Potent and durable antibacterial activity of ZnO-dotted nanohybrids hydrothermally derived from ZnAl-layered double hydroxides

Colloids and Surfaces B: Biointerfaces 181 (2019) 585–592

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Potent and durable antibacterial activity of ZnO-dotted nanohybrids hydrothermally derived from ZnAl-layered double hydroxides

T

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Mengxue Lia, Zhi Ping Xub, , Yasmina Sultanbawac, Weiyu Chenb, Jianyong Liua, ⁎ Guangren Qiana, a

School of Environmental and Chemical Engineering, Shanghai University, No. 333 Nanchen Rd., Shanghai, 200444, PR China Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, 4072, Australia c Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, 4072, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Zinc oxide nanocomposite Layered double hydroxides In situ hydrothermal treatment Potent and durable antibacterial activity

The search for effective alternatives to traditional antibiotics to avoid antibiotic resistant bacteria is growing worldwide. ZnO nanoparticles are found to effectively inhibit growth and proliferation of bacteria, and ZnObased layered double hydroxides (ZnO-based LDHs) have been intensively investigated for this purpose. However, the nanocomposites are made in a multi-step preparation process with severe agglomeration and limited bactericidal ability. In this research, ZnO-dotted nanohybrids using Zn3Al-LDHs as precursors (ZnOdotted LDHs or ZnO/LDHs) were synthesized under facile hydrothermal conditions. An understanding of the transformation of the LDH precursors to the ZnO/LDHs was conducted with TEM/HRTEM/XRD/FTIR. ZnO/ LDHs can be transformed from ZnAl-LDHs, with more ZnO nanodots generated upon heating at 150 and 200 °C for 2 h (Zn3Al-150, Zn3Al-200). Zn3Al-200 nanohybrids showed potent antibacterial activity towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) at 100–300 μg/mL for 4 days. Antibacterial activity of Zn3Al200 may be attributed to the synergistic effects (ROS, leached Zn2+ and physical interaction). This research thus suggests a potential economic approach to prepare ZnO/LDH nanocomposites for avoiding the antibiotic resistant bacteria in environmental engineering or clinic fields.

1. Introduction More antibiotics have been explored to deal with various bacterial infections. However, the occurrence of drug resistant bacteria restricts the development of conventional antibacterial agent [1]. In medical and industrial area, inorganic metal oxide nanoparticles (NPs) (e.g. CuO, TiO2, Al2O3, Ag and ZnO) are being explored as bactericidal agents [2–4]. Among inorganic metal oxides, ZnO materials are believed to be full of promise for biochemical sensors, anti-bacterial agents, food packaging and photocatalysts [5]. Antimicrobial efficacy of ZnO has also been studied with or without light. Layered double hydroxides (LDHs), one type of two-dimensional inorganic layered materials, are widely explored for applications in catalysis, biomedicine and environmental remediation [6]. [M1−x2+Mx3+(OH)2]x+[An−]x/n·zH2O expressed their general formula, in which M3+ and M2+ represent trivalent and divalent metal ions, respectively. An− anions in the interlayers compensate for the brucitelike layers’ positive charge. Owing to uniform distribution of metal cations in interlayer, the LDH is an excellent precursor for preparing

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nanocomposite materials containing ZnO [7]. Some previous studies have reported that ZnO-based materials thermally transformed from ZnAl-LDH precursors (ZnO-based LDHs) via calcining for more than 2 h or calcination at more than 200 °C [8,9]. The calcined ZnO-based LDHs have been examined for the antibacterial activity for 24 h [10]. However, as-prepared ZnO-nanoparticle (NP) composites are solid nanomaterials with low crystallinity and severe agglomeration. The agglomeration leads to smaller surface area for interactions with bacterial membranes and solubilization of zinc ions, which may limit the capability of ZnO NPs for potent and long-term antibacterial effect. To avoid these shortcomings, ZnO-dotted LDHs has been proposed to be tailored with a facile hydrothermal treatment of Zn3Al-LDH precursors in this research, yielding more uniform ZnO nanodots on the LDHs framework (ZnO-dotted LDHs, or ZnO/LDHs) with more surface exposure to integrate efficient antibacterial effects, which has not been reported yet. For practical antibacterial applications in environmental engineering or clinic fields, facile synthesis of uniform ZnO/LDH nanohybrids with optimal exposure of ZnO and excellent performance against Escherichia coli and Staphylococcus aureus (E. coli and S. aureus)

Corresponding authors. E-mail addresses: [email protected] (Z.P. Xu), [email protected], grqian@staff.shu.edu.cn (G. Qian).

https://doi.org/10.1016/j.colsurfb.2019.06.013 Received 5 February 2019; Received in revised form 3 June 2019; Accepted 6 June 2019 Available online 07 June 2019 0927-7765/ © 2019 Published by Elsevier B.V.

