Evaluation of the antibacterial power and biocompatibility of zinc oxide nanorods decorated graphene nanoplatelets: new perspectives for antibiodeteriorative approaches
© The Author(s) 2017
Received: 25 May 2017
Accepted: 21 July 2017
Published: 1 August 2017
Nanotechnologies are currently revolutionizing the world around us, improving the quality of our lives thanks to a multitude of applications in several areas including the environmental preservation, with the biodeterioration phenomenon representing one of the major concerns.
In this study, an innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was tested for the ability to inhibit two different pathogens belonging to bacterial genera frequently associated with nosocomial infections as well as biodeterioration phenomenon: the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. A time- and dose-dependent bactericidal effect in cell viability was highlighted against both bacteria, demonstrating a strong antimicrobial potential of ZNGs. Furthermore, the analysis of bacterial surfaces through Field emission scanning electron microscopy (FESEM) revealed ZNGs mechanical interaction at cell wall level. ZNGs induced in those bacteria deep physical damages not compatible with life as a result of nanoneedle-like action of this nanomaterial together with its nanoblade effect. Cell injuries were confirmed by Fourier transform infrared spectroscopy, revealing that ZNGs antimicrobial effect was related to protein and phospholipid changes as well as a decrease in extracellular polymeric substances; this was also supported by a reduction in biofilm formation of both bacteria. The antibacterial properties of ZNGs applied on building-related materials make them a promising tool for the conservation of indoor/outdoor surfaces. Finally, ZNGs nanotoxicity was assessed in vivo by exploiting the soil free living nematode Caenorhabditis elegans. Notably, no harmful effects of ZNGs on larval development, lifespan, fertility as well as neuromuscular functionality were highlighted in this excellent model for environmental nanotoxicology.
Overall, ZNGs represent a promising candidate for developing biocompatible materials that can be exploitable in antimicrobial applications without releasing toxic compounds, harmful to the environment.
Nowadays, an ever-growing interest is focused on nanoscience that works with and/or creates promising materials characterized by nanostructured dimensions. Nanotechnologies have extensively been developed in the last years, expanding more and more the range of possible applications. At the present, nanotechnology has implications in a plethora of areas including medicine, food industry and environmental field, depending on specific nanomaterial features such as mechanical, thermal and chemical properties as well as large surface area [1–3]. Nevertheless, the antimicrobial power together with optical/light properties make some nanostructures particularly helpful in applications involved in the conservation of cultural heritage and/or building construction. In fact, historic buildings need to be preserved avoiding the risk of biodeterioration. Such process lead to unpleasant alteration of the material determined by the metabolism of bacteria, fungi, algae and lichens [4, 5]. Biodeteriorative activities determine severe damages to architectural surfaces, church frescoes or wall paintings that are found in catacombs and caverns. Among the bacterial isolates derived from wall paintings, Pseudomonas and Staphylococcus genera are the most predominant together with Bacillus, Streptomyces and Mycobacterium . The formation of bacterial biofilm on construction material plays a key role in the possible occurrence of pathogen infections in nosocomial environments as well as in building biodeterioration [7, 8]. In fact, bacterial growth on wall surface as well as on medical devices represents a severe concern in the health care system, taking into account that bacteria are becoming multiresistant to antibiotics. From this perspective, the development of surfaces able to kill or inhibit bacterial growth without the use of antibiotics/drugs is attracting a great interest, and new wall paint and coatings, containing nanoparticles that possess antimicrobial activity, represent an emerging approach in order to prevent both the spread of nosocomial infections and biodeteriorative activity .
Among nanomaterials, great interest is currently addressed to the synthesis and development of graphene-based nanocomposites as reported in [10–13]. In particular, decoration of graphene with metal oxide offers unique properties that extensively broaden its application in chemical, medical and pharmaceutical fields [14, 15].
Several studies reported impressive antimicrobial power for metal oxide-based nanoparticles [16, 17]. Moreover, it has been possible to grow ZnO nanostructures onto graphene, so that decoration or functionalization was typically achieved only over the exposed surface of graphene. In our recent study, the synthesis of ZnO nanorods (ZnO-NRs) with controlled shape and density onto unsupported multilayer graphene flakes (also known as graphene nanoplatelets GNPs) was reported .
