Synthesis, characterization, size and shape-dependent antibacterial activity and cytotoxicity profile of ZnO nano- and microparticles

Synthesis, characterization, size and shape-dependent antibacterial activity and cytotoxicity profile of ZnO nano- and microparticles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis, characterization, size and shape-dependent antibacterial activity and cytotoxicity profile of ZnO nano- and microparticles Nataliya Babayevska, Łucja Przysiecka, Igor Iatsunskyi, Grzegorz Nowaczyk, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1317820/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The aim of our work was the synthesis of ZnO nano- and microparticles and to study the effect of shapes and sizes on cytotoxicity towards normal and cancer cells and antibacterial activity toward two kinds of bacteria. We fabricated ZnO nano- and microparticles through facile chemical and physical routes. The crystal structure, morphology, textural properties, photoluminescent properties were characterized by powder X-ray diffraction, electron microscopies, nitrogen adsorption/desorption measurements, and photoluminescence spectroscopy. The obtained ZnO structures were highly crystalline and monodispersed with intensive green emission. ZnO nanoparticles and nanorods showed the strongest antibacterial activity against Escherichia coli and Staphylococcus aureus compared to microparticles due to their high surface area. However, the ZnO hierarchical particles at higher concentrations also strongly inhibited bacterial growth. S. aureus strain was more sensitive to ZnO particles than the E. coli. Nanoparticles and nanorods were more harmful to cancer cell lines than to normal ones at the same concentration. ZnO morphology crystal structure photoluminescence antibacterial activity cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Successful fabrication of materials with desired properties using simple and low-cost approaches attracts a great deal of attention. Materials properties are strongly dependent on their characteristics, mainly phase composition, crystallinity, shape and size, dispersion, etc. Materials morphology plays an important role in optics, photonics, sensors, solar cells to obtain high-quality devices 1 – 3 . For example, it is well-known that the photocatalytic activity of semiconductor materials (e.g. NiO, TiO 2 ) depends strongly on their particle sizes, basically due to enhancing specific surface area 4 , 5 . Materials for bioapplication, such as biomarkers, biosensors, or drug carriers with improved optical, sensing, and surface properties can be obtained due to changing their shape, size, and dimension 6 , 7 . Nanoparticles due to their small size have exciting physicochemical properties, such as a high surface-to-volume ratio, a size similar to the size of biomolecules allowing for their greater mobility and passing through the cell biological membranes 8 . Morphology and surface area affect the antibacterial activity, therefore, it is necessary to synthesize a material with a novel morphology having a higher amount of adsorption sites and surface area 9 – 11 . Thus, verifying the morphology of the materials and hence their surface area can show great potential for bioapplication because the properties can be tuned and improved. Decreasing the particle size (nanosized materials, such as 0D materials (e.g. quantum dots, QDs)) is one of the effective ways to increase the surface area. Despite many advantages of the nanoparticles, their main disadvantage is particle agglomeration. Modern approaches allow obtaining high-quality nanoparticles without any stabilizers and surfactants 12 , 13 . Anisotropic multidimensional and multicompartmental particles with intriguing morphologies are the subjects of extensive research. One dimension materials (1D) offer high surface area because of their unique morphologies. 3D materials are seen to be more suitable for many applications than other morphologies/structures as they may be assembled from lower dimensional materials and provide a large surface area and fast electron transport 14 . Metal oxide nanoparticles have great attention in materials science and nanotechnology for a large variety of applications. From semiconductor materials, zinc oxide (ZnO) is an important multifunctional material with broad applications in many scientific fields. As a promising semiconductor oxide (large exciton binding energy (60 meV and wide bandgap Eg = 3.37 eV) ZnO possess excellent physicochemical properties, such as high thermal and chemical stabilities, thermal conductivity, UV protection or electronic, photonic, unique optical, and luminescent properties. At the same time, ZnO is a low-cost material, its synthesis is simple and it can be obtained with a big variety of morphology using physical and chemical approaches. ZnO with different morphology and dimension ( e.g. bulk ZnO, nanoparticles, films, 3D hierarchical structures) has broad applications as sensors, catalysts, solar cells, optoelectronic devices 15 – 17 . Furthermore, zinc is a necessary element to our health and has high biocompatibility to human cells, low toxicity, and good antimicrobial activity 10 , 18 , 19 . All of these properties allow using ZnO in medicine as biomarkers, drug carriers, and therapeutic 20 , 21 . Thus, Warheit D.B. et al. studied the effect of ZnO morphology on the toxicity of lung cells 22 . They showed a low cytotoxic effect on rat L2 lung epithelial cells and primary rat lung alveolar macrophage treated with ZnO nanoparticles (NPs). Z. Li. et al. studied the cell viability of ZnO nanowires using two cell lines 23 . Results showed that ZnO nanowires were biocompatible to HeLa cells up to the highest concentration used (up to 100 µg/mL), whereas for L929 cells were deleterious even at lower concentrations. ZnO possesses unique antibacterial, antimicrobial and antifungal properties, ZnO-based materials can initiate antibacterial activity even without the presence of light 24 . In presented studies 19 , 25 , 26 authors obtained ZnO with different morphology and indicated that ZnO and its composites can be used as good antibacterial agents toward Gram-positive, as well as Gram-negative bacteria. Thus, despite intensive research in this field, the evaluation of ZnO antibacterial activity and biocompatibility due to changing the particle morphology is still important and can significantly expand the scope of this material in biomedical applications. The main aim of our work was a synthesis of ZnO nano- and microparticles, the study of their crystal structures, and the effect of morphology and surface area on luminescent properties, cytotoxicity, and antibacterial activity toward Gram-positive and Gram-negative bacteria. 2. Results 2.1. Fabrication of ZnO nano- and microparticles ZnO nano- and microparticles with different morphology were fabricated using facile chemical and physical methods. We performed the study of the shape and size of the obtained ZnO nano- and microparticles using the transmission (HRTEM) and scanning electron microscopes (SEM). Indeed, in nanoparticles synthesis, we obtained the highly crystalline and monodispersed ZnO nanoparticles (ZnO NPs) and nanorods (ZnO NRs) using the sol-gel approach without any additional surfactants or ligands. ZnO NPs had a perfect spherical shape and uniform size distribution with a mean nanoparticle’s size of approximately 7 nm (Fig. 1 a, S1a). The FFT digital diffractogram pattern presented in Fig. S1b from nanoparticles with [010] zone axis orientation, demonstrated individual point reflections that indicate that particles had a single crystalline structure. The interplanar distance between adjacent lattice fringes is 2.57 Å corresponding to d(002). Changing the synthesis parameters in ZnO NP synthesis (reagent concentration and synthesis time, and temperature) we obtained ZnO NRs and ZnO particles (ZnO Ps), (Fig. 1 b and 1 c). The mean size of ZnO NRs was approximately 5×17 nm (width×length), Fig. S1c. After annealing of ZnO NPs at 900°C for 2h, we obtained ZnO Ps with sizes of ~ 150 nm (Fig. 1 c). To obtain ZnO with the hierarchical structure (ZnO HSs) we used a solvothermal reaction. The SEM image in Fig. 1 d confirmed the formation of the flower-like (peony) structure of ZnO. All individual particles had a spherical shape, were highly crystalline, and monodispersed with sizes of approximately 4 µm and the thickness of the individual plates of about 100 nm (Fig. S1d). We also obtained ZnO in the shape of tetrapods using thermal evaporation in the air. Tetrapods were well faceted with four uniform legs connected to a central nucleus (Fig. 1 e, S1e). The legs had hexagonal morphology with a length of about 5.2 µm and approximately 170 nm in thickness. 2.2. Structural characterization of ZnO nano- and microparticles Next to determine the crystal structure and phase purity of the obtained ZnO materials we performed XRD measurements. Figure 2 shows the XRD pattern of ZnO particles with different morphology. X-ray powder diffraction confirmed the formation of a single-phase for all ZnO samples. Table 1 Crystallographic parameters of the obtained ZnO nano- and microstructures. Sample XRD Cell parameters a , nm c , nm V , nm 3 ZnO NPs 0.324 0.526 4.811 ZnO NRs 0.326 0.528 4.853 ZnO Ps 0.324 0.520 4.753 ZnO HSs 0.324 0.521 4.766 ZnO TPs 0.325 0.521 4.764 All diffraction peaks are indexed according to the hexagonal phase of ZnO wurtzite crystal structure with main (100), (002), (101), (102), (110), (103), (112), and small (201) and (202) crystal planes. No additional peaks indicating the presence of impurity phases were found in all studied samples. The broadening of the peaks for ZnO NPs and ZnO NRs samples (Fig. 2 , black and red lines) can be attributed to the small particle size of ZnO. Calculated crystallographic parameters of zinc oxide particles are presented in Table 1 . The results indicate higher lattice constant values for ZnO NPs and ZnO NRs samples compared with the others. The lowest lattice constant a and c are observed for ZnO Ps and are the results of their preparation condition (high-temperature annealing). Similar results were presented by Lupan O. et al. 27 indicating a decrease of the lattice constant of ZnO with the increase of the temperature of its treatment. 