Dr Holger Sieg
email: holger.sieg@bfr.bund.de

Caroline Braeuning
email: caroline.braeuning@mdc-berlin.de

Birgitta Maria Kunz
email: birgitta.kunz@bfr.bund.de

Hannes Daher
email: hannes.daher@bfr.bund.de

Claudia Kästner
email: claudia.kaestner@bam.de

Benjamin-Christoph Krause
email: benjamin-christoph.krause@bfr.bund.de

Thomas Meyer
email: thomas.meyer@medizin.uni-leipzig.de

Pégah Jalili
email: pegah.jalili@anses.fr

Kevin Hogeveen
email: kevin.hogeveen@anses.fr

Linda Böhmert
email: linda.boehmert@bfr.bund.de

Dajana Lichtenstein
email: dajana.lichtenstein@bfr.bund.de

Agnès Burel
email: agnes.burel@univ-rennes1.fr

Soizic Chevance
email: soizic.chevance@univ-rennes1.fr

Harald Jungnickel
email: harald.jungnickel@bfr.bund.de

Jutta Tentschert
email: jutta.tentschert@bfr.bund.de

Peter Laux
email: peter.laux@bfr.bund.de

Albert Braeuning
email: albert.braeuning@bfr.bund.de

Fabienne Gauffre
email: fabienne.gauffre@univ-rennes1.fr

Valérie Fessard
email: valerie.fessard@anses.fr

Jan Meijer
email: jan.meijer@uni-leipzig.de

Irina Estrela-Lopis
email: irina.estrela-lopis@medizin.uni-leipzig.de@andreas

Andreas F Thünemann
email: andreas.thuenemann@bam.de@andreas

Andreas Luch
email: andreas.luch@bfr.bund.de

Alfonso Lampen
email: alfonso.lampen@bfr.bund.de

Uptake and molecular impact of aluminum-containing nanomaterials on

Keywords: Nanoparticles, Aluminum, Gastrointestinal Tract, Microarray, Toxicology

Introduction

Aluminum (Al) is the most frequent metal in the earth crust and reaches the human gastrointestinal tract via oral uptake [START_REF] Willhite | Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water[END_REF]. However, the majority of Al is bound in minerals and oxides and therefore unavailable for the biological cycle [START_REF] Lote | Aluminium: gastrointestinal absorption and renal excretion[END_REF]. Nevertheless, a certain amount gets dissolved, promoted by industrial processes and acidification of the environment [START_REF] Exley | A biogeochemical cycle for aluminium?[END_REF][START_REF] Wagner | Canadian Minerals Yearbook[END_REF]. Chemical activity, gastrointestinal bioavailability and biochemical mode of action are dependent on the species and the surrounding media [START_REF] Powell | The chemistry of aluminium in the gastrointestinal lumen and its uptake and absorption[END_REF]Thompson, 1993, Exley, 2016). A fraction of the oral exposure to Al has particulate nature. Recently, Al-containing nanoparticles were found in food for the first time [START_REF] Loeschner | Detection and characterisation of aluminium-containing nanoparticles in Chinese noodles by single particle ICP-MS[END_REF]. After ingestion, Al species can change their physicochemical characteristics along the gastrointestinal tract. Previous results indicate a partial dissolution of Al in the acidic stomach environment and resorption in the proximal part of the duodenum (Froment et al., 1989a). Bioavailable Al gets mostly excreted via the kidney (95 %) and more slightly via bile (2 %) or is accumulated in bones and other organs [START_REF] Yokel | Influence of renal impairment, chemical form, and serum protein binding on intravenous and oral aluminum kinetics in the rabbit[END_REF]. The resulting biochemical and toxicological consequences are still disputed [START_REF] Willhite | Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts[END_REF][START_REF] Lidsky | Is the Aluminum Hypothesis Dead[END_REF]. Up to now, there is still a lack of knowledge concerning toxicological effects and the hazardous potential of Al, although it certainly plays a role in blood and bone diseases [START_REF] Alfrey | The dialysis encephalopathy syndrome. Possible aluminium intoxication[END_REF][START_REF] Vick | Aluminum-related osteomalacia in renal-failure patients[END_REF], Lin et al., 2013). Some researchers see evidence for a link of Al exposure with breast cancer and neurodegenerative disorders [START_REF] Darbre | Underarm antiperspirants/deodorants and breast cancer[END_REF][START_REF] Walton | Chronic aluminum intake causes Alzheimer's disease: applying Sir Austin Bradford Hill's causality criteria[END_REF]. The chemical mechanisms behind these effects have not been completely revealed yet. Some studies consider ionic Al to cause oxidative damages, with observation of reactive oxygen species, lipid peroxidation or mitochondrial dysfunction [START_REF] Gutteridge | Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts[END_REF][START_REF] Fraga | Effects of aluminum on brain lipid peroxidation[END_REF][START_REF] Zatta | Aluminium(III) as a promoter of cellular oxidation[END_REF]. There are also indications for Al-induced apoptosis [START_REF] Savory | Intracellular mechanisms underlying aluminum-induced apoptosis in rabbit brain[END_REF] and genotoxicity [START_REF] Kumar | Aluminium-induced oxidative DNA damage recognition and cell-cycle disruption in different regions of rat brain[END_REF] in vivo. Many publications, however, do not distinguish between the different chemical species of Al. Differences between the effects induced by ionic species and nanoparticles were already shown for nano-silver [START_REF] Hsiao | Trojan-horse mechanism in the cellular uptake of silver nanoparticles verified by direct intra-and extracellular silver speciation analysis[END_REF] and nano-copper [START_REF] Semisch | Cytotoxicity and genotoxicity of nano -and microparticulate copper oxide: role of solubility and intracellular bioavailability[END_REF], so a similar investigation on nano-aluminum is required to fill the knowledge gap concerning toxicological effects caused by Al.

Chemical conversions like partial dissolution, agglomeration and de novo particle formation have been observed during the passage of Al species through artificial digestion with gastrointestinal fluids [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF]. Therefore, it is crucial to compare different particulate and non-particulate species when regarding element-specific effects. In this study, we chose 3 representative Al species including metallic Al 0 nanoparticles, mineralic Al 2 O 3 nanoparticles and soluble AlCl 3 . These species are already known to exhibit different physicochemical characteristics [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF]. In this study, we determined physicochemical parameters like size distribution, ion release and protein corona of these particles in biological media. Furthermore, we addressed the intestinal cellular uptake of these Al species as well as possible mode of actions in human intestinal cells, to fill the lack of information. Therefore, the well-established intestinal model of differentiated Caco-2 cells has been used [START_REF] Fogh | One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice[END_REF][START_REF] Lampen | Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted mixture in human Caco-2 cells[END_REF]. These cells can mimic the intestinal epithelium and so can be used for in vitro uptake experiments [START_REF] Xia | Transport mechanism of lipid covered saquinavir pure drug nanoparticles in intestinal epithelium[END_REF], Lichtenstein et al., 2017[START_REF] Ye | Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers[END_REF], Lichtenstein et al., 2016[START_REF] Je | Release properties and cellular uptake in Caco-2 cells of size-controlled chitosan nanoparticles[END_REF]. As an enhancement of the Caco-2 model, cocultures, which are simulating the intestinal mucus [START_REF] Lesuffleur | Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells[END_REF][START_REF] Wen | Transport of PLGA nanoparticles across Caco-2/HT29-MTX co-cultured cells[END_REF][START_REF] Castiaux | Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells[END_REF] and M-cells [START_REF] Gullberg | Expression of specific markers and particle transport in a new human intestinal M-cell model[END_REF][START_REF] Des Rieux | An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells[END_REF][START_REF] Lichtenstein | Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells[END_REF], have been established. Cellular uptake mechanisms can differ between nanoparticles and ionic species. Ions are often taken up by receptor-mediated transport [START_REF] Rolfs | Metal ion transporters in mammals: structure, function and pathological implications[END_REF], but nanoparticles are described to be taken up in vesicles by "trojan horse" mechanisms [START_REF] Hsiao | Trojan-horse mechanism in the cellular uptake of silver nanoparticles verified by direct intra-and extracellular silver speciation analysis[END_REF][START_REF] Semisch | Cytotoxicity and genotoxicity of nano -and microparticulate copper oxide: role of solubility and intracellular bioavailability[END_REF]. In this work, the cellular uptake of the different Al species was investigated by several analytical and imaging methods like atomic absorption spectroscopy (AAS), transmission electron microscopy (TEM), ion beam microscopy (IBM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Toxic effects were addressed with in vitro methods including a transcriptome analysis on regulatory functions and the measurement of multiple toxicological endpoints like cell viability, cellular impedance, GSH-levels, ATP-levels, mitochondrial membrane potential, apoptosis, necrosis, IL8-release and NFκB activation.

Materials and Methods

Chemicals and nanoparticles

Most chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) in the highest available purity.

Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al 2 O 3 nanoparticles) were supplied by IoLiTec (Heilbronn, Germany). Al 0 nanoparticles were stored and weighted under an argon atmosphere. Both particle species were freshly dispersed according to the modified NanoGenoTOX protocol (dispersion in 0.05% BSA/water by ultrasonication with KE76 tip for 5'09'' with ~20% energy), BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%)

and AlCl 3 was supplied by Sigma-Aldrich (Hexahydrate, ≥97%).

Cell cultures and transport studies

Caco-2 cells and Raji B-lymphocytes were cultivated in DMEM High Glucose, 10 % FCS, 1% penicillin/streptomycin (GE Healthcare, Solingen, Germany). For cultivation of HT29-MTX, 1% non-essential amino acids (NEAA) was added. 5,000 Caco-2 cells were seeded into each well of 96-well plates. 1 million of cells were grown for microarray analysis per 75 cm² flask. In every format, medium was changed every 2-3 days for a differentiation time of 21 days. For the 12-well transwell system, 50,000 cells per well were seeded as previously described [START_REF] Lichtenstein | Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells[END_REF]. For the mucus coculture, Caco-2 and HT29-MTX were cocultivated (40,000/10,000 cells) in HT29-MTX medium. For the M-cell-model, transwell inserts were inverted into boxes 7 days after seeding. At day 16, 50,000 Raji cells were added from the basolateral side onto the inverted monolayer using a tube. At day 20, inserts were reinverted back into the transwell system and incubated with samples 1 day later. For testing monolayer integrity, transepithelial resistance (TEER) values were determined and 1 mg/ml FITC-Dextran was used for measuring the passage into the basolateral compartment with a TECAN Infinite F200 Plate reader (Tecan, Switzerland). TEER values above 800 Ωcm² and p App -values for FITC-Dextran below 10 -6 were defined as indicators of good monolayer stability.

