Green nanotechnology-based zinc oxide (ZnO) nanomaterials for biomedical applications: a review

Sol–gel techniques are mostly used for the fabrication of metal oxide NPs (Kumar et al 2015), described as the transition of a sol (e.g. the solution containing inorganic metallic salts) gradually into a solid 'gel' phase, over a series of hydrolysis and polymerization reactions. Finally, the gel is subjected to the vaporization of the solvents and a heating process to obtain the final product (Sajjadi 2005, Sakka 2013). Figure 2(A) shows a schematic illustration of the sol–gel technique. The advantages of sol–gel processing include simplicity of the procedure, a suitable control on the chemical composition (Al Abdullah et al 2017), and the production of a very fine powder-like structure of ZnONPs (Carter and Grant, Norton 2007). On the other hand, this technique does have its shortcomings in possible shrinkage, cracking while drying, and an often difficult porosity control (Kumar et al 2015). Despite the drawbacks, this technique is one of the most widely used because of the feasibility of the protocol and the quick generation of the valuable material, often a reason why it is mentioned numerous times in relevant literature. For instance, Hasnidawani et al synthesized rod-shaped ZnONPs by a sol–gel method in the range of 81.28–84.98 nm, after mixing zinc acetate dehydrate (Zn(CH₃COO)₂^.2H₂O) with ethanol used as a solvent (Hasnidawani et al 2016). Similarly, Jurablu et al generated ZnONPs by a sol–gel method with an average size of 28 nm and spherical morphology. In this process, an ethanol solution of zinc sulfate heptahydrate (ZnSO₄^.7H₂O) was used in the presence of a diethylene glycol (C₄H₁₀O₃) surfactant (Jurablu et al 2015). Other examples include the production of ZnONPs with an average particle size of 12–30 nm using a methanol (CH₃OH) solution of Zn(CH₃COO)₂^.2H₂O and ammonia (NH₃) (Al Abdullah et al 2017), and the production of spherical-shaped ZnONPs by a sol–gel method in the range of 50–60 nm using Zn(CH₃COO)₂^.2H₂O as a precursor (Alwan et al 2015).

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Hydrothermal processes take place in a specific closed reaction vessel commonly known as an autoclave, with high pressure and high temperature. The insoluble, or poorly soluble, materials under room temperature are dissolved and recrystallized under high pressure and a pre-set temperature (G. Yang and Park 2019). Different solvents can be used in such reactions: for instance, if water is employed, the process is known as a hydrothermal technique, while if organic solvents are used, such as ethanol or polyols, the approach is named as a solvothermal technique (Komarneni 2003, J. Li et al 2015). Figure 2(B) represents a schematic illustration of the hydrothermal technique. The advantages of hydrothermal techniques include high purity and a high degree of crystallinity of the product (Kolodziejczak-Radzimska and Jesionowski 2014), high control of the final nanostructure size, morphology and crystal phase (Li et al 2015), as well as minimum pollution owing to the closed system conditions. This is precisely why the approach is often considered an eco-friendly methodology, and why it is included as a green methodology for the production of ZnONPs. However, this technique is also prone to drawbacks, some of which include slow reaction kinetics at any given reaction temperature (Parhi et al 2008), need for expensive autoclaves, limitations in studying the reaction (for instance, the observation of the reaction process is not possible as the reactor cannot be kept open) (Sonawane et al 2018), and potential safety issues during the autoclave procedure (Rane et al 2018).

As observed with the sol–gel approach, hydrothermal/solvothermal approaches are considered straightforward and easy to set-up. Notable examples include the work of Bharti and Bharati, who synthesized ZnONPs using a hydrothermal method, with a size range of 15.8–25 nm along with different shapes (Bharti and Bharati 2017). Additionally, Reddy et al synthesized hexagonal shaped ZnONPs comprised of cylindrical pores of diameters ranging from 9 to 12 nm using zinc nitrate hexahydrate (Zn(NO₃)₂^.6H₂O) and sodium hydroxide (NaOH) as the starting precursors (Reddy et al 2015). Similarly, Wirunmongkol et al synthesized ZnONPs through a hydrothermal process using an autoclave unit, in which Zn(NO₃)₂^.6H₂O and NaOH were added as the starting precursors. The fabricated NPs had the morphology of a short prism and flower-like shapes, sized between 30 and 80 nm in width and 0.5–1 µm in length (Wirunmongkol et al 2013).

In this method, MNPs are fabricated by simultaneous nucleation, growth and finally agglomeration of the small nuclei. Figure 2(C) represents a schematic illustration of the co-precipitation technique. The advantages of this method include simplicity, no high temperature requirement, and overall energy efficiency (Rane et al 2018). Nevertheless, one of the major disadvantages of this method is that the fabricated NPs contain a large amount of adsorbed water molecules which may affect their properties (Pudovkin et al 2018). Additional drawbacks include challenges in batch-to-batch reproducibility (Rane et al 2018), poor particle size distribution, and extensive agglomeration (Wang et al 2013, Mostafavi et al 2015). Despite this, notable examples include the work of Costenaro et al who synthesized spherical ZnONPs with a particle size ranging from 2 to 10 nm through a co-precipitation method using a solution of zinc acetate in methanol (Costenaro et al 2013), while, Purwaningsih et al synthesized ZnONPs by a co-precipitation method using zinc acetate dihydrate, hydrochloric acid, and ammonia as reactants. The morphology of the ZnONPs was found to be approximately a pseudo-spherical shape with the average particle size in the range of 11–20 nm (Purwaningsih et al 2016). Likewise, Adam et al synthesized uniform ZnONPs using a co-precipitation method with an average size of around 140 nm (Adam et al 2018).

