In vitro toxicological evaluation of aerosols generated by a 4th generation vaping device using nicotine salts in an air-liquid interface system.

BACKGROUND: Electronic cigarettes (EC) have gained popularity, especially among young people, with the introduction of fourth-generation devices based on e-liquids containing nicotine salts that promise a smoother vaping experience than freebase nicotine. However, the toxicological effects of nicotine salts are still largely unknown, and the chemical diversity of e-liquids limits the comparison between different studies to determine the contribution of each compound to the cytotoxicity of EC aerosols. Therefore, the aim of this study was to evaluate the toxicological profile of controlled composition e-liquid aerosols to accurately determine the effects of each ingredient based on exposure at the air-liquid interface. METHODS: Human lung epithelial cells (A549) were exposed to undiluted aerosols of controlled composition e-liquids containing various ratios of propylene glycol (PG)/vegetable glycerin (VG) solvents, freebase nicotine, organic acids, nicotine salts, and flavoured commercial e-liquids. Exposure of 20 puffs was performed at the air-liquid interface following a standard vaping regimen. Toxicological outcomes, including cytotoxicity, inflammation, and oxidative stress, were assessed 24 h after exposure. RESULTS: PG/VG aerosols elicited a strong cytotoxic response characterised by a 50% decrease in cell viability and a 200% increase in lactate dehydrogenase (LDH) production, but had no effects on inflammation and oxidative stress. These effects occurred only at a ratio of 70/30 PG/VG, suggesting that PG is the major contributor to aerosol cytotoxicity. Both freebase nicotine and organic acids had no greater effect on cell viability and LDH release than at a 70/30 PG/VG ratio, but significantly increased inflammation and oxidative stress. Interestingly, the protonated form of nicotine in salt showed a stronger proinflammatory effect than the freebase nicotine form, while benzoic acid-based nicotine salts also induced significant oxidative stress. Flavoured commercial e-liquids was found to be cytotoxic at a threshold dose of ≈ 330 µg/cm². CONCLUSION: Our results showed that aerosols of e-liquids consisting only of PG/VG solvents can cause severe cytotoxicity depending on the concentration of PG, while nicotine salts elicit a stronger pro-inflammatory response than freebase nicotine. Overall, aerosols from fourth-generation devices can cause different toxicological effects, the nature of which depends on the chemical composition of the e-liquid.

Considerations on dosimetry for in vitro assessment of e-cigarette toxicity.

Electronic cigarettes (or e-cigarettes) can be used as smoking cessation aid. Some studies tend to show that they are less hazardous than tobacco cigarettes, even if it does not mean they are completely safe. The huge variation in study designs assessing in vitro toxicity of e-cigarettes aerosol makes it difficult to make comparisons and draw robust and irrefutable conclusions. In this paper, we review this heterogeneity (in terms of e-cigarette products, biological models, and exposure conditions) with a special focus on the wide disparity in the doses used as well as in the way they are expressed. Finally, we discuss the major issue of dosimetry and show how dosimetry tools enable to align data between different exposure systems or data from different laboratories and therefore allow comparisons to help further exploring the risk potential of e-cigarettes.

Toward Better Characterization of a Free-Base Nicotine Fraction in e-Liquids and Aerosols.

Given that nicotine salts are a growing market, methods are needed to characterize nicotine forms in e-cigarette vaping products. By lowering the free-base nicotine fraction (αfb) in favor of protonated forms, the addition of organic acids to the e-liquid mix greatly modulates nicotine pharmacokinetics and improves vapers' craving. This research investigated (1) the performance of pH measurement, liquid-liquid extraction (LLE), and acid/nicotine molar ratio calculation methods for αfb estimation in 6 nicotine benzoate and nicotine salicylate e-liquids and (2) nicotine protonation in the aerosol post vaporization. Aerosols were generated with a JUUL device and another mod-pod on a vaping machine to assess device effects. E-liquid and aerosol samples were then analyzed after further analytical optimization of previous methods and careful consideration of biases. Globally, performances were comparable between methods. αfb accounted for less than 5% of nicotine content regardless of experimental conditions. αfb were consistent between e-liquids and aerosols irrespective of e-cigarette devices. Hence, e-liquids are adequate surrogates for aerosols, facilitating the establishment of regulations. pH measurement is one of the most used methods and enables the establishment of relative scales for e-liquid classification but lacks automation possibility. Until now, the extent of sample dilution remained arbitrary. The dilution factor was fixed at 10, as usually achieved, since no effect of dilution was noted. pH values ranged from 5.3 to 6.3 in accordance with the literature. By contrast, LLE relies on the specificity of organic solvent for free-base nicotine extraction, causing discrepancies in previous studies. Here, the results were similar to αfb values from pH determination. Yet, LLE presented the highest variability and was the most time-consuming protocol. Finally, αfb calculation from molar ratio was the most robust and versatile method. Estimations can be made in silico from reported composition data and/or after liquid chromatography routine analysis.

