Antimicrobial Activity of Zinc Oxide Nano/Microparticles and Their Combinations against Pathogenic Microorganisms for Biomedical Applications: From Physicochemical Characteristics to Pharmacological Aspects

Zinc oxide (ZnO) nano/microparticles (NPs/MPs) have been studied as antibiotics to enhance antimicrobial activity against pathogenic bacteria and viruses with or without antibiotic resistance. They have unique physicochemical characteristics that can affect biological and toxicological responses in microorganisms. Metal ion release, particle adsorption, and reactive oxygen species generation are the main mechanisms underlying their antimicrobial action. In this review, we describe the physicochemical characteristics of ZnO NPs/MPs related to biological and toxicological effects and discuss the recent findings of the antimicrobial activity of ZnO NPs/MPs and their combinations with other materials against pathogenic microorganisms. Current biomedical applications of ZnO NPs/MPs and combinations with other materials are also presented. This review will provide the better understanding of ZnO NPs/MPs as antibiotic alternatives and aid in further development of antibiotic agents for industrial and clinical applications.


Introduction
Zinc oxide (ZnO) nanoparticles (NPs) have been studied for the development of next-generation nanoantibiotics against pathogenic microorganisms to combat multi-drug resistance [1,2]. These nanoparticles show unique physicochemical properties including morphology, particle size, crystallinity, and porosity [3]. Based on these characteristics, ZnO NPs have a wide spectrum of antimicrobial activity against microorganisms including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and the M13 bacteriophage [4][5][6][7]. They can be combined with antibiotic and anti-inflammatory drugs to enhance antimicrobial activity against pathogenic microorganisms without antibiotic resistance in non-clinical and clinical conditions [7,8]. Use of ZnO NPs as well as microparticles (MPs) has been extended to biomedical applications including antibiotic drugs or medical devices, theranostics, implants, and cosmetics for conventional uses in clinics [9][10][11][12].
The physicochemical characterization of ZnO NPs/MPs offers advantageous information regarding biological or biochemical responses to pathogenic microorganisms, enabling the prediction of antimicrobial and toxicological effects [13,14]. Generally, their properties, including morphology, particle size or particle size distribution, porosity, and specific surface area, can affect antimicrobial responses [14,15]. ZnO NPs/MPs of various shapes such as spheres, rods, needles, and platelets, and their average aspect ratios (defined as ratio of length to width) can influence the antimicrobial actions against microorganisms. Their particle size and particle size distribution levels are the key parameters that determine NP uptake into biomembranes and thereby influence antimicrobial activity against pathogenic microorganisms [16,17]. Intra-and interparticle pores of ZnO NPs/MPs also enhance nitrate solution or zinc chloride solution as precursors [40]. This technique is advantageous to achieve simple and cost-effective outcomes in large-surfaced substrate, which produces various structures of nanoneedle-like, prism-like, porous, and continuous shapes with appropriate thickness [39]. On silicon, ZnO deposition occurred from nonaqueous solution of dimethyl sulfoxide (DMSO) containing zinc chloride and potassium chloride, which films of 1-3 µm thick had pore channels (20 nm). Next, sacrificial anode electrolysis from two-electrode cells to thee-electrode cells along with potentiostatic control improves reproducibility of morphological dimension in ZnO NPs/MPs [41]. In the electrode-cell, metal ions are produced in anodic dissolution or added into electrochemical medium and cathodic reduction induces particle growth with tunable morphology. Near-spherical ZnO NPs (<25 nm) in an amorphous phase were produced using sacrificial anodic dissolution of pure zinc metal strip (2 × 6 × 1 cm 3 ) controlling against platinum mesh as the cathode in electrolyte of 0.1 M tetrabutlyammonium bromide solution and acetonitrile (4:1) [46]. In EDOC, sacrificial anodic oxidation, cathodic reduction, and oxidation processes were involved for the synthesis of spherical ZnO NPs (9 nm) [42]. Hybridizing electrochemical and thermal approaches, ZnO NPs/MPs were synthesized in aqueous sodium bicarbonate solution, generating zinc hydroxide species and further oxidized to ZnO in calcination process at >300 • C [44,45]. Specifically, anionic or cationic stabilizers can be used to tune physicochemical properties of ZnO NPs/MPs including morphology and particle size.