For the transport studies on the 3 coculture models, 500 µL medium containing 100 µg Al/ml or control medium were added to the apical compartment of the transwell and incubated for 24 h. Basolateral and apical compartments were collected and the monolayer was washed with 500 µl PBS. After washing, the membrane was cut out and all collected compartments were digested by 69 % nitric acid microwave digestion using an MLS Ethos-1600 high pressure microwave (MLS, Germany) according to a previously described protocol [START_REF] Lichtenstein | Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells[END_REF]. 4 independent experiments were performed in triplicates. The Alcontent was determined by AAS comparable to previous studies [START_REF] Lichtenstein | It takes more than a coating to get nanoparticles through the intestinal barrier in vitro[END_REF].

Small-angle X-ray scattering (SAXS) SAXS measurements were conducted in a flow-through capillary with a Kratky-type instrument (SAXSess from Anton Paar AG, Graz, Austria) at 21±1 °C. The SAXSess has a low sample-detector-distance of 0.309 m which is appropriate for the investigation of dispersions with low scattering intensities. The experiments were performed with 120 measurement cycles (each averaged over 10 s). The measurements were backgroundcorrected with the respective mixture of aqueous BSA solution without addition of aluminum species. Deconvolution (slit length desmearing) of the SAXS curves was performed with the SAXS-Quant software (Anton Paar AG). Samples analyzed with SAXS were used as prepared. Curve fitting was performed with the software McSAS (Monte Carlo method, version 1.0.1). This procedure was described before [START_REF] Kästner | Catalytic Reduction of 4-Nitrophenol Using Silver Nanoparticles with Adjustable Activity[END_REF].

Ion release measurements

Al species were incubated at 2 concentrations, 4 and 100 µg Al/mL. Aliquots were taken in duplicates after 0, 3, 23 and 47 h. Ion release of nanoparticles in serum-containing cell culture medium (DMEM high glucose, 10% FCS, 1% P/S) was determined by ultracentrifugation (100,000 x g for 1 h at 4 °C) followed up by acidic hydrolysis of the supernatant (69% HNO 3 , 180 °C for 20 min in an MLS-ETHOS Microwave system) and element analysis with AAS (Perkin Elmer AAnalyst 800, Shelton, USA) comparable to previous studies (Lichtenstein et al., 2016). Results are given as percentage of the initially used aluminum amount. Results are shown as mean values of 3 independent experiments with standard deviations and statistical analysis was performed with student's t-test indicated by asterisks (* p<0.05;** p<0.01; *** p<0.001).

Protein Corona Analysis

The Protein coronae were determined after 24 h incubation (37 °C, 5 % CO 2 ) of 100 µg Al/mL samples in serum-containing cell culture medium (CCM). Centrifugation was performed in a Beckman Optima L centrifuge (Beckman, Unterschleissheim, Germany) for 1 h at 48,250 x g. Supernatants were removed, pellets suspended in 10 mL 50 mM sodium bicarbonate and transferred into a fresh tube. This washing step was repeated 3 times before, in the last step, the pellet was resuspended in 1 mL and centrifuged for 1 h at 153,700 x g. With these pellets, 2D-SDS-PAGE was performed as described before [START_REF] Oberemm | Toxicogenomic analysis of Nnitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters[END_REF][START_REF] Oberemm | Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver[END_REF]. Isoelectric focusing occurred according to a published protocol with 7 M urea and a 2 M thiourea-containing lysis buffer. After a second dimension migration (SDS-PAGE), gels were stained with ruthenium and photographed using a VersaDoc imager (VersaDoc MP 4000, BioRad, USA, excitation 450 nm, emission 605 nm). Spots were picked by self-made SpotXpress Spotpicker and digested by tryptic digestion over night for MALDI-TOF protein identification (Bruker Ultraflex II, Bruker, Berlin, Germany). Semi-quantitative data evaluation was carried out by manually comparing 4 replicates per sample using a VersaDoc imager.

Transmission Electron Microscopy (TEM)

Following a 24 h treatment with NMs, cells were rinsed twice with PBS and with 0.15 M Na cacodylate buffer (pH 7.2) before fixation by dropwise addition of glutaraldehyde (2.5%) for 45 min. The cells were rinsed several times with 0.15 M Na cacodylate buffer and post fixed with 1% osmium tetroxide for 45 min. After further rinsing with cacodylate buffer, the samples were dehydrated through an ethanol gradient from 70% to 100% and infiltrated in a mixture of acetone-epon resin (50/50) for 3 h, then, in pure epon resin for 16 h. Finally, the samples were embedded in DMP30-epon for 24 h at 60°C. Ultra-thin sections (90 nm) were cut on a Leica UC7 ultracut, collected onto copper grids and double-stained with 4% uranyl acetate and then with lead citrate (Reynold solution). Examination was performed with JEOL 1400 electron microscope operated at 120 kV equipped with a 2k-2k camera from Gatan Orius 1000 (MRic-TEM platform, Biosit, Rennes). Time of flight secondary ion mass spectrometry (ToF-SIMS) ToF-SIMS depth profiles were acquired using a ToF-SIMS V instrument (ION-TOF GmbH, Münster, Germany) with a 30 keV nano-bismuth primary ion beam source (Bi)x(y+)-cluster ion source with a BiMn emitter. The ion currents were 0.5 pA at 5 kHz using a Faraday cup.

A pulse of 0.7 ns from the bunching system resulted in a mass resolution that usually exceeded 9,000 (full width at half-maximum) at m/z < 500 in positive ion mode. The primary ion dose was controlled below 10 12 ions cm -2 to ensure static SIMS conditions. Charge compensation on the sample was obtained by a pulsed electron flood gun with 20 eV electrons. The primary ion gun scanned a field of view of 200x200 μm applying a 512×512 pixel measurement raster. Once the primary ion gun was aligned, a ToF-SIMS mass spectrum was generated by summing the detected secondary ion intensities and plotting them against the mass channels. The analytical methodology was described in detail elsewhere [START_REF] Booth | Freshwater dispersion stability of PAAstabilised cerium oxide nanoparticles and toxicity towards Pseudokirchneriella subcapitata[END_REF][START_REF] Tentschert | TOF-SIMS analysis of cell membrane changes in functional impaired human macrophages upon nanosilver treatment[END_REF][START_REF] Haase | Application of laser postionization secondary neutral mass spectrometry/time-of-flight secondary ion mass spectrometry in nanotoxicology: visualization of nanosilver in human macrophages and cellular responses[END_REF][START_REF] Jungnickel | Application of TOF-SIMS with chemometrics to discriminate between four different yeast strains from the species Candida glabrata and Saccharomyces cerevisiae[END_REF][START_REF] Thompson | ToF-SIMS studies as a tool to discriminate between spores and vegetative cells of bacteria[END_REF]. All depth profiles were performed in dual beam mode on a ToF-SIMS V instrument (ION-TOF GmbH, Münster, Germany) of the reflectron-type, equipped with a 30 keV bismuth liquid metal ion gun (LMIG) as primary ion source, a 20 keV argon gas cluster ion source both mounted at 45° with respect to the sample surface and an electron flood gun. Bi 3+ was selected as primary ion by appropriate mass filter settings. Primary and sputter ion currents were directly determined at 200 μs cycle time (i.e. a repetition rate of 5.0 kHz) using a Faraday cup located on a grounded sample holder. Scanning area for analysis was 200 × 200 μm 2 with 512 × 512 pixels. The sputter area for each measurement was 1,000 µm x 1,000 µm. Surface charging was compensated by flooding with low energy electrons. ToF-SIMS depth profiles were acquired in positive ion mode. The mass scale was internally calibrated using a number of well-defined and easily assignable secondary ions (C 2 H 5 + , C 3 H 7 + and C 4 H 9 + ) keeping the error in calibration for all spectra below 5 ppm. The data were evaluated using the Surface Lab software (ION-TOF GmbH, Münster, Germany).

Ion Beam Microscopy (IBM)

IBM measurements were performed at the LIPSION nanoprobe. A proton beam with an energy of 2.25 MeV was applied by a singletron accelerator. To minimize interactions between gas molecules and projectiles from the beam a vacuum with a pressure of 5.0 x 10 - 5 to 10 -7 Torr was used. Micro-particle induced x-ray emission (µPIXE) element maps were recorded using a Canberra (Canberra, Connecticut, U.S.A.) PIXE detector consisting of a High Purity Germanium crystal with an area of 95 mm². To avoid interactions between the detector and backscattered protons the detector was shielded by a 60 µm polyethylene foil.

For cellular analysis areas of 50 x 50 µm² were scanned. µPIXE was used to visualize twodimensional distributions of aluminum, phosphorus and sulfur. GeoPIXE 5.1 (CSIRO Exploration and Mining, Victoria, Australia) was used for data analysis.

Cytotoxicity Assays

Cell viability was determined by the Cell Titer Blue assay (CTB, Promega, USA) followed by the MTT assay. Differentiated Caco-2 cells were incubated with 200 µL of different concentrations of Al species for 24 h and 48 h in 96-well-plates. After incubation time, 100 µL of the media was removed and 40 µL CTB reagent was added to the cells. After 20-30 min, fluorescence was determined with a Tecan plate reader (Ex. 560, Em. 590 nm). Next, 10 µL 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS was added for another 1 h. After that, whole media was removed and 130 µL pre-warmed desorption agent (0.7 % w/v SDS in isopropanol) was added. Plates were shaken for 30 min to RNeasy Midi Prep kit (Qiagen, Germany) and the RNA amount was measured using a NanoDrop spectrophotometer. For the microarrays, only the highest exposure conditions of each Al-species were chosen, while the qPCRs were performed with all available conditions to gain information on the concentration dependency of the effects. 100 ng RNA of each sample was sent to Eurofins (Eurofins, Denmark) for microarray analysis using the human transcriptome array HTA 2.0. Data was analyzed by Affymetrix TAC 2.0 software (Affymetrix, UK) using as cutoffs a │fold change│≥ 1.5 and a one way ANOVA p-value <0.05. P-values were calculated automatically by the TAC 2.0 software according to the manufacturer's standard settings for microarray evaluation. Genes were only considered when they were significantly regulated, compared to the medium controls, in all 3 out of 3 technical replicates. Data evaluation was done using the software VennDis (Kisslinger Laboratories, Canada), Ingenuity Pathway Analysis (IPA, Qiagen, Germany) and geneontology.org (GeneOntology consortium, USA). Selected microarray results were verified by quantitative real-time PCR (qPCR, Applied Biosystems 7900 HT, Thermo Fisher Scientific, Berlin). The qPCRs were performed in technical triplicates; data evaluation was performed according to a standard protocol with representative deregulated genes of different sample groups using the ΔΔcT method referring to the housekeeping gene (GAPDH) and untreated control samples, as previously described [START_REF] Buhrke | Comparative analysis of transcriptomic responses to repeated-dose exposure to 2-MCPD and 3-MCPD in rat kidney, liver and testis[END_REF]. The ΔΔcT values for each condition were averaged, marked with standard deviations and statistics were performed as Student's t-test indicated by asterisks (* p<0.05;** p<0.01; *** p<0.001).

