Colloids and Surfaces B: Biointerfaces 132 (2015) 281–289
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Study on the antibacterial mechanism of copper ion- and neodymium ion-modified ␣-zirconium phosphate with better antibacterial activity and lower cytotoxicity Xiang Cai a , Bin Zhang b , Yuanyuan Liang c,d , Jinglin Zhang b , Yinghui Yan a , Xiaoyin Chen a , Zhimin Wu a , Hongxi Liu a , Shuiping Wen a , Shaozao Tan b,∗ , Ting Wu a,∗ a
Department of Light Chemical Engineering, Guangdong Polytechnic, Foshan 528041, PR China Department of Chemistry, Jinan University, Guangzhou 510632, PR China c College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, PR China d College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China b
a r t i c l e
i n f o
Article history: Received 24 January 2015 Received in revised form 14 May 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Copper ion Neodymium ion ␣-Zirconium phosphate Antibacterial mechanism Cytotoxicity
a b s t r a c t To improve the antibacterial activity of Cu2+ , a series of Cu2+ and/or Nd3+ -modified layered ␣-zirconium phosphate (ZrP) was prepared and characterized, and the antibacterial activities of the prepared Cu2+ and/or Nd3+ -modified ZrP on Gram-negative Escherichia coli were investigated. The results showed that the basal spacing of ZrP was not obviously affected by the incorporation of Cu2+ , but the basal spacing of the modified ZrP changed into an amorphous state with increasing additions of Nd3+ . An antibacterial mechanism showed that Cu2+ and Nd3+ could enter into E. coli cells, leading to changes in ion concentrations and leakage of DNA, RNA and protein. The Cu2+ - and Nd3+ -modified ZrP, combining the advantages of Cu2+ and Nd3+ , displayed excellent additive antibacterial activity and lower cytotoxicity, suggesting the great potential application as an antibacterial powder for microbial control. Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.
1. Introduction Because bacterial resistance is a major concern in infection control, various antibacterial materials have been developed [1,2]. In general, inorganic antibacterial materials are superior to organic antibacterial materials in terms of their safety, heat resistance, persistence of antibacterial effect, etc. [3]. Among the different inorganic antibacterial materials, silver, particularly in the form of the free ion, has been studied widely as antibacterial material because of its lower toxicity and higher antibacterial activity [4,5]. Nevertheless, the application of silver-based antibacterial material is limited because of silver’s poor oxidation resistance, which results in the loss of antibacterial activity. In recent years, the antibacterial property of copper has been tested experimentally with good success [6]. Copper is a low-cost and effective material. Bacteria, yeasts and viruses can be rapidly killed on the surface of metallic copper, and the term “contact killing” has been coined for this process [7]. Compared with other
∗ Corresponding authors. Tel.: +86 0757 83106906; fax: +86 0757 83106906. E-mail addresses: [email protected] (S. Tan), [email protected] (T. Wu). http://dx.doi.org/10.1016/j.colsurfb.2015.05.027 0927-7765/Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.
forms of copper such as copper nanoparticles, copper ions (Cu2+ ) possess the advantages of broad-spectrum antibacterial activity and better oxidation resistance properties than silver in aqueous solutions [8–12]. Even so, the antibacterial activity of Cu2+ is still inferior to that of silver ions. Moreover, colliding Cu2+ and bacteria is quite difficult in aqueous solution, hence the content of Cu2+ in antibacterial material should increase [13]. To address these problems, many strategies have been carried out. On one hand, some metal ions have been associated with Cu2+ in antibacterial materials to enhance the antibacterial activity of Cu2+ [14,15]. On the other hand, numerous substances have served as carriers for Cu2+ to increase the content of Cu2+ in antibacterial materials, such as clinoptilolite [4], montmorillonite [12], ˇ-tricalcium phosphate [14] and hydroxyapatite [15]. However, the antibacterial activity and content of Cu2+ still need to improve in order to fulfil the widely -used application of Cu2+ -based antibacterial material. The neodymium ion (Nd3+ ) is one of the members of the rare earth ions. In aqueous solution, most of the rare earth elements are stable in the trivalent oxidation state. Recently, some studies have showed rare earth ions, such as Nd3+ , Sm3+ , Dy3+ and Ce3+ , have definite antibacterial activities [16–19], which are due to the release of many free hydroxyl radicals that kill microorganisms efficiently [20,21]. Therefore, the antibacterial property of metal ions
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can be improved by the addition of rare earth ions into antibacterial material [22,23]. Sodium zirconium phosphate (Na@ZrP) can be synthesized by various methods, such as hydrothermal synthesis and coprecipitation [24–26]. The structure of Na@ZrP is a flexible hexagonal crystal structure based on a three-dimensional network of (Zr2 P3 O12 )− units, which is formed by the corner sharing of PO4 tetrahedra with ZrO6 octahedra along the c-axis [27]. The Na@ZrP compound has attracted much attention for its potential application as a supporter because it possesses high thermal stability, high pressure stability and low solubility in water over a wide pH range. Recently, Na@ZrP has been proposed as host material for different types of ions [28,29]. In this study, Na@ZrP was used as the carrier of Cu2+ and Nd3+ and was associated with Cu2+ in aqueous solution to obtain a Cu2+ - and Nd3+ -modified ZrP hybrid (Cu-Nd@ZrP). Then, the characteristics, morphology, antibacterial activity and antibacterial mechanism of Cu-Nd@ZrP were investigated. The specific benefits of this novel hybrid included: (i) a green and facile synthetic method, (ii) high content of Cu2+ in the antibacterial material, and (iii) dose control to achieve the desired excellent antibacterial effect.
Cu-Nd@ZrP3 (0.020 mol Cu2+ and 0.020 mol Nd3+ ) and Nd@ZrP (0.020 mol Nd3+ ).
2.4. Characterizations The chemical compositions of the samples were analyzed with an energy dispersive X-ray spectrometer (EDX, Oxford ISIS-300, England) and the contents of Cu2+ and/or Nd3+ in the samples were measured by an inductively coupled plasma optical emission spectrometer (ICP, Optima 2000DV, America). X-Ray Diffraction (XRD) patterns were taken by a Rigaku D/max-1200 X-ray diffractometer using Cu K˛ radiation (K˛ = 0.15405 nm) at a scanning rate of 1◦ /min, a voltage of 40 kV and a current of 200 mA. The morphologies of the samples were investigated by scanning electron microscope (SEM, Philips XL-30, Netherlands) and transmission electron microscope (TEM, Philips Tecnai 10, Netherlands). The Brunauer–Emmett–Teller (BET) surface areas, pore volumes and pore sizes were determined from nitrogen adsorption and desorption isotherm data obtained at 77 K with a constant-volume adsorption apparatus (Mmk-TriStar3000), and the prepared samples were degassed at 573 K for 5 h before measurements were taken.