Physicochemical Characteristics of ZnO Materials
We introduce the physicochemical characteristics of ZnO NPs/MPs which antimicrobial functions have already been reported. Table 1 displays morphology, particle size, porosity, surface area, and synthesis technique of ZnO NPs/MPs with antimicrobial functions against pathogenic microorganisms. In general, high aspect ratio, small particle size, multilevel porosity, and large surface area of ZnO NPs/MPs can enhance antimicrobial performance, irrespective of synthesis techniques [3,5,14]. Chemically or biologically synthesized ZnO NPs/MPs have been extensively used to determine their antimicrobial activity against microorganisms, rather than microfluidically or electrochemically synthesized ZnO NPs/MPs, due to simplicity, reproducibility, and production scale, yield or rate for obtaining high-value ZnO NPs/MPs with required characteristics [5,6,8].
First, in the 0D structure of ZnO NPs/MPs, Raghupathi et al. [16] reported the manufacture of spherical ZnO NPs (12-307 nm) with slit-like pores (3.49-90.4 m 2 /g surface area) using solvothermal and room temperature syntheses. Using the solvothermal technique for ZnO NP synthesis, smaller ZnO NPs (12-25 nm) with larger surface area (42.8-90.4 m 2 /g) were obtained, compared to ZnO NP synthesis at room temperature (30-307 nm; 3.49-35.6 m 2 /g surface area). Green synthesized spherical ZnO NPs were reported, using Catharanthus roseus leaf extract and zinc acetate dehydrate [47]. In spherical ZnO NP synthesis, controllable reaction factors were optimized for pH 12, 30 • C, 0.01 M precursor metal ion, and 2 h of reaction time to achieve high-value ZnO NPs for enhanced antimicrobial performance. Using green synthesis and co-precipitation, sphere-shaped ZnO NPs (15-20 nm) were obtained reacting with Bambusa Vulgaris plant extract and zinc nitrate aqueous solution (0.5 mol/50 mL) [48]. Compared to Bambusa Vularis plant extract, Artabotrys Hexapetalu plant extract was used to synthesize mixed spherical and rod-like ZnO NPs (20-30 nm). In green synthesis using Sambucus ebulus leaf extract and 1 M zinc acetate dihydrate, spherical ZnO NPs were also prepared at 25-30 nm for transmission electron microscopy (TEM) and 65 ± 4 nm for dynamic light scattering [49]. Moreover, a self-assembled 3D network structure of spherical ZnO NPs (48.3 nm) on a solid plate was also analyzed, in which NPs were synthesized via sol-gel technique using an annealing process at 150 • C [18]. In the self-assembled ZnO NP network, mesopores at 5-6.25 nm and macropores at 2-6 µm were detected, conferring bimodal porosity for enhanced performance. Meanwhile, spherical nanostructured ZnO (63 nm) manufactured by soft chemical synthesis using micrometric ZnO and urea in glycerol, generated NP clusters (590 nm) with mesopores of 34 nm and a 20.72 m 2 /g surface area [19].
In the 1D structure of ZnO NPs/MPs, Bala et al. [50] reported dumbbell-shaped ZnO NPs (190-250 nm in length and 50-60 nm in breadth), which were prepared via green synthesis by reacting with Hibiscus subdariffa leaf extract and zinc acetate in water. The morphology of ZnO NPs was modified depending on drying temperature. After particle precipitation, dumbbell-shaped ZnO NPs were generated with further heating at 100 • C for 4 h. In contrast to the dumbbell-shaped ZnO NPs, sphere-shaped ZnO NPs (12-46 nm) resulted from drying at 60 • C. The submicron ZnO dumbbell structure was generated using a chemical bath deposition technique at low temperature (80 • C) [51]. The resulting ZnO dumbbells were 1-2 µm long, 200-300 nm wide at one end, and 250-400 nm wide at the other end.  [63] ZnO nanorods (523 nm in length and 47 nm in diameter) were reported as photocatalytic antibacterial agents by Singh et al. [52]. They were synthesized using the sol-gel technique by reacting with 1 M zinc nitrate and 1 M hexamethyl tetraamine (HMTA) at pH 6.5. Reactions carried out at pH 5.0-6.5 in mediated smaller ZnO nanorods. Using the green synthesis technique of reacting with egg white albumen and zinc acetate dihydrate in water (1 mmol/mL), the resulting ZnO nanorods had a highly crystalline structure at~2 µm in length and~50 nm in diameter for enhanced antimicrobial performance [53]. Calcined temperature was determined as a controllable parameter for high-value ZnO nanorod synthesis, ranged at 400-650 • C. ZnO nanorods calcined at the highest temperature (650 • C) displayed pure and highly crystalline products.