Assessment of High-Power Electronic Nicotine Delivery System as an Alternative Aerosol Device for Terbutaline Delivery.

PURPOSE: The performance of new-generation high-power electronic nicotine delivery system (ENDS) for the administration of inhaled terbutaline was assessed. METHODS: The formulation of e-liquid was carried out using terbutaline in combination with 1, 3- propanediol. Several terbutaline concentrations (from 0.3125 to 2.500 mg / mL) and power levels (from 15 to 35 W) were assessed using a box type ENDS. The respirable drug dose was determined using a Glass Twin Impinger and quantified by liquid chromatography coupled with a UV-detector. The Next Generation Impactor and the Dekati Low Pressure Impactor were used to measure the aerosol particle size distribution in drug mass. The results were compared with a jet nebulizer (Cirrus TM 2) similar to the usual clinical conditions (2 mL at [terbutaline] of 2.5 mg / mL). RESULTS: The optimal conditions to maximize terbutaline delivery using ENDS are a drug concentration at 1 mg/mL, and a power level at 30 W, to reach a respirable dose of 8.73 ± 0.90 µg/puff. By contrast, during a 5 min nebulization, the respirable dose of terbutaline was 1040 ± 33 µg whatever the cascade impactors and the aerosol devices used. The mass median aerodynamic diameter (MMAD) remains similar for jet nebulizer and ENDS in the 1.74-2.07 µm range. CONCLUSION: Compared to the jet nebulizer, a same respirable dose of terbutaline at the same range of aerosol size distribution was delivered by ENDS if 120 puffs were performed. The ENDS can be considered as an alternative aerosol device for terbutaline delivery.

Évolution des comportements tabagique et de vapotage d'adolescents français de 15 à 18 ans (2018-2020).

Introduction : Longitudinal studies make it possible to study the evolution of a class of individuals over time. However, they are rarely used in France to observe the same population of young adolescents according to their grade level over the course of several years.Purpose of research : Based on the longitudinal monitoring of repeated annual cross-sectional studies on young adolescents (from 15 to 18 years old) conducted at the Honoré d’Urfé public high school (Saint-Étienne, Loire, France) involving 336 10th grade pupils in 2018, 360 11th grade pupils in 2019 and 273 12th grade pupils in 2020, we observed and studied students’ smoking and vaping behavior over a period of three years.Results : Between the ages of 15 and 18 years, the prevalence of tobacco experimentation increased almost linearly (39.10%, 47.50%, and 53.11%), while the prevalence of ‘non-smoking’ decreased by 4.5%. In addition, the prevalence of e-cigarette experimentation was stable (46.38%, 49.44%, 47.62%), whereas the prevalence of daily vaping increased by 5.0%.Conclusions : The prevalence of tobacco experimentation tends to increase with age and to catch up with that of e-cigarette experimentation, which remains stable at this stage of adolescence. Introductions to these two products seem to be rather recreational, playful, and experimental in nature, and do not have a significant connection with the development of their daily use or propensity to addiction.

Graphene-Based Materials In Vitro Toxicity and Their Structure-Activity Relationships: A Systematic Literature Review.