2. Experimental 2.5. Sterilizing rate 2.1. Materials Copper nitrate [Cu(NO3 )2 ·3H2 O] and disodium hydrogen phosphate (Na2 HPO4 ·12H2 O) were supplied by Guangzhou Chemical Industry Co., Ltd (Guangzhou, China). Zirconium sulfate tetrahydrate [Zr(SO4 )2 ·4H2 O] was provided by Deqing Xinkang Chemical Co., Ltd. Neodymium nitrate [Nd(NO3 )3 ·5H2 O] was purchased from Tianjing Standard Chemical Industry Co., Ltd (Tianjing, China). Escherichia coli (E. coli) ATCC 8099 and Staphylococcus aureus (S. aureus) ATCC 6538 were supplied by Guangdong Institute of Microbiology (Guangzhou, China). All aqueous solutions were prepared with ultrapure water (>18 M) from a Milli-Q Plus system (Millipore). 2.2. Preparation of Na@ZrP The Na@ZrP was synthesized as follows. Briefly, 140 g Na2 HPO4 ·12H2 O and 100 g Zr(SO4 )2 ·4H2 O were dissolved in deionized water in a 500 mL three-neck flask, and then the solution was reacted at 50 ◦ C for 0.5 h under vigorous stirring. After that, the pH of solution was adjusted to 5.5 and the refluxing was proceeded at 110 ◦ C for 12 h. The resultant product was washed with deionized water several times and dried at 120 ◦ C. Finally, the obtained sample was smashed and sifted through a 300-mesh sieve. The resulting powder was designated Na@ZrP. 2.3. Preparation of M@ZrP (M = Cu2+ or/and Nd3+ ) The M@ZrP complex was prepared as follows. Briefly, 15.0 g Na@ZrP and 90.0 g deionized water were mixed in a 250 mL threeneck flask at 50 ◦ C for 0.5 h under stirring. Then, different amounts of Cu2+ and/or Nd3+ were added into the solution. The reaction was kept stirring at 65 ◦ C for 6 h. After that, the resultant specimen was washed with deionized water until no Cu2+ and/or Nd3+ were detected. Afterwards, the resulting product was dried at 105 ◦ C for 12 h, treated at 700 ◦ C in a calcining furnace and pulverized to pass through a 300-mesh sieve. According to different additions of Cu2+ and/or Nd3+ , the final samples were designated Cu@ZrP (0.020 mol Cu2+ ), Cu-Nd@ZrP1 (0.015 mol Cu2+ and 0.005 mol Nd3+ ), Cu-Nd@ZrP2 (0.010 mol Cu2+ and 0.010 mol Nd3+ ),
The new prepared sample (0.2 mg) was dispensed into 10 mL of a sterile 0.8 wt.% saline water containing approximately 107 cfu/mL of E. coli or S. aureus, and then the solution was shaken at 37 ± 1 ◦ C for 6 h or 24 h. After that, 0.1 mL of the suspension was taken out from the test tube and diluted to a certain volume (to ensure the bacterial colonies grown could be counted easily and correctly) by a ten-fold dilution. The diluted solution was plated on Luria Bertani broth agar plate in triplicate and incubated at 37 ± 1 ◦ C for 24 h. The number of bacterial colonies on each plate was counted. The killing rate () was relative to the viable bacteria counts as follows: = (Y − X) × 100%/Y, where Y was the number of microorganism colonies on the control tube (a sterile 0.8 wt.% saline water without sample) and X was the number of microorganism colonies on the sample.
2.6. Minimum inhibitory concentration tests The minimum inhibitory concentration (MIC) of each sample against E. coli and S. aureus was measured by a two-fold diluting method. Briefly, the sample was suspended into Mueller–Hinton broth medium to form a homogeneous suspension and two-fold diluted into different concentrations. Each 1 mL of culture medium containing various concentrations of the test sample was inoculated with 0.1 mL of 106 cfu/mL bacterial suspension and cultured at 37 ◦ C for 24 h under shaking. Afterwards, the growth of the bacteria was observed. When no growth of bacteria was observed in the lowest concentration of test sample, the MIC of the sample was defined as this value of dilution. The test for every MIC of sample was repeated three times.
2.7. Leakage contents of DNA, RNA and proteins of E. coli 50 mL of sample (1 mg/mL) was dispensed into a certain volume of a sterile 0.8 wt.% saline solution containing approximately 108 cfu/mL of E. coli. Then, the solution was shaken for 0.5, 1.0, 1.5, 2.0 and 3.0 h. After that, 1 mL of the suspension was taken out and filtered by a Millipore filter (220 nm). Finally, the optical density (O.D.) of the filtrate was measured at 260 nm and 280 nm.
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2.8. Ion concentration analysis 80 mL of sample (1 mg/mL) was dispersed into a certain volume of a sterile 0.8 wt.% saline solution containing approximately 108 cfu/mL of E. coli. The solution was incubated at 37 ◦ C for 3 h with continuously shaking and then centrifuged. The centrifugation procedure was repeated many times to separate the sample and the E. coli thoroughly. The obtained E. coli was washed with deionized water twice and then dried. Finally, the E. coli was dispersed in 10 mL deionized water and ruptured by ultrasonic treatment for 1 h. Two drops of 10 mol L−1 nitric acid were added into the solution. The ion content in the solution was measured by an inductively coupled plasma optical emission spectrometer (ICP, Optima 2000DV, America).
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in Cu-Nd@ZrP1 reached 10.23% and 10.39% even if other types of ion were coexistent, indicating the high content of Cu2+ . This result was perhaps due to the reduction of the standard free energy of exchange between Na+ and Cu2+ when using Na@ZrP as a carrier. In addition, the samples were digested in aqua regia under a high temperature condition, and the total contents of Cu2+ and/or Nd3+ were measured using ICP analysis. As shown in Table 1, the results of the ICP analysis demonstrated that the content of Nd3+ was much greater than that of Cu2+ in the Cu-Nd@ZrP, even though the addition of Nd3+ was equal to or less than that of Cu2+ . These results implied that the combination mode between Nd3+ and carrier might be different from that between Cu2+ and carrier, and the more considerable combination of Na@ZrP towards Nd3+ was shown. However, judging by the total contents of ions in the samples, combinations between ions and carrier were favourable.
