In vitro immunotoxicological assessment of a potent microbicidal nanocomposite based on graphene oxide and silver nanoparticles.

Graphene oxide (GO) and silver nanoparticles (AgNPs) can be formed into a hybrid nanomaterial, known as GOAg nanocomposite, which presents high antibacterial activity. The successful translation of this nanomaterial into medical use depends on critical information about its toxicological profile. In keeping with a Safe-by-design approach, we evaluated the immunotoxicity of GOAg using J774 and primary murine macrophages. The interaction between GOAg and macrophages was investigated with a scanning electron microscope (SEM). High-throughput technologies were employed to evaluate cell viability, apoptosis/necrosis, mitochondrial depolarization and lipid peroxidation. The inflammogenicity of nanomaterials was predicted after quantification of the cytokines IL-1ß, TNF-α and IL-10 before and after stimulation with interferon-γ (IFN-γ). The ratio between CD80 and CD206 macrophage populations were also estimated. In addition, the production of nitric oxide (NO) was investigated. SEM surveys revealed the potential of GOAg to induce frustrated phagocytosis. GOAg induced a dose-dependent mitochondrial depolarization, apoptosis and lipid peroxidation to J774 macrophages. GOAg toxicity was not modified in an inflammatory microenvironment, but its toxicity was within the range of concentrations used in bacterial inactivation. GOAg did not induce primary macrophages to significantly produce inflammatory cytokines, and previous macrophage stimulation did not enhance GOAg inflammogenicity. Additionally, the pristine nanomaterials and GOAg do not shift macrophages polarization towards M1. Sublethal concentrations of GOAg did not impair macrophages NO production. Finally, we suggest options for improvement of GOAg nanocomposite in ways that may help minimize its possible adverse outcomes to human health.

Comparative in vitro toxicity of a graphene oxide-silver nanocomposite and the pristine counterparts toward macrophages.

BACKGROUND: Graphene oxide (GO) is a highly oxidized graphene form with oxygen functional groups on its surface. GO is an excellent platform to support and stabilize silver nanoparticles (AgNP), which gives rise to the graphene oxide-silver nanoparticle (GOAg) nanocomposite. Understanding how this nanocomposite interacts with cells is a toxicological challenge of great importance for future biomedical applications, and macrophage cells can provide information concerning the biocompatibility of these nanomaterials. The cytotoxicity of the GOAg nanocomposite, pristine GO, and pristine AgNP was compared toward two representative murine macrophages: a tumoral lineage (J774) and peritoneal macrophages collected from Balb/c mouse. The production of reactive oxygen species (ROS) by J774 macrophages was also monitored. We investigated the internalization of nanomaterials by transmission electron microscopy (TEM). The quantification of internalized silver was carried out by inductively coupled plasma mass spectrometry (ICP-MS). Nanomaterial stability in the cell media was investigated overtime by visual observation, inductively coupled plasma optical emission spectrometry (ICP OES), and dynamic light scattering (DLS). RESULTS: The GOAg nanocomposite was more toxic than pristine GO and pristine AgNP for both macrophages, and it significantly induced more ROS production compared to pristine AgNP. TEM analysis showed that GOAg was internalized by tumoral J774 macrophages. However, macrophages internalized approximately 60 % less GOAg than did pristine AgNP. The images also showed the degradation of nanocomposite inside cells. CONCLUSIONS: Although the GOAg nanocomposite was less internalized by the macrophage cells, it was more toxic than the pristine counterparts and induced remarkable oxidative stress. Our findings strongly reveal a synergistic toxicity effect of the GOAg nanocomposite. The toxicity and fate of nanocomposites in cells are some of the major concerns in the development of novel biocompatible materials and must be carefully evaluated.

