Co-directeur de thèse i ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisor Prof. Valérie Forest and to my co-advisor Prof. Jérémie Pourchez for their continuous support of my PhD study and related research, for their patience, motivation, and immense knowledge.

Besides my advisors, I would like to thank my thesis jury: Prof. Marie Carrière, Prof. Anna Bencsik, Prof. Jean-François Hochepied, and Prof. Francesco Turci.

I will forever be thankful to Prof. Marie Carrière, who has supported me and was a reference for me when I applied to this PhD project. From the very beginning to the end of my thesis, I am very grateful for her advice and suggestions.

My sincere thanks also go to Naila-Besma Belblidia and Prof Jean-François Hochepied for their support and help since they assisted in the synthesis and characterization of nanoparticles forming the base of my research. They always very helpful and supportive.

A special group from the University of Turin, Chemistry Department is not mentioned yet, because they deserve their own part. I praise the enormous amount of help by Prof.

Francesco Turci and Prof. Maura Tomatis throughout my mobility at their department.

Without their precious support, it would not be possible to improve this research. During the 3 months I spent there, I learned a lot from them, had a great time, and I was always greeted with so much smile and positivity. I thank the BioPI team, especially to Dr. Lara Leclerc a wonderful person who always supports and helps in many ways, and to my great office mate Yoann Mountigaud.

I took part of many other activities, with countless people teaching and helping me every step. Thank you to everyone from CIS, especially Françoise Bresson stands out notoriously as an amazing source of support and to my best friend Cyriac Azefack, your friendship was irreplaceable and has always been very precious. There are no words to convey how much I enjoyed the time we spent. He has been a true and great friend.

I also would like to thank Prof. Bedri Kurtulus for the precious support he gave me throughout my life.

I would like to thank my family: my parents, my brother, and my sister who are by my side with their love and support at every stage of my life.

TABLES Table Page

Table 1. Physical and chemical properties of different nanoparticles (Adapted from [START_REF]Nanotechnologies -Vocabulary -Part 1: Core terms[END_REF]). 6

Table 2. Workplace measurements in TiO2 depending on the activity types [START_REF] Sun | Dynamic Probabilistic Modelling of Environmental Emissions of Engineered Nanomaterials Supplementary Information[END_REF][START_REF] Tsai | Exposure assessment of nano-sized and respirable particles at different workplaces[END_REF][START_REF] Methner | Nanoparticle emission assessment technique (NEAT) for the identification and measurement of potential inhalation exposure to engineered nanomaterials-part b: Results from 12 field studies[END_REF]. ...... 10 Table 3. In vitro studies in literature releated to NPs size impact on its toxicity. ............. 23 Table 4. Protocols for determining optimal conditions for THP-1 differentiation ............ 39

INTRODUCTION

Nanoparticles, an increasingly important product of nanotechnology, are defined as objects with at least one dimension measuring less than 100 nanometers (nm) [START_REF]EU, Definition -Nanomaterials -Environment -European Commission[END_REF].

Metallic and poorly soluble nanoparticles such as titanium dioxide (TiO2) are among the most widely used nanoparticles in the world [START_REF] Anses | TITANIUM DIOXIDE CLH report Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008[END_REF]. In addition to its unique physicochemical properties such as small particle size, TiO2 has received much attention due to its chemical stability, high refractive index and photocatalytic activity [START_REF] Haider | Exploring potential Environmental applications of TiO2 Nanoparticles[END_REF][START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. TiO2 nanoparticles are used in many consumer products such as paints, antiseptic and antimicrobial compositions, deodorization (purifying/deodorizing indoor air), inks, toothpastes, ceramics, anti-reflective coating in silicon solar cells and many thin film optical devices [START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. The abundance of these products has raised concerns about human exposure and health consequences.

The main route of exposure to TiO2 nanoparticles is by inhalation. It mainly concerns the worker, the consumer being more probably concerned by the oral route. The environmental and occupational atmospheres are not well characterized for nanoparticles, which may explain the lack of epidemiological studies on the relationship between human exposure to nanoparticles and its adverse effects.

Despite this, to show associations between exposure to fine and ultrafine particles in the atmosphere and their adverse health effects, their potential toxicity has been investigated in animal and cellular models. Two important chronic inhalation studies reported elevated risk of lung cancer after exposure to fine [START_REF] Lee | Pulmonary response of rats exposed to titanium dioxide (TiO2) by inhalation for two years[END_REF] or ultrafine [START_REF] Heinrich | Chronic inhalation exposure of wistar rats and two different strains of mice to diesel engine exhaust, carbon black, and titanium dioxide[END_REF] TiO2 in rats and the International Agency for Research on Cancer (IARC) has classified TiO2 as a possible human carcinogen (Group 2B) [START_REF] Iarc | IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Carbon Black, Titanium Dioxide, and Talc[END_REF]. This classification applies to all forms of TiO2 without considering the size criterion and therefore the nanometric forms.

However, given their specificities, the nanometric structure could give rise to more concern regarding health and safety.

still specific, between the different physicochemical characteristics of exposure to TiO2 NPs and their impact on human health Due to the reported adverse effects of nano TiO2, its use in food products in the form of the additive E171 has been suspended for one year in France since January 1, 2020 [START_REF]portant suspension de la mise sur le marché des denrées contenant l[END_REF]. Consequently, the French government has sought to reduce the exposure of workers, consumers and the environment to TiO2 NPs following the notice published by the French National Agency for Food, Environmental and Occupational Health Safety (ANSES) [START_REF]Anses -Agence nationale de sécurité sanitaire de l'alimentation[END_REF]. As a result, the French government sought to reduce exposure to nano-TiO2 to workers, consumers and the environment.

With regard to safety, it is important to know what property makes a nanomaterial more or less safe, especially when producing and designing nanomaterials (NMs) according to a "safer by design" approach [START_REF] Kraegeloh | Implementation of safe-by-design for nanomaterial development and safe innovation: Why we need a comprehensive approach[END_REF]. Therefore, it is necessary to evaluate the toxic effects of TiO2 nanoparticles and to study how the physical and chemical properties of these nanoparticles determine their biological effects.

Depending on their physicochemical properties, TiO2 nanoparticles can cross the biological membranes, circulate in the human body, and interact with biomolecules once inhaled. Due to their interaction with biomolecules, their toxicity usually results in damaged organelle structures, DNA/RNA break, membrane structure destruction, mitochondrial dysfunction, oxidative stress, or inflammation [START_REF] Buzea | Nanomaterials and nanoparticles: Sources and toxicity[END_REF]. All these toxic manifestations induce many respiratory diseases such as chronic bronchitis, asthma, chronic obstructive pulmonary disease, or cystic fibrosis [START_REF] Geiser | Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells[END_REF][START_REF] Osman | Effect of nanoparticles on human cells from healthy individuals and patients with respiratory diseases[END_REF][START_REF] Ahn | Titanium dioxide particle -Induced goblet cell hyperplasia: Association with mast cells and IL-13[END_REF] and even cause injuries in other tissues.

The objective of this thesis work was to systematically study the in vitro toxicity of

INTRODUCTION

Les nanoparticules (NP), produits de plus en plus importants de la nanotechnologie, se definissent comme des objets dont au moins une dimension mesure moins de 100 nanomètres (nm) [START_REF]EU, Definition -Nanomaterials -Environment -European Commission[END_REF].

Les NPs métalliques et faiblement solubles comme les NPs de dioxyde de titane (TiO2) sont parmi les nanomatériaux (NMs) les plus utilisés au monde [START_REF] Anses | TITANIUM DIOXIDE CLH report Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008[END_REF]. Outre ses propriétés physicochimiques uniques telles que la taille des particules, le TiO2 est un nanomatériau très répandu en raison de sa stabilité chimique, de son indice de réfraction élevé et de son activité photocatalytique [START_REF] Haider | Exploring potential Environmental applications of TiO2 Nanoparticles[END_REF][START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. Les NPs de TiO2 sont utilisées dans de nombreux produits de consommation comme les peintures, les compositions antiseptiques et antimicrobiennes, la désodorisation (purifier/désodoriser l'air intérieur), les encres, les dentifrices, les céramiques, le revêtement anti-reflet dans les cellules solaires en silicium et de nombreux dispositifs optiques à couches minces [START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF].

L'abondance de ces produits a soulevé des inquiétudes concernant l'exposition humaine et les conséquences sur la santé.

an en France depuis le 1er janvier 2020 [START_REF]portant suspension de la mise sur le marché des denrées contenant l[END_REF]. Par conséquent, le gouvernement français a cherché à réduire l'exposition des travailleurs, consommateurs et de l'environnement aux NPs de TiO2, suite à l'avis publié par l' Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES) [START_REF]France bans Titanium Dioxide in food products[END_REF].

Par conséquent, le gouvernement français a cherché à réduire l'exposition au TiO2 des travailleurs, des consommateurs et de l'environnement.

En ce qui concerne la sécurité, il est important de savoir quelle propriété rend un nanomatériau plus ou moins sûr, en particulier lors de sa production et de sa conception selon une approche dite safer by design [START_REF] Kraegeloh | Implementation of safe-by-design for nanomaterial development and safe innovation: Why we need a comprehensive approach[END_REF].

BIBLIOGRAPHY 1. Nanomaterials

Up to date, there is no universal definition for nanomaterials (NMs). The European Union (EU) adopted a definition of a nanomaterial in 2011 as ''A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm -100 nm'' [START_REF]EU, Definition -Nanomaterials -Environment -European Commission[END_REF].

According to ISO/TS 80004 [START_REF]Nanotechnologies -Vocabulary -Part 1: Core terms[END_REF], material with any external dimension in the nanoscale or having an internal structure or surface structure in the nanoscale, with nanoscale defined as the length range approximately from 1 nm to 100 nm is defined as a nanomaterial. This includes both nano-objects, and nanostructured materials, which have internal or surface structure on the nanoscale; a nanomaterial may be a member of both these categories.

Nanomaterial properties and applications

Due to NMs' unique properties and their atomic sequences which can be changed according to the purpose, they are extremely suitable for both industrial and scientific applications and uses [START_REF] Buzea | Nanomaterials and nanoparticles: Sources and toxicity[END_REF].