2.9. Cytotoxicity assay The cytotoxicity of the samples was tested using an MTT assay based on the cellular uptake of MTT and its subsequent reduction in the mitochondria of living cells to dark blue MTT formazan crystals. The mouse fibroblast cell line NIH-3T3 was seeded on 96-well plates (1.5–2 × 104 cells/well) in corresponding medium. Then, the NIH-3T3 cells were treated with the samples for 24 h. After that, MTT (5 mg/mL in PBS) was added to each well and incubated for an additional 4 h (37 ◦ C, 5% CO2 ). The cells were then lysed in dimethyl sulfoxide (150 L/well) and the plates were allowed to stay in the incubator (37 ◦ C, 5% CO2 ) to dissolve the purple formazan crystals. The colour intensity, reflecting the cell viability, was read at 490 nm using a Model-550 enzyme-linked immunosorbent microplate (Bio-Rad, USA), and the morphologic changes of the NIH-3T3 cells were photographed by an IX-70 inverted phase contrast microscope (Olympus, Japan). All of the experiments were repeated four times, and Statistical Product and Service Solutions software was used to assess the statistical significance of the differences among the treatment groups. 2.10. Statistical analysis A statistical analysis was performed using Statistical Product and Service Solutions software (SPSS) statistical software (SPSS 11.0, United States). The differences among the groups were assessed using the analysis of variance test. The results were considered statistically significant when the P value was 10,000 650 350 1550
– – – 0.764 –
a b
FIC
Concentrations, analyzed by an inductively coupled plasma optical emission spectrometer, ±SD, n = 3. ±SD, n = 3.
the “contact killing” process of Cu2+ when it contacted the bacteria [7]. What was more, compared with the sterilizing rates of other samples, Cu-Nd@ZrP1 had the highest antibacterial activity against E. coli and S. aureus. Such an enhancement of antibacterial effect might be thanks to the combined action based on the functions of Cu2+ and Nd3+ . Using the MICs (Table 2) to determine the fractional inhibitory concentration (FIC) and assess the antibacterial interaction was as follows. The FIC value of antibacterial A (FICA ) was calculated from the MIC of antibacterial A alone and the MIC of antibacterial A in combination: FICA = MIC of antibacterial A in combination/ MIC of antibacterial A alone
(3)
The FIC value of antibacterial B (FICB ) was calculated from the MIC of antibacterial B alone and the MIC of antibacterial B in combination: FICB = MIC of antibacterial B in combination/ MIC of antibacterial B alone
FIC = FICA + FICB
(4)
(5)
The value of the FIC index was then used to determine whether synergism, indifference or antagonism occurred between the antibacterial agents. The following values, according to accepted criteria [36], were used to interpret the nature of the interaction: ≤0.5, synergy; 0.5–1.0, additivity; 1.0–4.0, indifference; ≥4, antagonism. The Cu-Nd@ZrP1 showed obvious additive antibacterial effect on E. coli and S. aureus (Table 1). This was because the Cu-Nd@ZrP1 combined the antibacterial advantages of Nd3+ and Cu2+ on antibacterial activity, and the use of Cu-Nd@ZrP1 would be more efficient. On the other hand, compared with the Gram-positive species (S. aureus), the Gram-negative strain (E. coli) has an outer membrane outside the peptidoglycan layer, which is composed mainly of lipopolysaccharides and phospholipids. The outer membrane played a significant role in protecting the bacteria cell from attack by a foreign compound. So, all of the samples exhibited lower antibacterial activities against E. coli than against S. aureus in identical test condition [37,38].
3.6. Antibacterial mechanism To further investigate the details of the contacts between the samples and bacteria, the leakage contents of DNA, RNA and protein from E. coli were measured before and after the contact between the samples and the E. coli. Because of the existence of a conjugated double bond system between the pyrimidine ring and the purine ring, DNA and RNA have an ultraviolet absorption peak at approximately 260 nm, while due to the existence of a conjugated double bond system between tyrosine and tryptophan, protein has an ultraviolet absorption peak at approximately 280 nm. Hence, the contents of DNA, RNA and protein could be obtained by measuring the O.D. values at 260 nm and 280 nm, separately. As seen from the results (Fig. 3), after the contact between the E. coli and the samples, the higher antibacterial activities of samples would result in more leakage of DNA, RNA and protein at the same experiment time. Moreover, the leakage of DNA, RNA and protein all gradually increased with a prolonged contact time. In addition, the ICP-AES measurements of K+ , Na+ , Ca2+ , Mg2+ , 2+ Cu and Nd3+ in E. coli were also carried out. The results in Table 3 showed that after the contact with Cu@ZrP, Cu-Nd@ZrP1 or Nd@ZrP for 2 h, the concentrations of K+ , Na+ , Ca2+ and Mg2+ all reduced and the concentrations of Cu2+ and/or Nd3+ increased in the E. coli, indicating that Cu2+ or/and Nd3+ entered into the treated cell. These results suggested the cell membrane of E. coli had been ruptured, leading to the changes of ion concentrations and the leakages of DNA, RNA and protein. In this case, the rupture of the cell membrane might be caused by Cu2+ and/or Nd3+ . As the surface of the cell membrane was negatively charged, the structure and selectivity of the cell membrane would change after the combination with Cu2+ through an electrostatic interaction, resulting in the damage of the cell membrane and even death of cell. Such process was an important part of antibacterial mechanism of Cu-Nd@ZrP. Some other studies reported similar mechanisms [39,40]. In addition, the release of many free hydroxyl radicals from Nd3+ would bring about oxidative damage in the bacteria [7], which could kill the bacteria efficiently. So as to describe more clearly the change of the membrane of E. coli, TEM images of E. coli before and after the contacts with samples were taken. As seen in Fig. 4, E. coli had integrated outer membranes (OM) and cytoplasmic membranes (CM) even after the contact with Na@ZrP. However, after the contact with Cu@ZrP, Cu-Nd@ZrP and Nd@ZrP, the structures of OM and CM of E. coli changed, showing the separation between the OM and the CM, some distortions of the OM and/or bleaching of the CM. Such a change of structure would cause the changes in ion concentrations and the leakage of the cytoplasm, including DNA, RNA and protein.