Hollow ZnO nanotubes in nanopowders, ranging at~500 nm were manufactured via hydrothermal synthesis using polyvinyl alcohol (PVA) with a further annealing process at 500-900 • C [54]. After annealing, the surface area and pore size of the ZnO nanopowders were reduced from 17.8 m 2 /g and 278.6 nm to 2.4-16.7 m 2 /g and 53-255.4 nm, respectively, compared to as-synthesized nanopowders, owing to sintering and growing boundaries to form microscale aggregates. Lopez de Dicastillo et al. [55] described hollow ZnO nanotubes 5 µm in length and 59.5 nm in thickness with an internal diameter of 178.2 nm, prepared via template methods using electrospun PVA nanofibers through atomic layer deposition of ZnO NPs from water and diethyl zinc. Thermal treatment or hydrolysis was additionally performed to remove the PVA template from the hollow ZnO nanotubes. Besides the nanotubes, ZnO needles were also reported as antimicrobial agents for urinary tract pathogens [56]. These needle-shaped ZnO NPs were green synthesized using Berberis aristata leaf extract and 0.1 M zinc acetate dihydrate adjusting pH with 1 M sodium hydroxide. Their size range was~2 µm in length and 20-40 nm in diameter.
In 3D structured ZnO NPs/MPs such as antibiotic agents, flower-like ZnO MPs were synthesized using electrochemical-thermal method with poly-diallyl-(dimethylammonium) chloride (PDDA) and benzyl-dimethylammonium chloride (BAC) as a stabilizer, which were 200-500 nm or 1-2 µm long [45]. Flower-shaped ZnO NPs were also synthesized using the sol-gel method reacting with 5% zinc acetate dihydrate and 0.2 w/v% cetyltrimethyl ammonium bromide (CTAB) at pH 11.7-11.9, and 25-75 • C of near-room temperatures [59]. They showed enhanced antibacterial and antifungal performance via larger zones of growth inhibition when compared to ZnO powders and conventional products. Flower-like ZnO NPs were further reported by Quek et al. [60], which were sized at 700 nm-2.2 µm in diameter for visible light-responsive photocatalytic antimicrobial action. Talebian et al. [61] described higher antibacterial activity of flower-shaped ZnO NPs compared to those of rod-and sphere-shaped ZnO NPs. The flower-shaped ZnO NPs were synthesized using the solvothermal technique reacting with zinc acetate dihydrate in water (5.6 g/50 mL) and 0.5 M sodium hydroxide. They resulted in a smaller particle (45 nm) with larger surface area (28.8 m 2 /g) than ZnO NPs synthesized in 1-hexanol (rod shape; 76 nm; 15.5 m 2 /g surface area) and ethylene glycol (sphere shape; 65 nm; 19.2 m 2 /g surface area), predicting enhanced antimicrobial performance. At this point, it is believed that the solvent in the solvothermal technique is a critical parameter in the synthesis of ZnO NPs, which results in differentiated morphology. Star-shaped ZnO NPs were also synthesized at 31 nm using liquid precipitation for preparing microorganism-protective defense clothing on cotton or polyester fabrics due to their biocompatibility [62]. Using co-precipitation, flake-shaped ZnO NPs were prepared at 32 nm size for enhanced antimicrobial performance [63].
Therefore, irrespective of ZnO NP/MP variety, the physicochemical characteristics of ZnO NPs/MPs affect their antimicrobial activity against pathogenic microorganisms [14,64], and synthesis techniques can be controlled to improve the properties of ZnO NPs/MPs for enhanced antimicrobial performance [13,65].

Antimicrobial Mechanisms of ZnO Materials
Mechanisms of antimicrobial actions of ZnO materials have been explained in association with particular interaction based on their unique physicochemical properties of (a) Zn 2+ ion release, (b) adsorption, and (c) ROS generation [14,66], and the intracellular responses in microorganisms of (d) energy metabolism inhibition; (e) lipid peroxidation, and cell membrane damage; and (f) DNA replication disruption, and DNA break [67,68] ( Figure 1). The Zn 2+ ions that are released from ZnO NPs/MPs induce an antimicrobial response in microorganisms due to interference in metabolic processes and disturbance in enzymatic systems [69,70]. ZnO NPs/MPs also have functions of particle adsorption to the biomembrane via a charge-charge interaction, and ROS generation as photocatalysts under UV and visible light irradiation [3,12,71,72]. Positively charged surfaces of ZnO NPs/MPs interact with the negatively charged cell wall or biomembrane of microorganisms [73]. They are internalized into the microorganisms after adsorption resulting in loss of cell integrity based on cell wall or membrane rupture, and further mediate oxidative stress owing to lipid peroxidation leading to deoxyribonucleic acid (DNA) damage. Based on the fundamental mechanisms of action, ZnO NPs/MPs have a differential susceptibility against pathogenic microorganisms, affected by their physicochemical characteristics including morphology, particle size, and porosity [3,17,18,73]. In the photocatalytic activation of ZnO NPs/MPs for ROS generation linked to oxidative stress, electrons (e − ) are transmitted from the valence band to the conduction band by the photoexcitation over band gap energy (E = ∆hν) leaving positive holes (h + ) [14,65]. Holes in the valence band produce hydroxyl radicals (OH·), whereas electrons in the conduction band generate superoxide radical anions (O 2 · − ) in an aqueous environment. Reacting with electrons and hydrogens, hydroxyl peroxide radicals (HO 2 ·) and finally hydrogen peroxide (H 2 O 2 ) are formed. In addition, surface defects of ZnO NPs can enhance ROS production even in the dark for enhanced antimicrobial performance [74].