The physical and chemical properties of the nanoparticles are different from the properties of micro-sized particles. Although nanoparticles consist of the same atoms as microparticles, they have a different formation geometry. The smaller a material, the higher the surface to volume ratio, meaning that equal volumes of nanoparticles have a much greater surface activity than micro-sized materials [START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. The high surface activity gives exceptional properties to nanoparticles or manufactured products containing nanoparticles. In addition to their size and surface area, the other properties of nanoparticles such as surface properties, crystallinity, dispersion status, and shape are among the factors affecting their behavior. Nanoparticles come in varied shapes including spherical, triangular, cubic, prism, oval, rod, nanoshell, helical, tubes and fibers [START_REF] Gatoo | Physicochemical properties of nanomaterials: implication in associated toxic manifestations[END_REF] ( Figure 1). Nanoparticles are generally divided into several categories depending on their morphology, size and chemical properties. Carbon-based NMs containing fullerenes and carbon nanotubes (CNTs), metal NMs which are made by purely metal precursors, metal oxide semiconductor materials which possess properties between metals and nonmetals, polymeric and lipid-based nanoparticles are some of the wellknown nanoparticle classes [START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. The properties of few common nanoparticles are given in Table 1.

Table 1. Physical and chemical properties of different nanoparticles (Adapted from [START_REF]A review on the classification, characterisation, synthesis of nanoparticles and their application -NASA/ADS[END_REF]). Each nanoparticle in these categories depending on its own physicochemical properties has been used in many applications such as in mechanical, energy, electronic industries, environmental approach, manufacturing materials, and pharmaceuticals [START_REF] Tiwari | Zero-dimensional, one-dimensional, twodimensional and three-dimensional nanostructured materials for advanced electrochemical energy devices[END_REF]. With the help of this type of high-tech industrial applications, many daily products have been produced with nanotechnology in higher quality such as cosmetics, inks, food package, food additives, diet products, antibacterial coated clothes. The nanoparticles used in the industrial consumer products are metal oxide such as titanium dioxide (TiO2), aluminium oxide (Al2O3), zinc oxide (ZnO), and iron (III) oxide (Fe2O3) nanoparticles. TiO2 and ZnO nanoparticles which are present in almost all cosmetic products as a sunscreen and as an ingredient in interior-exterior paints due to their high refractive index and photocatalytic properties. Film coatings consisting of organic nanoparticles are frequently used in dietary products and drug coatings. CNTs are used in textile industry. Ag NPs with antibacterial properties are used to keep all kinds of fabric hygienic [START_REF] Ibrahim | Carbon nanotubes-properties and applications: a review[END_REF]. Ceramic nanocomposites, TiO2 and silica NPs owing color protecting, hydrophobic and durability enhancing properties are used on glass and ceramic surfaces and insulation materials. Nanoparticles are abundant in the products we contact everyday such as health products and medical treatments as well. Products like sunscreen, deodorant, and cosmetics all employ nanoparticles and nanotechnology [START_REF] Buzea | Nanomaterials and nanoparticles: Sources and toxicity[END_REF].

Titanium dioxide nanoparticles and their applications

Titanium dioxide is a metal oxide, white powder with a molecular weight of 79.87 g.

It melts at 1855˚C, boils at 2750˚C and has no smell. It is a cheap and abundant semiconductor and naturally exsists in three crystal structures as the two most common tetragonal crystallographic polymorphs rutile and anatase, the rarer orthorhombic third crystal structure is brookite (Figure 2). In pure TiO2 for photovoltaic and photocatalytic applications, the anatase phase shows superior catalytic activity with the 3.2 eV energy band gap than rutile (3.02 eV)

or brookite phases (2.96 eV) [START_REF] Koelsch | Comparison of optical and electrochemical properties of anatase and brookite TiO2 synthesized by the sol-gel method[END_REF]. Also, the increased photoreactivity of the anatase phase is associated with low oxygen adsorption capacity, increased hydroxylation degree and slightly higher Fermi level. The rutile phase also exhibits high refractive index and high optical absorptivity [START_REF] Mo | Electronic and optical properties of three phases of titanium dioxide: Rutile, anatase, and brookite[END_REF].

Upon UV light exposure to TiO2 nanoparticles, the electrons in the valence bands (VBs) gain energy and therefore are stimulated to the corresponding conduction bands (CBs), thereby produce the electrons (e -) on the CBs and forming holes (h + ) on these VBs. Formed holes, h + , interact with surrounding water and produce hydroxyl radical (HO•) radicals [START_REF] Fujishima | Titanium dioxide photocatalysis[END_REF][START_REF] Nam | Photocatalytic activity of TiO2 nanoparticles: A theoretical aspect[END_REF][START_REF] Qian | Charge carrier trapping, recombination and transfer during TiO2 photocatalysis: An overview[END_REF].

If there is oxygen in the environment, the electron in the CB reduces molecular oxygen to form a superoxide radical (O2•  ) [START_REF] Wang | Effect of UV irradiation on the aggregation of TiO2 in an aquatic environment: Influence of humic acid and pH[END_REF]. The superoxide radical can react with water and produce hydrogen peroxide (H2O2). This hydrogen peroxide can either react with light energy (hv) or electrons to produce HO• radicals. The produced HO• radicals then decompose the organic compounds (R) into water (H2O) and carbon dioxide (CO2) (Figure 3) [START_REF] Zhang | UV irradiation mediated aggregation of TiO2 nanoparticles in simulated aquatic system[END_REF].

The formation of such photocatalytic oxidation in organic compounds damages microorganisms in air or in water [START_REF] Hashimoto | TiO 2 Photocatalysis: A Historical Overview and Future Prospects[END_REF]. Generally, electrons in the TiO2 structure can switch from the valence band to the conductivity band due to the absorbed beam.

During recombination, electrons can return to their old energy levels in a short time such as 10 -6 -10 -11 s [START_REF] Hashimoto | TiO 2 Photocatalysis: A Historical Overview and Future Prospects[END_REF][START_REF] Sun | UV irradiation induced transformation of TiO2 nanoparticles in water: Aggregation and photoreactivity[END_REF][START_REF] Watanabe | Photocatalytic activity and photoinduced hydrophilicity of titanium dioxide coated glass[END_REF].

Titanium dioxide nanoparticles environmental release

The increase in production and use of nanoparticles leads to a potential increase in their release into air, water and/or soil through various processes. Nanoparticles can pass and accumulate into the soil compartments, contaminate water and air during their production, their use and disposal of nanoparticle-containing products (waste handling) [START_REF] Wagner | Spot the Difference: Engineered and Natural Nanoparticles in the Environment-Release, Behavior, and Fate[END_REF].

Especially in nanotechnology companies both in producer and user companies, workers are the largest group to have contact with nanoparticles. Work environment can be contaminated during processing of nanoparticle containing products, packaging, cleaning of industrial installations. Regarding their airborne release; the collection and sorting of materials during production are the most frequently described activities, followed by physical and chemical synthesis processes [START_REF] Bundschuh | Nanoparticles in the environment: where do we come from, where do we go to?[END_REF][START_REF] Sun | Dynamic Probabilistic Modelling of Environmental Emissions of Engineered Nanomaterials Supplementary Information[END_REF].

Nanoparticles also spread to the working environment during sonication, weighing, mixing [START_REF] Tsai | Exposure assessment of nano-sized and respirable particles at different workplaces[END_REF][START_REF] Methner | Nanoparticle emission assessment technique (NEAT) for the identification and measurement of potential inhalation exposure to engineered nanomaterials-part b: Results from 12 field studies[END_REF]. The quantities of nanoparticles emitted, in particular into the ambient air, can be significant and reach hundreds of thousands of particles per cm 3 of air and even values approaching the million particles per cm 3 in mass concentration. In the absence of collective or individual protection, workers can be potentially exposed in an acute and chronic manner to much greater amounts of NPs than in environmental exposure.

Some studies investigated the release of TiO2 nanoparticles in workplace area depending on the activities [START_REF] Methner | Nanoparticle emission assessment technique (NEAT) for the identification and measurement of potential inhalation exposure to engineered nanomaterials-part b: Results from 12 field studies[END_REF][START_REF] Plitzko | Workplace exposure to engineered nanoparticles[END_REF][START_REF] Ding | Airborne engineered nanomaterials in the workplace-a review of release and worker exposure during nanomaterial production and handling processes[END_REF]. Background concentrations at the workplace were analyzed by a wide-range aerosol spectrometer (range of particle size 5 nm to >32 µm). Measured parameters were particle number concentration as well as particle size distribution. No significant airborne release was observed in comparison to background levels during the production of TiO2 nanoparticles and cleaning process.

Cleaning consists of a pyrolysis system, vacuum cleaning and cleaning-up spilled materials from dumping operations in this study [START_REF] Plitzko | Workplace exposure to engineered nanoparticles[END_REF]. Twelve facilities were evaluated between December 2005 and January 2008 and an increased release of particles was found during spraying process [START_REF] Methner | Nanoparticle emission assessment technique (NEAT) for the identification and measurement of potential inhalation exposure to engineered nanomaterials-part b: Results from 12 field studies[END_REF]. Especially flame-spray processes which are used for coating often resulted in very small sizes of highly airborne particles (Table 2) [START_REF] Ding | Airborne engineered nanomaterials in the workplace-a review of release and worker exposure during nanomaterial production and handling processes[END_REF]. Another study revealed that TiO2 aerosols were released in diameters up to 6.0 µm during industrial packaging activities and the higher levels of the respirable particle mass and number concentrations were found to be near the packing site of the pigment TiO2 production factory [START_REF] Huang | Measurements of respirable dust and nanoparticle concentrations in a titanium dioxide pigment production factory[END_REF].

A study [START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF] using the dynamic probabilistic material flow model (DP-MFA) based on the recently developed method by Bornhöft et al. [START_REF] Bornhöft | A dynamic probabilistic material flow modeling method[END_REF], calculated the estimated air concentrations of four NPs in 2020. The predictions were based on the combined estimates of different market research companies and were grounded in the increase in the market in the last years. The predicted (accumulated) concentration of TiO2, ZnO, Ag and CNT NPs in the air was found as 5.48, 2.33, 0.03, and 0.05 ng/m 3 , respectively. Among these four nanoparticles, the particle expected to be in the highest concentrations in the environment is TiO2.

