Toxicological Effect of ZnO Nanoparticles Based on Bacteria

4140

Langmuir 2008, 24, 4140-4144

Toxicological Effect of ZnO Nanoparticles Based on Bacteria Zhongbing Huang,† Xu Zheng,† Danhong Yan,† Guangfu Yin,*,† Xiaoming Liao,† Yunqing Kang,† Yadong Yao,† Di Huang,† and Baoqing Hao*,‡ College of Materials Science and Engineering, Sichuan UniVersity, Chengdu, 610065, China and College of Life Science & Technology, Southwest UniVersity for Nationalities, No. 16, South 4th Section, 1st Ring Road, Chengdu, P.R. China ReceiVed NoVember 20, 2007. In Final Form: January 23, 2008 Streptococcus agalactiae and Staphylococcus aureus are two pathogenetic agents of several infective diseases in humans. Biocidal effects and cellular internalization of ZnO nanoparticles (NPs) on two bacteria are reported, and ZnO NPs have a good bacteriostasis effect. ZnO NPs were synthesized in the EG aqueous system through the hydrolysis of ionic Zn2+ salts. Particle size and shape were controlled by the addition of the various surfactants. Bactericidal tests were performed in an ordinary broth medium on solid agar plates and in liquid systems with different concentrations of ZnO NPs. The biocidal action of ZnO materials was studied by transmission electron microscopy of bacteria ultrathin sections. The results confirmed that bactericidal cells were damaged after ZnO NPs contacted with them, showing both gram-negative membrane and gram-positive membrane disorganization. The surface modification of ZnO NPs causes an increase in membrane permeability and the cellular internalization of these NPs whereas there is a ZnO NP structure change inside the cells.

Introduction Recently, nanomaterials have received enormous attention because they can be used to create new types of analytical tools for biotechnology and life sciences.1 However, toxicological studies of nanomaterials have also attracted attention. It is reported that nanoparticles (NPs) pose serious problems to the liver, lungs, and other organs.2 It was demonstrated that NPs cause more inflammation than larger respirable particles made from the same material when delivered in the same mass dose. This behavior has been observed for a range of different materials of generally low toxicity such as carbon black (CB) and TiO2.3 Highly ionic nanoparticle metal oxides are particularly interesting in that they can be prepared with extremely high surface areas and with unusual crystal morphologies that possess numerous edge/corner and other reactive surface sites.4 Many nanomaterials, such as Fe3O4, Al, MoO3, and TiO2, had no measurable effect at lower doses (10-50 µg/mL), whereas there was a significant effect at higher levels (100-250 µg/mL).2a With the increased presence of nanostructured materials in commercial products, a growing public debate and a biosafety problem are emerging for toxicological and environmental effects of direct and indirect exposure to these materials, and their impacts are not completely illuminated. For example, nanoscale (20-100 nm) ZnO can be * Corresponding authors. (G.Y.) E-mail: [email protected]. Tel/Fax: 86-28-85413003. (B.H.) E-mail: [email protected]. Tel: 86-2886714164. † Sichuan University. ‡ Southwest University for Nationalities. (1) (a) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (b) Taton, T.; Mirkin, C.; Letsinger, R. Science 2000, 289, 1757. (c) Cui, Y.; Wei, Q.; Park, H.; Lieber, C. Science 2001, 293, 1289. (2) (a) Hussain, S. M.; Hess, b, K. L.; Gearhart, J. M.; Geiss, K. T.; Schlager, J. J. Toxic. In Vitro 2005, 19, 975. (b) Pisanic T. R., II; Blackwell, J. D.; Shubayev, V. I.; Fin˜ones, R. R.; Jin, S. Biomaterials 2007, 28, 2572. (c) Brigger, I.; Dubernet, C.; Couvreur, P. AdV. Drug DeliVery ReV. 2002, 54, 631. (d) Forastiere, F. Toxicol. Lett. 2007, 164S, S33. (3) (a) Wang, J.; Zhou, G.; Chen, C.; Yu, H.; Wang, Ti.; Ma, Y.; Jia, G.; Gao, Y.; Li, B.; Sun, J.; Li, Y.; Jiao, F.; Chai, Y. Z. Toxicol. Lett. 2007, 168, 173. (b) Wilson, M. R.; Lightbody, J. H.; Donaldson, K.; Sales, J.; Stone, V. Toxicol. Appl. Pharmacol. 2002, 184, 172. (c) Kwak, S.; Kim, S. H.; Kim, S. S. EnViron. Sci. Technol. 2001, 35, 2388. (4) Klabunde, K. J.; Stark, J.; Koper, O.; Mohs, C.; Park, D.; Decker, S.; Jiang, Y.; Lagadic, I.; Zhang, D. J. Phys. Chem. 1996, 100, 12142.