In this method, the collision of water droplets in a microemulsion environment led to a fast reactant exchange, resulting in a precipitation reaction in nanodroplets and consequent nucleation growth, with NPs stabilized by the presence of a surfactant. Figure 2(D) shows a schematic illustration of the microemulsion technique. Simplicity, thermodynamic stability and minimal agglomeration are the advantages of this method. Unfortunately, microemulsion processes suffer from disadvantages such as the impact of external factors like temperature and pH on the microemulsion stability and the constant need for high concentrations of surfactants and/or cosurfactants that may generally cause irritation (Rane et al 2018). In the literature, some examples include the work of Wang et al who synthesized ZnONPs from microemulsions in a microchannel reactor system with an average size of 16 nm. The ZnONPs were obtained by drying the solid product at 130 °C for 2 hr, and consequent calcination at 550 °C for 3 hr (Wang et al 2014). Similarly, Li et al synthesized ZnONPs by a simple microemulsion process with different morphologies including columnar, needle-like, and spherical under 100 nm (X. Li et al 2009).

In a conventional laser ablation technique, the removal of metallic ions from a metal surface takes place while immersed in a small volume of liquid, such as methanol, ethanol, distilled water, using a laser beam. Figure 2(E) represents a schematic illustration of the laser ablation technique. The advantages of this method are simplicity and a highly safe protocol from the environmental perspective, leading to a method that is both efficient and easy to perform (Mintcheva et al 2018). Nonetheless, the role of pyrolysis byproducts (as a consequence of the laser ablation in the presence of organic compounds) on the stability of the colloidal system is not clear and needs to be elucidated (Amendola and Meneghetti 2009). Notable examples of this technique include the work of Al-Dahash et al who synthesized ZnONPs by laser ablation in a NaOH aqueous solution (with a particle size ranging from 80.76 to 102.54 nm) and spherical morphology (Al-Dahash et al 2018). Besides, Farahani et al synthesized ZnONPs by laser ablation from a zinc target in methanol and distilled water solution (with almost spherical morphology) and a particle size ranging from 1 to 30 nm (Farahani et al 2016). Similarly, Mintcheva et al reported the synthesis of laser-ablated rod-shaped ZnONPs with an average width of 30 nm, and length in the range of 40–110 nm (Mintcheva et al 2018).

A high energy ball milling technique is employed to produce fine metal NPs using stiff balls in a high-energy shaker mill (Hodaei et al 2015). Figure 2(F) shows a schematic illustration of the high-energy ball milling technique, whose main advantage is the significant potential for scale up in order to produce large quantities of material. On the other hand, the disadvantages of this method include the possibility of contamination from milling balls and/or the atmosphere (Edelstein 2001, L. Yang 2015), as well as the formation of irregular shaped NPs (Piras et al 2019). Despite this, important examples include the work of Prommalikit et al who applied commercial grade ZnO powder with an average particle size of 0.8 μm as the starting material to synthesize ZnONPs by the high-energy ball milling technique. The final particle size after the milling process was found in the range of 200–400 nm (Prommalikit et al 2019). Similarly, Mohammadi et al reported the synthesis of rod-shaped ZnONPs by a high-energy ball milling process in the range of 20–90 nm (Mohammadi et al 2015). Likewise, Salah et al used a microcrystalline powder of ZnO to produce ZnONPs by the aforementioned high energy ball milling process. The samples were ball milled for 2, 10, 20, and 50 h. The results showed a time-dependent particle size. The more the ball milling time, the less the particle size. The sample milled for 50 h showed spherically shaped ZnONPs with a particle size of around 30 nm (Salah et al 2011).

A large array of diseases caused by bacterial pathogens and the origination of multidrug resistance provokes the need for developing new vectors or novel drug molecules for effective drug delivery and thus, better treatments. Nanotechnology has emerged as a novel approach to fight bacterial diseases. The use of NPs as an antibacterial agent has given rise to rich literature on the subject, with excellent studies showing how MNPs like silver (Ag), gold (Au), copper (Cu) or iron (Fe), and metal oxide nanoparticles (MoNPs) like ZnO, copper oxide (CuO), titanium oxide (TiO₂) and iron oxide (FeO) NPs, can be used for the effective treatment of pathogenic bacterial diseases (Chwalibog et al 2010, Ivask et al 2014).

The antimicrobial properties of 'NMs' arise from their inherent properties, such as high reactivity and large surface area to volume ratio, which allows them to bond of a large number of ligands on the NPs surface and hence, with receptors present on the bacterial surface (Brown et al 2018, Medina Cruz et al 2019, Medina-Cruz et al 2019, Lomelí-Marroquín et al 2019). Among all the NMs, the effect of ZnONPs is remarkable when combined with green synthesis approaches. Different natural raw materials, such as bacteria or plants, have been used for the production of ZnONPs which show a powerful antimicrobial effect. However, among all the green synthesis approaches, plant-mediated or phytochemical production flourish in the literature, due to the wide range of raw materials, and the possibility of generating a synergetic nanostructure combining the antimicrobial properties of both the plant extract and the NPs themselves (Jalal et al 2010, Gunalan et al 2012, Agarwal et al 2017).

