Biological response to purification and acid functionalization of carbon nanotubes

The final publication is available at Springer via: http://link.springer.com/article/10.1007/s11051-014-2507-yInternational audienceAcid functionalization has been considered as an easy way to enhance the dispersion and biodegradation of carbon nanotubes (CNT). However, inconsistencies between toxicity studies of acid functionalized CNT remain unexplained. This could be due to a joint effect of the main physicochemical modifications resulting from an acid functionalization: addition of surface acid groups and purification from catalytic metallic impurities. In this study, the impact on CNT biotoxicity of these two physiochemical features was assessed separately. The in vitro biological response of RAW 264.7 macrophages was evaluated after exposure to 15-240 µg x mL−1 of two types of multi-walled CNT. For each type of CNT (small: 20 nm diameter, and big: 90 nm diameter), three different surface chemical properties were studied (total of six CNT samples): pristine, acid functionalized and desorbed. Desorbed CNT were purified by the acid functionalization but presented a very low amount of surface acid groups due to a thermal treatment under vacuum. A Janus effect of acid functionalization with two opposite impacts is highlighted. The CNT purification decreased the overall toxicity, while the surface acid groups intensified it when present at a specific threshold. These acid groups especially amplified the pro-inflammatory response. The threshold mechanism which seemed to regulate the impact of acid groups should be further studied to determine its value and potential link to the other physicochemical state of the CNT. The results suggest that, for a safer-design approach, the benefit-risk balance of an acid functionalization has to be considered, depending on the CNT primary state of purification. Further research should be conducted in this direction

Thermal annealing of carbon nanotubes reveals a strong toxicological impact of the structural defects

Le document inséré dans cette page comprend des données supplémentaire à l'article " Thermal annealing of carbon nanotubes reveals a toxicological impact of the structural defects" (DOI: 10.1007/s11051-015-2999-0) paru dans Journal of Nanoparticles ResearchInternational audienceThe biological response to pristine and annealed multi-walled carbon nanotubes (MWCNT) was assessed on murine macrophages (RAW 264.7). First, the physicochemical features of the as-produced MWCNT and annealed at 2125 °C for 1 h were fully characterized. A decrease in structural defects, hydrophobicity and catalytic impurities was detected after annealing. Thereafter, their impact on cytotoxicity, oxidative stress, and pro-inflammatory response was investigated at concentrations ranging from 15 to 120 µg mL−1. No effect of the 2125 °C treatment was detected on the cytotoxicity. In contrast, the annealed carbon nanotubes showed a significant increase of the pro-inflammatory response. We assumed that this behavior was due to the reduction in structural defects that may modify the layer of adsorbed biomolecules. Surprisingly, the purification of metallic catalysts did not have any significant impact on the oxidative stress. We suggested that the structural improvements from the 2125 °C treatment can decrease the carbon nanotube scavenging capacity and thus allow a higher free radical release which may counterbalance the decrease of oxidative stress due to a lower content of metallic impurities

