, indicating that the

prepared Co-doped ZnO particles have a good cytocompatibility and a potential in several biomedical applications.

[95]

ZnO nanosheets

Highly crystalline ZnO hexagonal nanosheets, with an average edge length of 46.6±8.5nm can be obtained under

mild reaction conditions. The obtained ZnO nanosheets own the visible yellow-orange emission, allowing the

bio-imaging applications and showing the potential prospects in the biomedical fields.

[96]

Biomedical

applications

ZnxCa5-x(PO4)3OH

Zinc doped hydroxyapatite is used especially for bone grafts materials and dental applications, zinc being co-factor

of many enzymes including alkaline phosphates (directly involved in bone metabolism). Zinc doped hydroxyapatite

is also used as drug delivery system for different drugs, such as ciprofloxacin. Zinc based calcium phosphates are

also interesting because they allow a slow release of ionic zinc.

[78, 85,

97]

Photocatalytic

applications

ZnO-TiO2 electrospun

materials

ZnO-TiO2 based hierarchical flower shaped mesostructures have been successfully synthesized by electrospinning

their precursors followed by calcinations.

It was demonstrated ZnO-TiO2 flower that, the photocatalytic activity exhibited by the ZnO-TiO2 flowers was supe-

rior to that of pure ZnO nanoparticles

[98]

Solid state gas

sensor

Flower-like ZnO

microstructures

The flower-like ZnO microstructures materials were tested for solid state gas sensor. Gas sensing device was pre-

pared by thick film technology and tested for NO2, CO, ethanol and acetaldehyde gases at different testing tempera-

tures as well as concentrations, and increased with gas concentration as well as temperature (except 400 C). The

response for NO2 was higher than the CO, ethanol and acetaldehyde at low operating temperatures.

[99]

Oriented hierarchical

ZnO flower-rod

architectures

Oriented hierarchical ZnO flower-rod arrays (FRs) were prepared on indium doped tin oxide (ITO) glass using a

facile solution-based method assisted with ZnO seed layer. The as prepared ZnO FRs/ITO materials can be used as a

photocatalytic device. The ZnO FRs are pure wurtzite phase. The length of flower petal and rod is 1.2-1.5 and 0.9-

1.1 m, respectively. The ZnO FRs are single crystal grown along the c axis and the flowers are cone-like building

blocks. The photoluminescence (PL) spectra illustrate that there are more oxygen vacancies on the surface of ZnO

FRs compared with ZnO nanoparticles (NPs).

For ZnO FRs, the higher photo-induced currents under UV irradiation and current density prove that the recombina-

tion of electron-hole pairs is restrained with oxygen vacancies, and the lower charge transfer resistance suggests that

the charges could move quickly through ZnO oriented structures. The photocatalytic activity of ZnO FRs is better as

compared to the activity of other ZnO NPs, against Rhodamine B (RhB), degradation efficiency of ZnO FRs being

nearly 100% on UV irradiation for 1.5 h.

[100]

Photovoltaic

application

Aligned CdSe@ZnO

flower-rod core-shell

nanocable

CdSe@ZnO flower-rod core-shell nanocable arrays (CSZFRs) were prepared by a two steps procedure meaning

ZFRs growth on ITO surface followed by CdSe deposition onto the ZFRs by ion exchange. The core-shell configura-

tion, the type II band alignment, and the favorable absorption properties of CdSe contribute to high photovoltaic

performance (higher open-circuit photovoltage -0.93 VAg/AgCl and short-circuit photocurrent 1.00 mA/cm2).

[101]

4 Current Organic Chemistry, 2014, Vol. 18, No. 2 Oprea et al.

Thati et al. (2010) reported that the antibacterial activity of various classes of antibiotics (penicillin, amoxyclav, amikacin, methicillin, oxacillin, amphicilin, amoxicillin, genthamycin, clox-icillin, cephaxine, cefotoxime, ceftazidime, vancomycin, strepto-mycin, erythromycin, clindamycin, tetracycline, ciprofloxacin, rifampicin, sulphozidime, cotrimaxizole, chloramphinical, norflax-acin and claritromycin) is greatly enhanced in the presence of sub inhibitory concentration of zinc nano particles against Staphylocco-

cus aureus strain, [126]. Also Voicu et al. (2013) reported that the ZnO-gentamicin hybrid material has a synergic activity on Bacillus

Cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococ-

cus aureus and Listeria monocytogenes, the increasing of inhibition diameter ranging between 15% and 51%, very good results being obtained for Gram-positive bacteria [127].