These zinc oxide nanorods-decorated graphene nanoplatelets (ZNGs) were characterized by the ability to kill the bacterium causing dental caries, namely Streptococcus mutans. ZNGs were found to efficiently kill and to control S. mutans cells by inhibiting both planktonic and biofilm growth . This hybrid nanomaterial combines the remarkable electrical and antimicrobial properties offered by GNPs together with optical features and the highly effective killer action against both Gram-positive and Gram-negative bacteria of ZnO-NRs. Moreover, the characteristic grey color of graphene based nanomaterials is mitigated by ZnO whitening effect, making this hybrid nanostructure a promising candidate for the development of novel nanofiller-based wall paint in the field of building construction and cultural heritage.
Herein, ZNGs were used to inhibit two pathogens belonging to genera frequently associated to biodeterioration: the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa; a mechanical mode of action against both bacteria has been suggested. Environmental nanotoxicity was assessed through the soil free-living nematode Caenorhabditis elegans.
Production of nanostructures and suspensions
Bacterial strains and media
Pseudomonas aeruginosa ATCC 15692 and Staphylococcus aureus ATCC 25923 were the bacterial strains used in this study. They were grown in LB (Luria–Bertani) broth at 37 °C.
Cells viability test
Viability was evaluated in both suspensions and solid substrates. For liquid assay, bacteria were incubated at 37 °C under gentle shaking in H2Odd suspensions of ZNGs at various concentrations (ranging from 0.1 to 50 µg/mL). The bacterial concentration inoculated was 5 × 107 cells/mL. Both microbial strains were exposed to increasing concentrations of ZNGs and compared to the respective untreated controls. The experiments were carried out at 2 and 24 h of treatment.
In the case of antimicrobial test on solid surfaces, ZNGs applied on plywood samples (2.5 cm × 2.5 cm) covered or not by a commercial paint were drop casted with 150 µL of a ZNG suspension (250 µg/mL) and air-dried. After a 30 min of UV-sterilization, 200 µL of S. aureus suspension (6 × 105 cell/mL) were spotted onto the plywood surfaces. Cells were extracted at the initial time of contamination (t0) and after 4 h of incubation at 25 °C by washing plywood substrates in a sterile bag with 10 mL of sterile H2Odd.
The ability of bacterial survival was assessed by the colony count method (Colony Forming Unit, CFU) for both types of tests, by spreading the diluted samples onto LB agar plates.
Evaluation of biofilm formation
The biofilm growth in 96-well microtiter plate was estimated by using the Crystal Violet (CV) assay. In the case of S. aureus, each well was inoculated with 200 µL of a suspension containing S. aureus cells (final concentration 1 × 107 cell/mL), the Tryptic Soy Broth medium (TSB, Becton–Dickinson and Company, Franklin Lakes, NJ, USA) with 2% glucose (to stimulate biofilm formation) and ZNGs, present or not at various concentrations (in triplicate). For P. aeruginosa, 100 µL of a suspension of LB broth and ZNGs inoculated with bacterial aliquot (0.5 OD600) were placed in every well. After incubation of the plates under stirring (25 rpm) at 37° C for 24 h, the culture medium was removed and the wells were washed twice with H2Odd with the purpose to remove cells not adhered. Plates were then kept at 65 °C for 20 min. Finally, every well was stained with 0.3% Crystal Violet (Sigma-Aldrich) and incubated at RT for 15 min. After several washes with H2Odd, plates were left to dry and wells were then treated with 200 µL of 96% EtOH for CV elution. Absorbance at 600 nm was then measured by using a multiplate reader (Promega, GloMax multi+ detection system).
Pyocyanin assay in P. aeruginosa
For this test, 12-well microtiter plates were used. Each well was filled with 900 µL of LB broth containing or not different concentration of ZNGs (in triplicate) and inoculated with P. aeruginosa cells at a final concentration of 5 × 107 cell/mL from an overnight growth culture, reaching a final volume of 1.5 mL (adding sterile H2Odd). Plates were incubated at 37 °C overnight without agitation. Next, ON cultures were centrifuged and the supernatant absorbance was measured at 380 nm.
Preparation of bacterial cells for FE-SEM imaging
Treated and untreated cells of P. aeruginosa were incubated at 37 °C for 1 h, while S. aureus ones for 30 min. Short treatment times were chosen to obtain images in which the effects of ZNGs on bacterial cells were clearly visible. The tested concentration of ZNGs was 50 µg/mL in 1 mL of sterile water. The protocol for samples preparation was performed as described in Olivi et al. . Imaging was performed using a Zeiss Auriga FE-SEM, operated at an accelerating voltage of 5 kV.
