Assessing the inhaled dose of nanomaterials by nanoparticle tracking analysis (NTA) of exhaled breath condensate (EBC) and its relationship with lung inflammatory biomarkers.

The widespread and increasing use of nanomaterials has resulted in a higher likelihood of exposure by inhalation for nanotechnology workers. However, tracking the internal dose of nanoparticles deposited at the airways level, is still challenging. To assess the suitability of particle number concentration determination as biomarker of internal dose, we carried out a cross sectional investigation involving 80 workers handling nanomaterials. External exposure was characterized by portable counters of particles DISCminiTM (Testo, DE), allowing to categorize 51 workers as exposed and 29 as non-exposed (NE) to nanoparticles. Each subject filled in a questionnaire reporting working practices and health status. Exhaled breath condensate was collected and analysed for the number of particles/ml as well as for inflammatory biomarkers. A clear-cut relationship between the number of airborne particles in the nano-size range determined by the particle counters and the particle concentration in exhaled breath condensate (EBC) was apparent. Moreover, inflammatory cytokines (IL-1ß, IL-10, and TNF-α) measured in EBC, were significantly higher in the exposed subjects as compared to not exposed. Finally, significant correlations were found between external exposure, the number concentration of particles measured by the nanoparticle tracking analysis (NTA) and inflammatory cytokines. As a whole, the present study, suggests that NTA can be regarded as a reliable tool to assess the inhaled dose of particles and that this dose can effectively elicit inflammatory effects.

Advanced materials - Food grade melatonin-loaded Lipid Surfactant Submicron Particles (LSSP)-environmental impacts.

Lipid-based nanoparticles (LNPs) are advanced materials (AdMa), particularly relevant for drug delivery of poorly water-soluble compounds, while also providing protection, stabilization, and controlled release of the drugs/active substances. The toxicological data available often focus on the specific applications of the LNPs-drug tested, with indication of low toxicity. However, the ecotoxicological effects of LNPs are currently unknown. In the present study, we investigated the ecotoxicity of a formulation of Lipid Surfactant Submicron Particles (LSSPs) loaded with melatonin at 1 mg/mL. The LSSPs formulation has been developed to be fully compliant with regulatory for its potential use in the market and all components are food additives. The same formulation without the thickening agent xanthan gum (stabilizer in water phase) designated as LSSP-xg, was also tested. Two soil model invertebrate species were tested in LUFA 2.2 soil: Enchytraeus crypticus (Oligochaeta) and Folsomia candida (Collembola). Effects were assessed based on the OECD standard guideline (28 days) and its extension, the longer-term exposure (56 days). Assessed endpoints were survival, reproduction, and size. LSSPs and LSSP-xg were toxic to E. crypticus and F. candida reducing their survival and reproduction in a dose-dependent way: e.g., 28-day exposure: E. crypticus: LC/EC50 = 30/15 mg LSSPs/kg soil and F. candida LC/EC50 = 55/44 mg LSSPs/kg soil, with similar values for LSSP-xg. Size was also reduced for F. candida but was the least sensitive endpoint. There were no indications that toxicity increased with longer term exposure. The results provide relevant information on ecotoxicity of a AdMa and highlights the need for awareness of the potential risks, even on products and additives usually used in food or cosmetic industry. Further information on single components and on their specific assembly is necessary for the interpretation of results, as it is not fully clear what causes the toxicity in this specific AdMa. This represents a typical challenge for AdMa hazard assessment scenario.

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

A New Paclitaxel Formulation Based on Secretome Isolated from Mesenchymal Stem Cells Shows a Significant Cytotoxic Effect on Osteosarcoma Cell Lines.

BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.

Photoresponsive Inorganic Nanomaterials in Oncology.

The diagnosis and treatment of cancer are continuously evolving in search of more efficient, safe, and personalized approaches. Therapies based on nanoparticles or physical stimuli-responsive substances have shown great potential to overcome the inherent shortcomings of conventional cancer therapies. In fact, nanoparticles may increase the half-life of chemotherapeutic agents or promote the targeting in cancer tissues while physical stimuli-responsive substances are more effective and safer with respect to traditional chemotherapeutic agents because of the possibility to be switched on only when needed. These 2 approaches can be combined by exploiting the ability of some inorganic nanomaterials to be activated by light, ultrasounds, magnetic fields, or ionizing radiations. Albeit the development of stimuli-responsive materials is still at the early stages, research in this field is rapidly growing since they have important advantages with respect to organic nanoparticles or molecular substances, like higher stability, and higher efficiency in converting the stimulus in heat or, in some cases, reactive oxygen species. On the other hand, the translation process is slowed down by issues related to safety and quality of the formulations. This literature review summarizes the current advancements in this research field, analysing the most promising materials and applications.