Overall, the actual knowledge suggests that ZnO nanoparticles can be used externally to control the spreading of bacterial infec-tions. It would also be interesting to determine which (if any) de-rivatives of ZnO nanoparticles with various chemical groups or bioagents are more effective at eliminating different microorgan-isms.

Nanotechnology is being envisioned as a dynamically develop-ing field, it has potential to revolutionize food systems and improve the conditions of the food quality [128-130], but it also influences domains as paints industry, textile industry or medical applications.

As different bacterial strains become increasingly resistant to antibiotics, the search for new strategies to deal with the diseases causing bacteria intensifies. Researchers need to identify and de-velop the next generation of drugs or agents to control bacterial infections [131].

Another major concern is the growth of fungal pathogens which presents the main cause of economic loss during the post harvest handling of crops. It is difficult to control fungal growth because fungi have developed resistance to many conventional fungicides such as benzimidazoles and dicarboximides. To overcome this re-sistance, it is important to explore novel antifungal agents, which may replace current control strategies.

The most important pathogenic agent in food industry, Salmo-

nella, is the causal agent of a significant range of foodborne ill-nesses [132, 133].

Staphylococcus is also a major group of the foodborne patho-gens which is mostly associated with community acquired and nosocomial infections, and may be life threatening in immunodefi-cient conditions. The most important representative is Staphylococ-

cus aureus, which is a natural inhabitant of human and animal skin, but can sometimes cause infections that affect many organs [134].

Botrytis cinerea and Penicillium expansum can cause severe postharvest fruit diseases including grey and blue mould even when the most advanced postharvest technologies are applied [135]. Botrytis cinerea is considered as one of the most important diseases of table grapes [136], while Penicillium expansum primarily causes the rot of stored apples and pears [137]. Furthermore, Penicillium

expansum is regarded as the major producer of a mycotoxin, patu-lin, which is commonly found in rotting apples. U.S. Food and Drug Administration (FDA) limits patulin to 50 mg/l in apple juices [138].

There is a need to develop new antimicrobials to ensure food safety and extend shelf life. The use of antimicrobial agents directly added to foods or through antimicrobial packaging is one effective approach. ZnO has high biocompatibility and fast electron transfer kinetics, such features promote the use of this material as a biomimic membrane to immobilize and modify the biomolecules [139].

According to [104, 140], there are two classes of antibacterial agents used in the food industry: organic and inorganic agents. Among these, the inorganic antibacterial agents are more stable at high temperatures and pressures compared with the organic materi-als, and the metallic oxide powders could be suggested as powerful antimicrobial agents in this field.

Since ZnO is classified as a safe material for human sand ani-mal shaving superior durability, greater selectivity and heat resis-tance compared to it sorganic/ inorganic analogues and owing to its stability under harsh processing conditions [105, 141-143], it be-comes a promising antibacterial agent in food industry.

Moreover, there are some studies [93, 144, 145] that have indi-cated that ZnO nanoparticles exhibit minimal effect on human cells while having selective toxicity to bacteria, which recommend their prospective uses in agricultural and food industries.

The antibacterial and antifungal activities of bulk ZnO powders have been demonstrated already [146, 147]. In agriculture, zinc compounds are mainly used as fungicides [148].

Among metal oxides nanoparticles (zinc, aluminium, silicon and titanium) Jiang et al. (2009) [149] reported that ZnO nanoparti-cles were the most toxic antibacterial agents against Bacillus sub-

tilis, E. coli and Pseudomonas fluorescens. Also Liu et al. (2009), [150], indicated that ZnO nanoparticles can be used as an effective antibacterial agent against foodborne pathogens (e.g. E. coli

O157:H7) thus being a promise candidate for agricultural and food safety.

