Quantification of cellular associated graphene and induced surface receptor responses.

The use of graphene for biomedical and other applications involving humans is growing and shows practical promise. However, quantifying the graphitic nanomaterials that interact with cells and assessing any corresponding cellular response is extremely challenging. Here, we report an effective approach to quantify graphene interacting with single cells that utilizes combined multimodal-Raman and photoacoustic spectroscopy. This approach correlates the spectroscopic signature of graphene with the measurement of its mass using a quartz crystal microbalance resonator. Using this technique, we demonstrate single cell noninvasive quantification and multidimensional mapping of graphene with a detection limit of as low as 200 femtograms. Our investigation also revealed previously unseen graphene-induced changes in surface receptor expression in dendritic cells of the immune system. This tool integrates high-sensitivity real-time detection and monitoring of nanoscale materials inside single cells with the measurement of induced simultaneous biological cell responses, providing a powerful method to study the impact of nanomaterials on living systems and as a result, the toxicology of nanoscale materials.

A genomic characterization of the influence of silver nanoparticles on bone differentiation in MC3T3-E1 cells.

Silver nanoparticles (AgNPs) have been widely used in a variety of biomedical applications. Previous studies demonstrated that AgNPs significantly enhanced bone cell mineralization and differentiation in MC3T3-1 cells, a model in vitro system, when compared to several other NPs. This increased bone deposition was evaluated by phenotypic measurements and assessment of the expression of miRNAs associated with regulation of bone morphogenic proteins. In the present study, we used RNA-seq technology, a more direct measurement of gene expression, to investigate further the mechanisms of bone differentiation induced by AgNP treatment. Key factors associated with the osteoclast pathway were significantly increased in response to AgNP exposure including Bmp4, Bmp6 and Fosl1. In addition, genes of metabolism and toxicity pathways were significantly regulated as well. Although this study suggests the potential for AgNPs to influence bone morphogenesis in injury or disease applications, further investigation into the efficacy and safety of AgNPs in bone regeneration is warranted.

Modifying Dendritic Cell Activation with Plasmonic Nano Vectors.

Dendritic cells (DCs) can acquire, process, and present antigens to T-cells to induce an immune response. For this reason, targeting cancer antigens to DCs in order to cause an immune response against cancer is an emerging area of nanomedicine that has the potential to redefine the way certain cancers are treated. The use of plasmonically active silver-coated gold nanorods (henceforth referred to as plasmonic nano vectors (PNVs)) as potential carriers for DC tumor vaccines has not been presented before. Effective carriers must be able to be phagocytized by DCs, present low toxicity, and induce the maturation of DCs-an early indication of an immune response. When we treated DCs with the PNVs, we found that the cell viability of DCs was unaffected, up to 200 µg/ml. Additionally, the PNVs associated with the DCs as they were phagocytized and they were found to reside within intracellular compartments such as endosomes. More importantly, the PNVs were able to induce expression of surface markers indicative of DC activation and maturation, i.e. CD40, CD86, and MHC class II. These results provide the first evidence that PNVs are promising carriers for DC-based vaccines and warrant further investigating for clinical use.

p53-competent cells and p53-deficient cells display different susceptibility to oxygen functionalized graphene cytotoxicity and genotoxicity.

Due to the distinctive physical, electrical, and chemical properties of graphene nanomaterials, numerous efforts pursuing graphene-based biomedical and industrial applications are underway. Oxidation of pristine graphene surfaces mitigates its otherwise hydrophobic characteristic thereby improving its biocompatibility and functionality. Yet, the potential widespread use of oxidized graphene derivatives raises concern about adverse impacts on human health. The p53 tumor suppressor protein maintains cellular and genetic stability after toxic exposures. Here, we show that p53 functional status correlates with oxygen functionalized graphene (f-G) cytotoxicity and genotoxicity in vitro. The f-G exposed p53-competent cells, but not p53-deficient cells, initiated G0 /G1 phase cell cycle arrest, suppressed reactive oxygen species, and entered apoptosis. There was p53-dependent f-G genotoxicity evident as increased structural chromosome damage, but not increased gene mutation or chromatin loss. In conclusion, the cytotoxic and genotoxic potential for f-G in exposed cells was dependent on the p53 functional status. These findings have broad implications for the safe and effective implementation of oxidized graphene derivatives into biomedical and industrial applications. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

The role of surface chemistry in the cytotoxicity profile of graphene.

