Insight into Biological Effects of Zinc Oxide Nanoflowers on Bacteria

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New insight into biological effects of zinc oxide nanoflowers on bacteria: Why morphology matters Qian Cai, Yangyang Gao, Tianyi Gao, Shi Lan, Oudjaniyobi Jacob Simalou, Xinyue Zhou, Yanling Zhang, Chokto Harnoode, Ge Gao, and Alideertu Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11573 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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ACS Applied Materials & Interfaces

New Insight into Biological Effects of Zinc Oxide Nanoflowers on Bacteria: Why Morphology Matters Qian Cai,†,§ Yangyang Gao,†,§ Tianyi Gao,†,§ Shi Lan,‡ Oudjaniyobi Simalou,║ Xinyue Zhou,† Yanling Zhang,† Chokto Harnoode,† Ge Gao,┴ Alideertu Dong*,† †

College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot 010021, People’s

Republic of China ‡

College of Science, Inner Mongolia Agricultural University, Hohhot 010018, People’s Republic of

China ║

Département de Chimie, Faculté Des Sciences (FDS), Université de Lomé (UL), BP 1515 Lome, Togo

┴

College of Chemistry, Jilin University, Changchun 130021, People’s Republic of China

ACS Paragon Plus Environment

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ABSTRACT: Zinc oxides have gained exciting achievements in antimicrobial fields because of their advantageous properties, while their biological effects on bacteria are currently underexplored. In this study, biological effects of flower-shaped nano zinc oxides on bacteria were systematically investigated. Zinc oxide nanoflowers with controllable morphologies (viz., rod flowers, fusiform flowers, and petal flowers) were synthesized by modulating merely base type and concentration using the hydrothermal process. Their antibacterial power is in an order of petal flowers > fusiform flowers > rod flowers because of their differences in microscopic parameters such as specific surface area, pore size, and Znpolar plane, ect. More importantly, the role of morphology in influencing biological effect on bacteria was examined, focusing on the morphology-induced effect on integrality of cell wall, permeability of cell membrane, DNA cleavage, etc. As for cytotoxicity, all petal flowers, fusiform flowers, and rod flowers show trivial cytotoxicity to the Hela cells. This work provides a guide for enhancing biological effect of the biocides on pathogenic bacteria by the morphological modulation. KEYWORDS: Morphology, Biological effect, Zinc oxide nanoflower, Antibacterial, Bacteria


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INTRODUCTION Crucial interplay among environmental pollution and energy exploitation has impelled enormous research focus in these decades.1,2 Environmental pollution arising from pathogenic bacteria threatens human health and as a result poses unprecedented challenge to the bactericidal research.3 Development of effective sterilizing techniques to control pathogenic disease hence seems to be extremely urgent. As a reliable approach, designing novel and effective antibacterial materials has widely been applied for the prevention and removal of bacterial pathogen from environmental pollution.4 A large variety of antimicrobial agents thus have emerged for curing and preventing diseases in public health hygiene and antifouling in biomedical industry.5-8 Nanomaterials accepted widely for antibacterial property since the ancient times offer us a trustworthy handle on the microbial contamination problem.9-14 Unlike bulk powders, nanomaterials possess unique properties because of their smaller size and larger surface nature. A great deal of effort has been devoted to biocidal nanostructures and respectable achievements have been obtained. To date, nanomaterials such as silver nanoparticles (Ag NPs), titania nanoparticles (TiO2 NPs), and zinc oxide nanoparticles (ZnO NPs) have been developed prosperously as powerful bactericidal agents.15-17 Among them, ZnO NPs have been widely studied owing to their advantages over conventional biocides, including better stability within wide range of temperature and moisture, higher shelf-life, reusability, ease of storage and transportation, etc.1821

Thanks to these advantageous properties, ZnO NPs have been adopted widely in biomedical

arena including drug delivery system, cancer therapy, biological fluorescent imaging, biosensors, etc.22-24 In particular, ZnO NPs can induce cytotoxicity in a cell-specific and proliferation dependent manner, with rapidly dividing cancerous cells being more susceptible than quiescent cells.25 Such an order of magnitude difference in toxicity makes ZnO NPs potential candidate for cancer therapy.25 The synthesis of ZnO NPs is of great importance for both the fundamental


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study and the practical applications.26 As a result, a variety of synthetic strategies have been developed for the synthesis of ZnO NPs for biological applications. Hydrothermal method is one of the typical solution approaches for producing ZnO complex architectures having a uniform morphology.27 Recently, the modified hydrothermal techniques have been developed to modulate morphology by adding extra dopants.28-31 Many researchers have devoted to dope ZnO with dopants since they can reduce the native point defect densities in ZnO.28-31 These methods are highly desirable because of its easily controllable condition, relatively cheap equipment, environmental benignity kindness, and user-friendly feature. Therefore, hydrothermal technique has been extensively studied for designing ZnO products with diverse morphologies. Despite impressive advances, most previous studies focus merely on the synthesis condition and morphological modulation.32-34 Systematic investigations on the morphology effect on antibacterial properties of ZnO architectures are quite rare. As a smart biocide, antimicrobial capability of ZnO is strongly dependent on their microstructures, such as morphology, shape, composition, phase, crystal size, crystalline density, specific surface, and orientation of architectures.35 Previous reports have revealed that exquisite control over the microstructures is the key for determining the bactericidal activities of ZnO architectures.36 Recent efforts have focused on the morphological modulation of ZnO complex structures including sphere, rod, needle, wire, cheerios, tube, fusiform, urchin, flower, etc.37 Flower-like ZnO is one of the most favorite candidates for antibacterial applications because such architectures can construct continuous micronetworks with higher specific surface and distinguishable porosity providing enhanced biological effect. Also extensive researches have been done on the therapeutic application of zinc oxide nanoflowers for the medical purpose.38-40 Nevertheless, explicit biological action induced by flower-like ZnO towards pathogenic bacteria is not quite clear. Herein, we fabricated zinc oxides with flower-like morphology via the hydrothermal