Tayel A. et al. (2006), [104], tested ZnO nanoparticles and bulk ZnO against 9 strains (Staphyloccocus aureus, E. coli, E. coli

O157:H7, Bacillus cereus, Pseudomonas fluorescens, Pseudo-

monas aeruginosa, Salmonella enteritidis, Salmonella typhimurium,

Enterobacter cloacae). Their study indicates that for all strains tested (2 Gram-positive and 7 Gram-negative) at dimensions of ~50nm ZnO nanoparticles are more efficient than bulk ZnO (~5 m), and the best activity was recorded against Gram-positive strains. The influence of concentration and contact time were also accounted for. The diameter of growth inhibition zone increased with the decrement of required MIC from ZnO for each strain, but with no clear linear correlation between the used assays. The most sensitive strain was Bacillus cereus while the most resistant one was Pseudomonas spp. Regarding the contact time influence for MIC (minimum inhibitory concentration) to be effective the study indicates that Salmonella typhimurium required almost double val-ues vs. Staphyloccocus aureus (8h vs 4 h), after one hour of expos-ing the death rate being 3 times greater for the latest.

Jin et al. (2009) [103] recommended several approaches (pow-der, Polyvinylpyrolidone (PVP) capped, film and coating) for incorporation of ZnO into food systems. They concluded that ZnO nanoparticles exhibit antimicrobial effects against Listeria monocy-

togenes and Salmonella enteritidis in the white, liquid part of egg and in culture media. ZnO sedimentation in a liquid system should be reduced or avoided. The dispersion of ZnO nanoparticles in liq-uid media is very important. As ZnO sedimentation occurs, in the low concentrated solution the ZnO nanoparticles dispersed well and exhibited better antibacterial activity compared to its high concen-trated analogue [103]. This is to confirm that intimate contact be-tween ZnO nanoparticles and cell membrane is required for antibac-terial activity expression.

Similar results were obtained for ZnO-PVP coating system. If ZnO-PVP gel is applied as coating on the wall of the recipient, poor results will be obtained. The direct addition of ZnO-PVP nanoparti-cles (3.2 mg ZnO/mL) in the liquid egg white resulted in 5.3 lo-

ZnO Applications and Challenges Current Organic Chemistry, 2014, Vol. 18, No. 2 5

greduction of Listeriamonocytogenes and 6.0 logreduction of E.

coli O157:H7 after 48 h incubation, as compared to the controls [103].

To eliminate the sedimentation process some authors investi-gated the antibacterial behavior of ZnO nanofluids [144, 151, 152]. The results show that the use of two types of dispersants (Polyeth-ylene Glycol - PEG 400 and PVP) does not affect much the antibac-terial activity of ZnO nanofluids, in the dark, but enhances the sta-bility of the suspensions.

The same conclusion was formulated by Tam et al. (2008). The presence of surfactant (3-aminopropyltrimethoxysilane) or Au nanoparticles on the ZnO surface did not affect the antibacterial properties of ZnO nanoparticles [108].

The SEM images of the bacteria treated with ZnO nanofluids show considerable damage to some E. coli and that the damage has caused the breakdown of the membrane of the bacteria [144].

The storage time and the influence of light conditions over anti-bacterial activity were also investigated for ZnO nanofluids [151]. Best activity was obtained for nanofluids stored for 120 days, under light, most probably due to increased quantities of H2O2 produced in this time.

When ZnO was bound to a polystyrene film (ZnO-PS), no anti-bacterial activity was noticed, for concentrations up to 1% ZnO, as the nanoparticles were not released from the film into the growth broth or agar. The SEM image reflected the smooth surface of the ZnO-PS film, suggesting that the ZnO molecules were tightly bound within the film, which prevented ZnO release and expression of the antimicrobial action [103].

Other studies have shown that ZnO powder coated PVC (poly-vinyl chloride) films exhibit antibacterial activity against food borne pathogens like Escherichia coli and Staphylococcus aureus, but no antifungal activity [153]. The inhibition diameter was greater for Staphylococcus aureus (18.5 mm) than for Escherichia coli

(17.5 mm) at a concentration of 93.75 g cm-2, which is consistent with the fact that Gram-positive bacteria are more sensible to ZnO action than Gram-negative ones.

