Graphene oxide activates canonical TGFß signalling in a human chondrocyte cell line via increased plasma membrane tension.

Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway - TGFß and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFß response genes, while the use of a TGFß signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cell-material interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFß in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.

First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

Delivery of graphene oxide nanosheets modulates glutamate release and normalizes amygdala synaptic plasticity to improve anxiety-related behavior.

Graphene oxide nanosheets (GO) were reported to alter neurobiological processes involving cell membrane dynamics. GO ability to reversibly downregulate specifically glutamatergic synapses underpins their potential in future neurotherapeutic developments. Aberrant glutamate plasticity contributes to stress-related psychopathology and drugs which target dysregulated glutamate represent promising treatments. We find that in a rat model of post-traumatic stress disorder (PTSD), a single injection of GO to the lateral amygdala following the stressful event induced PTSD-related behavior remission and reduced dendritic spine densities. We explored from a mechanistic perspective how GO could impair glutamate synaptic plasticity. By simultaneous patch clamp pair recordings of unitary synaptic currents, live-imaging of presynaptic vesicle release and confocal microscopy, we report that GO nanosheets altered the probability of release enhancing the extinction of synaptic plasticity in the amygdala. These findings show that the modulation of presynaptic glutamate release might represent an unexplored target for (nano)pharmacological interventions of stress-related disorders.

Graphene Oxide Nanosheets Hamper Glutamate Mediated Excitotoxicity and Protect Neuronal Survival In An In vitro Stroke Model.

Small graphene oxide (s-GO) nanosheets reversibly downregulate central nervous system (CNS) excitatory synapses, with potential developments as future therapeutic tools to treat neuro-disorders characterized by altered glutamatergic transmission. Excitotoxicity, namely cell death triggered by exceeding ambient glutamate fueling over-activation of excitatory synapses, is a pathogenic mechanism shared by several neural diseases, from ischemic stroke to neurodegenerative disorders. In this work, CNS cultures were exposed to oxygen-glucose deprivation (OGD) to mimic ischemic stroke in vitro, and it is show that the delivery of s-GO following OGD, during the endogenous build-up of secondary damage and excitotoxicity, improved neuronal survival. In a different paradigm, excitotoxicity cell damage was reproduced through exogenous glutamate application, and s-GO co-treatment protected neuronal integrity, potentially by directly downregulating the synaptic over-activation brought about by exogenous glutamate. This proof-of-concept study suggests that s-GO may find novel applications in therapeutic developments for treating excitotoxicity-driven neural cell death.

Lung Persistence, Biodegradation, and Elimination of Graphene-Based Materials are Predominantly Size-Dependent and Mediated by Alveolar Phagocytes.

Graphene-based materials (GBMs) have promising applications in various sectors, including pulmonary nanomedicine. Nevertheless, the influence of GBM physicochemical characteristics on their fate and impact in lung has not been thoroughly addressed. To fill this gap, the biological response, distribution, and bio-persistence of four different GBMs in mouse lungs up to 28 days after single oropharyngeal aspiration are investigated. None of the GBMs, varying in size (large versus small) and carbon to oxygen ratio as well as thickness (few-layers graphene (FLG) versus thin graphene oxide (GO)), induce a strong pulmonary immune response. However, recruited neutrophils internalize nanosheets better and degrade GBMs faster than macrophages, revealing their crucial role in the elimination of small GBMs. In contrast, large GO sheets induce more damages due to a hindered degradation and long-term persistence in macrophages. Overall, small dimensions appear to be a leading feature in the design of safe GBM pulmonary nanovectors due to an enhanced degradation in phagocytes and a faster clearance from the lungs for small GBMs. Thickness also plays an important role, since decreased material loading in alveolar phagocytes and faster elimination are found for FLGs compared to thinner GOs. These results are important for designing safer-by-design GBMs for biomedical application.

Defect-free graphene enhances enzyme delivery to fibroblasts derived from patients with lysosomal storage disorders.

