Minh Quan Le

Liang Ye

Valentina Bernasconi

PhD Rodolphe Carpentier

François Fasquelle

Nils Lycke

Peter Staeheli

Didier Betbeder

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine

Keywords: Intranasal, vaccine, nanoparticle, influenza, CTA1-DD

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Influenza is a highly-contagious respiratory infectious disease which causes public health and socio-economic problems worldwide [START_REF] Gasparini | Clinical and socioeconomic impact of seasonal and pandemic influenza in adults and the elderly[END_REF]. Many studies have focused on the development of vaccines against the Influenza virus, and this has led to the commercialization of 'flu' vaccines over the last fifty years [START_REF] Yusuf | Current prospects and future challenges for nasal vaccine delivery[END_REF]. Among the ongoing vaccination strategies, the intranasal route has become very promising: over and above the fact that it is a needle-free and painless method, intranasal vaccination has been found superior to systemic immunizations for stimulating strong mucosal IgA and resident memory T cell responses and further results in systemic activation of the immune system via the nasal associated lymphoid tissue (NALT). This tissue, located inside and underneath the nasal epithelium, contains epithelial microfold cells (M cells) that overlay antigen presenting cells (APC) such as dendritic cells (DCs), macrophages and immature B cells [START_REF] Davis | Nasal vaccines[END_REF].

The M cells are able to transcytose antigens from the environment to the underlying APC, and mature APC will present antigen to naïve T cells in the draining lymph nodes. While B cell activation will lead to plasma cell development and antibody production, T cell activation will generate CD4 and/or CD8 T cells against the invading pathogen. Both humoral and cellular responses can occur locally at the administration site, but also throughout the whole body via a colonization of the spleen and lymph nodes [START_REF] Brandtzaeg | Mucosal immunity: induction, dissemination, and effector functions[END_REF][START_REF] Neutra | Mucosal vaccines: the promise and the challenge[END_REF].

Currently available influenza vaccines are either live, attenuated viruses or subunit/split viral antigens [START_REF] Tregoning | Adjuvanted influenza vaccines[END_REF]. The attenuated vaccines are highly immunogenic but are often unstable and can cause adverse reactions, particularly in immunocompromised individuals [START_REF] Bernstein | Clinical reactions and serologic responses after vaccination with whole-virus or split-virus influenza vaccines in children aged 6 to 36 months[END_REF], while subunit/split vaccines confer only limited immunogenicity [START_REF] Grohskopf | Prevention and Control of Seasonal Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices-United States, 2018-19 Influenza Season[END_REF][START_REF] Neuzil | Immunogenicity and reactogenicity of 1 versus 2 doses of trivalent inactivated influenza vaccine in vaccine-naive 5-8-year-old children[END_REF]. It is nevertheless possible to enhance this immunogenicity, while avoiding high antigen doses, through the use of immune modulators such as adjuvants or vaccine delivery systems.

The cholera toxin (CT) is one of the most common adjuvants used in veterinary vaccines [START_REF] Boyaka | Inducing Mucosal IgA: A Challenge for Vaccine Adjuvants and Delivery Systems[END_REF]. It binds ganglioside GM1-receptors present on APC, and when administered with antigens it augments antigen-specific mucosal sIgA and systemic IgG productions [START_REF] Cox | Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa[END_REF][START_REF] Giri | Is intranasal vaccination a feasible solution for tuberculosis?[END_REF]. Owing to its toxicity, however, CT cannot be used as an adjuvant in human vaccines [START_REF] Akhiani | The nontoxic CTA1-DD adjuvant enhances protective immunity against Helicobacter pylori infection following mucosal immunization[END_REF]. Consequently, a non-toxic derivative of CT was developed, where the CTA1 subunit is expressed in a fusion protein together with a dimer of the D-fragment of proteinA from Staphylococcus aureus bacteria (DD) [START_REF] Agren | Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit[END_REF]. Candidate vaccines containing CTA1-DD have been shown to induce strong mucosal responses [START_REF] Lycke | ADP-ribosylating enterotoxins as vaccine adjuvants[END_REF] and to enhance immunity to HIV-1, rotavirus, Influenza A virus and other pathogens, confirming its adjuvant properties [START_REF] Sundling | CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization[END_REF].

Finally, nanodelivery systems made from mucoadhesive polymers such as starch, chitosan or hyaluronic acid offer significant potential for enhancing the immunogenicity of mucosallyadministered antigens [START_REF] Cordeiro | Nanoengineering of vaccines using natural polysaccharides[END_REF][START_REF] Sharma | Polymer nanotechnology based approaches in mucosal vaccine delivery: challenges and opportunities[END_REF]. Such carriers can prolong the antigen residence time in the nasal mucosa, protect the antigens against proteolysis and specifically target APC. Porous and cationic maltodextrin-based nanoparticles with a phospholipid core (NPL) have received particular attention for the delivery of antigens via the nasal route. Previous studies showed that NPL were able to be loaded with large amounts of proteins, and that their residence time in the first layer of the nasal mucosa after administration was longer than antigens alone. Moreover, NPL have already been safely and successfully used as a nanocarrier for mucosal vaccines [START_REF] Bernocchi | Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles[END_REF]Carpentier et al., 2018;[START_REF] Dimier-Poisson | Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection[END_REF][START_REF] Ducournau | Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice[END_REF].

Most vaccines against influenza infection are generally administered intramuscularly using inactivated influenza virus antigens (IIV). They are painful, sometimes adjuvantsupplemented and not fully effective [START_REF] Poehling | 2015-2016 Vaccine Effectiveness of Live Attenuated and Inactivated Influenza Vaccines in Children in the United States[END_REF]. As an alternative, the intranasally administered Flumist® vaccine, a live attenuated influenza vaccine (LAIV), underlines the potential of mimicking a natural influenza infection able to trigger protective mucosal immune response. However, the inconsistent effectiveness of nasal LAIV coupled with production and storage issues have resulted in the withdrawal of recommendations for its use in the United States [START_REF] Furlow | US authorities discourage use of nasal influenza vaccine, citing ineffectiveness[END_REF]. Moreover, the American Center for Disease Control and prevention (CDC) recently reported an overall effectiveness of flu vaccines of only around 45% for the 2019-2020 season (https://www.cdc.gov/flu/vaccines-work/2019-2020.html), meaning there is an important gap to be filled in terms of flu vaccine efficiency.

To combine the advantages of both the IIV and LAIV-Flumist® approaches, and to increase effectiveness, we previously developed an influenza vaccine comprising a chimeric fusion protein (CTA1-M2e(3x)-DD) associated with NPL [START_REF] Bernasconi | Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections[END_REF] and showed that this combination decreased both the virus's mortality and its transmission. However, we did not demonstrate complete protection of this vaccine against infection.

Consequently, to increase the protection offered by our nanoparticle-assisted influenza nasal vaccine, in this study we used the full set of flu antigens (including seasonal and conserved antigens). We evaluated the immunogenicity of an intranasal vaccine against the influenza virus, comprised of split Udorn virus admixed or not with CTA1-DD adjuvant [START_REF] Lycke | Recent progress in mucosal vaccine development: potential and limitations[END_REF] and encapsulated in NPL. The viral antigen loading in NPL and the delivery studies were first performed in vitro in nasal mucosal cells. Immune protection was then examined in vivo in intranasally immunized mice by measuring serum Udorn-specific IgG titers, and the viral titers in the snouts and lungs after a nasal infection with influenza. Finally, the transmission of infectious influenza virus was examined by measuring the viral titers in unvaccinated mice co-housed with vaccinated and infected animals.

MATERIALS AND METHODS

Animals

Six-to eight-week-old BALB/c and DBA/2J mice were purchased from Janvier Laboratories (Strasbourg, France). Mice were kept under specific-pathogen-free conditions in the local animal facility. All experiments were approved by the local animal welfare committee and were performed in accordance with the guidelines of the Federation for Laboratory Animal Science Associations (FELASA), the national animal welfare body, and complied with the German animal protection law.

NPL synthesis

The NPL were prepared as described previously [START_REF] Le | Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles[END_REF][START_REF] Paillard | Positivelycharged, porous, polysaccharide nanoparticles loaded with anionic molecules behave as 'stealth' cationic nanocarriers[END_REF].

Maltodextrin was dissolved in 2 N sodium hydroxide with magnetic stirring at room temperature and epichlorhydrin and glycidyltrimethylammonium chloride were added to make a cationic polysaccharide gel. The gel was then neutralized with acetic acid and sheared with a high-pressure homogenizer (LM 20, Microfluidics, France). The particles thus obtained were purified by tangential flow ultra-filtration (AKTA flux 6, GE Healthcare, France) using a 750 kDa membrane (GE Healthcare, France) and mixed with 70% 1,2-Dipalmitoyl-snglycero-3-phospho-rac-(1-glycerol) (weight %) above the gel-to-liquid phase transition temperature to produce NPL.

Virus production, inactivation and labeling

Udorn virus (Influenza A virus A/Udorn/307/72, H3N2) strain was amplified using MDCK (Madin-Darby Canine Kidney) cells in serum-free Dulbecco's Modified Eagle medium (DMEM), supplemented with non-essential amino acids, 2 mM L-glutamine and 0.4 mM sodium pyruvate, at 37 °C in 5% CO2. Ninety six hours after virus inoculation, the culture medium was collected by centrifugation for 10 min at 2500 g and 4 °C, and the virus was pelleted from the supernatants by overnight centrifugation at 30,000 g and 4 °C. Viral titers were determined by plaque formation on MDCK cells [START_REF] Kolpe | Passively transferred M2e-specific monoclonal antibody reduces influenza A virus transmission in mice[END_REF].

Udorn virus was inactivated following incubating with β-propiolactone (β-BPL) for 16 hours at 4 ℃, then subsequently incubated at 37 ℃ for 2 hours to facilitate hydrolysis of β-BPL.

