Shape-Versatile Fixed Cellular Materials for Multiple Target Immunomodulation.

Therapeutic cells are usually administered as living agents, despite the risks of undesired cell migration and acquisition of unpredictable phenotypes. Additionally, most cell-based therapies rely on the administration of single cells, often associated with rapid in vivo clearance. 3D cellular materials may be useful to prolong the effect of cellular therapies and offer the possibility of creating structural volumetric constructs. Here, the manufacturing of shape-versatile fixed cell-based materials with immunomodulatory properties is reported. Living cell aggregates with different shapes (spheres and centimeter-long fibers) are fixed using a method compatible with maintenance of structural integrity, robustness, and flexibility of 3D constructs. The biological properties of living cells can be modulated before fixation, rendering an in vitro anti-inflammatory effect toward human macrophages, in line with a decreased activation of the nuclear factor kappa B (NF-κB) pathway that preponderantly correlated with the surface area of the materials. These findings are further corroborated in vivo in mouse skin wounds. Contact with fixed materials also reduces the proliferation of activated primary T lymphocytes, while promoting regulatory populations. The fixation of cellular constructs is proposed as a versatile phenotypic stabilization method that can be easily implemented to prepare immunomodulatory materials with therapeutic potential.

Engineering optical tools for remotely controlled brain stimulation and regeneration.

Neurological disorders are one of the world's leading medical and societal challenges due to the lack of efficacy of the first line treatment. Although pharmacological and non-pharmacological interventions have been employed with the aim of regulating neuronal activity and survival, they have failed to avoid symptom relapse and disease progression in the vast majority of patients. In the last 5 years, advanced drug delivery systems delivering bioactive molecules and neuromodulation strategies have been developed to promote tissue regeneration and remodel neuronal circuitry. However, both approaches still have limited spatial and temporal precision over the desired target regions. While external stimuli such as electromagnetic fields and ultrasound have been employed in the clinic for non-invasive neuromodulation, they do not have the capability of offering single-cell spatial resolution as light stimulation. Herein, we review the latest progress in this area of study and discuss the prospects of using light-responsive nanomaterials to achieve on-demand delivery of drugs and neuromodulation, with the aim of achieving brain stimulation and regeneration.

A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.

Messenger RNA (mRNA)-based therapies offer enhanced control over the production of therapeutic proteins for many diseases. Their clinical implementation warrants formulations capable of delivering them safely and effectively to target sites. Owing to their chemical versatility, polymeric nanoparticles can be designed by combinatorial synthesis of different ionizable, cationic, and aromatic moieties to modulate cell targeting, using inexpensive formulation steps. Herein, 152 formulations are evaluated by high-throughput screening using a reporter fibroblast model sensitive to functional delivery of mRNA encoding Cre recombinase. Using in vitro and in vivo models, a polymeric nanoformulation based on the combination of 3 specific monomers is identified to transfect fibroblasts much more effectively than other cell types populating the skin, with superior performance than lipid-based transfection agents in the delivery of Cas9 mRNA and guide RNA. This tropism can be explained by receptor-mediated endocytosis, involving CD26 and FAP, which are overexpressed in profibrotic fibroblasts. Structure-activity analysis reveals that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. These results highlight the use of high-throughput screening to rapidly identify chemical features towards the design of highly efficient mRNA delivery systems targeting fibrotic diseases.

Engineering graphene-based electrodes for optical neural stimulation.

Graphene-based materials (GBMs) have been investigated in recent years with the aim of developing flexible interfaces to address a range of neurological disorders, where electrical stimulation may improve brain function and tissue regeneration. The recent discovery that GBM electrodes can generate an electrical response upon light exposure has inspired the development of non-genetic approaches capable of selectively modulating brain cells without genetic manipulation (i.e., optogenetics). Here, we propose the conjugation of graphene with upconversion nanoparticles (UCNPs), which enable wireless transcranial activation using tissue-penetrating near-infrared (NIR) radiation. Following a design of experiments approach, we first investigated the influence of different host matrices and dopants commonly used to synthesize UCNPs in the electrical response of graphene. Two UCNP formulations achieving optimal enhancement of electrical conductivity upon NIR activation at λ = 780 or 980 nm were identified. These formulations were then covalently attached to graphene nanoplatelets following selective hydroxyl derivatization. The resulting nanocomposites were evaluated in vitro using SH-SY5Y human neuroblastoma cells. NIR activation at λ = 980 nm promoted cell proliferation and downregulated neuronal and glial differentiation markers, suggesting the potential application of GBMs in minimally invasive stimulation of cells for tissue regeneration.

JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport.