US National Institute for Occupational Safety and Health, Organisation for Economic

Co-operation and Development, International Organization for Standardization, and many other institutions have established the general rules of the protection of workers.

 The recommended exposure limit (REL) for ultrafine and nano TiO2 is set to 0.3 mg/m 3 (10 h TWA-time weighted average-per day during a 40-h work week) by US NIOSH [START_REF]Occupational Exposure to Titanium Dioxide[END_REF].

 Maximum admissible concentration-the time-weighted average (MAC-TWA) is proposed as 300 μg/m 3 for nano TiO2 by the Polish Nofer Institute of Occupational Medicine [START_REF] Świdwińska-Gajewska | Titanium dioxide nanoparticles: Occupational exposure limits[END_REF].

 Nano reference values (NRV) which is a risk management tool for unknown occupational exposure limits (OELs) of NPs, is defined for particles with a density lower than 6 g/cm 3 NRV like metal oxides (e.g. TiO2, ZnO, Al2O3), fullerenes, dendrimers, nanoclay, polystyrene, SiO2, carbon black, etc., NRV is 40,000 particles/cm 3 [START_REF] Oberbek | Inhalation exposure to various nanoparticles in work environment-contextual information and results of measurements[END_REF].

 Following an in-depth analysis of all the available toxicity data, the ANSES is recommending a chronic TRV by inhalation for the P25 form of TiO2-NP of 0.12 µg/m -3 . This reference value will be used when conducting health risk assessments as part of the management of industrial facilities and sites in France. It is also the first TRV developed for a nanomaterial in France [START_REF]Titanium dioxide in nanoparticle form: ANSES defines a toxicity reference value (TRV) for chronic inhalation exposure | Anses -Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail[END_REF]. Considering the area it covers, the skin can be described as the most exposed organ to the nanoparticles. Therefore, the dermal exposure of nanoparticles should not be neglected, especially for lipophilic and low molecular weight materials. However, in the absence of organic solvents, the interaction with the cells of the skin barrier remains limited because the nanoparticles hardly cross the upper layer of skin known as Stratum corneum. It has been shown on healthy volunteers that the TiO2 nanoparticles present in sunscreens did not cross the Stratum corneum [START_REF]Literature review on the safety of titanium dioxide and zinc oxide nanoparticles in sunscreens | Therapeutic Goods Administration (TGA)[END_REF].

However, it has been shown that NPs can pass through the damaged skin (e.g. eczema) [START_REF] Yah | A review of nanoparticles toxicity and their routes of exposures, Iran[END_REF][START_REF] Yokel | Engineered nanomaterials: Exposures, hazards, and risk prevention[END_REF]. Therefore, special attention should be paid to the safety assessment prior to human use.

Ocular

Ocular exposure is possible after applying cosmetics to the eyes or accumulating aerosolized NMs. However, the research on this route of exposure is limited [START_REF] Yokel | Engineered nanomaterials: Exposures, hazards, and risk prevention[END_REF].

Oral

Due to the fact that food products contain nanoparticles, especially when considering Food grade TiO2 (E171) as an ingredient in many products, such as sweets, candies and chewing gum, the digestive system is also an entry route of NPs of concern. If the route of administration is different, the biokinetic/biodistribution patterns vary significantly.

Titanium dioxide nanoparticles are generally fecally cleared after oral administration.

Nanoparticles are absorbed by the gastrointestinal tract and can translocate to tissues and organs such as the liver, spleen, kidneys, and lung tissues, and where they can lead to lung injury, nephrotoxicity and hepatic injury [START_REF] Ellender | Effect of bronchopulmonary lavage on lung retention and clearance of particulate material in hamsters[END_REF].

In addition to consuming foods containing nanoparticles, their oral intake can occur from unintentional hand to mouth transfer of materials for example during handling of materials that contain nanoparticles. Ingestion may also accompany inhalation exposure because particles which enter the organism by the respiratory route can pass to the digestive tract by mucocillary clearance [START_REF] Yah | A review of nanoparticles toxicity and their routes of exposures, Iran[END_REF].

Inhalation

Taking into account the significant nanoparticle concentrations released into the work environment, the most common route of exposure is by inhalation.

Once inhaled, depending on their size, the nanoparticles can penetrate deep into the lungs and can pass into the blood and/or translocate into other organs. The retention time of nanoparticles in the compartments depends on the exposure time. The NMs are cleared with alveolar macrophages slowly in the alveoli and then drained into the lymph nodes [START_REF] Muralidharan | Inhalable nanoparticulate powders for respiratory delivery[END_REF]. On the other hand, NPs can be rapidly cleared by the mucociliary escalator.

Adverse effects of inhaled nanoparticles

General anatomy of the respiratory system and nanoparticle interaction

The respiratory system is divided into two parts: the upper and lower respiratory tracts. The upper respiratory tract consists of a nasal layer, which trap harmful substances in the air, pharynx and larynx. The inhaled air passes from the larynx to the trachea, reaching the right and left bronchi. The bronchi eventually split into smaller airways called bronchioles. The diameter and length of each airway branch decreases progressively from the trachea to the bronchioles. At the end, the air reaches the alveoli where gas exchange occurs. The lower respiratory tract consists of trachea, bronchi, bronchioles and lungs (including alveoli) (Figure 5) [START_REF] Kling | A Review of Respiratory System Anatomy, Physiology, and Disease in the Mouse, Rat, Hamster, and Gerbil[END_REF]. Particles inhaled in different sizes show deposits in different regions of the human respiratory system. Ultra-fine particles with a diameter lower than 100 nm were observed in all regions, particles smaller than 10 nm in the tracheobronchial region, and particles from 10 to 20 nm in the alveolar region [START_REF] Asgharian | Deposition of Ultrafine (NANO) Particles in the Human Lung[END_REF]. It has been shown that 22

nm inhaled TiO2 NPs deposited in different compartments of lung tissue, capillaries, and on the luminal surface of the airways and alveoli [START_REF] Oberdorster | Correlation between particle size, in vivo particle persistence, and lung injury[END_REF]. The relocation of particles from the lung surface to lymphatic drainage might result in entering blood vessels and translocate easily in the circulatory system. Translocation of NPs cause their accumulation in several major organs such as the heart, liver, immune system, liver etc. (Figure 6) [START_REF]Literature review on the safety of titanium dioxide and zinc oxide nanoparticles in sunscreens | Therapeutic Goods Administration (TGA)[END_REF].

The retention of particles starts with their wetting and displacement from the air into the aqueous phase by surfactant. In general, it has been shown in rodents, that microparticles (0.5 -10 μm) remain on the epithelial surface in airways and alveoli and are accessible to BAL [START_REF] Ellender | Effect of bronchopulmonary lavage on lung retention and clearance of particulate material in hamsters[END_REF]. The same can be expected for nanoparticles even more However, clearance can be long in deeper region of lung due to the high mucus velocity. As we discuss in detail in the following section, the interaction of particles with macrophages results in phagocytic uptake that is also a key factor for particle clearance in airways and alveoli.

Cellular organization of the respiratory system and nanoparticle interaction

The respiratory tract is lined by different epithelial cells. There are thirteen different cells known, eleven of these are epithelial cells and two are mesenchymal cells. Most of the epithelium cells are defined as ciliated and mucus secreted cells (e.g. goblet cells). The ciliated, brush, basal, club, pulmonary neuroendocrine (PNEC) cells have been found in airways. The area from the trachea to the bronchi is usually covered with ciliated pseudostratified columnar epithelium. Epithelial cells take more cubic shape as they are directed towards the bronchioles to alveoli, while mesenchymal cells cover the lung tissue [START_REF] Crystal | Airway epithelial cells: Current concepts and challenges[END_REF].

Alveolar epithelium is made up of two cell types, Type I Alveolar Epithelial Cells (AECs I, Type I pneumocytes) and Type II Alveolar Epithelial Cells (AEC2s, Type II pneumocytes). The major epithelial cell types of the gas exchanging region are cuboidal AEC2s that are specialized for surfactant protein production and secretion, Type I cells are characterized by a narrow and extensive cytoplasm and are joined together by tight junctions (Figure 7). These cells allow passive gas diffusion while making the alveolar epithelium fairly impermeable to other substances [START_REF] Knight | The airway epithelium: Structural and functional properties in health and disease[END_REF]. The phagocytic activity of macrophages is considered as the primary mechanism for the intake of nanoparticles into the cell. In the biological fluids, proteins adsorb on the surface of the nanoparticles (forming the so-called protein corona, see 2.2.1).

Proteins from the corona (especially immunoglobulins or complement proteins) are recognized by cell membrane receptors of macrophages. This initiates a signaling cascade that can trigger actin assembly, the formation of cell surface extensions, and forming a zipper-like structure and subsequent engulfing and internalization of particles (Figure 9) [START_REF] Aderem | Mechanisms of phagocytosis in macrophages[END_REF]. In general, reorganization of cytoskeleton occurs in many cell types but especially in certain cells called as professional phagocytes. These cells are: macrophages, neutrophils, dendritic cells, monocytes and include endothelial and secretory epithelial cells in some special cases [START_REF] Aderem | Mechanisms of phagocytosis in macrophages[END_REF]. During the phagocytosis of NPs by macrophages, inflammatory markers such as cytokines and pro-inflammatory chemokines can be released [START_REF] Monks | Epithelial cells as phagocytes: Apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF]. The release of such chemokines leads to inflammatory cascades in the alveolar milieu, which are an important part of innate immunity. Chronic inflammation could represent an environment favorable to the development of lung diseases.

Protein corona formation

When nanoparticles interact with the biological environment, proteins in the biological fluids, sugars, lipids, and other macromolecules adsorb to the nanoparticle surface. Since proteins are largely predominant in biological fluids, this nanoparticleprotein structure is called the protein corona [START_REF] Cedervall | Understanding the nanoparticle-protein corona using methods to quntify exchange rates and affinities of proteins for nanoparticles[END_REF]. But the understanding of the formation and composition of the protein corona remains poor.