Enzyme replacement therapy shows remarkable clinical improvement in treating lysosomal storage disorders. However, this therapeutic approach is hampered by limitations in the delivery of the enzyme to cells and tissues. Therefore, there is an urgent, unmet clinical need to develop new strategies to enhance the enzyme delivery to diseased cells. Graphene-based materials, due to their dimensionality and favourable pattern of interaction with cells, represent a promising platform for the loading and delivery of therapeutic cargo. Herein, the potential use of graphene-based materials, including defect-free graphene with positive or negative surface charge and graphene oxide with different lateral dimensions, was investigated for the delivery of lysosomal enzymes in fibroblasts derived from patients with Mucopolysaccharidosis VI and Pompe disease. We report excellent biocompatibility of all graphene-based materials up to a concentration of 100 µg mL-1 in the cell lines studied. In addition, a noticeable difference in the uptake profile of the materials was observed. Neither type of graphene oxide was taken up by the cells to a significant extent. In contrast, the two types of graphene were efficiently taken up, localizing in the lysosomes. Furthermore, we demonstrate that cationic graphene flakes can be used as carriers for arylsulfatase B enzyme, for the delivery of the lacking enzyme to the lysosomes of Mucopolysaccharidosis VI fibroblasts. Arylsulfatase B complexed with cationic graphene flakes not only retained the enzymatic activity, but also exerted biological effects almost twice as high as arylsulfatase B alone in the clearance of the substrate in Mucopolysaccharidosis VI fibroblasts. This study lays the groundwork for the potential use of graphene-based materials as carriers for enzyme replacement therapy in lysosomal storage disorders.

Graphene Oxide Nanosheets Reduce Astrocyte Reactivity to Inflammation and Ameliorate Experimental Autoimmune Encephalomyelitis.

In neuroinflammation, astrocytes play multifaceted roles that regulate the neuronal environment. Astrocytes sense and respond to pro-inflammatory cytokines (CKs) and, by a repertoire of intracellular Ca2+ signaling, contribute to disease progression. Therapeutic approaches wish to reduce the overactivation in Ca2+ signaling in inflammatory-reactive astrocytes to restore dysregulated cellular changes. Cell-targeting therapeutics might take advantage by the use of nanomaterial-multifunctional platforms such as graphene oxide (GO). GO biomedical applications in the nervous system involve therapeutic delivery and sensing, and GO flakes were shown to enable interfacing of neuronal and glial membrane dynamics. We exploit organotypic spinal cord cultures and optical imaging to explore Ca2+ changes in astrocytes, and we report, when spinal tissue is exposed to CKs, neuroinflammatory-associated modulation of resident glia. We show the efficacy of GO to revert these dynamic changes in astrocytic reactivity to CKs, and we translate this potential in an animal model of immune-mediated neuroinflammatory disease.

Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor.

The gut microbiome produces metabolites that interact with the aryl hydrocarbon receptor (AhR), a key regulator of immune homoeostasis in the gut1,2. Here we show that oral exposure to graphene oxide (GO) modulates the composition of the gut microbiome in adult zebrafish, with significant differences in wild-type versus ahr2-deficient animals. Furthermore, GO was found to elicit AhR-dependent induction of cyp1a and homing of lck+ cells to the gut in germ-free zebrafish larvae when combined with the short-chain fatty acid butyrate. To obtain further insights into the immune responses to GO, we used single-cell RNA sequencing to profile cells from whole germ-free embryos as well as cells enriched for lck. These studies provided evidence for the existence of innate lymphoid cell (ILC)-like cells3 in germ-free zebrafish. Moreover, GO endowed with a 'corona' of microbial butyrate triggered the induction of ILC2-like cells with attributes of regulatory cells. Taken together, this study shows that a nanomaterial can influence the crosstalk between the microbiome and immune system in an AhR-dependent manner.

Graphene Oxide Nanoscale Platform Enhances the Anti-Cancer Properties of Bortezomib in Glioblastoma Models.

