Impact of Nanoparticles on Male Fertility: What Do We Really Know? A Systematic Review.

The real impact of nanoparticles on male fertility is evaluated after a careful analysis of the available literature. The first part reviews animal models to understand the testicular biodistribution and biopersistence of nanoparticles, while the second part evaluates their in vitro and in vivo biotoxicity. Our main findings suggest that nanoparticles are generally able to reach the testicle in small quantities where they persist for several months, regardless of the route of exposure. However, there is not enough evidence that they can cross the blood-testis barrier. Of note, the majority of nanoparticles have low direct toxicity to the testis, but there are indications that some might act as endocrine disruptors. Overall, the impact on spermatogenesis in adults is generally weak and reversible, but exceptions exist and merit increased attention. Finally, we comment on several methodological or analytical biases which have led some studies to exaggerate the reprotoxicity of nanoparticles. In the future, rigorous clinical studies in tandem with mechanistic studies are needed to elucidate the real risk posed by nanoparticles on male fertility.

Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells.

Liquid deposit mimicking surface aerosolization in the airway is a promising strategy for targeting bronchopulmonary tumors with reduced doses of nanoparticle (NPs). In mimicking and studying such delivery approaches, the use of human in vitro 3D culture models can bridge the gap between 2D cell culture and small animal investigations. Here, we exposed airway epithelia to liquid-apical gadolinium-based AGuIX® NPs in order to determine their safety profile. We used a multiparametric methodology to investigate the NP's distribution over time in both healthy and tumor-bearing 3D models. AGuIX® NPs were able to target tumor cells in the absence of specific surface functionalization, without evidence of toxicity. Finally, we validated the therapeutic potential of this hybrid theranostic AGuIX® NPs upon radiation exposure in this model. In conclusion, 3D cell cultures can efficiently mimic the normal and tumor-bearing airway epitheliums, providing an ethical and accessible model for the investigation of nebulized NPs.

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.

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.

Toxicity of boehmite nanoparticles: impact of the ultrafine fraction and of the agglomerates size on cytotoxicity and pro-inflammatory response.

Boehmite (γ-AlOOH) nanoparticles (NPs) are used in a wide range of industrial applications. However, little is known about their potential toxicity. This study aimed at a better understanding of the relationship between the physico-chemical properties of these NPs and their in vitro biological activity. After an extensive physico-chemical characterization, the cytotoxicity, pro-inflammatory response and oxidative stress induced by a bulk industrial powder and its ultrafine fraction were assessed using RAW264.7 macrophages. Although the bulk powder did not trigger a significant biological activity, pro-inflammatory response was highly enhanced with the ultrafine fraction. This observation was confirmed with boehmite NPs synthesized at the laboratory scale, with well-defined and tightly controlled physico-chemical features: toxicity was increased when NPs were dispersed. In conclusion, the agglomerates size of boehmite NPs has a major impact on their toxicity, highlighting the need to study not only raw industrial powders containing NPs but also the ultrafine fractions representative of respirable particles.

Quantitative Flow Cytometric Evaluation of Oxidative Stress and Mitochondrial Impairment in RAW 264.7 Macrophages after Exposure to Pristine, Acid Functionalized, or Annealed Carbon Nanotubes.

Conventional nanotoxicological assays are subjected to various interferences with nanoparticles and especially carbon nanotubes. A multiparametric flow cytometry (FCM) methodology was developed here as an alternative to quantify oxidative stress, mitochondrial impairment, and later cytotoxic and genotoxic events. The experiments were conducted on RAW264.7 macrophages, exposed for 90 min or 24 h-exposure with three types of multiwalled carbon nanotubes (MWCNTs): pristine (Nanocyl™ CNT), acid functionalized (CNTf), or annealed treatment (CNTa). An original combination of reactive oxygen species (ROS) probes allowed the simultaneous quantifications of broad-spectrum ROS, superoxide anion (O2•-), and hydroxyl radical (•OH). All MWCNTs types induced a slight increase of broad ROS levels regardless of earlier antioxidant catalase activity. CNTf strongly stimulated the O2•- production. The •OH production was downregulated for all MWCNTs due to their scavenging capacity. The latter was quantified in a cell-free system by electron paramagnetic resonance spectroscopy (EPR). Further FCM-based assessment revealed early biological damages with a mitochondrial membrane potential collapse, followed by late cytotoxicity with chromatin decondensation. The combined evaluation by FCM analysis and cell-free techniques led to a better understanding of the impacts of MWCNTs surface treatments on the oxidative stress and related biological response.

