Nano zinc oxide – Understanding the bactericidal mechanism and specific properties

Zinc oxide is an essential component of many enzymes, sunscreens, and ointments for pain and itching relief. Its microcrystals are very effective light absorbers in the UVA and UVB spectral regions due to their wide frequency range. The effects of zinc oxide on biological functions depend on its morphology, particle size, contact time, concentration, pH and biocompatibility. They are more effective against microorganisms such as Bacillus subtilis , Bacillus megaterium , Staphylococcus aureus , Sarcina lutea , Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumonia , Pseudomonas vulgaris , Candida albicans . The mechanism of action is attributed to the activation of Nano zinc oxide by light, which penetrates the bacterial cell wall through diffusion. From SEM and TEM images of bacterial cells, it was confirmed that Nano zinc oxide degrade cell membranes and accumulate in the cytoplasm, where they interact with biomolecules causing cell death. lead to cell death.

Bactericidal mechanism of nano zinc oxide

(Copyright by NanoCMM Technology)

INTRODUCTION

Nanotechnology deals with the production and application of materials up to 100 nm in size. They are widely used in a number of processes including materials science, agriculture, food industry, cosmetics, medical and diagnostic applications. Nanoscale inorganic compounds have shown remarkable antibacterial activity at very low concentrations due to their high surface area to volume ratio and unique chemical and physical features [ 11 ]. In addition, these particles are also more stable at high temperature and pressure [12 ]. Some of them are recognized as non-toxic and even contain mineral elements that are essential to the human body [ 13 ]. It has been reported that the inorganic materials with the highest antibacterial activity are metal nanoparticles and metal oxide nanoparticles such as silver, gold, copper, titanium oxide and zinc oxide [ 14 , 15 ].

Zinc is an essential trace element for the human system, without zinc many enzymes such as carbonic anhydrase, carboxypeptidase and alcohol dehydrogenase become inactive, while two other members, cadmium and mercury belong to the same group. groups of elements with the same electron configuration are toxic. . It is essential for eukaryotes because it regulates many physiological functions [ 16 , 17 ]. Bamboo salt, which contains zinc, is used as an herbal medicine to treat inflammation by modulating caspase-1 activity. Zinc oxide nanoparticles have been shown to reduce the mRNA expression of inflammatory cytokines by inhibiting the activation of NF-kB (nuclear factor kappa B cells) [ 18 ].

Globally, bacterial infections are recognized as a serious health problem. New bacterial mutations, antibiotic resistance, outbreaks of pathogenic strains, etc. is increasing, and therefore the development of more effective antibacterial agents is the need of the times. Zinc oxide is known for its antibacterial properties since time immemorial [ 19 ]. It was used in the regime of the Pharaohs, and historical records show that zinc oxide was used in many ointments to treat wounds and boils even as early as 2000 BC [ 20 ] . It is still used in sunscreen lotions, as a supplement, photoconductive materials, LEDs, transparent transistors, solar cells, memory devices [ 21 , 22 ], cosmetics [ 23 , 24], and catalyst [ 25 ]. Although a significant amount of ZnO is produced annually, very small amounts are used medicinally [ 26 ]. The U.S. Food and Drug Administration has recognized zinc oxide (21 CFR 182.8991) as safe [ 27 ]. It is characterized by photocatalytic and photooxidative properties against biochemicals [ 28 ].

Zinc oxide has been classified under the EU hazard classification of N; R50-53 (toxic to ecology). Zinc compounds are toxic to mammals and plants in trace form [ 29 , 30 ]. The human body contains about 2-3 g of zinc, and the daily requirement is 10–15 mg [ 29 , 31 ]. There are no reports demonstrating carcinogenicity, genotoxicity, and reproductive toxicity in humans [ 29 , 32 ]. However, zinc powder inhaled or ingested can produce a condition called zinc fever followed by chills, fever, cough, etc.

The morphology of zinc oxide nanoparticles depends on the synthesis process. They can be nanorods, nanosheets [ 33 , 34 , 35 ], nanospheres [ 36 ], nanoboxes [ 35 ], hexagons, tripods [ 37 ], tetrapods [ 38 ], nanowires, nanotubes, wires nano [ 39 , 40 , 41 ], nanocages , and nano flowers [ 42 , 43 ]. Zinc oxide nanoparticles were more active against gram-positive bacteria than other NPs of the same element group. Processed foods are more susceptible to contamination with Salmonella , Staphylococcus aureus, and E. coli which pose a major challenge to food quality and safety. Anti-microbial compounds are built into packaged foods to prevent them from spoiling. Antimicrobial packaging containing non-toxic materials that inhibit or retard the growth of microorganisms present in the food or packaging material [ 44 ]. Antibacterial agents for human use must have the following properties.

a) It will not be toxic.
b) It should not react with food or containers.
c) It must have good taste or tasteless.
d) It should not have an unpleasant smell.