Graphene-based 2D nanomaterials possess unique physicochemical characteristics which can be utilized in various biomedical applications, including the transport and presentation of chemotherapeutic agents. In glioblastoma multiforme (GBM), intratumorally administered thin graphene oxide (GO) nanosheets demonstrate a widespread distribution throughout the tumor volume without impact on tumor growth, nor spread into normal brain tissue. Such intratumoral localization and distribution can offer multiple opportunities for treatment and modulation of the GBM microenvironment. Here, the kinetics of GO nanosheet distribution in orthotopic GBM mouse models is described and a novel nano-chemotherapeutic approach utilizing thin GO sheets as platforms to non-covalently complex a proteasome inhibitor, bortezomib (BTZ), is rationally designed. Through the characterization of the GO:BTZ complexes, a high loading capacity of the small molecule on the GO surface with sustained BTZ biological activity in vitro is demonstrated. In vivo, a single low-volume intratumoral administration of GO:BTZ complex shows an enhanced cytotoxic effect compared to free drug in two orthotopic GBM mouse models. This study provides evidence of the potential that thin and small GO sheets hold as flat nanoscale platforms for GBM treatment by increasing the bioavailable drug concentration locally, leading to an enhanced therapeutic effect.

Graphene oxide modulates dendritic cell ability to promote T cell activation and cytokine production.

An important aspect of immunotherapy is the ability of dendritic cells (DCs) to prime T cell immunity, an approach that has yielded promising results in some early phase clinical trials. However, novel approaches are required to improve DC therapeutic efficacy by enhancing their uptake of, and activation by, disease relevant antigens. The carbon nano-material graphene oxide (GO) may provide a unique way to deliver antigen to innate immune cells and modify their ability to initiate effective adaptive immune responses. We have assessed whether GO of various lateral sizes affects DC activation and function in vitro and in vivo, including their ability to take up, process and present the well-defined model antigen ovalbumin (OVA). We have found that GO flakes are internalised by DCs, while having minimal effect on their viability, activation phenotype or cytokine production. Although adsorption of OVA protein to either small or large GO flakes promoted its uptake into DCs, large GO interfered with OVA processing. In terms of modulation of DC function, delivery of OVA via small GO flakes significantly enhanced DC ability to induce proliferation of OVA-specific CD4+ T cells, promoting granzyme B secretion in vitro. On the other hand, delivery of OVA via large GO flakes augmented DC ability to induce proliferation of OVA-specific CD8+ T cells, and their production of IFN-γ and granzyme B. Together, these data demonstrate the capacity of GO of different lateral dimensions to act as a promising delivery platform for DC modulation of distinct facets of the adaptive immune response, information that could be exploited for future development of targeted immunotherapies.

Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile.

BACKGROUND: A key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, has a role in the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to measure DNA damages in both epithelial and immune cells, after either single or repeated exposure. RESULTS: GO induced DNA damages were size and dose dependent, in both exposure scenario. After single exposure to a high dose, both USGO and LGO induced significant DNA damage in the lung parenchyma, but only during the acute phase response (p < 0.05 for USGO; p < 0.01 for LGO). This was followed by a fast lung recovery at day 7 and 28 for both GOs. When evaluating the chronic impact of GO after repeated exposure, only a high dose of LGO induced long-term DNA damages in lung alveolar epithelia (at 84 days, p < 0.05). Regardless of size, low dose GO did not induce any significant DNA damage after repeated exposure. A multiparametric correlation analysis of our repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damage was correlated to efficient recovery from acute inflammation (i.e., significant secretion of SAA3, p < 0.001; infiltration of neutrophils, p < 0.01). In contrast, the persistence of LGO in lungs was associated to a long-lasting presence of multinucleated macrophages (up to 84 days, p < 0.05), an underlying inflammation (IL-1α secretion up to 28 days, p < 0.05) and the presence of persistent DNA damages at 84 days. CONCLUSIONS: Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace.