The unique properties of graphene-based materials (GBMs) placed them among the most exciting nanomaterials of the past decade. Scientists and industry are looking forward to working with not only efficient but also safe, sustainable GBMs. Designing a safer-by-design GBM implies to acquire the knowledge of which physicochemical characteristics (PCCs) can increase toxicity. In this systematic review, we extracted data from the literature to provide the available information about the structure-activity relationship of GBMs. 93 papers studying a total of 185 GBMs are included. Graphene oxides (GOs) and few-layer graphenes (FLGs) are the most studied GBMs. While reduced graphene oxides were often classified as poorly oxidant and weakly cytotoxic, graphene quantum dots were mostly moderately or highly cytotoxic. FLGs demonstrated relationships between median size and oxidative stress, between lateral size and both cytotoxicity and oxidative stress, and between thickness and cytotoxicity. We also underline relationships between median size, lateral size, and thickness of GOs and oxidative stress. However, it appears difficult to highlight clear structure-activity relationships for most PCCs and biological end points because despite a large amount of available data, the GBMs are often too poorly characterized in terms of PCCs descriptors and the biological end points investigation is not standardized enough. There is an urgent need for a better standardization of the experimental investigation of both PCCs and biological end points to allow research teams to play a part in the collaborative work toward the construction of a safer-by-design GBM through a better understanding of their key toxicity drivers.

Short Preirradiation of TiO2 Nanoparticles Increases Cytotoxicity on Human Lung Coculture System.

Anatase titanium dioxide nanoparticles (TiO2 NPs) are used in a large range of industrial applications mainly due to their photocatalytic properties. Before entering the lung, virtually all TiO2 NPs are exposed to some UV light, and lung toxicity of TiO2 NPs might be influenced by photoexcitation that is known to alter TiO2 surface properties. Although the TiO2 NPs toxicity has been extensively investigated, limited data are available regarding the toxicity of TiO2 NPs that have been pre-exposed to UV light, and their impact on humans remains unknown. In this study, five types of TiO2NPs with tailored physicochemical features were characterized and irradiated by UV for 30 min. Following irradiation, cytotoxicity, pro-inflammatory response, and oxidative stress on a human lung coculture system (A549 epithelial cells and macrophages differentiated from THP-1 cells) were assessed. The surface charge of all samples was less negative after UV irradiation of TiO2 NPs, and the average aggregate size was slightly increased. A higher cytotoxic effect was observed for preirradiated TiO2 NPs compared to nonirradiated samples. Preirradiation of TiO2 NPs had no significant impact on the pro-inflammatory response and oxidative stress as shown by a similar production of IL-8, TNF-α, and reactive oxygen species.

Assessing biological oxidative damage induced by graphene-based materials: An asset for grouping approaches using the FRAS assay.

Graphene-based materials (GBMs) are extremely promising and their increasing number urges scientists to conduct more and more toxicity studies. However, case-by-case approaches are rarely the best options in the earliest phases of industrial processes. Grouping can show great assets in this context: it is defined as the process of gathering substances into a common group. Oxidative stress being a major mechanism of nanotoxicity, an important grouping criterion is the surface reactivity, for which a relevant assessment is the FRAS (ferric reducing ability of the serum) assay. However, the application of the FRAS to GBMs is questioned due to their hydrophobicity. In this study, we explored the relevance and feasibility of the FRAS for grouping, working on 22 GBMs and 2 carbon blacks. We concluded that with few adjustments, the FRAS method appeared perfectly adapted to these materials and allowed a classification as "reactive" or "non-reactive" in agreement with results of ROS production for 84% of our GBMs. While not self-sufficient for toxicity assessment, the FRAS presents interesting qualities: it is fast, cheap, and simple. Therefore, we recommend studying GBMs using the FRAS as a step of a grouping process, a complement to other assays or as an early screening tool.

Occupational exposure during metal additive manufacturing: A case study of laser powder bed fusion of aluminum alloy.