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technique. The morphological modulation of ZnO flowers can be achieved simply by regulating base type, concentration, and aging time. The antimicrobial test proved that ZnO flowers kill bacteria in a morphology-dependent manner. More significantly, the biological effect of zinc oxide with different morphology on bacteria were systematically discussed. This study provide a new insight into the development of novel biocide with unique morphology for extensive biological applications. EXPERIMENTAL SECTION Synthesis of ZnO nanoflowers. Flower-like ZnO architectures were prepared by the typical lowtemperature hydrothermal technique.41-43 Typically, zinc acetate (Beijing Chemical Company) and base (Tianjin Chemical Reagent Plant) were mixed within the deionized water (40 mL), and the mixture was placed into a Teflon stainless autoclave and sealed tightly. Hydrothermal treatment was carried out at 70 o

C, and the precipitate was filtered, washed with deionized water, and dried at 40 oC for 24 h.

Morphological modulation of the ZnO products was readily achieved by tuning the base type (KOH, NaOH, and NH3), concentration (0.2 M, 0.3 M, and 0.6 M), and aging time (24, 72, and 96 h) as shown in Table 1.41 Characterization of ZnO nanoflowers. SEM images were taken on a Shimadzu SSX-550 field emission scanning electron microscope at 15.0 kV. A drop of well dispersed samples in ethanol was cast onto a piece of silicon wafer and air dried. A thin gold coating was utilized to avoid charging during scanning and a detailed microscopic study was carried out. TEM and HRTEM images were taken on a Hitachi H-8100 transmission electron microscope at 200 kV. The sample was prepared by dripping suitable volume of the sample suspension on copper grid and air dried. Surface area and porosity were performed on an ASAP 2010 Brunauer-Emmett-Teller (BET) analyzer. The BET specific surface area was determined by the multipoint BET method. The XRD patterns were obtained with a Siemens model D5000 diffractometer equipped with a copper anode producing X-rays with a wavelength of 1.5418 Å. Data was collected in continuous scan mode from 10 to 80o with a 0.02o sampling interval. UV-visible


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absorption spectra of samples prepared by thoroughly sonicating in water for a few hours were recorded in a quartz cuvette using a SPECORD50 UV-vis spectrophotometer. FTIR spectra were captured by using a Thermo Nicolet Avatar 370 FTIR spectrometer within the wavenumber range from 400 to 4000 cm-1 on thoroughly dried samples using KBr pellets. The amount of zinc ions was determined using a ZA3000 Hitachi atomic absorption spectrometry. Bacterial culture. In all experiments, Escherichia coli (E. coli, Gram-negative bacteria, ATCC 8099) and Staphylococcus aureus (S. aureus, Gram-positive bacteria, ATCC 25923) were purchased from China General Microbiological Culture Collection Center (CGMCC), and grown overnight at 37 oC under agitation (250 rpm) in Luria Bertani (LB, 10 g of tryptone and 5 g of yeast extract/liter) growth medium. Inhibition zone study. The antibacterial behavior of the ZnO products was assessed by a modified Kirby-Bauer (KB) technique.44 The surface of Luria Bertani agar plate and tryptic soy agar plate was overlaid with 1 mL of 108-9 CFU/mL of E. coli (ATCC 8099) and S. aureus (ATCC 25923), respectively. The plates were then allowed to stand at 37 oC for 4 h. The ZnO products were placed onto the surface of each of the bacteria-containing agar plate, and gently pressed with a sterile forceps to ensure full contact between the sample and the agar. After incubation at 37 oC for 24 h, the inhibition zone around the sample was measured. Plate counting method. S. aureus and E. coli were also used as model microorganisms to test the antibacterial activities of the samples. Bacteria were grown overnight at 37 oC in Luria-Bertani medium (LB). Cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS, NaCl, 8.0 g/L; KCl, 0.20 g/L; Na2HPO4·12H2O, 3.49 g/L; KH2PO4, 0.2 g/L; pH 7.4), and diluted to concentrations of 106-7 colony-forming units/mL. ZnO powders were dispersed in the sterilized distilled water, vortexed, and then sonicated for 30 min to obtain ZnO suspension with concentration of 6.25 mg/mL. In antibacterial test, 50 µL of bacteria suspension and 450 µL of sample suspension were well mixed, and the mixture was incubated under constant shaking. The resulting mixture was mixed well,