X. Cai et al. / Colloids and Surfaces B: Biointerfaces 132 (2015) 281–289
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Fig. 3. Leakage contents of (a) DNA and RNA and (b) protein of E. coli (±SD, n = 3).
Table 3 The concentrations of K+ , Na+ , Ca2+ , Mg2+ , Cu2+ and Nd3+ in the E. coli after contact for 2 h with samples. Samples
Concentrationsa (mg L−1 ) K+
Blank sample Cu@ZrP Cu-Nd@ZrP1 Nd@ZrP a
1.339 0.644 0.393 0.467
Na+ ± ± ± ±
0.012 0.004 0.005 0.003
1.313 0.161 0.169 0.264
Ca2+ ± ± ± ±
0.007 0.003 0.006 0.001
1.262 1.202 0.096 0.966
Mg2+ ± ± ± ±
0.002 0.011 0.000 0.004
0.374 0.089 0.097 0.106
Cu2+ ± ± ± ±
0.005 0.001 0.003 0.001
0.008 2.304 0.336 0.014
Nd3+ ± ± ± ±
0.001 0.009 0.008 0.000
0.001 0.001 0.030 0.690
± ± ± ±
0.000 0.000 0.005 0.007
±SD, n = 3.
According to the above results, we proposed the antibacterial mechanism of Cu-Nd@ZrP as follows: First, Cu2+ and Nd3+ both had antibacterial activities, but their attack targets against bacteria might be different from each other. When the bacteria simultaneously contacted the Cu2+ and Nd3+ on Cu-Nd@ZrP, the Cu2+ contacted the cell membrane through an electrostatic interaction and the Nd3+ brought about oxidative damage towards bacteria through releasing many free hydroxyl radicals, causing the damage of cell membrane, changes of ion concentrations and leakage of DNA, RNA and protein. Such processes had an additive antibacterial effect (Table 2) to give rise to the death of bacteria more efficiently,
suggesting Cu-Nd@ZrP as antibacterial powder had great potential applications. 3.7. Cytotoxicity test We also carried out a cytotoxicity test on the sample. The MTT assays (Fig. 5A) showed that Na@ZrP (100 g/mL) exhibited a slight cytotoxicity (∼5%) to NIH-3T3 within 24 h of incubation. Cu2+ and Nd3+ exhibited a serious cytotoxicity to NIH-3T3 within 24 h of incubation (the cell viability of NIH-3T3 reduced to 9.9% and 25.4% with 100 g/mL Cu2+ and 100 g/mL Nd3+ , respectively). However,
Fig. 4. TEM images of E. coli (a) before and after contacts with (b) Na@ZrP, (c) Cu@ZrP, (d) Cu-Nd@ZrP1 and (e) Nd@ZrP.
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Fig. 5. (A) Cytotoxicity of (a) Cu2+ , (b) Nd3+ , (c) Cu-Nd@ZrP1, (d) Cu@ZrP, (e) Nd@ZrP and (f) Na@ZrP on NIH-3T3 cells (±SD, n = 3), the concentrations of the total Cu2+ and Nd3+ in the samples were set at 0.04, 0.2, 1, 5, 25, 100, and 500 g/mL. (B) When the concentrations of the total Cu2+ and Nd3+ in the samples were set at 500 g/mL, after interaction for 24 h, the morphologic changes of NIH-3T3 cells: In the (a) Na@ZrP group, there was no Cu2+ and Nd3+ , the NIH-3T3 cells had good shape, presented long fusiform or polygon. In the (b) Cu@ZrP group, (c) Cu-Nd@ZrP1 group and (d) Nd@ZrP group, the NIH-3T3 cells shape became irregular. In the (e) Cu2+ group and (f) Nd3+ group, the number of NIH-3T3 cells decreased significantly, and the shapes of the majority of the cells were seriously injured.
the cell viability of NIH-3T3 reduced to 56.0%, 57.2% and 61.8% with 100 g/mL Cu-Nd@ZrP1, 100 g/mL Cu@ZrP and 100 g/mL Nd@ZrP, respectively. Therefore, the cytotoxicity of Cu-Nd@ZrP1, Cu@ZrP and Nd@ZrP was significantly lower than those of Cu2+ and Nd3+ , and the use of Cu-Nd@ZrP1, Cu@ZrP and Nd@ZrP would be safer than the direct use of Cu2+ and Nd3+ , which was in accordance with the results of the inverted phase contrast microscope measurements (Fig. 5B). What was more, the antibacterial activity of Cu-Nd@ZrP1 was higher than those of Cu@ZrP and Nd@ZrP, so the Cu-Nd@ZrP1 showed the great potential application as an antibacterial powder. Compared with other reports [41–44], we concluded that Cu-Nd@ZrP was a relatively biocompatible nanomaterial with slight cytotoxicity. 4. Conclusions In this paper, the carrier (ZrP) was synthesized by a hydrothermal method, and a series of Cu2+ - or/and Nd3+ -modified ZrP was prepared. The basal spacing of ZrP was not obviously affected by the incorporation of Cu2+ , but the basal spacing of the modified ZrP changed into an amorphous state with the increasing addition of Nd3+ . Moreover, the combination mode between Nd3+ and carrier might be different from that between Cu2+ and carrier, and the more considerable combination of Na@ZrP towards Nd3+ was shown. Following the imports of Cu2+ and/or Nd3+ , the z-average size increased and the particle of the modified ZrP showed more agglomeration. On the other hand, ZrP displayed no antibacterial activity. Compared with Cu@ZrP and Nd@ZrP, Cu-Nd@ZrP showed better antibacterial activity due to the additive antibacterial effects of Cu2+ and Nd3+ . The antibacterial mechanism showed that Cu2+ and/or Nd3+ could enter into the E. coli cell, and the cell membrane of E. coli had been ruptured, leading to the changes of ion concentrations and the leakage of DNA, RNA and protein. The Cu-Nd@ZrP powder, combining the advantages of Cu2+ and Nd3+ , displayed higher antibacterial activity and lower cytotoxicity, suggesting the great potential application as an antibacterial powder. Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (51172099, 51203134, 21476052 and 21271087), the Foundation of EnterpriseUniversity-Research Institute Cooperation from Guangdong
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Study on the antibacterial mechanism of copper ion- and neodymium ion-modified ␣-zirconium phosphate with better antibacterial activity and lower cytotoxicity Xiang Cai a , Bin Zhang b , Yuanyuan Liang c,d , Jinglin Zhang b , Yinghui Yan a , Xiaoyin Chen a , Zhimin Wu a , Hongxi Liu a , Shuiping Wen a , Shaozao Tan b,∗ , Ting Wu a,∗ a
Department of Light Chemical Engineering, Guangdong Polytechnic, Foshan 528041, PR China Department of Chemistry, Jinan University, Guangzhou 510632, PR China c College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, PR China d College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China b
a r t i c l e
i n f o
Article history: Received 24 January 2015 Received in revised form 14 May 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Copper ion Neodymium ion ␣-Zirconium phosphate Antibacterial mechanism Cytotoxicity
a b s t r a c t To improve the antibacterial activity of Cu2+ , a series of Cu2+ and/or Nd3+ -modified layered ␣-zirconium phosphate (ZrP) was prepared and characterized, and the antibacterial activities of the prepared Cu2+ and/or Nd3+ -modified ZrP on Gram-negative Escherichia coli were investigated. The results showed that the basal spacing of ZrP was not obviously affected by the incorporation of Cu2+ , but the basal spacing of the modified ZrP changed into an amorphous state with increasing additions of Nd3+ . An antibacterial mechanism showed that Cu2+ and Nd3+ could enter into E. coli cells, leading to changes in ion concentrations and leakage of DNA, RNA and protein. The Cu2+ - and Nd3+ -modified ZrP, combining the advantages of Cu2+ and Nd3+ , displayed excellent additive antibacterial activity and lower cytotoxicity, suggesting the great potential application as an antibacterial powder for microbial control. Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.