Cellular Effects

Cellular effects were determined in 96-well-plates with differentiated Caco-2 cells as described before.

For GSH-levels, after incubation with substances and controls, the medium was removed prior to a 20-30 min incubation with 250 µL 40 µM monochlorobimane (MCB) in PBS. After that, cells were washed with 250 µL PBS and incubated with 150 µL desorption agent (see above) and shaken for 30 min. Fluorescence was measured with a plate reader (Ex. 380 nm, Em. 460 nm). Mean values were determined as described above. 100 µM buthioninesulfoximine was used as positive control.

Mitochondrial depolarization was determined by JC1 assay. Caco-2 cells were grown, differentiated and incubated as described before. After incubation, incubation media were removed and 100 µL 1 mg/ml JC1 in PBS was added for 20 min. After that, cells were washed twice with 250 µl PBS and finally 100 µL PBS were added for fluorescence measurements (Green: Ex. 485 nm, Em. 535 nm, Red: Ex. 535 nm, Em. 595 nm).

Fluorescence ratio (Red / Green) was determined and mean values were normalized to untreated controls. As a positive control, 1 mg/mL valinomycine was used and added 2 h before the end of incubation time.

Relative ATP content was determined by Cell Titer Glo assay (Promega, USA) according to a standard protocol. Medium was removed except 100 µL per well. Following, 100 µL of ready-to-use reagent were added and shaken for 10 min. Luminescence was quantified for 1 second measuring time Mean values were determined as described before. A previously determined substance specific correction factor was taken into account to identify treatmentspecific effects and to eliminate assay interactions [START_REF] Lehmann | Disturbance of firefly luciferase-based bioassays by different aluminum species[END_REF]. As positive control, 1 µM oligomycine was used. Statistics were performed as Student's t-test indicated by asterisks (* p<0.05;** p<0.01; *** p<0.001).

IL-8 release from cell via ELISA assay

The levels of interleukin-8 (IL-8) in cell media were measured using an enzyme-linked immunosorbent assay (ELISA). Primary IL-8 antibody (M801), biotin-conjugated human IL-8 (M802B), recombinant IL-8 cytokine, HRP-Conjugated Streptavidin (N100), SuperBlock blocking buffer, 3,3',5,5'-tetramethylbenzidine (TMB), Tumor necrosis Factor alpha (TNF-α)

and Tween 20 were obtained from Thermofisher scientific. 20 ng/mL Tumor necrosis Factor alpha (TNF-α) was used as positive control. Following 24 h incubation with particles, media were collected and frozen at 20 °C until analysis. 96 well microplates (Nunc) were coated with human recombinant IL-8 primary antibodies at 1 µg/mL and incubated overnight at 4 °C.

Between each step, wells were washed with PBS-Tween 20 (0.05%). After saturation with SuperBlock for 1 h, samples and standards were added into the wells and incubated at room temperature for 2 h. Biotin-conjugated human IL-8 antibodies (0.1 µg/ml) were then added for 1 h followed by 100 µL of HRP 1:10,000 labeling for 45 min. Finally 100 µL of the chromogenic substrate TMB was added and the reaction was stopped with 100 µL of H 2 SO 4

(1 M). Plates were read at 405 nm. The concentrations of IL-8 expressed in fold of negative control were calculated against a standard curve prepared in duplicate.

NFⱩB via High Content Screening (HCS)

The primary and secondary antibodies were purchased from Abcam (Cambridge, UK): rabbit monoclonal anti NFⱩB (ab16502), and goat anti-rabbit IgG H&L Alexia Fluor 647 (ab150079), prepared in blocking solution and filtered with a 0.2 μm syringe filter. After 1, 3 and 6 hours of treatment, cells were fixed with 4 % paraformaldehyde in PBS for 10 min and permeabilized with 0.2 % Triton X-100. Plates were then incubated in a blocking solution (PBS with 1% BSA and 0.05 % Tween-20) for 30 min before addition of the primary antibody (1/1000) overnight at 4 °C. . After 3 washes with PBS + 0.05 % Tween20, the secondary antibody (1/1000) was incubated for 45 min at room temperature. After two washes with PBS 1%, nuclear DAPI (1 μg/mL-1) staining was used for automated cell identification by HCA.

Plates were scanned with the Thermo Scientific ArrayScan VTI HCS Reader (Thermo Scientific, Waltham, USA) and analyzed using the target activation module of the BioApplication software. For each well, 7 fields (20×magnification, white bar = 100 μm)

were scanned and analyzed for immunofluorescence quantification at 647 nm. The nuclei/cytoplasm percentage ratio of NFⱩB was quantified. Cytotoxicity was determined by cell counting from DAPI staining and expressed as a percentage of cells compared to the control.

Results

Chemical characterization

The particles which were used in this study have been characterized before extensively in two studies [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF][START_REF] Krause | Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials using a combination of methods for particle surface and size analysis[END_REF]. These publications describe the physicochemical parameters of the applied particles in stock dispersions and artificial digestion fluids by a big variety of methods. These results, which form the analytical basis of the following experiments, are summed up in Supplementary Table 1. Both Al-containing nanoparticles showed a size distribution between 10 and 100 nm in stock dispersion. The spherical Al 0 nanoparticles turned out as rather broadly distributed. These particles consisted of metallic particle cores with an oxide layer on the particle surface due to the manufacturing process, which could be shown by TEM and XRD measurements [START_REF] Krause | Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials using a combination of methods for particle surface and size analysis[END_REF]. To avoid further oxidation, the particles were stored, weighted and diluted under an Argon atmosphere immediate prior to use. Al 2 O 3 nanoparticles formed loose agglomerates consisting of elongated single particles with core radii between 4 and 10 nm and lengths between 20 and 40 nm. Both species were rather stable in artificial digestion fluids, showed little ion release and agglomeration in gastric fluid as well as reassembled in intestinal fluid. In addition, AlCl 3 formed nanoscaled de novo particles in the intestinal fluid [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF]. A thorough characterization of the nanoparticles in biological systems was still missing but is also required to allow a meaningful interpretation of the results from cellular in vitro assays.

Therefore, Figure 1 shows results of the characterization of the particles in cell culture medium.

The size distributions of the different nanoparticles and the ion release from these Al species in cell culture medium were investigated. Figure 1A shows nanoscaled particle core size distributions for all applied Al species, derived by SAXS measurements. Distributions were species specific and quite stable over a time period of 48 h. Figure 1B shows ion release of the 3 used Al-species in cell culture medium for 2 different concentrations (4 and 100 µg Al/ml) over a time period of 48 h. Metallic Al nanoparticles showed a significantly higher ion release than Al 2 O 3 nanoparticles in both cases. At the low concentration, around 15 % of the totally loaded Al from metallic nanoparticles was released as free ions, while ion release from Al 2 O 3 nanoparticles was not detectable over the medium background, which was determined as 0.19 µg/mL in cell culture medium (4.75 % of applied 4 µg/mL, 0.19 % of applied 100 µg/mL). At the higher concentration, the relative ion release by metallic nanoparticles was much lower (around 1 %), which was still significantly above the released fraction from the and lipid interacting proteins (APOA1) was notable. AlCl 3 led to an enriched agglomeration of all detected proteins compared to the basic composition in the serum-containing cell culture medium.

Cellular uptake and transport

Cellular uptake and transport of the different Al species were investigated in transwell experiments with three different coculture models: the Caco-2 monoculture, a mucusproducing model and an M-cell model (Figure 2A). Incubation of 24 h with Al species was followed by element analysis. The amount of Al detected in the basolateral compartment was defined as transported, while the amount that remained in the washed membrane fraction was defined as taken up by the cells. The apical fraction (apical medium and wash solution) were used for recovery measurements (not shown). Figure 2B and C show no significant transport of Al above the medium Al-background irrespective of the Al species and cell model.

However, differences between the Al species were noticed in the uptake. Both Al-containing nanoparticles were taken up into the cells at fractions below 4 % of 50 µg applied Al with minor differences among the cellular models. This was a significantly higher uptake of particulate Al species, as compared to the ionic species which was not taken up to a remarkable degree. This implicates that Al is predominantly taken up as particles, whereas the uptake of dissolved Al ions was avoided through cellular mechanisms. Aluminum recovery, as a sum of all generated fractions, added up to around 50 µg (corresponding to 100 %) with measurement-related standard deviations (Supplementary Figure 1) while the control samples corresponded to the determined Al background of 0.12 -0.17 µg as a sum of all fractions.

Since there were no substantial differences among the cell models, differentiated Caco-2 monocultures were used for the subsequent experiments.

To confirm the uptake of Al and Al 2 O 3 nanoparticles and to precisely localize them within the Caco-2 cells, TEM experiments were carried out. Slices cut at mid-height of cells incubated with the nanoparticles or with the ionic control were inspected (Fig 2D). Control and treated Caco-2-cells were lipid droplets rich as expected. Intracytoplasmic nanoparticle uptake was observed for both particle species. Nanoparticles were identified in many clusters, delimitated by a membrane which is typical for late endosomes as expected from an endocytose pathway.