Nano zinc oxide is one of the inorganic metal oxides that fulfills all the above requirements, and therefore it can be safely used as a drug, a packaging preservative and an antibacterial agent. 45 , 46 ]. It easily diffuses into food materials, kills bacteria and prevents human diseases. According to the European Union regulations 1935/2004/EC and 450/2009/EC, active packaging is defined as active material in contact with food that has the potential to change the composition of the food or its atmosphere. the air around it [ 47 ]. Therefore, it is often used as a preservative and incorporated in polymeric packaging materials to prevent food materials from being damaged by bacteria [ 48]. Nano zinc oxide have been used as an antibacterial agent against Salmonella typhi and S. aureus in vitro. Among all metal oxide nanoparticles studied to date, zinc oxide nanoparticles have the highest toxicity to microorganisms [ 49]. It has also been shown from SEM and TEM images that zinc oxide nanoparticles first damage the bacterial cell wall, then penetrate, and finally accumulate in the cell membrane. They interfere with the metabolic functions of microorganisms causing their death. All properties of zinc oxide nanoparticles depend on particle size, shape, concentration and contact time with bacterial cells. Furthermore, biodistribution studies of Nano zinc oxide were also examined. For example, Wang et al. [ 50] investigated the effects of long-term exposure to zinc oxide nanoparticles on zinc biodistribution and metabolism in rats for 3 to 35 weeks. Their results showed minimal toxicity to rats when they were exposed to dietary zinc oxide nanoparticles of 50 and 500 mg/kg. At doses higher than 5000 mg/kg, zinc oxide nanoparticles reduced body weight but increased the weight of pancreas, brain and lungs. In addition, it increased serum glutamic-pyruvic transaminase activity and mRNA expression of genes involved in zinc metabolism such as metallothionein. Biodistribution studies indicate an adequate accumulation of zinc in the liver, pancreas, kidneys and bones. The absorption and distribution of zinc oxide nanoparticles/zinc oxide microparticles depends largely on the particle size. Li and associates. [ 51] studied the biodistribution of zinc oxide nanoparticles that were fed orally or intraperitoneally to 6-week-old rats. No obvious adverse effects were detected in the orally treated zinc oxide nanoparticles during the 14-day study. However, intraperitoneal injection of 2.5 g/kg body weight to rats showed an accumulation of zinc in the heart, liver, spleen, lungs, kidneys and testes. An almost ninefold increase in zinc oxide nanoparticles in the liver was observed after 72 h. Zinc oxide nanoparticles have been shown to be more effective in biodistribution of liver, spleen, and kidney than mice that were fed orally. Since zinc oxide nanoparticles are harmless at low concentrations, they stimulate certain enzymes in human and plant bodies and prevent disease. Singh et al. [ 52] has also been reviewed recently on the biosynthesis of zinc oxide nanoparticles, their absorption, translocation and bioconversion in plant systems.

In this review, we have tried to compile all the information regarding zinc oxide nanoparticles as antibacterial agents. The interaction mechanism of zinc oxide nanoparticles against a variety of bacteria has also been discussed in detail.

Antimicrobial activity of Nano zinc oxide

Everyone knows that zinc oxide nanoparticles are antibacterial and inhibit the growth of microorganisms by penetrating the cell membrane. Oxidative stress damages lipids, carbohydrates, proteins and DNA [ 53 ]. Lipid peroxidation is clearly the most important factor leading to cell membrane alterations, ultimately disrupting important cellular functions [ 54 ]. It was supported by the oxidative stress mechanism involving zinc oxide nanoparticles in  Escherichia coli [ 55 ]. However, for bulk zinc oxide suspensions, extrinsic induction of H 2 O 2  has been suggested to characterize anti-microbial properties [ 56]. In addition, the toxicity of nanoparticles, which release toxic ions, was considered. Since zinc oxide is amphoteric in nature, it reacts with both acids and alkalis to produce Zn 2+  ions.

Free Zn2+  ions are immediately bound to biomolecules such as proteins and carbohydrates, and all vital functions of the bacteria cease. The toxicity of zinc oxide, Nano zinc oxide and ZnSO4·7H2O was tested (Table 1 ) against Vibrio fischeri . It was found that ZnSO4·7H2O was six times more toxic than zinc oxide and zinc oxide nanoparticles. The nanoparticles are actually dispersed in the solvent, not being dissolved and therefore, they cannot release Zn 2+  ions. The bioavailability of Zn 2+ions is not always 100% and may always vary with physiological pH, redox potential and its associated anions such as Cl-  or SO4 2− .