Several studies have reported that ZnONPs are effective at inhibiting both Gram-positive and -negative bacteria. While the crystalline structure and particle shape have a very small effect, the concentration of NPs highly impacts the antibacterial behavior of the structures. Additionally, a number of mechanisms have also been proposed to interpret these experimental observations, including the production of reactive oxygen species (ROS) due to the presence of the NPs, damage of the membrane cell wall through adhesion on the cell membrane, penetration through the membrane cell wall and cellular internalization of the NPs. However, the dominant mechanisms have not been identified. Importantly, there is no substantial discussion about the green production of ZnONP suspensions where the presence of additives such as stabilizers and capping agents might affect the interaction between the NPs and the cells (Li et al 2009, Liu et al 2009, L. Zhang et al 2010, Emami-Karvani and Chehrazi 2011).

In the following section, remarkable examples of both bacteria and plant mediated ZnONPs are discussed, focusing on the improvements of these nanostructures compared to their physiochemically or traditionally produced NPs.

4.1.1. Microorganism-mediated synthesis of ZnONPs with antimicrobial properties

4.1.2. Plant extract-mediated synthesis of ZnONPs with antimicrobial properties

Inarguably, the use of plants for the production of ZnONPs is among the most widely employed approaches for NP production (Senthilkumar and Sivakumar 2014, Hussain et al 2019, Rauf et al 2019, Ruddaraju et al 2019), due to the accessibility of a huge variety of plant species and the formation of a synergetic effect between the antibacterial activity of the plants extracts and the NPs themselves (figure 3 and table 1) (Khalil et al 2017, Akhter et al 2018, Gupta et al 2018, Chemingui et al 2019).

Table 1. Antibacterial applications of green-synthesized ZnONPs.

Platform	Raw material	System	Size	Targeted bacteria
Bacteria-mediated	Bacillus megaterium (NCIM 2326) cell free extract (Saravanan et al 2018)	ZnONPs	45–95 nm	H. pylori
	Bacillus licheniformis Dahb1 exopolysaccharides (EPS) (Abinaya et al 2018)	ZnONPs	10–100 nm	P. aeruginosa Proteus vulgaris Bacillus subtilis Bacillus pumilus
Plant-mediated	Cassia fistula plant extract (D. Suresh et al 2015)	ZnONPs	5–15 nm	Klebsiella aerogenes E. coli Plasmodium desmolyticum S. aureus
	Trifolium pretense flower extract (Dobrucka and Długaszewska 2016)	ZnONPs	60–70 nm	P. aeruginosa E. coli S. aureus
	Boerhavia diffusa leaves (Joseph et al 2016)	ZnONPs	140 nm	MRSA
	Artocarpus gomezianus fruit extract (Anitha et al 2018)	ZnONPs	39, 35, 31 nm prepared with 5, 10 and 15 ml of 10% extract	S. aureus
	Sechium edule leaf extract (Elavarasan et al 2017)	ZnONPs	30–70 nm	Bacillus subtilis Klebsiella pneumonia
	Azadirachta indica (Neem) leaf extract (Bhuyan et al 2015)	ZnONPs	9.6–25.5 nm	Streptococcus pyogenes E. coli S. aureus
	Azadirachta indica (Neem) extract (Madan et al 2016)	ZnONPs	9–40 nm	Klebsiella aerogenes S. aureus E. coli P. aeruginosa
	Acalypha indica leaf extract (Karthik et al 2017)	ZnONPs	20 nm	E. coli S. aureus
	Tabernaemontana divaricata green leaf extract (Raja et al 2018)	ZnONPs	20–50 nm	E. coli S. aureus Salmonella paratyphi
	Aqueous leaf extract of Laurus nobilis (Vijayakumar et al 2016)	ZnONPs	47.27 nm	P. aeruginosa S. aureus
	Ruta graveolens aqueous stem extract (Lingaraju et al 2016)	ZnONPs	28 nm	Klebsiella aerogenes P. aeruginosa E. coli S. aureus
	Aristolochia indica extract (Steffy et al 2018b)	ZnONPs	22.5 nm	Multi-drug Resistant Organisms (MDROs) isolated from pus samples of DFU patients
	Aqueous extracts of garlic (Allium sativum), rosemary (R. officinalis) and basil (Ocimum basilicum) (Steffy et al 2018b)	ZnONPs	14 and 27 nm	S. aureus Bacillus subtilis L. monocytogenes E. coli Salmonella typhimurium P. aeruginosa
	Bauhinia tomentosa leaf extract (G. Sharmila et al 2018)	ZnONPs	22–94 nm	E. coli P. aeruginosa
	Ulva lactuca seaweed extract (Ishwarya et al 2018)	ZnONPs	10–50 nm	Bacillus licheniformis Bacillus pumilis E. coli Proteus vulgaris
	Amaranthus spinosus leaf extract (Aiswarya Devi et al 2017)	Undoped and Fe-doped ZnONPs	243 nm undoped/197 nm 1%-Fe-ZnONPs	E. coli Bacillus safensis
	Hibiscus rosa-sinensis leaf extracts (Chai et al 2019)	Fe-doped ZnONPs	15–170 nm	E. coli
	Gymnema sylvestre (G. sylvestre) leaves extract (Karthikeyan et al 2019)	Lanthanum, cerium and neodymium doped ZnONPs	138 nm, 52 nm, 59 nm, and 63 nm for undoped, La, Ce and Nd-doped	S. aureus Streptococcus pneumonia Klebsiella pneumoniae Shigella dysenteriae E. coli P. aeruginosa Proteus vulgaris