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serially diluted, and then 100 µL of each dilution was dispersed onto LB agar plates. Colonies on the plates were counted after incubation at 37 oC for 24 h. Microstructure of bacteria. The bacteria treated with zinc oxide nanoflowers were washed with 0.1 M phosphate buffer, and then fixed with 4% glutaraldehyde. The sample was then dehydrated in graded ethanol series. Subsequently, tert-butyl alcohol replaces ethanol as transfer solution. After drying, the sample was mounted on aluminum stubs using a double-sided adhesive carbon tape. The microstructures of bacteria were observed by SEM at an accelerating voltage of 5 KV. Membrane permeability assay. We incubated logarithmic phase 1 mL of E. coli (108 CFU/mL) with 1 mL of ZnO suspension at room temperature for 320 min. The bacteria were collected by centrifugation, and stained by contacting 0.5 mL of propidium iodide (PI, 3 µmol/L) for 30 min. The stained bacterial suspensions (5 µL) were collected by centrifugation and purified by several cycles of PBS solution washing. The bacteria suspended in 1 mL of PBS solution were trapped between a glass slide and cover slide. The confocal image was obtained by Zeiss 510 Meta laser scanning confocal microscope, and the fluorescence was monitored with excitation at 620 nm and emission at 670 nm, respectively. The equal volume of water instead of ZnO solution is a control. DNA cleavage study. DNA cleavage experiment was done according to the previous literature by the agarose gel electrophoresis technique.45 Nutrient broth (peptone 10 g·L-1, yeast extract 5 g·L-1, NaCl 10 g·L-1) was used as the media. 0.25 g of agarose was dissolved in 25 mL of TAE buffer (4.84 g tris base, pH 8.0, 0.5 M EDTA/1 L) by boiling. When the gel attained 55 oC, it was poured into the gel cassette fitted with comb and left to get it solidified. The comb was carefully removed and gel was placed in the electrophoresis chamber flooded with TAE buffer. 20 µL of DNA sample was loaded (mixed with bromophenol blue dye 1:1 ratio), carefully into the wells, along with standard DNA marker and a constant 60 V of electricity was passed for around 50 min. The gel was removed and carefully stained with ETBR solution (1 µg·L-1) for 10 min and the bands were observed under UV trans-illuminator. The results were then compared with standard DNA marker.


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Cytotoxicity assay. In vitro cytotoxicity assays for ZnO nanoflowers were carried out by exposing Henrietta Lacks strain of cancer cells (Hela cells, obtained from Perking Union Medical College, China) in microtiter plates to various dilutions of ZnO in aqueous saline solution. Concentrations ranging from 0.00 mg·mL-1 to 1.00 mg·mL-1 of ZnO were taken in saline solution and thoroughly sonicated. These are then pipetted into 96-well microtiter plates. Further 0.1 mL Hela cells cultured as monolayer in Dulbecco’s Modified Eagle Medium to active growth phase were added and incubated at 35 oC for 24 h in 5 % CO2 atmosphere. Wells containing an equal number of cells, without ZnO, were left as control. After incubation, the detached cells, medium, and ZnO were removed by vigorous shaking. The remaining live cells were fixed in 2 % solution of formalin in 0.067 M phosphate-buffered saline (PBS) (pH 7.2) for 1 min. The plates were stained with 0.4 % trypan blue dye-PBS solution for 20 min. Excess stain was removed through rinsing with water, and the plates were air dried. For quantification, the stain was eluted from the wells with four successive 50 mL samples of 50 % ethanol and diluted in 0.9 mL of PBS. Absorbance of the pooled washings was recorded at 595 nm in a spectrophotometer. Percentage of cells surviving the treatment was computed for various doses of ZnO by considering the optical density for control as a hundred percent. RESULTS AND DISCUSSION Using different type of base and concentration, the FE-SEM images of the products show different flower-like morphologies (Figure 1). The samples obtained with 0.6 M KOH in Figure 1A-1 are nanorod built flowers with average rod length of ~870 nm and average width of ~260 nm. A close-up view confirms the radical structure of the flowers with rods projecting out, as shown in Figure 1A-2. By contrast, wider corollas were formed when NH3 was applied as base source (Figure 1B and 1C). Figure 1B-1 and B-2 show that the structure of ZnO corresponding to 0.2 M NH3 is fusiform composed flower. The average fusiform length is about ~1.5 µm, and the average width is ~650 nm at the body. Interestingly, the corollas of ZnO flowers broaden to produce petal-based flowers after increasing NH3 concentration to 0.3 M (Figure 1C-1). Unlike the former two products, the enlarged SEM images in