Inactivated virus were stored at -80 ℃. Triton X-100 was added at a final concentration of 10% (v:v) to split the Udorn virus.

The split Udorn virus was then covalently labeled with 0.7% FITC (Fluorescein isothiocyanate, weight %) in 0.1 M bicarbonate buffer (pH 8.3), and the solution was mixed for 6 h in the dark at room temperature. The labeled virus was purified by gel filtration on a PD-10 Sephadex column and the concentration of the labeled protein was then evaluated using the Micro BCA Protein Assay Kit following the supplier's instructions (ThermoFisher Scientific, France).

Protein loading into nanoparticles

CTA1-DD mucosal adjuvant has been described previously [START_REF] Bernasconi | Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections[END_REF]. Based on their protein concentrations, CTA1-DD and split Udorn virus were loaded into sterile NPL by mixing both components at room temperature obtaining 1:5 split Udorn:NPL or 1:1:5 split Udorn:CTA1-DD:NPL (mass ratios) vaccine formulations.

Characterization of the nanoparticle and formulations

The hydrodynamic diameter (Z-average) of nanoparticles was measured by dynamic light scattering in 23 mM NaCl. The zeta potential was measured by electrophoretic mobility in pure water. Measurements were carried out in triplicate using a Zetasizer nanoZS (Malvern Instruments, France) with a NPL concentration of 100 µg/ml.

Characterization of the association of proteins and split virus with NPL

The protein association of CTA1-DD and the split Udorn virus with NPL was evaluated using native polyacrylamide gel electrophoresis (native PAGE). Formulations were supplemented with a non-denaturing buffer (125 mm Tris-HCL (pH 6.8), 10% glycerol and 0.06% bromophenol blue) and run on a 10% acrylamide-bisacrylamide gels stained by the silver nitrate method. Gels were scanned and quantify using the ImageJ software. Under these conditions, non-associated proteins entered the gel while NP-associated proteins did not.

Intracellular delivery of split Udorn by NPL

The H292 (NCI-H292, ATCC #CRL-1848) and PMA-differentiated [START_REF] Le | Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles[END_REF] THP-1 (ATCC #TIB-202) cells were maintained in RPMI supplemented with 10 % heat-inactivated, fetal calf serum (FCS), 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine at 37 o C in an humidified, 5% CO2 atmosphere. The SRDC [START_REF] Le | Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles[END_REF][START_REF] Ruiz | A novel CD4-CD8alpha+CD205+CD11b-murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis[END_REF] were cultured in IMDM supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine at 37 °C in an humidified, 5% CO2 atmosphere. Cells were plated at a density of 5x10 5 cells for 3 days in 6-well plates and treated for 3 h with 1 µg of split Udorn-FITC either free or loaded into nanoparticles. The cells were then collected by trypsinization and analyzed by flow cytometry using an Accuri™ C6 CFlow Sampler flow cytometer (BD Bioscience, USA). The cells were gated using the SSC/FSC parameters and the FL1 channel (laser 488nm, filter 533/30nm) was used to analyze the split Udorn-FITC intracellular delivery. Untreated cells were used as an auto-fluorescence negative control.

Vaccination of mice

Six-to eight-week-old female BALB/c mice were immunized three times by the intranasal route (i.n.), 10 days apart, with 10 μl (5 μl per nostril) containing either (a) 1 μg of split Udorn alone, (b) 1 µg of split Udorn formulated with 5 µg of NPL or (c) 1 µg of split Udorn and 1 µg of CTA1-DD formulated with 5 µg of NPL. Ten days after the first and the second immunizations, serum samples were collected in order to measure the levels of anti-HA specific antibodies by ELISA.

Detection of anti-Udorn antibodies in serum

To determine Udorn-specific antibody titers, microtiter plates (MaxiSorp, Nunc) were coated with split Udorn virus, and incubated overnight at 4 °C. After washing, the plates were blocked for 2 h with 5% BSA in PBS. After blocking, a series of 1/3 dilutions of the different serum samples were loaded on the peptide-coated plates. The bound antibodies were measured with a horseradish peroxidase-labeled antibody directed against mouse IgG, diluted 1/2000 (Thermo Fisher Scientific, USA). After washing, the microtiter plates were incubated for 20 min with a TMB substrate (Tetramethylbenzidine, Life technologies). The reaction was stopped by adding 0.5 M H2SO4 and absorbance was measured at 450 nm. The endpoint titers were defined as the highest dilution resulting in an O.D. value twice that of the background (pre-immune serum). The snouts and the lungs of the index and contact mice were collected after 4 days post cohabitation and viral loads were determined by viral titration assay.

Virus transmission study in naive mice

Six

Viral titration

The MDCK cells were maintained in DMEM supplemented with 10% FCS, non-essential amino acids, 2 mM L-glutamine and 0.4 mM sodium pyruvate and maintained at 37 °C in a 5% CO2 atmosphere. Cells were grown to a confluent layer in 12-well plates for the titration assay. Snouts and lungs were homogenized in 800 μl cold PBS using FastPrep® spheres and tubes in an homogenizer (3 cycles at 6.5 m/s for 20 seconds). Samples were then centrifuged for 10 min at 9,000 rpm and 4 °C, and supernatants were diluted 10-fold in OptiMEM (Thermo Fisher Scientific) supplemented with 0.3% BSA. A volume of 450 μl of these diluted samples were added to confluent MDCK cells for 2 h at room temperature. After removing the inoculum, cells were overlaid for 72 h with 1.5% Avicel (FMC BioPolymer) in DMEM supplemented with 10% BSA. Cells were then fixed with 3.7% formaldehyde and stained with 1% crystal violet solution for 15 min. Plaques were used to calculate virus titers in plaque-forming units (pfu) per ml.

Statistics

Data were analyzed by one-way and two-way ANOVA using GraphPad Prism version 6.01

for Windows (GraphPad Software, San Diego, CA).

RESULTS

Characterization of NPL and formulations

The NPL had a hydrodynamic diameter of 38 nm while the average size of the split Udorn Table 1).

Formulations Percentage of split Udorn loading

Split Udorn/NPL 100% Split Udorn/CTA1-DD/NPL 90% 2) were analyzed by densitometry using ImageJ. The percentages of association with the NPL were calculated over the 100% Split Udorn input and reported in the table.

Cellular delivery of split Udorn by NPL

We investigated the delivery of split Udorn by NPL into 3 different airway mucosa cell lines.

Using fluorescently-labeled Udorn viral proteins, flow cytometry analysis showed that NPL significantly increased the uptake of split Udorn antigens by H292 airway epithelial cells, SRDC dendritic cells and THP-1 macrophages. The delivery was 4 times higher in H292 and SRDC, and 5 times higher in THP-1 macrophages (Fig 3).

Udorn-specific antibody titers after intranasal vaccination

To investigate the efficacy of NPL-formulated vaccines, we compared the serum antibodies in

Protection against virus transmission

We then determined whether intranasal administration of NPL-encapsulated, CTA1-DDadjuvanted, split Udorn vaccine was sufficient to induce mucosal immunity and protection against influenza virus infection. Two weeks after final vaccination, we challenged

DISCUSSION

NPL are cationic maltodextrin nanoparticles with a phospholipid core. They are highly and rapidly endocytosed by airway epithelial cells, dendritic cells (DCs) and macrophages (Le et al., 2018) and significantly improve protein delivery into those cells, even at low doses [START_REF] Bernocchi | Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles[END_REF][START_REF] Le | Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles[END_REF]. In addition, vaccination studies against toxoplasmosis infection have shown that NPL-based vaccines protect animals against chronic and congenital infection, confirming their efficiency as vaccine delivery systems [START_REF] Dimier-Poisson | Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection[END_REF][START_REF] Ducournau | Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice[END_REF]. Moreover CTA1-DD has been used as an adjuvant for mucosal vaccines [START_REF] Lycke | ADP-ribosylating enterotoxins as vaccine adjuvants[END_REF][START_REF] Sundling | CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization[END_REF] and it was originally developed to circumvent the toxicity of CT holotoxin; it was found to augment mucosal and systemic immune responses comparable to that of CT, but without toxic side effects [START_REF] Dhere | A pandemic influenza vaccine in India: from strain to sale within 12 months[END_REF][START_REF] Lycke | Recent progress in mucosal vaccine development: potential and limitations[END_REF]. Hence this study aimed to evaluate the effect of NPL as a delivery system of vaccine antigens and mucosal adjuvants.

The positive surface charge and lipid core of NPL facilitates both electrostatic and hydrophobic interactions between the nanocarrier and antigens. Moreover, their porous structure allows NPL to be loaded with high amount of antigens [START_REF] Dimier-Poisson | Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection[END_REF][START_REF] Le | Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles[END_REF]. Split Udorn virus and the CTA1-DD adjuvant used in this study were shown to be >90% loaded into the NPL by Native PAGE assays (Fig 1 and2, Table 1). Importantly, NPL very efficiently delivered split Udorn into 3 main cell types present in the nasal mucosa.

The improvement in uptake (Fig 3) and processing [START_REF] Bernasconi | Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections[END_REF] of the associated Udorn antigens by antigen-presenting cells make NPL an ideal nanodelivery system for intranasal vaccines.

Many examples have shown that using an adjuvant in combination to a nanodelivery system is sometimes necessary to create significant protection against the influenza virus. It was demonstrated that recombinant HA nanoparticle-based influenza vaccine required a saponinbased adjuvant to induce protection against influenza A(H3N2) strains [START_REF] Smith | Novel hemagglutinin nanoparticle influenza vaccine with Matrix-M adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted A(H3N2) subtypes[END_REF].

