Caco-2 in vitro model of human gastrointestinal tract for studying the absorption of titanium dioxide and silver nanoparticles from seafood.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in industry as a white pigment (paints, paper industry and toothpastes), photocatalysts (environmental decontamination and photovoltaic cells), inorganic UV filter (sunscreens and personal care products) and as a food additive (E171) and antimicrobial food packaging material. Silver nanoparticles (Ag NPs) are used in photonics, microelectronics, catalysis and medicine due to their catalytic activity, magnetic and optical polarizability, electrical and thermal conductivities and enhanced Raman scattering. They also have antibacterial, antifungal and antiviral activities, as well as anti-inflammatory potential. The huge increase in the use of nano-based products, mainly metallic NPs, implies the presence of nanomaterials in the environment, and hence, the unintentional human ingestion through water or foods (gastrointestinal tract is the main pathway of NPs intake in humans). The presence of TiO2 NPs and Ag NPs in seafood samples was firstly established using an ultrasound assisted enzymatic hydrolysis procedure and sp-ICP-MS analysis. Several clams, cockles, mussels, razor clams, oysters and variegated scallops, which contain TiO2 NPs and Ag NPs, were subjected to an in vitro digestion process simulating human gastrointestinal digestion in the stomach and in the small and large intestine to determine the bioaccessibility of these NPs. Caco-2 cells were selected as model of human intestinal epithelium for transport studies because of the development of membrane transporters that are responsible for the uptake of chemicals. Parameters as transepithelial electrical resistance (TEER) and permeability of Lucifer Yellow were studied for establishing cell monolayer integrity. TiO2 NPs and Ag NPs transport as well as total Ti and Ag concentrations passing through the gastrointestinal epithelial barrier model (0-2 h) were assessed by sp-ICP-MS and ICP-MS in several molluscs.

AF4-UV-ICP-MS for detection and quantification of silver nanoparticles in seafood after enzymatic hydrolysis.

A method based on asymmetric flow field-flow fractionation (AF4) coupled to ultraviolet-visible (UV-vis) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) has been developed for silver nanoparticles (Ag NPs) detection and quantification in bivalve molluscs. Samples were pre-treated using a conventional enzymatic (pancreatin and lipase) hydrolysis procedure (37 °C, 12 h). AF4 was performed using a regenerated cellulose (RC) membrane (10 kDa, 350 µm spacer) and aqueous 5 mM Tris-HCl pH = 7.4 as carrier. AF4 separation was achieved with a program that included a focusing step with tip and focus flows of 0.20 and 3.0 mL min-1, respectively, and an injection time of 4.0 min. Elution of different size fractions was performed using a cross flow of 3.0 mL min-1 for 15 min, followed by linear cross flow decrease for 7.5 min, and a washing step for 9.4 min with no cross flow. Several bivalve molluscs (clams, oysters and variegated scallops) were analysed for total Ag content (ICP-MS after microwave assisted acid digestion), and for Ag NPs by the method presented here. Results show that Ag NPs are detected at the same elution time than proteins (UV monitoring at 280 and 405 nm), which suggests a certain interaction occurred between Ag NPs with proteins in the enzymatic extracts. AF4-UV-ICP-MS fractograms also suggest different Ag NPs size distributions for selected samples. Membrane recoveries, determined by peak area comparison of fractograms with and without application of cross flow, were within the 49-121% range. Confirmation of the presence Ag NPs in the investigated enzymatic extracts was demonstrated by SEM after an oxidative pre-treatment based on hydrogen peroxide and microwave irradiation.

Biopersistence rate of metallic nanoparticles in the gastro-intestinal human tract (stage 0 of the EFSA guidance for nanomaterials risk assessment).

The European Food Safety Authority has published a guidance regarding risk assessment of nanomaterials in food and feed. Following these recommendations, an in vitro gastrointestinal digestion has been applied to study the biopersistence of TiO2 and Ag NPs in standards, molluscs and surimi. TiO2 NPs standards and TiO2 NPs/ TiO2 microparticles from E171 were not found to be degraded. Ag NPs proved to be more degradable than TiO2 NPs, but the biopersistence rates were higher than 12%, which means that Ag NPs are also biopersistent. Findings for seafood are quite similar to those obtained for TiO2 NPs and Ag NPs standards, although the calculation of the biopersistence rate proposed by the EFSA was not found to be straightforward for foodstuff (the use of the NPs concentration in the sample instead of the NPs concentration at initial time (sample mixed with the gastric solution before enzymatic hydrolysis) has been proposed.

Size characterization and quantification of titanium dioxide nano- and microparticles-based products by Asymmetrical Flow Field-Flow Fractionation coupled to Dynamic Light Scattering and Inductively Coupled Plasma Mass Spectrometry.