Antimicrobial active packaging is a new generation of nano food packaging based on metal oxide nanocomposites which are made by incorporating the oxide nanoparticles into polymer films [102]. Because of the thermal stability metal oxide nanoparticles can be incorporated in an LDPE film as a contacting layer in the package. Emamifar et al. (2010) reported an extended shelf life for orange juice up to 28 days when used package was embedded with ZnO and Ag nanoparticles (vs. a normal 14 days shelf life) [102]. Emamifar et al. (2011) continued their studies using nanocomposite packaging containing Ag and ZnO on inactivation of Lactobacillus

plantarum in orange juice up to 128 days [154]. Nanosilver was found to have a higher antimicrobial activity on Lactobacillus plan-

tarum, yeast and moulds compared with ZnO nanoparticles, espe-cially for longer storage times. These results can help the industry to combine pasteurization with antimicrobial nanocomposite pack-ages, developing new cost-effective pasteurization method to con-trol microorganisms and to preserve the desirable food qualities [155].

The antifungal activity of ZnO nanoparticles has not received same attention, as there are a limited number of studies carried out. There are reports of antifungal activity against Aspergillus niger [128, 147], Botrytis cinerea and Penicillium expansum [105], Sac-

charomyces cerevisiae, Candida albicans and Rhizopus stolonifer [147], Fusarium sp. [156], and Aspergillus brasiliensis [157].

Li X et al. (2009) reported that antifungal activity of the ZnO-PVC coated films against Aspergillus flavus and Penicillium ci-

trinum was not observed. They concluded that it is likely due to the complexity of the fungal cell wall and the ZnO nanoparticles with-out irradiation or the insufficient amount of nanoparticles at the interface [153].

Sawai and Yoshikawa (2004) reported that the minimum inhibi-tory concentration of bulk ZnO powder against Saccharomyces

cerevisiae, Candida albicans, Aspergillus niger, and Rhizopus

stolonifer was over 100 mg ml-1 by an indirect conductimetric assay [147].

On the other hand, Kasemets et al. (2009) found that nano and bulk ZnO were of comparable toxicity against Saccharomyces cer-

evisiae [158].

Solutions that contain 12mmol l-1 exhibit a reduction rate of fungal growth of 80% for Botrytis cinerea and 91% for Penicillium

expansum. For Aspergillus niger, the growth inhibition was total for 0.4 mg l-1 [128].

Sharma D. et al. (2010) reported a good antifungal activity of ZnO nanoparticles against Fusarium sp. At concentration of 0.1M ZnO, they reported a better antifungal activity than that by the stan-dard CuSO4 and proposed that ZnO nanoparticles lead to the rup-ture of the fungal cell membrane resulting the possible decrease of fungal enzymatic activity [156].

Vlad et al. (2012) reported antifungal activity of a membrane ZnO-PU (polyurethane) at concentration of 5% and 10% ZnO up to 60 days (Fig. 1). Such modified ZnO membranes have important antifungal properties and can be successfully used in food or bio-medical applications, blocking the formation of biofilms [157].

Textile industry is one domain that can largely benefit from the antibacterial and antifungal activity of ZnO nanoparticles. One objective is to impact the additional value on natural textiles by adding to them ultraviolet (UV) absorption and antimicrobial pro-tection properties with ZnO nano-level coatings It was found that the treatment with ZnO formulation caused a net reduction in bacte-rial cells of 78% and 62% in the case of treated cotton and cot-ton/polyester fabrics while the net reduction in fungi was calculated to be 80.7% and 32%, respectively [159-161].

