Evaluation of the antibacterial power and biocompatibility of zinc oxide nanorods decorated graphene nanoplatelets: new perspectives for antibiodeteriorative approaches

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Field emission scanning electron microscopy (FE-SEM) images of a pristine graphene nanoplatelets (GNPs) and b ZnO-NRs-decorated GNPs (ZNG) (scale bar 1 µm)

Fig. 2

Effect of ZNGs on Pseudomonas aeruginosa viability. Bacteria were treated or not (UT) with different concentrations of ZNGs for a 2 h or b 24 h and bacterial survival was evaluated by CFU counting analysis. A one-way ANOVA analysis with the Bonferroni post-test was used to assess statistical significance (ns not significant; *p < 0.05, **p < 0.01 and ***p < 0.001 with respect to UT)

Fig. 3

Staphylococcus aureus survival after exposure with ZNGs for a 2 h and b 24 h in comparison with untreated cells (UT). Statistical analysis was performed by one-way ANOVA method coupled with the Bonferroni post-test (ns not significant; **p < 0.01 and ***p < 0.001 with respect to UT)

Fig. 4

FE-SEM micrographs of bacterial cells after exposure to zinc oxide nanorods-decorated GNPs. S. aureus cells incubated with a H₂O_dd or b ZNGs suspension (50 µg/mL). P. aeruginosa c untreated cells are shown in comparison to d the same bacteria exposed to ZNGs (50 µg/mL) (scale bar 400 nm)

Fig. 5

Biofilm formation was analyzed by Crystal violet binding assay in a P. aeruginosa or b S. aureus cells. The production of bacterial biomass was evaluated after exposure with the indicated concentrations of ZNGs and expressed as biofilm formation relative to untreated cells (UT). Asterisks indicate statistical significance (ns not significant; *p < 0.05 and ***p < 0.001 with respect to UT)

Fig. 6

Effect of ZNGs treatment on P. aeruginosa cells from FTIR spectroscopy. a Dried samples: comparison between the untreated (UT) sample FTIR spectrum (black line) and the treated sample one (red line) in the 1800–1300 cm⁻¹ spectral range. The difference between the two spectra is also reported (green line). Data relative to the 1300–900 cm⁻¹ are shown in the inset. b Liquid samples (D₂O solution): comparison between the untreated (UT) sample FTIR spectrum (black line) and the treated sample one (red line) in the 1800–1300 cm⁻¹ spectral range. For the purpose of comparing the shape of different spectra, data were scaled with respect to the low wavenumbers side of the Amide II band (~1543 cm⁻¹) in the case of dried samples or the Amide IIʹ band (~1450 cm⁻¹) in the case of deuterated liquid samples

Fig. 7

Survival of Staphyloccocus aureus cells on the indicated materials drop casted with ZNGs. Bacterial viability after a 4h-exposure is expressed as percentage of CFUs relative to those obtained at the initial time of contamination (t0). Data are presented as mean ± SD and asterisks indicate statistical significance (**p < 0.01)

Fig. 8

Effect of ZNGs on nematode lifespan, body size and fertility rate. Kaplan–Mèier survival plots of worms treated or not with ZNGs starting from a adult or b larval stages; n = 60 for single experiments. The abbreviation ‘ns’ indicates that results are not significant in comparison with control (log-rank test). c Average embryos production per worm of animals exposed to ZNGs with respect to untreated nematodes. Bars represent the mean of three independent experiments. d Effect of ZnO NR-decorated GNPs on C. elegans larval development. Worms were grown in the presence of E. coli OP50 supplemented or not with ZNGs and their length was measured from head to tail at the indicated time points. Statistical analysis of c and d was evaluated by one-way ANOVA method with the Bonferroni post-test (ns not significant)

Fig. 9

Analysis of neuromuscular functionality of C. elegans exposed to ZNGs. a Evaluation of pumping rates in nematodes treated or not with ZNGs, by measuring the number of pharynx contractions in 30 s. b analysis of locomotion behavior following ZNGs treatment by counting nematodes bending in the time interval of 1 min. Statistical analysis was evaluated by one-way ANOVA method with the Bonferroni post-test (ns not significant)