It is known that materials with different morphology can be obtained due to changing the synthesis parameters. ZnO NPs and NRs were obtained by facile sol-gel approach, which is based on the hydrolysis of zinc acetate with the addition of NaOH in a methanol solution. The mixing of the main precursors led to fast sol formation and then to the white homogeneous colloid solution (gel) of ZnO according to reactions (Eq. 1,2).
Zn 2+ + OH− → Zn(OH)2↓ (1)
Zn(OH) 2 ↓ → ZnO↓ + H 2 O (2)
The obtaining of larger ZnO nanoparticles (ZnO Ps with sizes approximately 150 nm) was performed due to Ostwald ripening, where small crystals dissolve and redeposit onto larger crystals using high temperatures 29.
ZnO HSs were formed from ZnO seeds by their aggregation to rods, assembly to plates, and finally to the spherical framework. The formation of the hierarchical structures is due to the use of “structural director” (generally organic polar molecules). In our case, trisodium citrate dihydrate was used for this purpose.
Obtaining the ZnO TPs was due to the fast formation of the ZnO seeds and then the formation of legs with preferred growth direction along the c-axis of the hexagonal unit cell. It is generally accepted that the nucleation and growth of ZnO tetrapods are understood to occur in the vapor phase during synthesis 30. The growth mechanism of the tetrahedral ZnO particles can be explained by the growth model proposed by Alsultany F.H. et al. and Markushev V.M. et al. 31,32. In our case, where no metal catalyst is used, the growth of ZnO TPs can be divided into several steps - nucleation and growth. Extremely high temperature leads to the reaction of the metal Zn (Znmet) and then Zn gas (Zng) with O2 gas (O2g) to form ZnO gas seeds (ZnOg), (Eq. 3,4). Then during constant heating, gaseous ZnO or unoxidized Zn will condense into a liquid state of Znl or ZnOl (Eq. 5), and finally following O2 absorption leads to the crystallization of ZnO solid seeds (ZnOs).
Zn met → Zng (1000 ºC) (3)
Zn g + O2g → 2ZnOg (4)
ZnO g (Zng) → ZnOl (Znl) (5)
ZnO l (Znl) + O2g → ZnOs (6)
According to the octa-twin nucleus model proposed by Iwanaga H. et al. 33, the ZnO seeds have an octahedral shape. These octahedral seeds are substrates for the following growth of ZnO TPs.
Obtained all ZnO nano- and microparticles were highly crystalline. It is well known that particles in nanorange or materials with unique structures. Including hierarchical structure possess high specific surface area. Often the specific surface area is strongly related to the particle size and increased with decreasing particle size 34. These results are consistent with the results of particle size estimated by SEM/TEM analysis. The samples with the highest surface areas (ZnO NPs and ZnO NRs) showed narrow pore size distribution with most of the pores in the size range from 3 to 10 nm, whereas the samples with lower surface areas possess additionally pores with a diameter above 10 nm.
Analysis of PL spectra for different semiconductor nanostructures is a powerful tool to investigate their morphology features, defects, and even chemical composition 35. Typical ZnO nanostructures, such as nanoparticles, nanorods, etc., exhibit two luminescence bands located at the UV region (the near band emission - NBE) and a broad long-wavelength band at the Visible spectrum (the deep-level emission - DLE) 35. The unique optical properties are very important for medical and biological visualization applications 36. Depending on the PL intensity, PL peaks position, and the ratio INBE/IDLE, one can conclude about the structural features of produced ZnO nanostructures. It is well known that the DLE is associated with different ZnO defects, such as zinc vacancies (Zni ++), single (Vo+), and double (Vo++) ionized oxygen vacancies, neutral oxygen vacancies (Vo), and oxygen interstitials (Oi). According to previous studies, there are three main defects involved in the DLE: Vo+ (2,45 eV), Vo++ (2,23 eV), Oi (2 eV) 35. Besides, in our case, the ratio INBE/IDLE indicates the good crystallinity of produced nanostructures. It was shown previously, that the increased concentration of oxygen sites in the ZnO leads to the variation of antibacterial properties of produced nanoparticles 37,38. Therefore, it is expectable that the different ZnO nanostructures would demonstrate various antibacterial behaviors.
The OD measurement is mainly used as a quick and affordable method to monitor the growth of bacteria during their culture in liquid media but can also be applied for testing antibacterial properties of different nanostructures and nanomaterials 39–41. The higher the number of bacteria in the solution, the greater the OD570 value, and thus the lower antibacterial activity of the added material, ZnO nano- and microparticles in this case.
The high antibacterial activity towards E. coli bacteria showed ZnO NPs, NRs, and HSs (Fig. 5). This could be related to the surface area, which is the largest for 3D heterostructures, and volume to surface ratio, the highest for nanoparticles and nanorods, respectively. The lowest surface area of ZnO Ps and TPs led to a decrease in antibacterial activity. Even at the highest concentration (1 mg/mL), their viability reached about 62% and 76%, respectively.