1. Introduction Because bacterial resistance is a major concern in infection control, various antibacterial materials have been developed [1,2]. In general, inorganic antibacterial materials are superior to organic antibacterial materials in terms of their safety, heat resistance, persistence of antibacterial effect, etc. [3]. Among the different inorganic antibacterial materials, silver, particularly in the form of the free ion, has been studied widely as antibacterial material because of its lower toxicity and higher antibacterial activity [4,5]. Nevertheless, the application of silver-based antibacterial material is limited because of silver’s poor oxidation resistance, which results in the loss of antibacterial activity. In recent years, the antibacterial property of copper has been tested experimentally with good success [6]. Copper is a low-cost and effective material. Bacteria, yeasts and viruses can be rapidly killed on the surface of metallic copper, and the term “contact killing” has been coined for this process [7]. Compared with other
∗ Corresponding authors. Tel.: +86 0757 83106906; fax: +86 0757 83106906. E-mail addresses: [email protected] (S. Tan), [email protected] (T. Wu). http://dx.doi.org/10.1016/j.colsurfb.2015.05.027 0927-7765/Crown Copyright © 2015 Published by Elsevier B.V. All rights reserved.
forms of copper such as copper nanoparticles, copper ions (Cu2+ ) possess the advantages of broad-spectrum antibacterial activity and better oxidation resistance properties than silver in aqueous solutions [8–12]. Even so, the antibacterial activity of Cu2+ is still inferior to that of silver ions. Moreover, colliding Cu2+ and bacteria is quite difficult in aqueous solution, hence the content of Cu2+ in antibacterial material should increase [13]. To address these problems, many strategies have been carried out. On one hand, some metal ions have been associated with Cu2+ in antibacterial materials to enhance the antibacterial activity of Cu2+ [14,15]. On the other hand, numerous substances have served as carriers for Cu2+ to increase the content of Cu2+ in antibacterial materials, such as clinoptilolite [4], montmorillonite [12], ˇ-tricalcium phosphate [14] and hydroxyapatite [15]. However, the antibacterial activity and content of Cu2+ still need to improve in order to fulfil the widely -used application of Cu2+ -based antibacterial material. The neodymium ion (Nd3+ ) is one of the members of the rare earth ions. In aqueous solution, most of the rare earth elements are stable in the trivalent oxidation state. Recently, some studies have showed rare earth ions, such as Nd3+ , Sm3+ , Dy3+ and Ce3+ , have definite antibacterial activities [16–19], which are due to the release of many free hydroxyl radicals that kill microorganisms efficiently [20,21]. Therefore, the antibacterial property of metal ions
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can be improved by the addition of rare earth ions into antibacterial material [22,23]. Sodium zirconium phosphate (Na@ZrP) can be synthesized by various methods, such as hydrothermal synthesis and coprecipitation [24–26]. The structure of Na@ZrP is a flexible hexagonal crystal structure based on a three-dimensional network of (Zr2 P3 O12 )− units, which is formed by the corner sharing of PO4 tetrahedra with ZrO6 octahedra along the c-axis [27]. The Na@ZrP compound has attracted much attention for its potential application as a supporter because it possesses high thermal stability, high pressure stability and low solubility in water over a wide pH range. Recently, Na@ZrP has been proposed as host material for different types of ions [28,29]. In this study, Na@ZrP was used as the carrier of Cu2+ and Nd3+ and was associated with Cu2+ in aqueous solution to obtain a Cu2+ - and Nd3+ -modified ZrP hybrid (Cu-Nd@ZrP). Then, the characteristics, morphology, antibacterial activity and antibacterial mechanism of Cu-Nd@ZrP were investigated. The specific benefits of this novel hybrid included: (i) a green and facile synthetic method, (ii) high content of Cu2+ in the antibacterial material, and (iii) dose control to achieve the desired excellent antibacterial effect.
Cu-Nd@ZrP3 (0.020 mol Cu2+ and 0.020 mol Nd3+ ) and Nd@ZrP (0.020 mol Nd3+ ).
2.4. Characterizations The chemical compositions of the samples were analyzed with an energy dispersive X-ray spectrometer (EDX, Oxford ISIS-300, England) and the contents of Cu2+ and/or Nd3+ in the samples were measured by an inductively coupled plasma optical emission spectrometer (ICP, Optima 2000DV, America). X-Ray Diffraction (XRD) patterns were taken by a Rigaku D/max-1200 X-ray diffractometer using Cu K˛ radiation (K˛ = 0.15405 nm) at a scanning rate of 1◦ /min, a voltage of 40 kV and a current of 200 mA. The morphologies of the samples were investigated by scanning electron microscope (SEM, Philips XL-30, Netherlands) and transmission electron microscope (TEM, Philips Tecnai 10, Netherlands). The Brunauer–Emmett–Teller (BET) surface areas, pore volumes and pore sizes were determined from nitrogen adsorption and desorption isotherm data obtained at 77 K with a constant-volume adsorption apparatus (Mmk-TriStar3000), and the prepared samples were degassed at 573 K for 5 h before measurements were taken.