In vitro toxicity and physico-chemical properties of carbon nanotubes

Les propriétés exceptionnelles des nanotubes de carbone (CNT) attirent de nombreux industriels dans les domaines de la microélectronique, des matériaux ou de la nanomédecine. Néanmoins, le risque sanitaire lié à ce nanomatériau reste encore mal compris. Des profils toxicologiques différents, dépendant des caractéristiques physico-Chimiques des CNT, ont été mis en évidence. Une approche « safer by design » est proposée, afin d’identifier les paramètres pouvant, dès la conception des CNT, pour limiter le risque sanitaire. Dans ce contexte, cette thèse avait pour objectif d’étudier l’impact sur la réponse in vitro d’une lignée de macrophages murins (RAW 264.7) de deux traitements de post-Production de CNT : la fonctionnalisation acide et le recuit haute température.Les groupements acides en surface des CNT fonctionnalisés ont entrainé une augmentation de la réponse pro-Inflammatoire sans influencer significativement la cytotoxicité. D’un autre côté, la fonctionnalisation acide, principalement par l’élimination des impuretés métalliques, a permis de diminuer le stress oxydant. Les CNT recuits à haute température étaient à l’origine d’une réponse pro-Inflammatoire plus importante que les CNT bruts, confirmant lasensibilité de cette réponse biologique à la chimie de surface. En revanche, le recuit n’a pas diminué significativement le stress oxydant malgré la purification des CNT, suggérant l’importance des défauts de structure sur cette réponse biologique. La fonctionnalisation acide de nano-Graphite et de noir de carbone a eu un impact similaire à celle des CNT sur l’activité biologique des macrophages. La comparaison de ces trois nanomatériaux fonctionnalisés semble s’accorder avec le paradigme mettant en exergue la toxicité spécifique des fibres et des plaquettes. Enfin, afin de compléter ces résultats, des études exploratoires sur les interférences entre les tests de toxicité et les CNT, ainsi que sur le stress oxydant, ont été conduites.Due to their exceptional properties, carbon nanotubes (CNT) have aroused a huge interest among in industrial fields such as microelectronics, material science and nanomedicine. Nevertheless, the health impacts of this nanomaterial still remain not well understood. The first toxicological studies pointed out that there is no unique response regarding the healthimpact of the CNT, but different toxicological profiles according to their various physicochemical properties. A safer by design approach is thus proposed to identify the parameters decreasing from their production the CNT biological impacts. In this context, this work aimed at studying the impact on the in vitro response from a macrophage cell line (RAW 264.7) of two post-Production treatments: acid functionalization and high temperature annealing.Surface acid groups from functionalized CNT enhanced the pro-Inflammatory response although the cytotoxicity remained stable. On the other hand, acid functionalization, through the elimination of metallic impurities, significantly decreased the oxidative stress. Annealed CNT increased the pro-Inflammatory response compared to the pristine CNT. It thus confirmed the sensitivity of this response for the changes in surface chemistry. However, the high temperature annealing did not influence the oxidative stress, despite of the CNT purification. It suggested that structural defects are also of importance for this response. Besides, the acid functionalization of nano-Graphite and carbon black displayed trends in the macrophage response similar to the acid functionalization of CNT. The comparison of these three carbon-Based nanomaterials seemed to conform to the fibre and platelets paradigm. Eventually, exploratory studies have also been conducted on the interferences between CNT and the toxicity assays, and on the oxidative stress