Table 1 Toxicity (30 min EC50 , EC20  and NOEC, and MIC) of CuSO4  and ZnSO4 .7H2O metal oxide aqueous suspensions against bacteria Vibrio fischeri [ 59 ]

Comparison of nano zinc oxide and zinc sulfate toxicity

The solubility of zinc oxide (1.6–5.0 mg/L) in aqueous medium is higher than that of Nano zinc oxide (0.3–3.6 mg/L) in the same medium [ 57 ] toxic to algae and crustaceans. Both nano zinc oxide and bulk zinc oxide are less toxic than ZnSO 4 40–80 times for  V. fischeri . The higher antibacterial activity of ZnSO 4  is directly proportional to its solubility releasing Zn 2+  ions, which have higher mobility and greater affinity [ 58 ] for intracellular biomolecules bacteria due to the positive charge on Zn 2+  and the negative charge on biomolecules.

Since zinc oxide and its nanoparticles have limited solubility, they are less toxic to microorganisms than ZnSO4·7H 2 O. However, entry of metal oxide nanoparticles into bacterial cells is not required for toxicity [ 59 ]. The contact between the nanoparticles and the cell wall is sufficient to cause toxicity. If true, then a large amount of metal nanoparticles is required for the bacterial cell to be completely enclosed and shielded from its environment, not giving nutrients a chance to be absorbed to continue the process of life. . Since nanoparticles and metal ions are smaller than bacterial cells, they are more likely to disrupt cell membranes and inhibit their growth.

Several nano-sized metal oxides such as ZnO, CuO, Al2O3 , ​​La2O3 , ​​Fe2O3 , ​​SnO2 , and TiO2  have been shown to be the most toxic to  E. coli [ 49 ]. Nano zinc oxide are used externally to treat mild bacterial infections, but zinc ion is an essential trace element for some viruses and humans, increasing viral enzymatic activity. integrated [ 45 , 60 , 61]. It was also supported by an increase in infectious pancreatic necrosis virus to 69.6% when treated with 10 mg/L Zn [ 46 ]. It may be because the solubility of Zn ions is greater than that of ZnO alone. SEM and TEM images have shown that zinc oxide nanoparticles damage bacterial cell walls [ 55 , 62 ] and increase permeability, followed by their accumulation in  E. coli  preventing their multiplication [ 63 ].

In the recent past, the antibacterial activity of Nano zinc oxide has been studied against 4 known gram-positive and gram-negative bacteria, namely Staphylococcus aureus , E. coli , Salmonella typhimurium and Klebsiella pneumoniae . It was observed that the dose inhibiting the growth of zinc oxide nanoparticles was 15 μg/ml, although in the case of  K. pneumoniae  it was as low as 5 μg/ml [ 63 , 64]. It was found that with increasing concentration of nanoparticles, the inhibition of microbial growth increased. When they were incubated for 4–5 h with a maximum concentration of zinc oxide nanoparticles of 45 μg/ml, growth was strongly inhibited. It is expected that if incubation time is increased, growth inhibition will also increase without much change in the mechanism of action [ 63 ].

It has been reported that metal oxide nanoparticles first damage the bacterial cell membrane and then permeate it [ 64 ]. It has also been suggested that the release of H2O2  could be an alternative to the antimicrobial activity [ 65 ]. However, this proposal requires experimental evidence because the mere presence of zinc oxide nanoparticles is not sufficient to generate H2O2 . Zinc nanoparticles or extremely low concentrations of zinc oxide nanoparticles cannot be toxic to the human body. Daily zinc supplementation through food is required to perform regular metabolic functions. Zinc oxide is known to protect the stomach and intestinal tract from harmful E. coli [65 ]. The pH in the stomach varies from 2 to 5, and so the zinc oxide in the stomach can react with the acid to produce Zn2+  ions. They can help activate the enzymes carboxy peptidase, carbonic anhydrase and alcohol dehydrogenase that help digest carbohydrates and alcohol. Premanathan et al. [ 66 ] reported the toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic cells. The MICs of zinc oxide nanoparticles against  E. coli ,  Pseudomonas aeruginosa  and  S. aureus were 500 and 125 μg/ml, respectively. Two mechanisms of action have been proposed for the toxicity of zinc oxide nanoparticles, namely (1) generation of ROS and (2) induction of apoptosis. Metal oxide nanoparticles induce ROS production and expose cells to oxidative stress causing damage to cellular components, i.e., lipids, proteins and DNA [ 67 , 68 , 69 ]. Thus, zinc oxide nanoparticles induce toxicity through apoptosis. They are relatively more toxic to cancer cells than normal cells, although they cannot distinguish between them.

Recently, Pati et al. [ 70 ] have shown that zinc oxide nanoparticles disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity, and regulate transcription of oxidative stress resistance genes in bacteria. . They enhance the killing of intracellular bacteria by generating ROS. These nanoparticles disrupt biofilm formation and inhibit hemolysis by the hemolysin toxin produced by the pathogen. The use of zinc oxide nanoparticles in the skin was found to significantly reduce skin infection and inflammation in mice and also improve the structure of infected skin.