To investigate the antimicrobial properties of ZNGs, Fourier Transform Infrared (FTIR) spectroscopy was used. The comparison of the FTIR spectra of untreated bacterial cells and of bacterial cells treated with this nanocomposite allowed to assess whether the treatment induced alterations of the bacterial cell structure and surface components. Briefly, about 5 × 108 cell/mL of overnight grown cultures of P. aeruginosa and S. aureus were incubated in 1 mL of sterile H2Odd at 37° C for 90 min under gentle agitation, with or without ZNGs (10 µg/mL). Both ZNGs concentration and time of exposure were chosen in order to have a high cellular survival and to appreciate the early structural changes in treated bacteria. Cells were withdrawn and then fixed with 1 mL of a freshly prepared 4% (v/v) formaldehyde solution. After incubation for 1 h in the dark, the samples were washed three times and the cells were initially suspended in 20 μL of H2O (water suspension) or of D2O (deuterium oxide suspension). FTIR spectra have been collected either on dried samples or on liquid samples. Dried samples were prepared by drop-casting 20 μL of a bacterial suspension onto a CaF2 window and then leaving the liquid suspension to air-drying. Measurements on liquids were performed by placing 50 µL of a bacterial suspension in deuterated water between two CaF2 windows separated by a 50 μm Teflon spacer. In both cases, FTIR spectra of untreated bacterial samples and of treated bacterial samples have been acquired and then analyzed. FTIR measurements were carried out with a Bruker Vertex 70 spectrometer equipped with a DTGS (doped triglycine sulfate) detector. During data collection the sample was at room temperature and the sample compartment was under continuum purging with dry N2 gas. Each spectrum is an average over 256 scans and has a spectral resolution of 2 cm−1. In the case of dried samples, the intensity transmitted by the CaF2 substrate was used as a reference to obtain the sample absorbance. In the case of liquid samples, absorbance was calculated using the intensity transmitted by the CaF2 cell filled with pure D2O as a reference.
Method of cultivation for C. elegans
In this study the C. elegans wild type strain N2 was used. It was maintained at 16 °C on agar plates of the Nematode Growth Medium (NGM) covered by a layer of bacterial suspension of Escherichia coli OP50 as feeding source .
Nematode treatment with ZNGs was performed starting on 1-day adults or newly hatched L1 larvae, resulting from synchronized cultures that were transferred to NGM-OP50 plates with ZNGs at the indicated concentrations. Every day nematodes were placed onto freshly prepared plates and 100 µL of ZNG suspensions were distributed before worms seeding. The nematodes were monitored daily for their survival with respect to untreated nematodes, and were considered dead when there was no response to the delicate touch of a platinum wire. At least 60 nematodes per condition were used in each experiment.
OP50-NGM plates containing or not ZNGs were seeded with adult worms (in triplicate) and were incubated at 16 °C, allowing embryos laying. Next, each animal was transferred onto a fresh plate every day, and the number of progeny was recorded for 4 days until the worm stopped laying eggs.
Body length analysis
Nematode larvae exposed to ZNGs starting from embryos hatching, were photographed at the indicated time points by using a Leica MZ10F stereomicroscope with a Jenoptik CCD camera. Length of worm body was determined by using the Delta Sistemi IAS software. An average of 30 nematodes were imaged on at least three independent experiments.
Pumping rate measurements
The pharyngeal pumping rate was measured in C. elegans individuals exposed or not to ZNGs starting from their larval development as described in lifespan assay. About 10 worms for each experimental condition were analyzed for the number of their pharyngeal contractions during a time interval of 30 s. This analysis was repeated at the indicated time points.
Body bending evaluation
The locomotion behavior of nematodes, treated with ZNGs starting from embryos hatching, was analyzed by body bending counting at the indicated time points. After several washes in M9 buffer to remove bacteria, nematodes were placed in 10 μL of M9 buffer allowing them to swim freely. About 10 worms for each experimental condition were monitored for the number of head thrashes within a minute.
All experiments were performed at least in triplicate. Data are presented as mean ± SD. The statistical significance was determined by Student’s t test or one-way ANOVA analysis coupled with a Bonferroni post test (GraphPad Prism 5.0 software, GraphPad Software Inc., La Jolla, CA, USA), and defined as *p < 0.05, **p < 0.01, and ***p < 0.001.