Occupational exposure during metal additive manufacturing (Laser Powder Bed Fusion) using an aluminum alloy (AlSi10Mg) was assessed. Background aerosols before manufacturing, powder sieving, machine loading, manufacturing, machine unloading, powder unpacking, and machine cleaning were analyzed. Measurements were taken simultaneously at the source, in the near field, and on the operator during five manufacturing cycles. Aerosol measurement devices and physico-chemical techniques were used to determine the particle number or mass concentration (DiSCmini, core particle counter and sampling cassette), particle size distribution (NanoScan, optical particle detector and impactor), and the shape/size and chemical compositions of the inhalable particles (laser diffraction, inductively coupled plasma spectroscopy, scanning electron microscopy, energy dispersive X-ray microanalysis, and Brunauer-Emmett-Teller Method). The laser powder-bed fusion machine emitted in the additive manufacturing room an inhalable fraction of 2.37 ± 0.35 mg/m3, with an aerosol number concentration ranging from 2 × 104 to 105 #/cm3 and a mass mean aerodynamic diameter of 318 nm. A relatively low concentration level was observed in the near field of the machine with an aerosol number concentration of ∼104 #/cm3. A higher concentration level on the operator was attained during the unpacking and cleaning steps, showing an inhalable fraction of 1.73 ± 0.30 mg/m3. Al and Mg nanoparticles were aerosolized at the source (inside the laser powder-bed fusion machine) with a particle size distribution of 153 nm for Al and 117 nm for Mg and an aerosol number concentration reaching ten times that of the background aerosol level. The number or mass concentration of particles in the room atmosphere was increased to double that of the background aerosol level at specific workstations during manufacturing. Metal additive manufacturing is a source of potential occupational exposure to airborne metal nanoparticles. Particle-counting instruments showed high numbers of nanoparticles and some important peaks of particles ranging from 10 nm to 10 µm or larger at specific work tasks in the Additive Manufacturing (AM) environment. A multimetric approach was used to characterize the particle emissions resulting from this type of additive manufacturing.

Three-dimensional quantitative MRI of aerosolized gadolinium-based nanoparticles and contrast agents in isolated ventilated porcine lungs.

PURPOSE: The objective of this study is to evaluate the suitability and performance of ultra-short echo time (UTE) sequences for imaging and quantifying the deposition of nebulized MRI contrast agents in human-sized lungs. METHODS: Nebulization of clinically used contrast agent or gadolinium-based nanoparticles were performed using a commercial jet nebulizer in isolated and ventilated porcine lungs connected to a 3D-printed human upper airways replica. MR images of isolated lungs were acquired on a 3T clinical MR scanner using 3D UTE sequences at different flip angles. RESULTS: 3D acquisitions with isotropic millimetric resolution were obtained in less than 4 min. Images exhibit homogeneous and large MR signal enhancement (above 200%) following nebulization of both types of aerosols. Deposition of aerosol down to the level of the bronchi of secondary lobules was visualized. T1 values and the concentration of nanoparticles obtained by MRI were found to correlate with the amount of nebulized gadolinium3+ ions. CONCLUSION: The distribution of aerosolized gadolinium-based contrast agent or nanoparticles can be visualized and quantified using UTE MRI in large animal ventilated lung model on a clinical MRI scanner. This protocol can be used for assessing and quantifying aerosol regional deposition with high spatial resolution (1 mm 3D isotropic) without ionizing radiation and could be applied in the future for diagnostic or therapeutic applications in patients.

Impact of the Physicochemical Features of TiO2 Nanoparticles on Their In Vitro Toxicity.

The concern about titanium dioxide nanoparticles (TiO2-NPs) toxicity and their possible harmful effects on human health has increased. Their biological impact is related to some key physicochemical properties, that is, particle size, charge, crystallinity, shape, and agglomeration state. However, the understanding of the influence of such features on TiO2-NP toxicity remains quite limited. In this study, cytotoxicity, proinflammatory response, and oxidative stress caused by five types of TiO2-NPs with different physicochemical properties were investigated on A549 cells used either as monoculture or in co-culture with macrophages differentiated from the human monocytic THP-1 cells. We tailored bulk and surface TiO2 physicochemical properties and differentiated NPs for size/specific surface area, shape, agglomeration state, and surface functionalization/charge (aminopropyltriethoxysilane). An impact on the cytotoxicity and to a lesser extent on the proinflammatory responses depending on cell type was observed, namely, smaller, large-agglomerated TiO2-NPs were shown to be less toxic than P25, whereas rod-shaped TiO2-NPs were found to be more toxic. Besides, the positively charged particle was slightly more toxic than the negatively charged one. Contrarily, TiO2-NPs, whatever their physicochemical properties, did not induce significant ROS production in both cell systems compared to nontreated control groups. These results may contribute to a better understanding of TiO2-NPs toxicity in relation with their physicochemical features.