Furthermore PEG-liposomes containing CRX-601 adjuvant and methylglycol chitosan also increased the murine sublingual immune response to influenza vaccination [START_REF] Oberoi | PEG modified liposomes containing CRX-601 adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination[END_REF]. Here, the in vivo study showed that intranasal administration of a split Udorn/NPL vaccine enhances Udorn-specific IgG antibody response as compared to mice receiving split Udorn alone. Moreover, adding CTA1-DD into the split Udorn/NPL formulation tended to improve the level of Udorn-specific IgG compared with administration of split Udorn alone (Fig 4), but not significantly more so than the split Udorn/NPL formulation. This is in agreement with our recently published results [START_REF] Bernasconi | Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections[END_REF] and suggests that, combined with split Udorn antigens, an efficient delivery system (NPL) is sufficiently effective even in the absence of adjuvant. This pattern was also seen in the transmission study we performed (Fig 5).

Noteworthy, influenza virus protection should be assessed in the snout and lungs as these are the entry point for infection [START_REF] Kohlmeier | Immunity to respiratory viruses[END_REF]. We therefore analyzed the protection induced by NPL-formulated influenza vaccine in snouts and lungs of infected and immunized mice. Correlated with the production of Udorn-specific IgG (Fig 4 ), the NPL appears essential to limit both viral infection and transmission, an effect enhanced by the CTA1-DD adjuvant in the case of Udorn antigens compared with vaccination using the split virus alone (Fig 5). In addition, mucosal immunity is a key objective when seeking to limit influenza infection and transmission. We have previously demonstrated that NPL, associated with a chimeric antigen of influenza virus, induced the mucosal secretion of specific IgA while antigen alone could not [START_REF] Bernasconi | Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections[END_REF]. Here, our strategy was designed to mimic a natural infection and to deliver antigens into mucosa cells. Further experiments will be performed to characterize the mucosal immunity triggered by our NPL-based vaccine, especially measuring humoral response and T-cell activation.

In these first experiments using NPL associating the total split Udorn virus, we immunized mice three times while human vaccinations led by public health authorities are often singledose (IIV or LAIV). However, experimental mice are naïve to flu virus infection and booster administrations are required, contrary to humans that have been previously infected by flu viruses having shared conserved epitopes.

We conclude that the use of an adjuvant in combination with a nanocarrier did not appear to be an absolute necessity for conferring 100% protection against influenza infection which agrees with a NPL-assisted anti-Toxoplasma gondii vaccine [START_REF] Dimier-Poisson | Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection[END_REF][START_REF] Ducournau | Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice[END_REF] that does not require the use of any additional adjuvant. Finally, this study emphasizes the interest of using NPL as a vaccine delivery system for influenza antigens and adjuvant.

CONCLUSION

Intranasal administration of a split Udorn virus using an NPL nanocarrier, with or without the CTA1-DD adjuvant, is an efficient vaccine strategy for stimulating immunity against influenza virus. Importantly, the mice vaccinated with NPL formulations were not only protected against infection, but did also not transmit the influenza infection, an often overlooked parameter of immune protection.

FIGURE CAPTIONS

  -to eight-week-old female BALB/c mice were immunized three times (10 days apart) by the intranasal route with either (a) 1 μg of split Udorn alone, (b) 1 µg of split Udorn formulated with 5 µg of NPL, (c) 1 µg of split Udorn and 1 µg of CTA1-DD formulated with 5 µg of NPL, or (d) with a mock vaccine composed of 1 µg of ovalbumin (OVA) loaded into 5 µg of NPL. Five weeks after the final immunization, all immunized BALB/c mice were infected intranasally with 20 μl containing 3x10 4 PFU H3N2 Udorn virus (A/Udorn/307/1972 -H3N2) and were used as index animals. At 24 h post-infection, these index mice were cohoused one-to-one with uninfected, 6-week-old DBA/2 mice as contact mice in a clean cage.

  was 111 nm. Moreover NPL possessed a positive surface charge (+38 mV) while split Udorn virus had a negative charged (-20 mV). The association of the split Udorn with NPL at a mass ratio of 1:5 (protein:NPL) yielded formulation of the same size as NPL alone (40 nm), with a positive surface charge (+26 mV) which implied that split Udorn antigens were successfully loaded inside the NPL (Fig 1). Similarly, the association between split Udorn, CTA1-DD and NPL (mass ratio 1:1:5) showed no increase of the size of NPL and a zeta potential of +26 mV, again confirming the encapsulation (Fig 1). In addition to size and zeta potential analysis, the loading of proteins into NPL was assessed by native PAGE (Fig 2). Splitting the virus did not modify the protein pattern of the Udorn virus. The split Udorn was completely encapsulated by NPL as no free protein was observed, while the combination of split Udorn and CTA1-DD with NPL resulted in a loading efficacy of 90% of split Udorn. Taken together, these data added further evidence for the encapsulation of the majority of Udorn antigens and the mucosal adjuvant by NPL at mass ratio 1:1:5 (Fig 2 and

  mice after intranasal vaccination. As a negative control, mice were vaccinated with OVA/NPL, otherwise mice were immunized intranasally with split Udorn alone, split Udorn/NPL, or split Udorn/CTA1-DD/NPL. The levels of Udorn-specific serum IgG of vaccinated mice are shown in Figure 4. Vaccination with all of the above formulations increased Udorn-specific IgG antibody production, and it was enhanced by a second immunization. After the second immunization, a significant increase of Udorn-specific IgG was induced by split Udorn/NPL compared with split Udorn alone. Moreover, adding the CTA1-DD adjuvant to the split Udorn/NPL formulation induced a greater increase of the specific serum immune response, though not significantly different from the split Udorn/NPL formulation. Altogether, these results reveal that both NPL and CTA1-DD have immuneenhancing effects after intranasal administration.

  immunized mice (referred to as index mice) intranasally with 20 μl containing 3*10 4 PFU/animal of H3N2 Udorn virus and co-housed them 24 hours later with naïve, nonimmunized, contact mice. Virus transmission was assessed by monitoring the snouts and lungs for viral titers of both index and contact mice after 4 days of one-to-one co-habitation. Index mice immunized with split Udorn/NPL exhibited significantly lower viral titers in the snout compared with non-immunized mice (OVA/NPL group) and this was further enhanced with the split Udorn/CTA1-DD/NPL formulation. In the lungs of index mice, only the immunization with intranasal split Udorn/CTA1-DD/NPL significantly reduced the Udorn viral titers (Fig 5a). Though index mice vaccinated with split Udorn alone were poorly protected from virus infection (Fig 5a), they showed a limited virus transmission (25% of mice compared to 100% transmission using OVA/NPL immunization). Importantly, index mice vaccinated with split Udorn/NPL formulations, with or without CTA1-DD, did not transmit the influenza virus to contact mice (100% of virus transmission prevention, Fig 5b). This demonstrates that NPL were effective in limiting virus transmission.

Figure 1 :

 1 Figure 1: Size, PDI and zeta potential of NPL, split Udorn, CTA1-DD and the related formulations. The hydrodynamic diameter (left), the polydispersity index -PDI-(middle) and the zeta potential (right) of NPL, split Udorn, CTA1-DD, split Udorn/NPL, split Udorn/CTA1-DD/NPL as measured by dynamic light scattering and electrophoretic mobility, respectively. The graphs represent the mean +/-SD of three independent measurements.

Figure 2 :

 2 Figure 2: Characterization of the split Udorn/NPL and split Udorn/CTA1-DD/NPL. The encapsulation of antigens and adjuvant by NPL was characterized by non-denaturing polyacrylamide gel electrophoresis (PAGE) using formulations of 1(:1):5 (mass ratio of split Udorn:(CTA1-DD):NPL) ratios. Unbound proteins were revealed by silver nitrate staining.

Figure 3 :

 3 Figure 3: Split Udorn delivery by NPL in H292 airway epithelial cells, SRDC dendritic cells and THP-1 macrophages. Split Udorn was labeled with fluorescein and loaded or not into NPL at a mass ratio protein/NPL of 1:5. Cells were treated for 3 hours and the protein delivery was evaluated by flow cytometry. Statistical analysis: Two-way ANOVA; asterisks indicate p-values: *** p<0.001.

Figure 4 :

 4 Figure 4: Serum Udorn-specific IgG titers from vaccinated mice. Udorn-specific IgG titers were measured by ELISA in serum from mice immunized 3 times intranasally (a primary immunization followed by two boosters, 10 days apart) with OVA/NPL as negative control, split Udorn, split Udorn/NPL or split Udorn/CTA1-DD/NPL (mass ratio 1:(1:)5). Statistical analysis: Two-way ANOVA. P-values: ** p<0.01 between indicated groups; **** p<0.0001 between indicated groups; $ p<0.0001 OVA/NPL vs. all other groups.

Figure 5 :

 5 Figure 5: Viral transmission from vaccinated mice. Index mice were immunized with OVA/NPL as negative control, split Udorn, split Udorn/NPL or split Udorn/CTA1-DD/NPL (mass ratio 1:(1:)5) and infected before being individually co-housed with non-vaccinated contact mice. A) The viral titers in index mice snouts and lungs; B) The viral titers in contact mice snouts. Statistical analysis: One-way or two-way ANOVA; asterisks indicate p-values: p<0.0001 (****), p<0.001, p< 0.01 (**) and p<0.05 (*).

  FIGURE 3

Table 1 : Loading efficacy of split Udorn and split Udorn/CTA1-DD in NPL

 1

. Non-denaturing PAGE (from figure

ACKNOWLEDGEMENTS

This research has received funding from the People Program (Marie Curie Actions) of the European Union Seventh Framework Program FP7/2007-2013/ under REA grant agreement no. [607690]. We are grateful to Dr. Mike Howsam for English proofreading and the Flow core facility of BICeL campus Hospitalo-universitaire for their support and technical assistance.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

LOW RESOLUTION FIGURES FOR REVIEWING PURPOSE ONLY

High resolution figures are supplied in separated TIFF files


Elodie Germani

Camille Maumet

Institutions

FMRI data analysis: How does analytical variability vary with sample size?