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Fig. 1. XRD patterns for LDHs treated in different conditions. Right curves were enlarged x5 times in intensity. Table 1 Structural parameters of LDH samples treated in different conditions. Structural parameters

Zeta potential (mV) pH of suspension LDH (nm)

Samples

1

a110 c003 2 Thickness in c-axis d(100) d(002) d(101) 3 c002/a100 2 Crystallite size d(200) d(400) d(111) d(220) d(400) d(422) 1

ZnO (nm)

Zn5(OH)8(NO3)2·2H2O (nm) ZnAl2O4 (nm)

Zn3Al-LDH

Zn3Al-80

Zn3Al-150

Zn3Al-200

20.4 ± 0.4 8.11 0.306 2.66 7.35 – – – – – – – – – – –

16.6 ± 0.6 6.64 0.307 2.65 28.9 0.281 0.261 0.247 1.61 35.1 ± 9.6 0.973 0.489 0.489 – 0.207 –

19.8 ± 1.2 6.15 – 2.67 49.6 0.281 0.261 0.247 1.61 33.0 ± 9.9 0.969 0.484 0.484 0.289 0.205 0.167

24.6 ± 0.2 6.17 – – – 0.281 0.261 0.247 1.61 36.9 ± 9.3 0.965 0.483 0.483 0.289 0.205 0.167

a110 = 2d110; c003 = 3d-spacing; 2d110·sinθ110 = λ; λ = 0.15406 nm; d-spacing = (d003 + 2*d006 + 3*d009 + … + n*d00(3n))/n. For LDH, the thickness in c-axis calculated by the Scherrer’s equation with (003) reflection; For ZnO, the crystallite size was the average dimension calculated from the FWHM of peaks (100), (002), (101), (102), (110) and (112). 3 a100 = 2*3−0.5d100, c002 = 2d002 for ZnO. 1 2

2. Experimental section

is highly expected. Therefore, the purposes in this research were to: (1) prepare uniform ZnO-dotted LDH nanohybrids using LDHs as precursors (ZnO/ LDHs) by facile hydrothermal method and investigate the transformation of LDHs into ZnO/LDHs; (2) determine whether ZnO/LDHs had potent and durable antibacterial activity (e.g. 4 days); (3) reveal antibacterial action of ZnO/LDHs. This research has demonstrated that ZnO/LDHs upon hydrothermal treatment at 200 °C for 2 h significantly improved antimicrobial efficacy against E. coli and S. aureus, and sustained the potent effect for up to 4 days.

2.1. Preparation of LDH nanoparticles Zn3Al-LDH nanomaterials as precursors were synthesized via coprecipitation. Zn(NO3)2·6H2O (3.6 mmol) and Al(NO3)3·9H2O (1.2 mmol) in 10 mL solution were added quickly to 40 mL of NaOH solution (9.6 mmol) with vigorous stirring for 10 min at 25 °C. Then the nanomaterials was centrifuged and washed and redispersed in 30 mL of distilled water. ZnO/LDHs were then synthesized by hydrothermal treatment of Zn3Al-LDH precursors. Based on previous reports [8,9,11], it is not benefited for the formation of ZnO at low temperature for a short time in the treatment for ZnAl-LDH precursors. Thus, Zn3Al-LDH suspension was with hydrothermal treatment in a 50-mL Teflon-lined stainless steel 586

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[12], filter paper disks (∼6 mm diameter) containing 0–300 μg of ZnO/ LDH nanoparticles were put on an agar plate with uniform bacteria (107 CFU/mL). Then, the agar plate was cultured at 37 °C for 24 h under a light or dark condition. After that, the plates were imaged to examine the formation of zone inhibition around the disk. The inhibition zone diameter around the disk was recorded using a vernier scale.

2.4. Statistical analysis All data were calculated as mean ± SD based on three independent experiments. One-way ANOVA and Tukey's multiple comparison tests were used to analyze the data. The levels of significant differences were shown as following：*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001.