2. Experimental 2.5. Sterilizing rate 2.1. Materials Copper nitrate [Cu(NO3 )2 ·3H2 O] and disodium hydrogen phosphate (Na2 HPO4 ·12H2 O) were supplied by Guangzhou Chemical Industry Co., Ltd (Guangzhou, China). Zirconium sulfate tetrahydrate [Zr(SO4 )2 ·4H2 O] was provided by Deqing Xinkang Chemical Co., Ltd. Neodymium nitrate [Nd(NO3 )3 ·5H2 O] was purchased from Tianjing Standard Chemical Industry Co., Ltd (Tianjing, China). Escherichia coli (E. coli) ATCC 8099 and Staphylococcus aureus (S. aureus) ATCC 6538 were supplied by Guangdong Institute of Microbiology (Guangzhou, China). All aqueous solutions were prepared with ultrapure water (>18 M) from a Milli-Q Plus system (Millipore). 2.2. Preparation of Na@ZrP The Na@ZrP was synthesized as follows. Briefly, 140 g Na2 HPO4 ·12H2 O and 100 g Zr(SO4 )2 ·4H2 O were dissolved in deionized water in a 500 mL three-neck flask, and then the solution was reacted at 50 ◦ C for 0.5 h under vigorous stirring. After that, the pH of solution was adjusted to 5.5 and the refluxing was proceeded at 110 ◦ C for 12 h. The resultant product was washed with deionized water several times and dried at 120 ◦ C. Finally, the obtained sample was smashed and sifted through a 300-mesh sieve. The resulting powder was designated Na@ZrP. 2.3. Preparation of M@ZrP (M = Cu2+ or/and Nd3+ ) The M@ZrP complex was prepared as follows. Briefly, 15.0 g Na@ZrP and 90.0 g deionized water were mixed in a 250 mL threeneck flask at 50 ◦ C for 0.5 h under stirring. Then, different amounts of Cu2+ and/or Nd3+ were added into the solution. The reaction was kept stirring at 65 ◦ C for 6 h. After that, the resultant specimen was washed with deionized water until no Cu2+ and/or Nd3+ were detected. Afterwards, the resulting product was dried at 105 ◦ C for 12 h, treated at 700 ◦ C in a calcining furnace and pulverized to pass through a 300-mesh sieve. According to different additions of Cu2+ and/or Nd3+ , the final samples were designated Cu@ZrP (0.020 mol Cu2+ ), Cu-Nd@ZrP1 (0.015 mol Cu2+ and 0.005 mol Nd3+ ), Cu-Nd@ZrP2 (0.010 mol Cu2+ and 0.010 mol Nd3+ ),
The new prepared sample (0.2 mg) was dispensed into 10 mL of a sterile 0.8 wt.% saline water containing approximately 107 cfu/mL of E. coli or S. aureus, and then the solution was shaken at 37 ± 1 ◦ C for 6 h or 24 h. After that, 0.1 mL of the suspension was taken out from the test tube and diluted to a certain volume (to ensure the bacterial colonies grown could be counted easily and correctly) by a ten-fold dilution. The diluted solution was plated on Luria Bertani broth agar plate in triplicate and incubated at 37 ± 1 ◦ C for 24 h. The number of bacterial colonies on each plate was counted. The killing rate () was relative to the viable bacteria counts as follows: = (Y − X) × 100%/Y, where Y was the number of microorganism colonies on the control tube (a sterile 0.8 wt.% saline water without sample) and X was the number of microorganism colonies on the sample.
2.6. Minimum inhibitory concentration tests The minimum inhibitory concentration (MIC) of each sample against E. coli and S. aureus was measured by a two-fold diluting method. Briefly, the sample was suspended into Mueller–Hinton broth medium to form a homogeneous suspension and two-fold diluted into different concentrations. Each 1 mL of culture medium containing various concentrations of the test sample was inoculated with 0.1 mL of 106 cfu/mL bacterial suspension and cultured at 37 ◦ C for 24 h under shaking. Afterwards, the growth of the bacteria was observed. When no growth of bacteria was observed in the lowest concentration of test sample, the MIC of the sample was defined as this value of dilution. The test for every MIC of sample was repeated three times.
2.7. Leakage contents of DNA, RNA and proteins of E. coli 50 mL of sample (1 mg/mL) was dispensed into a certain volume of a sterile 0.8 wt.% saline solution containing approximately 108 cfu/mL of E. coli. Then, the solution was shaken for 0.5, 1.0, 1.5, 2.0 and 3.0 h. After that, 1 mL of the suspension was taken out and filtered by a Millipore filter (220 nm). Finally, the optical density (O.D.) of the filtrate was measured at 260 nm and 280 nm.
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2.8. Ion concentration analysis 80 mL of sample (1 mg/mL) was dispersed into a certain volume of a sterile 0.8 wt.% saline solution containing approximately 108 cfu/mL of E. coli. The solution was incubated at 37 ◦ C for 3 h with continuously shaking and then centrifuged. The centrifugation procedure was repeated many times to separate the sample and the E. coli thoroughly. The obtained E. coli was washed with deionized water twice and then dried. Finally, the E. coli was dispersed in 10 mL deionized water and ruptured by ultrasonic treatment for 1 h. Two drops of 10 mol L−1 nitric acid were added into the solution. The ion content in the solution was measured by an inductively coupled plasma optical emission spectrometer (ICP, Optima 2000DV, America).
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in Cu-Nd@ZrP1 reached 10.23% and 10.39% even if other types of ion were coexistent, indicating the high content of Cu2+ . This result was perhaps due to the reduction of the standard free energy of exchange between Na+ and Cu2+ when using Na@ZrP as a carrier. In addition, the samples were digested in aqua regia under a high temperature condition, and the total contents of Cu2+ and/or Nd3+ were measured using ICP analysis. As shown in Table 1, the results of the ICP analysis demonstrated that the content of Nd3+ was much greater than that of Cu2+ in the Cu-Nd@ZrP, even though the addition of Nd3+ was equal to or less than that of Cu2+ . These results implied that the combination mode between Nd3+ and carrier might be different from that between Cu2+ and carrier, and the more considerable combination of Na@ZrP towards Nd3+ was shown. However, judging by the total contents of ions in the samples, combinations between ions and carrier were favourable.