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Figure 1C-2 shows lumpish petal with the average length of ~600 nm and the average width of ~650 nm at the center. The petal surfaces are not smooth and are covered by large amount of nanoparticles. Difference of the products in morphology implied us that the control design is advisable by tuning base type and concentration. TEM technique was utilized as well to verify that the hydrothermal precipitates can produce flowerlike ZnO nanostructures with different morphologies. On general observation from low-magnified TEM images (Figure 1A-3, B-3 and C-3), one would see that all products are constructed from nanocrumb emanated from the centers to obtain nanoflower. Such striking structures well matched with those captured in SEM images, giving rod (Figure 1A-3), fusiform (Figure 1B-3), and petal (Figure 1C-3) based flowers, respectively. The high-resolution TEM (HRTEM) shows the lattice fringes of width 0.26 nm corresponding to the distance between the (002) plane, suggesting that the all the corollas assembled orderly along the ‹0001› direction.46,47 Such ordered lattice fringe of the three products implied the single crystalline nature of ZnO. Nitrogen adsorption/desorption measurement shown was used to characterize the Brunauer-EmmettTeller (BET) specific surface area and pore property (Figure 2A). Similar as results reported previously, the as-synthesized products showed type Ⅳ isotherms with type H3 hysteresis loops.48 The information of surface area and pore diameter are obtained and are summarized in Table 2. The surface area of petal based flowers is as large as 7.21 m2·g-1 and much higher than that of rod (3.28 m2·g-1) and fusiform (2.72 m2·g-1) based flowers. Such difference may be assigned to the morphological variation. Interestingly, unlike surface area, there is no tremendous difference detected among the rod, fusiform, and petal built flowers in the average pore diameter. The XRD patterns of the ZnO nanoflowers obtained under different conditions are illustrated in Figure 2B. All these samples show characteristic (100), (002), (101), (102), (110), (103), (200), (112), (201), and (202) peaks matched well with the hexagonal ZnO with the wurtzite structure.49 No other impurities such as Zn(OH)2 are obtained, suggesting that these samples are pristine ZnO phase.


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Nevertheless, the intensities of the diffraction peaks increased to different degrees with the base type and concentration, which are generally related to the enhanced crystallization, formation of large crystallites, and well-ordered orientation. The intensity ratio of I(100)/I(002) of the products were calculated, and the results are also listed in Table 2. ZnO prepared using KOH possesses the highest I(100)/I(002) value of 1.47, and NH3·H2O gives smaller I(100)/I(002) value of 1.41 with 0.2 M and 1.38 with 0.3 M. As reported, I(100)/I(002) value, to a great extent, is reflecting the formation mechanism of zinc oxide towards different morphology. The lower I(100)/I(002) value represents the corollas oriented along the c-axis with (0001) end faces, and conversely the corollas along the c-axis is shortened with increasing I(100)/I(002) value.50 Such a significant variation suggests that the oriented growth of ZnO corollas can by well controlled by adjusting the base type and concentration, therefore tuning the morphology. The optical absorption of ZnO flowers is recorded within the UV-visible (UV-vis) range as shown in Figure 2C. All these samples show obvious, unimodal, and intense absorption within the narrow range of 350-450 nm, attributing to the excitonic absorption of ZnO.51 The UV-vis absorption position is highly dependent on the ZnO structure and size.52 The nanorod built flowers shows a sharp absorption peak at the wavelength of ~370 nm, which is similar as that value in other literature.51 The absorption band of fusiform and petal based flowers is significantly red-shifted and the maximum is seen at ~385 nm and ~400 nm, respectively. Most possible reason is explained as follows. In TEM and SEM, nanorod-based ZnO flowers have more gracile corolla than other two samples. It is widely accepted that the shrinking in structure can constrain the electrons and holes, and result in a shift of the absorption edge toward lower wavelengths. Therefore, the optical absorption of fusiform and petal composed ZnO flowers show the blue shift from that of nanorod-based counterparts. Intensity ratio of I(100)/I(002) is another possible explanation for the optical absorption difference of nanoflowers. A small I(100)/I(002) ratio reflecting an intense (002) peak can indicate the vigorous growth of petal oriented along the c-axis. Conversely, a high I(100)/I(002) ratio can shorten c-axis oriented growth. It is obvious that the oriented


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growth of petal oriented along the c-axis is determining the structural development, thereby influencing the optical absorption. Table 2 reveals that the nanopetal-formed ZnO flowers possess the lowest I(100)/I(002) value, which is beneficial to broaden the corolla leading to the red shift of the absorption band. FTIR analysis was performed to further confirm the existence of the difference among three structures. As shown in Figure 2D, the intense peaks appeared at about 3440 and 1635 cm-1 are attributed to the -OH bond stretching and bending vibration from the hydroxyl groups and residual water, respectively.53 Besides, all these products have two characteristic peaks at approximately 500 and 430 cm-1, corresponding to the vibration of Zn-O-H bending and Zn-O stretching, respectively.50,54 The presence of relative intense Zn-O-H and Zn-O vibration acts as the specific markers for ZnO, suggesting that all products are the exact ZnO component despite of structural difference. The slight change in the vibration position and intensity with the different base type and concentration indicates the crystallization enhancement in well agreement with the XRD and UV data. The spread plate technique as an intuitive method for microbiological detection was herein applied to examine the antibacterial performance of the ZnO products against both S. aureus and E. coli.55 The susceptibility of the bacteria towards the biocides can be informed from the survival case on the cultural plate. Figure 3A shows photographs of the bacterial culture plates, visualizing the survival case of bacteria after 80 min exposure to the control and ZnO nanostructures. Both the survival S. aureus and E. coli on the culture plates are visible as small white dots. The control provides too dense bacterial colonies on the cultural plate to make fully distinction between two dots. Yet the population of bacterial colonies drastically decreases as contacting the ZnO flowers, showing that all the products with different morphology have the high toxicity both to S. aureus and E. coli. No significant difference was detected among three morphologies just from Figure 3A, namely these findings could not prove which morphology is the most powerful one. To differentiate their bactericidal ability, the killing kinetics following incubating of both E. coli and