Biomineralization: A new tool for developing eco-sustainable Ti-doped hydroxyapatite-based hybrid UV filters.

It is well known that the prolonged exposure to UV radiation from sunlight can compromise human health and is particularly damaging to the skin, leading to sunburn, photo-aging and skin cancer. Sunscreen formulations containing UV-filters present a barrier against solar UV and help to mitigate the harmful effects however, concern about their safety for both human and environmental health is still a much-debated topic. EC regulations classify UV-filters depending on their chemical nature, particle size, and mechanism of action. Furthermore, it regulates their use in cosmetic products with specific limitations in terms of concentration (organic UV filters) and particle size and surface modification to reduce their photo-activity (mineral UV filters). The regulations have prompted researchers to identify new materials that show promise for use in sunscreens. In this work, biomimetic hybrid materials composed of titanium-doped hydroxyapatite (TiHA) grown on two different organic templates, derived from animal (gelatin - from pig skin) and vegetable (alginate - from algae) sources. These novel materials were developed and characterized to obtain sustainable UV-filters as a safer alternative for both human and ecosystem health. This 'biomineralization' process yielded TiHA nanoparticles that demonstrated high UV reflectance, low photoactivity, good biocompatibility and an aggregate morphology which prevents dermal penetration. The materials are safe for topical application and for the marine environment; moreover, they can protect organic sunscreen components from photodegradation and yield long-lasting protection.

Nano- and microplastics: a comprehensive review on their exposure routes, translocation, and fate in humans.

Contamination of the environment with nano-and microplastic particles (NMPs) and its putative adverse effects on organisms, ecosystems, and human health is gaining increasing scientific and public attention. Various studies show that NMPs occur abundantly within the environment, leading to a high likelihood of human exposure to NMPs. Here, different exposure scenarios can occur. The most notable exposure routes of NMPs into the human body are via the airways and gastrointestinal tract (GIT) through inhalation or ingestion, but also via the skin due to the use of personal care products (PCPs) containing NMPs. Once NMPs have entered the human body, it is possible that they are translocated from the exposed organ to other body compartments. In our review article, we combine the current knowledge on the (1) exposure routes of NMPs to humans with the basic understanding of the potential (2) translocation mechanisms into human tissues and, consequently, their (3) fate within the human body. Regarding the (1) exposure routes, we reviewed the current knowledge on the occurrence of NMPs in food, beverages, personal care products and the air (focusing on indoors and workplaces) and found that the studies suggest an abundant presence of MPs within the exposure scenarios. The overall abundance of MPs in exposure matrices relevant to humans highlights the importance of understanding whether NMPs have the potential for tissue translocation. Therefore, we describe the current knowledge on the potential (2) translocation pathways of NMPs from the skin, GIT and respiratory systems to other body compartments. Here, particular attention was paid to how likely NMPs can translocate from the primary exposed organs to secondary organs due to naturally occurring defence mechanisms against tissue translocation. Based on the current understanding, we conclude that a dermal translocation of NMPs is rather unlikely. In contrast, small MPs and NPs can generally translocate from the GIT and respiratory system to other tissues. Thus, we reviewed the existing literature on the (3) fate of NMPs within the human body. Based on the current knowledge of the contamination of human exposure routes and the potential translocation mechanisms, we critically discuss the size of the detected particles reported in the fate studies. In some cases, the particles detected in human tissue samples exceed the size of a particle to overcome biological barriers allowing particle translocation into tissues. Therefore, we emphasize the importance of critically reading and discussing the presented results of NMP in human tissue samples.

Inhibitory Effect against Listeria monocytogenes of Carbon Nanoparticles Loaded with Copper as Precursors of Food Active Packaging.

Human listeriosis is a serious foodborne disease of which outbreaks are occurring increasingly frequently in Europe. Around the world, different legal requirements exist to guarantee food safety. Nanomaterials are increasingly used in the food industry as inhibitors of pathogens, and carbon nanomaterials are among the most promising. In the present study, novel carbon nanoparticles loaded with copper (CNP-Cu) were prepared, and their antimicrobial activity against Listeria monocytogenes was assessed. CNPs of two sizes were synthesized and characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and electron microscopy (EM). The minimum inhibitory concentration (MIC) of CNP-Cu was determined in accordance with the available standard. To get insights into its mechanism of action, the release of copper ions into a cell media was assessed by inductively coupled plasma optical emission (ICP-OE), and the ability of loaded CNPs to generate cytotoxic reactive oxygen species (ROS) was evaluated by EPR spectroscopy. Finally, the extent of release of copper in a food simulant was assessed. The results demonstrated the antimicrobial effectiveness of CNP-Cu, with growth inhibition up to 85% and a release of copper that was more pronounced in an acidic food simulant. Overall, the results indicate CNP-Cu as a promising agent for the design of active food packaging which is able to improve food shelf-life.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Molecular Aspects of the Interaction with Gram-Negative and Gram-Positive Bacteria of Hydrothermal Carbon Nanoparticles Associated with Bac8c2,5Leu Antimicrobial Peptide.