2.3. Textural properties of ZnO nano- and microparticles The next stage was the study of the textural properties of the investigated samples and we performed their N 2 adsorption-desorption analysis on powdered ZnO samples at -196°C. The specific surface area, pore volume, and pore size distribution of samples were specified. The results are summarized in Table 2 and presented in Fig. 3 . The adsorption/desorption isotherms of investigated ZnO materials correspond to type IV typical for mesoporous materials. The estimated BET specific surface area of the obtained ZnO nano- and microparticles were 83.5 m 2 /g (NPs), 83.8 m 2 /g (NRs), 3.0 m 2 /g (Ps), 4.5 m 2 /g (HSs), and 29.4 m 2 /g (TPs). The lowest surface area, as well as pore volume, showed ZnO Ps and ZnO HSs. Table 2 Surface area and pore volume of ZnO samples with different morphology. Samples S BET , m 2 /g Pore volume, cm 3 /g ZnO NPs 83.5 0.18 ZnO NRs 83.8 0.17 ZnO Ps 3.0 0.08 ZnO HSs 4.5 0.04 ZnO TPs 29.4 0.04 2.4. Photoluminescence properties of ZnO nano- and microparticles The PL spectra of ZnO nanostructures are shown in Fig. 4 a. All investigated ZnO nanostructures demonstrate the intense and broad peak in the visible range. The intense emission for ZnO TPs and Ps is mostly associated with oxygen vacancies (Fig. 4 b), whereas the PL for ZnO NPs, NRs, and HSs is attributed to oxygen interstitials (Fig. 4 c). 2.5. Antibacterial properties of ZnO nano- and microparticles As a next important step, to use our ZnO-based materials as potential antibacterial agents, the antimicrobial activity of ZnO nano- and microstructures was evaluated against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacterial strains using optical density measurements after 24-hour incubation, as summarized in Fig. 5 . By measuring the optical density at 570 nm (OD 570 ), the growth of bacteria can be quantified, based on turbidity resulting from light scattering 28 . For all samples, the bacteria number decreased in a concentration-dependent manner. The most effective towards E. coli bacterial strains were ZnO nanoparticles and nanorods, as well as ZnO hierarchical structures at the highest concentration, and the least antibacterial effect was observed for ZnO Ps and TPs. The Gram-positive S. aureus bacteria strains were more sensitive to the antimicrobial action of ZnO nano- and microparticles than E. coli . However, also here, the greatest lowering of the bacteria number was visible after incubation with ZnO NPs, NRs, and HSs with the highest surface areas. 2.6. Cytotoxicity studies of ZnO nano- and microparticles The impact of distinct ZnO nano- and microparticles on human cells was evaluated by cytotoxicity analysis by WST-1 assay, performed on cervical cancer cell line (HeLa) and normal human fibroblasts (MSU1.1) (Fig. 6 ). Five different ZnO nano- and microparticles concentrations of 0.1, 1.0, 10, 100, and 1000 µg/mL were employed, and the cell viability was determined after 24h of treatment. The cell viability data revealed that for both cell types at the concentration up to 10 µg/mL all kinds of ZnO particles were biocompatible, maintaining a high level of viability (up to 80%). However, at a higher concentration range (100–1000 µg/mL) the cell viability declines to 40%, indicating a high level of toxicity. Interestingly, at a concentration of 100 µg/mL, a similar level of viability was observed for all samples on MSU1.1 cells, while for HeLa cells significant differences were visible. 3. Discussion 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 (Zn met ) and then Zn gas (Zn g ) with O 2 gas (O 2g ) to form ZnO gas seeds (ZnO g ), (Eq. 3,4). Then during constant heating, gaseous ZnO or unoxidized Zn will condense into a liquid state of Zn l or ZnO l (Eq. 5), and finally following O 2 absorption leads to the crystallization of ZnO solid seeds ( ZnO s ). Zn met → Zn g (1000 ºC) (3) Zn g + O 2g → 2ZnO g (4) ZnO g (Zn g ) → ZnO l (Zn l ) (5) ZnO l (Zn l ) + O 2g → ZnO s (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 I NBE /I DLE , 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 (Zn i ++ ), single (V o + ), and double (V o ++ ) ionized oxygen vacancies, neutral oxygen vacancies (V o ), and oxygen interstitials (O i ). According to previous studies, there are three main defects involved in the DLE: V o + (2,45 eV), V o ++ (2,23 eV), O i (2 eV) 35 . Besides, in our case, the ratio I NBE /I DLE 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 OD 570 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 OD 570 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 Zn 2+ 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. 4. Conclusion The simple chemical and physical methods were used to obtained ZnO nano- and microparticles with different morphologies. The morphology effect on luminescent and surface properties, antibacterial activity toward Gram-positive ( Staphyloccocus aureus ) and Gram-negative ( Escherichia coli ) bacteria, and cytotoxicity toward normal and cancer cells was studied. All obtained ZnO materials were highly crystalline with hexagonal wurtzite crystal structures and monodispersed. N 2 adsorption-desorption analysis showed that ZnO in nanorange (nanoparticles and nanorods) had a higher surface area. All samples performed high green emission caused by structural defects in ZnO. Due to high crystallinity ZnO tetrapods (TPs) showed the strongest photoluminescence. The antimicrobial activity measurements of ZnO nano- and microparticles indicated that for all samples, the bacteria number decreased in a concentration-dependent manner. Results showed that surface area has a significant impact on antibacterial performance. The high surface area of ZnO nanoparticles led to the most effective antibacterial activity towards E. coli bacterial strains. Among used bacteria, the Gram-positive S. aureus strains were more sensitive to the antimicrobial action of ZnO nano- and microparticles than E. coli . All kinds of ZnO materials were biocompatible towards cervical cancer cell line (HeLa) and normal human fibroblasts (MSU1.1), maintaining a high level of viability (up to 100 µg/mL). As we can see materials of different sizes and varieties of morphology can be used in biomedical fields. According to our present results, highly luminescent and biocompatible ZnO particles in nanorange (especially ZnO NPs and NRs) are planned to be used for cancer diagnostic and therapy. At the same time, the microparticles will be used for biosensors, drug adsorption, and release experiments. 5. Material And Methods 5.1. Materials Zinc acetate dihydrate (Zn(CH 3 COO) 2 ×2H 2 O, Zn(Ac) 2 , Sigma Aldrich), sodium hydroxide (NaOH, Stanlab), methanol (MeOH, Sigma Aldrich), ethanol (EtOH, Sigma Aldrich), tri-sodium citrate dihydrate (HOC(COONa)(CH 2 COONa) 2 ×2H 2 O, Sigma Aldrich), Zn powder (Sigma Aldrich) were all of the analytical grades and used as starting materials. 5.2. Synthesis of ZnO nano- and microparticles 5.2.1 Synthesis of ZnO nanoparticles ZnO nano- and microparticles (nanoparticles (ZnO NPs), nanorods (ZnO NRs), hierarchical flower-like structures (ZnO HSs), and tetrapods (ZnO TPs)) were synthesized using chemical and physical approaches presented in schematic illustration (Fig.S2). The ZnO NPs and ZnO NRs were obtained by the modified low-temperature sol-gel route described in 58 . The proposed synthesis approach is facile and allows to obtaining of the crystalline monodisperse ZnO NPs (Fig.S2a) at a temperature below 100 °C. In ZnO NPs synthesis Zn(Ac) 2 was dissolved in methanol at a constant temperature of 60 °C. Then the solution of NaOH in methanol was added to Zn(Ac) 2 solution under vigorous stirring. The complete hydrolysis of zinc acetate with the addition of NaOH in a methanol solution results in the formation of ZnO colloid (gel). After about 3h of stirring, white ZnO nanoparticles were separated from the mother liquor, washed with methanol twice, and dried. In the synthesis of ZnO NRs, the reaction mixture (prepared in the same manner as for ZnO NPs synthesis) was concentrated 10 times by solvent evaporation. After about 12h of stirring white precipitate was separated, washed, and dried as described above. ZnO particles (ZnO Ps) were obtained using ZnO NPs (obtained earlier) by their annealing at 900 °C for 2h (Fig.S2b). 5.2.2. Synthesis of ZnO microparticles The ZnO 3D hierarchical structures (ZnO HSs) (Fig.S2c) were prepared by a template-free solvothermal approach according to Fang et al. 59 . First, Zn(Ac) 2 and sodium citrate were dissolved in H 2 O. Separately NaOH (1g) was dispersed in EtOH at 60 °C. After complete dissolution of both precursors, NaOH solution was added dropwise to Zn 2+ solution. The resulting mixture was agitated at room temperature for 1h. Then, for solvothermal reaction (150 °C for 24h) the resulting mixture was transferred into a Teflon-lined stainless steel autoclave. After cooling down final product was centrifuged, washed with H 2 O, and dried at 60 °C for 12h before characterization. ZnO tetrapods (ZnO TPs), (Fig.S2d) were obtained by the simple catalyst-free oxidative-metal-vapor-transport method. The method was based on thermal evaporation of Zn powder at 1000 °C for 1h in the air 29 . 