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S. aureus with increasing contacting period was monitored. The kinetic antibacterial curves, shown in Figure 3B and 3C, provide the biocidal activity by contacting E. coli and S. aureus with the ZnO products with different morphologies. The number of the survival bacterial colonies was counted as a function of the contact time ranged from 0 min to 320 min. The survival case of the bacteria without treating with the ZnO is comparatively examined with the same period as control. As expected, remarkable population decrease of the bacterial colonies is visible as treating with ZnO, whereas the control almost shows horizontal development trend for both E. coli and S. aureus within the whole contact time range. So we are quite sure that three types products possess capability of disinfection. In a closer view, different survival indication emerges for the flower-like products with different morphologies. The petal flowers show the fastest bactericidal speed, the rod flowers have the slowest killing speed. Namely, the biocidal power is in an order of petal > fusiform > rod flowers. The microstructural variability is the conclusive explanation for the difference. The comprehensive effect of the parameters such as specific surface, Zn-polar plane, pore size etc., leads to the order of the antibacterial capability. It can be expected that ZnO nanostructures have potent antibacterial capabilities, which is strongly dependent on their microstructures. Biocidal mechanism of ZnO can mainly be classified into ion exudation and light catalysis.50,56 Herein, the relationship between the microstructures and biological effects of ZnO was studied systematically to provide the exact evidence for the antibacterial mechanism. Zone of inhibition study (DIZ) is one effective method for determining the bactericidal mechanism of the biocides, which is directly proportionate to the bactericidal activity.57 DIZ test of ZnO architectures with different morphologies was performed also selecting E. coli and S. aureus as the representative microorganism by a disk diffusion test. Figure 3D shows the optical images of the DIZ result of ZnO against E. coli and S. aureus. A robust growth of bacteria scattering in culture dish is detected in the control test without giving any aseptic area. By contrast, all ZnO products show relative different degree of DIZ value, demonstrating that Zn2+ diffusion might be relatively important parameter determining


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antimicrobial activity. The average DIZ value for the rod flowers, fusiform flowers, and petal flowers is 26.3 mm, 27.7 mm, and 31.5 mm for E. coli and 21.0 mm, 23.3 mm, and 24.8 mm for S. aureus, respectively. The microstructural factors such as intensity ratio of I(100)/I(002), pore size, and surface area of ZnO flowers can be probable explanation for the antibacterial variability. As expected, specific surface area is conclusive element influencing the biocidal effect of the biocides. The ZnO flowers composed from petal-like corolla are calculated to have the larger surface area of 7.21 m2·g-1, offering the more functional sites for antibacterial behavior. The nanorod built flowers have the highest I(100)/I(002) value of 1.47, providing a greater proportion of (0001) Zn-polar faces therefore enhancing the Zn2+ exudation. The porosity may also be a determining factor contributing to the antibacterial capability of the ZnO nanoarchitectures. The fusiform based flowers give the larger pore diameter of 46.43 nm, providing beneficial transport path for Zn2+ ion diffusion. In order to confirm the ion release from zinc oxides, we quantified zinc ions in the suspension of zinc oxide with different morphology using atomic absorption spectrometry. It can be seen from Table 3 that all three morphologies release zinc ions, and the corresponding release amount is in an order of rod > fusiform > petal flowers. Interestingly, this order is the same with that of intensity ratio of I(100)/I(002) in Table 2, which further confirms that the proportion of (0001) Zn-polar face is proportional to the Zn2+ exudation. As proven in Figure 3, the biocidal power is in an order of petal > fusiform > rod flowers, showing inversely proportional to the order of Zn2+ release. Such an opposite order suggests that zinc oxide kill bacteria not only by Zn2+ release but also in other ways. To find out the real cause, we designed a test to compare the antibacterial activity of zinc oxide (petal flowers as the representatives) with the zinc ions (zinc nitrate and zinc acetate as model). For comparison, the traditional biocide sodium hypochlorite was applied as the positive control. The antibacterial results in Figure 4 show that zinc oxides and sodium hypochlorite have quite high activity against both E. coli and S. aureus, while the zinc nitrate and zinc acetate are less active. These results further proved that the antibacterial activity of zinc oxide flowers is codetermined by Zn2+ release, light catalysis, adsorption or complex function


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rather than only one of them.50 The germicidal actions of zinc oxides on bacteria are well known but their biological effects on bacteria are still not fully understood.58,59 Here an attempt is also made to give a systematic study based on our own studies focusing the integrality of cell wall, permeability of cell membrane, and DNA cleavage induced by zinc oxide with different morphologies (as shown in Figure 5). Keeping integrality of cell wall is necessary for the bacterial survival. The damage of cell wall could unavoidably cause bacterial death. Herein, we examine the integrality of cell wall by testing structural changes of bacteria after zinc oxide treatment by SEM technique. Figure 6A shows SEM images of E. coli and S. aureus after treatment respectively with rod, fusiform, and petal flowers for 320 min. Compared with intact cells with smooth surfaces, both E. coli and S. aureus treated with ZnO flowers show somewhat rugged appearances, and even small holes are detected (blue arrow). These results suggest that all three products could destroy the bacterial surface structures. On a magnified observation, we can easily find that rod flowers and fusiform flowers can make more serious holey surface, which is especially clearly seen form E. coli. We infer that the rod flowers and fusiform flowers have more sharp leaves compared with petal flowers. These sharp leaves can be act as surgical knife to lancinate bacteria in an easy way. Thereby, it can be concluded that rod and fusiform flowers are more destructive toward bacteria than petal flowers. To understand the real effect of zinc oxide on bacterial structure, we explored the structure of E. coli after a quite long-term treatment with fusiform flower-shaped zinc oxide. As can be seen from the left of Figure 6B, there are some fragments scattered onto flowers. To identify their chemical components, EDS spectra were utilized. The EDS spectrum corresponding to the red dotted line region in upper image of Figure 6B mainly shows peaks for Zn, O, Si, and C element. Element Zn and O are form the zinc oxide, and element Si is from the Si substrate used for sample immobilization. Most importantly, C elemental peak in EDS spectrum is possibly from the fragment of treated E. coli. While EDS spectrum of smooth region on zinc oxide (red dotted line region in down image of Figure 6B) shows almost no C