Solubility and concentration- Activity dependent of Nano zinc oxide

Nanoparticles have also been used as a carrier to deliver therapeutic agents to treat bacterial infections [ 1 , 9 ]. Since zinc oxide nanoparticles with concentrations up to 100 μg/ml are harmless to normal body cells, they can be used as an alternative to antibiotics. It was found that 90% of the colonies died after exposing them to doses of 500–1000 μg/ml of zinc oxide nanoparticles for only 6 hours. Even drug-resistant S. aureus , Mycobacterium smegmatis and Mycobacterium bovisWhen treated with zinc oxide nanoparticles in combination with a low dose of the anti-tuberculosis drug, rifampicin (0.7 μg/ml), their growth was significantly reduced. tell. These pathogens were completely destroyed by incubation for 24 h with 1000 μg/ml zinc oxide nanoparticles. Therefore, it is concluded that if the same dose is repeated, patients suffering from such infectious diseases can be completely cured. It was also noted that the size of the zinc oxide nanoparticles between 50 and 500 nm had an identical effect on the inhibition of bacterial growth.

The cytotoxicity of zinc oxide has been studied by many researchers in a variety of microorganisms and plant systems [ 71 , 72 , 73 , 74 ]. The toxicity of zinc oxide nanoparticles is concentration and solubility dependent. It has been shown that the maximum exposure concentration of zinc oxide suspension (125 mg/l) releases 6.8 mg/l Zn 2+  ions. Toxicity is the synergistic effect of zinc oxide nanoparticles and Zn 2+ ions released in the aqueous medium. However, minimal effect of metal ions was detected, which suggests that the inhibition of bacterial growth is mainly due to the interaction of zinc oxide nanoparticles with microorganisms. The cytotoxic effect of a specific metal oxide nanoparticle that is species-sensitive is reflected by the growth inhibition zone for some bacteria [ 75 ].

It has been suggested that the inhibition of bacterial cell growth occurs mainly by Zn 2+  ions generated by extracellular lysis of zinc oxide [ 76 ] nanoparticles. Cho and associates. [ 77 ] concluded from their studies in mice that zinc oxide nanoparticles remained intact at around neutral or biological pH but rapidly dissolved under acidic conditions (pH 4.5) in the bacterial lysosomes. organisms leading to their death. This is true because under acidic conditions, zinc oxide dissolves and produces Zn 2+  ions, which bind to biomolecules inside bacterial cells, inhibiting their growth.

Nano zinc oxide have been shown to be cytotoxic against various primary immune competent cells. Transcriptional analysis revealed that the nanoparticles had a common genomic feature with an upregulation of metallothionein genes corresponding to the dissolution of the nanoparticles [ 78 ]. However, it cannot be ascertained whether the absorbed zinc is Zn 2+  or zinc oxide or both, although the smaller zinc oxide nanoparticles have greater blood concentrations than the larger ones (19). and > 100 nm). The effectiveness of zinc oxide nanoparticles mainly depends on the reaction environment to form Zn 2+  and their penetration into the cell.

Chiang et al. [ 79 ] reported that dissociation of zinc oxide nanoparticles leads to destruction of cellular Zn homeostasis. The specific properties of nanoparticles and their impact on biological functions are completely different from those of bulk materials [ 80 ]. The aggregation of nanoparticles affects the cytotoxicity of macrophages, and their concentration helps to regulate the aggregation of nanoparticles. Zinc oxide nanoparticles were ineffective at low concentrations, but at higher concentrations (100 μg/ml) they exhibited cytotoxicity that varied from one pathogen to another.

Inadvertent use of zinc oxide nanoparticles can sometimes adversely affect living systems. Apoptotic processes and their genotoxicity in human hepatocytes and cytotoxicity were investigated. It was found that a decrease in the viability of hepatocytes occurred when they were exposed to 14–20 μg/ml of zinc oxide nanoparticles for 12 h. It also causes DNA damage due to oxidative stress. Sawai et al. [ 56 ] demonstrated that ROS generation was proportional to the concentration of zinc oxide powder. ROS induces a decrease in mitochondrial membrane potential leading to apoptosis [ 81 ]. Cell uptake of nanoparticles is not required for cytotoxicity to occur.

Size-dependent antibacterial activity of Nano zinc oxide

In one study, Azam et al. [ 82 ] reported that antibacterial activity against both gram-negative ( E. coli  and  P. aeruginosa ) and gram-positive ( S.  and  Bacillus subtilis ) bacteria increased with increasing surface-to-volume ratio due to decrease particle size of zinc oxide nanoparticles. Furthermore, in this study, zinc oxide nanoparticles were shown to inhibit the maximum bacterial growth (25 mm) against  B. subtilis  (Figure  1 ).