Importance of Choosing Relevant Biological End Points To Predict Nanoparticle Toxicity with Computational Approaches for Human Health Risk Assessment.

Because it is impossible to assess in vitro or in vivo the toxicity of all nanoparticles available on the market on a case-by-case basis, computational approaches have been proposed as useful alternatives to predict in silico the hazard potential of engineered nanoparticles. Despite promising results, a major issue associated with these mathematical models lies in the a priori choice of the physicochemical descriptors and the biological end points. We performed a thorough bibliographic survey on the biological end points used for nanotoxicology purposes and compared them between experimental and computational approaches. They were found to be disparate: while conventional in vitro nanotoxicology assays usually investigate a large array of biological effects using eukaryotic cells (cytotoxicity, pro-inflammatory response, oxidative stress, genotoxicity), computational studies mostly focus on cell viability and also include studies on prokaryotic cells. We may thus wonder the relevance of building complex mathematical models able to predict accurately a biological end point if this latter is not the most relevant to support human health risk assessment. The choice of biological end points clearly deserves to be more carefully discussed. This could bridge the gap between experimental and computational nanotoxicology studies and allow in silico predictive models to reach their full potential.

Ex vivo detection and quantification of gold nanoparticles in human seminal and follicular fluids.

Increasing consumption of engineered nanoparticles and occupational exposure to novel, ultrafine airborne particles during the last decades has coincided with deterioration of sperm parameters and delayed fecundity. In order to prevent possible adverse health effects and ensure a sustainable growth for the nanoparticle industry, the ability to investigate the nanosized, mineralogical load of human reproductive systems is becoming a real clinical need. Toward this goal, the current study proposes two methods for the detection and quantification of engineered nanoparticles in human follicular and seminal fluid, developed with the use of well-defined 60 nm Au particles. Despite the complexity of these biological fluids, simple physical and chemical treatments allow for the precise quantification of more than 50 and 70% wt of the spiked Au nanoparticles at low µg ml-1 levels in follicular and seminal fluids, respectively. The use of electron microscopy for the detailed observation of the detected analytes is also enabled. The proposed method is applied on a small patient cohort in order to demonstrate its clinical applicability by exploring the differences in the metal and particulate content between patients with normal and low sperm count.

Biological Monitoring of Inhaled Nanoparticles in Patients: An Appealing Approach To Study Causal Link between Human Respiratory Pathology and Exposure to Nanoparticles.

Although necessary, in vitro and in vivo studies are not fully successful at predicting nanomaterials toxicity. We propose to associate such assays to the biological monitoring of nanoparticles in clinical samples to get more relevant data on the chemical and physical nature and dose of nanoparticles found in humans. The concept is to establish the load of nanoparticles in biological samples of patients. Then, by comparing samples from different patient groups, nanoparticles of interest could be identified and a potential link between a given nanoparticle type and toxicity could be suggested. It must be confirmed by investigating the biological effects induced by these nanoparticles using in vitro or in vivo models (mechanistic or dose-response studies). This translational approach from the bedside to the bench and vice versa could allow a better understanding of the nanoparticle effects and mechanisms of toxicity that can contribute, at least in part, to a disease.

Micron-sized and submicron-sized aerosol deposition in a new ex vivo preclinical model.

BACKGROUND: The knowledge of where particles deposit in the respiratory tract is crucial for understanding the health effects associated with inhaled drug particles. METHOD: An ex vivo study was conducted to assess regional deposition patterns (thoracic vs. extrathoracic) of radioactive polydisperse aerosols with different size ranges [0.15 µm-0.5 µm], [0.25 µm-1 µm] and [1 µm-9 µm]. SPECT/CT analyses were performed complementary in order to assess more precisely the regional deposition of aerosols within the pulmonary tract. Experiments were set using an original respiratory tract model composed of a human plastinated head connected to an ex vivo porcine pulmonary tract. The model was ventilated by passive expansion, simulating pleural depressions. Aerosol was administered during nasal breathing. RESULTS: Planar scintigraphies allowed to calculate the deposited aerosol fractions for particles in the three size ranges from sub-micron to micron The deposited fractions obtained, for thoracic vs. extra-thoracic regions respectively, were 89 ± 4 % vs. 11 ± 4 % for [0.15 µm-0.5 µm], 78 ± 5 % vs. 22 ± 5 % for [0.25 µm-1 µm] and 35 ± 11 % vs.65 ± 11 % for [1 µm-9 µm]. CONCLUSION: Results obtained with this new ex vivo respiratory tract model are in good agreement with the in vivo data obtained in studies with baboons and humans.

Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages.

To improve the biological properties of calcium phosphate (CaP) bone substitute, new chemical compositions are under development. In vivo such materials are subject to degradation that could lead to particles release and inflammatory reactions detrimental to the bone healing process. This study aimed at investigating the interactions between a murine macrophage cell line (RAW 264.7) and substituted hydroxyapatite particles presenting promising biological properties. Micron size particles of stoichiometric and substituted hydroxyapatites (CO3 substitution for PO4 and OH; SiO4 substitution for PO4; CO3 and SiO4 co-substitution) were obtained by aqueous precipitation followed by spray drying. Cells, incubated with four doses of particles ranging from 15 to 120 µg/mL, revealed no significant LDH release or ROS production, indicating no apparent cytotoxicity and no oxidative stress. TNF-α production was independent of the chemistry of the particles; however the particles elicited a significant dose-dependent pro-inflammatory response. As micron size particles of these hydroxyapatites could be at the origin of inflammation, attention must be paid to the degradation behavior of substituted hydroxyapatite bone substitute in order to limit, in vivo, the generation of particulate debris.

A new Strategy to Improve Drug Delivery to the Maxillary Sinuses: The Frequency Sweep Acoustic Airflow.

PURPOSE: Enhancement of intranasal sinus deposition involves nebulization of a drug superimposed by an acoustic airflow. We investigated the impact of fixed frequency versus frequency sweep acoustic airflow on the improvement of aerosolized drug penetration into maxillary sinuses. METHODS: Fixed frequency and frequency sweep acoustic airflow were generated using a nebulizing system of variable frequency. The effect of sweep cycle and intensity variation was studied on the intranasal sinus deposition. We used a nasal replica created from CT scans using 3D printing. Sodium fluoride and gentamicin were chosen as markers. RESULTS: Studies performed using fixed frequency acoustic airflow showed that each of maxillary sinuses of the nasal replica required specific frequency for the optimal aerosol deposition. Intranasal sinus drug deposition experiments under the effect of the frequency sweep acoustic airflow showed an optimal aerosol deposition into both maxillary sinus of the nasal replica. Studies on the effect of the duration of the sweep cycle showed that the shorter the cycle the better the deposition. CONCLUSIONS: We demonstrate the benefit of frequency sweep acoustic airflow on drug deposition into maxillary sinuses. However further in vivo studies have to be conducted since delivery rates cannot be obviously determined from a nasal replica.

Testicular biodistribution of silica-gold nanoparticles after intramuscular injection in mice.

With the continuing development of nanomaterials, the assessment of their potential impact on human health, and especially human reproductive toxicity, is a major issue. The testicular biodistribution of nanoparticles remains poorly studied. This study investigated whether gold-silica nanoparticles could be detected in mouse testes after intramuscular injection, with a particular focus on their ability to cross the blood-testis barrier. To that purpose, well-characterized 70-nm gold core-silica shell nanoparticles were used to ensure sensitive detection using high-resolution techniques. Testes were collected at different time points corresponding to spermatogenesis stages in mice. Transmission electron microscopy and confocal microscopy were used for nanoparticle detection, and nanoparticle quantification was performed by atomic emission spectroscopy. All these techniques showed that no particles were able to reach the testes. Results accorded with the normal histological appearance of testes even at 45 days post sacrifice. High-resolution techniques did not detect 70-nm silica-gold nanoparticles in mouse testes after intramuscular injection. These results are reassuring about the safety of nanoparticles with regard to male human reproduction, especially in the context of nanomedicine.

Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.

Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes…). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification.

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

Carbon nanotube (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 for nanosized materials. Indeed, because of their unique physicochemical 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 under 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 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 K(ads) and n(eq) 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.