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An interesting approach was shown by Suresh et al when they synthesized ZnONPs using Cassia fistula plant extracts as capping agents, composed of reducing components such as polyphenols (11%) and flavonoids (12.5%), which allowed for easy production of ZnONPs with sizes between 5 and 15 nm and a hexagonal wurtzite structure (with a clear absorption band at 370 nm), assigned to the intrinsic band-gap absorption of ZnO. Significant antioxidant activity was exhibited by NPs through scavenging of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals, while excellent bactericidal activity was observed toward Klebsiella aerogenes, E. coli, Plasmodium desmolyticum and S. aureus (D. Suresh et al 2015). Similarly, the biological production of ZnONPs from zinc acetate was recently demonstrated using the flower extract of Trifolium pratense as a unique reducing and capping agent. The prepared ZnONPs, with a size range of 60–70 nm, showed antimicrobial efficacy against clinical and standard strains of S. aureus and P. aeruginosa, as well as a standard strain of E. coli. The inhibitory effect of ZnONPs increased with a rise in concentration, with comparable results to the ones obtained with the antibiotic gentamicin. However, the ZnONPs rendered a better antimicrobial effect against Psudomonas aeruginosa than gentamicin (Dobrucka and Długaszewska 2016). Alternatively, an aqueous extract of Boerhavia diffusa leaves was employed to generate stable ZnONPs with an average size of 140 nm. The antibacterial and antibiofilm properties were evaluated showing a significant antibacterial effect towards Methicillin resistant S. aureus (MRSA) strains (Joseph et al 2016).

In a different report, spherical ZnONPs were synthesized by an eco-friendly green combustion method using a citrate containing Artocarpus gomezianus fruit extract. TEM studies demonstrated that the particles were highly uniform, spherical in shape and loosely agglomerated, with sizes of 39, 35, and 31 nm for the ZnONPs prepared with 5, 10, and 15 ml of 10% fruit extract, respectively. The antimicrobial effect was evaluated through the zone of inhibition method, showing that the NPs exhibited significant antibacterial activity against S. aureus at a concentration range of 10–500 μg ml⁻¹ (Anitha et al 2018). Another example reported the use of Sechium edule leaf extracts as a raw material, producing NPs with a specific bandgap at 362 nm, spherical-shape and a size between 30 and 70 nm. Furthermore, compared with chemically synthesized ZnONPs, the biosynthesized NPs showed significant cytotoxicity when cultured with Bacillus subtilis and Klebsiella pneumonia at concentrations of 10–40 µg ml⁻¹. Additionally, it was shown that the antibacterial behavior could be due to the chemical interactions between hydrogen peroxide and the membrane proteins, or between other chemical species produced in the presence of ZnONPs and the outer lipid bilayer of the targeted bacteria (Elavarasan et al 2017).

Another interesting result was found in the low-cost and green synthesis of ZnONPs using an Azadirachta indica (Neem) leaf extract, producing pure, predominantly spherical NPs with a size ranging from 9.6 to 25.5 nm. The antibacterial activity of the samples was determined towards S. aureus, Streptococcus pyogenes and E. coli using the shake flask method, with results revealing that the bacterial growth decreased with an increased concentration. In addition, Gram-positive bacteria seemed to be more sensitive to the NPs than Gram-negative (Bhuyan et al 2015). Similarly, a low temperature solution combustion mechanism was recently reported for the production of ZnONPs using Neem extracts. The NPs revealed a pattern which confirmed a hexagonal wurtzite structure, while SEM images indicated the transformation of mushroom-like hexagonal disks to bullets, buds, cones, bundles and closed pinecone structures all over the samples, with an increase in the concentration of the neem extract and sizes between 9 and 40 nm. The antibacterial studies indicated that the ZnONPs had a significant antibacterial activity on Klebsiella aerogenes and S. aureus, but not against E. coli and P. aeruginosa. Further, the NPs exhibited significant antioxidant activity against scavenging DPPH free radicals (Madan et al 2016). In addition, Karthik et al produced ZnONPs using an Acalypha indica leaf extract as a unique reducing and capping agent from zinc acetate. The prepared NPs were calcined at three different temperatures (100, 300, and 600 °C) and the results showed that those NPs calcined at 600 °C exhibited a higher surface area (230 m² g⁻¹) and smaller particle size (around 20 nm), proving to be effective against E. coli and S. aureus at a concentration of 100 mg ml⁻¹. The study concluded that optimizing the production of ZnONPs by varying calcination temperature may render an effective strategy to produce favorable antibacterial agents employing plants as raw materials (Karthik et al 2017). Alternatively, Raja et al reported the green synthesis of 20–50 nm spherical ZnONPs using an aqueous extract of a Tabernaemontana divaricata green leaf. The FTIR analysis showed that the NPs were stabilized through the interactions of steroids, terpenoids, flavonoids, phenyl propanoids, phenolic acids and enzymes present in the leaf extract. On the other hand, the NPs showed antibacterial activity against bacterial strains of Salmonella paratyphi, E. coli, and S. aureus (Raja et al 2018).