A procedure for the size characterization and quantification of titanium dioxide (TiO2) nano- and microparticles by Asymmetric Flow Field-Flow Fractionation (AF4) coupled to Dynamic Light Scattering (DLS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is described. Different strategies for size characterization with size standards and the use of the DLS signal for the estimation of hydrodynamic diameters are evaluated. The procedure has been applied to the characterization of TiO2 nanoparticles in photocatalytic products and crab sticks (surimis), where TiO2 is present as E171 food additive. Sizes in the range of 50-90 nm and 160-170 nm were estimated in the different photocatalytic products by AF4-DLS, in good agreement with the sizes predicted by calibration versus SiO2 and polystyrene standards. In surimis, sizes between 140 and 350 nm were estimated by AF4-DLS, similar to those reported in literature for E171 additive. These results were also compared to those obtained by single particle ICP-MS, which allowed the detection of a nano-sized fraction of TiO2 present in the four surimis analyzed. Titanium contents in one of the photocatalytic products determined by AF4-ICP-MS was 16.86 ± 2.54 mg g-1, whereas the alkaline extraction followed by AF4-ICP-MS allowed the determination of TiO2 content in four surimis at concentration levels in the range of the µg g-1 (from 3.14 ± 0.10 to 14.55 ± 1.46 µg Ti g-1), with channel recoveries above 85% in all cases. The method has been validated by comparison with the Ti content determined by ICP-OES after microwaved assisted acid digestion of all the samples. The methodology proposed allows the complete quantification of the (nano)particulate forms of titanium in complex matrices together with their size characterization.

Enzymatic hydrolysis as a sample pre-treatment for titanium dioxide nanoparticles assessment in surimi (crab sticks) by single particle ICP-MS.

A reliable sample pre-treatment based on enzymatic hydrolysis has been fully optimized and validated for TiO2 NPs isolation and determination/characterization in surimi (crab sticks). Efficient extractions have been found when using a pancreatin/lipase mixture at pH 7.4 and 37 °C for 12 h under continuous stirring. The proposed sample pre-treatment procedure has been found not to change TiO2 NPs size distribution, therefore guaranteeing TiO2 NPs integrity. TiO2 NPs determination (TiO2 NPs concentration) and TiO2 NPs characterization (size distribution) were assessed by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) working with dwell times in the microsecond range (high frequency of data acquisition). Method validation was performed for TiO2 NPs concentrations and TiO2 NPs sizes. Good repeatability (25% and 8% for TiO2 NPs concentration and TiO2 NPs most frequent size), and sensitivity (limit of detection of 5.28 × 105 NPs g-1for TiO2 NPs concentrations, and 31.3-37.1 nm for TiO2 NPs size) were obtained. Accuracy, calculated through analytical recovery was adequate. Recoveries for TiO2 NPs standards of 50 and 100 nm were 108 ±â€¯5 and 105 ±â€¯4%, respectively. The proposed methodology was applied to several surimi samples for assessing TiO2 NPs concentrations and size distribution. Some surimi samples were found to contain TiO2 NPs (concentrations from 1.40 × 107 to 1.19 × 109 NPs g-1). TiO2 NPs size distributions were very different among the samples, and some of them showed wide size ranges (the most frequent size varied from 53.8 to 62.1 nm; whereas, the mean size values were within the 73.4-217.5 nm range).

Ultrasound assisted enzymatic hydrolysis for isolating titanium dioxide nanoparticles from bivalve mollusk before sp-ICP-MS.

Applicability of single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) using dwell times equal to or shorter than 100 µs has been tested for assessing titanium dioxide nanoparticles (TiO2 NPs) in bivalve mollusks. TiO2 NPs isolation from fresh mollusk tissues was achieved by ultrasound assisted enzymatic hydrolysis procedure using a pancreatin/lipase mixture. Optimum extraction conditions imply ultrasonication (60% amplitude) for 10 min, and 7.5 mL of a solution containing 3.0 g L-1of pancreatin and lipase (pH 7.4). The developed method was found to be repeatable (repeatability of 17% for the over-all procedure, TiO2 NPs concentration of 5.33 × 107 ±â€¯8.89 × 106, n = 11), showing a limit of detection of 5.28 × 106 NPs g-1, and a limit of detection in size of 24.4-30.4 nm, based on the 3σ criteria, and on the 3σ/5 σ criteria, respectively. The analytical recovery within the 90-99% range (use of TiO2 NPs standards of 50 nm at 7 and 14 µg L-1 as Ti). Several bivalve mollusks (clams, cockles, mussels, razor clams, oysters and variegated scallops) were analyzed for total titanium (ICP-MS after microwave assisted acid digestion), and for TiO2 NPs by the proposed method. TiO2 NPs concentrations were within the 2.36 × 107-1.25 × 108 NPs g-1 range, and the most frequent sizes were from 50 to 70 nm.