Figure 1

The antibacterial activity and/or zone of inhibition produced by zinc oxide nanoparticles against gram-positive and gram-negative bacterial strains, namely Escherichia coli , b Staphylococcus aureus , c Pseudomonas aeruginosa , and d Bacillus subtilis [ 82 ]

nano zinc oxide kills 4 strains of bacteria

It has been reported that the smaller size of Nano zinc oxide exhibits greater antibacterial activity than the microsized particles [ 83 ]. For example, Au 55  nanoparticles with a size of 1.4 nm have been shown to interact with the major grooves of DNA, causing its toxicity [ 84 ]. Although conflicting results have been reported, many workers show a positive effect of zinc oxide nanoparticles on bacterial cells. However, Brayner et al. [ 63] from TEM images showed that 10-14 nm zinc oxide nanoparticle was penetrated inside (when exposed to bacteria) and damaged the bacterial cell membrane. It is also important that the zinc/zinc oxide nanoparticles are not toxic to humans as they are toxic to T cells above 5 mM [ 85 ] and to neuroblastoma cells above 1.2 mM [ eighty six ]. Nair et al. [ 87 ] exclusively explored the effect of the size of zinc oxide nanoparticles on bacterial and human cell toxicity. They investigated the effects of zinc oxide nanoparticles on both gram-positive and gram-negative bacteria and osteoblastic cancer cell lines (MG-63).

It is well known that the antibacterial activity of zinc oxide nanoparticles is inversely proportional to their size and directly proportional to their concentration [ 88 ]. It has also been noticed that it does not need UV light to activate; it works under normal or even diffused sunlight. The cytotoxic activity presumably includes the production of ROS and the accumulation of nanoparticles in the cytoplasm or on the outer cell membrane. However, the production of H 2 O 2 and its involvement in the activation of nanoparticles cannot be ignored. Raghupathi et al. [ 88 ] synthesized zinc oxide nanoparticles from different zinc salts and observed that the nanoparticles obtained from Zn(NO 3 ) 2 had the smallest size (12 nm) and the largest in terms of surface area. (90.4). The authors showed that the inhibition of growth of  S. aureus  at 6 mM concentration of zinc oxide nanoparticles was size dependent. It has also been shown from the determination of surviving cells during bacterial cell exposure to zinc oxide nanoparticles that the number of recovered cells is significantly reduced when the size of the zinc nanoparticles is reduced. oxide. Jones and associates. [ 89 ] showed that zinc oxide nanoparticles with a diameter of 8 nm inhibited the growth of  S. aureus ,  E. coli  and  B. subtilis. Zinc oxide nanoparticles ranging in size from 12 to 307 nm were were selected and confirmed the relationship between antibacterial activity and their size. Their toxicity to microorganisms is attributed to the formation of Zn 2+  ions from zinc oxide when it is suspended in water and to some extent small changes in pH. Since Zn 2+  ions are rarely released from zinc oxide nanoparticles, the antibacterial activity is mainly due to the smaller zinc oxide nanoparticles. When the size is 12 nm, it inhibits the growth of  S. aureus , but when the size exceeds 100 nm, the inhibitory effect is minimal [ 89 ].

Shape, composition and cytotoxicity of Nano zinc oxide

Nano zinc oxide have shown concentration- and cell-type dependent cytotoxicity due to different sensitivities [ 90 , 91 ]. Sahu et al. [ 90 ] highlighted differences in cytotoxicity between particle sizes and different cellular sensitivities to particles of the same composition. In another recent study, Ng et al. [ 91] examined concentration-dependent cytotoxicity in human lung MRC5 cells. The authors reported the uptake and intrinsic of zinc oxide nanoparticles into MRC5 cells in human lung using TEM investigation. These particles were detected in the cytoplasm of the cell as electron-dense clusters, which were further observed to be enclosed by vesicles, whereas zinc oxide nanoparticles were not found in the cells. untreated control cells. Papavlassopoulos et al. [ 92] synthesized zinc oxide nanoparticle tetrapods entirely by a novel pathway known as the “Flame Transport Synthetic Method”. Tetrapods are morphologically different from conventionally synthesized Nano zinc oxide. Their interactions with mammalian fibroblast cells in vitro have shown that their toxicity is significantly lower than that of spherical zinc oxide nanoparticles. The tetrapod exhibits a hexagonal wurtzite crystal structure with alternating Zn 2+  and O 2−  ions having a three-dimensional geometry. They prevent virus entry into living cells and are further enhanced by precisely illuminating them with UV radiation. Because zinc oxide tetrapods have oxygen vacancies in their structure,  Herpes simplexvirus is attached via heparan sulfate and is denied entry into body cells. Thus, they prevent HSV-1 and HSV-2 infection in vitro. Therefore, zinc oxide tetrapods can be used as a prophylactic agent against these viral infections. The cytotoxicity of Nano zinc oxide also depends on the proliferation rate of mammalian cells [ 66 , 93 ]. Surface reactivity and toxicity can also be altered by controlling the oxygen gap in zinc oxide tetrapods. When they are exposed to UV light, the amount of free oxygen in tetrapods easily increases. Alternatively, empty oxygen can be reduced by heating them in an oxygen-rich environment. Thus, the unique properties of zinc oxide tetrapods can be altered at will, thereby altering their antimicrobial efficacy.