Graphene and its derivative, because of their unique physical, electrical and chemical properties, are an important class of nanomaterials being proposed as foundational materials in nanomedicine as well as for a variety of industrial applications. A major limitation for graphene, when used in biomedical applications, is its poor solubility due to its rather hydrophobic nature. Therefore, chemical functionalities are commonly introduced to alter both its surface chemistry and biochemical activity. Here, we show that surface chemistry plays a major role in the toxicological profile of the graphene structures. To demonstrate this, we chemically increased the oxidation level of the pristine graphene and compared the corresponding toxicological effects along with those for the graphene oxide. X-ray photoelectron spectroscopy revealed that pristine graphene had the lowest amount of surface oxygen, while graphene oxide had the highest at 2.5% and 31%, respectively. Low and high oxygen functionalized graphene samples were found to have 6.6% and 24% surface oxygen, respectively. Our results showed a dose-dependent trend in the cytotoxicity profile, where pristine graphene was the most cytotoxic, with decreasing toxicity observed with increasing oxygen content. Increased surface oxygen also played a role in nanomaterial dispersion in water or cell culture medium over longer periods. It is likely that higher dispersity might result in graphene entering into cells as individual flakes ~1 nm thick rather than as more cytotoxic aggregates. In conclusion, changes in graphene's surface chemistry resulted in altered solubility and toxicity, suggesting that a generalized toxicity profile would be rather misleading. Copyright © 2016 John Wiley & Sons, Ltd.

Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity.

Autophagy is a cellular process that facilitates nutrient turnover and removal of expended macromolecules and organelles to maintain homeostasis. The recycling of cytosolic macromolecules and damaged organelles by autophagosomes occurs through the lysosomal degradation pathway. Autophagy can also be upregulated as a prosurvival pathway in response to stress stimuli such as starvation, hypoxia or cell damage. Over the last two decades, there has been a surge in research revealing the basic molecular mechanisms of autophagy in mammalian cells. A corollary of an advanced understanding of autophagy has been a concurrent expansion of research into understanding autophagic function and dysfunction in pathology. Recent studies have revealed a pivotal role for autophagy in drug toxicity, and for utilizing autophagic components as diagnostic markers and therapeutic targets in treating disease and cancer. In this review, advances in understanding the molecular basis of mammalian autophagy, methods used to induce and evaluate autophagy, and the diverse interactions between autophagy and drug toxicity, disease progression and carcinogenesis are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of silver nanoparticles on human and rat embryonic neural stem cells.

Silver nano-particles (Ag-NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products will almost certainly increase environmental silver levels, resulting in increased exposures and the potential for increased adverse reactions including neurotoxic effects. In the present study, embryonic neural stem cells (NSCs) from human and rat fetuses (gestational day-16) were used to determine whether Ag-NPs are capable of causing developmental neurotoxicity. The NSCs were cultured in serum free medium supplemented with appropriate growth factors. On the eighth day in vitro (DIV 8), the cells were exposed to Ag-NPs at concentrations of 1, 5, 10, and 20 µg/ml for 24 h. The cultured cells then were characterized by NSC markers including nestin and SOX2 and a variety of assays were utilized to determine the effects of Ag-NPs on NSC proliferation and viability and the underlying mechanisms associated with these effects. The results indicate that mitochondrial viability (MTT metabolism) was substantially attenuated and LDH release was increased significantly in a dose-dependent manner. Ag-NPs-induced neurotoxicity was further confirmed by up-regulated Bax protein expression, an increased number of TUNEL-positively stained cells, and elevated reactive oxygen species (ROS). NSC proliferation was also significantly decreased by Ag-NPs. Co-administration of acetyl-L-carnitine, an antioxidant agent, effectively blocked the adverse effects associated with Ag-NP exposure.