employed as a safe physical sunscreen because it scatters and reflects UV in sunlight. Crystalline ZnO is a semiconductor and a piezoelectrical material with a gap energies of about 3.3 eV,5a which is very close to that of TiO2 anatase. ZnO NPs are believed to be nontoxic, biosafe, and biocompatible5b and have been used in many applications in daily life, such as drug carriers and in cosmetics and fillings in medical materials.5c,d However, few reports are available on the toxicological effect of ZnO NPs. In this article, we also report the use of ZnO NPs as a sunscreen semiconductor, which is a versatile material that has achievable applications in biosensors,6 biogenerators,7 bioelectrodes,8 electroluminescent devices,9 and ultraviolet laser diodes.10 In this article, we report the initial interactions that govern the bactericidal activity of ZnO NPs as well as the effect of the complex surface chemistry of NPs on the bacterial membranes. Streptococcus agalactiae and Staphylococcus aureus are two etiological agents of several infective diseases in humans. Our experimental results showed that high concentrations of ZnO NPs were toxic to S. agalactiae and S. aureus cultured cells and also showed the cellular internalization of these NPs and the structural change of ZnO NPs inside the cells. Materials and Methods ZnO NPs were synthesized in an ethylene glycol (EG) medium by the hydrolysis of zinc acetate.11,12 Zn(CH3COO)2‚2H2O (99%, 10.0 g) was dissolved in 50 EG by heating to 80 °C. Thereafter, (5) (a) Jiang, P.; Zhou, J. J.; Fang, H. F.; Wang, C. Y.; Wang, Z. L.; Xie, S. S. AdV. Funct. Mater. 2007, 17, 1303. (b) Zhou, J.; Xu, N. S.; Wang, Z. L. AdV. Mater. 2006, 18, 2432. (c) Ito, M. Biomaterials 1991, 12, 41. (d) Ørstavik, D.; Hongslo, J. K. Biomaterials 1985, 2, 129. (6) Lin, F. C.; Takao, Y.; Shimizu, Y.; Egashira, M. Sens. Actuators, B 1995, 24-25, 843. (7) (a) Wang, Z. L.; Song, J. H. Science 2006, 312, 242. (b) Wang, X. D.; Song, J. H.; Liu, J.; Wang, Z. L. Science 2007, 316, 102. (8) Hou, X. Q.; Ren, X. L.; Tang, F. Q.; Chen, D. Chin. J. Anal. Chem. 2006, 34, 303. (9) Chen, S.; Liu, Y.; Shao, C.; Mu, R.; Lu, Y.; Zhang, J.; Shen, D.; Fan, X. AdV. Mater. 2005, 17, 586. (10) Huang, M. H.; Mao, S.; Feick, H.; Yan, H. Q.; Wu, Y. Y.; Kind, H.; Weber, E.; Russo, R.; Yang, P. D. Science 2001, 292, 1897. (11) Xia, H. L.; Tang, F. Q. J. Phys. Chem. B 2003, 107, 9175. (12) Feldmann, C. Adv. Funct. Mater. 2003, 13, 101.