As it was mentioned above, the OD570 is proportional to the total number of bacteria, however, it does not provide any information regarding their viability. Thus, additionally, the LIVE/DEAD BacLight staining with the use of confocal laser scanning microscopy was carried out. To recognize live and dead bacteria two fluorescence dyes were used. SYTO 9 stains in green both live and dead cells, and propidium iodide (PI) stains in red dead cells, that have lost membrane integrity. Confocal images of E. coli and S. aureus were shown in Figs. 7 and 8, respectively. All of the untreated bacterial cells showed green fluorescence, due to the viable cells, indicating intact cell wall structure.
As can be seen in Fig. 7, the co-incubation of E. coli cells with ZnO nano- and microparticles for 2h was enough to influence the bacteria viability. In all cases, the number of bacterial cells decreased. Moreover, the red signals, indicating dead cells appeared. The counting of both signals, collected from nine randomly selected images for all samples, allowed us to determine the percentage of live and dead cells. Based on this, we can conclude that ZnO NRs and ZnO HSs were the most effective towards E. coli, with the number of dead cells above 50%. As it turned out after optical density measurements, also here, the least effective were ZnO particles and tetrapods.
For the S. aureus strain (Fig. 8) ZnO materials exhibited a stronger antibacterial effect than for E. coli. The number of bacterial cells was significantly reduced compared to the non-treated control. Moreover, after ZnO nanoparticles treatment the number of dead cells was about 50:50 to living cells, whereas, for nanorods and heterostructures, the percentage of viable cells decreased to about 20%. Also, the ZnO TPs decreased cells viability, however in this case the general number of cells seems to be higher. As in the case of E. coli, the ZnO particles show the weakest antibacterial activity. The obtained results for viability analysis were compatible and comparable with optical density measurements. As it was mentioned before the antibacterial effect could be related to the surface-to-volume ratio of ZnO materials, which is consistent with Azam A. et al. 42 whose indicated that the antimicrobial activity increased due to a decrease in particle size of zinc oxide nanoparticles, as well as with Yamamoto O. 43, who indicated that smaller size of zinc oxide nanoparticles exhibits greater antibacterial activity than microscale particle.
Four mechanisms of action have been proposed as responsible for the antibacterial properties of zinc oxide particles, namely the production of reactive oxygen species (ROS) 44,45, the loss of cellular integrity after contact of ZnO materials and the cell wall 46, ZnO NPs internalization 47, as well as the release of Zn2+ ions 48,49. The mechanism of nanomaterials toxicity is not specific, and thus bacteria are not able to get the resistance for nanoparticles treatment 50. The differences in antimicrobial activity of ZnO materials used in this study depending on their size and shape could be related to the distinct mechanism of action. The smallest nanoparticles and nanorods probably internalize bacterial cells, whereas particles of micrometer size like tetrapods and heterostructures can interact with cell walls, through ion diffusion and free radicals generation, which further enter the cells, destroying cellular components such as DNA, proteins, and lipids.
Generally is it thought that Gram-negative bacteria are more susceptible than Gram-positive to attack by external factors, such as metal nanoparticles like it was observed for silver 51 and gold nanoparticles 52. As the main reason for differences in bacterial susceptibility and resistance the bacterial cell walls composition is suggested. In the case of Gram-negative bacteria, bacterial cells are covered by a layer of lipopolysaccharides (1–3 µm thick) and thin peptidoglycans (~ 8 nm thick), whereas Gram-positive bacteria possess a peptidoglycan layer (~ 80 nm thick) with covalently attached teichoic and teichuronic acids 53. However, here we observed that Gram-negative Escherichia coli were less susceptible to ZnO materials than Gram-positive S. aureus, which is consistent with Tayel and co-workers 54, who showed that the inhibition of Gram-negative bacteria requires higher concentrations of ZnO NPs. This is likely because the peptidoglycan layer that surrounds Gram-positive bacteria can promote ZnO attack inside the cell, while the cell wall components of Gram-negative bacteria, such as lipopolysaccharides, can counter this attack. Similar results were found by d’Agua R.B. et al. 55, who showed that Gram-positive bacteria were more sensitive to peroxide hydrogen than Gram-negative bacteria. It was also seen in our earlier studies with gelatin-ZnO nanofibers 56.
At cytotoxicity studies (Fig. 6), results showed that all nan- and microparticles are biocompatible at low concentrations. The greatest decrease in viability above 100 µg/mL was seen with the administration of ZnO nanorods and nanoparticles, and then heterostructures. The differences could be related to the distinct mechanism of action, and different levels of nanoparticles internalization. The toxicity mechanism is comparable with antibacterial action, which means that the ROS generation, mechanical harm due to direct interaction of ZnO materials with the cells, cells internalization, as well as zinc ions releasing could be responsible for cytotoxic activity towards human cells. Moreover, Cho W-S. et al. 57 indicated that zinc oxide nanoparticles rapidly dissolve under acidic conditions (pH 4.5), which may occur after absorption of nanoparticles into lysosomes in the process of endocytosis, leading in turn to cell death.