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elemental peak. These further confirm that the carbon element is from E. coli other than zinc oxide, suggesting that zinc oxide could cut the E. coli structure into small fragments. The damage of original structure of bacteria could break/or inhibit the balance of the normal metabolic process, resulting in the bacterial death. Therefore, the structural destruction of bacteria triggered by zinc oxide might be one possible way to killing bacteria. Along with the change in bacterial structure, the morphology of zinc oxide could also change when overly contacting. In the right of Figure 6B, some flowers lose their original holonomic appearances, and even some destroyed leaves of zinc oxide flowers are detected in the green dotted line region. The antibacterial kinetic test (Figure 3B and 3C) reveals that long-term contacting could realize the complete killing of bacteria, but over-contacting might also cause the damage of zinc oxides themselves. Figure 6A shows that we can get the goal of complete killing without destroying the zinc oxide if we can control the contact time based on antibacterial kinetic pattern (Figure 3B and 3C). These findings are powerful evidences for the structural changes in bacterial cells and zinc oxides after the contact between them. To shed light on the mechanistic aspect of the biological effect of zinc oxide on cell membrane, we carried out the confocal study using propidium iodide (PI), which was an effective tool to visualize the dead bacteria by monitoring the color changes. PI is capable of penetrating the disrupted merely the membrane of dead bacteria and specifically binding DNA (or RNA) to acquire enhanced fluorescence.60 Based on such feature, intracellular staining of PI can identify dead cells, and PI is always utilized as marker to verify whether the permeability of cell membranes changed in the presence of the biocides. For the confocal test in this study, E. coli and S. aureus was treated with ZnO nanoflowers, and subsequently stained with PI. Confocal images are shown in Figure 7. The PI stained bacteria in confocal image is detected as red dots. The drastic increases in red dots after treating with zinc oxide indicate that the rise in the permeability of cell membranes. The change in the permeability of cell membranes is an alternative reason for the killing action of zinc oxide.


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Morphology-dependent effect of zinc oxide on cell membrane was also seen from Figure 7. Compared with the control, the red dots were obviously detected after treatment with rod flowers, which reflects that relative amount of bacteria were killed. More dense red dots are found when fusiform and petal flowers were used. Even for petal flowers, it is hardly to discern the aggregated dead bacteria. The gradual increase of red dots from rod flowers to fusiform flowers then to petal flowers is attributed to the increasing membrane permeability in accord with the result of antibacterial kinetic test. Such a difference induced by morphological variability further proves the above statement for antibacterial test. These results suggest that ZnO can show wide-spectrum activities by the increase in the permeability of cell cytoplasmic membranes. The disruptions of the bacterial structure and cell membrane might cause the leakage of the intracellular contents, e.g., DNA. So the effect of zinc oxide on DNA induced by direct or indirect contacting between zinc oxide and DNA caught our attentions. To investigate the effect, we treat E. coli with different ZnO nanoflowers, and then DNA was extracted from E. coli for testing DNA cleavage. DNA cleavage was analyzed by the agarose gel electrophoresis.61 Figure 8A reveals slight DNA wreck induced by three ZnO flowers, which is seen as thinner band in exposed samples compared with thicker band for the control (DNA without treating with ZnO). So, it might be recognized that the ZnO flowers possess capability of attacking DNA, but we can’t realize whether this is the very reason for killing bacteria. In a detailed view, the thickness of DNA band show a regulation with the change in morphology, suggesting the intensity of biological effect on DNA. The rod flowers shows weakest, and the effect aggravates by contacting the fusiform flowers, and even DNA displays most serious exhaustion towards petal flowers. A mixture sample by physical mixing containing all rod, fusiform, and petal flowers (mrod : mfusiform : mpetal = 1 : 1 : 1) was also treated with E. coli for further confirmation of morphology effect. It is vivid that the extent of DNA damage for the mixture falls in between that of the rod and petal flowers, further indicating that the petal flowers are the most destructive and rod flowers are relatively