10.1021/la7035949 CCC: $40.75 © 2008 American Chemical Society Published on Web 03/15/2008

Toxicological Effect of ZnO Nanoparticles

Langmuir, Vol. 24, No. 8, 2008 4141

Figure 1. SEM images of ZnO NPs prepared in different molecules of 1.5 wt %: (a) Tween-20, (b) SDS, (c) PVA, (d) PVA 2.0 wt %. under vigorous stirring 50 mL H2O was added, and the solution was heated to 95 °C. This temperature was maintained for 4 h. After precipitation and washing three times, respectively, ZnO particles were resuspended by ultrasonic treatment in water to obtain a final concentration from 0.01 to 0.5 mol/L. To control the particle size and shape, small molecules and macromolecules such as sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA), and polyoxyethylene sorbitan monolaurate (Tween 20) were added to zinc acetate and the EG aqueous medium at concentrations of 0.05 M. The mixture was then heated under gentle stirring to a temperature varying from 70 to 90 °C. In this study, S. agalactiae and S. aureus with a Meticillinresistant strain were used because of their strong antibiotic resistance. They have been used as the bacterial strain reference for genome sequencing. Two bacteria were grown aerobically at 37 °C overnight with shaking (200 rpm) in an ordinary broth medium containing 3 g/L beef extract, 10 g/L peptone, and 5 g/L NaCl. The saturated cultures were first diluted in fresh sugar medium and incubated at 37 °C overnight in Erlenmeyer flasks with vigorous shaking at 200 rpm as well as on solid agar plates, which needed to be sterilized at 120 °C for 20 min before inoculation. Bacteriological tests were performed on solid agar plates with different concentrations of ZnO NPs (from 10-1 to 10-4 M). The inoculated cells were estimated to 200 cfu (colony forming units) per plate, and all results were compared to a control without ZnO NPs. Bacteria and ZnO NPs in the EG aqueous medium cultured on solid agar plates were incubated overnight at 37 °C, and the number of colonies was counted. ZnOfree solid agar plates were used as a control. Bacteriological tests were also performed on solid agar plates partially coated by filter paper mixed with ZnO NPs, and a ZnO-free filter on solid agar plates was used as a control. Transmission electron microscopy (TEM, JEOL-200, 160 kV) and scanning electron microscopy (SEM, JEOL-5900LV, 20 kV) with accompanying selected-area electron diffraction (SAED) were used to characterize the particle structure, size, and morphology. TEM analyses of bacteria thin sections were used to study the biocidal action of ZnO NPs. X-ray diffraction (XRD) patterns were recorded on a Philips X’Pert MDP diffractometer with Cu KR radiation. The rigid particles that precipitated by hydrolysis were rinsed with distilled water before being subjected to XRD measurements. Fourier transform infrared (FTIR) spectra were recorded using a Nexus 670 spectrometer. Bacteria samples, grown in a liquid culture medium at 37 °C for 24 h, were then diluted in a fresh broth medium. In the exponential

phase, 10-2 M PVA-coated ZnO NPs in an EG aqueous medium was added to the culture medium. The pH value of the medium in the test and control groups was maintained at 7.2 during culturing. The final solution was then incubated overnight and consisted of a sample containing 1.5 × 106 bacteria fixed in a mixture containing 2.5% glutaraldehyde, 1.0% acrolein, and 0.1% ruthenium red in a phosphate buffer (0.1 M, pH 7.2). All samples for TEM were prepared following standard procedures for fixing and embedding biological samples.12