Pharmacological characterization of the 3D MucilAir™ nasal model.

The preclinical evaluation of nasally administered drug candidates requires screening studies based on in vitro models of the nasal mucosa. The aim of this study was to evaluate the morpho-functional characteristics of the 3D MucilAir™ nasal model with a pharmacological focus on [ATP]-binding cassette (ABC) efflux transporters. We initially performed a phenotypic characterization of the MucilAir™ model and assessed its barrier properties by immunofluorescence (IF), protein mass spectrometry and examination of histological sections. We then focused on the functional expression of the ABC transporters P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)1, MRP2 and breast cancer resistance protein (BCRP) in bidirectional transport experiments. The MucilAir™ model comprises a tight, polarized, pseudo-stratified nasal epithelium composed of fully differentiated ciliated, goblet and basal cells. These ABC transporters were all expressed by the cell membranes. P-gp and BCRP were both functional and capable of actively effluxing substrates. The MucilAir™ model could consequently represent a potent tool for evaluating the interaction of nasally administered drugs with ABC transporters.

Chronic Oral Exposure to Synthetic Amorphous Silica (NM-200) Results in Renal and Liver Lesions in Mice.

INTRODUCTION: Silicon dioxide, produced as synthetic amorphous silica (SAS), is made of nanoparticles (NPs), either present as such or as agglomerates and aggregates, and is widely used in many types of food processes and products as an additive. To assess whether repeated, long-term exposure to SAS NPs may result in adverse effects, mice were exposed for 18 months via drinking water to NM-200, one of the reference nanostructured silica used for applications related to food, at 4.8 mg NM-200/kg body weight per day, a dose relevant to the estimated dietary exposure to SAS in humans. METHODS: The experiment focused on the kidney and liver as target organs and was carried out in parallel using 3 mouse lines (wild type and transgenic) differing for the expression of α-synuclein, that is, murine and human mutated (A53T). Sensitive determination of silicon revealed higher contents in liver and kidneys of NM-200-exposed mice compared with unexposed aged-matched controls. RESULTS: Histological abnormalities, such as vacuolization of tubular epithelial cells, were detected in all kidneys, as well as inflammatory responses that were also detected in livers of exposed animals. Less frequent but more deleterious, amyloidosis lesions were observed in glomeruli, associated with perivascular amyloid accumulation in liver. CONCLUSION: These histological findings, in conjunction with the observation of detectable deposition of silica, highlight that chronic oral intake of SAS may pose a health risk to humans and need to be examined further.

A method for the quantitative extraction of gold nanoparticles from human bronchoalveolar lavage fluids through a glycerol gradient.

Bronchoalveolar lavage (BAL) is a diagnostic procedure which samples the cellular and non-cellular components of the pulmonary epithelial surface. The inherent biological noise of BAL fluids inhibits their direct mineralogical analysis while currently available particle retrieval protocols are suspected to impose quantitative and qualitative bias on the studied particle load. This study presents a simple method for the near-lossless extraction of citrate-capped gold nanoparticles from human BAL fluids at sub-ppm levels which enables their quantitation and surface characterization. This procedure was modeled according to fundamental principles of particle sedimentation and liquid-liquid interdiffusion and was evaluated by a battery of analytical techniques. The extraction yield of gold nanoparticles ranged from 61 to 86%, with a quantitation limit at 0.5 µg ml-1, as measured by inductively-coupled optical emission spectroscopy. Dynamic light scattering could resolve the hydrodynamic size distribution of extracted particles which returned significantly different photon count rates at various concentrations. Their shape and primary size were easily observable by electron microscopy while atomic force microscopy, Auger electron spectroscopy and X-ray photoelectron spectroscopy could respectively probe the particles' biomolecular corona, detect surface-adsorbed S- and N- species, and identify carbon-based covalent bonds.

Impact of silica nanoparticle surface chemistry on protein corona formation and consequential interactions with biological cells.