ANTIBACTERIAL AND ANTIFUNGAL ACTION MECHA-

NISMS OF ZnO NANOPARTICLES

Tam et al. (2008) revealed that ZnO nanorods in the liquid phase (without UV light) lead to bacterial cell death which is due to the cell membrane damage [108]. TEM images present cells with obviously damaged membrane in addition to partially leaked out contents. Also, some of the cells exhibit shrinkage of cytoplasmatic material inside the cell wall. Internalization of the ZnO rod or ZnO nanoparticles can be observed in some cases, but this is not a gen-eral occurrence. Therefore the mechanical damage of the cell mem-brane cannot be considered as the main mechanism of antibacterial activity of ZnO. In the case of Bacillus atrophaeus, different ZnO morphologies cause similar types of cell damage. Cells with par-tially leaked out contents can be observed, and there exist very few intact cells. A TEM image of a single cell was published in [108], to more clearly observe the cell damage in E. coli and Bacillus at-

rophaeus cells exposed to ZnO nanoparticles. For Bacillus atro-

phaeus cells, can be observed multiple breaches of the cell walls, while this is not the case for E. coli. Tam’s study has demonstrated that the ZnO is more effective for killing Gram-positive than Gram-negative bacteria because they have simpler cell membrane struc-ture.

6 Current Organic Chemistry, 2014, Vol. 18, No. 2 Oprea et al.

Repeated incubation tests result in eventual loss of antibacterial activity of ZnO nanoparticles. This is likely due to the fact that it is necessary for nanoparticles to be in contact with the cells to present antibacterial activity. Thus no antibacterial activity will be observed once contamination by dead bacteria residue etc. has covered the surface of nanoparticles [108].

The interactions between the nanomaterial and the individual cell components can elucidate the impact of nanomaterials on bac-teria. The first interaction that can occur between a nanoparticle and a cell is at the membrane interface. Some studies, [162], suggested that nanoparticles were able even to embed themselves in the cell membrane.

Similar results were reported by Liu et al. (2009). ZnO nanopar-ticles may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacte-

rial cells. The inhibitory effects against E. coli O157:H7 increase as the concentration of ZnO nanoparticles increases [150].

A study conducted by Tayel A. et al. (2011), [104], based on scanning electron micrographs of the treated bacterial cells (Salmo-

nella typhimurium and Staphyloccocus aureus) revealed that ZnO nanoparticles attached to the cellular wall, softening and broadening it, having as result that the cells were lysed and their internal com-ponents began to release.

As Gram-positive bacteria were more sensitive than Gram-negative bacteria to ZnO nanoparticles, this can be explained by ZnO interaction with specific cell wall compounds; these com-pounds may be found or increased in Gram-positive rather than in Gram-negative bacteria. The cell wall structure for Gram negative bacteria is also relatively more complex than that of Gram positive cells [163].

Fig. (1). SEM images: fungal growing profile of the samples PUDMPA-0 (0% nano-ZnO), PUDMPA-2 (5% nano-ZnO) and PUDMPA-4 (10% nano-ZnO) [157].

ZnO Applications and Challenges Current Organic Chemistry, 2014, Vol. 18, No. 2 7

Jeng et al. (2006), [164], proposed that nanomaterials that can physically attach to a cell can be bactericidal if they come into con-tact with this cell. If the membrane of a cell is compromised, the cell may repair itself or, if the damage is severe, the cell component may release and eventually the cell will die [165].

To explain the adhesion of ZnO nanoparticles to the cell wall, Tayel A. et al. (2006) in [104] indicated as potential candidates the outer thick peptidoglycan layer and its amino acid constituent, sur-face proteins and teichoic acids plus lipoids (forming lipoteichoic acids), which act as chelating agents. Among the compounds that can block the adhesion of ZnO (compounds found only in Gram-negative bacteria) the possible nominees include the extra outer membranes and the pathogen-associated molecular patterns which include lipopolysaccharide (consisting of lipid A, core polysaccha-ride and O antigen), porins and particular fragments of peptidoglycan.

Leone et al. (2007) proposed that carboxyl, amide, phosphate, hydroxyl groups and carbohydrate-related moieties in the bacterial cell wall may provide sites for the molecular-scale interactions with the oxide nanoparticles [166].

The exact nature of the interaction of the nanoparticles with bacterial membrane and possible permeation of the nanoparticles into the bacterial cells is still under investigation as exact informa-tion and consensus are lacking [149].