Animal studies have shown an increase in lung inflammation, oxidative stress, etc. with inhalation of nanoparticles [ 94 ]. Yang et al. [ 95 ] investigated the cytotoxicity, genotoxicity and oxidative stress of zinc oxide nanoparticles on primary mouse embryonic fibroblast cells. It was observed that Nano zinc oxide induced significantly greater cytotoxicity than that caused by carbon and SiO 2  nanoparticles. It was further confirmed by measuring glutathione depletion, malondialdehyde production, superoxide dismutase inhibition, and ROS generation. The potential cytotoxic effects of various nanoparticles are attributed to their shape.

Polymer coated nanoparticles

Many bacterial infections are transmitted by contact with doorknobs, keyboards, faucets, bathtubs, and phones; therefore, it is essential to grow and coat such surfaces with inexpensive advanced antimicrobial agents to prevent their growth. It is important to use such concentrations of antibacterial agents so that they can kill pathogens but save lives. This is only possible if they are coated with a low cost, biocompatible hydrophilic polymer. Schwartz et al. [ 96 ] reported the preparation of a novel antibacterial composite material hydrogel by mixing poly( N-isopropylacrylamide) with zinc oxide nanoparticles. The SEM image of the composite film shows a uniform distribution of zinc oxide nanoparticles. It exhibits antibacterial activity against  E. coli  at very low concentrations of zinc oxide (1.33 mM). In addition, the coating was found to be non-toxic to the mammalian cell line (N1H/3T3) over a period of 1 week. Zinc oxide/hydrogel nanocomposites can be safely used as biomedical coatings to prevent people from getting bacterial infections.

Although the zinc oxide nanoparticles were very stable, they were further stabilized by coating them with various polymers such as polyvinyl pyrolidone (PVP), polyvinyl alcohol ( PVA ), poly (α, γ, L -glutamic acid) ) (PGA), polyethylene glycol (PEG), chitosan and dextran [ 97 , 98 ]. The antibacterial activity of engineered zinc oxide nanoparticles was examined against gram-negative and gram-positive pathogens, namely E. coli and S. aureus and compared with commercial zinc oxide powder. Polymer-coated spherical zinc oxide nanoparticles showed maximum bacterial cell destruction compared with bulk zinc oxide powder [ 99]. Since polymer-coated nanoparticles are less toxic due to their low solubility and sustained release, their cytotoxicity can be controlled by coating them with an appropriate polymer.

Effect of particle size and shape of polymer coated nanoparticles on antibacterial activity

Ecoli and  S. aureus exposed to different concentrations of poly ethylene glycol (PEG) coated zinc oxide nanoparticles (1–7 mM) of different sizes (401 nm – 1.2 μm) showed that the activity The antibacterial activity increased with decreasing size and increasing concentration of nanoparticles. However, the effective concentration in all of these cases was above 5 mM. There was a strong change in the cellular morphology of the  E. coli  surface that could be seen from the SEM images of the bacteria before and after they were exposed to the Nano zinc oxide [ 84 ]. It has been demonstrated nicely by Nair et al. [ 87] that PEG-coated zinc oxide particles and zinc oxide nanorods were toxic to human osteoblastic cancer (MG-63) cells at concentrations above 100 μM. The PEG starch coated nanorods/nanoparticles did not injure healthy cells.

In Vivo and In Vitro antibacterial activity for wound dressing

Among all natural and synthetic wound dressing materials, the chitosan hydrogel microfiber dressing impregnated with zinc oxide nanoparticles was developed by Kumar et al. [ 100 ] is highly effective in treating burns, wounds and diabetic foot ulcers. Nanoparticles about 70–120 nm in size are dispersed on the surface of the ice. The degradation products of chitosan were identified as  D -glucosamine and glycosamine glycan. They are not toxic to cells because they are already present in our body to heal wounds. Wounds containing  P. aeruginosa ,  S. intermedicus , and  S. hyicus were also identified from rat antlers and were successfully treated with chitosan zinc oxide dressings for about 3 weeks [ 100 ].

Effect of doping on the toxicity of Nano zinc oxide

Doping of zinc oxide nanoparticles with iron reduces toxicity. The concentration of  zinc oxide and Zn 2+ nanoparticles is also an important factor for toxicity. Concentrations that reduced viability by 50% in microbial cells exposed to zinc oxide nano and microsize were very close to that of Zn 2+  that reduced viability by 50% in cells treated with zinc oxide. Zn 2+ [ 101 , 102 ].