The aqueous leaf extract of Laurus nobilis was used for the co-precipitation synthesis of ZnONPs with a crystalline and flower like structure, which showed a hexagonal wurtzite structure with a mean particle size of 47.27 nm. The antibacterial activity of the NPs was greater against S. aureus than P. aeruginosa at a range of concentrations between 25 and 75 μg ml⁻¹, while the cytotoxicity studies revealed that NPs showed no effect on normal murine RAW264.7 macrophage cells; and hence, becoming biocompatible at the same range of concentrations (Vijayakumar et al 2016). Similarly, Lingaraju et al employed an aqueous stem extract of Ruta graveolens as a unique reducing agent for the formation of ZnONPs with sizes around 28 nm, alongside a hexagonal phase with a wurtzite structure. Significant antibacterial activity was observed on S. aureus and P. demolyticum and moderate activity was observed on K. aerogenes and E. coli, with concentrations of 200 and 400 μg/well, determined by the agar well diffusion method. Furthermore, the ZnONPs effectively inhibited the scavenging of DPPH free radicals (as an IC50 value of 9.34 mg ml⁻¹ suggests) (Lingaraju et al 2016). More recently, the bactericidal effects of ZnONPs generated from Aristolochia indica were evaluated against Multi-drug Resistant Organisms (MDROs) isolated from the pus of patients attending a tertiary care hospital in South India. The ZnONPs, with a size of 22.5 nm and a zeta potential of −21.9 ± 1 mV, exhibited remarkable bactericidal activity with MIC/MBC ranging from 25 to 400 μg ml⁻¹ with a significant reduction in viable counts from 2 h onwards. Furthermore, the protein leakage and flow cytometric analysis confirmed that the bacterial death was associated with the presence of the NPs in bacterial media (Steffy et al 2018b).

Another interesting study presented the production of ZnONPs by co-precipitation using aqueous extracts of garlic (Allium sativum), rosemary (R. officinalis), and basil (Ocimum basilicum). The X-ray diffraction studies revealed that all ZnONPs had a hexagonal wurtzite structure with a particle size between 14 and 27 nm, the variations of which were dependent on the synthesis method and the type of extracts. Importantly, the green synthesized ZnONPs showed good bactericidal activity against S. aureus, Bacillus subtilis, L. monocytogenes, E. coli, Salmonella typhimurium, and P. aeruginosa bacterial strains at a standard concentration of 100 µg ml⁻¹. Moreover, the NPs were found to exhibit enhanced antibacterial and antioxidant activities as compared to chemically-produced ZnONPs (Stan et al 2016). More recently, a facile and eco-friendly synthesis of ZnONPs employing a Bauhinia tomentosa leaf extract was reported by Sharmila et al with a hexagonal morphology observed and a size range of 22–94 nm. The NPs showed significant antibacterial activity against P. aeruginosa and E. coli. The results of the study demonstrated that the leaf extract contained phytochemicals, such as alkaloids, terpenoids, flavonoids, tannins, carbohydrates, and sterols, which were utilized as unique redactor agents for the generation of the ZnONPs, revealing no need of artificial or chemical agents to stabilize the nanostructures, in advantage to traditional methods (G. Sharmila et al 2018). In a similar manner, the use of a Ulva lactuca seaweed extract as a reducing and capping agent was postulated as an efficient method for the production of ZnONPs with average crystallite sizes between 10 and 50 nm, as well as excellent bactericidal activity was shown by the NPs on Bacillus licheniformis, Bacillus pumilis, E. coli, and Proteus vulgaris bacteria at a concentration of 50 μg ml⁻¹, along with high antibiofilm potential (Ishwarya et al 2018).

Intriguingly, plant extracts can be used for the successful production of bimetallic nanostructures. For instance, in 2017, an innovative study showed the production of both undoped and Fe-doped ZnONPs, synthesized using Amaranthus spinosus leaf extracts as a unique reducing agent. The results revealed that the ZnONPs were rod shaped with hexagonal phase structure and crystal size of 243 nm and 197 nm for undoped and 1%-Fe doped ZnONPs, respectively. Further, the authors demonstrated that the dissolubility and aggregation decreased with Fe doping of NPs under conditions that are difficult using traditional synthesis. The antibacterial activity of NPs was tested against E. coli and Bacillus safensis using disc diffusion, minimum inhibitory concentration, and growth curve methods, revealing a powerful bactericidal activity of Fe-doped ZnONPs. This effect was more prominent when cultured with E. coli than with Bacillus safensis bacteria, when compared to undoped ZnONPs, which showed a reduced inhibitory effect. Additionally, the cytotoxicity of the NPs was studied against MCF-7 cells by an MTT assay, with IC50 values for undoped and 1 wt% Fe-doped ZnONPs of 400 and 600 μg ml⁻¹, respectively. Therefore, the cell viability with Fe-doped ZnONPs was higher than that of undoped ZnONPs, revealing that the doping of these nanostructures helped enhance their biomedical applications (Aiswarya Devi et al 2017). Similarly, the green synthesis of magnetic Fe-doped ZnONPs was developed using Hibiscus rosa-sinensis leaf extracts, with sizes ranging between 15 and 170 nm. While the FTIR analysis confirmed the presence of phytochemicals in the leaf extracts (which helped the formation of NPs), the ferromagnetic behavior was assessed by a magnetization study. After characterization, the NPs were tested as antimicrobial agents towards E. coli, demonstrating excellent antibacterial activity, as compared to those made of pure ZnO and commercial ZnO (Chai et al 2019). Very recently, another interesting report showed the production of ZnONPs doped with rare earth metals (REM) such as lanthanum (La), cerium (Ce) and neodymium (Nd), synthesized by Gymnema sylvestre leaf extracts. The average particle size was observed to be 138 nm, 52 nm, 59 nm, and 63 nm for undoped, La, Ce, and Nd-doped samples, respectively. The synthesized NPs samples were tested against clinical pathogens such as S. aureus, Streptococcus pneumonia, Klebsiella pneumoniae, Shigella dysenteriae, E. coli, P. aeruginosa and a Proteus vulgaris bacterial strain using a well diffusion method. Results revealed that La-coated ZnONPs had a higher antibacterial effect when compared to uncoated, Ce- and Nd-coated samples. In addition, the in vitro cytotoxicity effect was analyzed using a A498 (human kidney carcinoma) cell line and a normal Vero (African monkey kidney) cell line, revealing IC50 values of 41.74, 35.86, 40.05 and 63.08 μg ml⁻¹, respectively. On the other hand, IC50 values were quantified at 55.27, 49.69, 56.83 and 51.10 μg ml⁻¹ for undoped, La-, Ce- and Nd-coated ZnONPs samples (against A498 and Vero cell lines) (Karthikeyan et al 2019).