Introduction:

Neuroimaging workflows are highly flexible leaving researchers with many possible choices at each step of their analysis [START_REF] Carp | On the Plurality of (Methodological) Worlds: Estimating the Analytic Flexibility of fMRI Experiments[END_REF]. Recents studies [START_REF] Bhagwat | Understanding the impact of preprocessing pipelines on neuroimaging cortical surface analyses[END_REF][START_REF] Botvinik-Nezer | Variability in the analysis of a single neuroimaging dataset by many teams[END_REF][START_REF] Bowring | Exploring the impact of analysis software on task fMRI results[END_REF] have demonstrated how different analytical choices can substantially impact neuroimaging results, effectively leading to a "vibration of effects" [START_REF] Ioannidis | Why Most Discovered True Associations Are Inflated[END_REF]. This observation is not limited to brain imaging and was also made across many scientific fields (Hoffmann et al., 2020). In psychology, [START_REF] Klau | Comparing the vibration of effects due to model, data pre-processing and sampling uncertainty on a large data set in personality psychology[END_REF] showed that the vibration of effects decreases and stabilizes as sample sizes increase.

We built on the results of [START_REF] Botvinik-Nezer | Variability in the analysis of a single neuroimaging dataset by many teams[END_REF], in which a functional Magnetic Resonance Imaging (fMRI) "many analyst" study was conducted with 70 teams. In this study, each team used their favorite pipeline to analyze the same dataset and answer 9 pre-defined hypotheses. Here, after reproducing the pipelines used by 4 teams, we observe the impact of varying sample sizes on the vibration of effects.

Methods:

Using descriptions provided in the original study, we reproduced the SPM pipelines of 4 teams: 2T6S, V55J, Q6O0 and C88N. For each team, we assessed the quality of the reproduction by comparing our results with the statistic maps published on NeuroVault [START_REF] Gorgolewski | NeuroVault.org: a web-based repository for collecting and sharing unthresholded statistical maps of the human brain[END_REF].

After this validation step, we focused our experiments on the results of Hypothesis 5 for which 80% of the teams agreed on a positive answer in the original paper. We randomly selected 20, 40, 60 and 80 participants (each set being a superset of the smaller ones) among the full dataset of 108 participants. We replicated the results of each pipeline with each subset of participants and with the full dataset.

Results:

1) Validation of the reproductions For each team, we compared the reproduced and the original maps visually and quantitatively using Pearson's correlations (unthresholded maps) and Dice scores (thresholded maps). Fig. 1.A and B present the statistic maps for an example team (2T6S). We found similar activation patterns and the same observation was made for all reproduced teams. All correlations were above 0.98 (Fig. 1.C) for all teams. There were differences in the number of activated voxels between the original and reproduced maps and sometimes many non-overlapping voxels with Dice scores ranging 0.63-0.81. Multiple attempts were necessary to obtain these results.

2) Analytical variability with varying sample sizes

The thresholded maps obtained for each sample size and each team are presented in Fig. 2.A. Overall, the thresholded maps presented a lot of variability. For some pipelines, more participants were necessary to find activations in the ventro-medial prefrontal cortex (vmPFC) (which was the region of expected activation for H5). In particular, for the team Q6O0 with 60 participants there was no activation for H5 while all other teams had detected a significant activation.

For each result, we also looked at the mean activation value in the vmPFC. With smaller sample sizes (N=20 and N=40), for a majority of pipelines, there was no activation in the vmPFC (and hence a mean activation value of 0) (Fig 2.B). This value increased with larger sample size, except for 2T6S maps for which it was above 0 for all sizes, highlighting the higher sensibility of this pipeline, probably due to the absence of multiple testing correction. However, with growing sample sizes, mean activations seemed to converge, consistent with the previous observation that the vibration of effects reduces and stabilizes with larger sample sizes.

Conclusions:

With this work, our objective was to show the impact of sample size on analytical variability. Our findings show that, in fMRI data analysis, the vibration of effects decreases with sample size. Our results also suggest that some variability remains even for large sample sizes. Further work will be needed in order to include more pipelines and investigate which part of the pipelines are the most impactful.


Andrea Carboni
email: carboni@cerege.fr

Alexandre Gélabert

Gaëlle Charron

Stéphane Faucher

Gaetane Lespes

Yann Sivry

Marc F Benedetti

A Gelabert

Mobility and transformation of CdSe/ZnS quantum dots in soil: Role of the capping ligands and ageing effect

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Engineered nanoparticles (NPs) are currently incorporated in a large number of consumer products and their production is expected to increase dramatically in the next years [START_REF] Roco | The long view of nanotechnology development: the National Nanotechnology Initiative at 10 years[END_REF]. Quantum Dots (QDs) are among the most promising materials in emerging nanotechnologies due to the unique high-yield and size-tunable electrical and optical properties [START_REF] Bruchez | Semiconductor nanocrystals as fluorescent biological labels[END_REF][START_REF] Bera | Quantum dots and their multimodal application: a review[END_REF], which make them ideal candidates for applications in photovoltaics and biomedical imaging as well as in the construction of sensors and electronics in general [START_REF] Alivisatos | The use of nanocrystals in biological detection[END_REF][START_REF] Kamat | Quantum Dot solar cells. Semiconductor nanocrystals as light harvesters[END_REF][START_REF] Anikeeva | Quantum Dot light-emitting devices with electroluminescence tunable over the entire visible spectrum[END_REF][START_REF] Kalita | Graphene Quantum Dots Soil Moisture Sensor[END_REF]. Such properties are correlated to QDs distinctive structure where a semiconductive metal core, usually composed of CdTe, CdSe or InP, is surrounded by a shell (e.g. ZnS, CdSe). The resulting core-shell nanocrystals have diameters in the 2-100 nm size range and are commonly functionalized with organic polymer coatings in order to increase their stability and solubility in aqueous media, i.e. their applicability [START_REF] Dabbousi | CdSe/ZnS core-shell Quantum Dots: synthesis and characterization of a size series of highly luminescentnanocrystallites[END_REF][START_REF] Bruchez | Semiconductor nanocrystals as fluorescent biological labels[END_REF][START_REF] Bera | Quantum dots and their multimodal application: a review[END_REF][START_REF] Karakoti | Surface functionalization of quantum dots for biological applications[END_REF]. There is concern for the possible negative effects arising from the wide usage of engineered nanomaterials and in the last two decades several countries and organizations (e.g. European Commission; US Environmental Protection Agency) have launched campaigns for the study of the associated risks for humans and the ecosystem [START_REF] Dunphy | Environmental risk of nanotechnology: national nanotechnology initiative funding, 2000-2004[END_REF][START_REF] Owen | Formulating the problems for environmental risk assessment of nanomaterials[END_REF]U.S. EPA, 2011;[START_REF] Oomen | Risk assessment frameworks for nanomaterials: Scope, link to regulations, applicability, and outline for future directions in view of needed increase in efficiency[END_REF]. In particular, QDs may display peculiar reactivity and persistency and their toxicity could be related to the NPs form, the release of potentially toxic metals and enhanced by the organic ligands [START_REF] Hardman | A toxicologic review of Quantum Dots: toxicity depends on physicochemical and environmental factors[END_REF] as highlighted by recent works showing the noxious effects on bacteria and eukariota (e.g. [START_REF] Khare | Adverse effect of CdTe quantum dots on the cell membrane of Bacillus subtilis: insight from microscopy[END_REF].

QDs release in the environment can occur from point and diffuse sources, such as the use and disposal of consumer and biomedical products but also the waste production and land application of biosolids. Thus, soil may act as a sink for NPs accumulation and understanding their behavior in the terrestrial ecosystems is of utmost importance for risk assessment and management [START_REF] Cornelis | Fate and bioavailability of Engineered nanomaterials in soils: a review[END_REF]. However, most research on QDs fate in environmental systems has been focused on freshwater [START_REF] Xiao | Aggregation, sedimentation Dissolution and Bioavailability of Quantum Dots in estuarine systems[END_REF] and the number of studies addressing their behavior in other compartments is still limited. Research has been conducted to investigate the deposition of QDs onto porous media employing quartz crystal microbalance (QCM; [START_REF] Quevedo | Influence of solution chemistry on deposition and detachment kinetics of a CdTe Quantum Dot examined using a Quartz Crystal Microbalance[END_REF][START_REF] Quevedo | Deposition kinetics of Quantum Dots and polystyrene latex nanoparticles onto alumina: role of water chemistry and particle coating[END_REF] and packed-bed soil column systems, with the latter being mostly applied. In general, although the comparison between these studies is complicated by the differences in experimental settings and materials utilized (e.g. soil matrices, NPs compositions and coatings), a high mobility of the QDs was observed at low ionic strength and neutral pH in unsaturated conditions representative of natural subsurface systems [START_REF] Usuyur | Coupled effects of solution chemistry and hydrodynamics on the mobility and transport of Quantum Dot nanomaterials in the vadose zone[END_REF] as well as in saturated granular matrices consisting of either standardized porous media (e.g. quartz sand) or real soil samples [START_REF] Torkzaban | Transport and deposition of functionalized CdTe nanoparticles in saturated porous media[END_REF][START_REF] Al-Salim | Quantum Dot transport in soil, plants and insects[END_REF]. However, retention of the QDs during column experiments was greatly enhanced in presence of divalent cations, likely due to cation bridging between the negatively charged capping groups [START_REF] Quevedo | Mobility of functionalized Quantum Dots and model polystyrene nanoparticle in saturated quartz sand and loamy sand[END_REF][START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF], and when the collector surface presented impurities or was coated with iron [START_REF] Torkzaban | Transport and deposition of functionalized CdTe nanoparticles in saturated porous media[END_REF]. [START_REF] Wang | Effect of surface coating composition on Quantum Dot mobility in porous media[END_REF] systematically studied the effect of ionic strength, pH and NPs coating on the transport of QDs and reported enhanced mobility at decreasing ionic strength and increasing pH as well as a diminished retention of the carboxylated compared to the non-ionic coated NPs. These works agreed that QDs coating will ultimately affect their behavior in the real terrestrial systems and that its interaction with the natural components will determine the stability and environmental fate of the NPs. However, alteration of the nanomaterials pristine state during the transport experiments was scarcely investigated and may include ligand desorption, oxidation and aggregation/precipitation of the QDs, which have been reported in aqueous systems as a consequence of irradiation and depending on solution chemistry [START_REF] Aldana | Photochemical instability of CdSe nanocrystals coated by hydrophilic thiols[END_REF]Zhang et al., 2008;Zhang et al., 2008b;[START_REF] Lead | Nanomaterials in the environment: behavior, fate, bioavailability, and effects-an updated review[END_REF]. In soil, [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF] found evidences of degradation during leaching studies in presence of organic matter and suggested that the stability of these chemicals over the long period is questionable whereas, more recently, [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF] reported dissolution and aggregation of the QDs in soil solutions.