Antimicrobial peptides (AMPs) are widely studied as therapeutic agents due to their broad-spectrum efficacy against infections. However, their clinical use is hampered by the low in vivo bioavailability and systemic toxicity. Such limitations might be overcome by using appropriate drug delivery systems. Here, the preparation of a drug delivery system (DDS) by physical conjugation of an arginine-rich peptide and hydrothermal carbon nanoparticles (CNPs) has been explored, and its antimicrobial efficacy against Eschericia coli (E. coli) and Staphylococcus aureus investigated in comparison with the unloaded carrier and the free peptide. The mechanism of interaction between CNPs and the bacteria was investigated by scanning electron microscopy and a combined dielectrophoresis-Raman spectroscopy method for real-time analysis. In view of a possible systemic administration, the effect of proteins on the stability of the DDS was investigated by using albumin as a model protein. The peptide was bounded electrostatically to the CNPs surface, establishing an equilibrium modulated by pH and albumin. The DDS exhibited antimicrobial activity toward the two bacterial strains, albeit lower as compared to the free peptide. The decrease in effectiveness toward E. coli was likely due to the rapid formation of a particle-induced extracellular matrix. The present results are relevant for the future development of hydrothermal CNPs as drug delivery agents of AMPs.

Biological interactions of ferromagnetic iron oxide-carbon nanohybrids with alveolar epithelial cells.

Iron oxide nanoparticles (IONPs) have been largely investigated in a plethora of biological fields for their interesting physical-chemical properties, which make them suitable for application in cancer therapy, neuroscience, and imaging. Several encouraging results have been reported in these contexts. However, the possible toxic effects of some IONP formulations can limit their applicability. In this work, IONPs were synthesized with a carbon shell (IONP@C), providing enhanced stability both as colloidal dispersion and in the biological environment. We conducted a careful multiparametric evaluation of IONP@C biological interactions in vitro, providing them with an in vivo-like biological identity. Our hybrid nanoformulation showed no cytotoxic effects on a widely employed model of alveolar epithelial cells for a variety of concentrations and exposure times. The IONP@C were efficiently internalized and TEM analysis allowed the protective role of the carbon shell against intracellular degradation to be assessed. Intracellular redistribution of the IONP@C from the lysosomes to the lamellar bodies was also observed after 72 hours.

A Biomonitoring Pilot Study in Workers from a Paints Production Plant Exposed to Pigment-Grade Titanium Dioxide (TiO2).

Among particulate matter composing paints, titanium dioxide (TiO2) forms about 20% of the final suspension. Although TiO2 is broadly used in many applications, TiO2 powders represent an established respiratory hazard for workers with long-term exposure. In 35 workers of a paints production plant (15 exposed and 20 not exposed), we assessed pro-inflammatory cytokines (IL-1ß, TNF-α, IL-10, IL-17), surfactant protein D (SP-D) and Krebs von den Lungen-6 glycoprotein (KL-6) in exhaled breath condensate (EBC). In urine samples, we measured 8-isoprostane (Isop) and Malondialdehyde (MDA) as biomarkers of oxidative stress, and Titanium (Ti-U) as a biomarker of exposure. Health status, habits and occupational history were recorded. Airborne respirable dusts and Ti were quantified. Particle number concentration and average diameter (nm) were detected by a NanoTracer™ monitoring device. Ti was measurable in filters collected at the respiratory breathing zone (0.11−0.44 µg/m3 8-h TWA). IL-1ß and IL-10 values were significantly higher in exposed workers, whereas SP-D was significantly lower (p < 0.001). KL-6 was significantly higher in workers than in controls (p < 0.01). MDA levels were significantly increased in exposed workers and were positively correlated with Ti-U. Exposure to TiO2 in paint production is associated with the subtle alterations of lung pathobiology. These findings suggest the need for an integrated approach relying on both personal exposure and biomarker assessment to improve the hazard characterisation in occupational settings.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate.