2.9. Cytotoxicity assay The cytotoxicity of the samples was tested using an MTT assay based on the cellular uptake of MTT and its subsequent reduction in the mitochondria of living cells to dark blue MTT formazan crystals. The mouse fibroblast cell line NIH-3T3 was seeded on 96-well plates (1.5–2 × 104 cells/well) in corresponding medium. Then, the NIH-3T3 cells were treated with the samples for 24 h. After that, MTT (5 mg/mL in PBS) was added to each well and incubated for an additional 4 h (37 ◦ C, 5% CO2 ). The cells were then lysed in dimethyl sulfoxide (150 L/well) and the plates were allowed to stay in the incubator (37 ◦ C, 5% CO2 ) to dissolve the purple formazan crystals. The colour intensity, reflecting the cell viability, was read at 490 nm using a Model-550 enzyme-linked immunosorbent microplate (Bio-Rad, USA), and the morphologic changes of the NIH-3T3 cells were photographed by an IX-70 inverted phase contrast microscope (Olympus, Japan). All of the experiments were repeated four times, and Statistical Product and Service Solutions software was used to assess the statistical significance of the differences among the treatment groups. 2.10. Statistical analysis A statistical analysis was performed using Statistical Product and Service Solutions software (SPSS) statistical software (SPSS 11.0, United States). The differences among the groups were assessed using the analysis of variance test. The results were considered statistically significant when the P value was 10,000 650 350 1550
– – – 0.764 –
a b
FIC
Concentrations, analyzed by an inductively coupled plasma optical emission spectrometer, ±SD, n = 3. ±SD, n = 3.
the “contact killing” process of Cu2+ when it contacted the bacteria [7]. What was more, compared with the sterilizing rates of other samples, Cu-Nd@ZrP1 had the highest antibacterial activity against E. coli and S. aureus. Such an enhancement of antibacterial effect might be thanks to the combined action based on the functions of Cu2+ and Nd3+ . Using the MICs (Table 2) to determine the fractional inhibitory concentration (FIC) and assess the antibacterial interaction was as follows. The FIC value of antibacterial A (FICA ) was calculated from the MIC of antibacterial A alone and the MIC of antibacterial A in combination: FICA = MIC of antibacterial A in combination/ MIC of antibacterial A alone
(3)
The FIC value of antibacterial B (FICB ) was calculated from the MIC of antibacterial B alone and the MIC of antibacterial B in combination: FICB = MIC of antibacterial B in combination/ MIC of antibacterial B alone
FIC = FICA + FICB
(4)
(5)
The value of the FIC index was then used to determine whether synergism, indifference or antagonism occurred between the antibacterial agents. The following values, according to accepted criteria [36], were used to interpret the nature of the interaction: ≤0.5, synergy; 0.5–1.0, additivity; 1.0–4.0, indifference; ≥4, antagonism. The Cu-Nd@ZrP1 showed obvious additive antibacterial effect on E. coli and S. aureus (Table 1). This was because the Cu-Nd@ZrP1 combined the antibacterial advantages of Nd3+ and Cu2+ on antibacterial activity, and the use of Cu-Nd@ZrP1 would be more efficient. On the other hand, compared with the Gram-positive species (S. aureus), the Gram-negative strain (E. coli) has an outer membrane outside the peptidoglycan layer, which is composed mainly of lipopolysaccharides and phospholipids. The outer membrane played a significant role in protecting the bacteria cell from attack by a foreign compound. So, all of the samples exhibited lower antibacterial activities against E. coli than against S. aureus in identical test condition [37,38].
3.6. Antibacterial mechanism To further investigate the details of the contacts between the samples and bacteria, the leakage contents of DNA, RNA and protein from E. coli were measured before and after the contact between the samples and the E. coli. Because of the existence of a conjugated double bond system between the pyrimidine ring and the purine ring, DNA and RNA have an ultraviolet absorption peak at approximately 260 nm, while due to the existence of a conjugated double bond system between tyrosine and tryptophan, protein has an ultraviolet absorption peak at approximately 280 nm. Hence, the contents of DNA, RNA and protein could be obtained by measuring the O.D. values at 260 nm and 280 nm, separately. As seen from the results (Fig. 3), after the contact between the E. coli and the samples, the higher antibacterial activities of samples would result in more leakage of DNA, RNA and protein at the same experiment time. Moreover, the leakage of DNA, RNA and protein all gradually increased with a prolonged contact time. In addition, the ICP-AES measurements of K+ , Na+ , Ca2+ , Mg2+ , 2+ Cu and Nd3+ in E. coli were also carried out. The results in Table 3 showed that after the contact with Cu@ZrP, Cu-Nd@ZrP1 or Nd@ZrP for 2 h, the concentrations of K+ , Na+ , Ca2+ and Mg2+ all reduced and the concentrations of Cu2+ and/or Nd3+ increased in the E. coli, indicating that Cu2+ or/and Nd3+ entered into the treated cell. These results suggested the cell membrane of E. coli had been ruptured, leading to the changes of ion concentrations and the leakages of DNA, RNA and protein. In this case, the rupture of the cell membrane might be caused by Cu2+ and/or Nd3+ . As the surface of the cell membrane was negatively charged, the structure and selectivity of the cell membrane would change after the combination with Cu2+ through an electrostatic interaction, resulting in the damage of the cell membrane and even death of cell. Such process was an important part of antibacterial mechanism of Cu-Nd@ZrP. Some other studies reported similar mechanisms [39,40]. In addition, the release of many free hydroxyl radicals from Nd3+ would bring about oxidative damage in the bacteria [7], which could kill the bacteria efficiently. So as to describe more clearly the change of the membrane of E. coli, TEM images of E. coli before and after the contacts with samples were taken. As seen in Fig. 4, E. coli had integrated outer membranes (OM) and cytoplasmic membranes (CM) even after the contact with Na@ZrP. However, after the contact with Cu@ZrP, Cu-Nd@ZrP and Nd@ZrP, the structures of OM and CM of E. coli changed, showing the separation between the OM and the CM, some distortions of the OM and/or bleaching of the CM. Such a change of structure would cause the changes in ion concentrations and the leakage of the cytoplasm, including DNA, RNA and protein.