In 3D structures of ZnO NPs/MPs followed by aggregate formation or nanostructure generation, multiple scattering can be one of several antimicrobial mechanisms to improve photocatalytic performance based on enhanced mass transfer and exchange [18,75]. The bimodal porous self-assembled network of ZnO NPs, with mesopores and macropores was reported to enhance the photocatalytic antimicrobial action caused by multiple scattering under dual UV irradiation [18]. Hierarchically porous self-forming structured aggregates of polydispersed ZnO NPs showed a submicron size of 100-500 nm in diameter, which enhanced the multiple scattering phenomenon [75]. Thus, the hierarchically porous structure of ZnO NPs/MPs mediating multiple scattering can counteract the undesirable photocatalytic performance, which prevents large particles from retaining a small surface area.
Compared to other metal or metal oxide NPs/MPs, ZnO NPs/MPs have multiple functional mechanisms of antimicrobial performance [4]. In antibiotic metals such as silver (Ag) and gold (Au), NPs have the primary functions of metal ion release and membrane adsorption, respectively. In general, titanium dioxide, cupric oxide (CuO), and magnesium oxide (MgO) NPs display ROS generation, NP internalization, and membrane damage, respectively. Regardless of the susceptibility of pathogenic microorganisms to antimicrobial activity, ZnO NPs/MPs can combat microbial regrowth to overcome multi-drug resistance of conventional antibiotics based on targeting antibiotic-resistant pathways in a non-specific manner, irrespective of NP penetration into microorganisms [7,30,76].

ZnO Materials and Combinations Based on Antimicrobial Functions
ZnO NPs/MPs have been studied to enhance their antimicrobial activity against pathogenic microorganisms [33,92]. In addition to improving their physicochemical characteristics, antimicrobial activities of combinations of ZnO NPs/MPs with other biomaterials have been explored for enhanced antimicrobial performance [93,94]. Table 2 presents the antimicrobial activity of (i) ZnO NPs/MPs, (ii) ZnO NPs/MPs with antibiotic or antiinflammatory drugs, (iii) ZnO NPs/MPs with other metal oxide NPs/MPs or metal doping, (iv) ZnO NPs/MPs with other polymeric biomaterials or films, and (v) ZnO QDs.

ZnO Materials
The antimicrobial activity of various-sized or -shaped ZnO NPs/MPs has been investigated against pathogenic bacteria as well as viruses [3,69,95]. ZnO NPs/MPs show various forms as spheres, cubes, pyramids, rods, tubes, donuts, platelets, mixtures, and 3D architectures of clusters or networks, ranging from 3.4 nm to 30 µm in size. Size-dependent antimicrobial activity of spherical ZnO NPs of 12-307 nm was described against S. aureus, Proteus vulgaris, Salmonella typhimurium, Shigella flexneri, and Bacillus cereus [16]. Smaller NPs (12-30 nm) showed a superior antimicrobial growth inhibition (~94.05%) than larger NPs at the same concentration (6 mM). In addition, three types of ZnO NPs shaped like hexagons, spheres, and cubes or rods showed antimicrobial activity against E. coli, S. aureus, and B. subtilis [17]. The minimum inhibitory concentration (MIC) levels of ZnO NPs at 63 nm,~65 nm and 60-180 nm, and 40-45 nm were determined as 1562 µg/mL against E. coil, 391-781 µg/mL against S. aureus, and 195-391 µg/mL against B. subtilis, respectively. ZnO nanopyramids at~15.4 nm in a segment showed an MIC of 333 µg/mL against MRSA [96]. DeLucas-Gil et al. [19] illustrated that nanostructured ZnO of spherical NP clusters at 590 nm in diameter, had superior antimicrobial activity against E. coli and S. aureus at >3.5 log (control/sample) value than the ZnO NPs at 63 nm in diameter.