All these studies successfully revealed that the use of both bacteria and plants allow for the noncomplex, environmentally friendly, and cost-effective production of valuable ZnONPs with antimicrobial properties. These green ZnONPs displayed a comparative advantage over traditionally synthesized ZnONPs, with this behavior clearly making an impact in the research around piezoelectric NMs, shifting efforts towards the employment of these green approaches instead of physicochemical methods.

Cancer is a conjunction of diseases characterized by the abnormal growth of tissue that might lead to the development of tumors that can spread into other tissues and cause severe effects in the patient, with complications and severities potentially causing death ((US) and Study 2007). In 2019, cancer was cataloged as the second leading cause of death in the US with approximately 2 million people being diagnosed every year (Siegel et al 2019). Current approaches such as chemotherapy and radiotherapy, although functional, are not completely effective and present significant drawbacks in the form of severe side effects, such as immunosuppression, anemia, sickness, or even death. In fact, it has been reported in the literature that some cancer cells have become resistant to treatments, leading to the appearance of chemotherapy-resistant tumors, making those treatments no longer a viable option for this kind of patient (Pavlopoulou et al 2016). As a consequence, significant efforts have been made towards the development of new approaches. Consequently, the use of nanotechnology has become more popular, as it can be applied towards cancer treatment and overcome substantial drawbacks (of the traditional treatment options) without experiencing toxicity to healthy tissues (Blanco et al 2015, Mostafavi et al 2019). For instance, different NPs have been shown to effectively encapsulate chemotherapy drugs and deliver them directly to the tumor site, diminishing (or in some cases, completely eradicating) the possible side effects (Ahmed et al 2018). Moreover, the use of NMs allows for a more efficient permeability to the tumor site, when compared to the free drugs (Su and Hu 2018, Asadi et al 2018). NPs can also be functionalized to target specific cancerous cells by attaching different molecules, such as aptamers, proteins or antibodies, and the NPs can specifically bind to the carcinogenic structures (Vahed et al 2019).

ZnO has been investigated in the past few years for anticancer treatments. Since Zn is present in the body as an essential trace element (in tissues such as brain, bones or skin), it often does not compromise cell viability (Jiang et al 2018). On the other hand, ZnONPs are known to induce the production of ROS upon contact with the cells, which leads to mitochondrial damage, triggering cell death in cancer tissue (J. Wang et al 2018). Moreover, ZnONPs can be used in combination with immunotherapy, since they are rapidly taken up by the immune cells because of their electrostatic properties. The surface charge of these NPs is normally neutral but can be modulated depending on the pH; hence at biological conditions, ZnONPs are positively charged. This fact allows for their easy digestion by negatively-charged immune cells (P. Sharma et al 2019). Furthermore, ZnONPs have the ability to showcase luminescence, therefore they could be used as diagnostic tools or targeted drug delivery for cancer diseases. Commonly, ZnONPs can be doped with materials such as Mg or mixed with quantum dots (QD) in order to increase these luminescent properties (Martínez-Carmona et al 2018). As an example, ZnO-QD NPs were loaded with doxorubicin (DOX) to deduce a pH-responsive drug delivery for lung cancer (Cai et al 2016). From the variety of methods for synthesizing ZnONPs, green methodologies offer an ecofriendly and cost-effective approach that overcomes significant drawbacks of traditional synthesis. Different materials have been employed for a considerable amount of time for the production of green ZnONPs with anticancer properties, with plants and algae extracts acting as one of the most common reducing agents, owing to their high natural sugar concentration (Hameed et al 2019). Once generated, the green-synthesized ZnONPs show different cell death mechanisms (figure 4) which are associated with the combination of the metallic ions present on the nanostructures and the natural compounds coming from the natural raw materials employed, leading to some interesting examples in the literature (table 2).