Coating zinc oxide nanoparticles with mercaptopropyl trimethoxysilane or SiO 2  reduced their cytotoxicity [ 103 ]. In contrast, Gilbert et al. [ 104 ] showed that in BEAS-2B cells, absorption of zinc oxide nanoparticles was the main mechanism of zinc accumulation. In addition, they suggested that fully soluble zinc oxide nanoparticles generate Zn 2+  ions that are bound to target cell biomolecules. However, the toxicity of zinc oxide nanoparticles depends on their uptake and subsequent interaction with target cells.

INTERACTION MECHANISM OF INTERACTIVE NANOPOLS

Nanoparticles may be toxic to some microorganisms, but they may be essential nutrients for some [ 55 , 105 ]. Nanotoxicity is basically related to damage to microbial cell membranes leading to penetration of nanoparticles into the cytoplasm and their accumulation [ 55 ]. The impact of nanoparticles on the growth of bacteria and viruses largely depends on the particle size, shape, concentration, agglomeration, colloidal formula and pH of the medium [ 106 , 107 , 108 ] . The mechanism of antibacterial action of zinc oxide nanoparticles has been described in Fig.

Figure 2

Mechanism of antibacterial action of zinc oxide nanoparticles

Antibacterial mechanism of nano zinc oxide

Zinc oxide nanoparticles are generally less toxic than silver nanoparticles at a wide concentration range (20 to 100 mg/l) with an average particle size of 480 nm [ 55 , 62 , 63 ]. Metal oxide nanoparticles damage cell membranes and bacterial DNA [ 63 , 109 , 110 , 111 ] through diffusion. However, the production of ROS via photocatalysis induced bacterial cell death [ 112 ]. UV-Vis spectra of zinc oxide nanoparticle suspension in aqueous medium exhibit peaks between 370 and 385 nm [ 113]. It has been shown that it generates ROS (hydroxyl radicals, superoxide and hydrogen peroxide) in the presence of moisture, which obviously reacts with bacterial cell materials such as proteins, lipids and DNA, eventually inducing apoptosis. Xie et al. [ 114 ] examined the effect of zinc oxide nanoparticles on the Campylobacter jejuni  cell morphology using SEM images (Figure  3 ). After 12 h of treatment (0.5 mg/ml),  C. jejuni  was found to be extremely sensitive and the cells changed from helical to coccoid. SEM studies revealed the growth of coccoid forms in the treated cells and displayed the formation of irregular cell surfaces and cell wall plaques (Figure  3a). Furthermore, these coccoid cells remain intact and possess sheathed polar flagella. However, SEM images of untreated cells clearly showed helical shapes (Figure  3b ). Overall, it has been shown from SEM and TEM images of bacterial cells treated with zinc oxide nanoparticles that they break apart and, in many cases, the nanoparticles damage the cell wall forcing them to must infiltrate it [ 114 , 115 ].

Figure 3

SEM image of  Campylobacter jejuni . a Untreated cells from the same growth conditions were used as controls. C. jejuni  cells in the middle stage of growth were treated with 0.5 mg/ml zinc oxide nanoparticles for 12 h under microaerobic conditions [ 114 ]

0.5 ppm nano zinc oxide after 12 h exposure to campylobacter jejuni

Zinc oxide nanoparticles have strong effects on the cell surface and can be activated upon exposure to UV-Vis light to induce ROS (H2O2 ) that permeate the cell body while the ROS species are negatively charged such as O 2 2−  remain active on the cell surface and affect their integrity [ 116 , 117 ]. Antimicrobial activity of zinc oxide nanoparticles against many other bacteria has also been reported [ 1 , 5 , 114 , 115 ]. From the TEM images it was shown that the nanoparticles have a strong effect on the cell surface (Figure  4 ).

Figure 4

TEM image of untreated normal salmonella typhimurium cells. b Effect of nanoparticles on cells (marked with arrows). c , d Microscopic images of attenuated and ruptured S. typhimurium  cells treated with zinc oxide nanoparticles [ 115 ]

TEM image of bacterial cells when exposed to nano zinc oxide

Sinha and associates. [ 118 ] also showed the effect of zinc oxide and silver nanoparticles on growth, membrane structure and their accumulation in the cytoplasm of (a) mesophiles: Enterobacter sp. (gram negative) and  B. subtilis(gram positive) and (b) halophiles: color-loving bacteria sp. (gram positive) and Marinobacter sp. (negative gram). Nanotoxicity of zinc oxide nanoparticles against aerobic gram-negative Marinobacter species and gram-positive aerobic bacterial species showed an 80% growth inhibition. It has been shown that zinc oxide nanoparticles with concentrations below 5 mM are not effective against bacteria. Bulk zinc oxide also did not affect growth rate and survival numbers, although they showed a significant reduction in these parameters. Enterobacter species showed significant changes in cell morphology and size reduction when treated with zinc oxide.