ZnO nanorods, at <100 nm or ranging from 500 nm to 1 µm, also showed an antimicrobial activity against pathogenic microorganisms [97,98]. Reddy et al. [99] reported antimicrobial activity of ZnO nanorods at 88.7 nm against K. pneumoniae, for which the MIC was 40 µg/mL. These NPs were synthesized by the precipitation method using 2mercaptoethanol as a capping agent. Tahizdeh et al. [100] reported that ZnO nanorods at an MIC of 250 µg/mL had antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and Enterococcus faecalis, excluding E. faecalis which was resistant to ZnO nanorods. These nanorods were small crystals 3.4 nm in size and the NPs were 150 nm in length and 21 nm in width (aspect ratio, 7.14). Nanometric ZnO were synthesized using ethylenediamine and citric acid monohydrate as capping agents; the antimicrobial activity of the resulting nanorod-shaped particles (500 nm to 1.0 µm) was affected by the synthesis technique [101]. ZnO nanorods synthesized with ethylenediamine showed antimicrobial activity against E. coli but not against Micrococcus luteus. Conversely, ZnO nanorods synthesized using citric acid monohydrate showed growth inhibition for M. luteus but not for E. coli.
Elkady et al. [54] reported antimicrobial activity against E. coli, S. aureus, P. aeruginosa, and B. subtilis using as-synthesized ZnO nanopowders of hexagonal hollow tube shapes 500 nm in length with 17.8 m 2 /g surface area and 278.6 nm average pore size. Their MIC values were found to be 0.0585 mg/mL for E. coli (zone of inhibition, 13 mm), 0.234 mg/mL for S. aureus (zone of inhibition, 14 mm), 0.234 mg/mL for P. aeruginosa (zone of inhibition, 18 mm), and 0.938 mg/mL for B. subtilis (zone of inhibition, 15 mm) using disk diffusion. Hollow nanotubes of ZnOs were also reported by López de Dicastillo et al. [55]. They were coated on acrylic polymer/extruded 32 µm-polyethylene (PE) substrate at 1% (wt) and were~5 µm in length, 59.5 nm in thickness, and 178.2 nm in internal diameter. The hollow nanotubes reduced microbial growth against E. coli by 4.67 log (cells/cm 2 ) and S. aureus by 2.46 log (cells/cm 2 ).
Three-dimensional structured ZnO NPs also displayed antimicrobial activity against pathogenic microorganisms [14,18]. Jin et al. [18] presented the antimicrobial activity of self-assembled networks of spherical ZnO NPs at~3 µm against E. coli under dual UV irradiation for 30 s. No colonies were detected at 0.05 mg/mL of self-assembled ZnO networks, whereas colonies at 2.8-3.0 log (CFU/mL) were grown after no particle treatment.
Farzana et al. [79] reported MIC levels of ZnO NPs with ciprofloxacin against E. coli and K. pneumonia at 0.2-1 mg/mL compared to 0.08 and 0.05 mg/mL for ZnO NPs alone, respectively. However, ZnO NPs with imipenem had no growth inhibition with decreasing antimicrobial effect except for one K. pneumonia strain.
Sharma et al. [106] described antimicrobial activity of ZnO NPs (25 nm) with ampicillin/sulbactam against E. coli, K. pneumoniae, P. aeruginosa, S. typhi, and S. aureus. MIC levels of ampicillin/sulbactam were 50 µg/mL against E. coli, S. typhi, and S. aureus; and 100 µg/mL against K. pneumoniae and P. aeruginosa, whereas MIC levels of ZnO NPs alone were 25-200 µg. Although the MIC level of ZnO NPs was 25 µg and that of ampicillin/sulbactam was 100 µg/mL against K. pneumoniae, the MIC level of the ZnO NP-ampicillin/sulbactam conjugated form against K. pneumoniae was 6.25 µg/mL, suggesting antimicrobial activity enhancement.
In 3D porous architectures, CuZnO NPs on mesoporous silica SBA-3 had highly ordered mesoporous structures of near-spherical NPs~2 µm in size, 3.6274 nm in pore size, and 829 m 2 /g in specific surface area with a relatively rough surface [117]. They showed growth inhibition against E. coli and S. aureus at MIC levels of 25 mg/mL (0.558 mg/mL as CuZnO NPs) and 6.25 mg/mL (0.139 mg/mL as CuZnO NPs), respectively.