However, the extent and kinetic of these phenomena remain unclear, especially regarding QDs dissolution, which may cause the release and mobilization of novel potentially toxic species such as Cd 2+ , Se 2-and SeO3 2-. Furthermore, such processes may be affected by QDs ageing and general alteration of their pristine state. For instance, increased toxicity was reported on Zebra Fish and bacteria as a consequence of QDs weathering [START_REF] Mahendra | Quantum Dots weathering results in microbial toxicity[END_REF][START_REF] Wiecinski | Toxicity of oxidatively degraded Quantum Dots[END_REF] whereas [START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF] found a diminished release of deposited QDs from the soil with increasing ageing time. However, to date most of the studies on QDs environmental fate were focused on pristine as-produced nanomaterials, which physico-chemical properties may be representative of the behavior occurring at manufacturing and processing sites but not upon release in the environment [START_REF] Lowry | Transformations of nanomaterials in the environment[END_REF]. In this context, [START_REF] Mitrano | The need for a life-cycle based aging paradigm for nanomaterials: importance of real-world test systems to identify realistic particle transformation[END_REF] recently stressed the need for novel research focused on the transformation and ageing of nanoparticles, in order to better represent the real conditions at which they are found in the ecosystem and allow a more precise and life-cycle based assessment of these nanomaterials.

In this work we examined the mobility and stability of two QDs nanoparticles presenting nearly identical nanocrystalline structure but different organic coating. A series of soil leaching experiments were conducted to study the behavior of the two QDs in quartz sand, with regard to leached but also deposited amounts, which have often been neglected in literature. The comparison between fluorescence emission and mass spectrometric measurements allowed an estimation of NPs transformation during the experiments whereas a further assessment of the NPs dissolution was provided by ultrafiltration of the leached samples. A second set of experiments was devoted to understanding the effect of NPs alteration and ageing on these processes. In particular, we investigated the effect of I) increased Ca 2+ -mediated homo-aggregation of the QDs in the spiking solution and II) the ageing of the NPs in the column systems, i.e. their incubation in quartz sand for 15 days prior the experiments. The main aim of this study is to provide observation of QDs behavior in simplified environmental systems, which can provide the basis for future works in more complex matrices. These results will help the understanding of nanomaterials behavior and fate in soils, with particular regard to small NPs such as QDs, as well as the effects of their transformation and ageing in environmental systems.

Materials and Methods

Chemicals and Quantum Dots synthesis

The chemicals used for QDs nanocrystals and organic coating synthesis were purchased from Sigma Aldrich (unless specified) and used without further purification: sulfur (99.9%), cadmium oxide (99.9%), selenium (99.9%), anhydrous zinc acetate (99.9%), oleic acid (90%), trioctylphospine (90%), 1octadecene (90%), chloroform (99%), thioglycolic acid (99%), poly(maleic anhydride-alt-1-octadecene) and ethanolamine (99%, Alfa Aesar). Concentrated nitric acid (65%) for samples acidification and digestion and hydrogen peroxide (30%) were purchased from VWR whereas ultra-pure milli-Q water (UPW) was produced in the laboratory with a Millipore purification system (Merck, Fontenay-sous-Bois, France). The core-shell CdSe/ZnS QDs were synthesized in our group according to the single-step protocol reported by Bae et al., (2008) with minor modifications, i.e. the use of zinc oxide instead of zinc acetate as precursor. Purification of the QDs was achieved as following: first, the nanocrystals were dissolved in chloroform and isolated by precipitation after centrifugation at 6000 rpm for 10 min (5810 R centrifuge; Eppendorf Amburg, Germany). Then, the excess of reagents was removed with consecutive steps of centrifugation (6000 rpm, 10 min) and filtration (PTFE 0.2 μm, Sigma Aldrich).

After the synthesis, different batches of QDs nanocrystals were functionalized with organic polymers, in order to enhance their solubility in aqueous media. Two different coatings were selected based on synthesis reproducibility and yield. Specifically, the relatively simple thioglycolic acid, consisting of a short organic chain presenting both thiol and carboxylic acid functional groups (TGA, [START_REF] Zhu | Synthesis and Optical Properties of Thiol Functionalized CdSe/ZnS (Core/Shell) Quantum Dots by Ligand Exchange[END_REF] was compared to the more complex organic polymer poly(maleic anhydride-alt-1-octadecene) (POAMA, [START_REF] Pellegrino | Hydrophobic nanocrystals coated with an amphiphilic polymer shell: a general route to water soluble nanocrystals[END_REF]. According to Faurier et al., (2017), the QDs are expected to have a CdS gradient between the CdSe core and the ZnS shell whereas the yellowish color and fluorescence emission in the green portion of the visible spectra indicated NPs diameter of 7 nm, with core size of circa 3.4 nm. The final QDs stock solutions in ultrapure water and at pH 11 were stored at 4C in the dark and a visual assessment of their homogeneity and limpidity was routinely performed with a BTCP-20.MC compact transilluminator (Vilber Lourmat, Collegien, France).

Soil leaching experiments

IOTA high purity quartz sand from Unimin Corporation (Le Sueur, MN, US) was used as the porous media for all the soil leaching experiments. Grain size observed with Scanning Electron Microscopy coupled to Energy Dispersive X-Ray Analysis (SEM-EDX) ranged from 280 to 340 μm (n=20), with average grain diameter of 300 μm. In order to remove metals and organic impurities from the quartz sand, a cleaning protocol was performed similar to what reported by [START_REF] Wang | Transport and retention of fullerene nanoparticles in natural soils[END_REF]. Briefly, the sand was soaked in 10% HNO3 placed in an ultrasonic bath (Ultrasonic Cleaner, VWR) at 60 Hz for 30 min and stirred every 5 min. The same procedure was repeated once with UPW, then once with 5% H2O2 and eventually twice with UPW. The whole wash/rinse protocol was carried out three times and the sand was eventually rinsed with 1 mM Ca(NO3)2 until pH equilibration, then rinsed with UPW and oven dried at 65 C. The column apparatus consisted of custom-made 23 mL volume polycarbonate columns (internal diameter 2.3 cm, length 4 cm) equipped with 25 mm polycarbonate filter holders (Sartorius GhbH, Goettingen, Germany) sealed with polytetrafluoroethylene (PTFE) seal tape to prevent leaking. Glass fiber filters (HA type; VWR) were placed at both extremities and approximately 23 g of quartz sand were added to each column with 0.5 cm increments in wet-packing conditions. A background electrolyte, consisting of 1 mM Ca(NO3)2 solution at pH 7 was flushed in bottom-up mode through the vertically oriented columns at a constant flow of 0.5 mL/min using a peristaltic pump (Minipuls3; Gilson, Middleton, WI, USA). The resulting pore-water velocity of 4.5 m/day is representative of typical coarse textured porous media and has been previously applied to study the transport of NPs in saturated column systems [START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF]. At these conditions, the porosity and pore volume (PV) were about 0.4 and 6.5 mL respectively, with PV residence time in the column of 13 min. Each soil leaching experiment was performed in duplicate and the columns were covered with aluminum foil to prevent exposure to light. The columns were first equilibrated with the background electrolyte solution for at least 24 hrs (circa 100 PV) before introducing 0.5 mL of QDs spiking solutions and 0.5 mL of a 120 mg/L Br -tracer solution with the procedure reported by [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF]. A scheme of the experimental set-up is provided in the supplementary info, fig. S1. 0.5 ml/min; 4.5 m/day). Spiking solution of the QDs in 1 mM Ca(NO3)2 and pH 7.0 were obtained from dilutions of the stock solution for a concentration of 10.5 ± 0.1 μg/mL Cd. This corresponded to 8.5 ± 1.5 × 10 13 particles/mL (14.1 ± 2.5 nM) based on a CdSe density of 5.82 g/cm 3 and assuming NPs spherical shape with diameter of 3.4 nm. For experiments with altered (aggregated) QDs, spiking solution was prepared in 100 mM Ca(NO3)2 solution, stored at room temperature in the dark for 4 hrs and then ultrasonicated for 15 min before introduction. For ageing experiments the QDs spiking solution was applied into the column, the peristaltic pumps were stopped, the tubing blocked with clamps and the experiments re-started after 15 d of incubation. In each experiment, after introduction of the QDs, 7 PV of background electrolyte were run through the column and 2 mL eluent samples were collected into 5 mL polypropylene vials. This corresponded to a resolution of circa 3 samples per PV. At the end of the experiments, the columns were first frozen at -18 °C overnight and then the quartz sand was extracted and cut into thin sections of 0.3 to 0.5 mm thick.