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3's interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3's RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bidirectional organelle transport.

Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications.

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

The impact of graphene oxide sheet lateral dimensions on their pharmacokinetic and tissue distribution profiles in mice.

Although the use of graphene and 2-dimensional (2D) materials in biomedicine has been explored for over a decade now, there are still significant knowledge gaps regarding the fate of these materials upon interaction with living systems. Here, the pharmacokinetic profile of graphene oxide (GO) sheets of three different lateral dimensions was studied. The GO materials were functionalized with a PEGylated DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), a radiometal chelating agent for radioisotope attachment for single photon emission computed tomography (SPECT/CT) imaging. Our results revealed that GO materials with three distinct size distributions, large (l-GO-DOTA), small (s-GO-DOTA) and ultra-small (us-GO-DOTA), were sequestered by the spleen and liver. Significant accumulation of the large material (l-GO-DOTA) in the lungs was also observed, unlike the other two materials. Interestingly, there was extensive urinary excretion of all three GO nanomaterials indicating that urinary excretion of these structures was not affected by lateral dimensions. Comparing with previous studies, we believe that the thickness of layered nanomaterials is the predominant factor that governs their excretion rather than lateral size. However, the rate of urinary excretion was affected by lateral size, with large GO excreting at slower rates. This study provides better understanding of 2D materials in vivo behaviour with varying structural features.

A high-throughput screening platform to identify nanocarriers for efficient delivery of RNA-based therapies.

RNA-based therapies are highly selective and powerful regulators of biological functions. Non-viral vectors such as nanoparticles (NPs) are very promising formulations for the delivery of RNA-based therapies but their cell targeting, cell internalization and endolysomal escape capacity is rather limited. Here, we present a methodology that combines high-throughput synthesis of light-triggerable NPs and a high-content imaging screening to identify NPs capable of efficiently delivering different type of RNAs. The NPs were generated using polymers synthesized by Michael type addition reactions and they were designed to: (i) efficiently complex coding (mRNAs) and non-coding (miRNAs and/or lncRNAs) RNA molecules, (ii) allow rapid cell uptake and cytoplasmic release of RNA molecules and (iii) target different cell types based on their composition. Furthermore, light-responsive domains were attached to the polymers by distinctive methods to provide diverse disassembly strategies. The most efficient formulations were identified using cell-based assays and high-content imaging analysis. This strategy allows precise delivery of RNA-based therapies and provides an effective design approach to address critical issues in non-viral gene delivery.

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration.

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of ß-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aß oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

Graphene oxide nanosheets modulate spinal glutamatergic transmission and modify locomotor behaviour in an in vivo zebrafish model.

Graphene oxide (GO), an oxidised form of graphene, is widely used for biomedical applications, due to its dispersibility in water and simple surface chemistry tunability. In particular, small (less than 500 nm in lateral dimension) and thin (1-3 carbon monolayers) graphene oxide nanosheets (s-GO) have been shown to selectively inhibit glutamatergic transmission in neuronal cultures in vitro and in brain explants obtained from animals injected with the nanomaterial. This raises the exciting prospect that s-GO can be developed as a platform for novel nervous system therapeutics. It has not yet been investigated whether the interference of the nanomaterial with neurotransmission may have a downstream outcome in modulation of behaviour depending specifically on the activation of those synapses. To address this problem we use early stage zebrafish as an in vivo model to study the impact of s-GO on nervous system function. Microinjection of s-GO into the embryonic zebrafish spinal cord selectively reduces the excitatory synaptic transmission of the spinal network, monitored in vivo through patch clamp recordings, without affecting spinal cell survival. This effect is accompanied by a perturbation in the swimming activity of larvae, which is the locomotor behaviour generated by the neuronal network of the spinal cord. Such results indicate that the impact of s-GO on glutamate based neuronal transmission is preserved in vivo and can induce changes in animal behaviour. These findings pave the way for use of s-GO as a modulator of nervous system function.

Size-Dependent Pulmonary Impact of Thin Graphene Oxide Sheets in Mice: Toward Safe-by-Design.

Safety assessment of graphene-based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here, the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice are compared. Micrometer-sized GO induces stronger pulmonary inflammation than nanometer-sized GO, despite reduced translocation to the lungs. Genome-wide RNA sequencing also reveals distinct size-dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggers the formation of granulomas that persists for up to 90 days, no pulmonary fibrosis is observed. These latter results can be partly explained by Raman imaging, which evidences the progressive biotransformation of GO into less graphitic structures. The findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometer-sized GO should be avoided in the production plant or applications, where aerosolized dispersions are likely to occur. These results are important toward the implementation of a safer-by-design approach for GBM products and applications, for the benefit of workers and end-users.