Moreover, ZnO MPs with TiO 2 MPs showed antimicrobial activity against E. coli, Streptococcus pyogenes, and K. pneumoniae under UV and visible light irradiation [118]. Using ZnO MPs at 0.25% and TiO 2 MPs at 1%, growth inhibition levels were 76.8% against E. coli, 70.2% against S. pyrogenes, and 80.8% against K. pneumoniae. ZnO MPs synergistically enhanced antimicrobial activity against pathogenic microorganisms compared to UV and visible light-irradiated TiO 2 MPs alone. The ZnO MPs in combinations had near-spherical shape, with sizes of 3.17-10.3 µm compared to 2.15-37.1 µm of the TiO 2 MPs.
Among natural products, chitosan has been extensively studied as a pharmaceutical excipient for drug delivery carriers in biomedical applications owing to their biocompatibility and physicochemical characteristics [113,114]. It is beneficial to have inherent therapeutic functions including antimicrobial activity and anti-inflammatory activity for accelerated wound healing, hemostasis, and reduced fat absorption to decrease plasma and liver lipids and increase fecal excretion [114,130]. Regarding the biomedical functions of chitosan, the antimicrobial mechanisms are explained by cell binding via charge-charge interaction owing to the polycationic nature of chitosan, leading to disruption of the membrane and inhibition of nucleic acid processes, thereby causing cell death. As a chelating agent, chitosan also interacts with trace metal elements, which are toxic to cells, inducing microbial growth inhibition. It is applied for wound dressing/healing, cotton fabric, daily food packaging, and paper packaging as an antimicrobial agent, mostly in films [113,[131][132][133][134]. Therefore, chitosan can be extended to the combinations with ZnO NPs/MPs [135].
Chitosan-ZnO NP-loaded gallic acid (C-ZnO@gal) films showed antimicrobial activity against E. coli and B. subtilis [28]. ZnO@gal NPs were near-rod-shape at~19.2 nm, and C-ZnO@gal films included homogeneously distributed ZnO@gal NPs on chitosan matrixes (2 g chitosan/70 mg ZnO@gal) at a thickness of 0.10 mm. Using C-ZnO@gal films at 0.5 mg/mL, growth inhibition zone levels were reported as 28 mm against E. coli and 25 mm against B. subtilis. Al-Nabulsi et al. [82] described the antimicrobial activity of ZnO NP-containing chitosan against E. coli O157:H7, which was used as a biocompatible coating agent for smart storage. The ZnO NPs had a near-spherical shape at~65 nm and were uniformly distributed in chitosan matrixes. The growth reduction levels against E. coli O157:H7 at 4 • C were 2.5 log (CFU/g) for chitosan (2.5%, w/v) and 2.8 log (CFU/g) for ZnO NP (1%, w/v)-containing chitosan (2.5%, w/v). Although combinations of ZnO NPs in a chitosan matrix did not show a significant improvement of antimicrobial activity against E. coli O157:H7 (p > 0.05) compared to ZnO NPs or chitosan alone, the ZnO NP combination in the chitosan matrix presented better antimicrobial results.
Kumar et al. [91] reported growth inhibition levels of chitosan/gelatin hybrid nanocomposite films containing ZnO NPs against E. coli and S. aureus along with their improved structural integrity. In these films, ZnO NPs had various shapes such as polyhedrons, quasi-spheres, or rods of 20-40 nm, 500-1000 nm, and 200-400 nm, respectively, which were evenly distributed on smooth, compact, and heterogeneous surfaces with 86-92 µm in thickness. Depending on the ZnO NP contents in the films, their antimicrobial activity against E. coli presented a 10.5 mm zone of growth inhibition for ZnO NPs at 1% and 2% in the films and a 10.7 mm zone of growth inhibition for ZnO NPs at 4% in the films. The films showed comparable growth inhibition results based on inherent antimicrobial activity of chitosan. Unlike the effects against E. coli, no prominent results were obtained for S. aureus.
In particular, Javed et al. [137] reported a biodental materials for orthodontics, composed of star-shaped, chitosan-based ZnO NPs (20-25 nm), which showed enhanced antimicrobial activity against K. pneumoniae (13 mm zone of inhibition, the highest); E. coli, P. aeruginosa; and B. subtillis, S. aureus, and MRSA (6 mm zone of inhibition, the lowest), compared to those composed of ZnO NPs or chitosan alone. In the synthesis of chitosanbased ZnO NPs via co-precipitation, zinc acetate dihydrate (0.06 M) and chitosan were used as a precursor and a capping agent, respectively. Conventionally used adhesive was coated using chitosan-based ZnO NPs at 2-10%. The pure ZnO NPs had a spherical shape with a diameter of 25-30 nm.