3. Results and discussion Fig. 2. FT-IR spectra for LDH samples treated in different conditions.

3.1. Structure and composition of ZnO/LDH nanoparticles autoclave at 80 °C for 24 h, 150 or 200 °C for 2 h, respectively. Three kinds of hybrid materials (ZnO/LDHs) were obtained and denoted as Zn3Al-80, Zn3Al-150 and Zn3Al-200, respectively.

As shown in Fig. 1, Zn3Al-LDH displays four typical symmetric diffractions of LDHs, indicating the formation of a crystal with layered structure [13,14]. The calculated parameter a110 and c003 were 0.306 and 2.66 nm (Table 1), quite similar to that reported elsewhere [13]. The XRD patterns of Zn3Al-80 and Zn3Al-150 show the reflection peaks (003) and (006). The estimated lattice parameters (c003) of Zn3Al-80 and Zn3Al-150 had a similar value (2.65–2.67 nm, Table 1). Consequently, the original structure of LDHs was partly preserved. The disappeared peaks of (003), (006), (012) and (110) diffractions in Zn3Al200 nanohybrids confirmed that the LDH layer structure was already decomposed. The XRD patterns of sample Zn3Al-80, Zn3Al-150 and Zn3Al-200 also clearly show new sharper reflection peaks. These new diffractions correspond well to planes (100), (002) and (101) of ZnO wurtzite phase, demonstrating its formation after hydrothermal treatment [10]. Peaks of ZnO diffractions in ZnO/LDHs treated at 80 °C for 24 h were not obvious. So peaks of ZnO diffractions in ZnO/LDHs treated at 80 °C for 2 h would be even weaker. Thus, 80 °C for 24 h, 150 and 200 °C for 2 h in the hydrothermal treatments were used to demonstrate the influence of the heating temperature and the heating time. The intense and sharp reflections of crystalline ZnO showed the improved crystallinity in the samples treated at a higher thermal temperature or for a longer duration [15]. For Zn3Al-80, Zn3Al-150 and Zn3Al-200, the c002/ a100 ratio value (1.61) was very close to 1.63, which was consistent with wurtzite structure (Table 1). In addition, the crystallite size of ZnO crystals was 35.1 ± 9.6, 33.0 ± 9.9 and 36.9 ± 9.3 nm after heating at 80 °C for 24 h, 150 °C for 2 h and 200 °C for 2 h (Table 1), respectively. These size is similar to the reports (the crystallite size of ZnO: 25–62 nm) where precursor Zn3Al-LDHs were calcined at 500–900 °C [7,9]. Detail inspection of the XRD patterns reveals that zinc hydroxide nitrate (ZHN), including Zn5(OH)8(NO3)2·2H2O [16], and ZnAl2O4 were possibly formed in Zn3Al-80, Zn3Al-150 and Zn3Al-200 samples, while their amount seemed very limited as their weaker diffraction peaks compared with that of the ZnO phase (Table 1). The zeta potential of these ZnO/LDHs was around 20 mV in aqueous suspension at room temperature (Table 1). The positive charge may be mainly attributed to that of LDH, ZnO and ZHN [17–19], which may benefit for ZnO/LDH adhesion onto bacterial surfaces. When freshly prepared LDHs slurry was treated hydrothermally, the pH values decreased from 8.11 to 6.17 with increasing the thermal temperature (Table 1). As ZnO is the major phase in ZnO/LDHs, the lower pH of the suspension may be attributed to ZnO (isoelectric points of ZnO > 8.0) with positive charges, adsorbing some OH− on the surface [20].