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4.2.1. Fungi-mediated synthesis of ZnONPs with anticancer properties

4.2.2. Algae and plants extract-mediated synthesis of ZnONPs with anticancer properties

4.2.3. Natural biomolecule-mediated synthesis of ZnONPs with anticancer properties

Apart from its antimicrobial and anticancer properties, ZnO nanostructures present antineoplastic, wound healing, ultraviolet scattering, and angiogenic properties, which are often employed in tissue engineering applications (Ghazali et al 2018, Kalantari et al 2020). Particularly, osteogenesis and angiogenesis effects from the ZnONPs have effectively been demonstrated in numerous studies, due to their remarkable thermostability, marked antimicrobial activity, and low cost (Manuja and Raguvaran 2015, Laurenti and Cauda 2017, Medina et al 2020).

Although the rise of ZnONPs has led to new and promising antibacterial and anticancer properties, there has been only select research published highlighted the role of ZnO nanostructures in promoting cell growth, proliferation, differentiation, and increasing the metabolic activity of different cell lines for tissue engineering and regenerative medicine applications (figure 5). Consequently, the implementation of green technology using ZnONPs in tissue engineering has turned into an even narrower field. Nevertheless, a few examples have emerged in the past few years introducing the idea that the pro-angiogenic properties of ZnONPs can be extremely useful enhancing the integration of the scaffolds to host tissue. However, a much better understanding for the mechanism of their selective cytotoxic action, as well as the lack of appropriate biocompatible dispersion protocols, is required in this regard.

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For example, Shubha et al reported the use ZnONPs synthesized by gallic acid, as isolated from the Phyllanthus emblica aqueous extract. The physical, chemical, in vitro, and in vivo toxicity of their products were compared with a clinically advocated ZnONPs. The group used balb mice 3T3 fibroblasts in vitro as one of the key cells of connective tissue (which assist in tissue repair and regeneration). The in vitro and in vivo results revealed that at the highest concentration, green synthesized ZnONPs were less toxic than the commercially available nanostructures. This factoid effectively implies that ZnONPs could be a potential candidate to be applied in the vicinity of connective tissue cells (Shubha et al 2019). Alternatively, an environmentally friendly and affordable synthesis of ZnONPs was reported using Prosophis fracta and coffee extracts as unique raw materials with an average size 26 nm. In vitro tests revealed a strong antibacterial effect of these NPs when cultured with Acinetobacter baumannii and P. aeruginosa cultures. These ZnONPs were impregnated on cotton wound bandages, conferring patches with antimicrobial properties. This property can potentially be used for treating and covering infection-sensitive wounds, namely diabetic or burns wounds (Khatami et al 2018). Similarly, ZnONPs were synthesized using Barleria gibsoni aqueous leaf extracts, which acted as both a reducing and protective agent, because of the presence of polyphenols, flavonoids and amino acids in the extracts. The NPs were characterized as hexagonal wurtzite structures with a size range between 30 and 80 nm. Acting as an efficient and superior tropical antimicrobial formulation, they were tested for their antibacterial properties for healing burn infections. Moreover, the ZnONPs exhibited a remarkable wound healing potential in rats after an in vivo study (Shao et al 2018).

In a similar work, Kumar et al studied the phytochemical effect of a Raphanus sativus (white radish) root extract for the production of ZnONPs, with the aim to develop an effective antimicrobial agent towards E. coli for wound healing applications. While the ZnONPs were synthesized using the root extract, commercially available ZnONPs were bio-functionalized using the same extract, with the goal of analyzing any possible differences in their biomedical applications. Both NPs showed enhanced antimicrobial activity compared to their pure ZnONPs counterparts. After an extensive characterization, it was concluded that the NPs showed a high potential as wound healing agents, with the need of subsequent studies (Kumar et al 2019). Steffy et al reported the synthesis of ZnONPs using Strychnos nux-vomica extracts acting as a powerful agent capable of tackling antibiotic-resistant bacterial pathogens, and for treating a non-healing ulcer. The ZnONPs (with a size range of 10–12 nm) exhibited significant bactericidal potency at a concentration of 100–200 μg ml⁻¹, against MDR–Methicillin-resistant S. aureus, MDR–E. coli, MDR–P. aeruginosa, and MDR–Acinetobacter baumannii. These ZnONPs also exhibited significant bactericidal potency (at a similar concentration range) against the standard bacterial strains S. aureus, E. coli, P. aeruginosa and Enterococcus faecalis. The wound-healing properties were assessed by a scratch assay on a mouse L929 fibroblastic cell line, in order to quantify cell migration towards the injured area. Cytotoxicity was assessed using 3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay on the L929 cell line and a human embryonic kidney epithelial (HEK-293) cell line. This experiment resulted in ZnONPs exhibiting wound-healing and reduced cytotoxic properties at active antimicrobially-favored concentrations (Steffy et al 2018a)

An impediment in the process of wound healing can be attributed to reactive oxygen species and inflammation. Yadav et al assessed the use of ZnONP synthesized by Trianthema portulacastrum Linn, with sizes of 10–20 nm. An in vitro anti-inflammatory activity study of the NPs was conducted by membrane stabilization and albumin denaturation (along with proteinase inhibitory assays), resulting in a significant wound contraction rate, epithelialization and histopathology of the healed tissues of rats, which confirmed the promising wound healing property of the nanostructures. In addition, inflammatory markers and the profile of antioxidant enzymes also support the wound healing potential of NMs, revealing a potential use in wounds via antioxidant and anti-inflammatory activity (Yadav et al 2018).