Out of the five isoforms of human flavin-containing monooxygenase (hFMO), FMO1 and FMO3 are the most relevant to Phase I drug metabolism. They are involved in the oxygenation of xenobiotics including drugs and pesticides using NADPH and FAD as cofactors. Majority of the characterization of these enzymes has involved hFMO3, where intermediates of its catalytic cycle have been described. On the other hand, research efforts have so far failed in capturing the same key intermediate that is responsible for the monooxygenation activity of hFMO1. In this work we demonstrate spectrophotometrically the formation of a highly stable C4a-hydroperoxyflavin intermediate of hFMO1 upon reduction by NADPH and in the presence of O2. The measured half-life of this flavin intermediate revealed it to be stable and not fully re-oxidized even after 30 min at 15 °C in the absence of substrate, the highest stability ever observed for a human FMO. In addition, the uncoupling reactions of hFMO1 show that this enzyme is <1% uncoupled in the presence of substrate, forming small amounts of H2O2 with no observable superoxide as confirmed by EPR spin trapping experiments. This behaviour is different from hFMO3, that is shown to form both H2O2 and superoxide anion radical as a result of ∼50% uncoupling. These data are consistent with the higher stability of the hFMO1 intermediate in comparison to hFMO3. Taken together, these data demonstrate the different behaviours of these two closely related enzymes with consequences for drug metabolism as well as possible toxicity due to reactive oxygen species.

Efficacy, biocompatibility and degradability of carbon nanoparticles for photothermal therapy of lung cancer.

Aim: To investigate near infrared-induced phototoxicity toward lung cancer cells, and the biodegradability and effect on immune cells of glucose-derived carbon nanoparticles (CNPs). Methods: The human A549 lung adenocarcinoma cell line was used as a model to study the phototoxicity of CNPs. The biodegradability and the effect on immune cells was demonstrated in primary human neutrophils and macrophages. Results: Near infrared-activated CNPs elicited rapid cell death, characterized by the elevation of heat shock proteins and the induction of DNA damage. CNPs were found to be noncytotoxic toward primary human macrophages and were susceptible to biodegradation when cocultured with human neutrophils. Conclusions: Our results identify CNPs as promising platforms for photothermal therapy of lung cancer.

Occupational Exposure to Carbon Nanotubes and Carbon Nanofibres: More Than a Cobweb.

Carbon nanotubes (CNTs) and carbon nanofibers (CNFs) are erroneously considered as singular material entities. Instead, they should be regarded as a heterogeneous class of materials bearing different properties eliciting particular biological outcomes both in vitro and in vivo. Given the pace at which the industrial production of CNTs/CNFs is increasing, it is becoming of utmost importance to acquire comprehensive knowledge regarding their biological activity and their hazardous effects in humans. Animal studies carried out by inhalation showed that some CNTs/CNFs species can cause deleterious effects such as inflammation and lung tissue remodeling. Their physico-chemical properties, biological behavior and biopersistence make them similar to asbestos fibers. Human studies suggest some mild effects in workers handling CNTs/CNFs. However, owing to their cross-sectional design, researchers have been as yet unable to firmly demonstrate a causal relationship between such an exposure and the observed effects. Estimation of acceptable exposure levels should warrant a proper risk management. The aim of this review is to challenge the conception of CNTs/CNFs as a single, unified material entity and prompt the establishment of standardized hazard and exposure assessment methodologies able to properly feed risk assessment and management frameworks.

Mechanistic Insights into the Role of Iron, Copper, and Carbonaceous Component on the Oxidative Potential of Ultrafine Particulate Matter.

Transition metals play a key role in the pathogenic potential of urban particulate matter (PM). However, air quality regulations include exposure limits only for metals having a known toxic potential like Pb, As, Cd, and Ni, neglecting other transition metals like Fe and Cu. Fe and Cu are mainly found in the water-soluble fraction of PM. However, a fraction of the ions may persist strongly bound to the particles, thus potentially acting as surface reactive sites. The contribution of surface ions to the oxidative potential (OP) of PM is likely different from that of free ions since the redox activity of metals is modulated by their local chemical environment. The aim of this study was to investigate how Fe and Cu bound to carbonaceous particles affect the OP and associated toxicity of PM toward epithelial cells and macrophages. Carbonaceous nanoparticles (CNPs) having well-defined size were loaded with controlled amounts of Cu and Fe. The effect of Cu and Fe on the OP of CNPs was evaluated by electronic paramagnetic resonance (EPR) spectroscopy associated with the spin-trapping technique and correlated with the ability to induce cytotoxicity (LDH, WST-1), oxidative stress (Nrf2 translocation), and DNA damage (comet assay) on lung macrophages (NR8383) and/or epithelial cells (RLE-6TN). The release of pro-inflammatory cytokines (TNF-α, MCP-1, and CXCL2) by macrophages and epithelial cells was also investigated. The results indicate a major contribution of surface Cu to the surface reactivity of CNPs, while Fe has a minor role. At the same time, Cu increases the cytotoxicity of CNPs and their ability to induce oxidative stress and DNA damage. In contrast, surface Fe increases the release of pro-inflammatory cytokines by macrophages. Overall, these results confirm the role of Cu and Fe in PM toxicity and suggest that the total metals content in PM might be a better indicator of pathogenicity than water-soluble metals.