The antibacterial activity can be explained also by a mechanism proposed by Yamamoto et al. (2001), [146], which relies on the generation of hydrogen peroxide (H2O2). H2O2 that generates from the surface of ZnO is considered as an effective mean for the inhibi-tion of bacterial growth. It can be assumed that the concentration of H2O2 generated from the surface increases with decreasing particle size. As the size of particles decreases the surface increases, so more H2O2 will be generated. Li et al., (2009) reported that genera-tion of H2O2 from ZnO leads to the penetration of particles into the cell membrane of bacteria, that leads to the formation of injuries and finally the death of bacterium [153]. Same mechanism was reported for antibacterial activity against E. coli and Pseudomonas

aeruginosa [128].

The size of ZnO nanoparticles also directly influences the anti-bacterial activity. On one hand while the nanoparticles size de-creases, the surface/volume ratio increases. On the other hand, smaller nanoparticles tend to have a larger surface defect density, which leads to better photocatalytic activity by generating increased levels of reactive oxygen species (ROS) [167].

Electron resonance measurements show that aqueous suspen-sion of ZnO nanoparticles generates increased level of ROS [110]. Theoretically, ROS could be produced as a consequence of ZnO contact with some bacterial electron carriers. Applerot et al., (2009) found that an increased oxidative stress is detected after the antibac-terial treatment, beyond the level that is yielded by the ZnO itself. Moreover, the quantity of ROS detected was different for various bacterial strains. When ZnO slurry was added to E. coli, an increase of 124% in the level of ROS was detected, but when ZnO was added with Staphyloccocus aureus, an immediate decrease of ROS level with 31% was noticed [110]. These findings were explained by authors in the light of a relatively new hypothesis “the bacterial suicide response” [168]. The central idea is that when bacteria ex-perience continuous stress conditions, cells may eventually suffer growth arrest while maintaining their metabolism. An excessive burst of free-radical production then results from this uncoupling of growth from metabolism, and it is this free-radical burst that is lethal to the cells, rather than the stress per se. This mode of trigger-ing bacterial cell death can differ for various kinds of bacteria, ex-

plicating the difference between E. coli and Staphyloccocus aureus. As Staphyloccocus aureus is a more antioxidant protected bacte-rium, any burst of free-radical production can be neutralized more easily, ending up, in the short term, with smaller net number of hydroxyl radicals [110].

Also at it was demonstrated by Yamamoto et al. (2001) that for ZnO powders, the influence of particle size on Staphyloccocus

aureus was less than that on E. coli. The structures and chemical compositions of the cell surface of the bacteria used in the study are quite different. Thin layers of lipid A, lipopolysaccharide and pep-tidoglycan are present on the cell surface of E. coli, whereas there is only a peptidoglycan layer for Staphyloccocus aureus. However, the differences in antibacterial action towards Staphyloccocus

aureus and E. coli are assumed to be due to the different sensitivi-ties towards H2O2 [146].

According to Tam et al. (2008), ZnO nanoparticles were con-siderably more effective against Bacillus atrophaeus than against E.

coli (at 5 mM vs. 15 mM concentration, respectively, showing zero viable cell count). For both bacterial strains, damage of the cell membranes was found, and the effect was more pronounced for Bacillus atrophaeus. At the same time, significant differences were observed in the effect of H2O2 and ZnO nanostructures on Bacillus

atrophaeus. TEM investigation of the effects of H2O2 (30 mg/ mL) on the E. coli and Bacillus atrophaeus reveals that no damage of Bacillus atrophaeus cells is observed at concentrations causing damage to E. coli. This is different from the behavior observed with all ZnO morphologies, indicating existence of a different mecha-nism of antibacterial activity. While the release of H2O2 from ZnO may contribute to the cell damage of E. coli, cell damage of Bacil-

lus atrophaeus is caused by a different mechanism since no effect of H2O2 is observed in this case [108]. They also reported that an-nealing the ZnO nanorods array used for antibacterial test, changed the luminescence spectra, altering the surface defects density re-sponsible for green emission, but with no effect on the antibacterial activity. This suggested that there are other mechanisms, beside those mediated by H2O2, contributing to the antibacterial activity of ZnO nanoparticles [108].

Another possible mechanism for ZnO antibacterial activity is the release of Zn2+ ions [169]. The study of Doménech and Prieto

(1986), [170], indicates that the Zn2+ ion concentration is increased when H2O2 is produced. The dissolution of ZnO nanoparticles into Zn ions was found to be size dependent. Thus engineered ZnO nanostructures may change their toxicity by influencing their disso-lution rate [167, 171-177].