2.2. Characterization The zeta potential of synthetic samples in distilled water was characterized with a Zeta-Sizer Nano-ZS instrument at room temperature. The morphological examination of samples was performed with transmission electron microscopy (HRTEM/TEM) (JEOL JEM-2100) and scanning electron microscopy (SEM) (JEOL JSM-7500). X-ray powder diffraction (XRD) patterns of the ZnO/LDHs were investigated by XRD-6000 coupled to Cu Kα radiation in the 2θ range of 5-70°. Fourier transform infrared (FT-IR) spectra were recorded in a Nicolet 6700 spectrometer from 4000 to 400 cm−1. The ultraviolet–visible (UV–Vis) spectra were investigated in a U-3010 spectrophotometer (Hitachi Co., Japan). Electron spin resonance (ESR) spectrum was collected with a Bruker A300 spectrometer at room temperature and 5,5dimethyl-1-pyrroline-N-oxide (DMPO) was used as the spin trap to verify the formation of hydroxyl radical. ZnO/LDH samples (30 mg of total zinc) in a 30-kD dialysis bag were added into 50 mL medium with shaking at 37 °C for 1–4 days. A 1-mL aliquot was treated in 4 mL of 5% HNO3 solution. The solution containing Zn2+ was detected by inductively coupled plasma - optical emission spectrometry (Prodigy7 model, ICP-OES, Leeman, USA). 2.3. Bactericidal activity evaluation Escherichia coli and Staphylococcus aureus (E. coli and S. aureus) were grown at 37 °C in nutrient broth with shaking. At the exponential phase, bacterial stock suspension in deionized water (dH2O) was prepared after washed with deionized water (dH2O) twice via centrifugation. Then, the suspension was diluted to 109 CFU/mL according to the values of optical density at 600 nm (OD600). The antibacterial efficacy of ZnO/LDHs was judged using broth and disk tests. The broth tests were done by diluting nanoparticle dispersion with the nutrient broth. Then, 50 μL bacterial solution (109 CFU/mL) was added to 5.0 ml of a series of ZnO/LDH dispersions. The mixtures were shaken at 37 °C for 24 h in visible light. The viability of bacteria was confirmed by OD600. The medium only with ZnO/LDH nanoparticles in the absence of bacteria was logged as a background to subtract turbidity resulting from particles. OD reading (subtracted background) thus presents the bacterial viability. Disk tests were done to determine sensitivity towards samples, as marked by their growth inhibition zone. As reported by Ruparelia et al. 587

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Fig. 3. TEM, HRTEM images and schematic illustration of the morphology and size changes of LDH samples heated from room temperature (A), 80 (B) and 150 (C) to 200 °C (D).

and/or ZHN at 200 °C [9]. Formation of ZHN and ZnO may be due to the one oxygen of the NO3- strongly interacted with the Zn2+ in the LDH layers. The structure of Zn3Al-200 was characterized with symmetry degeneration of the nitrate group. The TEM and HRTEM images (Fig. 3) indicate that the pristine Zn3Al-LDHs were thin platelet-like particles, and some with a clear hexagonal shape and the lateral size of around 100 nm. The thin crystallites seemed to form big agglomerates. Interestingly, heat treatment of pristine Zn3Al-LDH at 80 °C for 24 h (Zn3Al-80) dispersed aggregates into nearly-homogeneous LDH crystallites and increased the lateral size to around 200 nm (Fig. 3.B1). Moreover, there were indistinct dots on the LDH sheet surface, as confirmed with HRTEM image (Fig. 3.B2 and Fig. S1). HRTEM image of Zn3Al-80 shows that the matrix was crystalline with a lattice spacing of about 0.26 nm, consistent with the distance between the (002) plane in ZnO (Table 1) [21]. There seemed no obvious ZnAl2O4 phase in the HRTEM image of Zn3Al-80, in consistence with its XRD pattern. Many dots were observed to locate on the LDH surface in the Zn3Al-

3.2. Phase composition and morphology of LDH nanohybrids The FT-IR spectra (Fig. 2) further confirmed the typical phase of Zn3Al-LDH, Zn3Al-80, Zn3Al-150 and Zn3Al-200. The peak of the pristine Zn3Al-LDH at 1644 cm−1 corresponds to δH-O-H deformation mode. The bands of the pristine Zn3Al-LDH at 1340 cm-1, 1050 cm-1 and 820 cm-1 correspond to ν3, ν1 and ν2 stretching mode of nitrate group, respectively. A few peaks of the pristine Zn3Al-LDH at 596, 549 and 422 cm-1 correspond to MeOH and MeO vibrations [13]. The relative intensity of 596, 549 and 422 cm-1 bands was clearly high for Zn3Al-80, but became weaker for Zn3Al-150 and Zn3Al-200. This may suggest that the specific MeOH and MeO bonds in the LDH layers changed because of the existence of ZnO, ZHN and ZnAl2O4. The increased intensity of 1050 cm−1 band suggests that the NO3- group possessed C3v symmetry instead of D3h symmetry [13], probably owing to the formation of ZHN. Moreover, the 1340 cm−1 vibration band was split into two peaks at 1340 and 1425 (ν (NO2, as)) as the hydrothermal condition increased to 200 °C, which is tentatively due to the distortion of the NO3- groups 588