5.3. Characterization Structural characteristics of the obtained ZnO nano- and microparticles were measured by powder X-ray diffraction (XRD). The studies were carried out on powdered samples using an Empyrean (PANalytical) diffractometer with Cu Kɑ radiation (λ=0.154 nm), reflection-transmission spinner (sample stage), and PIXcel 3D detector, operating in the Bragg-Brentano geometry. Scans were recorded at room temperature in angles ranging from 20° to 80° (2θ) with a step size of 0.006° and continuous scan mode. The morphology of the obtained samples was studied by high-resolution transmission electron microscopy (HRTEM; JEOL ARM 200F) and scanning electron microscopy (SEM, JEOL, JSM-7001F). For the determination of the specific surface area of the samples, the N 2 adsorption/desorption isotherms were measured at -196 °C on a Quantachrome Nova 1000 apparatus. The samples were outgassed at 150 °C for 15h in a vacuum before the measurements. The specific surface area was determined using the BET method. The total volume of pores was calculated using the single point mode (at p/p 0 = 0.98). The pore size distribution was determined by applying the Barrett-Joyner-Halenda (BJH) method from the desorption branch of the isotherm. The photoluminescence (PL) of the samples was measured at room temperature using Kimmon HeCd laser. The excitation wavelength was 325 nm, and the power was around 2 mW. The PL spectra were recorded in the range from 360 to 1000 nm by Ocean Optics Spectrometer QE65 pro. 5.4. Biological characterization 5.4.1 Cell line and cell culture conditions Human cervical cancer cell line HeLa was obtained from American Type Culture Collection (ATCC). Human fibroblast cell line MSU1.1 was obtained from Prof. C. Kieda (CBM, CNRS, Orléans, France). Cells were cultured in a complete medium Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/mL streptomycin, and grown at 37 °C in a humidified atmosphere containing 5 % CO 2 . 5.4.2 Cytotoxicity analysis Cervical cancer cells (HeLa) and normal fibroblasts (MSU1.1) were used for in vitro cellular toxicity studies of ZnO nano- and microparticles. Cells (1×10 3 cells/well) were seeded onto 96-well plates and incubated overnight at 37 °C under a 5 % CO 2 atmosphere. The medium in the wells was then replaced with a fresh medium containing increasing concentrations of ZnO particles (from 0.1 μg/mL to 1 mg/mL) and incubation was continued for 24h. The medium without ZnO particles was used as a negative control. The effect of the ZnO nano- and microparticles on cell proliferation and viability was determined by WST-1 assay according to the manufacturer’s instructions. Briefly, 10 μL of WST-1 solution was added to each well and the plates were further incubated. After 2h the absorbance was measured with a microplate reader (AnthosZenyth 340rt) at 450 nm and 650 nm as reference. The mitochondrial function and, by extension, the relative cell viability (%) related to the negative control was calculated by test sample/negative control ×100 %. Data are reported as the average ± standard deviation (SD) of wells performed in quadruplicate. 5.4.3 Bacterial growth inhibition study Stock cultures of bacterial strains E. coli ATCC 35218 and S. aureus ATCC 29213 were stored in 30 % glycerol. The strains were cultured in LB Broth Lennox at 37 °C with constant agitation at 230 rpm for 24h. The bacterial cultures were then diluted between 2.5×10 5 and 5×10 5 cells/mL in LB broth medium. 100 μL of the cell suspension was then added to each well of a 96-well plate. Appropriate concentrations of freshly prepared ZnO particles solutions (10, 100, 250, 500, 1000 μg/mL) were added and placed at 37 °C in an incubator. Turbidity of the suspension, as a measure of bacteria growth, was recorded spectrophotometrically at 570 nm (OD 570 ) with a microplate reader (Anthos Zenyth 340rt) after 24h. To avoid potential interference during optical measurements caused by the light scattering properties of the solutions, the same liquid medium without microorganisms, but containing the same concentration of studied samples, were used as blank controls. The positive control was bacterial cultures without ZnO particles treatment. All experiments were performed in triplicates. 5.4.4 Bacterial cells viability analysis To visualize the effect of S. aureus and E. coli cells' interaction with ZnO particles with different morphology, the fluorescence assay LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies) was applied and observed under a confocal laser scanning microscope (Olympus, FV1000). In brief, the S. aureus and E. coli overnight cultures were used to inoculate of fresh LB medium. At the mid-log phase of bacterial growth, the ZnO nano- and microparticles solutions were added to the final concentration of 500 μg/mL and allowed to grow for 3h. From these cultures, 1 mL of each bacterial solution was centrifuged at 5000 rpm for 10 minutes. The pellets were resuspended in HEPES buffer, centrifuged, and washed with HEPES buffer three times more. Finally, the pellets were resuspended in 500 μL of HEPES buffer, and the combination of fluorescent dyes SYTO9 and PI were mixed in identical volumes. 1.5 μL of their mixture was added to each bacterial suspension and incubated in dark for 15 minutes. Fluorescence images were taken by trapping 5 μL of stained bacterial samples mounted on glass slides with mounting medium and cover with coverslips. For each sample, nine randomly selected images were captured by the microscope, and live/dead cells were counted to ascertain percentage viability. Data presented are live (green) and dead (red) cells as a percentage of the total cell number (live + dead). Declarations Acknowledgements The authors gratefully acknowledge the financial support by the following projects: H2020-MSCA-RISE-2017, CanBioSe 778157 (II), SONATA BIS 6 UMO-2016/22/E/ST3/00458 (GN, ŁP), WPC2/nanoHEART/2021 (NB, SJ), and SPUB - 41/E-336/SPUB/SP/2019 (MJ, SJ, GN). Authors' contributions N.B. wrote the first version of this manuscript and prepared all samples. N.B., L.P., I.I, G.N., M.J., E.J., and SJ reviewed and prepared the final version of the manuscript. L.P. performed bacterial culture and all bacteria-based experiments and human cell cultures. M.J. and N.B. performed XRD analysis of the obtained samples. G.N. and I.I. performed scanning and transmittance electron microscopy. E.J. provided and characterized the textural properties of the obtained samples. I.I. studied the photoluminescence properties. All authors commented and have approved the final version of the manuscript. Conflict of interests There is no conflict of interests. References Boopathi Raja, R., Parthibavarman, M. Reagent induced formation of NiCo 2 O 4 with different morphologies with large surface area for high performance asymmetric supercapacitors. Chem. Phys. Lett. 755 , 137809 (2020). Sharma, N., Sharma, S. K., Sachdev, K. 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Influence of particle size on the antibacterial activity of zinc oxide. Int. J. Inorg. Mater. 3 , 643-646 (2013). Guo, B-L. et al . The antibacterial activity of Ta-doped ZnO nanoparticles. Nanoscale Res. Lett . 10(1) , 1047 (2015). Raghupathi, K. R., Koodali, R. T., Manna, A. C. Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir . 27 , 4020-4028 (2011). Zhang, L., Jiang, Y., Ding, Y., Povey, M., York, D. Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). J Nanopart Res . 9 , 479-489 (2007). Brayner, R., Ferrari-Iliou, R., Brivois, N., Djediat, S., Benedetti, M. F., Fiévet, F. Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Lett . 6(4) , 866-870 (2006). Li, M., Zhu, L., Lin, D. Toxicity of ZnO nanoparticles to Escherichia coli: mechanism and the influence of medium components. Environ. Sci. 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Antibacterial action of zinc oxide nanoparticles against foodborne pathogens. J. Food Saf. 31(2) , 211-218 (2011). d’Água, R.B. et al . Efficient coverage of ZnO nanoparticles on cotton fibers for antibacterial finishing using a rapid and low cost in situ synthesis. New J. Chem . 42 , 1052-1060 (2018). Babayevska, N. et al. Fabrication of gelatin-ZnO nanofibers for antibacterial applications, Materials . 14 , 103 (2021). Cho, W-S. et al. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn 2+ dissolution inside lysosomes. Part Fibre Toxicol . 8 , 27 (2011). Beek, W. J. E., Wienk, M. M., Kemerink, M., Yang, X., Janssen, R. A. J. Hybrid zinc oxide conjugated polymer bulk heterojunction solar cells. J. Phys. Chem. B . 109 , 9505-9516 (2005). Fang, L., Wu, W., Huang, X., He, J., Jiang, P. Hydrangea-like zinc oxide superstructures for ferroelectric polymer composites with high thermal conductivity and high dielectric constant. Compos. Sci. Technol . 107 , 67-74 (2015). Additional Declarations No competing interests reported. Supplementary Files supportinginformation.doc Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 24 Mar, 2022 Reviews received at journal 23 Mar, 2022 Reviews received at journal 15 Mar, 2022 Reviewers agreed at journal 15 Mar, 2022 Reviewers agreed at journal 14 Mar, 2022 Reviewers invited by journal 10 Mar, 2022 Editor assigned by journal 10 Mar, 2022 Editor invited by journal 12 Feb, 2022 Submission checks completed at journal 12 Feb, 2022 First submitted to journal 01 Feb, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1317820","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":83321964,"identity":"6ebd8cc2-f468-41f2-8963-f1679c33fbca","order_by":0,"name":"Nataliya 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13:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1317820/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1317820/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18216138,"identity":"04ea3156-77ca-49ad-a395-e9af78b8cdbc","added_by":"auto","created_at":"2022-02-15 00:48:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495255,"visible":true,"origin":"","legend":"\u003cp\u003eTEM and SEM images of ZnO nano- and microparticles: nanoparticles (a), nanorods (b),\u003cem\u003e \u003c/em\u003eparticles\u003cem\u003e \u003c/em\u003e(b),\u003cem\u003e \u003c/em\u003ehierarchical structures (d),\u003cem\u003e \u003c/em\u003etetrapods (e).