Biotransformation of Food-Grade and Nanometric TiO2 in the Oral-Gastro-Intestinal Tract: Driving Forces and Effect on the Toxicity toward Intestinal Epithelial Cells.

Background: Oral exposure to titanium dioxide (TiO2) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO2. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO2. This might be due to the different biological identity of TiO2 in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO2 and two nanometric TiO2 samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering-DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering-ELS-, Electron Paramagnetic Resonance Spectroscopy-EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO2 particles surface and reactivity. Pristine and treated TiO2 nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.

Identification of physicochemical properties that modulate nanoparticle aggregation in blood.

Inorganic materials are receiving significant interest in medicine given their usefulness for therapeutic applications such as targeted drug delivery, active pharmaceutical carriers and medical imaging. However, poor knowledge of the side effects related to their use is an obstacle to clinical translation. For the development of molecular drugs, the concept of safe-by-design has become an efficient pharmaceutical strategy with the aim of reducing costs, which can also accelerate the translation into the market. In the case of materials, the application these approaches is hampered by poor knowledge of how the physical and chemical properties of the material trigger the biological response. Hemocompatibility is a crucial aspect to take into consideration for those materials that are intended for medical applications. The formation of nanoparticle agglomerates can cause severe side effects that may induce occlusion of blood vessels and thrombotic events. Additionally, nanoparticles can interfere with the coagulation cascade causing both pro- and anti-coagulant properties. There is contrasting evidence on how the physicochemical properties of the material modulate these effects. In this work, we developed two sets of tailored carbon and silica nanoparticles with three different diameters in the 100-500 nm range with the purpose of investigating the role of surface curvature and chemistry on platelet aggregation, activation and adhesion. Substantial differences were found in the composition of the protein corona depending on the chemical nature of the nanoparticles, while the surface curvature was found to play a minor role. On the other hand, large carbon nanoparticles (but not small carbon nanoparticles or silica nanoparticles) have a clear tendency to form aggregates both in plasma and blood. This effect was observed both in the presence or absence of platelets and was independent of platelet activation. Overall, the results presented herein suggest the existence of independent modes of action that are differently affected by the physicochemical properties of the materials, potentially leading to vessel occlusion and/or formation of thrombi in vivo.

Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS).

The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO2, SiO2, and CeO2 were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1ß production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO2 and CeO2 were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1ß releases were found for the SiO2, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

Pro- and anti-oxidant properties of near-infrared (NIR) light responsive carbon nanoparticles.

Elemental carbon nanomaterials (ECNMs) are redox active agents that can be exploited to purposely modify the redox balance of cells. Both pro- or antioxidant properties have been reported. However, to the best of our knowledge, there are not comprehensive studies exploring both properties on the same material in view of a potential application in medicine. At the same time, the effect of the bulk structure on the pro/antioxidant properties is poorly known. Here, carbon nanoparticles (CNPs) derived by glucose with definite size and shape have been prepared, and their redox properties evaluated in cell free systems in the dark or following activation with a Near Infrared (NIR) laser beam (945 nm, 1.3 W/cm2). We found that, when irradiated with NIR, CNPs efficiently generate heat and singlet oxygen (1O2), a property that can be exploited for dual photo-thermal (PT)/photodynamic (PD) therapy in cancer. On the other hand, in the absence of photo-activation, CNPs react with both oxidant (hydroxyl radicals) and antioxidant (glutathione) species. When tested on a murine macrophages cell line (RAW 264.7) CNPs were clearly antioxidant. Furthermore, albeit efficiently internalized, CNPs do not exert cytotoxic effect up to 80 µg/ml and do not exacerbate TNF-α-mediated inflammation. Overall, the results reported herein suggest that CNPs may represent a new class of safe nanomaterials with potential applications in medicine.