Graphene oxide-silver nanocomposite as a promising biocidal agent against methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has been responsible for serious hospital infections worldwide. Nanomaterials are an alternative to conventional antibiotic compounds, because bacteria are unlikely to develop microbial resistance against nanomaterials. In the past decade, graphene oxide (GO) has emerged as a material that is often used to support and stabilize silver nanoparticles (AgNPs) for the preparation of novel antibacterial nanocomposites. In this work, we report the synthesis of the graphene-oxide silver nanocomposite (GO-Ag) and its antibacterial activity against relevant microorganisms in medicine. MATERIALS AND METHODS: GO-Ag nanocomposite was synthesized through the reduction of silver ions (Ag(+)) by sodium citrate in an aqueous GO dispersion, and was extensively characterized using ultraviolet-visible absorption spectroscopy, X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, and transmission electron microscopy. The antibacterial activity was evaluated by microdilution assays and time-kill experiments. The morphology of bacterial cells treated with GO-Ag was investigated via transmission electron microscopy. RESULTS: AgNPs were well distributed throughout GO sheets, with an average size of 9.4±2.8 nm. The GO-Ag nanocomposite exhibited an excellent antibacterial activity against methicillin-resistant S. aureus, Acinetobacter baumannii, Enterococcus faecalis, and Escherichia coli. All (100%) MRSA cells were inactivated after 4 hours of exposure to GO-Ag sheets. In addition, no toxicity was found for either pristine GO or bare AgNPs within the tested concentration range. Transmission electronic microscopy images offered insights into how GO-Ag nanosheets interacted with bacterial cells. CONCLUSION: Our results indicate that the GO-Ag nanocomposite is a promising antibacterial agent against common nosocomial bacteria, particularly antibiotic-resistant MRSA. Morphological injuries on MRSA cells revealed a likely loss of viability as a result of the direct contact between bacteria and the GO-Ag sheets.

Fabrication of transparent and ultraviolet shielding composite films based on graphene oxide and cellulose acetate.

Graphene oxide (GO) has been considered a promising filler material for building polymeric nanocomposites because of its excellent dispersibility and high surface area. In this work, we present the fabrication and characterization of transparent and ultraviolet (UV) shielding composite films based on GO and cellulose acetate (CA). GO sheets were found to be well-dispersed throughout the CA matrix, providing smooth and homogeneous composite films. Moreover, the GO sheets were completely embedded within the CA matrix and no presence of this nanomaterial was found at the surface. Nevertheless, CAGO composite films offered an improved high energy light-shielding capacity when compared to pristine CA films. Particularly for UVC irradiation, the CAGO film containing 0.50wt% GO displayed a UV-shielding capacity of 57%, combined with 79% optical transparency under visible light. These CAGO composite films can be potentially applied as transparent UV-protective coatings for packing biomedical, pharmaceutical, and food products.

Anti-adhesion and antibacterial activity of silver nanoparticles supported on graphene oxide sheets.

This work reports on the preparation, characterization and antibacterial activity of a nanocomposite formed from graphene oxide (GO) sheets decorated with silver nanoparticles (GO-Ag). The GO-Ag nanocomposite was prepared in the presence of AgNO3 and sodium citrate. The physicochemical characterization was performed by UV-vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), Raman spectroscopy and transmission electron microscopy (TEM). The average size of the silver nanoparticles anchored on the GO surface was 7.5 nm. Oxidation debris fragments (a byproduct adsorbed on the GO surface) were found to be crucial for the nucleation and growth of the silver nanoparticles. The antibacterial activity of the GO and GO-Ag nanocomposite against the microorganism Pseudomonas aeruginosa was investigated using the standard counting plate methodology. The GO dispersion showed no antibacterial activity against P. aeruginosa over the concentration range investigated. On the other hand, the GO-Ag nanocomposite displayed high biocidal activity with a minimum inhibitory concentration ranging from 2.5 to 5.0 µg/mL. The anti-biofilm activity toward P. aeruginosa adhered on stainless steel surfaces was also investigated. The results showed a 100% inhibition rate of the adhered cells after exposure to the GO-Ag nanocomposite for one hour. To the best of our knowledge, this work provides the first direct evidence that GO-Ag nanocomposites can inhibit the growth of microbial adhered cells, thus preventing the process of biofilm formation. These promising results support the idea that GO-Ag nanocomposites may be applied as antibacterial coatings material to prevent the development of biofilms in food packaging and medical devices.