Over the past few decades, ZnONPs have shown unprecedented performance as a biosensor, owing to a high isoelectric point, biocompatibility and other multifunctional characteristics. This notion explains why ZnONPs have extensively been studied as a transduction material for biosensor development. Besides the fascinating properties of ZnO (already discussed), ZnONPs help retain the biological activity of the immobilized biomolecule, achieving enhanced sensing performance, and allowing for a successful label-free electrical detection of biomarkers (and other molecules of interest). The combination of green nanotechnology with ZnO at the nanoscale has rendered some outstanding examples (table 3 and figure 6).

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Recently, a non-enzymatic glucose biosensor was developed by using a ZnONP-synthesized Ocimum tenuiflorum leaf extract. While the XRD analysis of the NPs revealed a crystalline and hexagonal wurtzite structure, the UV–vis absorption spectrum estimated the ZnONPs band gap of 2.82–3.45 eV with an average size ranging from 10 to 20 nm. Once characterized, a glassy carbon electrode (GCE) glucose sensor was fabricated by coating the electrode with the ZnONPs. The electrode showed superior electrocatalytic activity with a reproducible sensitivity of 631.30 μA mM⁻¹ cm⁻² and a linear dynamic range from 1 to 8.6 mM, coupled with a low detection limit of 0.043 μM. Moreover, the response time was found to be below 4 s (Dayakar et al 2017). In a similar study, ZnONPs synthesized by a Corymbia citriodora leaf extract acting as a unique reducing and stabilizing agent were employed for the construction of an electrochemical H₂O₂ biosensor on a GCE. The sensor exhibited a stronger ability to reduce H₂O₂, contrasted with that of a bare GCE and a chemically modified GCE with ZnONPs. A linear relationship between the current response and H₂O₂ concentration was observed between 0.1 and 150 μM, with a lower detection limit at 0.07 μM. The study also concluded that there was no interference with other molecules, such as uric, ascorbic acids, and glucose, which are three electro-active molecules that commonly coexist in biological systems and could interfere with the electrochemical determination of H₂O₂. The authors were able to demonstrate that a 20-fold excess of the acids and glucose produced negligible current responses, suggesting that the electrode presented an excellent ability to detect H₂O₂, among species prone to interference (Zheng et al 2016).

Alternatively, ZnONPs, with an average size of 4–8 nm, were synthesized by using a Carica papaya seed extract, while using oleic acid as an important capping agent. Later, the authors investigated the electrochemical application of the ZnONPs as a sensor for silymarin, by integrating them with multi-walled carbon nanotubes (MWCNTs) on a GCE. While the electrochemical signals obtained from the electrode were 2-fold higher than both MWCNTs/GCE and bare GCE electrodes, the electrochemical detection could detect 122 mg of silymarin in the quoted concentration, confirming a detection efficiency of approximately 76%. At a molecular level, the authors found that the charge transfer from ZnO clusters to OH groups (of silymarin) strengthened the interactions between the silymarin molecule and ZnO clusters. The obtained energies indicated the most probable position and functional groups in silymarin, which led to its detection and binding with a linear relationship found between 0.014 and 0.152 mg l⁻¹, with a lower detection limit of 0.08 mg l⁻¹ (D. Sharma et al 2018). A stable enzymatic glucose sensor, of a highly selective environmentally friendly and sensitive nature, was fabricated on GCE using ZnONPs embedded in a nitrogen-doped carbon sheet (together with glucose oxidase (GOx)). The production of these sheets was achieved by a simple hydrothermal method, wherein Zn powder, aqueous ammonia, and peach extract served as predecessors for the ZnONPs, nitrogen and carbon, respectively. Once built, the fabricated biosensor exhibited a high and reproducible sensitivity of 231.7 μA mM⁻¹ cm⁻², a linear range from 0.2 to 12 mM, along with a correlation coefficient R² of 0.998 and the lowest detection limit of 6.3 μM. The authors found the biosensor to be generally stable and selective, which allowed for its successful application to human blood serum for quantitative monitoring (Muthuchamy et al 2018). On a similar note, an ecofriendly method for the synthesis of self-assembled hexagonal pyramids of ZnONPs was created through the use of thermal decomposition of zinc nitrate. These novel pyramids were thoroughly characterized, and tested for their features as a biosensor, towards the detection of dopamine at trace levels. These synthesized ZnONPs showed a linear range for dopamine up to 300 μM, with a detection limit of 1 μM. While efforts to reproduce results over periods of several months took place, its electrochemical performance indicated no significant deviation (Udayabhanu et al 2016). Correspondingly, the green synthesis of ZnONPs decorated MWCNTs–graphene hybrid composite was treated with focused sunlight (a clean energy source) in the form of a convex lens of 90 mm diameter (exposed for 1–2 min). Upon coating with a GCE, the composite was evaluated for its application as a transducer candidate as a organophosphorus biosensor. The hybrid composite showed noteworthy electrochemical activity, owing largely to its large electrochemically-active surface area, in comparison to the ZnONP-decorated graphene prepared by the same root. Later, this phenomenon was discovered due to the presence of MWCNTs between the graphene layers. These graphene layers effectively prevent its restacking, thereby increasing the likelihood of accessing the surface area of interest. Using this composite as a transducer candidate, this fabricated biosensor presented a high affinity to acetyl cholinesterase (AChE) enzyme with an evident Michaelis–Menten constant (Km) value of 0.8 mM. Further, a linear response for Paraoxon detection was exhibited from 1 to 26 nM, with a detection limit of 1 pM (Nayak et al 2013).