Calcium-channel blocking and nanoparticles-based drug delivery for treatment of drug-resistant human cancers.

BACKGROUND: Cancer cell chemoresistance is one of the major limitations to successful cancer treatment and one of the factors that is responsible for the possible recurrence of the disease. Here, we aimed to combine a calcium-channel blocker, verapamil, with an alternative delivery of the anti-cancer drug, doxorubicin, using nanostructural materials. This approach could reduce the cellular resistance to chemotherapeutics agents. RESULTS: The outcome of this complex approach on cellular viability was investigated by using various assays in both a time- and concentration-dependent manner: WST-1, flow cytometry cell viability assay, fluorescence microscopy, DNA fragmentation, and TUNEL labeling of apoptotic cells. CONCLUSION: All of these analytical assays confirmed the ability to reduce the chemoresistance of the cancer cells based on the proposed procedure.

Nanomaterials for targeted drug delivery to cancer stem cells.

Recent developments in cancer biology have identified the existence of a sub-poplulation of cells - cancer stem cells (CSC) that are resistant to most traditional therapies (e.g. chemotherapy and radiotherapy) and have the ability to repair their damaged DNA. These findings have necessitated a break with traditional oncology management and encouraged new perspectives concerning cancer treatment. Understanding the functional biology of CSCs - especially the signaling pathways that are involved in their self-renewal mechanisms - is crucial for discovering new forms of treatment. In this review, we highlight current and future prospects for potential cancer therapies based on the use of nano-sized materials. Nanomaterials could revolutionize cancer management because of their distinctive features - unique surface chemistry, strong electronic, optic, and magnetic properties - that are found neither in bulk materials nor in single molecules. Based on these distinct properties, we believe that nanomaterials could be excellent candidates for use in CSC research in order to optimize cancer therapeutics. Moreover, we propose these nanomaterials for the inhibition of the self-renewal pathways of CSCs by focusing on the Hedgehog, Notch, and Wnt/ß-catenin self-renewal mechanisms. By introducing these methods for the detection, targeting, and destruction of CSCs, an efficient alternative treatment for the incurable disease of cancer could be provided.

The use of genomics in model in vitro systems.

Traditional toxicological tests generally provide descriptive information regarding the potential toxicity of chemicals, drugs and physical agents and are limited in their ability to assess risk to humans because they use model systems that are nonhuman in origin. Upon completion of the sequencing of the human genome, new tools were established that identify early biomarkers of toxicity and disease not only in model organisms but also in man. Gene expression profiling led to the development of a new subdiscipline of toxicology termed toxicogenomics. This new subdiscipline combines the emerging technologies of genomics, proteomics and bioinformatics to identify and characterize mechanisms of action of known and suspected toxicants. This chapter describes some advances in the area of toxicogenomics and discusses several models to study chemical-induced liver injury.

Electrically conductive gold-coated collagen nanofibers for placental-derived mesenchymal stem cells enhanced differentiation and proliferation.

Gold-coated collagen nanofibers (GCNFs) were produced by a single-step reduction process and used for the growth and differentiation of human adult stem cells. The nanomaterials were characterized by a number of analytical techniques including electron microscopy and spectroscopy. They were found to be biocompatible and to improve the myocardial and neuronal differentiation process of the mesenchymal stem cells isolated from the placental chorionic component. The expression of specific differentiation markers (atrium, natriuretic peptide, actin F and actin monomer, glial fibrilary acidic protein, and neurofilaments) was investigated by immunocytochemistry.

Nanostructural materials increase mineralization in bone cells and affect gene expression through miRNA regulation.

We report that several nanomaterials induced enhanced mineralization (increased numbers and larger areas of mineral nests) in MC3T3-E1 bone cells, with the highest response being induced by silver nanoparticles (AgNPs). We demonstrate that AgNPs altered microRNA expression resulting in specific gene expression associated with bone formation. We suggest that the identified essential transcriptional factors and bone morphogenetic proteins play an important role in activation of the process of mineralization in bone cells exposed to AgNPs.

Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells.