The adsorption of proteins on the surface of the nanoparticles is mainly driven by electrostatic interactions, as well as H-bond, van der Waals interactions, and salt bridge formations. The protein corona is composed of two main layers: the hard corona, which is the first and richer layer of proteins, characterized by direct The evolution and dynamics of hard corona formation are relatively well studied [START_REF] Cedervall | Understanding the nanoparticle-protein corona using methods to quntify exchange rates and affinities of proteins for nanoparticles[END_REF][START_REF] Weiss | In Situ Characterization of Protein Corona Formation on Silica Microparticles Using Confocal Laser Scanning Microscopy Combined with Microfluidics[END_REF]. The current understanding is that the hard corona proteins-with their long residence time-give the nanoparticles a biological identity by presenting receptorbinding sites for cellular interactions with a biologically relevant timescale. As soft corona proteins by definition have a shorter residence time on nanoparticles than hard corona proteins making them difficult to isolate from free proteins of the mother liquid, their potential biological impacts through specific and/or nonspecific interactions have often been ignored [START_REF] Walczyk | What the cell "sees" in bionanoscience[END_REF].

The protein corona evolves when nanoparticle-adsorbed-protein complexes are moved from one biological environment into another, with some proteins from the original corona being replaced by proteins from the new biological fluid. It is intriguing that while the corona evolves, it retains a fingerprint of its prior history.

This may turn out to be an important paradigm, for understanding in vitro and in vivo transport. Besides this, however, the protein-particle complex may contain a historydependent set of protein markers or signals that can be elucidated via the protein corona of particles recovered from their final subcellular location [START_REF] Lundqvist | The evolution of the protein corona around nanoparticles: A test study[END_REF].

The physicochemical properties of NPs have an impact on the formation and composition of the protein corona [START_REF] Forest | CHAPTER 2: Biological Significance of the Nanoparticles Protein Corona[END_REF]. The size and surface area of the NPs are the main factors that affect the amount of proteins that can adsorb to its surface. The surface charge, surface chemistry, shape of NPs also have a great effect on the composition of the corona. The protein interaction can also alter the physicochemical properties of NPs. Proteins adsorbed on the nanoparticle surface may change their conformation, agglomeration status and surface charge [START_REF] Monopoli | Physical-Chemical aspects of protein corona: Relevance to in vitro and in vivo biological impacts of nanoparticles[END_REF].

All these are parameters can affect the contact of nanoparticles with the cells. The proteins on the nanoparticle surface can be recognized by the receptors on the cell membrane and the nanoparticle-cell membrane interaction begins. After this point, nanoparticles can be taken up into the cell and their fate is determined [START_REF] Corbo | The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery[END_REF].

Mechanisms of nanoparticle toxicity

Following the uptake of nanoparticles into the cell, the next important consideration is the determination of their cellular outcomes. Nanoparticles show their toxicity mainly by inducing oxidative stress or by causing inflammation.

Oxidative stress

Aerobic organisms use the mitochondria organelle as the main energy producer to perform vital functions [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. Mitochondria produce adenosine triphosphate (ATP) through oxidation and reduction reactions, and the produced ATP goes through to the tricarboxylic acid cycle by the electron transport chain [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. This situation occurs by oxidation of food produced in different metabolic pathways such as glycolysis, B oxidation and Krebs cycle, and nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2) reduction-oxidation [START_REF] Castellucci Estevam | A Historical Perspective on Oxidative Stress and Intracellular Redox Control[END_REF].

However, these reactions lead to the production of extremely highly reactive oxygen species (ROS), which are unstable due to their unpaired electrons. ROS try to stabilize their unpaired electrons by attacking nucleic acids, proteins, carbohydrates and the consequence is an alteration of the functions of these biomolecules. A complex antioxidant defense system often balances this attack. However, in some cases, this balance may be disrupted. Oxidative stress is a physiological response to a cellular stress or aggression, it becomes deleterous when it is uncontroled [START_REF] Sies | On the history of oxidative stress: Concept and some aspects of current development[END_REF].

The transport chain of the mitochondrial electron is the main source of the endogenous ROS; molecular oxygen is gradually reduced to a number of intermediate types: hydroperoxyl radical, superoxide radical anion, hydrogen peroxide (H2O2), hydroxide ion and hydroxyl radical. Gradual molecular oxygen reduction is shown as follows [START_REF] Gutteridge | Biological origin of free radicals, and mechanisms of antioxidant protection[END_REF]. The oxidative stress induced by NPs can be caused by the active redox cycle on the surface of metal-based NPs, and the interaction of NPs with the cell. Reduced particle size causes structural defects and alteration of electronic properties on the particle surface, forming reactive groups on the NP surface [START_REF] Gonzalez | Genotoxicity of engineered nanomaterials: A critical review[END_REF][START_REF] Fu | Mechanisms of nanotoxicity: Generation of reactive oxygen species[END_REF]. Within these reactive regions, electron donor or acceptor active sites interact with molecular O2 to form O2• - which can generate additional ROS via Fenton-type reactions [START_REF] Fu | Mechanisms of nanotoxicity: Generation of reactive oxygen species[END_REF]. ROS can be produced by photocatalytic NPs such as TiO2. As electrons migrate from the valence band to the conduction band, during their stimulation with light, they can react with molecular O2 and H2O to form hydroxyl radicals (see Figure 3) [START_REF] Hoffmann | Environmental Applications of Semiconductor Photocatalysis[END_REF].

Besides being oxidative by themselves, NPs can induce cellular ROS production by interacting with cells, affecting NADPH-like enzyme systems and mitochondrial mechanism. In particular, they can affect the cellular redox systems of alveolar macrophages and neutrophils. Professional phagocytic cells including neutrophils and alveolar macrophages of the immune system induce substantial ROS upon internalization of NPs via the NADPH oxidase enzyme system [START_REF] Manke | Mechanisms of nanoparticle-induced oxidative stress and toxicity[END_REF].

Pro-inflammation

The immune system works with blood and lymphatic circulatory systems that interact and communicate via chemical conductors to regulate an immune response against a particular stimulus. T and B lymphocytes, natural killer cells and phagocytic cells are the main cells of the immune system. Macrophages are essential parts of the immune system that function as cleansers or antigen presenting cells. Phagocytic cells are found in many tissues. Monocytes and neutrophils circulate throughout the body through blood, therefore they can detect pathogens, remove them and synthesize and release the cytokines necessary for the immune system [START_REF] Dwivedi | Impact of nanoparticles on the immune system[END_REF].

Many cytokines, such as interleukin 1 (IL-1), IL-6, IL-8 and tumor necrosis factor alpha TNF-α, activate inflammatory cells during inflammatory responses. While TNFα is stimulated by endothelial cells, IL-8 is a chemokine that stimulates the activation of neutrophils. Many cytokines act together to initiate and regulate the inflammation process [START_REF] Elsabahy | Cytokines as biomarkers of nanoparticle immunotoxicity[END_REF].

When the NPs enter the systemic circulation, the immune system can detect many components of the NPs (e.g. shell, core, surface-decorating moieties) as foreign entities which stimulate the immune system through a complex process [START_REF] Moyano | Modulation of Immune Response Using Engineered Nanoparticle Surfaces[END_REF].

Role of physicochemical features in nanoparticle toxicity

Cellular internalization of nanoparticles depends on their physicochemical properties, especially nanoparticle size, surface area, shape, surface charge. Therefore, it is clear that nanoparticle physicochemical properties determine their toxicity. The toxicityinducing abilities of certain physicochemical properties are discussed below.

Effect of size and surface area

Particle size and surface area are important in the interaction of nanoparticles with biological systems. When the particle size decreases and the surface area increases, the nanomaterial surface becomes more reactive in the biological environment.

In general, the toxicity of the NPs can be attributed to the ability to enter biological systems, to pass through the cell membrane more easily, and then to change the structure of various biomolecules, thereby interfering with critical biological functions [START_REF] Gatoo | Physicochemical properties of nanomaterials: implication in associated toxic manifestations[END_REF]. Therefore, in nanoparticle toxicity, their cellular internalization is the major step.

In that case nanoparticle size has an important impact on its cellular internalization [START_REF] Powers | Characterization of the size, shape, and state of dispersion of nanoparticles for toxicological studies[END_REF].

The fact is that small NPs can pass through the cell membrane more easily than large particles. In studies supporting this idea, a size-dependent internalization in different cell lines was observed for Au [START_REF] Arnida | Cellular uptake and toxicity of gold nanoparticles in prostate cancer cells: a comparative study of rods and spheres[END_REF][START_REF] Chitra | Induction of oxidative stress by bisphenol A in the epididymal sperm of rats[END_REF][START_REF] Huang | Size-dependent localization and penetration of ultrasmall gold nanoparticles in cancer cells, multicellular spheroids, and tumors in vivo[END_REF][START_REF] Elbakry | Layer-by-Layer Coated Gold Nanoparticles: Size-Dependent Delivery of DNA into Cells[END_REF], mesoporous silica [START_REF] Lu | Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles[END_REF], polystyrene [START_REF] Varela | Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells[END_REF] and iron oxide NPs [START_REF] Huang | Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidonecoated iron oxide nanoparticles[END_REF]. On the other hand, some studies reported that larger nanoparticles exhibited higher cellular uptake or no any size releated internalization in various cell lines [START_REF] Huang | Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidonecoated iron oxide nanoparticles[END_REF][START_REF] Sun | Uniform TiO2 nanoparticles induce apoptosis in epithelial cell lines in a size-dependent manner[END_REF].

The maximum intracellular uptake size range was observed as 30-50 nm [START_REF] Huang | Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidonecoated iron oxide nanoparticles[END_REF]. The internalization of <200 nm size nanoparticles is mediated by clathrine-dependent endocytosis, however bigger than 200 nm size nanoparticle internalization is found evident in caveolae-mediated endocytosis [START_REF] Rejman | Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis[END_REF]. However, it should be noted that this process is highly dependent on the cell type. Because internalization requires compatible formation of NP-receptor interactions. Small NPs have less ligandreceptor interactions than large NPs; therefore, many small NPs are expected to interact with nearby receptors simultaneously, in order to triger the membrane wrapping. A study showed that thermodynamically 50-60 nm NP has the capacity to have enough receptors to successfully trigger internalization [START_REF] Shang | Engineered nanoparticles interacting with cells: Size matters[END_REF].