Next-Generation Sequencing Reveals Differential Responses to Acute versus Long-Term Exposures to Graphene Oxide in Human Lung Cells.

Numerous studies have addressed the biological impact of graphene-based materials including graphene oxide (GO), yet few have focused on long-term effects. Here, RNA sequencing is utilized to unearth responses of human lung cells to GO. To this end, the BEAS-2B cell line derived from normal human bronchial epithelium is subjected to repeated, low-dose exposures of GO (1 or 5 µg mL-1 ) for 28 days or to the equivalent, cumulative amount of GO for 48 h. Then, samples are analyzed by using the NovaSeq 6000 sequencing system followed by pathway analysis and gene ontology enrichment analysis of the differentially expressed genes. Significant differences are seen between the low-dose, long-term exposures and the high-dose, short-term exposures. Hence, exposure to GO for 48 h results in mitochondrial dysfunction. In contrast, exposure to GO for 28 days is characterized by engagement of apoptosis pathways with downregulation of genes belonging to the inhibitor of apoptosis protein (IAP) family. Validation experiments confirm that long-term exposure to GO affects the apoptosis threshold in lung cells, accompanied by a loss of IAPs. These studies reveal the sensitivity of RNA-sequencing approaches and show that acute exposure to GO is not a good predictor of the long-term effects of GO.

Graphene oxide as a 2D platform for complexation and intracellular delivery of siRNA.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches to the bedside. Graphene oxide (GO) could help bypass such bottlenecks, thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10 : 1, 20 : 1 and 50 : 1 GO : siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO : siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. The time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effects (i.e. gene silencing) achieved by GO : siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow the effective release of the nucleic acid and achieve efficient gene silencing.

Antifungal and anti-inflammatory potential of eschweilenol C-rich fraction derived from Terminalia fagifolia Mart.

ETHNOPHARMACOLOGICAL RELEVANCE: Folk knowledge transmitted between generations allows traditional populations to maintain the use of medicinal plants for the treatment of several diseases. In this context, the species Terminalia fagifolia Mart., native to Brazil, is used for the treatment of chronic and infectious diseases. Plants rich in secondary metabolites, such as this species and their derivatives, may represent therapeutic alternatives for the treatment of diseases that reduce the quality of life of people. AIM OF THE STUDY: The aim of this study was to evaluate the antifungal and anti-inflammatory potential of aqueous fraction from ethanolic extract of T. fagifolia, with in silico study of the major compound of the fraction. MATERIAL AND METHODS: The phytochemical study of the aqueous fraction was performed by HPLC, LC/MS and NMR. The antifungal activity was evaluated against yeasts, by determination of the minimum inhibitory concentration and minimum fungicidal concentration. The effect on Candida albicans was analyzed by AFM. The antibiofilm potential against biofilms of C. albicans was also tested. The anti-inflammatory potential of the aqueous fraction was evaluated in vivo by the carrageenan-induced paw edema and peritonitis. A microglial model of LPS-induced neuroinflammation was also studied. Further insights on the activation mechanism were studied using quantum chemistry computer simulations. Toxicity was evaluated in the Galleria mellonella and human erythrocytes models. RESULTS: Eschweilenol C was identified as the major constituent of the aqueous fraction of the ethanolic extract of T. fagifolia. The aqueous fraction was active against all Candida strains used (sensitive and resistant to Fluconazole) with MICs ranging from 1000 to 0.4 µg/mL. By AFM it was possible to observe morphological alterations in treated Candida cells. The fraction significantly (p < 0.05) inhibited paw edema and decreased levels of malondialdehyde induced by carrageenan. In a microglial cell model, aqueous fraction demonstrated the ability to inhibit NF-κB after induction with lipopolysaccharide. The theoretical studies showed structural similarity between eschweilenol C and indomethacin and an excellent antioxidant potential. The aqueous fraction did not present toxicity in the studied models. CONCLUSION: The results indicate that the aqueous fraction of T. fagifolia has potential for biomedical applications with low toxicity. This finding can be attributed to the predominance of eschweilenol C in the aqueous fraction.

3D Organotypic Spinal Cultures: Exploring Neuron and Neuroglia Responses Upon Prolonged Exposure to Graphene Oxide.

Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model in vitro tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 µg/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death.

The Interaction between the Drosophila EAG Potassium Channel and the Protein Kinase CaMKII Involves an Extensive Interface at the Active Site of the Kinase.

The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. We have characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase.