Nanoparticles (NP) physico-chemical features greatly influence NP/cell interactions. NP surface functionalization is often used to improve NP biocompatibility or to enhance cellular uptake. But in biological media, the formation of a protein corona adds a level of complexity. The aim of this study was to investigate in vitro the influence of NP surface functionalization on their cellular uptake and the biological response induced. 50nm fluorescent silica NP were functionalized either with amine or carboxylic groups, in presence or in absence of polyethylene glycol (PEG). NP were incubated with macrophages, cellular uptake and cellular response were assessed in terms of cytotoxicity, pro-inflammatory response and oxidative stress. The NP protein corona was also characterized by protein mass spectroscopy. Results showed that NP uptake was enhanced in absence of PEG, while NP adsorption at the cell membrane was fostered by an initial positively charged NP surface. NP toxicity was not correlated with NP uptake. NP surface functionalization also influenced the formation of the protein corona as the profile of protein binding differed among the NP types.

Detection and analysis of nanoparticles in patients: A critical review of the status quo of clinical nanotoxicology.

On the cusp of massive commercialization of nanotechnology-enhanced products and services, the physical and chemical analysis of nanoparticles in human specimens merits immediate attention from the research community as a prerequisite for a confident clinical interpretation of their occurrence in the human organism. In this review, we describe the caveats in current practices of extracting and isolating nanoparticles from clinical samples and show that they do not help truly define the clinical significance of detected exogenous nano-sized objects. Finally, we suggest a systematic way of tackling these demanding scientific tasks. More specifically, a precise and true qualitative evaluation of nanoparticles in human biological samples is still hindered by various technical reasons. Such a procedure is more refined when the nature of the pollutants is known, like in the case of nano-sized wear debris originating from biomedical prostheses. Nevertheless, nearly all available analytical methods provide unknown quantitative accuracy and qualitative precision due to the challenging physical and chemical nature of nanoparticles. Without trustworthy information to describe the nanoparticulate load of clinical samples, it is impossible to accurately assess its pathological impact on isolated cases or allow for relevant epidemiological surveys on large populations. Therefore, we suggest that the many and various specimens stored in hospitals be used for the refinement of methods of exhaustive quantitative and qualitative characterization of prominent nanoparticles in complex human milieu.

In vitro toxicity of carbon nanotubes, nano-graphite and carbon black, similar impacts of acid functionalization.

Carbon nanotubes (CNT) and nano-graphite (NG) are graphene-based nanomaterials which share exceptional physicochemical properties, but whose health impacts are unfortunately still not well understood. On the other hand, carbon black (CB) is a conventional and widely studied material. The comparison of these three carbon-based nanomaterials is thus of great interest to improve our understanding of their toxicity. An acid functionalization was carried out on CNT, NG and CB so that, after a thorough characterization, their impacts on RAW 264.7 macrophages could be compared for a similar surface chemistry (15 to 120 µg·mL(-1) nanomaterials, 90-min to 24-h contact). Functionalized nanomaterials triggered a weak cytotoxicity similar to the pristine nanomaterials. Acid functionalization increased the pro-inflammatory response except for CB which did not trigger any TNF-α production before or after functionalization, and seemed to strongly decrease the oxidative stress. The toxicological impact of acid functionalization appeared thus to follow a similar trend whatever the carbon-based nanomaterial. At equivalent dose expressed in surface and equivalent surface chemistry, the toxicological responses from murine macrophages to NG were higher than for CNT and CB. It seemed to correspond to the hypothesis of a platelet and fiber paradigm.

Metals distribution in colorectal biopsies: New insight on the elemental fingerprint of tumour tissue.

BACKGROUND: Some studies have linked colorectal cancer to metal exposure. AIMS: Our objective was to evaluate the element distribution in colorectal adenocarcinoma biopsies, adjacent non-tumour tissues, and healthy controls. METHODS: The study is a case-control study which compared the element distribution in colon biopsies from two groups of patients: with colorectal cancer (2 types of samples: colorectal cancer biopsies and adjacent non-tumour tissues) and healthy controls. Fifteen metal concentrations (Aluminium, Boron, Cadmium, Chromium, Copper, Iron, Magnesium, Manganese, Nickel, Lead, Selenium, Silicon, Titanium, Vanadium, and Zinc) were quantified by using inductively coupled plasma atomic emission spectrometry. RESULTS: 104 patients were included: 76 in the colorectal cancer group, 28 in the healthy control group. Among the 15 elements analyzed, only boron, chromium, zinc, silicon and magnesium were found at clearly detectable concentrations. Colorectal tumour biopsies had significantly higher concentrations of magnesium as compared to adjacent non-tumour or healthy tissues. Zinc concentration followed the same trend but differences were not statistically significant. In addition, concentration of silicon was higher in colorectal cancer tissue than in healthy non-cancer tissue, while chromium was mostly found in adjacent non-tumour tissue. CONCLUSION: Magnesium, chromium, zinc and silicon were found in noteworthy concentrations in colorectal tumour. Their potential role in colorectal carcinogenesis should be explored.