TEM images shown by Akbar and Anal [ 115 ] showing disrupted cell membranes and accumulation of zinc oxide nanoparticles in the cytoplasm (Figure   4 ) were further confirmed by FTIR, XRD and SEM . It is thought that Zn 2+  ions are attached to biomolecules in bacterial cells through electrostatic force. They are actually coordinated with protein molecules via the single electron pair on the nitrogen atom of the protein fraction. Despite the significant effects of zinc oxide nanoparticles on both aquatic and terrestrial microbiota as well as human systems, it has not been possible to determine whether it is due to single nanoparticles or to an effect. combined action of zinc oxide nanoparticles and Zn 2+ [ 55, 106 , 109 , 119 ] ions. The antimicrobial effects of metal oxide nanoparticles include its diffusion into bacterial cells, followed by release of metal ions and DNA damage leading to cell death [ 63 , 109 , 110 , 111 ]. The generation of ROS via photocatalysis is also a reason for the antimicrobial activity [ 62 , 112 ]. Wahab et al. [ 120] have shown that when zinc oxide nanoparticles are ingested, their surface area increases, which in turn increases their ability to absorb and interact with both pathogens and enzymes. Therefore, zinc oxide nanoparticles can be used in preventing biological systems from infection. It is clear from the TEM images (Figures  5a,b ) of  E. coli incubated for 18 h with the MIC of zinc oxide nanoparticles that they adhered to the bacterial cell wall. The rupture of the outer cell membrane leads to cell lysis. In some cases, microbial cell cleavage was unnoticed, but zinc oxide nanoparticles could still be seen penetrating the inner cell wall (Fig. Its consequence is that intracellular material leaks out leading to cell death, regardless of the thickness of the bacterial cell wall.

Figure 5

TEM images of  Escherichia coli ( a ), Nano zinc oxide with  E. coli  at different stages ( b  and inset), Klebsiella pneumoniae ( c ), and zinc oxide nanoparticles with  K. pneumoniae ( d  and inset) [ 120 ]

Nano zinc oxide and ecoli . exposure stages

lls has been shown below [ 120 ]. Zinc oxide absorbs UV-Vis light from the sun and separates the components of water.

The dissolved oxygen molecules are converted to a superoxide, O2 – , which in turn reacts with H + to give the radical HO2 and, after colliding with electrons, produces the hydrogen peroxide anion, HO2 – . They then react with H + ions to produce H2O2 .

It is thought that negatively charged hydroxyl radicals and superoxide ions cannot penetrate cell membranes. Free radicals are so reactive that they cannot stay in the free and so they can form a molecule or react with an ion of opposite sign to produce another molecule. However, it is true that zinc oxide can absorb sunlight and help break down water molecules that can combine in many ways to produce oxygen. The mechanism of oxygen production in the presence of zinc oxide nanoparticles still needs experimental evidence.

Zinc oxide at a dose of 5 μg/ml has been shown to be highly effective against all microorganisms which can be used as the minimal inhibitory dose.

CONCLUSION

Zinc is an indispensable inorganic element widely used in medicine, biology and industry. Its daily intake in an adult is 8–15 mg/day, of which about 5-6 mg/day is lost through urine and sweat. In addition, it is an essential component of bones, teeth, enzymes and many functional proteins. Metallic zinc is an essential trace element for human, animal, plant and bacterial growth while zinc oxide nanoparticles are toxic to many fungi, viruses and bacteria. People with an inherent genetic deficiency of the soluble zinc-binding protein have acrodermatitis enteropathica, an inherited disease characterized by rough and scaly skin. Although there have been many conflicting reports about nanoparticles due to their unintentional use and disposal, some metal oxide nanoparticles are useful to humans, animals and plants. Essential nutrients become harmful when they are consumed in excess. The mutagenic potential of zinc oxide has not been well studied in bacteria, although DNA damage has been reported. It is true that zinc oxide nanoparticles are activated by absorbing UV light without affecting other rays. If the zinc oxide nanoparticles produce ROS, they can damage the skin and cannot be used as a sun screen. The antibacterial activity could be catalyzed by sunlight, but hopefully, it could prevent ROS formation. Nano zinc oxide and zinc nanoparticles coated with soluble polymers can be used to treat wounds, ulcers, and many microbial infections in addition to being used as agents drug delivery in cancer treatment. It has great potential as a safe antibacterial drug that could replace antibiotics in the future. The application of zinc oxide nanoparticles in various fields of science, medicine and technology shows that it is an indispensable and equally important substance for humans and animals. However, prolonged exposure to higher concentrations can be harmful to living systems.

 

Reference source:

Properties of Zinc Oxide Nanoparticles and Their Activity Against Microbes

Khwaja Salahuddin Siddiqi 1, Aziz Ur Rahman 2, Tajuddin 2, Azamal Husen 3
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