Overall, the nanoparticle internalization highly depends on the cell type, and nanoparticle characteristics. Also most of the uptake studies involving living cells are carried out in a cell culture medium supplemented with protein mixtures of different compositions, it is not surprising that in some cases the apparently "identical" studies give contradictory results.

Following the internalization, smaller sized nanoparticles showed greater cytotoxicity than larger ones in several studies but there are also conflicting results (Table 3). No cytotoxicity observed for either size [START_REF] Schübbe | Sizedependent localization and quantitative evaluation of the intracellular migration of silica nanoparticles in Caco-2 cells[END_REF] The critical size of many NPs is defined as 30 nm for toxicity. Surface reactions increase the likelihood of toxicity as nanoparticle surface energy increases at sizes lower than 30 nm. The results of one study showed that Ag NPs with smaller dimensions (4 nm) stimulated the production of ROS in human myeloid leukaemia cell line (U937) and caused more severe pulmonary inflammation than Ag

NPs with larger dimensions (20 nm and 70 nm) [START_REF] Park | Size dependent macrophage responses and toxicological effects of Ag nanoparticles[END_REF]. Another study showed that 1.4 nm gold NPs caused more severe toxicity in connective tissue, fibroblasts, epithelial cells, macrophages and melanoma cells than the smaller (0.8 nm and 1.2 nm) or larger (1.8 nm and 15 nm) particles [START_REF] Pan | Jahnen-Dechent, Size-Dependent Cytotoxicity of Gold Nanoparticles[END_REF].

Overall, there is no definitive conclusion about the effect of particle size on toxicity.

Toxic effect of TiO2 NPs may be related to their structural characteristics as suggested by Zhang et al. [START_REF] Zhang | Cytotoxicity of different sized TiO2nanoparticles in mouse macrophages[END_REF].

Effect of particle shape

Shape-related toxicity is mainly associated with the membrane's ability to wrap particles during endocytosis [START_REF] Verma | Effect of surface properties on nanoparticle-cell interactions[END_REF]. For example, studies showed that the cellular uptake of spherical gold nanoparticles by murine macrophages was higher than that of rod gold nanoparticles [START_REF] Chithrani | Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells[END_REF][START_REF] Champion | Role of target geometry in phagocytosis[END_REF]. The reason might be associated with membrane wrapping time which is longer for the larger rod-shaped nanoparticles due to the varying number of receptor localization [START_REF] Behzadi | Cellular uptake of nanoparticles: Journey inside the cell[END_REF].

On the other hand, it was observed that the fiber-shaped TiO2 NPs were more cytotoxic than the spherical-shaped ones [START_REF] Hsiao | Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells[END_REF]. Due to the unusual aerodynamics of fiber materials such as asbestos fibers or carbon nanotubes, long fibers penetrate and accumulate beyond the ciliated airways. Short fibers could be completely covered by cell membrane and can be cleared with phagocytosis. Longer fibers, on the other hand, cannot be enclosed. Therefore it leads to 'frustrated phagocytosis' which can cause chronic inflammation, oxidative stress, direct cell damage, and chromosomal abnormalities [START_REF] Donaldson | The inhalation toxicology of p-aramid fibrils[END_REF] .

In a study, among the rods (39×18 nm), stars (215 nm) and spheres (6 nm) gold nanoparticles, spherical caused less cytotoxicity than others [START_REF] Steckiewicz | Impact of gold nanoparticles shape on their cytotoxicity against human osteoblast and osteosarcoma in in vitro model. Evaluation of the safety of use and anti-cancer potential[END_REF]. However, it should be considered that the examined nanoparticles had a wide variety of dimensions.

Effect of surface charge

The surface charge of nanoparticles, which is of great importance in interacting with biological systems, plays an important role in toxicity.

Positively charged particles show significant cellular uptake compared to negatively charged and neutral NPs due to their increased opsonization by plasma proteins [START_REF] Goodman | Toxicity of gold nanoparticles functionalized with cationic and anionic side chains[END_REF].

Considering that the surface charge of the cell membrane is negative, positively charged particles may pass through the membrane easily. However, studies in our laboratory in the previous years showed the positively charged nanoparticles were more uptaken by cells than neutral or negatively charged nanoparticles [START_REF] Forest | Electrostatic interactions favor the binding of positive nanoparticles on cells: A reductive theory[END_REF][START_REF] Forest | Preferential binding of positive nanoparticles on cell membranes is due to electrostatic interactions: A too simplistic explanation that does not take into account the nanoparticle protein corona[END_REF].

Therefore, nanoparticles with different surface charge may cause different toxic manifestations. In a study using molecular simulation [START_REF] Li | Thermodynamics of charged nanoparticle adsorption on chargeneutral membranes: A simulation study[END_REF], the electrostatic attraction effect between cationic nanoparticles and the cell membrane was investigated. Charged nanoparticles showed a more favorable thermo-dynamic interaction than their uncharged counterparts. In another molecular dynamic simulation study, the interaction of cationic, hydrophobic and anionic Au nanoparticles with the cell membrane was investigated [START_REF] Lin | Penetration of lipid membranes by gold nanoparticles: Insights into cellular uptake, cytotoxicity, and their relationship[END_REF]. They showed that the internalization increased with increasing charge density. They also reported membrane disruptions with higher nanoparticle charge density.

Effect of agglomeration

The aggregation status of NPs also influences their toxicity. Generally, the aggregation status of nanoparticles depends on size, surface charge, and chemical composition among others.

Conclusion

Once nanoparticle are uptaken into human body by inhalation, ingestion or dermal route, they can circulate in the bloodstream. Nanoparticles can interact with cells, tissues and organs, and depending on their physico-chemical properties they can internalize through the cell membrane, interacting with macromolecules and altering their structure and function [START_REF] Park | Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro[END_REF][START_REF] Wang | Oxidative stress contributes to silica nanoparticle-induced cytotoxicity in human embryonic kidney cells[END_REF][START_REF] Lin | In vitro toxicity of silica nanoparticles in human lung cancer cells[END_REF][START_REF] Waters | Macrophage responses to silica nanoparticles are highly conserved across particle sizes[END_REF]. The typical changes in cell functions and structures play an important role in the pathogenesis of many respiratory diseases such as chronic bronchitis, asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), and such natural and engineered particles, fibrous materials and inhaled chemicals can be caused by exposure to daily lung inhalants (Figure 11) [START_REF] Geiser | Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells[END_REF][START_REF] Osman | Effect of nanoparticles on human cells from healthy individuals and patients with respiratory diseases[END_REF].

Therefore, the NP toxicity strongly depends on their physical and chemical properties, such as the size, shape, surface charge, chemical composition, etc. It also should be noted that, the physicochemical properties of nanoparticles has an impact on protein corona formation since it is known that the protein corona has an impact on the internalization of NPs by cells and thus potentially on the cytotoxicity.

4.

In vivo and in vitro titanium dioxide toxicity

In vivo studies

In vivo studies have shown that after inhalation or after instillation of TiO2 NPs, undesirable effects might occur at high doses, generally higher than 0.5 mg/kg in rats and mice [START_REF] Baisch | Equivalent titanium dioxide nanoparticle deposition by intratracheal instillation and whole body inhalation: The effect of dose rate on acute respiratory tract inflammation[END_REF][START_REF] Wang | Lung injury induced by TiO2 nanoparticles depends on their structural features: Size, shape, crystal phases, and surface coating[END_REF][START_REF] Silva | Biological Response to Nano-Scale Titanium Dioxide (TiO 2 ): Role of Particle Dose, Shape, and Retention[END_REF].

In particular, a rapid inflammatory response characterized by a significant increase in the number of phagocytes in the broncho-alveolar lavage fluids as well as by an increase in pro-inflammatory cytokines production, oxidative stress cytotoxicity, and lung tissue structural damage have been demonstrated in several studies [START_REF] Baisch | Equivalent titanium dioxide nanoparticle deposition by intratracheal instillation and whole body inhalation: The effect of dose rate on acute respiratory tract inflammation[END_REF][START_REF] Wang | Lung injury induced by TiO2 nanoparticles depends on their structural features: Size, shape, crystal phases, and surface coating[END_REF]. After chronic exposure to large doses of TiO2, genotoxic and carcinogenic effects have been demonstrated [START_REF] Wang | Lung injury induced by TiO2 nanoparticles depends on their structural features: Size, shape, crystal phases, and surface coating[END_REF][START_REF] Shi | Titanium dioxide nanoparticles: A review of current toxicological data[END_REF]. Some studies are categorized depending on their exposure duration.

Acute studies

According to Noël et al. [START_REF] Noël | Rat pulmonary responses to inhaled nano-TiO2: Effect of primary particle size and agglomeration state[END_REF], bronchoalveolar lavage fluid indicated large aerosols (>100 nm) caused an acute inflammatory response, as shown by the significantly enhanced number of neutrophils, while small aerosols (5, 10-30 or 50 nm) produced significant oxidative stress damages and cytotoxicity in the lungs of male rats exposed to TiO2 NPs at 20 mg/m 3 for 6 h. In both aerosols, the 10-30 nm TiO2 NPs induced the significant pro-inflammatory effects in comparison to the controls. Noël et al.

(2012) also showed that an acute (6 hours) inhalation of 5 nm TiO2 (anatase) with two distinct agglomeration states, smaller or larger than 100 nm, induced mild pulmonary effects at 7 mg/m 3 [START_REF] Noël | Effects of inhaled nano-TiO2 aerosols showing two distinct agglomeration states on rat lungs[END_REF].

Sub-acute studies

Oberdorster et al. [START_REF] Oberdörster | Acute Pulmonary Effects of Ultrafine Particles in Rats and Mice[END_REF] found a marked increase in pulmonary inflammation due to 20 nm TiO2 NPs in rats and mice, expressed by the increase of total protein in BALF, and LDH activity. Li et al. [128] studied the effects of intratracheally instilled 3-nmsized TiO2 NPs once a week for 28 days in mice after a total dose of 13.2 mg/kg body weight. Lung damage and change in the permeability of the alveolar-capillary barrier were observed. A 12-week inhalation experiment in rats resulted in a similar mass deposition of the two particle types in the lower respiratory tract. The ultrafine particles elicited a persistently high inflammatory reaction in the lungs of the animals compared to the larger-sized particles [START_REF] Oberdorster | Correlation between Particle Size[END_REF].