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/0b67324b721bc140a9d44bc8.png"},{"id":18215983,"identity":"d8cc8a3a-67ba-4877-aa8c-8414dec0bba0","added_by":"auto","created_at":"2022-02-15 00:45:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162351,"visible":true,"origin":"","legend":"\u003cp\u003eTypical XRD pattern of ZnO nano- and microparticles.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/8130dd5f91e0dc8ece97738a.png"},{"id":18215775,"identity":"664a72d7-e730-4ec5-a126-37ba96b09a7f","added_by":"auto","created_at":"2022-02-15 00:42:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":426143,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen physisorption isotherms and pore size distribution for indicated samples.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/c58eb941af4a3f1434af4511.png"},{"id":18215770,"identity":"d16f47a4-9b4e-45bb-8a9a-884b29925e95","added_by":"auto","created_at":"2022-02-15 00:42:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":571403,"visible":true,"origin":"","legend":"\u003cp\u003ePL spectra of ZnO nano- and microparticles.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/2aaa1f37e55183adf9172e1a.png"},{"id":18216139,"identity":"3782b99e-414e-4fd4-9124-8d4a2b1b01df","added_by":"auto","created_at":"2022-02-15 00:48:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":263433,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial activity of ZnO nano- and microparticles against \u003cem\u003eE. coli\u003c/em\u003e (a) and \u003cem\u003eS. aureus\u003c/em\u003e (b) evaluated by measuring optical density at 570 nm (OD \u003csub\u003e570 nm\u003c/sub\u003e) after incubation at 37\u0026nbsp;°C, 220 rpm for 24h. Data presented as mean ± SD of three independent experiments, each performed in triplicates.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/e2f1d82fa6d38830b60be68a.png"},{"id":18215774,"identity":"4f0e925d-1074-401c-b87f-cef995b9bc0a","added_by":"auto","created_at":"2022-02-15 00:42:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":297930,"visible":true,"origin":"","legend":"\u003cp\u003eWST-1 assay performed on normal human fibroblasts MSU1.1 (a) and cervical cancer HeLa cells (b) after 24h of incubation with indicated ZnO materials.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/4bc71de37fc77216f47f3a99.png"},{"id":18215777,"identity":"a04a6709-962d-48d1-aa31-d2fb00684abf","added_by":"auto","created_at":"2022-02-15 00:42:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2086154,"visible":true,"origin":"","legend":"\u003cp\u003eBAC Live/dead assay. \u003cem\u003eE. coli \u003c/em\u003ebacteria (AATC: 35218 \u003cem\u003eE. coli \u003c/em\u003estrain) after 2h incubation with ZnO nano- and microparticles on distinct morphology at concentration 500\u0026nbsp;μg/mL. Scale bar: 20\u0026nbsp;μm.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/d486f137fe8e00b6031875f8.png"},{"id":18216192,"identity":"09bd4a03-16df-43f9-9b8b-bdb04a2f93fc","added_by":"auto","created_at":"2022-02-15 00:51:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1100125,"visible":true,"origin":"","legend":"\u003cp\u003eBAC Live/dead assay. \u003cem\u003eS. aureus \u003c/em\u003ebacteria (AATC: 29213 \u003cem\u003eS. aureus \u003c/em\u003estrain) after 2h incubation with ZnO nano- and microparticles on distinct morphology at concentration 500\u0026nbsp;μg/mL. Scale bar: 10\u0026nbsp;μm.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/74af0684c085a2458ec65c37.png"},{"id":18216193,"identity":"01c8ed88-742c-4abe-ab9e-5b5f2b551bab","added_by":"auto","created_at":"2022-02-15 00:51:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1007544,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/6dc65b14-9eba-4c40-ab22-6dbb1fda97c4.pdf"},{"id":18215986,"identity":"fb83e547-cb5c-41c1-b1e5-47bc1a250468","added_by":"auto","created_at":"2022-02-15 00:45:20","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6453248,"visible":true,"origin":"","legend":"","description":"","filename":"supportinginformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-1317820/v1/49f94a3e5eb54afd21ca42c8.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis, characterization, size and shape-dependent antibacterial activity and cytotoxicity profile of ZnO nano- and microparticles","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSuccessful fabrication of materials with desired properties using simple and low-cost approaches attracts a great deal of attention. Materials properties are strongly dependent on their characteristics, mainly phase composition, crystallinity, shape and size, dispersion, etc. Materials morphology plays an important role in optics, photonics, sensors, solar cells to obtain high-quality devices \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For example, it is well-known that the photocatalytic activity of semiconductor materials (e.g. NiO, TiO\u003csub\u003e2\u003c/sub\u003e) depends strongly on their particle sizes, basically due to enhancing specific surface area \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMaterials for bioapplication, such as biomarkers, biosensors, or drug carriers with improved optical, sensing, and surface properties can be obtained due to changing their shape, size, and dimension \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nanoparticles due to their small size have exciting physicochemical properties, such as a high surface-to-volume ratio, a size similar to the size of biomolecules allowing for their greater mobility and passing through the cell biological membranes \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Morphology and surface area affect the antibacterial activity, therefore, it is necessary to synthesize a material with a novel morphology having a higher amount of adsorption sites and surface area \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, verifying the morphology of the materials and hence their surface area can show great potential for bioapplication because the properties can be tuned and improved.\u003c/p\u003e \u003cp\u003eDecreasing the particle size (nanosized materials, such as 0D materials (e.g. quantum dots, QDs)) is one of the effective ways to increase the surface area. Despite many advantages of the nanoparticles, their main disadvantage is particle agglomeration. Modern approaches allow obtaining high-quality nanoparticles without any stabilizers and surfactants \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Anisotropic multidimensional and multicompartmental particles with intriguing morphologies are the subjects of extensive research. One dimension materials (1D) offer high surface area because of their unique morphologies. 3D materials are seen to be more suitable for many applications than other morphologies/structures as they may be assembled from lower dimensional materials and provide a large surface area and fast electron transport \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMetal oxide nanoparticles have great attention in materials science and nanotechnology for a large variety of applications. From semiconductor materials, zinc oxide (ZnO) is an important multifunctional material with broad applications in many scientific fields. As a promising semiconductor oxide (large exciton binding energy (60 meV and wide bandgap Eg\u0026thinsp;=\u0026thinsp;3.37 eV) ZnO possess excellent physicochemical properties, such as high thermal and chemical stabilities, thermal conductivity, UV protection or electronic, photonic, unique optical, and luminescent properties. At the same time, ZnO is a low-cost material, its synthesis is simple and it can be obtained with a big variety of morphology using physical and chemical approaches. ZnO with different morphology and dimension (\u003cem\u003ee.g.\u003c/em\u003e bulk ZnO, nanoparticles, films, 3D hierarchical structures) has broad applications as sensors, catalysts, solar cells, optoelectronic devices \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Furthermore, zinc is a necessary element to our health and has high biocompatibility to human cells, low toxicity, and good antimicrobial activity \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. All of these properties allow using ZnO in medicine as biomarkers, drug carriers, and therapeutic \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Thus, Warheit D.B. \u003cem\u003eet al.\u003c/em\u003e studied the effect of ZnO morphology on the toxicity of lung cells \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. They showed a low cytotoxic effect on rat L2 lung epithelial cells and primary rat lung alveolar macrophage treated with ZnO nanoparticles (NPs). Z. Li. \u003cem\u003eet al.\u003c/em\u003e studied the cell viability of ZnO nanowires using two cell lines \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Results showed that ZnO nanowires were biocompatible to HeLa cells up to the highest concentration used (up to 100 \u0026micro;g/mL), whereas for L929 cells were deleterious even at lower concentrations.