Results and Discussion In general, there are two factors that influence the size and shape of ZnO materials synthesized by hydrolysis in an EG aqueous medium: (i) the nature of the molecular template added during ZnO formation and (ii) the hydrolysis ratio.10,11 Figure 1 shows SEM images of ZnO NPs obtained with different template molecules. For ZnO prepared with the addition of a template of Tween 20 (Figure 1a), spherical NPs with a narrow size distribution of around 150 nm were obtained. However, the irregular sheetlike-organized ZnO particles were observed by electrostatic stabilization with SDS as a template additive (Figure 1b). The advantage of SDS used as a template additive is its surfactive and nontoxic properties. A very narrow size distribution of ZnO nanorods of 100 nm diameter was obtained with the addition of PVA as a template molecule (Figure 1c,d). Under this reactive condition, PVA acts as a template and also as a linker in the self-assembly of ZnO NPs.12 During PVA-coated ZnO preparation, when PVA addition concentration increased to 2.0%, ZnO NPs of about 60 nm diameter were observed (Figure 1d), indicating that ZnO isotropic morphologies were obtained by the coalescence of spherical NPs in the presence of PVA macromolecules. Figure 2 shows the XRD pattern of as-synthesized ZnO NPs in PVA. The results show the good wurtzite crystallinity of the obtained ZnO NPs, and the diffraction peaks of other minerals were not detected, revealing that the obtained NPs have a singlecrystal structure. The wurtzite synthesized in PVA exhibits seven obvious diffraction peaks in the (100), (002), (101), (102), (110),

4142 Langmuir, Vol. 24, No. 8, 2008

Huang et al.

Figure 5. Images of bactericidal tests (S. aureus) as a function of different ZnO suspensions after incubation at 37 °C overnight: (left) 0 wt % and (right) 0. 12 wt %. Figure 2. XRD pattern of ZnO NPs obtained in PVA solution.

Figure 3. FTIR spectrum of the PVA-coated ZnO NPs.

Figure 4. Images of S. agalactiae incubated for 24 h at 37 °C together with filter paper: (a) with ZnO NPs and (b) without ZnO NPs.

Figure 6. Bactericidal tests as a function of the PVA-coated ZnO concentration after incubation at 37 °C overnight: (a) S. agalactiae and (b) S. aureus.

(103), and (112) planes, with higher intensity in the (101) plane indicating that the ZnO NPs may be grown along the [0001] direction. We chose ZnO NPs obtained in PVA for bacteriological testing because of their narrow size distribution of rodlike NPs. Figure 3 illustrates the FTIR spectra of the prepared PVA-coated ZnO particles with DMSA. A peak at ∼418 cm-1 is the stretching vibration of the Zn-O bond in ZnO particles. Similarly, two peaks at 2921 and 2857 cm-1 are assigned to the vibration of the C-H bond in PEG. A broad absorption band at 3423 cm-1 and a peak at 1420 cm-1 in the IR spectra of ZnO particles can be seen, and these are attributed to the hydroxyl groups in PEG. A peak at ∼1140 cm-1 is the stretching vibration of the C-O bond in PVA. These peaks reveal that there are many PVA molecules coated on the surface of ZnO particles. Figure 4 shows the images of bacteriological tests performed on solid agar plates that are partially covered by filter paper with and without ZnO NPs. The results show that there are a few S. agalactiae colonies (only about 10-15) inside an open circle around the filter paper with ZnO, in comparison to many colonies (more than 70) inside an open circle around the filter paper without ZnO NPs, indicating that the ZnO NPs could obviously restrain

the proliferation of S. agalactiae. Figure 5 shows the image of bacteriological tests of S. aureus on solid agar plates without ZnO NPs and with 0.12 M ZnO suspensions. The results show that a number of S. aureus colonies appeared on the solid agar plates without ZnO NPs; however, several S. aureus colonies are observed on the solid agar plates with 0.12 M ZnO, revealing that ZnO NPs synthesized in PVA solution can also restrain S. aureus proliferation. The bactericidal effect of PVA-coated ZnO on gram-negative bacteria is presented in Figure 6. All tests were repeated three times after culture incubation at 37 °C overnight for statistical studies. The numbers of bacterial colonies grown on test plates with S. agalactiae and S. aureus were quite similar. It is reported that SDS surfactant could inhibit bacterial growth12 as a result of the denaturalization of bacterial protein by SDS. PVA, a similar sugar structure because of the many C-O bonds and -OH groups, may be metabolized to short-chain molecules by bacteria, and glucose may be present in the culture medium.13 Obviously, these molecules could promote bacterial growth after their metabolization. This effect was more important for S. agalactiae (13) Bechtel, D. B.; Bulla, L. A., Jr. J. Bacteriol. 1976, 127, 1472.