Immunological impact of graphene oxide sheets in the abdominal cavity is governed by surface reactivity.

Graphene oxide (GO) is an oxidised form of graphene that has attracted commercial interest in multiple applications, including inks, printed electronics and spray coatings, which all raise health concerns due to potential creation of inhalable aerosols. Although a number of studies have discussed the toxicity of GO sheets, the in vivo impact of their lateral dimensions is still not clear. Here, we compared the effects of large GO sheets (l-GO, 1-20 µm) with those of small GO sheets (s-GO, < 1 µm) in terms of mesothelial damage and peritoneal inflammation, after intraperitoneal (i.p.) injection in mice. To benchmark the outcomes, long and rigid multi-walled carbon nanotubes (MWCNTs) that were shown to be associated with asbestos-like pathogenicity on the mesothelium were also tested. Our aim was to assess whether lateral dimensions can be a predictor of inflammogenicity for GO sheets in a similar fashion as length is for MWCNTs. While long MWCNTs dispersed in 0.5% BSA induced a granulomatous response on the diaphragmatic mesothelium and immune cell recruitment to the peritoneal cavity, GO sheets dispersed under similar conditions did not cause any response, regardless of their lateral dimensions. We further interrogated whether tuning the surface reactivity of GO by testing different dispersions (5% dextrose instead of 0.5% BSA) may change the biological outcome. Although the change of dispersion did not alter the impact of GO on the mesothelium (i.e. no granuloma), we observed that, when dispersed in protein-free 5% dextrose solution, s-GO elicited a greater recruitment of monocytic cells to the peritoneal cavity than l-GO, or when dispersed in protein-containing solution. Such recruitment coincided with the greater ability of s-GO to interact in vivo with peritoneal macrophages and was associated with a greater surface reactivity in comparison to l-GO. In conclusion, large dimension was not a determining factor of the immunological impact of GO sheets after i.p. administration. For an equal dose, GO sheets with lateral dimensions similar to the length of long MWCNTs were less pathogenic than the MWCNTs. On the other hand, surface reactivity and the ability of some smaller GO sheets to interact more readily with immune cells seem to be key parameters that can be tuned to improve the safety profile of GO. In particular, the choice of dispersion modality, which affected these two parameters, was found to be of crucial importance in the assessment of GO impact in this model. Overall, these findings are essential for a better understanding of the parameters governing GO toxicity and inflammation, and the rational design of safe GO-based formulations for various applications, including biomedicine.

Redox tuning of Ca2+ signaling in microglia drives glutamate release during hypoxia.

Hypoxia causes oxidative stress and excitotoxicity, culminating in neuronal damage during brain ischemia. Hypoxia also activates microglia, the myeloid resident cells of the brain parenchyma. Upon activation, microglia release high amounts of the neurotransmitter glutamate, contributing for neuronal excitotoxicity during brain insults. Here, we reveal a signaling pathway controlling glutamate release from human microglia during hypoxia. We show that hypoxia-mediated redox imbalance promotes the activation of endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP3) receptors leading to Ca2+ mobilization into the cytosol. Increasing cytosolic Ca2+ signaling in microglia activates the non-receptor protein tyrosine kinase Src at the plasma membrane. Src activation enhances the permeability of microglial gap junctions promoting the release of glutamate during hypoxia. Preventing the hypoxia-triggered redox imbalance, using the dietary antioxidants neochlorogenic acid or vitamin C, inhibits InsP3-dependent Ca2+ signaling and abrogates the release of glutamate. Overall, modulating microglial Ca2+ signaling in response to changes in the redox microenvironment might be critical for controlling glutamate excitotoxicity during hypoxia.

Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic.

Neutrophils were previously shown to digest oxidized carbon nanotubes through a myeloperoxidase (MPO)-dependent mechanism, and graphene oxide (GO) was found to undergo degradation when incubated with purified MPO, but there are no studies to date showing degradation of GO by neutrophils. Here we produced endotoxin-free GO by a modified Hummers' method and asked whether primary human neutrophils stimulated to produce neutrophil extracellular traps or activated to undergo degranulation are capable of digesting GO. Biodegradation was assessed using a range of techniques including Raman spectroscopy, transmission electron microscopy, atomic force microscopy, and mass spectrometry. GO sheets of differing lateral dimensions were effectively degraded by neutrophils. As the degradation products could have toxicological implications, we also evaluated the impact of degraded GO on the bronchial epithelial cell line BEAS-2B. MPO-degraded GO was found to be non-cytotoxic and did not elicit any DNA damage. Taken together, these studies have shown that neutrophils can digest GO and that the biodegraded GO is non-toxic for human lung cells.