Relatively large amounts of Al 0 and Al 2 O 3 nanoparticles were observed inside the cells, consistent with the results from the element analysis. Both particulate forms showed a similar behavior with a compartmentalization of the nanoparticles into late endosomes. In the case of Al nanoparticles, however, only large clusters were observed, whereas as Al 2 O 3 nanoparticles were found both as large agglomerates and as isolated particles. In addition, the Al 2 O 3 particles are possibly slightly released and/ or deteriorated within the endosomes. In contrast, no particle uptake was observed for the control samples, endosomes without dark content were infrequently observed. Using TOF-SIMS (Figure 2E), the 3D depth profile of the cells,

Cellular and molecular effects

Initially, cell viability testings such as CTB, MTT, apoptosis-/ necrosis-and cellular impedance measurements were done to determine applicable experimental conditions (Figure 3). None of the Al-species showed increased cytotoxicity on differentiated Caco-2 cells over 48 h of treatment. Only a weak toxic effect was observed with a high concentration of 200 µg Al/mL of ionic AlCl 3 . Similarly, no induction of apoptosis or necrosis, as well as no effects on cellular impedance was observed. After determination of cytotoxicity, regulatory effects of Al-containing nanoparticles and controls were investigated by an untargeted transcriptome screening analysis via microarrays under non-toxic conditions. Figure 4A shows Venn-diagrams of the deregulated genes of differentiated Caco-2 cells by each Al species. While there was only a moderate number of deregulated sequences for the particulate Al species (298 for Al 0 , 401 for Al 2 O 3 ), there were much more deregulated sequences resulting from treatment with ionic Al (1336). Many of those deregulated sequences were not annotated because they belong to unknown sequence segments. Some of them belong to regulatory elements, miRNAs, snRNPs, spliceosomal subunits or pseudogenes and only a smaller amount stands for protein coding sequences. Our results highlight that effects of Al-containing nanoparticles are rather low compared to the considerable effects caused by ionic species. These effects were obtained by in silico and in vitro testing methods. The gene regulation of 8 significantly regulated genes of different treatment groups was verified via qPCR, whichis shown in Figure 4B and Supplementary Figure 2.

The results of the in silico evaluation of the transcriptome analysis is shown in Figure 4C and Table 1. IPA analysis (Table 1) gives indications for involved signaling pathways. Predictions include, besides others, canonical pathways and toxicological mechanisms. With respect to the underlying array data, the predictions vary in their significance. Especially because the transcriptome analysis showed rather moderate fold changes and hit numbers, some of the predictions do not reach the significance level and therefore should be interpreted with care.

Basically, there are more meaningful predictions for the AlCl 3 treatment group, where the highest number of deregulated genes was found as well. IPA was only able to predict involvement of the relevant pathways, but not the direction of the regulation, i.e. up-or downregulation of the involved pathways. The data from treatment with ionic AlCl 3 pointed towards the involvement of xenobiotic response pathways such as CAR and PXR/RXR, or mechanisms that indicate stress reactions like glutathione (GSH) level changes or mitochondrial dysfunction. The predictions for elementary Al 0 nanoparticles focused as well on glutathione depletion and CAR/RXR activation. In this group, the significance levels were much lower and the predictions more limited because of the lower effects given from the microarrays. The predictions caused by Al 2 O 3 nanoparticles concentrated on xenobiotic metabolism and the cytochrome P450 panel, which was predicted for AlCl 3 as well with a higher significance. Effects on xenobiotic metabolism were found in all treatment groups, from which the predictions in the AlCl 3 group showed the strongest significance and also the involvement of the PXR/RXR signaling pathway.

The program "geneontology" (Figure 4C) indicated grouping of the regulated genes in relation to their cellular localization, protein function and involvement into biological processes. The bars show the fraction of the identified genes of each group which are related to the individual process, function or cellular localization. This provides hints about cellular processes which are influenced by the respective treatment. Although the predictions have to be interpreted with caution, tendencies were observable. Many detected sequences refer to stress response, metal ion interaction or metal binding activities. The relative amounts of each protein group did not differ much among the different species, which speaks for almost similar mode of actions that are only in a lower extent for particulate species, and therefore less significant, but following the same mechanistic principles. Probably the weak cellular response observed with Al-containing nanoparticles is related to the lower amount of free ions that are released from the particles compared to the ionic controls. Interestingly, protein fractions assigned to the predicted functions and processes caused my Al 0 nanoparticles were even lower than for both other species, what corresponds to the lower effects detected in the microarrays. This does not correspond to the ion release, which is higher than the ion release from Al 2 O 3 nanoparticles. The predictions for cellular localization appeared not to prefer any protein class and distributed equally over the different compartments. Here, the weakest predictions are made for Al 0 nanoparticles again. This is also valid for the protein functions.

Our results suggest that both particulate species did not induce any particle-specific reaction. Table 1: Results of predicted influence on canonical pathways and TOX-lists by IPA analysis. Alspecies were compared to the medium control, Significance defined by 3 out of 3 replicates for genes with a fold change > 1.5 up or down and ANOVA p-value < 0.05. Pathway predictions with a p-value above 0.05 are not statistically significant and therefore indicated in italics.

To see, if the predicted molecular effects were reflected on the cellular level, we chose several toxicological endpoints like GSH level, ATP level, mitochondrial membrane potential, IL-8 release and NFκB nuclear translocation, shown in Figure 5A-E. No effects on GSH levels or mitochondrial membrane potential were measured, irrespective of the Al-species. ATP level measurements showed a significant and concentration dependent decrease of ATP at high concentrations of ionic Al in contrast to the particulate species at the same concentrations. An interaction of Al with this luciferase testing system was already published and a correction factor was taken into account to subtract this interaction as described in the referred article. The levels of IL-8 release were studied by an ELISA assay which showed a non-significant increase compared to the control after 24 h of treatment.

Similarly, the transcription factor NFκB, which is translocated into the nucleus of cells as early event of inflammatory stimulation, showed only a slight increase with Al 0 nanoparticles (at 3 h and 6 h) and Al 2 O 3 nanoparticles (at 1 h for the highest concentration). The effect of AlCl 3 depicted a large variability but without any dose-response-relationship. Those results confirmed the absence of an inflammatory effect of Al and Al 2 O 3 nanoparticles and AlCl 3 .

Discussion

In this work, several analytical and toxicological questions concerning orally taken up Al were addressed. First, the issue of species-dependent ion release from Al-containing nanoparticles in presence of biological media was investigated. In nature, Al is mostly bound in minerals forming oxide species [START_REF] Yokel | Aluminium toxicokinetics: an updated minireview[END_REF]. Metallic species are mainly produced by humans and are so transferred into the biological cycles. In addition, industrial processes acidify the environment and increase therewith the availability of dissolved, ionic

Al [START_REF] Exley | A biogeochemical cycle for aluminium?[END_REF][START_REF] Martin | Aluminum: A Neurotoxic Product of Acid Rain[END_REF]. Ionic Al also derives from food packaging and kitchen ware [START_REF] Liukkonen-Lilja | Leaching of aluminium from aluminium dishes and packages[END_REF]Piepponen, 1992, Verissimo et al., 2006) or gets dissolved along the gastrointestinal tract [START_REF] Schönholzer | Intestinal absorption of trace amounts of aluminium in rats studied with 26aluminium and accelerator mass spectrometry[END_REF], Froment et al., 1989a). Therefore, to investigate effects of Al, it is necessary to have detailed knowledge about the chemical state of the Al that is observed. The physico-chemical parameters of the 3 Al species used in this study have been measured with complementary methods.

The volume-weighted distributions of the core gyration radii were determined by SAXS measurements. The advantage of SAXS is that a measurement direct in complex matrices like cell culture medium is possible. Since the aluminum nanoparticles scatter much stronger than the surrounding cell culture medium, SAXS is an appropriate method for the determination of the core radii. For the Al 2 O 3 nanoparticles a bimodal size distribution consisting of small particles and aggregates is depicted (Figure 1A). After an incubation time of 24 h in cell culture medium, the primary particles showed a radius of 13.6 ± 0.6 nm, whereas the aggregates exhibited radii > 30 nm. A longer incubation (48 h) resulted in a slight increase in the small particles' radii to 15.7 ± 0.8 nm. For the Al 0 nanoparticles a distinct differentiation of different size populations was not possible. Instead, the size distribution after 24 h showed particles with radii > 10 nm, which remained the same for an incubation time of 48 h. As already discussed in the preliminary study of the stock solutions [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF] the incubation of ionic Al in form of AlCl 3 in cell culture medium resulted in a nanoparticle formation. During the incubation time of 24 h and 48 h the formed particles showed radii in the range of 2 -60 nm with a broad size distribution. All in all, the particles size distribution for all three Al species did not change significantly upon incubation in cell culture medium for 24 h and 48 h.

We showed that metallic Al 0 particles are more susceptible to ion release in biological media than Al 2 O 3 nanoparticles (Figure 1). This effect was concentration-dependent as the fraction of released Al-ions was much higher at low Al-concentrations (15 % at 4 µg Al/mL and only 1 % at 100 µg Al/mL). This process can be two-directional, meaning that free ions can also form de novo particles or aggregates. This effect was described before for other elements [START_REF] Lichtenstein | Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells[END_REF], Juling et al., 2016[START_REF] Hansen | Characterization of Silver Nanoparticles in Cell Culture Medium Containing Fetal Bovine Serum[END_REF] and also for Al species in artificial digestion fluids [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF]. AlCl 3 was present in the form of free ions only in a proportion of around 20 -30 % irrespective of the loaded concentration. The present composition of Al species has an influence on bioavailability and cellular uptake of Al. Generally, soluble Al shows more systemic bioavailability than Al from solid species [START_REF] Van Oostdam | Canadian perspectives on aluminum[END_REF], Yokel et al., 2005[START_REF] Krewski | Human health risk assessment for aluminium, aluminium oxide, and aluminium hydroxide[END_REF]. Most Al is resorbed by the proximal section of the human duodenum (Froment et al., 1989b). In our experiment, we investigated the species-specific uptake and passage of Al into the intestinal barrier.