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Fig. 4. (a) and (b) Antimicrobial activities of LDHs at 200 μg/mL for 24 h in light. (c) and (d) Antimicrobial activities of Zn3Al-200 at different concentrations for 24 h in light. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Table 2 Comparison of antibacterial activity of samples in literatures. Samples

Bacteria tested

Inhibition time (h)

Applied dosage (μg/mL)

Mechanism

References

Zn3Al-200 Calcined Zn-LDH LDHs

E. coli and S. aureus E. coli and S. aureus E. coli and S. aureus E. coli and S. aureus E. coli and S. aureus S. aureus E. coli and S. aureus S. aureus E. coli P. aeruginosa S. aureus E. coli E. coli No papers reported No papers reported

96 24 24 – – 24 24 24 9-16 48 6-10 2 24

100-300 (˜67-200 ZnO) > 18-53 50-1250 > 2500 14-10000 8000-14000 16-18 80 150-250 80-344 400 250-500 800

Adhesion; ROS and Zn2+; penetration Adhesion; ROS and metal ions Adhesion; metal ions

This study [10] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]

ZnO

Zn5(OH)8(NO3)2·2H2O ZnAl2O4

Adhesion; ROS and Zn2+; penetration

ZnAl2O4 phase [21]. The results were consistent with the analysis of XRD pattern and FT-IR spectra. For Zn3Al-200, small nanoparticle dots were well dispersed with the clearer contrast (Fig. 3.D1). The dots were mostly ZnO nanoparticles with the size of around 30–40 nm, as reflected by the fringe (0.26 nm) of the (002) plane (Fig. 3.D2 and Fig. S1). This change may indicate that the initial Zn3Al-LDH sheets were thermally converted to ZnO as

150 TEM image (Fig. 3.C1). The HRTEM image reveals that these dots were mostly ZnO nanoparticles with the size of around 30 nm (Fig. 3.C2 and Fig. S1). The spacing of lattice fringe was 0.26 nm, consistent with the (002) facet of formed ZnO. Clearly, the lateral size of LDH framework seemed to increase to nearly ˜1 μm after heat-treatment at 150 °C for 2 h. In addition, ZnAl2O4 phase seemingly appeared as a fringe spacing of 0.48 nm was observed, consistent with the (111) facet of 589

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ZnO crystals. As shown in Fig. 4c and d, Zn3Al-200 exhibited a dose-dependent antimicrobial performance. Zn3Al-200 started to show the antibacterial activity at 100 μg/mL against both bacteria. The growth of both bacteria was mostly inhibited at 200 μg/mL and completely inhibited at 300 μg/mL of Zn3Al-200. In this study, ZnO in 300 μg/mL of Zn3Al-200 contained about 200 μg/mL ZnO, and Zn3Al-200 achieved the complete bacterial inhibition for 24 h. These demonstrate that the antibacterial activity of Zn3Al-200 is obviously better than that of calcined Zn-LDH and several LDHs (Table 2). Moreover, Zn3Al-200 nanohybrids possess stronger antibacterial ability compared to some naked ZnO nanoparticles in some cases (Table 2). To further compare the antimicrobial efficacy of Zn3Al-200, SEM was employed to visualize morphological changes of E. coli and S. aureus upon exposure to Zn3Al-200 at different concentrations for 24 h (Fig. S2). The bacterial wall of E. coli remained relatively intact after treatment with 100 μg/mL Zn3Al-200, with partial membrane disintegrated but more parts invaginated (Fig. S2b). Serious injuries and bacterial membrane breaks were observed for E. coli treated with 200 μg/mL of Zn3Al-200 (Fig. S2c). Similar situation was noted for S. aureus. When S. aureus were treated with Zn3Al-200 at 50 μg/mL, nanohybrids seemed just to adhere onto the bacterial cell wall without breaks (Fig. S2e), consistent with the limited antibacterial activity shown in Fig. 4d. When treated with 200 μg/mL of Zn3Al-200, most bacterial cell wall was lysed (Fig. S2f). Another set of bacterial tests of the Zn3Al-200 was performed by applying the disk diffusion method for the inhibition zone of coated disks. The inhibition zone indicates bacterial sensitivity towards samples. Fig. S3 shows the photographs of the inhibition zone and Table S1 lists its diameter. Both the bacterial strains were sensitive to Zn3Al-200 nanohybrids in a dose-dependent manner (Fig. S3 and Table S1). Furthermore, the inhibition zone diameter was obviously larger under daylight condition ((Fig. S3a and c) than under dark condition ((Fig. S3b and d), with some cases being significantly different (Table S1), demonstrating that the contribution of light irradiation to the antibacterial activity. 3.4. Durable antibacterial efficiency of Zn3Al-200 Fig. 5. Bacterial growth kinetics in the presence of Zn3Al-200 samples in light. (a) E. coli, (b) S. aureus. Asterisks represent the significant difference between bacteria in the absence and presence of Zn3Al-200 samples.