Innate but Not Adaptive Immunity Regulates Lung Recovery from Chronic Exposure to Graphene Oxide Nanosheets.

Graphene has drawn a lot of interest in the material community due to unique physicochemical properties. Owing to a high surface area to volume ratio and free oxygen groups, the oxidized derivative, graphene oxide (GO) has promising potential as a drug delivery system. Here, the lung tolerability of two distinct GO varying in lateral dimensions is investigated, to reveal the most suitable candidate platform for pulmonary drug delivery. Following repeated chronic pulmonary exposure of mice to GO sheet suspensions, the innate and adaptive immune responses are studied. An acute and transient influx of neutrophils and eosinophils in the alveolar space, together with the replacement of alveolar macrophages by interstitial ones and a significant activation toward anti-inflammatory subsets, are found for both GO materials. Micrometric GO give rise to persistent multinucleated macrophages and granulomas. However, neither adaptive immune response nor lung tissue remodeling are induced after exposure to micrometric GO. Concurrently, milder effects and faster tissue recovery, both associated to a faster clearance from the respiratory tract, are found for nanometric GO, suggesting a greater lung tolerability. Taken together, these results highlight the importance of dimensions in the design of biocompatible 2D materials for pulmonary drug delivery system.

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.

Graphene Oxide Nanosheets Interact and Interfere with SARS-CoV-2 Surface Proteins and Cell Receptors to Inhibit Infectivity.

Nanotechnology can offer a number of options against coronavirus disease 2019 (COVID-19) acting both extracellularly and intracellularly to the host cells. Here, the aim is to explore graphene oxide (GO), the most studied 2D nanomaterial in biomedical applications, as a nanoscale platform for interaction with SARS-CoV-2. Molecular docking analyses of GO sheets on interaction with three different structures: SARS-CoV-2 viral spike (open state - 6VYB or closed state - 6VXX), ACE2 (1R42), and the ACE2-bound spike complex (6M0J) are performed. GO shows high affinity for the surface of all three structures (6M0J, 6VYB and 6VXX). When binding affinities and involved bonding types are compared, GO interacts more strongly with the spike or ACE2, compared to 6M0J. Infection experiments using infectious viral particles from four different clades as classified by Global Initiative on Sharing all Influenza Data (GISAID), are performed for validation purposes. Thin, biological-grade GO nanoscale (few hundred nanometers in lateral dimension) sheets are able to significantly reduce copies for three different viral clades. This data has demonstrated that GO sheets have the capacity to interact with SARS-CoV-2 surface components and disrupt infectivity even in the presence of any mutations on the viral spike. GO nanosheets are proposed to be further explored as a nanoscale platform for development of antiviral strategies against COVID-19.

Graphene oxide prevents lateral amygdala dysfunctional synaptic plasticity and reverts long lasting anxiety behavior in rats.

Engineered small graphene oxide (s-GO) sheets were previously shown to reversibly down-regulate glutamatergic synapses in the hippocampus of juvenile rats, disclosing an unexpected translational potential of these nanomaterials to target selective synapses in vivo. Synapses are anatomical specializations acting in the Central Nervous System (CNS) as functional interfaces among neurons. Dynamic changes in synaptic function, named synaptic plasticity, are crucial to learning and memory. More recently, pathological mechanisms involving dysfunctional synaptic plasticity were implicated in several brain diseases, from dementia to anxiety disorders. Hyper-excitability of glutamatergic neurons in the lateral nucleus of the amygdala complex (LA) is substantially involved in the storage of aversive memory induced by stressful events enabling post-traumatic stress disorder (PTSD). Here we translated in PTSD animal model the ability of s-GO, when stereotaxically administered to hamper LA glutamatergic transmission and to prevent the behavioral response featured in long-term aversive memory. We propose that s-GO, by interference with glutamatergic plasticity, impair LA-dependent memory retrieval related to PTSD.

Intracerebral Injection of Graphene Oxide Nanosheets Mitigates Microglial Activation Without Inducing Acute Neurotoxicity: A Pilot Comparison to Other Nanomaterials.