Therefore, we used three different cell culture models (Caco-2 monoculture, mucus model, M-cell model). Al uptake and transport were measured by element analysis. While there was no transport through the membrane detectable for all species (Figure 2B), we could observe differences in cellular uptake (Figure 2C). Among all three culture models, particulate species were measurable taken up into the (washed) membrane fraction. There was no measurable difference among the models and also not between both particle species, but there were strong differences to ionic Al species, which were taken up rarely into the cell fraction. While no transport through the in vitro intestinal models for the 3 Al forms was detected, results on cellular uptake highlighted that both NP forms were taken up by cells but rarely AlCl 3 . This particle-specific cellular uptake was already known for other nanoparticles and described as "trojan-horse-effect" [START_REF] Hsiao | Trojan-horse mechanism in the cellular uptake of silver nanoparticles verified by direct intra-and extracellular silver speciation analysis[END_REF][START_REF] Semisch | Cytotoxicity and genotoxicity of nano -and microparticulate copper oxide: role of solubility and intracellular bioavailability[END_REF] In Caco-2 cells, which were exposed to ionic aluminum (AlCl 3 ) only very few very small aluminum clusters could be detected within the whole culture, and these clusters seemed to localize in specific areas. However, TEM showed relatively similar patterns for Al and Al 2 O 3 nanoparticle clustering. In case of µPIXE analysis, the Al detection limit was higher than the natural Al background of control cells. Caco-2 cells exposed to Al 2 O 3 didn't reveal an uptake larger than the detection limit. The aluminum pattern for cells exposed to Al 0 nanoparticles exhibited a clear uptake of aluminum. This is in good consistence to TEM and TOF-SIMS results.

The absence of transport across the transwell membrane could be due to experimental issues.

Transwell systems show elevated barrier function and so limit paracellular crossing compared to the in vivo situation [START_REF] Artursson | Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells[END_REF]. In addition, persorption due to intestinal regeneration processes are not mimicked by this system [START_REF] Volkheimer | Passage of particles through the wall of the gastrointestinal tract[END_REF].

Nevertheless, transwell experiments often show a suitable control for intestinal uptake and transport of some nanoparticle species [START_REF] Gullberg | Expression of specific markers and particle transport in a new human intestinal M-cell model[END_REF][START_REF] Des Rieux | Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium[END_REF][START_REF] Walczak | Translocation of differently sized and charged polystyrene nanoparticles in in vitro intestinal cell models of increasing complexity[END_REF], Lichtenstein et al., 2016[START_REF] Lin | Cellular transport pathways of polymer coated gold nanoparticles[END_REF][START_REF] Bouwmeester | Characterization of translocation of silver nanoparticles and effects on whole-genome gene expression using an in vitro intestinal epithelium coculture model[END_REF].

Another influencing parameter on particle stability, agglomeration and biological interaction is the composition of the specific protein corona (Figure 1C). Our results show differences of protein identities between both particle species. Metallic Al nanoparticles showed a slightly complex protein distribution, where mainly β-actin was enriched. Al 2 O 3 nanoparticles showed a more complex variety of corona proteins. It was already shown, that serum proteins can have influence on the toxicity of metal ions [START_REF] Haase | The biochemical effects of extracellular Zn(2+) and other metal ions are severely affected by their speciation in cell culture media[END_REF]. In this context, proteins like serum albumin (Uniprot abbreviation: ALBU), transferrin (TRFE), apolipoprotein A1

(APOA1) and α-glycoprotein (FETUA) and β-actin (ACTB) were shown to interact with metal homeostasis and endocytosis activity [START_REF] Waheed | HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron[END_REF][START_REF] El Hage Chahine | Uptake and release of metal ions by transferrin and interaction with receptor 1[END_REF][START_REF] Yang | Biomimetic, synthetic HDL nanostructures for lymphoma[END_REF][START_REF] Lewis | Enhancement of human monocyte phagocytic function by alpha 2HS glycoprotein[END_REF][START_REF] Conner | Regulated portals of entry into the cell[END_REF], that have been detected in this study, too.

Recently, the toxicological effects of Al have been largely discussed. Dialyzed patients suffering encephalopathy and osteomalacia were correlated with elevated Al levels in blood and organs (Alfrey et al., 1976, Vick and[START_REF] Vick | Aluminum-related osteomalacia in renal-failure patients[END_REF]. The cation chelator deferoxamin which complexes Al ions was found to alleviate the symptoms [START_REF] Felsenfeld | Desferrioxamine therapy in hemodialysis patients with aluminum-associated bone disease[END_REF].

Current research focusses on a link with breast cancer [START_REF] Darbre | Underarm antiperspirants/deodorants and breast cancer[END_REF]) and Alzheimer's disease [START_REF] Walton | Chronic aluminum intake causes Alzheimer's disease: applying Sir Austin Bradford Hill's causality criteria[END_REF][START_REF] Percy | Towards the prevention of potential aluminum toxic effects and an effective treatment for Alzheimer's disease[END_REF], which although is disputed [START_REF] Lidsky | Is the Aluminum Hypothesis Dead[END_REF]. In this study, a broad concentration range from 4 -200 µg Al/mL was chosen. With an estimated Al consumption of 0.2 -2.3 mg / kg bw/week and a TWI of 1 mg / kg bw/week (EFSA, 2011), the lower part of the scale (4 -10 µg Al/mL) corresponds to a realistic oral Al exposure. The upper end of the scale corresponds to the highest possible exposure derived from the maximal Al contents found in food products such as mollusks, paprika, cocoa powder and blackboard salt with more than 100 mg/kg food mass [START_REF] Stahl | Aluminium content of selected foods and food products[END_REF][START_REF] Arnich | Dietary exposure to trace elements and health risk assessment in the 2nd French Total Diet Study[END_REF][START_REF] Millour | Pb, Hg, Cd, As, Sb and Al levels in foodstuffs from the 2nd French total diet study[END_REF]. Above 200 µg Al/mL, toxic effects of Al on Caco-2 cells are described [START_REF] Zhou | The chemical species of aluminum influences its paracellular flux across and uptake into Caco-2 cells, a model of gastrointestinal absorption[END_REF]. 100 µg Al/mL were found out to be non-toxic on Caco-2 cells with multiple assays (Figure 3) and so could be used for cellular uptake experiments. To avoid unspecific stress reactions in the very sensitive transcriptome analysis, a lower concentration of 50 µg Al/mL was used for the microarrays. The microarray study showed rather moderate deregulation of genes, what nevertheless allowed predicting the involvement of xenobiotic metabolism, oxidative stress response and metal ion homeostasis, mainly in the treatment group with ionic AlCl 3 . In vitro, these effects caused by Al 3+ ions were described on cellular level as oxidative damage [START_REF] Zatta | Aluminium(III) as a promoter of cellular oxidation[END_REF], indicated by membrane lipid peroxidation [START_REF] Gutteridge | Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts[END_REF][START_REF] Fraga | Effects of aluminum on brain lipid peroxidation[END_REF]. Comparable effects were also described for particulate Al 2 O 3 [START_REF] Zhang | In vivo toxicity of nano-alumina on mice neurobehavioral profiles and the potential mechanisms[END_REF]. This leads further to a destabilization of the cell membrane [START_REF] Amador | Lipid peroxidation facilitates aluminum accumulation in rat brain synaptosomes[END_REF]. Another described key effect of Al is the competition with essential metal ions such as iron [START_REF] Mujika | A QM/MM study of the complexes formed by aluminum and iron with serum transferrin at neutral and acidic pH[END_REF], magnesium [START_REF] Martin | Aluminum: A Neurotoxic Product of Acid Rain[END_REF] and calcium [START_REF] Mundy | Aluminum potentiates glutamate-induced calcium accumulation and iron-induced oxygen free radical formation in primary neuronal cultures[END_REF], due to their chemical resemblance [START_REF] Zatta | Aluminium(III) as a promoter of cellular oxidation[END_REF]. Al has a strong affinity to essential cellular compounds such as phosphate and influences enzymatic functions of the antioxidative system [START_REF] Zatta | Aluminium(III) as a promoter of cellular oxidation[END_REF]. Al has a strong affinity to essential cellular compounds such as phosphate and influences enyzmatic functions of the antioxidative system [START_REF] Zatta | Aluminium(III) as a promoter of cellular oxidation[END_REF][START_REF] Kong | Aluminum(III) facilitates the oxidation of NADH by the superoxide anion[END_REF][START_REF] Hasanoglu | The relationship between erythrocyte superoxide dismutase activity and plasma levels of some trace elements (Al, Cu, Zn) of dialysis patients[END_REF][START_REF] Atienzar | Effect of aluminum on superoxide dismutase activity in the adult rat brain[END_REF]. Our in silico methods predicted that metal ion homeostasis, xenobiotic metabolism and oxidative stress response could be affected (Figure 4, Table 1). Nevertheless, our in vitro results showed that at least for the differentiated Caco-2 cell model, most relevant endpoints were not affected. A significant change in cellular ATP levels that can result from oxidative stress was only reported with AlCl 3 . Even if the cellular uptake of Al-containing nanoparticles was higher, no specific nanoparticle effect which was detected. We conclude, that effects are mainly caused by the released Al ions, what is underlined by the in silico microarray predictions.

Conclusion

Cellular uptake, bioavailability, metabolism and toxicological effects of Aluminum are species-specific and controversially discussed. We have shown for the first time that intestinal cellular uptake of Al occurs preferably in the particle form, while toxicological effects appear to be ion-related. Regarding this, in biological systems, such as the gastrointestinal tract, there occurs a transition of particulate to ionic Al species as well as reverse. The proteomic composition of the biological interface is also species-specific, as well as the quantity of released Al ions from different nanoparticles. Toxicological effects of Al target cellular metal ion interaction, oxidative damage and xenobiotic metabolism. Acute toxic effects were mainly found with high AlCl 3 concentrations in intestinal cells. Systemic exposure is still under debate as no in vitro crossing of the intestinal barrier was detected using 3 different human intestinal cell models. With contemplation to these results in question of uptake, cellular effects and toxicological potential, the resultant impact of Al on human health is not completely foreseeable. Small acute effects imply investigations of long term effects with regard to the respective Al species, which can vary in their quality and quantity of effects.

Therefore, Al will stay in the focus of research and risk assessment in the following years.