The extended antimicrobial activity is highly expected to prevent bacterial infection. Thus, the growth patterns of bacteria treated with Zn3Al-200 at concentrations of 100–300 μg/mL in a 4-day period were assayed (Fig. 5). E. coli and S. aureus grew well up to 4 days in the absence of Zn3Al-200, with the OD reading in 1.6-1.8. The presence of Zn3Al-200 inhibited the bacterial growth depending on the nanohybrid concentration. In particular, Zn3Al-200 showed a weak inhibition to the growth of E. coli at 100–150 μg/mL and a moderate inhibition at 200 μg/mL in the 4-day test. Remarkably, 300 μg/mL of Zn3Al-200 nanohybrids maintained nearly 100% inhibition towards E. coli continuously 4 days (Fig. 5a). Most S. aureus’ growth was inhibited at 100 μg/mL of Zn3Al-200, and nearly 100% inhibition of the growth of bacteria was achieved at 200 μg/mL of Zn3Al-200 for 4 days (Fig. 5b). S. aureus bacteria were seemingly more sensitive to Zn3Al-200 compared to E. coli, which may be attributed to their characteristics of the cell walls [34]. The cell wall of Gram-negative bacteria (E. coli) is composed of a peptidoglycan inner membrane and an outer membrane that is constituted of lipoprotein, phospholipids and lipopolysaccharide. However, the cell wall of Grampositive bacteria (S. aureus) is solely made up of peptidoglycan with plenty of pores. Thus the cell wall structure of S. aureus may allow for the close surface contact with exposed ZnO in Zn3Al-200 hybrids and make Zn3Al-200 enter into the cell more easily, which may more severely affect the bacterial biological functions. The durable antibacterial activity was also reflected in the disk diffusion tests. The diameters of the inhibition zone were maintained the same for both 1-day and 4-day tests in the daylight. Compared with

well as a small amount of ZnAl2O4 and ZHN [22], which was consistence with its XRD pattern and FT-IR spectra. On the basis of the above characterization results, the possible phase transformation of ZnO/LDHs may occur as follows. Heat-treatment of Zn3Al-LDHs seems to initiate the formation of ZnO and ZHN at 80 °C (Fig. 3.B3) while the Zn3Al-LDH framework is still kept. With increasing the hydrothermal temperature to 150 °C, better crystallized ZnO and amorphous ZnAl2O4 are formed and well dispersed on the LDH/ZHN framework (Fig. 3.C3). The increasing thickness in c-axis of LDH/ZHN (Table 1) from Zn3Al-LDH to Zn3Al-150 may be due to the LDH/ZHN crystal growth as a result of increasing hydrothermal temperature. Heat-treatment at 200 °C seems to destroy the LDH framework into well crystallized ZnO and ZnAl2O4 onto ZHN frame (Fig. 3.D3).

3.3. Antibacterial effect of ZnO/LDH nanohybrids The antibacterial effects of ZnO/LDHs against E. coli and S. aureus were investigated by the broth dilution and disk diffusion methods. Pristine Zn3Al-LDH and Zn3Al-80 had no impact on bacterial activity at the concentration of up to 200 μg/ml for both bacteria (Fig. 4a and b). Zn3Al-200 displayed the strong antibacterial activity for both bacteria while Zn3Al-150 is only active against S. aureus. The antibacterial activity of Zn3Al-150 and Zn3Al-200 could be mainly attributed to smaller 590

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Scheme 1. Schematic diagram describing the antibacterial effects of Zn3Al-200 towards E. coli.