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faintish. Despite this order is the same with that of antibacterial activity, we can’t affirm that the DNA damage is the real cause for antibacterial action, it can only be proven the morphology-induced effect on DNA. From the results of the agarose gel electrophoresis, the definite destruction of zinc oxide flowers on DNA is not quite explicit. We speculate that the zinc oxides were mainly exhausted by bacterial outer structures, e.g., cell wall and membrane, other than DNA. So the main question is whether zinc oxide can affect DNA when there is a direct contacting between them for enough periods. Based on this consideration, we extract DNA from E. coli firstly, and then the naked DNA was treated with different zinc oxide flowers. The agarose gel electrophoresis was also utilized to determine the effect on the treated DNA as shown in Figure 8B. Similar trend as Figure 8A was found in Figure 8B, viz., no significant DNA damage was found as well. Thus we judge that the effect of zinc oxides on DNA is not quite palpable in the present experimental conditions. Yet we are sure that the influence of zinc oxide on DNA is not the main approach to killing bacteria. Also a conclusion regarding morphology-dependent effect on DNA is advisable, keeping an order of petal > fusiform > rod flowers. As for the practical clinical application, therapeutic materials always require excellent biocompatibility, should not elicit any adverse response or toxicity to the normal cells.62 Taking such stringent clinical prerequisites into consideration, the cell toxicity of the ZnO products obtained in this study need to be systematically assessed. Release of uncontrolled amount of Zn2+ from the ZnO products is a very crucial and delicate factor contributing for the cell toxicity, which could stimulate immune system eliciting toxic effect to cells. Thereby, the cytotoxicity of the ZnO products was assessed by using normal cultured cells. Hela cells were treated with zinc oxide samples for 24 h, and then cell toxicity was evaluated.63 As shown in Figure 9, all ZnO products display a certain degree of toxicity to the Hela cells, showing certain reduction in cell viability. Such a result successfully reveals that Zn2+ released from the ZnO flowers more or less toxicity to Hela cell. Noteworthy, the toxicity changes subtly with the morphological variability. Nanorod and nanofusiform based flowers show lower


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toxicity compared with the nanopetal counterpart. Relative higher toxicity of the petal flowers well annotates the higher capability of Zn2+ exudation from ZnO body, which is fitted in well with the results of the antibacterial tests. Furthermore, as expected, the cell survival corresponding to all three products shows reduction trend with increasing concentration from 0 mg·mL-1 to 1.0 mg·mL-1. As the concentration reaches as high as 1.0 mg·mL-1, nanopetal built flowers give 63 % viability, higher than those of rod and fusiform based flowers at the same concentration. In general, we can analogize that the as-prepared ZnO products have trivial cytotoxicity to the normal cell within the selected concentration range. CONCLUSIONS We established a facile approach for the synthesis of the flower-like ZnO nanoarchitectures with different morphology by the hydrothermal technique. All products exhibited powerful bactericidal capability both against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. The relationship between morphology and biological effect was constructed systematically. From the spread plate method, biocidal kinetic test, and zone of inhibition study, the biocidal power is in an order of petal > fusiform > rod flowers, because they have different microscopic parameters such as specific surface area, pore size, and Zn-polar plane, ect. All the petal, fusiform, and rod built ZnO flowers showed disrupting capability of structure of bacterial cells and cell membranes, while the definite effect on DNA is not quite clear. Noteworthy, all the petal, fusiform, and rod ZnO flowers revealed trivial cytotoxicity to Hela cells. We are firmly convinced that the present study opens up the possibility for extensive investigation of the antimicrobial ZnO, tuning their biological activity by tuning their morphology. AUTHOR INFORMATION Corresponding Author E-mail: [email protected], Tel.: +86 471 4992982. Author Contributions §

These authors contribute equally to this work.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21304044), the Natural Science Foundation of Inner Mongolia Autonomous Region (2015MS0520), and the Supported by Program of Higher-level Talents of Inner Mongolia University (30105-125136). REFERENCES (1) Dai, Y.; Yang, D.; Ma, P.; Kang, X.; Zhang, X.; Li, C.; Hou, Z.; Cheng, Z.; Lin, J. Doxorubicin Cconjugated NaYF4:Yb3+/Tm3+ Nanoparticles for Therapy and Sensing of Drug Delivery by Luminescence Resonance Energy Transfer. Biomaterials 2012, 33, 8704-8713. (2) Treccani, L.; Klein, T. Y.; Meder, F.; Pardun, K.; Rezwan, K. Functionalized Ceramics for Biomedical, Biotechnological and Environmental Applications. Acta Biomater 2013, 9, 7115-7150. (3) Song, J.; Kim, H.; Jang, Y.; Jang, J. Enhanced Antibacterial Activity of Silver/PolyrhodanineComposite-Decorated Silica Nanoparticles. ACS Appl Mater Interfaces 2013, 5, 11563-11568. (4) Fox, S.; Wilkinson, T. S.; Wheatley, P. S.; Xiao, B.; Morris, R. E.; Sutherland, A.; Simpson, A. J.; Barlow, P. G.; Butler, A. R.; Megson, I. L.; Rossi, A. G. NO-loaded Zn2+-Exchanged Zeolite Materials: A Potential Bifunctional Anti-bacterial Strategy. Acta Biomater 2010, 6, 1515-1521. (5) Rizzello, L.; Pompa, P. P. Nanosilver-Based Antibacterial Drugs and Devices: Mechanisms, Methodological Drawbacks, and Guidelines. Chem Soc Rev 2014, 43, 1501-1518. (6) Shukla, A.; Fleming, K. E.; Chuang, H. F.; Chau, T. M.; Loose, C. R.; Stephanopoulos, G. N.; Hammond, P. T. Controlling the Release of Peptide Antimicrobial Agents from Surfaces. Biomaterials 2010, 31, 2348-2357. (7) Huo, D.; He, J.; Li, H.; Yu, H.; Shi, T.; Feng, Y.; Zhou, Z.; Hu, Y. Fabrication of Au@Ag CoreShell NPs as Enhanced CT Contrast Agents with Broad Antibacterial Properties. Colloid Surf B 2014, 117, 29-35.