List of abbreviations

  and absorption was measured by a plate reader (570 nm) with subtraction of the background absorption (630 nm). Results were normalized on untreated controls after subtraction of equally treated cell free reference wells. As a positive control, 0.01% Triton X-100 was used.Statistics were performed as Student's t-test indicated by asterisks (* p<0.05;** p<0.01; *** p<0.001).Cellular impedance was determined by an xCELLigence system (xCELLigence, Roche, Germany). Caco-2 cells were differentiated in gold-coated 96-well E-plates, incubated with 200 µL testing substances and controls and measured over a time period of 48 h. The cell index was determined as mean values of at least 3 replicates with standard deviation. Cell indices were double-normalized to time point 0 and untreated controls. As a positive control, ZnO-Particles (IoLiTec, Heilbronn, Germany) were used.Apoptosis / Necrosis measurementsApoptosis / Necrosis measurements and cell cycle analysis were performed by flow cytometry with BD Accuri C6 (Accuri, Belgium). Caco-2 cells were cultivated and incubated as described before. After incubation, media were saved and cells were washed with 100 µl PBS, harvested with 50 µL trypsin/EDTA and collected in 200 µL warm, serum-containing CCM.Cells and media were reunited for each sample and centrifuged (3 min, 200 x g) to sediment the cells. Cells were washed with 800 µL cold PBS and then 800 µL annexin buffer afterwards (5 mM Hepes, 70 mM NaCl, 2.5 mM CaCl 2 , pH 7.4). After that, cells were stained with AnnexinV-FITC (1:20) and 7AAD (1:10) at the same time in annexin buffer for 30 min.Fluorescence was detected for FITC (Ex. 488, Em. 533/30 nm) and 7AAD (Ex. 488, Em. >670 nm) and data for 10,000 cells each sample was collected. Color compensation was performed to avoid interactions between both fluorophores. Background level was determined by untreated stained controls. Cells that were AxV-positive were defined as apoptotic; cells that were 7AAD-positive or double-positive were defined as late-apoptotic / necrotic. The relative cell counts of the populations were averaged over at least 3 independent experiments, error bars indicate standard deviations. Statistics were performed as student's t-test indicated by asterisks (* p<0.05;** p<0.01; *** p<0.001). As positive controls, 2 µM staurosporine (apoptosis) and 50 µM tert-butyl-hydroxyperoxide (tBOOH, necrosis) were used.Transcriptome analysisCaco-2 cells were grown and differentiated in 75 cm² cell culture flasks incubated for 24 h with different species and concentrations of Al by adding 1 mL of Al-containing nanoparticles and controls into 19 mL cell culture medium up to non-toxic final concentrations of 5, 25 and 50 µg Al/mL. Each condition was run in triplicates. After incubation, cell pellets were harvested by scraping into 1 mL PBS and cells were washed twice. Cell pellets were stored at -80 °C before use. RNA isolation was performed according

  Al 2 O 3 nanoparticles and the medium background. The ionic Al species showed an almost steady amount of 15 -25 % free ions in cell culture medium. The remaining proportion could exist as aggregates, de novo emerged particles, polymers and protein complexes. The determination of the protein corona composition surrounding the nanoparticles in serumcontaining cell culture medium could also influence interactions between nanoparticles and cells. Figure1Cshows the protein composition of the coronae surrounding both nanoparticle species, separated by 2D-PAGE and analyzed by tryptic digestion and MALDI-TOF-MS. The corona of metallic nanoparticles appeared less complex and contained predominantly enriched β-Actin while the other serum proteins got depleted and were barely detectable in the protein analysis. The corona of Al 2 O 3 nanoparticles displayed a more complex composition. The appearance of metal ion interacting proteins (FETA, TRFE, abbreviations defined in the table)

Figure 1 :

 1 Figure 1: Characterization of nanoparticles. (A) Particle core size as determined by SAXS in cell culture medium (DMEM) after 24 h and 48 h incubation time. (B) Ion release of Al-containing nanoparticles and controls. Quantification of ionic Al was performed via ultracentrifugation, microwave digestion and AAS after incubation of testing substances in serum-containing cell culture medium (CCM) over a time period of 48 h. Relative ionic fraction compared to initial concentration (4

  depicted as translucent green with Al 0 nanoparticles in dark blue color and Al 2 O 3 in light blue, showing clearly the intracellular localization of Al 0 nanoparticle aggregates. The cells, which were exposed to Al 2 O 3 nanoparticles are showing the cell monolayer in translucent green and with Al 2 O 3 nanoparticles in blue color, showing clearly the intracellular localization of Al 2 O 3 nanoparticle aggregates. IBM (Figure 2F) displays the element distributions of Al, phosphorous and sulfur among differentiated Caco-2 cells after incubation with Al species. In untreated control cells as well as in Caco-2 cells incubated with Al 2 O 3 nanoparticles, only a background level of Al was visible, which was lower than the methodical limit of detection and therefore not quantifiable. In contrast, cells incubated with Al 0 nanoparticles showed a clear association between the cells and Al.

Figure 2 :

 2 Figure 2: Analysis of transport and uptake of aluminum species in in vitro models. (A) Schematic overview over the used in vitro model. Quantification of transport into the basolateral compartment (B) and cellular uptake (C) of Al-containing nanoparticles and controls in transwell experiments with differentiated Caco-2 cells and cocultures after incubation with testing substances (100 µg Al/mL) for 24 h. Al-amount determined by microwave digestion of basolateral compartment (transport) and washed membrane fraction (uptake) followed up by AAS measurements. Each experiment was repeated at least 3 times with 3 replicates of each sample with standard deviation, significance

Figure 3 :

 3 Figure 3: Measurements of toxicological endpoints after 24 h incubation of differentiated Caco-2 cells after incubation with Al-species. (A and B) Relative viability after 48 h incubation with Al-species, shown by CTB (A) and MTT (B). 0.01 % Triton X-100 was used as positive control. (C) Apoptosis-/ necrosis measurements determined by flow cytometry with Annexin-V / 7-AAD staining. As positive controls, 2 µM staurosporine and 50 mM tert-butyl hydroxyperoxide were used. (D) Measurements of cellular impedance by xCELLigence technology after incubation with Al-species over a time period of 48 h. Relative cell indices normalized on initial cell index and medium control. Mean values calculated out of at least 3 replicates indicated by standard deviation. ZnO nanoparticles (100 µg Zn/ml, supplied by IoLiTec) were used as positive control. Results normalized on medium control as mean values of at least 3 independent experiments indicated by standard deviation. Significance determined via student's t-test indicated by asterisks.

Figure 4 :

 4 Figure 4: Results of microarray analysis. (A) Venn diagram comparison of deregulated primary hits (black numbers) and annotated sequences (grey numbers) determined by microarray analysis of differentiated Caco-2 cells incubated with Al species (50 µg Al/mL) for 24 h. (B) Quantitative real time RT-PCR for selected genes ABCC3 and COG7. Further genes are depicted in Supplementary Figure 2. (C) Results of in silico analysis of the fractions of regulated genes related to a specific cellular localization, protein function and biological processes by "geneontology" software. Significance defined by 3 out of 3 replicates with a fold change > 1.5 up or down and ANOVA pvalue < 0.05.

Figure 5 :

 5 Figure 5: Measurements of toxicological endpoints of differentiated Caco-2 cells after 24 h incubation with Al-species. (A) Determination of relative GSH-levels determined by monochlorobimane assay. (B) Relative cellular ATP-levels determined by Cell Titer Glo-assay. (C) Relative mitochondrial membrane potentials determined by JC1-assay. (D) IL-8 release from differentiated Caco2 cells after 24h nanoparticle exposition. As positive control, 20 ng/mL TNFα was used. Values are presented as the mean percentage ± SD (n = 3). *** indicate significantly higher levels compared to controls corresponding to p < 0,001, student's t-test. (E) Nuclear NFⱩB translocation after 1, 3 or 6 hours treatment with different concentrations of Al species. Positive control TNFα 20 ng/mL. Values are presented as the mean percentage ± SD (n = 3). *,**,*** indicate significantly higher levels compared to controls corresponding respectively to p < 0.05, p < 0.01, p < 0.001, Student's t-test. Results normalized on medium control as mean values of at least 3 independent experiments indicated by standard deviation. Significance was determined via student's t-test indicated by asterisks. As positive controls 100 µM BSO (A), 1 µM oligomycin (B), 1 mg/mL valinomycin (C), and TNFα (D and E) were used.

  . Particles were taken up incidentally while the cells have mechanisms to keep metal ions actively out. TEM experiments are consistent with elemental analysis, since they clearly show the uptake of the particles. This result is no contradiction to the previously preferred ionic intestinal bioavailability, because an uptake into epithelial cells does not necessarily mean systemic transport. Intestinal epithelial cells, with or without particles, have a lifetime of around 72 h and are excreted by the gut. So only transported Al is systemic bioavailable. The 3D TOF-SIMS depth profiles of the cells showed clearly the intracellular localization of Al 0 nanoparticle aggregates in cells exposed to Al 0 nanoparticles. The cells, which were exposed to Al 2 O 3 nanoparticles showed as well clearly the intracellular localization of Al 2 O 3 nanoparticle aggregates. No aluminum could be detected within unexposed Caco-2 control cells.

Acknowledgements

The authors are grateful to the MRic-TEM core facility of BIOSIT (CNRS-INSERM UMS 3480, US_S 018, University of Rennes 1, France) for the transmission electronic microscopy.

We would like to thank Rachelle Lanceleur for technical assistance in TEM uptake experiments.

Funding

This publication, as part of the German-French SolNanoTOX project was funded by the German Research Foundation DFG (Grant Numbers LA 3411/1-1), by the French "Agence Nationale de la Recherche" ANR (Project ID ANR-13-IS10-0005).

Availability of data and material

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Supplementary data

Supplementary Table 1: Particle characterization parameters. The particle characterization of the applied nanoparticles was already published [START_REF] Sieg | Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials[END_REF][START_REF] Krause | Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials using a combination of methods for particle surface and size analysis[END_REF] and is summed up in this table.

Supplementary Figure 1: Aluminum recovery from Al-containing nanoparticles and controls in transwell uptake experiments with differentiated Caco-2 cells and cocultures after incubation with testing substances (100 µg Al/mL) for 24 h. The Al amount was determined by microwave digestion of all compartments (apical, wash fraction, membrane and basolateral compartment) followed by AAS

Declarations

Competing interests

The authors declare that there is no competing of interest.


Fabrice Pointillart

Jiang-Kun Ou-Yang

Guglielmo Fernandez

Vincent Montigaud

Jessica Flores Gonzalez

Rémi Marchal

Ludovic Favereau

Federico Totti

Jeanne Crassous

Olivier Ca- Dor

Lahcène Ouahab

Boris Le Guennic

Tetrathiafulvalene-Based Helicene Ligand in the Design of Dysprosium Field-Induced Single-Molecule Magnet

The design of a coordination complex which involves a ligand combining both a tetrathiafulvalene core and helicene fragment was achieved thanks to the reaction between the new 2-{1-[2-methyl[6]helicene]-4,5-[4,5-bis(propylthio)tetrathiafulvalenyl]-1H-benzimidazol-2-yl}pyridine ligand (L) and the Dy(hfac) 3 ⋅2H 2 O metallo precursor. Magnetic investigations showed a field-induced Single-Molecule Magnet (SMM) behavior under an applied magnetic field of 1000 Oe for [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 while experimental oriented single crystal magnetic measurements allowed for the determination of the magnetic anisotropy orientation. The magnetic behavior was rationalized based on ab initio CASSCF/SI-SO calculations. This redox-active chiral field-induced SMM paves the route for designing switchable multi-properties SMMs.