properties of ZnO/LDHs. The disk inhibition tests showed that light irradiation enlarged the inhibition zone diameter (Fig. S3 and Table S1). The antibacterial activity of Zn3Al-200 nanohybrids may thus involve the production of ROS (hydroxyl radicals, singlet oxygen and hydrogen peroxide) from ZnO (Scheme 1.C). The radical production by Zn3Al-200 was reflected in Fig. S4c upon light irridiation. ROS may contribute to destructing membrane components such as lipids and enhance the internalization of Zn3Al-200. ROS may also damage functional proteins and DNAs, inhibiting the bacterial growth or causing bacterial death [35,38]. As reported previously, < 50 nm nanoparticles could be taken up by bacteria [35]. Some smaller ZnO NPs in Zn3Al-200 nanohybrids (Fig. 3) are possibly taken up by bacteria or penetrate into bacteria, similarly to naked ZnO nanoparticles (Table 2), which subsequently release Zn2+ and generate ROS within the bacteria, and more efficiently inhibit the bacterial growth (Scheme 1.C). Therefore, the high and durable bacterial inhibition property of Zn3Al-200 nanohybrids can be attributed to synergy of: (1) enhanced adhesion towards the membrane of bacteria, which blocks the channels for nutrient uptake and waste exclusion and (2) the enhanced local concentration of ROS and Zn2+ on the bacterial surface; (3) penetration of small Zn3Al-200 into bacteria, which interferences bacterial functions by releasing Zn2+ and producing ROS to inhibit the bacterial growth.

previous reports that had 2–66 h inhibition time (Table 2), ZnO/LDH nanohybrids exhibited a much longer inhibition time towards E. coli and S. aureus. Therefore, the particular structure and composition of Zn3Al-200 nanohybrids may be responsible for the observed high and durable antibacterial activity. 3.5. Mechanism of antibacterial activities Our antibacterial tests demonstrate that Zn3Al-200 had a high and durable antibacterial activity by the broth dilution and disk diffusion methods, more effective than Zn3Al-LDH, Zn3Al-80 and Zn3Al-150, calcined Zn-LDH, LDHs and some reported naked ZnO (Table 2), which can be attributed to the particular properties of Zn3Al-200. Zn3Al-200 are cationic nanohybrids (Table 1) and could adsorb to the negatively charged cell wall of E. coli and S. aureus via electrostatic interactions. Moreover, Zn3Al-200 with the ZnO nanoparticle size of ˜30 nm provide a large specific surface area to adsorb onto the surface of bacteria. Such Zn3Al-200 attached on the surface of bacteria may influence bacterial metabolism at the high concentration, which may result in the death or non-growth of bacteria (Scheme 1.A). The SEM results clearly show that Zn3Al-200 nanohybrids deposit on the bacterial exterior surface, and lead to damages to the bacterial cell wall (Fig. S2). On the other hand, Zn3Al-200 may also adsorb proteins, celluloses and starches on the large surface to reduce the biological availability of nutrients when they move closely to the cell surface, and thus hinder growth of bacteria [35]. Zn3Al-200 antibacterial activity may be also relevant to the release of Zn2+ ions as zinc ions may readily enter into the bacterial cells and cause protein denaturation and dysfunction (Scheme 1.B) [35]. The leached Zn2+ ion concentration from Zn3Al-200 nanohybrids for 1–4 days in LB medium (pH ∼7) was 69.8–96.2 μg/mL, due to easy dissolution of ZnO nanocrystals [36]. The leached zinc ions from Zn3Al200 would have a contribution to the antibacterial efficacy [37]. UV-visible light may be another contributor to the antimicrobial activity of Zn3Al-200 as ZnO can produce ROS under the light irradiation. As shown in Fig. S4a and b, the energy bandgap of Zn3Al-80, Zn3Al-150 and Zn3Al-200 was determined to be 3.26, 3.14 and 3.06 eV, respectively, which suggests that the optical and semiconductor

4. Conclusion In summary, ZnO/LDHs were prepared by a facile method using LDH as precursor under mild hydrothermal conditions. When ageing at 200 °C for 2 h, the phase transformation from LDHs to ZnO and ZnAl2O4 nanohybrids on ZHN layers was observed and ˜30 nm uniform of ZnO nanoparticles was obtained. Zn3Al-200 at 100–300 μg/mL resulted in higher antibacterial activity than some pure ZnO nanoparticles. In particular, Zn3Al-200 exhibited a prolonged bacterial inhibition for up to 4 days towards E. coli and S. aureus. The effective and durable antibacterial efficiency of Zn3Al-200 could be attributed to the synergistic effects (ROS, leached Zn2+ and physical interactions). The simple approach proposed in this research can promote the preparation of highly 591

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efficient and durable antibacterial inorganic nanoscale metal oxides based on LDHs.

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