Jose et al. [120] also reported growth inhibition of tungsten-doped polyethylene glycol (PEG)-capped ZnO (W-PEG-ZnO) NPs against E. coli and B. cereus, which were near-spherical at~40.46 nm. Compared to ampicillin at 100 µg/µL inhibiting growth at 35 mm against E. coli and 20 mm against B. cereus, W-PEG-ZnO NPs showed a 5-6 mm zone of growth inhibition against E. coli at 400-600 µg/µL and a 6-8 mm zone of growth inhibition against B. cereus at 300-600 µg/µL, suggesting B. cereus was more susceptible to W-PEG-ZnO NPs than E. coli.
Curcumin in turmeric is known to have beneficial anti-inflammatory activity, and is used as a multipurpose nutraceutical in various dosage forms of tablets, capsules, and ointments [123]. After loading curcumin on ZnO (C-ZnO) NPs, the antimicrobial activity of C-ZnO NPs against E. coli, S. epidermis, S. aureus, and B. cereus was determined [124]. Among the C-ZnO NPs, spheres (C-sZnO), rods (C-rZnO), javelin (C-jZnO), short petal nanoflowers (C-spZnO), and long petal nanoflowers (C-lpZnO) were detected with sizes of 40-100 nm, 600-900 nm (<300 nm of width), 300-600 nm (<300 nm of width), 2-4 µm, and~500 nm, respectively. Their microbial growth inhibition levels were confirmed via measurements of well diffusion zones of inhibition as follows: C-sZnO, 8 Graphene, graphene oxide (GO), and reduced GO (rGO) have also been studied as antibiotic agents in combinations of ZnO NPs with other biomaterials. Antimicrobial activity of graphene/ZnO nanocomposite films against Streptococcus mutans was reported by Kulshrestha et al. [84]. The ZnO NPs distributed in the graphene sheet films were near-spherical at 20-40 nm. The MIC level of graphene/ZnO nanocomposite films was 125 µg/mL. Oves et al. [85] described the antimicrobial activity of graphene/ZnO nanocomposites with curcumin against MRSA strains (ATCC 43300 and ATCC BAA-1708). In the nanocomposites, spherical ZnO NPs at 35 nm were homogenously distributed on graphene sheets. Compared to curcumin with an MIC of 125 µg/mL, the graphene/ZnO nanocomposite induced growth inhibition at 125 µg/mL against MRSA ATCC 43300 and at 250 µg/mL against MRSA ATCC BAA-1708. Use of curcumin together with the graphene/ZnO nanocomposites achieved growth inhibition against MRSA ATCC 43300 and MRSA ATCC BAA-1708 at 31.25 and 62.5 µg/mL, respectively. Against in vivo topical dermatitis infection, the growth of MRSA was inhibited by up to 64%.
GO/ZnO nanocomposites for wound care showed growth inhibition against E. coli, S. typhi, P. aeruginosa, and S. flexneri [86]. GO with smooth and wrinkled surface layers was used to prepare GO/ZnO nanocomposites. The ZnO NPs were well incorporated and distributed on the GO sheets generating agglomerates. Analysis of the zone of growth inhibition with GO/ZnO nanocomposites at levels of 25-100 µg/mL under dark or visible light irradiation, showed that the composites were more susceptible to E. coli, P. aeruginosa, S. typhi, and S. flexneri than with GO alone. In addition, Wang et al. [87] reported antimicrobial activity against E. coli of GO/ZnO composites, including rod-like ZnO NPs at 4 nm, homogenously attached on GO sheets. Using 1-4 µg GO/ZnO composites, growth inhibition zones were detected at 2 and 4 µg (MIC, 2 µg).
In the case of rGO, combination of ZnO NPs with rGO had antimicrobial activity against microorganisms [88]. Spherical ZnO NPs at 100-300 nm were attached to rGO sheets for manufacturing rGO/ZnO films with roughness at 159 nm, containing homogenously distributed ZnO NPs on rGO sheets. They displayed >99% (2-log) growth reduction against S. aureus at 1% (wt) rGO.