Chemists are working hand in hand with physicists to design Single-Molecule Magnets (SMMs) displaying magnetic bistability at temperatures as high as possible. Recently a mononuclear Dy III -SMM highlighted a blocking temperature of 60 K [START_REF] Guo | A Dysprosium Metallocene Single-Molecule Magnet Functioning at the Axial Limit[END_REF][START_REF] Goodwin | Molecular magnetic hysteresis at 60 kelvin in dysprosocenium[END_REF][3] jump-starting this kind of molecular objects suitable for potential applications in high-density data storage, quantum computing and spintronics. [START_REF] Mannini | Magnetic memory of a single-molecule quantum magnet wired to a gold surface[END_REF][START_REF] Pedersen | Toward Molecular 4f Single-Ion Magnet Qubits[END_REF][START_REF] Rocha | Towards Molecular Spintronics[END_REF] One more challenge is to combine the SMM behavior with others chemical, optical or physical properties such as ferroelectricity, redox-activity/conductivity, luminescence, nonlinear optics, chirality/chiroptical activity in order to open the route to more potential applications. [START_REF] Pointillart | Slow magnetic relaxation in radical cation tetrathiafulvalene-based lanthanide(III) dinuclear complexes[END_REF][START_REF] Long | A High-Temperature Molecular Ferroelectric Zn/Dy Complex Exhibiting Single-Ion-Magnet Behavior and Lanthanide Luminescence[END_REF][START_REF] Liu | Field-Induced Single-Ion Magnets Based on Enantiopure Chiral β-Diketonate Ligands[END_REF] In the specific case of chiral SMMs, the chirality can come from highly optically-active ligands such as [n]helicenes, [START_REF] Ou-Yang | Improved slow magnetic relaxation in optically pure helicene-based Dy III single molecule magnets[END_REF][START_REF] Fernandez-Garcia | Slow Magnetic Relaxation in Chiral Helicene-Based Coordination Complex of Dysprosium[END_REF] which are organic molecules with π-conjugated helical backbone made of ortho-fused aromatic rings with configurational stability for n ≥ 5. [START_REF] Shen | Helicenes Chemistry: From Synthesis to Applications[END_REF] Subsequently the [n]helicenes can be decorated by an electroactive unit such as organometallic, 13 quinone, [START_REF] Schweinfurth | Redox-Triggered Chiroptical Switching and Chiral Recognition of the Semiquinone Radical Anion Lithium Salt by Electron Nuclear Double Resonance Spectroscopy[END_REF] pyridinium [START_REF] Pospíšil | Intense Chiroptical Switching in a Dicationic Helicene-Like Derivative: Exploration of a Viologen-Type Redox Manifold of a Non-Racemic Hequat[END_REF] and tetrathiafulvalene [START_REF] Biet | Ethylenedithio-Tetrathiafulvalene-Helicenes: Electroactive Helical Precursors with Switchable Chiroptical Properties[END_REF] (TTF) derivatives. The latter TTF core is a well-known electroactive fragment mainly used to design conducting materials ranging from semiconductors to superconductors. [START_REF] Kobayashi | BETS as a source of molecular magnetic superconductors (BETS = bis(ethylenedithio)tetraselenafulvalene[END_REF][START_REF] Coronado | Coexistence of ferromagnetism and metallic conductivity in a molecule-based layered compound[END_REF] Its functionalization with a plethora of accepting groups allowed the TTF-based ligands to participate in the development of fluorescence switches, [START_REF] Zhang | A new Redox-Fluorescence Switch Based on a Triad with Tetrathiafulvalene and Antracene Units[END_REF] photovoltaic cells, [START_REF] Bendikov | and their buckminsterfullerene derivatives: the brick and mortar of organic electronics[END_REF] coordination of transition metals [START_REF] Lorcy | Tetrathiafulvalene-based group XV ligands: synthesis, coordination chemistry and radical cation salts[END_REF] and finally coordination of 4f elements. In particular, the combination of lanthanide ions with TTF-based ligands permitted to reach new classes of multi-properties compounds with redox-active luminescent SMM behaviour. [START_REF] Pointillart | Slow magnetic relaxation in radical cation tetrathiafulvalene-based lanthanide(III) dinuclear complexes[END_REF][START_REF] Pointillart | Lanthanide Ion and Tetrathiafulvalene-Based Ligand as a "magic" Couple toward Luminescence, Single-Molecule Magnets, and Magnetostructural Correlations[END_REF] To the best of our knowledge, no chiral SMM with a redox activity easily chemically accessible in common organic solvents has been reported to date. To reach such objective, we propose i) to combine both electro-active TTF and carbo [6]helicene in a unique ligand and ii) to coordinate such ligand to magnetically anisotropic Dy III ion. The functionalization of the molecular skeleton 4,5bis(propylthio)-tetrathiafulvalenyl]-1H-benzimidazol-2-yl}pyridine [START_REF] Wu | Imidazole-Annulated Tetrathiafulvalene Exhibiting pH-Tuneable Intramolecular Charge Transfer and Redox Properties[END_REF] with the 2-bromomethyl [6]helicene [START_REF] Storch | Synthesis and Characterization of a Helicene-Based Imidazolium Salt and Its Application in Organic Molecular Electronics[END_REF] in its racemic form (85% yield, see S.I.) led to the 2-{1-[2-methyl [6]helicene]-4,5-[4,5-bis(propylthio)-tetrathiafulvalenyl]-1H-benzimidazol-2-yl}pyridine (L) ligand which reacted with the precursor Dy(hfac) 3 ⋅2H 2 O (hfac -= 1,1,1,5,5,5-hexafluoroacetylacetonate anion) to give the mononuclear complex [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 .

Single-crystal X-ray diffraction confirms that this compound crystallizes in the triclinic space group P-1 (N°2) (Table S1). The molecular structure attests the success of the alkylation of the TTF-based molecular skeleton by the 2-methylenecarbo [6]helicene derivative (Figures 1 andS1). The Dy III ion is coordinated to the bischelating 1H-benzimidazol-2-yl}pyridine (bzip) moiety and to the three hfac -anions in a N 2 O 6 surrounding. In a strict structural point of view, the first neighboring atoms create a D 2d symmetry polyhedron (Table S2, SHAPE analysis [START_REF] Llunell | version 2.1)[END_REF] ). The neutrality of the ligand L is confirmed by the C9=C10 central bond length of 1.343(7) Å. The benzimidazol-tetrathiafulvalene fragment is planar while the planes formed by the benzimidazole and pyridine moieties have a twist angle of 18.0(2)° resulting from the steric hindrance of the [6]helicene arm and optimized ππ interactions between the terminal benzene ring of the helicene and the bzip coordinated fragment. A quick overview of the literature on this kind of TTF-based ligands shows a zero twist angle when no alkylation is realized (amine, NH) [START_REF] Cosquer | Slow Magnetic relaxation in Condensed versus Dispersed Dysprosium(III) Mononuclear Complexes[END_REF] and for methyl-2pyridine, [START_REF] Pointillart | Rational Design of a Lanthanide-Based Complex Featuring Different Single-Molecule Magnets[END_REF] 4-methylpyridine-N-oxide, [START_REF] Pointillart | Rational Design of a Lanthanide-Based Complex Featuring Different Single-Molecule Magnets[END_REF] 4,4'-dimethyl-2,2'bipyridine [START_REF] Speed | Lanthanide complexes involving multichelating TTF-based ligands[END_REF] as alkylating arms while a twist angle of 12.2(3)° is measured for a bulkier arm such as 2,6-di(pyrazol-1-yl)-4methylpyridyl [START_REF] Zadrozny | Slow magnetization dynamics in a series of two-coordinate iron(II) complexes[END_REF] . The crystal packing highlights the formation of a one-dimensional network of stacked L along the a axis which is formed by intramolecular π-π interactions between the helicene and bzip fragments and intermolecular π-π interactions between two "head-to-tail" ligands (Figure 1). The shortest intermolecular Dy-Dy distance is equal to 9.529 Å.

The redox properties of the ligand L and the complex [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 are investigated by cyclic voltammetry (Figure S2, Table S3). The cyclic voltammogram for the free ligand shows two mono-electronic oxidations at 0.53 V for the first oxidation and 0.93 V for the second oxidation, corresponding to the formation of a radical cation and a dication TTF fragment, respectively. [START_REF] Wu | Imidazole-Annulated Tetrathiafulvalene Exhibiting pH-Tuneable Intramolecular Charge Transfer and Redox Properties[END_REF][START_REF] Cosquer | Slow Magnetic relaxation in Condensed versus Dispersed Dysprosium(III) Mononuclear Complexes[END_REF][START_REF] Pointillart | Rational Design of a Lanthanide-Based Complex Featuring Different Single-Molecule Magnets[END_REF][START_REF] Speed | Lanthanide complexes involving multichelating TTF-based ligands[END_REF][START_REF] Zadrozny | Slow magnetization dynamics in a series of two-coordinate iron(II) complexes[END_REF] The coordination of the Dy(hfac) 3 fragment has almost no significant effect on the oxidation potentials (0.55 V and 0.94 V) as observed for similar compounds. [START_REF] Cosquer | Slow Magnetic relaxation in Condensed versus Dispersed Dysprosium(III) Mononuclear Complexes[END_REF][START_REF] Pointillart | Rational Design of a Lanthanide-Based Complex Featuring Different Single-Molecule Magnets[END_REF][START_REF] Speed | Lanthanide complexes involving multichelating TTF-based ligands[END_REF][START_REF] Zadrozny | Slow magnetization dynamics in a series of two-coordinate iron(II) complexes[END_REF] The electrochemical properties attest that the reversibility of the oxidation potentials and the redox-activity of the ligand are conserved after complexation.