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Tables Table 1. Morphology of zinc oxide products prepared with different base type, base concentration, and aging time by the hydrothermal technique41 Number

Base

1

KOH

Concentration Time

Morphology

(mol/L)

(h)

0.2

24

sheet

2

72

sheet

3

96

sheet

24

sheet

5

72

sheet

6

96

sheet

24

sheet block

8

72

sheet block

9

96

sheet block

24

rod block

11

72

rod block

12

96

rod block

24

rod flower

14

72

rod flower

15

96

rod block

24

sheet

17

72

Sheet

18

96

sheet

24

sheet block

72

sheet block

4

0.3

7

0.4

10

0.5

13

16

19 20

0.6

NaOH

0.2

0.3


27


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21

Page 28 of 42

96

sheet

24

rod block

23

72

Rod

24

96

rod

24

rod flower

26

72

rod block

27

96

rod

24

rod block

29

72

rod

30

96

rod

24

fusiform flower

32

72

fusiform flower

33

96

fusiform block

24

petal flower

35

72

petal flower

36

96

petal block

24

petal flower

38

72

petal block

39

96

petal block

24

rod block

41

72

rod

42

96

rod

24

rod block

44

72

rod

45

96

rod

22

0.4

25

0.5

28

31

34

37

40

43

0.6

NH3

0.2

0.3

0.4

0.5

0.6


28

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Table 2. Morphology, intensity ratio of I(100)/I(002), surface area, and pore diameter of zinc oxide prepared with different base types and concentrations TEM/SEM Base type (Con.)

Morphology

XRD Intensity ratio of I(100)/I(002)

BET Surface

Pore

area

diameter

(m2·g-1)

(nm)

KOH (0.6 M)

Rod flowers

1.47

3.28

42.63

NH3 (0.2 M)

Fusiform flowers

1.41

2.72

46.43

NH3 (0.3 M)

Petal flowers

1.38

7.21

38.31


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Table 3. The amount of zinc ions released from zinc oxides with different morphologies Zinc ions Concentrationa (mg/L) Percentageb (%)

Rod flowers

Fusiform flowers

Petal flowers

2.18

1.12

0.47

0.035

0.018

0.0075

a

Concentration of zinc ions released from 6.25 mg/mL zinc oxide with different morphologies

b

Percentage = (B/A) × 100 %; where A is concentration of zinc oxides, and B is the corresponding

concentration of zinc ions


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Figure captions Figure 1. SEM (1 and 2), TEM (3), and HRTEM (4) image of zinc oxide with rod (A), fusiform (B), and petal (C) built flower morphology. Figure 2. Nitrogen adsorption/desorption isotherms (A), powder XRD patterns (B), UV-vis spectra (C), and FTIR spectra (D) of zinc oxide with rod (1), fusiform (2), and petal (3) built flower morphology. Figure 3. (A) Photographs showing the bacterial culture plates of E. coli and S. aureus upon a 80 min exposure to the control and zinc oxide with different morphologies. (B and C) Antibacterial kinetic test graphs of zinc oxide with different morphologies against E. coli (B) and S. aureus (C). Survival (%) = b/a ×100%; where a is the number of surviving bacteria colonies on the control culture plate and b is that on the sample culture plate. (D) Photographs of the zone of inhibition against E. coli and S. aureus for zinc oxide with different morphologies. Figure 4. Photographs showing the bacterial culture plates of E. coli and S. aureus upon a 30 min exposure of the control, NaClO, petal flower-shaped ZnO, Zn(NO3)2, and Zn(OAc)2, respectively. Figure 5. Schematic illustration of biological effects of zinc oxide nanoflowers with different morphologies on integrality of bacterial cell wall, permeability of bacterial cell membrane, and bacterial DNA cleavage. Figure 6. (A) SEM images of E. coli and S. aureus treated with zinc oxide with different morphologies for 320 min. (B) SEM images and EDS spectra of E. coli long-term treated with fusiform flowers. Figure 7. Photographs showing permeability of cell membranes of E. coli and S. aureus treated with zinc oxide with different morphologies. The scale bar is 30 µm. Figure 8. (A) Agarose gel electrophoresis images of DNA extracted from E. coli (after treating E. coli with different zinc oxides). (B) Agarose gel electrophoresis images of DNA (extracted from E. coli) treated with different zinc oxides. M: Marker; C: Control; R: Rod flowers: F: Fusiform flowers; P: Petal flowers; Mix: Mixture of rod, fusiform, and petal flowers. Figure 9. Survival percentage of Hela cells after treated with zinc oxide with different morphologies


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and different concentrations.


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Figures

Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Figure 8


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Figure 9


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Table of Contents Graphic


42

Insight into Biological Effects of Zinc Oxide Nanoflowers on Bacteria

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