Sub-chronic studies Warheit et al. [START_REF] Warheit | Pulmonary Instillation Studies with Nanoscale TiO2 Rods and Dots in Rats: Toxicity Is not Dependent upon Particle Size and Surface Area[END_REF] compared several types of TiO2 fine particles and nanoparticles with different sizes, surface areas and crystal structures by intratracheal instillation of TiO2 NPs with the size of 25 or 100 nm and dose of 1 and 5 mg/kg body weight for 24 h, 1 week and 3 months into rats. In the comparison between these particles, even though the surface areas differed as large as 30-fold, the lung inflammation observed was almost similar for the two particle sizes. Therefore, they concluded that toxicity of TiO2 particles is not dependent on the particle size or surface area through lung instillation. Moreover, the same research group suggested that toxicity depends upon particle surface properties instead of surface areas. When mice were intratracheally instilled with single fixed doses (5, 50 and 500 μg) of TiO2 fine as well as rutile nanoparticles, Roursgaard et al. [START_REF] Roursgaard | Acute and subchronic airway inflammation after intratracheal instillation of quartz and titanium dioxide agglomerates in mice[END_REF] found an increase in interleukin-6 and total protein in BALF as well as airway inflammation at the highest doses in the acute phase by both fine as well as nanoparticles. Another sub-chronic inhalation study (13 weeks) showed that clearance was strongly impacted in rats and mice, more particularly at the highest concentration in aerosol (10 mg/m 3 ), but not in the hamster. More pronounced inflammatory effects were observed in the rat than in the mouse, with the consequence of greater tissue damage [START_REF] Bermudez | Long-Term Pulmonary Responses of Three Laboratory Rodent Species to Subchronic Inhalation of Pigmentary Titanium Dioxide Particles[END_REF]. Nanoparticles, compared to fine particles made of the same chemical substance, are less efficiently cleared. This participates to the different potential of toxicity of nanoparticles compared to fine particles of the same chemical composition. It might be related to the fact that most of inhaled TiO2 particles are eliminated from the lungs in a size dependent manner [START_REF] Oberdorster | Correlation between Particle Size, In Vivo Particle Persistence, and Lung Injury[END_REF].

Chronic studies

In the longer term, tissue damage may appear (fibrosis for example), due to the slow clearance of NPs [START_REF] Shi | Titanium dioxide nanoparticles: A review of current toxicological data[END_REF][START_REF] Bermudez | Pulmonary Responses of Mice, Rats, and Hamsters to Subchronic Inhalation of Ultrafine Titanium Dioxide Particles[END_REF]. Indeed, the presence of tissue damage is even more important for chronic or subchronic exposures, due to persistent inflammation. The exposure of mice to various shapes of anatase TiO2 (nanospheres, short belts of 1-5 μm, and long nanobelts of 4-12 μm) resulted in the lung deposition of 135 μg for the animals exposed to both nanospheres and long nanobelts. At 112 days after exposure, the lung burden was significantly lower in nanosphereexposed mice than in nanobelt-exposed mice [START_REF] Ding | Airborne engineered nanomaterials in the workplace-a review of release and worker exposure during nanomaterial production and handling processes[END_REF].

TiO2 has been classified by the IARC as a possible carcinogen for humans (Group 2B) by inhalation due to sufficient evidence from chronic animal studies in 2010 [START_REF] Iarc | [END_REF].

This classification is not specific to the nano forms of TiO2.

NIOSH also declined inhaled ultrafine TiO2 is a potential occupational carcinogen [START_REF]Occupational Exposure to Titanium Dioxide[END_REF][START_REF] Świdwińska-Gajewska | Titanium dioxide nanoparticles: Occupational exposure limits[END_REF]. This statement needs to be reviewed. On the other hand, what is important to clarify is that the NIOSH proposes exposure limit values which were established from (and therefore are strictly applicable only for) P25. NIOSH recommends airborne exposure limits of 2.4 mg/m 3 for fine TiO2 and 0. Several studies reported a greater inflammatory response against TiO2 NPs with comparison to its fine counterparts in acute or chronic exposure [START_REF] Ferin | Pulmonary retention of ultrafine and fine particles in rats[END_REF][START_REF] Oberdorster | Role of the alveolar macrophage in lung injury: Studies with ultrafine particles[END_REF]. Some studies have shown no correlation between nanoparticle agglomeration and pulmonary inflammation [START_REF] Gosens | Impact of agglomeration state of nano-and submicron sized gold particles on pulmonary inflammation[END_REF][START_REF] Schmid | Surface area is the biologically most effective dose metric for acute nanoparticle toxicity in the lung[END_REF]. Size-related impact studies revealed that smaller particles have a greater impact on inflammation than bigger nanoparticles [START_REF] Kobayashi | Comparative pulmonary toxicity study of nano-TiO2 particles of different sizes and agglomerations in rats: Different short-and long-term post-instillation results[END_REF][START_REF] Oberdörster | Correlation between particle size, in vivo particle persistence, and lung injury[END_REF].

Crystal structure (anatase or rutile) may influence TiO2 NPs reactivity in in vivo as shown by in vitro studies [START_REF] Uboldi | Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts[END_REF]. But until now there is no in vivo studies that would provide sufficient evidence to demonstrate which crystallinity could be the most toxic and to what extent. In order to evaluate the effect of surface chemistry on biocompatibility, there are a few studies investigating APTES modified NPs in vivo toxicity. To assess lung inflammation, C57BL/6 mice were administered bare or amine-functionalized silica NPs via intra-tracheal instillation. Two doses (0.1 and 0.5 mg NPs/mouse) were tested using the in vivo model. At the higher dose used, bare silica NPs elicited a significantly higher inflammatory response, as evidence by increased neutrophils and total protein in bronchoalveolar lavage fluid compared to amine-functionalized NPs. From this study, they conclude that functionalization of nonporous silica NPs with APTES molecules reduces murine lung inflammation and improves the overall biocompatibility of the nanomaterial [START_REF] Morris | Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs[END_REF].

The differences in species, application methods and physicochemical properties of the applied nanoparticles lead to contradictory results. There is still limited number of in vivo studies regarding TiO2 toxicity to draw a conclusion, additional researches seem highly necessary.

In vitro studies

It has been shown in in vitro studies that TiO2 nanoparticles can induce a proinflammatory response, oxidative stress, cytotoxicity, and genotoxicity on lung cells [START_REF] Shi | Titanium dioxide nanoparticles: A review of current toxicological data[END_REF][START_REF] Lanone | Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines[END_REF].

The size impact on toxicity was investigated in number of studies. Generally smaller TiO2 nanoparticles caused cytotoxicity [START_REF] Gandamalla | Nano titanium exposure induces dose-and size-dependent cytotoxicity on human epithelial lung and colon cells[END_REF][START_REF] Andersson | Polymorph-and Size-Dependent Uptake and Toxicity of TiO2 Nanoparticles in Living Lung Epithelial Cells[END_REF][START_REF] Ma | Titanium dioxide nanoparticles induce size-dependent cytotoxicity and genomic DNA hypomethylation in human respiratory cells[END_REF][START_REF] Hussain | Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: Role of particle surface area and internalized amount[END_REF][START_REF] Gurr | Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells[END_REF], however other studies showed contradictory results. For example, an interesting finding was that 25 nm anatase particles induced more cell death, higher LDH release, and ROS activities than 5 and 100 nm anatase particles [START_REF] Zhang | Cytotoxicity of different sized TiO2nanoparticles in mouse macrophages[END_REF]. Similar to this results, Braydich-Stolle et al. (2009) showed that 50 nm TiO2 NPs had a higher toxicity than TiO2 NPs of 6.3, 10, and 100 nm [START_REF] Braydich-Stolle | Crystal structure mediates mode of cell death in TiO2 nanotoxicity[END_REF]. Therefore, there is no unanimous conclusion about the effect of particles size on toxicity.

The effects of TiO2 NP phase on the toxicity were investigated on A549 cells among amorphous, anatase and anatase/rutile phases (18-53 nm) [START_REF] Hsiao | Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells[END_REF]. The toxicity order of NPs was amorphous >anatase >anatase-rutile TiO2. The amorphous and anatase NPs induced greater IL-8 pro-inflammatory response than anatase-rutile phase P25.

Although the anatase and P25 NPs had a similar surface area, size, and shape, they exhibited different toxicity responses, confirming that the phase of a TiO2 NP affects its cytotoxicity. Similarly, Sayes et al. [START_REF] Sayes | Correlating Nanoscale Titania Structure with Toxicity: A Cytotoxicity and Inflammatory Response Study with Human Dermal Fibroblasts and Human Lung Epithelial Cells[END_REF] also found that the anatase TiO2 was 100 times more toxic than an equivalent sample of rutile TiO2.