Applerot et al., (2009), [110] also suggested that ROS, at a very low concentration, are able to modulate biological processes in the bacterial cells. It has already been shown that low ROS fluxes (in contrast to high fluxes which have detrimental effects on cells) may act as messengers which activate cell processes such as transcrip-tion factor release, gene expression, and cell growth [178].

Green and Howman (2005), [179], indicated the probable pro-duction of free radicals under dark conditions as a mechanism for antibacterial activity. Tayel A. et al. (2006), [104], confirmed that the observed antibacterial activity reported by them was in the ab-sence of light.

On the other hand Applerot et al., (2009), [110], compared the generation of hydroxyl radicals in ZnO suspensions in the dark and before and after illumination with visible light. The results revealed that with dark prepared suspensions the antibacterial activity was to some extent reduced, but was still present. In contrast, upon brief illumination of the suspensions with visible light emitted from a

8 Current Organic Chemistry, 2014, Vol. 18, No. 2 Oprea et al.

lamp, the ROS level increased up to nearly 400% compared to sus-pensions without illumination. Such results may be advantageous for developing medical and water purification devices based on ZnO nanoparticles.

The mechanism of toxicity is still only partially understood [180]. However, the toxicity of ZnO nanoparticles seems not to be directly related to their entering into the cell, but rather to their intimate contact with the cell, contact that causes changes in the microenvironment in the vicinity of the organism-particle contact area to either increase metal solubilization or to generate ROS, that may ultimately damage cell membrane [172].

Moreover, the toxicity of ZnO nanoparticles is not only affected by the light via ROS production, but may also be affected in the dark although its mechanism is not yet defined [181].

There are also many studies that suggest that the mechanism of ZnO toxicity is related to its photosensitivity (or can be enhanced by it) and production of ROS under specific wavelength light. These ROS can impair the cell membrane architecture through lipid peroxidation. By increasing the irradiation time, the quantity of ROS formed on ZnO surface increases, yielding a better antibacte-rial activity. Brayner R. et al. (2006) reported damage and disor-ganization in the cell wall in the bacteria exposed to ZnO nanopar-ticles [93].

The generation of highly ROS such as OH , H2O2 and O22 is

explained as follows. Since ZnO with defects can be activated by both UV and visible light, electron-hole pairs (e-h+) can be created. The holes split H2O molecules (from the suspension of ZnO) into OH- and H+. Dissolved oxygen molecules are transformed to super-oxide radical anions (·O2

-), which in turn react with H+ to generate (HO2

·) radicals, which upon subsequent collision with electrons

produce hydrogen peroxide anions (HO2-). They then react with

hydrogen ions to produce molecules of H2O2. The generated H2O2 can penetrate the cell membrane and kill the bacteria. Since, the hydroxyl radicals and super oxides are negatively charged particles, they cannot penetrate into the cell membrane and must remain in direct contact with the outer surface of the bacteria; however, H2O2 can penetrate into the cell [107, 143].

Liu H.L. et al. (2003) reported that under UV irradiation with 365nm ZnO nanoparticles exhibit better antibacterial activity than TiO2 nanoparticles. Almost all the initial E. coli cells (108 CFU/ml) were inactivated in 40 min in the presence of 2 g/l ZnO. Photocata-lytic inactivation of bacteria was found to follow first order kinetics with the highest rate constants being 4.5x10-1 and 2.2x10-1/min for E. coli and Lactobacillus Helveticus, respectively, treated with 2 g/l ZnO. As Gram-negative bacteria was more sensitive to ZnO than Gram-positive one, authors hypothesized that no significant cell wall destruction by photocatalytic oxidation could be observed [107], but most probably cells may adapt different photocatalytic inactivation mechanism [110, 146].

The use of air as the purging gas provides a stronger oxidizing environment and gives a higher catalytic effect on bacteria inactiva-tion than that of nitrogen. Such methods are suitable not only for food industry but also for water treatment (simultaneous treatment of photolytic sterilization of microbial cells and photocatalytic de-composition of organic compounds) [107, 152, 182, 183].