The sections were transferred into 10 mL glass vials and freeze-dried with a Alpha 1-4 LD plus freeze drier (Bioblock Scientific, Rungis Complexe, France) operating at approximately -56 °C and 0.035 mbar for 48 hours.

Samples processing and analysis

In general, since fluorescence emission is peculiar of the QDs nanocrystals, at least with regard to the pristine nanoparticles andFigures CdSe cores (Pereira et al., 2016), it was used for estimation of the nanoparticle species during the experiments. Total Cd and Se were measured before and after ultrafiltration (3 KDa) in order to assess total and dissolved metals concentrations, respectively. In detail, the 2 mL column eluent samples underwent fluorescence emission analysis for estimation of the QDs concentration and metal determination to assess the total Cd and Se contents. For fluorescence analysis, 0.5 mL was transferred into 5 mL polypropylene tubes and diluted with 3.5 mL of 1 mM Ca(NO3)2 at pH 7 then analyzed within 3 min from the collection to minimize alteration of the analytes (e.g. dissolution with consequent loss of fluorescence). For metal content analysis, 0.5 mL samples was transferred into 14 mL Falcon tubes, diluted up to 10 mL with UPW and acidified for a final 1% HNO3 concentration. Additional 0.5 mL sample underwent ultrafiltration with 3 KDa regenerated cellulose centrifugal filters (Amicon 4-ultra, Merck), operated with centrifugation at 4000 rpm for 35 min. The filtrated samples were then diluted and acidified for Cd and Se analysis following a similar procedure than above. For time reasons, the ultrafiltration was carried twice during each experiment with sets of 9 samples each. The remaining amount was stored at 4°C in the dark.

Extraction of the QDs from the lyophilized quartz sand sections was achieved with 15 min ultrasonication of 0.5 g samples in 4 mL of 50 mM NaCl solution at pH 10 at room temperature. Specifically, Na + was employed to enhance the extraction of the deposited QDs via cation exchange onto the sand surface whereas the high pH prevented dissolution and aggregation/precipitation of the QDs in solution [START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF]. Recovery of extraction of the QDs from the quartz sand were 78 ± 3 % and 85 ± 2 % for TGA-QDs and POAMA-QDs, respectively. Acid digestion was carried out for the analysis of metals in the solid samples as following: first, 0.5 g of each sample was transferred into 50 mL falcon tubes and 3 ml of concentrated acid (1:1; HNO3:UPW) were added before undergoing ultrasonication for 30 min at room temperature. Then, 2 mL of H2O2 and 3 mL of UPW were added and the samples ultrasonicated again at the same conditions. Eventually, the samples were diluted 6 times with UPW for a final 2% HNO3 concentration. The recovery for Cd and Se extraction from the quartz sand was 96 ± 3 % and 92 ± 4 %, respectively. Fluorescence emission analysis of both eluent and soil extracts samples were performed with a Horiba Scientific FluoroMax-4 spectrofluorimeter, with excitation wavelength set at 310 nm and emission spectra recorded in the 480-600 nm range. The measurements were repeated at least two times per samples and quantitation was achieved with external calibration lines obtained from dilutions of the QDs stock solutions at concentrations ranging from 1 to 500 μg/L Cd (at least 6 points). Limit of quantitation for the QDs, based on Cd concentration, was 0.3 μg/L (equivalent to 2.1E+9 particles/mL). The metal content in the samples was analysed with a sector field inductively coupled plasma mass spectrometer (SF-ICP-MS, Element II, ThermoScientific, Bremen, Germany). Measurements of Se and Cd were carried out in high and low resolution modes, respectively. Quantitation was achieved analyzing standard solution at known concentrations of the elements ranging from 1 ng/L to 10 μg/L (n=5) and Indium at the concentration of 1 μg/L was continuously introduced during each analysis as internal standard. Limit of detection and quantitation were in the 100 ng/L and 10 ng/L ranges for Se and Cd, respectively. The data were processed with uFreasi software [START_REF] Tharaud | UFREASI: User friendly elemental data processing. A free and easy-to-use tool for elemental data treatment[END_REF]. Analysis of the metals digested from the quartz sand were done with an ICP-OES ICAP 6000 Series (ThermoScientific) equipped with an ASX-520 autosampler (Cetac, Omaha, NE, USA). Bromide tracer analysis were performed with a Dionex ICS 1100 ion chromatography system equipped with AS DV autosampler (ThermoScientific).

Results and discussion

Mobility and Transformation of QDs

The synthesized NPs presented very similar CdSe/ZnS core-shell structures, with Zn/Cd ratio of 2.8 and 3.0, and Zn/Se ratio of 7.8 and 8.7, for TGA-QDs and POAMA-QDs, respectively. Furthermore, the QDs displayed similar fluorescence spectra in the green portion of the visible spectrum (500-600 nm), with maximum yield at excitation wavelength of 310 nm and emission centered at 551 nm and 534 nm for TGA-QDs and POAMA-QDs, respectively (supplementary info, fig. S2). Thus, the main difference between the two NPs under investigation consisted in the ligand covalently attached to the surface, with TGA being a short organic chain and POAMA a more complex aromatic polymer (supplementary info, fig. S3 andS4). In this work, the fluorescence measurements were considered as indicative of a nanoparticles state, since the loss of emission is expected to correspond to a severe alteration of their core-shell structure (e.g. total dissolution). It must be noted that these could not necessarily discern between pristine and slightly altered nanomaterials (e.g. following partial dissolution of the ZnS shell). Fluorescence was compared with the ICP-MS measurements, which provided a quantitative assessment of the QDs metals but no information about their state (e.g. NPs or free ions). The mass balances for each experiment are provided in supplementary information (Table S5). Since Zn mass balances were generally >120% at the end of the experiments, Zn was not considered for further evaluation. Higher Zn concentrations may be due to background levels in the porous media or eluent solutions as well as incidental introduction during samples treatment. Thus, the results presented here are focused on Cd and Se analysis, which seemed not affected by Zn imbalances and presented recoveries generally > 90%.

The first aim of the present work was to study the QDs mobility during soil leaching experiments and to provide an assessment of their behavior and transformation. Figure 1 shows the breakthrough curves (BTC) obtained for POAMA-QDs (Fig. 1a) and TGA-QDs (Fig. 1b) in which NPs in the leachate (fluorescence, black lines) are shown together with Cd (red) and Se (green). Similar to what was reported by [START_REF] Al-Salim | Quantum Dot transport in soil, plants and insects[END_REF] during column studies in silt loam top-soil systems, the QDs were eluted after 1 PV at the beginning of the experiments and displayed little or no significant difference in the physical transport when compared to the conservative Br-tracer (supplementary info, fig. S6).

However, ICP-MS analysis revealed that different processes occurred during the transport due to differences between the NPs in the capping ligand stability. Specifically, POAMA-QDs (fig. 1a) metals BTCs were well correlated and displayed no significant difference with the measured fluorescence, suggesting that Cd and Se were transported as clusters and that QDs leached from the columns in a nanoparticulated form, at least with regard to the CdSe inner core liable for fluorescence emission.

The core integrity was further confirmed by the absence or low concentrations of Cd and Se after samples ultrafiltration (Fig. 1a, light lines), which is selective for the QDs and generally for particles larger than 3 KDa. Cd traces (in ultrafiltrates) were also measured immediately at the starting of the experiment and likely belonged to endogenous Cd, which eluted from the column before QDs. In contrast, TGA-QDs were slightly more retained in the column, with elution starting after 1.3 PV, and a significant difference between NPs and metals BTC was observed (fig. 1b). Indeed, while QDs concentrations were negligible, almost 40% of total Se and 0.5% of Cd were recovered in the leachate. This indicates that QDs transformation occurred during the experiment, resulting in the release of dissolved metal species, which transport was enhanced in comparison with the nanoparticulate forms. Also, since Se is confined into the inner portion of the QDs core-shell structure, its large concentration in the leachate necessarily implies at least a partial removal of both the ZnS shell and organic coating outer layers. In general, such degradation is cause of concern for mobilization of potentially toxic elements from NPs, including Cd(II), Se(-II), Se(IV) and several reactive oxygen species (ROS) [START_REF] Yaghini | Quantification of reactive oxygen species generation by photoexcitation of PEGylated quantum dots[END_REF]. In this context, the larger amount of Se in the leachate can be explained assuming the formation of novel oxyanions Se species (such as SeO3 2-), similar to those reported by [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF] during soil leaching studies, and considering the electrostatic repulsion with the negatively charged quartz sand. Accordingly, Cd divalent cation retention is in agreement with its fast sorption kinetic to soil. However, no Cd and only half of eluted Se were present in the samples after ultrafiltration (fig. 1b, light lines). Since the low fluorescence measured in the non-filtered leachate precluded the presence of large amounts of intact CdSe cores, the Se retention observed suggests the formation of colloidal metal phases withheld in the 3 KDa membrane. Interestingly, colloidal Se species (such as hydrogen selenide ion ,HSe -and colloidal elemental selenium) were reported to catalyze aerobic oxidation of thiols [START_REF] Nuttal | Hydrogen selenide ion and colloidal selenium in the catalytic oxidation of thiols[END_REF] and it could be expected that their released after a first dissolution of the TGA-QDs, may have further enhanced the ligand transformation, leading to the extensive degradation observed. In general, although the original assumption was of non-favorable deposition conditions, with minimal retention due to the electrostatic repulsion between NPs and quartz sand, both QDs displayed a strong retentive behavior and leached fractions were 2.1% and 0.1% only of the POAMA-QDs and TGA-QDs initial amounts, respectively. [START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF] reported deposition of 94% of the acrylic-capped CdTe QDs, which is similar to the Cd and Se POAMA-QDs deposition in this study (~93%), suggesting that enhanced NPs retention was due to the presence of divalent cations in solution (2 mM CaCl2). In detail, Ca 2+ -mediated deposition can be explained with the compression of the electrical double layer onto the NPs surface, which in turn determined a decreased repulsion between NPs and mineral surface. Furthermore, enhanced deposition of the QDs could result from the formation of homo-aggregates due to both the diminished mutual repulsion between NPs and the capacity of multivalent cations to complex the NPs by bridging the negatively charged capping groups (Quevedo and Tufenkji, 2011;[START_REF] Torkzaban | Release of Quantum Dot nanoparticles in porous media: role of cation exchange and aging time[END_REF].