Toxicité in vitro et propriétés physico-chimiques de nanotubes de carbone

Due to their exceptional properties, carbon nanotubes (CNT) have aroused a huge interest among in industrial fields such as microelectronics, material science and nanomedicine. Nevertheless, the health impacts of this nanomaterial still remain not well understood. The first toxicological studies pointed out that there is no unique response regarding the healthimpact of the CNT, but different toxicological profiles according to their various physicochemical properties. A safer by design approach is thus proposed to identify the parameters decreasing from their production the CNT biological impacts. In this context, this work aimed at studying the impact on the in vitro response from a macrophage cell line (RAW 264.7) of two post-Production treatments: acid functionalization and high temperature annealing.Surface acid groups from functionalized CNT enhanced the pro-Inflammatory response although the cytotoxicity remained stable. On the other hand, acid functionalization, through the elimination of metallic impurities, significantly decreased the oxidative stress. Annealed CNT increased the pro-Inflammatory response compared to the pristine CNT. It thus confirmed the sensitivity of this response for the changes in surface chemistry. However, the high temperature annealing did not influence the oxidative stress, despite of the CNT purification. It suggested that structural defects are also of importance for this response. Besides, the acid functionalization of nano-Graphite and carbon black displayed trends in the macrophage response similar to the acid functionalization of CNT. The comparison of these three carbon-Based nanomaterials seemed to conform to the fibre and platelets paradigm. Eventually, exploratory studies have also been conducted on the interferences between CNT and the toxicity assays, and on the oxidative stress.Les propriétés exceptionnelles des nanotubes de carbone (CNT) attirent de nombreux industriels dans les domaines de la microélectronique, des matériaux ou de la nanomédecine. Néanmoins, le risque sanitaire lié à ce nanomatériau reste encore mal compris. Des profils toxicologiques différents, dépendant des caractéristiques physico-Chimiques des CNT, ont été mis en évidence. Une approche « safer by design » est proposée, afin d’identifier les paramètres pouvant, dès la conception des CNT, pour limiter le risque sanitaire. Dans ce contexte, cette thèse avait pour objectif d’étudier l’impact sur la réponse in vitro d’une lignée de macrophages murins (RAW 264.7) de deux traitements de post-Production de CNT : la fonctionnalisation acide et le recuit haute température.Les groupements acides en surface des CNT fonctionnalisés ont entrainé une augmentation de la réponse pro-Inflammatoire sans influencer significativement la cytotoxicité. D’un autre côté, la fonctionnalisation acide, principalement par l’élimination des impuretés métalliques, a permis de diminuer le stress oxydant. Les CNT recuits à haute température étaient à l’origine d’une réponse pro-Inflammatoire plus importante que les CNT bruts, confirmant lasensibilité de cette réponse biologique à la chimie de surface. En revanche, le recuit n’a pas diminué significativement le stress oxydant malgré la purification des CNT, suggérant l’importance des défauts de structure sur cette réponse biologique. La fonctionnalisation acide de nano-Graphite et de noir de carbone a eu un impact similaire à celle des CNT sur l’activité biologique des macrophages. La comparaison de ces trois nanomatériaux fonctionnalisés semble s’accorder avec le paradigme mettant en exergue la toxicité spécifique des fibres et des plaquettes. Enfin, afin de compléter ces résultats, des études exploratoires sur les interférences entre les tests de toxicité et les CNT, ainsi que sur le stress oxydant, ont été conduites

Three-Dimensional in vitro Models of Healthy and Tumor Brain Microvasculature for Drug and Toxicity Screening

International audienceTissue vascularization is essential for its oxygenation and the homogenous diffusion of nutrients. Cutting-edge studies are focusing on the vascularization of three-dimensional (3D) in vitro models of human tissues. The reproduction of the brain vasculature is particularly challenging as numerous cell types are involved. Moreover, the blood-brain barrier, which acts as a selective filter between the vascular system and the brain, is a complex structure to replicate. Nevertheless, tremendous advances have been made in recent years, and several works have proposed promising 3D in vitro models of the brain microvasculature. They incorporate cell co-cultures organized in 3D scaffolds, often consisting of components of the native extracellular matrix (ECM), to obtain a micro-environment similar to the in vivo physiological state. These models are particularly useful for studying adverse effects on the healthy brain vasculature. They provide insights into the molecular and cellular events involved in the pathological evolutions of this vasculature, such as those supporting the appearance of brain cancers. Glioblastoma multiform (GBM) is the most common form of brain cancer and one of the most vascularized solid tumors. It is characterized by a high aggressiveness and therapy resistance. Current conventional therapies are unable to prevent the high risk of recurrence of the disease. Most of the new drug candidates fail to pass clinical trials, despite the promising results shown in vitro. The conventional in vitro models are unable to efficiently reproduce the specific features of GBM tumors. Recent studies have indeed suggested a high heterogeneity of the tumor brain vasculature, with the coexistence of intact and leaky regions resulting from the constant remodeling of the ECM by glioma cells. In this review paper, after summarizing the advances in 3D in vitro brain vasculature models, we focus on the latest achievements in vascularized GBM modeling, and the potential applications for both healthy and pathological models as platforms for drug screening and toxicological assays. Particular attention will be paid to discuss the relevance of these models in terms of cell-cell, cell-ECM interactions, vascularization and permeability properties, which are crucial parameters for improving in vitro testing accuracy

Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results about the cytotoxicity of these nanomaterials

International audienceCarbon nanotubes (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable to nano-sized materials. Indeed, due to their unique physico-chemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments in cell free conditions were performed and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and to the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The Kads and neq constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT

An in vitro self-organized three-dimensional model of the blood-brain barrier microvasculature

International audienceThe blood-brain barrier (BBB) protects the human brain from external aggression. Despite its great importance, very few in vitro models of the BBB reproducing its complex organization are available yet. Here we fabricated such a three-dimensional (3D) self-organized in vitro model of BBB microvasculature by means of a combination of collagen microfibers (CMF) and fibrin gel. The interconnected fibers supported human brain microvascular endothelial cell migration and the formation of a capillary-like network with a lumen diameter close to in vivo values. Fibrin, a protein involved in blood vessel repair, favored the further 3D conformation of the brain microvascular endothelial cells, astrocytes and pericytes, ensured gel cohesion and avoided shrinkage. The maturation of the BBB microvasculature network was stimulated by both the CMF and the fibrin in the hydrogel. The expression of essential tight-junction proteins, carriers and transporters was validated in regards to bidimensional simple coculture. The volume of gel drops was easily tunable to fit in 96-well plates. The cytotoxicity of D-Mannitol and its impacts on the microvascular network were evaluated, as an example of the pertinence of this 3D BBB capillary model for screening applications

A journey from the endothelium to the tumor tissue: distinct behavior between PEO-PCL micelles and polymersomes nanocarriers

Polymeric nanocarriers must overcome several biological barriers to reach the vicinity of solid tumors and deliver their encapsulated drug. This study assessed the in vitro and in vivo passage through the blood vessel wall to tumors of two well-characterized polymeric nanocarriers: poly(ethyleneglycol-b-ε-caprolactone) micelles and polymersomes charged with a fluorescent membrane dye (DiO: 3,3'-dioctadecyloxacarbo-cyanine perchlorate). The internalization and translocation from endothelial (human primary endothelial cells HUVEC) to cancer cells (human tumor cell line HCT-116) was studied in conventional 2D monolayers, 3D tumor spheroids, or in an endothelium model based on transwell assay. Micelles induced a faster DiO internalization compared to polymersomes but the latter crossed the endothelial monolayer more easily. Both translocation rates were enhanced by the addition of a pro-inflammatory factor or in the presence of tumor cells. These results were confirmed by early in vivo experiments. Overall, this study pointed out the room for the improvement of polymeric nanocarriers design to avoid drug losses when crossing the blood vessel walls

Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels

International audienceCell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber e fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60e30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the bloodbrain barrier functions

Biological Effects and Applications of Bulk and Surface Acoustic Waves on In Vitro Cultured Mammal Cells: New Insights

International audienceMedical imaging has relied on ultrasound (US) as an exploratory method for decades. Nonetheless, in cell biology, the numerous US applications are mainly in the research and development phase. In this review, we report the main effects on human or mammal cells of US induced by bulk or surface acoustic waves (SAW). At low frequencies, bulk US can lead to cell death. Under specific intensities and exposure times, however, cell proliferation and migration can be enhanced through cytoskeleton fluidization (a reorganization of the actin filaments and microtubules). Cavitation phenomena, frequencies of resonance close to those of the biological compounds, and mechanical transfers of energy from the acoustic pressure could explain those biological outcomes. At higher frequencies, no cavitation is observed. However, USs of high frequency stimulate ionic channels and increase cell permeability and transfection potency. Surface acoustic waves are increasingly exploited in microfluidics, especially for precise cell manipulations and cell sorting. With applications in diagnosis, infection, cancer treatment, or wound healing, US has remarkable potential. More mechanotransduction studies would be beneficial to understand the distinct roles of temperature rise, acoustic streaming and mechanical and electrical stimuli in the fiel