Graphitic nanomaterials such as graphene layers (G) and single-wall carbon nanotubes (SWCNT) are potential candidates in a large number of biomedical applications. However, little is known about the effects of these nanomaterials on biological systems. Here we show that the shape of these materials is directly related to their induced cellular toxicity. Both G and SWCNT induce cytotoxic effects, and these effects are concentration- and shape-dependent. Interestingly, at low concentrations, G induced stronger metabolic activity than SWCNT, a trend that reversed at higher concentrations. Lactate dehydrogenase levels were found to be significantly higher for SWCNT as compared to the G samples. Moreover, reactive oxygen species were generated in a concentration- and time-dependent manner after exposure to G, indicating an oxidative stress mechanism. Furthermore, time-dependent caspase 3 activation after exposure to G (10 microg/mL) shows evidence of apoptosis. Altogether these studies suggest different biological activities of the graphitic nanomaterials, with the shape playing a primary role.

Cytotoxicity and biological effects of functional nanomaterials delivered to various cell lines.

Functional nanomaterials that included gold, silver nanoparticles and single wall carbon nanotubes were delivered to two cell lines (MLO-Y4 osteocytic cells and HeLa cervical cancer cells) in various concentrations. The cells were found to uptake the nanomaterials in a relatively short time, a process that significantly affected the shape and the size of the cells. The percentage of cellular death, due to the delivery of these nanomaterials, was found to be the highest for carbon nanotubes and increased gradually with the concentration of these nanostructures. Moreover, when the nanomaterials were delivered to the cells combined with commonly used chemotherapeutic agents such as etoposide or dexamethasone, the number of the cells that died increased significantly (100-300%) as compared with the case when only the nanomaterials or the chemotherapeutic agents were delivered. The experimental results were confirmed by Caspase 3 studies, indicating a strong interaction between the nanomaterials used in this study and the protein structure of the cells, which allowed a more effective action of the apoptotic agents. These findings could be the foundation of a new class of cancer therapies that are composed of both chemotherapeutic agents and nanomaterials.

The Viracept-EMS case: impact and outlook.

This perspective first considers the potential impact of the Viracept-EMS case in the framework of the current understanding of the low-dose effects of DNA-reactive chemicals and the approaches used to estimate health risks from genotoxins occurring as impurities in pharmaceutical products or as contaminants in the environment or workplace. It also presents an outlook on the nature of additional research building upon the Viracept-EMS case to test assumptions underlying thresholded dose-response relationships and to establish biologically based risk assessment models in lieu of default models for DNA-reactive compounds.

Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization.

The assessment of human cancer risk from chemical exposure requires the integration of diverse types of data. Such data involve effects at the cell and tissue levels. This report focuses on the specific utility of one type of data, namely DNA adducts. Emphasis is placed on the appreciation that such DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information. As emerging technologies provide even more sensitive quantitative measurements of DNA adducts, integration that establishes links between DNA adducts and accepted outcome measures becomes critical for risk assessment. The present report proposes an organizational approach for the assessment of DNA adduct data (e.g., type of adduct, frequency, persistence, type of repair process) in concert with other relevant data, such as dosimetry, toxicity, mutagenicity, genotoxicity, and tumor incidence, to inform characterization of the mode of action. DNA adducts are considered biomarkers of exposure, whereas gene mutations and chromosomal alterations are often biomarkers of early biological effects and also can be bioindicators of the carcinogenic process.

Effect of p53 genotype on gene expression profiles in murine liver.

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the "guardian of the genome". Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53(-/-) and p53(+/-) mice. Six male mice from each genotype (p53(+/+), p53(+/-), and p53(-/-)) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53(+/+) and p53(+/-) or between p53(+/+) and p53(-/-) at the level of p < or = 0.05. Both genes with increased expression and decreased expression were identified in p53(+/-) and in p53(-/-) mice. Most notable in the gene list derived from the p53(+/-) mice was the significant reduction in p53 mRNA. In the p53(-/-) mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative.

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..

Proceedings of a workshop on DNA adducts: biological significance and applications to risk assessment Washington, DC, April 13-14, 2004.

In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled "Can low levels of DNA adducts predict adverse outcomes?" served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim "decision-logic" or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA adducts.