\u003c/p\u003e \u003cp\u003eZnO possesses unique antibacterial, antimicrobial and antifungal properties, ZnO-based materials can initiate antibacterial activity even without the presence of light \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In presented studies \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e authors obtained ZnO with different morphology and indicated that ZnO and its composites can be used as good antibacterial agents toward Gram-positive, as well as Gram-negative bacteria. Thus, despite intensive research in this field, the evaluation of ZnO antibacterial activity and biocompatibility due to changing the particle morphology is still important and can significantly expand the scope of this material in biomedical applications.\u003c/p\u003e \u003cp\u003eThe main aim of our work was a synthesis of ZnO nano- and microparticles, the study of their crystal structures, and the effect of morphology and surface area on luminescent properties, cytotoxicity, and antibacterial activity toward Gram-positive and Gram-negative bacteria.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Fabrication of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eZnO nano- and microparticles with different morphology were fabricated using facile chemical and physical methods. We performed the study of the shape and size of the obtained ZnO nano- and microparticles using the transmission (HRTEM) and scanning electron microscopes (SEM). Indeed, in nanoparticles synthesis, we obtained the highly crystalline and monodispersed ZnO nanoparticles (ZnO NPs) and nanorods (ZnO NRs) using the sol-gel approach without any additional surfactants or ligands. ZnO NPs had a perfect spherical shape and uniform size distribution with a mean nanoparticle\u0026rsquo;s size of approximately 7 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, S1a). The FFT digital diffractogram pattern presented in Fig. S1b from nanoparticles with [010] zone axis orientation, demonstrated individual point reflections that indicate that particles had a single crystalline structure. The interplanar distance between adjacent lattice fringes is 2.57 \u0026Aring; corresponding to d(002).\u003c/p\u003e\n \u003cp\u003eChanging the synthesis parameters in ZnO NP synthesis (reagent concentration and synthesis time, and temperature) we obtained ZnO NRs and ZnO particles (ZnO Ps), (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). The mean size of ZnO NRs was approximately 5\u0026times;17 nm (width\u0026times;length), Fig. S1c. After annealing of ZnO NPs at 900\u0026deg;C for 2h, we obtained ZnO Ps with sizes of ~\u0026thinsp;150 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003eTo obtain ZnO with the hierarchical structure (ZnO HSs) we used a solvothermal reaction. The SEM image in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed confirmed the formation of the flower-like (peony) structure of ZnO. All individual particles had a spherical shape, were highly crystalline, and monodispersed with sizes of approximately 4 \u0026micro;m and the thickness of the individual plates of about 100 nm (Fig. S1d).\u003c/p\u003e\n \u003cp\u003eWe also obtained ZnO in the shape of tetrapods using thermal evaporation in the air. Tetrapods were well faceted with four uniform legs connected to a central nucleus (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee, S1e). The legs had hexagonal morphology with a length of about 5.2 \u0026micro;m and approximately 170 nm in thickness.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Structural characterization of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eNext to determine the crystal structure and phase purity of the obtained ZnO materials we performed XRD measurements. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the XRD pattern of ZnO particles with different morphology. X-ray powder diffraction confirmed the formation of a single-phase for all ZnO samples.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCrystallographic parameters of the obtained ZnO nano- and microstructures.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\" style=\"width: 49.2632%;\"\u003e\n \u003cp\u003eXRD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 26.1053%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\" style=\"width: 49.2632%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ea\u003c/span\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ec\u003c/span\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eV\u003c/strong\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enm\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003e\u003cem\u003eZnO NPs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e4.811\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003e\u003cem\u003eZnO NRs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e4.853\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003e\u003cem\u003eZnO Ps\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e4.753\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003e\u003cem\u003eZnO HSs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.521\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e4.766\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 26.1053%;\"\u003e\n \u003cp\u003e\u003cem\u003eZnO TPs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e0.521\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 16.4211%;\"\u003e\n \u003cp\u003e4.764\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll diffraction peaks are indexed according to the hexagonal phase of ZnO wurtzite crystal structure with main (100), (002), (101), (102), (110), (103), (112), and small (201) and (202) crystal planes. No additional peaks indicating the presence of impurity phases were found in all studied samples. The broadening of the peaks for ZnO NPs and ZnO NRs samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, black and red lines) can be attributed to the small particle size of ZnO. Calculated crystallographic parameters of zinc oxide particles are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe results indicate higher lattice constant values for ZnO NPs and ZnO NRs samples compared with the others. The lowest lattice constant \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e are observed for ZnO Ps and are the results of their preparation condition (high-temperature annealing). Similar results were presented by Lupan O. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e indicating a decrease of the lattice constant of ZnO with the increase of the temperature of its treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Textural properties of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eThe next stage was the study of the textural properties of the investigated samples and we performed their N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption analysis on powdered ZnO samples at -196\u0026deg;C. The specific surface area, pore volume, and pore size distribution of samples were specified. The results are summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and presented in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The adsorption/desorption isotherms of investigated ZnO materials correspond to type IV typical for mesoporous materials. The estimated BET specific surface area of the obtained ZnO nano- and microparticles were 83.5 m\u003csup\u003e2\u003c/sup\u003e/g (NPs), 83.8 m\u003csup\u003e2\u003c/sup\u003e/g (NRs), 3.0 m\u003csup\u003e2\u003c/sup\u003e/g (Ps), 4.5 m\u003csup\u003e2\u003c/sup\u003e/g (HSs), and 29.4 m\u003csup\u003e2\u003c/sup\u003e/g (TPs). The lowest surface area, as well as pore volume, showed ZnO Ps and ZnO HSs.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSurface area and pore volume of ZnO samples with different morphology.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS\u003csub\u003eBET\u003c/sub\u003e, m\u003csup\u003e2\u003c/sup\u003e/g\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePore volume, cm\u003csup\u003e3\u003c/sup\u003e/g\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZnO NPs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZnO NRs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZnO Ps\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZnO HSs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZnO TPs\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Photoluminescence properties of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eThe PL spectra of ZnO nanostructures are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea. All investigated ZnO nanostructures demonstrate the intense and broad peak in the visible range. The intense emission for ZnO TPs and Ps is mostly associated with oxygen vacancies (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb), whereas the PL for ZnO NPs, NRs, and HSs is attributed to oxygen interstitials (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Antibacterial properties of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eAs a next important step, to use our ZnO-based materials as potential antibacterial agents, the antimicrobial activity of ZnO nano- and microstructures was evaluated against Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e and Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e bacterial strains using optical density measurements after 24-hour incubation, as summarized in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. By measuring the optical density at 570 nm (OD\u003csub\u003e570\u003c/sub\u003e), the growth of bacteria can be quantified, based on turbidity resulting from light scattering \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For all samples, the bacteria number decreased in a concentration-dependent manner. The most effective towards \u003cem\u003eE. coli\u003c/em\u003e bacterial strains were ZnO nanoparticles and nanorods, as well as ZnO hierarchical structures at the highest concentration, and the least antibacterial effect was observed for ZnO Ps and TPs. The Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e bacteria strains were more sensitive to the antimicrobial action of ZnO nano- and microparticles than \u003cem\u003eE. coli\u003c/em\u003e. However, also here, the greatest lowering of the bacteria number was visible after incubation with ZnO NPs, NRs, and HSs with the highest surface areas.