The temperature dependence of χ M T for a powdered sample of [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 shows a room temperature value of 14.06 cm 3 K mol -1 in agreement with isolated Dy III ion (Figure 2a). On cooling, χ M T decreases monotonically down to 12.17 cm 3 K mol -1 at 2 K. The first magnetization at 2 K highlights a classic behavior in the field range of 0-50 kOe for such isolated Dy III ion (inset Figure 2a). The same sample shows frequency dependence in zero external dc field (Figures 2b andS3) but with a maximum of the χ M '' vs. ν curve (ν the frequency of the ac oscillating field) that falls out of the frequency range of the experimental window. The application of an external dc field cancels the Quantum Tunneling of the Magnetization (QTM) and the maximum of the χ M '' vs. ν curve shifts to lower frequency (Figure 2b). The 1000 Oe value was chosen as the optimum field since the relaxation is slowest and more intense (Figure 2b). In such applied field, the out-of-phase component of χ M was observed in the temperature range of 2-8 K (Figures 2c andS4). The frequency dependence of the ac susceptibility can be analyzed in the framework of the extended Debye model both for field and temperature variations. The temperature dependence of the relaxation time at 1000 Oe is extracted from the extended Debye model between 2.0 and 6.0 K (Table S4). The Arrhenius plot of the relaxation time is well fitted by a Raman relaxation process τ=C×T n with C=2.4(4)×10 -2 and n=5.5(1) (Figure 2d) [START_REF] Zadrozny | Slow magnetization dynamics in a series of two-coordinate iron(II) complexes[END_REF] . Theoretically, the expected value of the n exponent for Kramers ions is 9 and can be decreased down to 4, depending on the energies of the ground state doublets. [START_REF] Dekker | Activated dynamics in a two-dimensional Ising spin glass: Rb 2Cu1-xCoxF4[END_REF][START_REF] Tang | Lanthanide Single Molecule Magnets[END_REF] Recently is was shown that such exponent could reach a value close to 2 due to the presence of both acoustic and optic phonons in specific ligand environments. [START_REF] Goodwin | Synthesis and Electronic Structures of Heavy Lanthanide Metallocenium Cations[END_REF] The Cole-Cole [START_REF] Cole | Dipersion and Absorption in Dielectrics I. Alternating Current Characteristics[END_REF] plots normalized to their isothermal value are represented in Figure S5. The Dy III ion lies in a N 2 O 6 environment with the two nitrogen atoms coming from a bischelating imidazol-2-ylpyridine derivative while the six oxygen atoms come from three hfac -anions. Thus the extracted dynamic parameters for the complex [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 can be compared with examples in which the common TTF-based skeleton is alkylated with different chemical arms. In these compounds and others of the litterature, [START_REF] Kishi | Luminescence and Single-Molecule Magnet Behaviour in Lanthanide Coordination Complexes Involving Benzothiazole-Based tetrathiafulvalene Ligands[END_REF] the effective energy barrier range from 18 K to 57 K. Nevertheless, starting from the same coordination polyhedron symmetry and N 2 O 6 environment, it is clear that the nature of the alkylated group and/or the positions of the hfac -anions (which can depend on the steric hindrance of the alkylated group) have a crucial importance on the value of the energy barrier. Since the complex [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 crystallizes in the triclinic P-1 space group and considering an effective spin ½ at low temperature, the orientation of the g-tensor can be determined by measuring the magnetization of a single crystal in the three perpendicular planes (Figure S6). This oriented single crystal measurements revealed an uniaxial magnetic anisotropy with a Landé factor of 19.6 (20 expected for a pure M J =±15/2 ground state) with the orientation of the main magnetic axis along the most negatively charged direction of the coordination surrounding i.e. perpendicular to the plane containing the nitrogen atoms (Figure 3). SA-CASSCF/SI-SO calculations were performed on [Dy(hfac) 3 (L)] to rationalize the observed magnetic properties (see computational details). Energy spectra and g tensors for the eight Kramer's doublets of the ground 6 H 15/2 multiplet of the Dy III ion are given in Table S5. The calculations confirm the axial character of the magnetic anisotropy tensor of the ground Kramer's doublet with a large g z value of 19.09 for the Dy III center and almost negligible g x and g y values. The decomposition in terms of pure |J=15/2> spin wavefunctions shows that the ground state has a majority (88%) |±15/2> character with a non-negligible (10%) |±11/2> component with a first excited state lying at more than 75 K above the ground state. Such discrepancy with the This orientation is further supported by the representation of the molecular electrostatic potential around the Dy III ion using the home-made CAMMEL program (Calculated Molecular Multipolar Electrostatics; the description of the code is detailed in the Supporting Information). The total electrostatic potential is represented for the ground state along with both experimental and calculated axes in Figure 3a. As one can see, the most negative potential, containing the calculated magnetic axis, appears along the plane containing the two hfac -ligands and perpendicular to the TTF plane. Moreover, the multipolar expansion of the total molecular electrostatic potential (Figures S7-S9) shows that quadrupolar contributions seem to be preponderant, as already observed in previous studies. [START_REF] Zhang | Tuning Magnetic Interactions in Dy(III)4 Single-Molecule Magnets[END_REF][START_REF] Huang | Magnetic slow relaxation in a Metal Organic Framework made of chains of ferromagnetically coupled Single-Molecule Magnets[END_REF] Regarding the static magnetic properties, this electronic structure leads to a calculated magnetization at 2 K in good agreement with the experimental M vs H and to a quantitative agreement in the thermal dependence of the χ M T product (Figure 2), with some small discrepancy at low temperatures that may be due to possible intermolecular dipolar interactions. The computed magnetization blocking barrier is reported in Figure 3b. Even though main relaxation mechanisms probably involve higher excited states (up to the third), calculations also support quantum tunneling relaxation mechanisms (QTM) within the ground state doublet. However, these transition moments allow only a qualitative evaluation of the magnetization barrier, since not all contributions (e.g. spin-phonon coupling) are included. [START_REF] Escalera-Moreno | Determining Key Local Vibrations in the Relaxation of Molecular Spin Qubits and Single-Molecule Magnets[END_REF][START_REF] Lunghi | Intra-molecular origin of the spin-phonon coupling in slow-relaxing molecular magnets[END_REF][START_REF] Lunghi | The role of anharmonic phonons in under-barrier spin relaxation of single molecule magnets[END_REF] In conclusion, a redox-active chiral field-induced SMM, [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 , has been successfully synthesized by combination of a redox-active TTF-based skeleton and a [6]Helicene derivative followed by the coordination reaction of the Dy(hfac) 3 precursor. This complex exhibits field-induced SMM behavior. This compound paves the route to the redox modulation of the electronic circular dichroism ECD signal and SMM behavior. To reach such aim, the synthesis of pure enantiomers of [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 and the oxidation of L are in progress.

A redox-active chiral Field-Induced Single-Molecule Magnet was achieved through the coordination reaction of the [Dy(hfac) 3 ] precursor to a ligand which combines a redox-active tetrathiafulvalene core and a chiral carbo [6]-helicene fragment. This multi-properties system was characterized using X-ray crystallography, magnetism and ab initio calculations.

Figure 1 .

 1 Figure 1. (Left) molecular structure of [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 , the hydrogen atoms and dichloromethane molecule of crystallization were omitted for clarity. Selected bond lengths (Å): Dy1-N1, 2.466(5); Dy1-N2, 2.562(5); Dy1-O1, 2.330(4); Dy1-O2, 2.366(4); Dy1-O3, 2.332(4); Dy1-O4, 2.351(4); Dy1-O5, 2.372(4); Dy1-O6, 2.323(4); C9-C10, 1.343(7); carbon (C, gray); fluorine (F, green). (Right) crystal packing of [Dy(hfac) 3 (L)] highlighting the π-π interactions along the a axis between the TTF-based molecular skeletons and the helicenic moieties (spacefill representation).

Figure 2 .

 2 Figure 2. (a) Thermal variation of χ M T for. In inset, first magnetization. Calculated curves are in red. (b) Scan field of the frequency dependence of χ M '' of at 2 K. (c) Frequency dependence of χ M " between 2 and 8 K at 1000 Oe. (d) Temperature variation of the relaxation time measured in an external field of 1000 Oe with the best-fitted curve (red line) in the temperature range of 2-6 K.

  hypothetic energy barrier coming from an Orbach process (21.1 K) supports the only operative Raman relaxation process in [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 . The calculated ground-state easy axis (Figure3) for the Dy III ion is oriented perpendicular to the plane formed by the {tetrathiafulvalenyl-1H-benzimidazol-2-yl}pyridine moiety in agreement with experiment.[START_REF] Da Cunha | Magnetic Poles Determinations and Robustness of Memory Effect upon Solubilization in a Dy III -Based Single Ion Magnet[END_REF]

Figure 3 .

 3 Figure 3. (a) Representation (two different orientations) of the ground state total molecular electrostatic potential around the Dy III ion. The black and red lines correspond to experimental and theoretical main anisotropy axes, respectively. (b) Computed magnetization blocking barriers. Numbers provided on each arrow are the mean absolute values for the corresponding matrix elements of the magnetic transition dipole moment.

ACKNOWLEDGMENT

This work was supported by Région Bretagne, Rennes Métropole, CNRS, Université de Rennes 1, the ANR (ANR-13-BS07-0022-01) and the ERC-CoG 725184 MULTIPROSMM (project n. 725184). G.F.G. and V. M. gratefully acknowledges the European Commission through the ERC-AdG 267746 MolNanoMas (project N. 267746) and the ANR (ANR-13-BS07-0022-01) for financial support. B.L.G., G.F.G. and V.M. thank the French GENCI/IDRIS-CINES center for high-performance computing resources.

ASSOCIATED CONTENT Supporting Information

Ortep view of [Dy(hfac) 3 (L)]⋅0.5CH 2 Cl 2 (Figure S1), crystallographic data (Table S1-2), cyclic voltamograms (Figure S2, Table S3), additional magnetic data (Figures S4-9 and Tables S4-5), Xray data in CIF format, experimental and material details and computational details. This material is available free of charge via the Internet at http://pubs.acs.org."

AUTHOR INFORMATION

Corresponding Author E-mail: fabrice.pointillart@univ-rennes1.fr E-mail: boris.leguennic@univ-rennes1.fr E-mail: Jeanne.crassous@univ-rennes1.fr ORCID Fabrice Pointillart: 0000-0001-7601-1927 Boris Le Guennic: 0000-0003-3013-0546 Jeanne Crassous: 0000-0002-4037-6067 Olivier Cador: 0000-0003-2064-6223 Ludovic Favereau 0000-0001-7847-2911

Notes

The authors declare no competing financial interests.