Toxicological Effect of ZnO Nanoparticles

Langmuir, Vol. 24, No. 8, 2008 4143

Figure 7. TEM images of bacterial thin sections with ZnO NP (1.6 × 10-3 M) internalization: (a, b) S. agalactiae and (c, d) S. aureus. The arrows point to ZnO NPs.

or S. aureus supplemented with PVA. The same test was conducted with only PVA-coated ZnO NPs (as Figure 1d shows) in the culture medium (Figure 6). The concentration of ZnO NPs was varied from 10-1 to 10-4 M. The presence of these NPs at a concentration of 10-1 M (0.12 M) caused more than 95% inhibition of bacterial growth. A concentration between 1.2 × 10-2 and 6 × 10-2 M could inhibit bacterial growth by 30%. For concentrations between 1.2 × 10-3 and 6 × 10-4 M, a slight decrease in the number of bacteria colonies was detected. Colonies could also result from the similar-sugar metabolism-dependent PVA because bacteria can metabolize Zn2+ as an ion of oligomer chains, showing that ZnO NPs of low concentrations are not toxic for S. agalactiae or S. aureus. Some of the nanomaterials settled rapidly and needed constant stirring to make a homogeneous suspension, and the full cellular dose/time of these materials would be larger. The other issue that is still not known is whether the cells internalize nanoparticles and, if so, which mechanisms are involved. It was reported by Roselli et al.14 that a ZnO NP concentration from 10-4 to 10-3 M did not affect cell permeability. In all cases, the process of bacterial growth during the devouring of NPs remains a significant challenge. A TEM image of bacterial sample ultrathin sections allowed us to observe morphological changes directly, resulting in bacterial upon contact with ZnO NPs (PVA-coated ZnO sample) at a concentration of 10-3 M in a liquid culture medium (Figure 7). The PVA-coated ZnO sample was chosen because it presents a narrow size distribution of NPs (Figure 1c). Moreover, it was demonstrated by Brayner et al.13 that polyol macromolecules do not prevent bacterial growth13 and Escherichia coli could grow well in a liquid medium. In this study, the bacteria are strong antibiotic-resistant gram-negative bacilli that present a tubular form and a spherical, aerobic, gram-positive form usually occurring in irregular clusters. The gram-negative cell wall of S. agalactiae is composed of an organized triple membrane (14) Ramnath, M.; Beukes, M.; Tamura, K.; Hastings, J. W. Appl. Environ. Microbiol. 2000, 66, 3098. (15) Brayner, R.; Ferrari-lliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fie´vet, F. Nano Lett. 2006, 6, 866. (16) Roselli, M.; Finamore, A.; Garaguso, I.; Britti, M. S.; Mengheri, E. Biochem. Mol. Actions Nutriments 2003, 4077.