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Cytotoxicity studies of ZnO nano- and microparticles\u003c/h2\u003e\n \u003cp\u003eThe impact of distinct ZnO nano- and microparticles on human cells was evaluated by cytotoxicity analysis by WST-1 assay, performed on cervical cancer cell line (HeLa) and normal human fibroblasts (MSU1.1) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Five different ZnO nano- and microparticles concentrations of 0.1, 1.0, 10, 100, and 1000 \u0026micro;g/mL were employed, and the cell viability was determined after 24h of treatment. The cell viability data revealed that for both cell types at the concentration up to 10 \u0026micro;g/mL all kinds of ZnO particles were biocompatible, maintaining a high level of viability (up to 80%). However, at a higher concentration range (100\u0026ndash;1000 \u0026micro;g/mL) the cell viability declines to 40%, indicating a high level of toxicity. Interestingly, at a concentration of 100 \u0026micro;g/mL, a similar level of viability was observed for all samples on MSU1.1 cells, while for HeLa cells significant differences were visible.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIt 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.\u0026nbsp;1,2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eZn\u003c/em\u003e \u003csup\u003e \u003cem\u003e2+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003e+ OH\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026rarr; Zn(OH)\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026darr; (1)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eZn(OH)\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e\u0026darr; \u0026rarr; ZnO\u0026darr; + H\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eO (2)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe 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 \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eZnO 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 \u0026ldquo;structural director\u0026rdquo; (generally organic polar molecules). In our case, trisodium citrate dihydrate was used for this purpose.\u003c/p\u003e \u003cp\u003eObtaining 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 \u003cem\u003ec\u003c/em\u003e-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 \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The growth mechanism of the tetrahedral ZnO particles can be explained by the growth model proposed by Alsultany F.H. \u003cem\u003eet al.\u003c/em\u003e and Markushev V.M. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 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 (Zn\u003csub\u003emet\u003c/sub\u003e) and then Zn gas (Zn\u003csub\u003eg\u003c/sub\u003e) with O\u003csub\u003e2\u003c/sub\u003e gas (O\u003csub\u003e2g\u003c/sub\u003e) to form ZnO gas seeds (ZnO\u003csub\u003eg\u003c/sub\u003e), (Eq.\u0026nbsp;3,4). Then during constant heating, gaseous ZnO or unoxidized Zn will condense into a liquid state of Zn\u003csub\u003el\u003c/sub\u003e or ZnO\u003csub\u003el\u003c/sub\u003e (Eq.\u0026nbsp;5), and finally following O\u003csub\u003e2\u003c/sub\u003e absorption leads to the crystallization of ZnO solid seeds (\u003cem\u003eZnO\u003c/em\u003e\u003csub\u003e\u003cem\u003es\u003c/em\u003e\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eZn\u003c/em\u003e \u003csub\u003e \u003cem\u003emet\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e\u0026rarr; Zn\u003c/em\u003e\u003csub\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(1000 \u0026ordm;C) (3)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eZn\u003c/em\u003e \u003csub\u003e \u003cem\u003eg\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e+ O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2g\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e\u0026rarr; 2ZnO\u003c/em\u003e\u003csub\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(4)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eZnO\u003c/em\u003e \u003csub\u003e \u003cem\u003eg\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e(Zn\u003c/em\u003e\u003csub\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) \u0026rarr; ZnO\u003c/em\u003e\u003csub\u003e\u003cem\u003el\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(Zn\u003c/em\u003e\u003csub\u003e\u003cem\u003el\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) (5)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eZnO\u003c/em\u003e \u003csub\u003e \u003cem\u003el\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e(Zn\u003c/em\u003e\u003csub\u003e\u003cem\u003el\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)\u0026thinsp;+\u0026thinsp;O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2g\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e\u0026rarr; ZnO\u003c/em\u003e\u003csub\u003e\u003cem\u003es\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(6)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAccording to the octa-twin nucleus model proposed by Iwanaga H. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, the ZnO seeds have an octahedral shape. These octahedral seeds are substrates for the following growth of ZnO TPs.\u003c/p\u003e \u003cp\u003eObtained 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 \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eAnalysis of PL spectra for different semiconductor nanostructures is a powerful tool to investigate their morphology features, defects, and even chemical composition \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. 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) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The unique optical properties are very important for medical and biological visualization applications \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Depending on the PL intensity, PL peaks position, and the ratio I\u003csub\u003eNBE\u003c/sub\u003e/I\u003csub\u003eDLE\u003c/sub\u003e, 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 (Zn\u003csub\u003ei\u003c/sub\u003e \u003csup\u003e++\u003c/sup\u003e), single (V\u003csub\u003eo\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e), and double (V\u003csub\u003eo\u003c/sub\u003e\u003csup\u003e++\u003c/sup\u003e) ionized oxygen vacancies, neutral oxygen vacancies (V\u003csub\u003eo\u003c/sub\u003e), and oxygen interstitials (O\u003csub\u003ei\u003c/sub\u003e). According to previous studies, there are three main defects involved in the DLE: V\u003csub\u003eo\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (2,45 eV), V\u003csub\u003eo\u003c/sub\u003e\u003csup\u003e++\u003c/sup\u003e (2,23 eV), O\u003csub\u003ei\u003c/sub\u003e (2 eV) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Besides, in our case, the ratio I\u003csub\u003eNBE\u003c/sub\u003e/I\u003csub\u003eDLE\u003c/sub\u003e 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 \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Therefore, it is expectable that the different ZnO nanostructures would demonstrate various antibacterial behaviors.\u003c/p\u003e \u003cp\u003eThe 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 \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The higher the number of bacteria in the solution, the greater the OD\u003csub\u003e570\u003c/sub\u003e value, and thus the lower antibacterial activity of the added material, ZnO nano- and microparticles in this case.\u003c/p\u003e \u003cp\u003eThe high antibacterial activity towards \u003cem\u003eE. coli\u003c/em\u003e bacteria showed ZnO NPs, NRs, and HSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eAs it was mentioned above, the OD\u003csub\u003e570\u003c/sub\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, respectively. All of the untreated bacterial cells showed green fluorescence, due to the viable cells, indicating intact cell wall structure.\u003c/p\u003e \u003cp\u003eAs can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the co-incubation of \u003cem\u003eE. coli\u003c/em\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e, 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.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003eS. aureus\u003c/em\u003e strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) ZnO materials exhibited a stronger antibacterial effect than for \u003cem\u003eE. coli.\u003c/em\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e, 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. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e whose indicated that the antimicrobial activity increased due to a decrease in particle size of zinc oxide nanoparticles, as well as with Yamamoto O. \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, who indicated that smaller size of zinc oxide nanoparticles exhibits greater antibacterial activity than microscale particle.