The literature data suggest a different mechanism for the inhibi-tory effect of ZnO nanoparticles on fungi compared to those re-ported for bacteria. For the antifungal activity of ZnO nanoparticles, the mechanisms proposed are related to the fungus nature.

The fine structure of Botrytis cinerea mycelia was preserved af-ter treatment with ZnO nanoparticles, but the surface of fungal hy-

phae was deformed. The mechanism of cell functions of Botrytis

cinerea was affected by ZnO nanoparticles due to increased produc-tion of nucleic acids and carbohydrates through the stress response in fungal hyphae, that leads to cells death [105]. The increase of carbohydrates may be due to the self-protecting mechanism against the ZnO nanoparticles [184].

ZnO nanoparticles treatment inhibited the conidial development and distorted the conidiophores of Penicillium expansum. Treated cells of Penicillium expansum release proteins, carbohydrates and lipids through the damaged cell membrane, that leads to cells death [105].

Several studies reported that integration of ZnO nanoparticles into bacterial cells may induce continuous release of membrane lipids and proteins, which changes the membrane permeability of bacterial cells [93, 185].

The antifungal and antibacterial activities reported by Emamifar et al. (2010) were recorded for samples kept in the dark at 4°C, which exclude the photocatalytic activity as a mechanism [102].

The literature data suggest that ZnO nanoparticles have a poten-tial application as a bacteriostatic agent, especially in visible light and may have future applications in the development of derivative agents to control the spread and infection in a variety of domains.

In the prevention and control of bacterial spreading and infec-tions, the main target is the cell wall structure. The cell wall of most pathogenic bacteria is composed of surface proteins for adhesion and colonization, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions [186]. These components are charged macromolecules; therefore, specific interactions to disrupt their main function and location may be triggered by introducing specific groups on the surface of the nanoparticles. It has been reported that certain long-chain polycations coated onto surfaces can efficiently kill on con-tact both Gram-positive and Gram-negative bacteria [187, 188]. These studies have indicated that families of unrelated hydrophobic groups are equally efficient at killing bacteria. Therefore, in the future, ZnO nanoparticles containing formulations may be utilized for external uses as antibacterial agents in ointments, lotions, mouthwashes, and surface coatings on various substrates to prevent microorganisms from attaching, colonizing, spreading, and forming biofilms in indwelling medical devices [106].

CONCLUSION

The main conclusions of these studies can be summarized as follows:

ZnO particles are effective for inhibiting both Gram-positive and Gram-negative bacteria. Usually Gram-positive bacteria are more sensible but there are few studies, especially on E. coli and S.

aureus, where Gram-negative bacteria are killed faster than Gram-positive ones. In such cases, authors proposed alternative mecha-nisms to try elucidate this behavior.

The antibacterial activity of ZnO nanoparticles appeared in close vicinity of the surface, or on the surface.

Smaller ZnO particles have a better antibacterial activity. High concentration and large surfaces lead to better antibacterial activity. Shape of the nanoparticles and their crystalline structure do not have any influence on the antibacterial activity. The antibacterial activity depends only on the surface area and concentration.

The mechanisms of the antibacterial activity of ZnO particles are not well understood although two major hypotheses try to eluci-date it:

ZnO Applications and Challenges Current Organic Chemistry, 2014, Vol. 18, No. 2 9

a) That the generation of hydrogen peroxide is the main factor of the antibacterial activity.

b) That the binding of the particles on the bacteria surface due to the electrostatic forces is driving force behind antibacterial ac-tivity.

Antibacterial activity was noticed, with some difference in in-tensity, in the dark and under UV/visible light. The antibacterial activity is in direct correlation with photocatalytic activity of ZnO.

High-temperature treatment of ZnO particles has a significant effect on their antibacterial activity. Treatment at a higher tempera-ture leads to a lower activity. Antibacterial activity has no direct connection with the luminescence spectra (the green emission is usually attributed to surface defects, so in theory a higher defect density should lead to better luminescence and better antibacterial activity). These sustain the idea that ZnO nanoparticles act through more than one simple mechanism against bacteria.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

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