Results and discussion
In the spectral region between 1300 and 900 cm−1 (inset of Fig. 6a), the band at about 1234 cm−1, attributed to phosphodiester functional groups of DNA/RNA polysaccharide backbone structures, is essentially unaffected by ZNGs treatment. On the other hand, the band at ~1069 cm−1, attributed to the symmetric stretching vibration of PO2 − groups in nucleic acids and to C–O–C and C–O–P stretching vibrations of various oligo- and poly-saccharides, becomes wider because of the appearance of a component at 1114 cm−1. This observation suggests that an alteration in bacterial polysaccharide structures (Extracellular polymeric substances, EPS) results from the interaction of the bacterial cell surface with ZNGs, in agreement with the biofilm results. Indeed, cells forming a biofilm are surrounded by EPS, which represents the immediate environment of these cells, thus playing a relevant role in nutrient acquisition and in the protection of the bacterial cells from environment and mechanical stresses. Consistent with this, Wang et al. suggested a protective role for bacterial EPS against ZnO nanoparticles killer action, via nanostructures sequestering . We can hypothesize that ZNGs act by lowering EPS production and thus by inhibiting cellular barrier mechanisms.
Fourier transform infrared spectroscopy data indicate that EPS reduction is more pronounced in S. aureus than in P. aeruginosa bacteria. This observation is in line with biofilm growth inhibition results (Fig. 5), confirming a stronger antimicrobial effect of ZnO NRs-decorated GNPs on S. aureus. Moreover, the FTIR spectrum of the treated S. aureus bacteria was, in all repeated experiments, always noisier with respect to the spectrum of the untreated ones. This result could be a further indication of the intensive interaction between the ZNGs and the external structure of the S. aureus bacteria. Similar overall changes (mainly alterations in the structure of proteins and polysaccharides) induced by the treatment with ZNGs are observed in the case of S. aureus (Additional file 1: Figure S2). However, in the 1300–900 cm−1 region, the band associated to saccharide structures underwent a bigger modification. Indeed, in the case of S. aureus, the appearance of two components, one at ~1119 cm−1 and the other at ~998 cm−1, was also observed. The FTIR results support the hypothesis that ZNGs exposure produces cell damages. In particular, the FTIR analysis suggests that the antimicrobial effect-related changes are associated with protein and phospholipid damages. This is consistent with the previously observed results demonstrating modifications in protein structures as well as membrane injuries in S. aureus cells treated with ZnO NRs . Moreover, several studies highlighted both partial protein unfolding and changes in phospholipids as a meaning of the interaction between cell wall biomolecules and nanomaterials surface [42–44]. Cell surface proteins play important roles in cellular physiological activities, including DNA stability and replication, which in turn may lead to DNA damages.
We have already demonstrated that the two components of ZNGs, namely ZnO NRs and GNPs, showed non-toxic effect in C. elegans [23, 24]. Moreover, the lack of cytotoxicity of ZnO nanorods has been assessed in different human cell lines . However, it has been reported that ZnO nanoparticles resulted to be toxic to different model systems including also C. elegans [39–41] and that the main components of their nanotoxicity resulted to be the reactive oxygen species production and the consequent release of zinc ion in suspensions . In our previous studies we demonstrated that Zn ion dissolution was negligible in ZnO NRs as well as in ZNGs [10, 15]. We can thus speculate that the lack of ZNGs toxicity in C. elegans could be ascribed to the low concentration of bioavailable Zn2+.
Our data are consistent with the observation that the two components of ZNGs, namely ZnO NRs and GNPs, showed no harmful effects in C. elegans [15, 16], and that ZnO NRs resulted to be not cytotoxic in different human cell lines . Although it has been reported that ZnO nanoparticles induced toxic effect in different model systems including also C. elegans [47–49], the main components of their nanotoxicity resulted to be the production of reactive oxygen species as well as the consequent release of zinc ion in suspensions . Remarkably, in our previous studies we demonstrated that Zn ion dissolution was negligible in both ZnO NR and ZNG suspensions [19, 23], suggesting that the low concentration of bioavailable Zn2+ may account for lack of harmful effects in C. elegans exerted by of ZNGs.
Conceived and designed the experiments: DU, MSS. Wrote the paper: EZ, DU, EB. Critical revision of manuscript: PM. Performed the nanomaterial fabrication: CRC, AB. Did bacterial and nematode experiments/treatments: EB. Performed SEM analysis: GDB. Performed FTIR experiments: MGS. Analyzed and supervised FTIR data: ML. All authors read and approved the final manuscript.
We thank Dr. Domenico Cavallini for helpful support.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Consent for publication
Ethics approval and consent to participate
The authors wish to thank Italian MIUR for funding by the PON R&C 2007–2013 program with the Project PON03PE_00214_1 Nanotechnologies and Nanomaterials for Cultural Heritages (TECLA, B62F14000560005).
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