\u003c/p\u003e \u003cp\u003eFour mechanisms of action have been proposed as responsible for the antibacterial properties of zinc oxide particles, namely the production of reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, the loss of cellular integrity after contact of ZnO materials and the cell wall \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, ZnO NPs internalization \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, as well as the release of Zn\u003csup\u003e2+\u003c/sup\u003e ions \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The mechanism of nanomaterials toxicity is not specific, and thus bacteria are not able to get the resistance for nanoparticles treatment \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eGenerally 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 \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and gold nanoparticles \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. 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\u0026ndash;3 \u0026micro;m thick) and thin peptidoglycans (~\u0026thinsp;8 nm thick), whereas Gram-positive bacteria possess a peptidoglycan layer (~\u0026thinsp;80 nm thick) with covalently attached teichoic and teichuronic acids \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. However, here we observed that Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e were less susceptible to ZnO materials than Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e, which is consistent with Tayel and co-workers \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, 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\u0026rsquo;Agua R.B. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, 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 \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt cytotoxicity studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), results showed that all nan- and microparticles are biocompatible at low concentrations. The greatest decrease in viability above 100 \u0026micro;g/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. \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e 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.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe simple chemical and physical methods were used to obtained ZnO nano- and microparticles with different morphologies. The morphology effect on luminescent and surface properties, antibacterial activity toward Gram-positive (\u003cem\u003eStaphyloccocus aureus\u003c/em\u003e) and Gram-negative (\u003cem\u003eEscherichia coli\u003c/em\u003e) bacteria, and cytotoxicity toward normal and cancer cells was studied. All obtained ZnO materials were highly crystalline with hexagonal wurtzite crystal structures and monodispersed. N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption analysis showed that ZnO in nanorange (nanoparticles and nanorods) had a higher surface area. All samples performed high green emission caused by structural defects in ZnO. Due to high crystallinity ZnO tetrapods (TPs) showed the strongest photoluminescence. The antimicrobial activity measurements of ZnO nano- and microparticles indicated that for all samples, the bacteria number decreased in a concentration-dependent manner. Results showed that surface area has a significant impact on antibacterial performance. The high surface area of ZnO nanoparticles led to the most effective antibacterial activity towards \u003cem\u003eE. coli\u003c/em\u003e bacterial strains. Among used bacteria, the Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e strains were more sensitive to the antimicrobial action of ZnO nano- and microparticles than \u003cem\u003eE. coli\u003c/em\u003e. All kinds of ZnO materials were biocompatible towards cervical cancer cell line (HeLa) and normal human fibroblasts (MSU1.1), maintaining a high level of viability (up to 100 \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eAs we can see materials of different sizes and varieties of morphology can be used in biomedical fields. According to our present results, highly luminescent and biocompatible ZnO particles in nanorange (especially ZnO NPs and NRs) are planned to be used for cancer diagnostic and therapy. At the same time, the microparticles will be used for biosensors, drug adsorption, and release experiments.\u003c/p\u003e"},{"header":"5. Material And Methods","content":"\u003cp\u003e\u003cstrong\u003e5.1. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZinc acetate dihydrate (Zn(CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e\u0026times;2H\u003csub\u003e2\u003c/sub\u003eO, Zn(Ac)\u003csub\u003e2\u003c/sub\u003e, Sigma Aldrich), sodium hydroxide (NaOH, Stanlab), methanol (MeOH, Sigma Aldrich), ethanol (EtOH, Sigma Aldrich), tri-sodium citrate dihydrate (HOC(COONa)(CH\u003csub\u003e2\u003c/sub\u003eCOONa)\u003csub\u003e2\u003c/sub\u003e\u0026times;2H\u003csub\u003e2\u003c/sub\u003eO, Sigma Aldrich), Zn powder (Sigma Aldrich) were all of the analytical grades and used as starting materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2. Synthesis of ZnO nano- and microparticles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.1 Synthesis of ZnO nanoparticles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZnO nano- and microparticles (nanoparticles (ZnO NPs), nanorods (ZnO NRs), hierarchical flower-like structures (ZnO HSs), and tetrapods (ZnO TPs)) were synthesized using chemical and physical approaches presented in\u0026nbsp;schematic illustration\u0026nbsp;(Fig.S2).\u003c/p\u003e\n\u003cp\u003eThe ZnO NPs and ZnO NRs were obtained by the modified low-temperature sol-gel route described in \u003csup\u003e58\u003c/sup\u003e.\u0026nbsp;The proposed synthesis approach is facile and allows to obtaining of the crystalline monodisperse ZnO NPs\u0026nbsp;(Fig.S2a) at a temperature below 100\u0026nbsp;\u0026deg;C.\u0026nbsp;In ZnO NPs synthesis Zn(Ac)\u003csub\u003e2\u003c/sub\u003e was dissolved in methanol at a constant temperature of 60 \u0026deg;C. Then the solution of NaOH in methanol was added to Zn(Ac)\u003csub\u003e2\u003c/sub\u003e solution under vigorous stirring. The complete hydrolysis of zinc acetate with the addition of NaOH in a methanol solution results in the formation of ZnO colloid (gel). After about 3h of stirring, white ZnO nanoparticles were separated from the mother liquor, washed with methanol twice, and dried.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the synthesis of ZnO NRs, the reaction mixture (prepared in the same manner as for ZnO NPs synthesis) was concentrated 10 times by solvent evaporation. After about 12h of stirring white precipitate was separated, washed, and dried as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZnO particles (ZnO Ps) were obtained using ZnO NPs (obtained earlier) by their annealing at 900\u0026nbsp;\u0026deg;C for 2h\u0026nbsp;(Fig.S2b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2.2. Synthesis of ZnO microparticles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ZnO 3D hierarchical structures (ZnO HSs) (Fig.S2c) were prepared by a template-free solvothermal approach according to Fang\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e \u003csup\u003e59\u003c/sup\u003e. First, Zn(Ac)\u003csub\u003e2\u003c/sub\u003e and sodium citrate were dissolved in H\u003csub\u003e2\u003c/sub\u003eO. Separately NaOH (1g) was dispersed in EtOH at 60\u0026nbsp;\u0026deg;C. After complete dissolution of both precursors, NaOH solution was added dropwise to Zn\u003csup\u003e2+\u003c/sup\u003e solution. The resulting mixture was agitated at room temperature for 1h. Then, for solvothermal reaction (150 \u0026deg;C for 24h) the resulting mixture was transferred into a Teflon-lined stainless steel autoclave. After cooling down final product was centrifuged, washed with H\u003csub\u003e2\u003c/sub\u003eO, and dried at 60 \u0026deg;C for 12h before characterization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZnO tetrapods (ZnO TPs), (Fig.S2d) were obtained by the simple catalyst-free oxidative-metal-vapor-transport method. The method was based on thermal evaporation of Zn powder at 1000 \u0026deg;C for 1h in the air \u003csup\u003e29\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3. Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural characteristics of the obtained ZnO nano- and microparticles were measured by powder X-ray diffraction (XRD). The studies were carried out on powdered samples using an Empyrean (PANalytical) diffractometer with Cu Kɑ radiation (\u0026lambda;=0.154 nm), reflection-transmission spinner (sample stage), and PIXcel 3D detector, operating in the Bragg-Brentano geometry. Scans were recorded at room temperature in angles ranging from 20\u0026deg; to 80\u0026deg; (2\u0026theta;) with a step size of 0.006\u0026deg; and continuous scan mode. The morphology of the obtained samples was studied by high-resolution transmission electron microscopy (HRTEM; JEOL ARM 200F) and scanning electron microscopy (SEM, JEOL, JSM-7001F).\u0026nbsp;For the determination of the specific surface area of the samples, the N\u003csub\u003e2\u003c/sub\u003e adsorption/desorption isotherms were measured at -196 \u0026deg;C on a Quantachrome Nova 1000 apparatus. The samples were outgassed at 150 \u0026deg;C for 15h in a vacuum before the measurements. The specific surface area was determined using the BET method. The total volume of pores was calculated using the single point mode (at p/p\u003csub\u003e0\u003c/sub\u003e = 0.98). The pore size distribution was determined by applying the Barrett-Joyner-Halenda (BJH) method from the desorption branch of the isotherm.\u003c/p\u003e\n\u003cp\u003eThe photoluminescence (PL) of the samples was measured at room temperature using Kimmon HeCd laser. The excitation wavelength was 325 nm, and the power was around 2 mW.