Carbon-based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial-based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain.

Splenic Capture and In Vivo Intracellular Biodegradation of Biological-Grade Graphene Oxide Sheets.

Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible.

Graphene oxide: A growth factor delivery carrier to enhance chondrogenic differentiation of human mesenchymal stem cells in 3D hydrogels.

Cartilage engineering with stem cells in 3D scaffolds is a promising future therapy to treat cartilage defects. One challenge in the field is to design carriers to efficaciously deliver biological factors in 3D scaffolds containing stem cells to appropriately guide differentiation of these cells in same scaffolds and promote specific tissue synthesis. Graphene-based 2D nanomaterials have recently attracted extensive interest for their biomedical applications as they can adsorb a plethora of biological molecules, thus offering high potential as delivery carriers. This study utilized graphene oxide (GO) flakes to adsorb transforming growth factor ß3 (TGF-ß3), which were then incorporated into a collagen hydrogel. Human mesenchymal stem cells (hMSCs) were encapsulated in the same gel and chondrogenic differentiation assessed. The study showed GO flakes adsorbed > 99% TGF-ß3 with <1.7% release. Adsorbed TGF-ß3 retained a similar conformation to its dissolved counterpart (free protein) but importantly demonstrated greater conformational stability. Smad2 phosphorylation was promoted, and higher chondrogenic gene expression and cartilage-specific extracellular matrix deposition were achieved compared to exogenously delivering TGF-ß3 in culture media. Effects were sustained in long-term 28-day culture. The results demonstrate GO flakes as highly-efficient for delivering GFs in 3D to guide cells in the same scaffold and induce tissue formation. The ability of GO flakes to provide sustained local delivery makes this material attractive for tissue engineering strategies, in particular for regionally-specific MSC differentiation (e.g. osteochondral tissue engineering). STATEMENT OF SIGNIFICANCE: Cartilage engineering involving stem cells in 3D scaffolds is a promising future therapy to treat cartilage defects which can lead to debilitating conditions such as osteoarthritis. However, this field faces the challenge to design delivery carriers to efficaciously deliver biological factors inside these 3D cell-containing scaffolds for appropriately-guided cell differentiation. Graphene-based 2D nanomaterials offer high potential as delivery carriers, but to date studies using them to deliver biological factors have been restricted to 2D substrates, non-scaffold cell masses, or acellular 3D scaffolds. Our study for the first time demonstrated simultaneously incorporating both human mesenchymal stem cells (hMSCs) and GO (graphene oxide)-adsorbed growth factor TGFß3 into a 3D scaffold, where GO-adsorbed TGFß3 enhanced chondrogenic differentiation of hMSCs and cartilage-tissue synthesis throughout the scaffold without needing to repeatedly supply TGFß3 exogenously.

Liposome-Indocyanine Green Nanoprobes for Optical Labeling and Tracking of Human Mesenchymal Stem Cells Post-Transplantation In Vivo.

Direct labeling of human mesenchymal stem cells (hMSC) prior to transplantation provides a means to track cells after administration and it is a powerful tool for the assessment of new cell-based therapies. Biocompatible nanoprobes consisting of liposome-indocyanine green hybrid vesicles (liposome-ICG) are used to safely label hMSC. Labeled hMSC recapitulating a 3D cellular environment is transplanted as spheroids subcutaneously and intracranially in athymic nude mice. Cells emit a strong NIR signal used for tracking post-transplantation with the IVIS imaging system up to 2 weeks (subcutaneous) and 1 week (intracranial). The transplanted stem cells are imaged in situ after engraftment deep in the brain up to 1 week in living animals using optical imaging techniques and without the need to genetically modify the cells. This method is proposed for efficient, nontoxic direct cell labeling for the preclinical assessment of cell-based therapies and the design of clinical trials, and potentially for localization of the cell engraftment after transplantation into patients.

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production.

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.