In cotton fabrics, ZnO NPs have been used as protective agents against textile degradation, unpleasant odor, and potential health risks caused by microbial growth after contact with the human body [128,130,138]. As an alternative to chemical biocides, ZnO NPs provide enhanced antimicrobial activity against pathogenic microorganisms in surfacemodified textiles during the manufacturing process. Noman, M.T. and M. Petrů [127] reported eight types of cotton-ZnO NP (C-nZnO) composites and their antimicrobial actions against E. coli and S. aureus. The ZnO NPs were spherical at 27 nm, and formed thick and dense layers on cotton surfaces in C-nZnO composites at 2.2%, 1.7%, 4.9%, 4.3%, 11.1%, 7.8%, 22.2%, and 16.7% (wt). They showed microbial growth reduction of 97-100% against E. coli and 96-98% against S. aureus. Subash et al. [129] also demonstrated microbial growth reduction by ZnO NP-coated fabric against E. coli and S. aureus. The ZnO-coated fabric comprised evenly distributed spherical ZnO NPs (200 nm) on the fabric surface. In a 4.8 cm-diameter circular cut of the fabric, E. coli and S. aureus growth levels were reduced by 80% and 99.99%, respectively. In the case of ZnO MPs, they were coated on cotton fabrics together with chitosan and formed a uniformly distributed dense microstructure of rods showing antimicrobial growth inhibition against E. coli [126]. Immersion of ZnCl 2 (4%) for ZnO MP synthesis on chitosan-loaded cotton fabrics (1-2%/1 g cotton fabric) resulted in a growth inhibition zone of 2.5 cm in disk diffusion against E. coli.

ZnO QDs
ZnO QDs have been studied for imaging and therapy as theranostic agents having photoluminescence properties with antimicrobial activity against pathogenic microorganisms [139,140]. They have superior characteristics including high quantum yield, high stability, and a narrow emission range.

Current Biomedical Applications
ZnO NPs/MPs are one of Zn-based materials that have been extensively used in various biomedical fields including antibiotic therapy, medical devices, theranostics, tissue engineering, and health care because of their antimicrobial functions against pathogenic microorganisms ( Figure 2) [11,[143][144][145][146][147][148]. In the human body, Zn plays a pivotal role in life cycle maintenance and Zn deficiency causes cell impairment and malignancy leading to severe diseases related to immune responses such as infection and cancer [69]. As a first aid antibiotic alternative for the protection of skin [143,147], ZnO ointment (40%) or cream (10%) is conventionally used. In addition, ZnO NPs/MPs and antibiotic drugs are applied as endo dressing in paste forms [107]. They are also used as ZnO surgical adhesives in medical devices [144,145]. Moreover, ZnO NPs/MPs have been utilized as postoperative dressing on leg excisions [148]. In theranostics, biocompatible ZnO QDs are explored as photodynamic therapeutics as well as diagnostic agents owing to their photoluminescence properties based on photochemical stability [11]. For dental tissue engineering, ZnO NPs/MPs are used as coating agents in orthopedic and dental implants [146]. ZnO NPs/MPs and eugenol are also extensively used as temporary cement for dental implants [108]. ZnO waxes or sunscreen lotions are used as health care products for skin protection against acne/blemish or UV and visible light [149,150]. Although ZnO NPs/MPs still have exposure risks to health via the inhalation route [151,152], ZnO is considered as "generally recognized as safe" and is approved by the U.S. Food and Drug Administration and the health risks of ZnO NPs/MPs are guided by the Organisation for Economic Co-operation and Development, based on their unique physicochemical characteristics via synthesis techniques [153]. Therefore, ZnO NPs/MPs have a promising potential for use in biomedical applications including medicine, medical devices, and cosmeceuticals connected with antibiotic functions overcoming drug resistance.

Conclusions
The antibiotic properties of ZnO materials have been highlighted for their biomedical applications in combating multi-drug resistance. Compared to currently available antibiotic drugs, they exhibit different mechanisms of antimicrobial actions; these mechanisms are mainly induced by the Zn 2+ ion, particle adsorption, ROS generation and photocatalytic responses based on physicochemical characteristics of these materials, which vary substantially according to their synthesis techniques. ZnO NPs, sometimes MPs, show enhanced antimicrobial performance even toward pathogenic viruses. Their combinations with other antibiotic drugs, metal oxide NPs/MPs or metal doping, and other biomaterials also have a wide-spectrum antimicrobial activity that can be customized for multi-therapeutic options and used to improve their applicability as industrial and clinical translation platforms. Moreover, ZnO QDs display enhanced antimicrobial actions as theranostic agents. Therefore, ZnO materials can be a next-generation antibiotic drug against multi-drug resistant pathogenic microorganisms and, in the near future, combinations of these can be developed further with industrial and clinical impacts.

Conflicts of Interest:
The authors declare no conflict of interest.