In fig. 2, soil profiles were plotted to show the retained QDs and metals distribution along the columns. With regard to the POAMA-QDs (fig. 2a), although 77% of the deposited NPs was found in the first 0.5 cm, gradually decreasing amounts in the further sections revealed that the QDs migrated along the soil profile. Similarly, the larger fractions of Cd (71%) and Se (57%) were retained in the top column with smaller concentrations detected in the further sections. The correlation between metals and QDs distributions suggested that most of the NPs were intact during migration, confirming the overall stability previously observed in the leachate. Despite this the NPs quantified on the basis of fluorescence emission, which is indicative of pristine structures and/or CdSe cores (Pereira et al., 2016) accounted for 76% of the initial amount, showing that also a portion of the POAMA-QDs were altered after or during deposition. This observation is further supported by the minor differences between Cd and Se concentrations in the column segments, with the latter moving deeper along the profile. As expected from the analysis of the leachate, such phenomena had a larger extent in TGA-QDs amended column (fig. 2b). Indeed, although the quasi totality of Cd and 55% of Se were recovered in the quartz sand, according with the amounts measured in the eluent, only 25.9% of these corresponded to pristine QDs. Furthermore, while Cd and Se had a distribution similar to that of POAMA-QDs, with >65% of the metals in the top-column and the rest distributed in the further sections, the NPs displayed no migration and were entirely (> 95%) accumulated in the first 0.5 cm.

The concentration of Se and QDs in this section were similar and within the measured error, indicating that Se was retained in the top column as NPs and that transport occurred only for the species resulting from NPs degradation. Thus, it can be assumed that in addition to exposing the coreshell nanocrystals to the outer environment, with consequent dissolution phenomena, the ligand removal also caused the immobilization of the QDs onto the porous media due to the loss of surface coatings negative charges and non-favorable deposition conditions. It must be noted that the instability of thiol-capped QDs, similar to those in this work, was investigated by previous studies in aqueous media, which reported a pH-dependent aggregation and capping desorption of the NPs as well as light-mediated oxidation with consequent precipitation of the nanocrystals [START_REF] Aldana | Photochemical instability of CdSe nanocrystals coated by hydrophilic thiols[END_REF]Zhang et al., 2008b). However, the same works highlighted the relative stability of QDs (homogeneous dispersion and no precipitation) at the experimental conditions we applied (neutral pH, no light exposure) suggesting that degradation during the leaching experiments was enhanced by their interaction with the quartz sand matrix. Nonetheless, similar transformation phenomena were often neglected during soil transport studies, given the assumption that QDs were stable in soil matrices and during the relatively short duration of typical column experiments (usually 1-3 hrs) (e.g. [START_REF] Wang | Effect of surface coating composition on Quantum Dot mobility in porous media[END_REF], and evidences of QDs degradation were only reported after long-term exposure of NPs in soil systems [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF]. Rather, we observed that a fast ligand desorption may occur in soil with consequent transformation of the nanomaterials in the short-term. In this context, similar processes may hinder both the qualitative (speciation) and quantitative (deposition, attachment efficiency) assessment of QDs fate. For instance, in the case of TGA-QDs in this study the analysis of QDs fluorescence only may lead to an overestimation of deposited amounts, whereas mass spectrometric measurements alone would result in the overestimation of the eluted QDs.

Effect of Ageing and Alteration of the QDs

Once released in the environment, NPs will undergo modification of their state and properties due to the interactions with the natural components and ageing in the natural media (Mitrano and Novak, 2017). Thus, a second set of soil leaching studies was carried out to investigate the effect that alteration of the pristine QDs may have on their behavior during their transport in soil.

In the first ageing experiment, POAMA-QDs spiking solution was prepared at a larger concentration of Ca 2+ and incubated in the dark for a few hours before being introduced in the column. As shown in fig.

3a, this resulted in a higher QDs sorption onto quartz sand, and their complete retention in the column, i.e. no fluorescence measured in the leachate. A fraction of Cd and Se (0.5+/-0.1 % and 6.9+/-2.4 %; respectively) eluted at the beginning of the experiment with leaching profiles similar to those of the pristine QDs (Fig. 1a). However, no significant difference was observed with the concentrations found after ultrafiltration of the samples, suggesting that these were free metal species formed either in the preparation and ageing of the spiking solution or during NPs transport in the column.

Homo-and hetero-aggregation of nanomaterials are important processes driving their environmental fate and effects [START_REF] Lead | Nanomaterials in the environment: behavior, fate, bioavailability, and effects-an updated review[END_REF] and their characterization (e.g. kinetics of aggregation, determination of attachment efficiency) is increasingly being used for predicting NPs behavior and transport models [START_REF] Geitner | Measuring Nanoparticle Attachment Efficiency in Complex System[END_REF]. Increasing ionic strength of multivalent cations in solution is known to cause increasing hydrodynamic diameters and reduced electrophoretic mobility of QDs [START_REF] Wang | Effect of surface coating composition on Quantum Dot mobility in porous media[END_REF]Quevedo and Tufenkji, 2011). For instance, Quevedo and Tufenkji (2011) reported up to 15-fold increase of CdSe QDs diameter (assessed by transmission electron microscopy, TEM) at Ca 2+ concentrations similar to those applied in the present study (2,5 mM). In this case, the hydrodynamic diameter of QDs, measured by dynamic light scattering (DLS) was circa 550 nm and resulted in enhanced retention and one order of magnitude increase in the attachment efficiency.

Although such measurements were not performed here, recent studies by some of the authors of this work showed aggregation of POAMA-QDs in aqueous suspensions and soil solutions [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF]. Thus, it can be assumed that homo-aggregation of the NPs prior introduction in the columns imposed a reduced transport due to the larger size and smaller charge density of QDs. This also resulted in a decreased mobility along the soil profile (fig. 2c), with 95% of the retained NPs accumulated in the top-columns together with the largest fractions of Cd and Se. The metals were recovered to a larger extent, specifically 94% and 97% for Cd and Se, respectively (SI, table S4).

Furthermore, in the quartz sand, 80% or the metals were found in NPs form, indicating the relative stability of the altered NPs, likely due to the decreasing of the surface to volume ratio in the aggregates, i.e. a smaller surface exposed to the outer environment. Based on these results, it can be expected that aggregation will determine an even more limited mobility and higher persistency of QDs in terrestrial systems. However, it must be noted that the present work investigated the effect of homo-aggregation processes, which may only be representative of specific environmental scenarios where the presence of high concentrations are expected (e.g. disposal of consumer products, hotspots of contaminations) and that future works will need to clarify the effect of hetero-aggregation phenomena (e.g. interaction with organic matter), which are generally considered more relevant after dispersion of engineered nanomaterials in real ecosystems.

In the final set of experiments, NPs were aged in contact with the quartz sand before leaching in order to determine changes in their behaviour and mobility over time. Thus, the experiments were carried out at the same experimental conditions as previously (see section 3.1), but elution started 15 days after the introduction of QDs in the columns. The incubation time of 15 days was chosen in order to represent short to medium ageing periods and was previously applied in environmental fate studies [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF]. In general, ageing resulted in a reduced NPs transport in comparison to the non-aged experiments, with neither of the two QDs types leaching from the columns (fig. 3b and3c, black lines), and with retained fractions being almost totally (98%) accumulated in the first section of the soil profile (fig. 2d and2e, fluorescence bars). This behavior can be explained by an enhanced time-dependent NPs sorption to the quartz sand, as well as the consequence of homo-aggregation induced by the presence of a high NPs concentration in the top column for a relatively long period. [START_REF] Navarro | Differences in soil mobility and degradability between water-dispersible CdSe and CdSe/ZnS Quantum Dots[END_REF] reported increased mobility of thiol coated CdSe QDs after ageing in soil matrices due to the presence of organic matter in the system (i.e. ethylenediaminetetraacetic acid in the eluent and native organic acids in the soil). Furthermore, the authors found evidences of NPs degradation over time and suggested that organic material acted as chelating agent for the metals species originated from the QDs. In this study, both QDs were transformed to a large extent but in the absence of organic matter the etching of the pristine structures (e.g. transformation/desorption of the capping ligands) likely contributed at enhancing their deposition onto the sand. Indeed, the overall recovery from the system was only 22 +/-5 % of the original POAMA-QDs (fig. 3b) and 8 +/-2 % for TGA-QDs. Notably, while a larger degradation of the latter was expected due to its lower stability (see section 3.1), severe transformation were also observed for POAMA-QDs, which instead remained almost intact in the non-aged experiments. Accordingly, the metal species resulting from NPs dissolution were more abundant in TGA-QDs leachate, and predominantly consisted of Se, due to preferential Cd sorption to the sand. As shown in fig. 3b and3c, the ageing also caused significant changes leaching profiles of the metals, which eluted immediately at the beginning of the experiments, probably because of Cd and Se species migration in the saturated system during the incubation time. Interestingly, the overall larger degradation observed in the aged columns did not result in a faster transport of metals in the liquid phase. Indeed, Se amounts in the leachate were lower than those found in the non-aged experiments and specifically 2.9 +/-0.6 % and 13.6 +/-5.2 % for POAMA-QDs (fig. 3b) and TGA-QDs (fig. 3c), respectively. However, similar concentrations were found after ultrafiltration, specifically 2.4 +/-0.1 for POAMA-QDs and 11.1 +/-4.5, for TGA-QDs, suggesting that only free metal ions (e.g. SeO3 2-) were transported and that the metal colloids previously observed in the TGA-QDs non-aged column leachate (see fig. 1b) were deposited onto the quartz sand. Consequently, the amount of Se retained in the columns increased significantly from 54% of the non-aged experiments to 79% in aged TGA-QDs experiments. Analysis of the soil column profiles (fig. 2d and2e) confirmed that Cd and Se did not consistently move as a cluster in the quartz sand confirming the disruption of the NPs form. Specifically, in both studies more than 90% of Cd was accumulated in the first 0.5 cm whereas Se was detected throughout the soil column profiles, with larger migration in TGA-QDs columns where >50% of Se migrated from the introduction spot to further segments. Nonetheless, fluorescence emission recorded in the top column sections extracts showed the presence of potentially intact CdSe QDs cores deposited. However, analysis of the spectra revealed that an 11 nm blue-shift was present in the emission maximum of the TGA-QDs whereas POAMA-QDs spectra had a 4 nm shift to the right (supplementary info, fig. S7 andS8). Interestingly, similar red-shift was also recorded for QDs recovered from the columns amended with altered spiking solution, i.e. where homo-aggregation was enhanced. Since QDs emission is correlated to their size, with emission wavelengths decreasing with the NPs diameters, these results suggest that the CdSe cores of TGA-QDs were partially altered and became smaller with ageing. On the contrary, deposited POAMA-QDs cores were likely intact also due to aggregation phenomena that enhanced the sorption onto the sand and prevented dissolution of the structure. Eventually, the results of the ageing experiments showed that QDs will undergo profound alteration in the soil over time independently of their synthetic route (i.e. the organic coating), but that the capping ligand will still determine the kinetic at which these transformations will occur.