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Fig. 3. Leakage contents of (a) DNA and RNA and (b) protein of E. coli (±SD, n = 3).
Table 3 The concentrations of K+ , Na+ , Ca2+ , Mg2+ , Cu2+ and Nd3+ in the E. coli after contact for 2 h with samples. Samples
Concentrationsa (mg L−1 ) K+
Blank sample Cu@ZrP Cu-Nd@ZrP1 Nd@ZrP a
1.339 0.644 0.393 0.467
Na+ ± ± ± ±
0.012 0.004 0.005 0.003
1.313 0.161 0.169 0.264
Ca2+ ± ± ± ±
0.007 0.003 0.006 0.001
1.262 1.202 0.096 0.966
Mg2+ ± ± ± ±
0.002 0.011 0.000 0.004
0.374 0.089 0.097 0.106
Cu2+ ± ± ± ±
0.005 0.001 0.003 0.001
0.008 2.304 0.336 0.014
Nd3+ ± ± ± ±
0.001 0.009 0.008 0.000
0.001 0.001 0.030 0.690
± ± ± ±
0.000 0.000 0.005 0.007
±SD, n = 3.
According to the above results, we proposed the antibacterial mechanism of Cu-Nd@ZrP as follows: First, Cu2+ and Nd3+ both had antibacterial activities, but their attack targets against bacteria might be different from each other. When the bacteria simultaneously contacted the Cu2+ and Nd3+ on Cu-Nd@ZrP, the Cu2+ contacted the cell membrane through an electrostatic interaction and the Nd3+ brought about oxidative damage towards bacteria through releasing many free hydroxyl radicals, causing the damage of cell membrane, changes of ion concentrations and leakage of DNA, RNA and protein. Such processes had an additive antibacterial effect (Table 2) to give rise to the death of bacteria more efficiently,
suggesting Cu-Nd@ZrP as antibacterial powder had great potential applications. 3.7. Cytotoxicity test We also carried out a cytotoxicity test on the sample. The MTT assays (Fig. 5A) showed that Na@ZrP (100 g/mL) exhibited a slight cytotoxicity (∼5%) to NIH-3T3 within 24 h of incubation. Cu2+ and Nd3+ exhibited a serious cytotoxicity to NIH-3T3 within 24 h of incubation (the cell viability of NIH-3T3 reduced to 9.9% and 25.4% with 100 g/mL Cu2+ and 100 g/mL Nd3+ , respectively). However,
Fig. 4. TEM images of E. coli (a) before and after contacts with (b) Na@ZrP, (c) Cu@ZrP, (d) Cu-Nd@ZrP1 and (e) Nd@ZrP.
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Fig. 5. (A) Cytotoxicity of (a) Cu2+ , (b) Nd3+ , (c) Cu-Nd@ZrP1, (d) Cu@ZrP, (e) Nd@ZrP and (f) Na@ZrP on NIH-3T3 cells (±SD, n = 3), the concentrations of the total Cu2+ and Nd3+ in the samples were set at 0.04, 0.2, 1, 5, 25, 100, and 500 g/mL. (B) When the concentrations of the total Cu2+ and Nd3+ in the samples were set at 500 g/mL, after interaction for 24 h, the morphologic changes of NIH-3T3 cells: In the (a) Na@ZrP group, there was no Cu2+ and Nd3+ , the NIH-3T3 cells had good shape, presented long fusiform or polygon. In the (b) Cu@ZrP group, (c) Cu-Nd@ZrP1 group and (d) Nd@ZrP group, the NIH-3T3 cells shape became irregular. In the (e) Cu2+ group and (f) Nd3+ group, the number of NIH-3T3 cells decreased significantly, and the shapes of the majority of the cells were seriously injured.
the cell viability of NIH-3T3 reduced to 56.0%, 57.2% and 61.8% with 100 g/mL Cu-Nd@ZrP1, 100 g/mL Cu@ZrP and 100 g/mL Nd@ZrP, respectively. Therefore, the cytotoxicity of Cu-Nd@ZrP1, Cu@ZrP and Nd@ZrP was significantly lower than those of Cu2+ and Nd3+ , and the use of Cu-Nd@ZrP1, Cu@ZrP and Nd@ZrP would be safer than the direct use of Cu2+ and Nd3+ , which was in accordance with the results of the inverted phase contrast microscope measurements (Fig. 5B). What was more, the antibacterial activity of Cu-Nd@ZrP1 was higher than those of Cu@ZrP and Nd@ZrP, so the Cu-Nd@ZrP1 showed the great potential application as an antibacterial powder. Compared with other reports [41–44], we concluded that Cu-Nd@ZrP was a relatively biocompatible nanomaterial with slight cytotoxicity. 4. Conclusions In this paper, the carrier (ZrP) was synthesized by a hydrothermal method, and a series of Cu2+ - or/and Nd3+ -modified ZrP was prepared. The basal spacing of ZrP was not obviously affected by the incorporation of Cu2+ , but the basal spacing of the modified ZrP changed into an amorphous state with the increasing addition of Nd3+ . Moreover, the combination mode between Nd3+ and carrier might be different from that between Cu2+ and carrier, and the more considerable combination of Na@ZrP towards Nd3+ was shown. Following the imports of Cu2+ and/or Nd3+ , the z-average size increased and the particle of the modified ZrP showed more agglomeration. On the other hand, ZrP displayed no antibacterial activity. Compared with Cu@ZrP and Nd@ZrP, Cu-Nd@ZrP showed better antibacterial activity due to the additive antibacterial effects of Cu2+ and Nd3+ . The antibacterial mechanism showed that Cu2+ and/or Nd3+ could enter into the E. coli cell, and the cell membrane of E. coli had been ruptured, leading to the changes of ion concentrations and the leakage of DNA, RNA and protein. The Cu-Nd@ZrP powder, combining the advantages of Cu2+ and Nd3+ , displayed higher antibacterial activity and lower cytotoxicity, suggesting the great potential application as an antibacterial powder. Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (51172099, 51203134, 21476052 and 21271087), the Foundation of EnterpriseUniversity-Research Institute Cooperation from Guangdong
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