In another study [START_REF] Ekstrand-Hammarström | Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B[END_REF], custom-made 9 nm rutile (R9, 270 m 2 /g), 5 nm rutile (R5, 11 m 2 /g) and 14 nm anatase (A14, 300 m 2 /g) NPs and commercial 60 nm anatase (A60, 40 m 2 /g) and 21 nm P25 (50 m 2 /g) were used to compare their cytotoxic potential on 16HBE cells, and human lung epithelial cell lines (A549 and BEAS-2B). None of the TiO2 NPs were found to have significant effect on cell viability in 16HBE, BEAS-2B and A549 cells. Whereas, a significant concentration-dependent increase in IL-8

release in all three-cell types was observed for P25, and it was more important compared to other particles. In A549 cells, P25 caused concentration-dependent ROS production whereas A14 particles did not cause significant ROS production. A60 and R9 also induced the ROS response. The researchers emphasized that the toxicity of NPs varies depending on the cell type and the physicochemical properties of NPs [START_REF] Ekstrand-Hammarström | Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B[END_REF]. Discussing the phase effect alone on toxicity is very difficult due to the different NP sizes used. However, this study shows that TiO2 NPs are able to induce ROS and IL-8 production without causing cell viability loss. Similar to this result, a study showed that TiO2 NPs caused a toxicity by triggering other pathways than decrease of cell viability. TiO2 (200 nm, 0-800 µg/mL) were exposed to alveolar macrophage-like THP-1, A549 and Human Pulmonary Microvascular Endothelial (HPMEC-ST1.6R) cells. Concentration-dependent ROS formation was observed in all cell lines at a 24hour exposure. However, the increase in ROS in A549 and HPMEC-ST1.6R cell lines was more pronounced at 4-hour exposures [START_REF] Hanot-Roy | Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro[END_REF] There are also studies showing that TiO2 toxicity depends on concentration and exposure time [START_REF] Ekstrand-Hammarström | Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B[END_REF]. A549 cell viability decreased in a concentration dependent manner when cells were exposed to 0.75-75 mg/L TiO2 NPs (<150 nm, anatase/rutile 80/20) for 24 hours [START_REF] Rosário | Unravelling the Potential Cytotoxic Effects of Metal Oxide Nanoparticles and Metal(Loid) Mixtures on A549 Human Cell Line[END_REF]. In a recent study published in 2020 is reported that 25, 50, 100 and 200 μg/mL TiO2 inhibited A549 cell proliferation depending on time and concentration [START_REF] Wang | Cytotoxicity, DNA damage, and apoptosis induced by titanium dioxide nanoparticles in human non-small cell lung cancer A549 cells[END_REF]. In subsequent studies, where only 48 hours of exposure were applied, DNA damage increased depending on the concentration. The mRNA levels of caspase-3 and caspase-9 genes increased significantly only in cells treated with high doses (100 and 200 µg/mL) of TiO2 NPs [START_REF] Wang | Cytotoxicity, DNA damage, and apoptosis induced by titanium dioxide nanoparticles in human non-small cell lung cancer A549 cells[END_REF]. These studies have shown the potential to cause a negative biological response in two cell lines related to the respiratory system.

However, there are also contradictory findings. In a study, the toxicity of TiO2 NPs (Evonik P25) on A549 was investigated. TiO2 NPs did not cause statistically significant cytotoxic effect even up to their highest tested concentration of 250 µg/mL as determined by WST-1 (cell metabolism) and LDH assay [START_REF] Rosário | Unravelling the Potential Cytotoxic Effects of Metal Oxide Nanoparticles and Metal(Loid) Mixtures on A549 Human Cell Line[END_REF]. In another study THP-1 differentiated macrophages were exposed for 24 hours to TiO2 (spherical/ellipsoidal 10 m 2 /g) at an average size of 190 nm at 1, 10, 25, 50 and 100 µg/mL concentrations.

Even at the highest concentrations, TiO2 NPs showed no significant reduction in THP-1 cell viability as determined by MTS method. TEM analyzes confirmed the cell internalization of NPs. Regarding the cytokine production, no changes were observed in the IL-1b, IL-8 and TNF-α production compared to the control group for all tested concentrations. In addition, intracellular ROS formation was not detected [START_REF] Brzicova | Molecular responses in THP-1 macrophage-like cells exposed to diverse nanoparticles[END_REF].

Differentiated THP-1 cells were exposed to 30-40 nm TiO2 at 0.1, 1, 10, 30 and 100 µg/mL concentrations for 24 h. MTT assay showed that TiO2 NPs exposure did not change THP-1 viability compared to the control group. Moreover, no IL-1b and TNFα cytokine expressions were detected [START_REF] Poon | Nano-sized zinc oxide and silver, but not titanium dioxide, induce innate and adaptive immunity and antiviral response in differentiated THP-1 cells[END_REF].

Overall, like in in vivo studies, it is hard to draw a conclusion on TiO2 nanoparticle in vitro toxicity due to the number of varieties in studies such as cell type, nanoparticle exposure time, concentration, NP preparation conditions (handling, sonication, suspension preparation) and variable NPs physicochemical properties.

Conclusion and Objectives of the Work

Due to the widespread use of TiO2 nanoparticles in the industry, concerns about their potential harmful effects on human health have increased. After exposure to TiO2 nanoparticles, they reach the target organs through inhalation or other routes described, where they can exert their toxic effects. TiO2 nanoparticle cytotoxicity can be summarized as follows;

 As a semi-conductor material, TiO2 nanoparticles can induce ROS production following the induction of electron-hole pairs under light.

 Cell membrane damage and cell membrane lipid peroxidation might occur due to the cell-nanoparticle interaction.

 Nanoparticles can interact with cellular organelles and disrupt their functions.

 TiO2 nanoparticles depending on their properties can lead to ROS production and induce up-regulation of stress releated genes, as well as inflammation releated genes [START_REF] Xia | Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm[END_REF].

Their biological effects are related to some basic physicochemical properties, namely particle size, charge, crystallinity, shape and agglomeration state. However, understanding the effect of these properties on TiO2 NPs toxicity is rather limited and although there is growing scientific evidence about TiO2 NPs toxicological properties, controversial data are still reported.

In the light of the information described in this bibliography part, the aim of this thesis was to evaluate the toxic effects of five types of TiO2 NPs with different and wellcontrolled physicochemical properties on three in vitro human lung cell systems:

A549 cell line, macrophages differentiated from THP-1 cells and coculture of A549 Étant donné que les macrophages THP-1 sont utilisés comme cellules cibles dans la toxicité des NP de TiO2 dans cette thèse, ce chapitre présente les caractéristiques générales des cellules THP-1 et les différents protocoles utilisés pour l'optimisation et la validation de leur différenciation en macrophages, et les résultats de ces protocoles.

A l'issue des études réalisées dans ce chapitre, les conditions les plus adaptées à la différenciation THP-1 ont été déterminées. Les résultats de cette section sont les résultats fondamentaux des conditions optimales de différenciation cellulaire requises pour effectuer des études de toxicité.

The THP-1 Cell Line and the Identification of Macrophage Differentiation in PMA-stimulated THP-1 Cells

The THP-1 cell line which is a human monocytic leukaemia cell line was initiated in 1980 by Tsuchiya et al. [START_REF] Tsuchiya | Establishment and characterization of a human acute monocytic leukemia cell line (THP-1)[END_REF] from the blood of a patient with acute monocytic leukaemia. THP- The cell adhesion, the first indicator of macrophage differentiation, was detected by microscope observation in all of the applied PMA concentrations.

However, decreases in cell viability were observed when the cells were enzymatically separated from the culture flask and collected for flow cytometry analysis, as reported in Table 5. Due to this low cell viability, it was not possible to get a fully efficient result from flow cytometry regarding increasing cell size and granularity. The cells were then collected by scrabing rather than enzymatic separation. However, similar results were obtained although the experiments were repeated at least twice. Afterwards, it has been investigated whether this low cell viability was due to the DMSO in which PMA stock solution was prepared. Therefore, DMSO content of 50, 100, 150, 200 ng/mL PMA concentrations in culture media were calculated as 0.0005%, 0.005%, 0.01%, 0.015% and 0.02%, respectively. These concentrations of DMSO were prepared in culture medium and applied to the cells for 72 hours. Cell viability was determined by the trypan blue method as shown in Figure 12. As the DMSO in the applied concentrations did not cause a decrease in cell viability, it was concluded that the low cell viability obtained from flow cytometry results was not due to the DMSO content in PMA working solutions.

Although there are studies that show successful results with flow cytometry analysis, some researchers [START_REF] Xia | Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: The NIEHS Nano go consortium[END_REF] reported an exessive cell death occurring upon scraping the differentiated THP-1 cells, like our observations. They also indicated that during the activation process PMA induced strong responses in THP-1 cells, resulting in severe clumping of the THP-1 cells [START_REF] Xia | Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: The NIEHS Nano go consortium[END_REF].

In consequence of the results described above, it was determined that investigation of size and granularity of cells by flow cytometry was not an ideal method because it requires the cell collection enzymatically or by scrabbing technique. It was also suspected that 72 hours of PMA exposure with 5 days of resting phase was not appropriate in our study although this length of exposure to PMA is commonly used in the literature [START_REF] Daigneault | The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages[END_REF][START_REF] Lund | The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus[END_REF][START_REF] Hayman | The effects of exogenous lipid on THP-1 cells: An in vitro model of airway aspiration?[END_REF][START_REF] Pinto | Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells[END_REF]. However, there is currently no standard protocol for differentiating THP-1 monocytes into macrophages using PMA. For this reason, to find the best conditions in our study further experiments were carried out by applying 24 and 48 hours PMA exposure without resting phase.

Protocol 2 and 3

5 x 10 6 , 7.5 x 10 6 , 10 x 10 6 cells were treated with 0, 5, 30, 50, 100 ng/mL PMA for 24 and 48 h in T25 cm 2 flask.

Cell morphology and adhesion were observed using optical microscopy and pictures were captured at 20 x magnification. The microscopic images of THP-1 after treatment with PMA are shown in Figure 13. It is observed that the adhesion rate of THP-1 cells varies depending on the PMA concentration and exposure time (Figure 13). 48 hours incubation of 5 ng/ml PMA showed less cell adhesion compared to other concentrations and incubation time.

Thus, the percentage of cell adhesion was determined by counting unattached THP-1 cells and substracting them from the initial/total cell amount. Results were expressed as percentage of cell adhesion in Figure 14. As shown in Figure 13, 24 hours PMA treatment showed slightly more cell adherence than 48 hours treatment for all tested PMA concentrations. Cell attachment was found unstable at 5 ng/mL PMA concentration for both treatment time showing that 5 ng/mL PMA was not enough to differentiate the THP-1 cells as confirmed by the microscope images (Figure 13). Similar to our results, Park et al. [START_REF] Park | Optimized THP-1 differentiation is required for the detection of responses to weak stimuli[END_REF] showed the increased cell adhesion at 48 h after 10-100 ng/mL PMA treatement (10 6 cells/60 mm dish qsp. 1.2 x 10 6 cells/25cm 2 flask). 30 ng/mL PMA for 24 hours treatment to 7.5 x 10 6 cells showed the highest THP-1 cell adhesion (96%). When these results and microscope images were evaluated together, PMA incubation time was selected as 24 hours.

The appropriate cell density was then determined by measuring the cell metabolic activity after PMA treatment by MTT method.