\u0026nbsp;The PL spectra were recorded in the range from 360 to 1000\u0026nbsp;nm by Ocean Optics Spectrometer QE65 pro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBiological characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.1 Cell line and cell culture\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman cervical cancer cell line HeLa was obtained from American Type Culture Collection (ATCC). Human fibroblast cell line MSU1.1 was obtained from Prof. C. Kieda (CBM, CNRS, Orl\u0026eacute;ans, France). Cells were cultured in a complete medium Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, 100 \u0026mu;g/mL streptomycin, and grown at 37\u0026nbsp;\u0026deg;C in a humidified atmosphere containing 5\u0026nbsp;% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.2 Cytotoxicity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCervical cancer cells (HeLa) and normal fibroblasts (MSU1.1) were used for \u003cem\u003ein vitro\u003c/em\u003e cellular toxicity studies of ZnO nano- and microparticles. Cells (1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) were seeded onto 96-well plates and incubated overnight at 37 \u0026deg;C under a 5 % CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The medium in the wells was then replaced with a fresh medium containing increasing concentrations of ZnO particles (from 0.1 \u0026mu;g/mL to 1 mg/mL) and incubation was continued for 24h. The medium without ZnO particles was used as a negative control. The effect of the ZnO nano- and microparticles on cell proliferation and viability was determined by WST-1 assay according to the manufacturer\u0026rsquo;s instructions. Briefly, 10 \u0026mu;L of WST-1 solution was added to each well and the plates were further incubated. After 2h the absorbance was measured with a microplate reader (AnthosZenyth 340rt) at 450 nm and 650 nm as reference. The mitochondrial function and, by extension, the relative cell viability (%) related to the negative control was calculated by test sample/negative control \u0026times;100 %. Data are reported as the average \u0026plusmn; standard deviation (SD) of wells performed in quadruplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBacterial growth inhibition study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStock cultures of bacterial strains \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eATCC 35218 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 were stored in 30 % glycerol. The strains were cultured in LB Broth Lennox at 37 \u0026deg;C with constant agitation at 230 \u0026thinsp;rpm for 24h. The bacterial cultures were then diluted between 2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 5\u0026times;10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells/mL in LB broth medium. 100\u0026nbsp;\u0026mu;L of the cell suspension was then added to each well of a 96-well plate. Appropriate concentrations of freshly prepared ZnO particles solutions (10, 100, 250, 500, 1000\u0026nbsp;\u0026mu;g/mL) were added and placed at 37\u0026nbsp;\u0026deg;C in an incubator. Turbidity of the suspension, as a measure of bacteria growth, was recorded spectrophotometrically at 570\u0026nbsp;nm (OD\u003csub\u003e570\u003c/sub\u003e) with a microplate reader (Anthos Zenyth 340rt) after 24h. To avoid potential interference during optical measurements caused by the light scattering properties of the solutions, the same liquid medium without microorganisms, but containing the same concentration of studied samples, were used as blank controls. The positive control was bacterial cultures without ZnO particles treatment. All experiments were performed in triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4.4 Bacterial cells viability analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize the effect of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e cells\u0026apos; interaction with ZnO particles with different morphology, the fluorescence assay LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies) was applied and observed under a confocal laser scanning microscope (Olympus, FV1000). In brief, the \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e overnight cultures were used to inoculate of fresh LB medium. At the mid-log phase of bacterial growth, the ZnO nano- and microparticles solutions were added to the final concentration of 500 \u0026mu;g/mL and allowed to grow for 3h. From these cultures, 1 mL of each bacterial solution was centrifuged at 5000 rpm for 10 minutes. The pellets were resuspended in HEPES buffer, centrifuged, and washed with HEPES buffer three times more. Finally, the pellets were resuspended in \u0026nbsp;500 \u0026mu;L of HEPES buffer, and the combination of fluorescent dyes SYTO9 and PI were mixed in identical volumes. 1.5 \u0026mu;L of their mixture was added to each bacterial suspension and incubated in dark for 15 minutes. Fluorescence images were taken by trapping 5 \u0026mu;L of stained bacterial samples mounted on glass slides with mounting medium and cover with coverslips. For each sample, nine randomly selected images were captured by the microscope, and live/dead cells were counted to ascertain percentage viability. Data presented are live (green) and dead (red) cells as a percentage of the total cell number (live + dead).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the financial support by the following projects:\u0026nbsp;H2020-MSCA-RISE-2017, CanBioSe 778157\u0026nbsp;(II),\u0026nbsp;SONATA BIS 6 UMO-2016/22/E/ST3/00458\u0026nbsp;(GN, ŁP),\u0026nbsp;WPC2/nanoHEART/2021\u0026nbsp;(NB, SJ), and SPUB -\u0026nbsp;41/E-336/SPUB/SP/2019\u0026nbsp;(MJ, SJ, GN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.B. wrote the first version of this manuscript and \u0026nbsp; prepared all samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN.B., L.P., I.I, G.N., M.J., E.J., and SJ reviewed and prepared the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eL.P. performed bacterial culture and all bacteria-based experiments and human cell cultures. M.J. and N.B. performed XRD analysis of the obtained samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG.N. and I.I. performed scanning and transmittance electron microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE.J. provided and characterized the textural properties of the obtained samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eI.I. studied the\u0026nbsp;photoluminescence properties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors commented and have approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoopathi Raja, R., Parthibavarman, M. Reagent induced formation of NiCo\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e with different morphologies with large surface area for high performance asymmetric supercapacitors. \u003cem\u003eChem. Phys. 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Technol\u003c/em\u003e. \u003cstrong\u003e107\u003c/strong\u003e, 67-74 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ZnO, morphology, crystal structure, photoluminescence, antibacterial activity, cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-1317820/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1317820/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of our work was the synthesis of ZnO nano- and microparticles and to study the effect of shapes and sizes on cytotoxicity towards normal and cancer cells and antibacterial activity toward two kinds of bacteria. We fabricated ZnO nano- and microparticles through facile chemical and physical routes.\u003c/p\u003e \u003cp\u003eThe crystal structure, morphology, textural properties, photoluminescent properties were characterized by powder X-ray diffraction, electron microscopies, nitrogen adsorption/desorption measurements, and photoluminescence spectroscopy.\u003c/p\u003e \u003cp\u003eThe obtained ZnO structures were highly crystalline and monodispersed with intensive green emission. ZnO nanoparticles and nanorods showed the strongest antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e compared to microparticles due to their high surface area. However, the ZnO hierarchical particles at higher concentrations also strongly inhibited bacterial growth. \u003cem\u003eS. aureus\u003c/em\u003e strain was more sensitive to ZnO particles than the \u003cem\u003eE. coli.\u003c/em\u003e Nanoparticles and nanorods were more harmful to cancer cell lines than to normal ones at the same concentration.\u003c/p\u003e","manuscriptTitle":"Synthesis, characterization, size and shape-dependent antibacterial activity and cytotoxicity profile of ZnO nano- and microparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-15 00:42:18","doi":"10.21203/rs.3.rs-1317820/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-03-24T05:26:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-03-23T15:08:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-03-15T12:21:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8da3dd10-6356-4d3b-91b5-a6b14e81dc95","date":"2022-03-15T04:16:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e6b5677d-c9cb-44d4-992d-52f4b27d06bb","date":"2022-03-14T10:39:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-11T00:44:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-10T23:40:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-02-12T08:35:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-02-12T08:10:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-02-01T13:25:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8e93a01-d083-4732-b42c-2d77904e85e7","owner":[],"postedDate":"February 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-05-03T04:29:06+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-15 00:42:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1317820","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1317820","identity":"rs-1317820","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}