containing a thin inner layer of peptidoglycan between an outer membrane. Damage and disorganization in the S. agalactiae cells wall were observed (Figure 7a,b). In Figure 7a, NPs have succeeded in penetrating inside the cells, thus damaging the membranes, while cellular division was represented for S. agalactiae grown in a liquid medium. In the last case, the new cells presented considerable damage with a very disorganized cell wall and an affected morphology. Obviously, TEM micrographs of bacteria grown in the presence of a PVA-coated ZnO sample showed preliminary results of the cellular internalization of ZnO NPs and cell wall disorganization (Figure 7a). The cell membrane in some S. agalactiae is damaged (Figure 7b). Figure 7 shows ZnO NPs inside and outside the cell, and the cellular internalization was also clearly observed with ZnO NPs. Similarly, ZnO NPs have also been devoured by gram-positive cells of S. aureus, and the partial cell membranes were also damaged whereas cellular division was represented for S. aureus growth (Figure 7c,d). It is reported17,18 that bactericides have a greatly improved efficiency when paired with alkaline substances. Alkaline compounds dissolve the external part of the cell membrane, which is the major protective barrier for the conventional bactericides. There are many hydroxyl groups and PVA macromolecules on the surface of coated metal oxide NPs because of their preparation by a wet chemical method,19 so this kind of ZnO NP was able to enter the cells. Our microscopic study results showed that ZnO NPs associate with cell membranes and that the cells internalize some of the particles. This is very evident in bacteria where nanoparticles are surrounded within cells. Figure 8 presents SAED patterns of ZnO NPs outside and inside the S. agalactiae cell, showing that ZnO NPs in the cellular matrix could retain the ZnO crystal wurtzite structure. This [100] electron diffraction pattern of an isolated species, recorded from a NP, indicates that the NP grows along [0001] and its side surfaces are defined by {0110} (Figure 8a). Our previous experiment’s results, which are not shown here, suggested that (17) Death, J. E.; Coates, D. J. Clin. Pathol. 1979, 32, 148. (18) Bloomfield, S. F.; Arthur, M. J. Appl. Bacteriol. Symp. Suppl. 1994, 76, 91S-104S. (19) Huang, Z. B.; Zhang, Y. Q.; Tang, F. Q. Chem. Commun. 2005, 342-344.

4144 Langmuir, Vol. 24, No. 8, 2008

Figure 8. SEAD patterns of ZnO NPs (a) outside the cell and (b) inside the cell (S. aureus).

ZnO NPs in pH 7.2 solutions were not changed and retain their shape and size for many days. However, NPs inside the cell could not remain this crystalline and started to change into an amorphous structure as a result of dipping the cell sap into ZnO NPs and partially damaging the NP crystal structure. Although this diffraction pattern is dark, the [002 ] and the [112] faces could be distinguished (Figure 8b). These results indicated not only that ZnO NPs possessed biocidal effects but also that the cell impacted the crystal structure of ZnO NPs inside the cell. Oberdo¨ster indicated in a recent study20 that nanomaterials induced oxidative stress in a fish brain, as demonstrated by a significant elevation of lipid peroxidation. Our results here show that there was a significant increase in the bactericidal test at different concentrations of ZnO NPs. The bacterial colony number decreased slightly with exposure to concentrations of up to 0.012 M, with a sharp decrease at 0.12 M. The decrease in bacteria at the higher-exposure concentrations may be a consequence of the leakage of intracellular contents from the cell (Figure 7b) because evidence of significant membrane damage was apparent at the highest concentration. Increased generation by ZnO NPs is likely (20) Oberdo¨ster, E. EnViron. Health Perspect. 2004, 112, 1058.

Huang et al.

to contribute to oxidative stress that may ultimately lead to the observed cytotoxicity. It is noted that the crystal structure of ZnO NPs changes significantly inside the cell. Many hydroxyl groups and PVA macromolecules on the surface of coated ZnO NPs are obviously correlated with their accumulation inside the cells. Because PVA-coated ZnO NPs are relatively new particles, it is necessary to investigate their toxicological behavior, and this first report provides such preliminary information in this direction.

Conclusions We report preliminary studies on the toxicological effect of ZnO NPs synthesized in an EG aqueous medium on S. agalactiae and S. aureus. Our experimental results revealed that low concentrations of ZnO NPs did not induce any cellular damage, as also demonstrated by other researchers.1a,12,13 Moreover, these cells, after contact with PVA-coated ZnO NP concentrations higher than 0.016 M in the EG aqueous medium, were damaged, and there was a great change in the crystal structure of ZnO NPs. Cellular internalization of these NPs was observed. These results indicate that ZnO NPs are good bactericidal agents. Acknowledgment. The support of Sichuan University through a Young Science Research Fund (JS20070406506415) and the Key Technologies Research and Development Program of Sichuan Province (2006Z08-001-1) is acknowledged with gratitude. This work was also supported by a grant from the Ph.D. Doctoral Programs Foundation of the Ministry of Education of China (no. 20070610131). We thank the Analytical & Testing Center, Sichuan University, for assistance with the microscopy and XRD work. LA7035949