Conclusions

The transport of core-shell CdSe/ZnS QDs in quartz sand appeared to be limited at all conditions tested: pristine, altered and aged NPs. However, it must be noted that the soil column systems presented here were necessarily simplistic in comparison to real environmental systems, with regard to porous media and eluent compositions. These factors will play a major role in determining NPs fate in soils [START_REF] Geitner | Harmonizing Across Environmental Nanomaterial Testing Media for Increased Comparability of Nanomaterial Datasets[END_REF] and experiments employing more complex matrices (e.g. in the presence of minerals and organic matter) are currently being performed in our team. The results presented suggest that QDs will be accumulated in surface soils upon release in the environment but dissolution occurring due to the alteration of the pristine structures (i.e. capping ligand desorption) will result in the formation of novel potentially toxic metal species, which mobility was enhanced in our experiments in comparison with intact NPs. Thus, the stability of the organic coatings-NPs complexes will be as important as their physico-chemical properties in dictating the eventual fate of QDs in soil matrices, with regard to both their speciation and partitioning. In particular, small capping ligands used in this study (e.g. thioglycolic acid, TGA) displayed faster desorption rates from the NPs surface, resulting in the immobilization of the nanocrystals onto the sand. The organic coating removal likely enhanced the exposure of ZnS shells and subsequently CdSe cores to the outer environment, resulting in a larger degradation of the NPs. On the contrary, the stability conferred by larger organic coatings, as the poly(maleic anhydride-alt-1-octadecene) (POAMA), will likely determine higher transport and accumulation of the pristine QDs. Although alteration of the NPs manufactured state, such as homo-aggregation, may prevent degradation and enhance their persistency, we observed severe NPs transformation over time independently of their surface ligand. It must be noted that although the characterization of as-produced NPs is fundamental for the understanding of their environmental behavior, most nanomaterials are currently being incorporated in a variety of matrices, in the case of QDs electronics and optics. Hence, future works will need to investigate the release of these nanomaterials in the presence of (embedded) matrices in order to represent more realistic release/mobility pathways and contamination scenarios [START_REF] Nowack | Meeting the Needs for Released Nanomaterials Required for Further Testing The SUN Approach[END_REF].

The results presented here suggest that the stability of the QDs in soils is questionable during both the short-and long-term periods and that transformation phenomena will eventually determine the removal of these nanomaterials from the soil compartments. Furthermore, degradation phenomena may hinder a proper estimation and assessment of the QDs during transport and fate experiments, and future works will need to address such issues. In this context, the use of multiple techniques has been often recommended for the NPs characterization in environmental media in order to gather information about their state (e.g. pristine, aggregated, dissolved). In this work, the comparison of mass spectrometry and fluorescence analysis as well as the use of ultrafiltration proved to be valuable tools in discerning the NPs forms among the metal species detected. Similar strategies were recently applied by our team to determine quantum dots at low concentrations and in complex biological and environmental matrices [START_REF] Supiandi | Isotopically labeled nanoparticles at relevant concentrations: how low can we go? The case of CdSe/ZnS QDs in surface waters[END_REF]2020). Nonetheless, future works will need to provide more qualitative information about the QDs speciation and to describe the occurrence of the degradation products in real soil media and their interaction with natural components.

1 Figure 1

 11 Figure captions

Figure 2 Figure 2 .

 22 Figure 2

Figure 3 Figure 3 .

 33 Figure 3

Acwnoledgements

The present study was funded by the Agence Nationale de la Recherche (ANR), France, project QUADOS: ANR-CE01-0013. The authors would like to acknowledge the support by IPGP


Alexandre Mielniczek¹
email: mielniczek@gmail.com

Charlotte Roux²

Florence Jacquinod¹

Toward enhanced ecodesign of urban project thanks to City Information Modelling

CLCA for district ecodesign : new data opportunity

Consequential CLCA, although useful for early ecodesign of urban project, is rarely used and there is still few literature (Roux 2020)  among challenges to overcome : CLCA requires many datasets and data sources Today, more data are available in City Information Models (CIM) [START_REF] Deprêtre | EVALUATING URBAN INTENSITY THROUGH A CITY INFORMATION MODEL -INTERMEDIATE RESULTS FROM AN ACTION RESEARCH PROJECT[END_REF] • Building Information Models (BIM),

• Urban open geodata, either public or private.

… when others have very few data, trees for instance

In a CIM trees as well as utilities and public spaces objects (bench, technical equipment, vegetation, etc.) are also modelled, but usually with limited sets of properties  There is no standard describing LCA requirements  If LCA requirements were defined, more properties could be added to these objects in the modelling phase

• Urban heat island studies or Building thermal simulations • Shading • Need leaf type (deciduous or not)

• Need tree height • Local air pollution (Canyon effect, photochemical ozone creation)

• Need tree specie (VOC emissions)

• Need tree height and canopee form • Need to evaluate tree stress (access to water, local climate, urban soil management, etc.)

Other calculation could also be performed : How to improve CIM usability for CLCA ?

Some objects have many linked data, walls for instance…

Walls are being modelled in BIM for many years. In this example of a wall of the disctrict CIM, at least 9 properties could be used for LCA.

As an example, « Analytical Properties » tab or « Structure » give precise informations about wall materials and dimensions that can be used in thermal studies which is a very important aspect of building LCA.  in order to fully exploit those CIM geodata, CLCA methodologies also need to be adapted As a wider framework for future research, we're aiming at developping CLCA at district scale thanks to CIM, which involves :

Toward enhanced ecodesign of urban project thanks to City Information Modelling

• Refine, develop and extend district CLCA thanks to geodata from CIM • Provide guidelines so as to improve CIM data quality • Explore the potential of exploratory geovisusalition for LCA scientists so as to allow them to explore, understand and better anticipate the use of complex geodatasets

Future work

  What are BIM and CIM ? BIM (Building Information Model) = 3D objects with properties (type, quantities, etc.) representing a building site • Standardized open exchange format with categorized objects (IFC) •  Numerous BIM-LCA coupling studies CIM (City Information Model) • District or city scale • Diverse 3D models (public spaces, underground networks) • Multiple data sources (GIS, Urban Open Datas, BIM, etc.) and heterogeneity of data formats •  Scarcity of CIM-LCA coupling studies In this poster, we explore the potential of CIM geodata for CLCA through the analysis of the relevance of an existing CIM for LCA Data quality discrepancy between CIM objects Case study : analysing a district CIM  Need to define requirements for the production of CIM easily usable for CLCA CIM data potentiel for LCA -some examplesDefine CLCA requirements as far as CIM geodata are concerned, so as to allow urban planners to order necessary data for CLCA at early design stages (i.e. Planning and Programming)

La Vallée" district in Châtenay Malabry, France

  Each model is dedicated to a specific building and a specific discipline ans is georeferenced so as to allow for the aggregation of all models into one at the district scale

Conputational Fluid Dynamic (CFD) study for air quality or wind analysis, noise calculation, solar exposition, comfort, urban intensity, … "• Large district : 20.6 Ha, started in 2018 and under construction until 2027 • Mixte uses Dwellings/ Offices/ Shops

• CIM of the entire district made by various stakeholders (real estate developers, general contractor, architects, urban planner, design offices, public actors, etc.) working together to develop more than 40 digital models •  The CIM contains 138.000 BIM objetcs provinding more than 1.2 millionproperties values offering huge analysis potential, although not always easily usable at the disctrict scale

[START_REF] Deprêtre | EVALUATING URBAN INTENSITY THROUGH A CITY INFORMATION MODEL -INTERMEDIATE RESULTS FROM AN ACTION RESEARCH PROJECT[END_REF]