Modulation of the multidrug resistance of glioma by glutathione levels depletion--interaction with Tc-99M-Sestamibi and Tc-99M-Tetrofosmin.

We have investigated the effect of glutathione (GSH) depletion on the chemosensitivity of human malignant glioma cell lines: G111, G5 and G152. All the cell lines showed a multidrug resistant (MDR) phenotype associated with MRP1 expression, high intracellular levels of GSH, and depolarized plasma membranes. Tc-99M-Sestamibi (MIBI) and Tc-99M-Tetrofosmin (Tfos) were used for monitoring the MDR mechanisms. Modulation of GSH content was performed with butoxysulfoximide (BSO) pre-treatment alone or in combination with GSH ethyl ester. MIBI and Tfos accumulation in the cells was inversely correlated to the GSH content, a higher accumulation was found after BSO pre-treatment and addition of GSH ethyl ester reversed this process. BSO could therefore play a role as a chemosensitizing drug and thus help to overcome MDR. However, higher accumulation of MIBI and Tfos was observed even in the sensitive cells suggesting another effect of BSO on the cell physiological characteristics. No sign of apoptosis has been found indicating a possible direct effect on the plasma membrane fluidity and permeability. MIBI and Tfos don't follow the expected behavior of a MDR probe in the glioma cells and given the particular morpho-physiological characteristics of these types of tumors, Tfos could be rather used as a marker of the tumor growth and proliferation.

In vitro cellular responses to silicon carbide particles manufactured through the Acheson process: impact of physico-chemical features on pro-inflammatory and pro-oxidative effects.

Silicon carbide (SiC) an industrial-scale product manufactured through the Acheson process, is largely employed in various applications. Its toxicity has been poorly investigated. Our study aims at characterizing the physico-chemical features and the in vitro impact on biological activity of five manufactured SiC powders: two coarse powders (SiC C1/C2), two fine powders (SiC F1/F2) and a powder rich in iron impurities (SiC I). RAW 264.7 macrophages were exposed to the different SiC particles and the cellular responses were evaluated. Contrary to what happens with silica, no SiC cytotoxicity was observed but pro-oxidative and pro-inflammatory responses of variable intensity were evidenced. Oxidative stress (H2O2 production) appeared related to SiC particle size, while iron level regulated pro-inflammatory response (TNFα production). To investigate the impact of surface reactivity on the biological responses, coarse SiC C1 and fine SiC F1 powders were submitted to different thermal treatments (650-1400 °C) in order to alter the oxidation state of the particle surface. At 1400 °C a decrease in TNFα production and an increase in HO·, COO(·-) radicals production were observed in correlation with the formation of a surface layer of crystalline silica. Finally, a strong correlation was observed between surface oxidation state and in vitro toxicity.

New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: study of TNF-α adsorption on alumina oxide nanoparticle.

Biomolecules can be adsorbed on nanoparticles (NPs) and degraded during in vitro toxicity assays. These artifactual phenomena could lead to misinterpretation of biological activity, such as false-negative results. To avoid possible underestimation of cytokine release after contact between NP and cells, we propose a methodology to account for these artifactual phenomena and lead to accurate measurements. We focused on the pro-inflammatory cytokine tumor necrosis factor TNF-α. We studied well-characterized boehmite engineered NP [aluminum oxide hydroxide, AlO(OH)]. The rate of TNF-α degradation and its adsorption (on boehmite and on the walls of wells) were determined in cell-free conditions by adding a known TNF-α concentration (1500 pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7 wt.% of the initial TNF-α was degraded over time, 6 wt.% adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. Finally, boehmite NP were incubated with murine macrophages (RAW 264.7 cell line). The release of TNF-α was assessed for boehmite NP and the experimental data were corrected considering the artifactual phenomena, which accounted for about 20-30% of the total.