Corresponding to the cell density used in T25 cm 2 flask in 96-well plate 6.5 x 10 4 cells, 9 x 10 4 cells, and 13 x 10 4 cells were seeded and treated with 0, 30, 50 and 100 ng/mL of PMA for only 24 hours. After, cell surface was washed with DPBS and 200 µL MTT solution (5 mg/mL in DMEM) were added in each well. Plates were incubated for 3 hours. After, MTT was removed and 200 µL DMSO were added in each well. Plates were placed on orbital shaker for 20 min. Optical density was read on a microplate reader at 570 nm (Multiskan RC; Thermolabsystems, Helsinki, Finland). An increase in number of living cells results in an increase in the total metabolic activity. This increase directly correlates to the amount of purple formazan crystals formed, as monitored by the absorbance. The condition that gives an absorbance between 0.8-1.0 was preferred. With the evaluation of all results obtained from these protocols, optimal incubation time and cell density were determined. The absorbance range for all cell densities were found between 0.68-0.90 O.D. units.

7.5 x 10 6 cells showed higher absorbance of all tested concentrations. Among the tested concentrations 30 ng/mL showed higher absorbance thus high metabolic activity at 7.5 x 10 6 cell density.

When all the results above were evaluated, it was decided to apply a 24-hour PMA exposure to 7.5 x 10 6 cells in T25 cm 2 flask (corresponding to 10 5 cells per well in 96-well plate).

Optimal PMA concentration determination by immunocytochemistry staining (Protocol 4)

CD11b expression was determined by the ICC method, since cells do not need to be separated from the culture flask surface in this method. ICC analysis of CD11b expression in THP-1 treated with 0, 30, 50, 100 ng/mL PMA for 24 h is shown in Figure 16. As observed by ICC analysis, the stimulation with PMA induced cell adhesion and the expression of CD11b (macrophage marker) in differentiated THP-1 was detected. This is in good agreement with other studies [START_REF] Starr | The phorbol 12myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium[END_REF][START_REF] Schwende | Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25dihydroxyvitamin D3[END_REF] which showed the increased CD11b surface levels by flow cytometry and ICC. Starr et al. [START_REF] Starr | The phorbol 12myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium[END_REF] detected the increased levels of CD11b in 2 and 3 days 20 ng/mL of PMA treated cells. Among tested concentrations, 30 and 50 ng/mL PMA showed more pronounced CD11b expression, supporting their differentiation into macrophages.

When all the results were evaluated, and as the studies recommending that PMA and THP-1 differentiation should be optimized to reduce the possibility of masking the effect caused by excessive PMA use [START_REF] Lund | The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus[END_REF][START_REF] Park | Optimized THP-1 differentiation is required for the detection of responses to weak stimuli[END_REF], 30 ng/mL was chosen as the optimal concentration, since 30 ng/mL PMA treated cells showed higher cell adhesion and metabolic activity than 50 ng/mL PMA treated cells.

Conclusion

We determined the optimal conditions for THP-1 cell differentiation into macrophages: 10 5 cells (per well in 96-well plate) exposed to 30 ng/mL PMA for

hours.

We validated the differentiation of THP-1 cells into macrophages by the determination of the expression of CD11b, one of the main surface marker of macrophages.

Nous avons déterminé les conditions optimales pour la différenciation des cellules THP-1 en macrophages: 10 5 cellules (par puits, en plaque 96 puits) exposées à 30 ng/mL de PMA pendant 24 heures.

Nous avons validé la différenciation des cellules THP-1 en macrophages par la détermination de l'expression de CD11b, l'un des principaux marqueurs de surface des macrophages.

Complementary Data

We also assessed the toxicity induced by the 5 TiO2 NPs in THP-1 macrophages differentiated from THP-1 monocytes. In parallel, we used further biological assays such as the LDH release and the MTT assay to better understand TiO2 NP toxicity. In order to assess cell membrane integrity after exposure to TiO2 NPs, the release of LDH was measured in the culture medium by using a commercial kit CytoTox-96™

Homogeneous Membrane Integrity Assay (Promega, Charbonnières-les-Bains, France), following the supplier's instructions. The fluorescence was measured at 490 nm using a spectrophotometer (Multiskan RC; Thermolabsystems, Helsinki, Finland). The value of each sample was expressed as a function of the maximum LDH released by the cells (measured after cell lysis), as a percentage of cellular integrity compared to the positive control (lysed cells) as presented in Figure 18.

Coculture

Titanium dioxide NPs did not induce significant LDH release in all cell systems. This is in accordance with previous studies, which demonstrated that P25 TiO2 NPs did not induce LDH release in THP-1 macrophages [START_REF] Xia | Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: The NIEHS Nano go consortium[END_REF], A549 cells, BEAS-2B cells [START_REF] Gea | Shape-engineered titanium dioxide nanoparticles (TiO 2 -NPs): cytotoxicity and genotoxicity in bronchial epithelial cells[END_REF],

However, other studies detected LDH increases in A549 cells especially after exposure to P25 [START_REF] Simon-Deckers | In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes[END_REF] and in THP-1 cells [START_REF] Tilton | Three human cell types respond to multiwalled carbon nanotubes and titanium dioxide nanobelts with cell-specific transcriptomic and proteomic expression patterns[END_REF].

Besides the contradictory results in the literature, our LDH results were not consistent with microscopic observations. The LDH activity was observed to lessen in TiO2 NPs treated cells compared to the control, and this loss of activity was probably due to the interaction between NPs and LDH enzyme. The possible interaction was evaluated in Chapter 5. Therefore, cytotoxicity was assessed by using other methods.

Cell viability-MTT assay

The 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) test is a colorimetric analysis used to evaluate the metabolic activity of cells [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. In this test, MTT (a yellow water-soluble tetrazolium salt) passes through the cell membrane and enters the cell mitochondria (Figure 19). Living cells use NAD(P)H-bound oxidoreductase enzymes to reduce the yellow MTT reagent to dark purple waterinsoluble formazan while this color formation is not observed in dead cells due to their lack of metabolic activity [START_REF] Stockert | Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives[END_REF][START_REF] Bernas | Mitochondrial and nonmitochondrial reduction of MTT: Interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes[END_REF]. Water insoluble formazan crystals formation can then be determined in DMSO by measuring the absorbance with a spectrophotometer at a specific wavelength (usually between 500 and 600 nm).

Coculture

After exposure to TiO2 NPs, cell-type dependent cytotoxicity was observed. Slight decreases in differentiated THP-1 cell viability were detected compared to the control group (unexposed to TiO2 NPs). TiO2 NPs cytotoxicity was more pronounced in A549 cells than in differentiated THP-1. In coculture, lower cell viability was observed in TiO2 NPs treated cells compared to the control group, except for S1. However, no statistically significant difference was found except for S2 at 120 µg/mL and S3 at 30 µg/mL. Similar to our results slight but significant decreases were observed after 10, 50 and 100 µg/mL < 25 nm TiO2 NPs exposure to A549 cells for 24 and 48 h (between 70-90% viability) [START_REF] Srivastava | Nano-titanium dioxide induces genotoxicity and apoptosis in human lung cancer cell line, A549[END_REF]. 250 and 500 μg/mL 25 nm TiO2 NPs exposure also caused significant cell viability decrease in A549 [START_REF] Aueviriyavit | Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells[END_REF]. In another study, cell viability of THP-1 macrophages decreased after exposure to 10-50 µg/mL of anatase 50 nm, 100 nm TiO2 particles [START_REF] Tada-Oikawa | Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells[END_REF]. However, 24 h exposure of 10, 50, 100 µg/mL P25 did not cause the decrease in cell viability of A549 and differentiated THP-1 [START_REF] Lankoff | The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells[END_REF], at the 0-200 µg/mL TiO2 exposure [START_REF] Kansara | TiO 2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells[END_REF] or wide range concentration (0-800 µg/mL) [START_REF] Hanot-Roy | Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro[END_REF]. Even long term exposure (2 months) to 50 µg/mL TiO2 NPs did not affect A549 cell viability [START_REF] Armand | Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents[END_REF]. The reason for these contradictory results in the literature may be due to the fact that possible interactions between NPs and MTT reagent were discussed and taken into account in some studies [START_REF] Srivastava | Nano-titanium dioxide induces genotoxicity and apoptosis in human lung cancer cell line, A549[END_REF][START_REF] Hanot-Roy | Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro[END_REF][START_REF] Armand | Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents[END_REF], while not in others.

There are studies reporting that commonly used cytotocicity tests, LDH, and tetrazolium-based tests (e.g. MTS and MTT) are interfered by a number of different NPs [START_REF] Ong | Widespread nanoparticle-assay interference: Implications for nanotoxicity testing[END_REF][START_REF] Kroll | Current in vitro methods in nanoparticle risk assessment: Limitations and challenges[END_REF]. In our study, as in the LDH experiment, the results obtained in the MTT test were not consistent with microscopic observations. For this reason, preliminary studies were conducted for the bias of MTT and LDH analysis and reported in Chapter 5. Because both methods might not reflect the true results of the TiO2 NPs cytotoxicity, the cell viability was measured by the trypan blue method as reported in the Article 1.

Pro-inflammatory response

TNF-α production after differentiated THP-1 cells were exposed to TiO2 NPs is shown in Figure 21. P25 caused a significantly increased TNF-α production compared to control group, whereas no any significant TNF-α production was observed for other samples.

Similar to our results, Morishige et al. showed no significant TNF- and IL-1β production after exposure to TiO2 NPs (<50 μm, <25 nm, 10 nm) in differentiated THP-1 cells with 0.5 μM PMA for 24 h [START_REF] Morishige | Titanium dioxide induces different levels of IL-1β production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B[END_REF]. Also, 100 nm anatase TiO2 NPs did not induce IL-1β, IL-8, TNF-α in differentiated THP-1 cells (100 nm PMA for 72 h) [START_REF] Brzicova | Molecular responses in THP-1 macrophage-like cells exposed to diverse nanoparticles[END_REF]. 20-30 nm 2.5 mg/mL TiO2 NPs in serum-free media also did not cause TNF-α production [START_REF] Müller | Oxidative stress and inflammation response after nanoparticle exposure: Differences between human lung cell monocultures and an advanced threedimensional model of the human epithelial airways[END_REF].

Altough Li and Mithran (2014) showed no trigerred pro-inflammatory response production upon P25 treatment [START_REF] Xia | Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium[END_REF], we found an increase in TNF- production after P25 treatment.

In THP-1 differentiated cells, a factor contributing to the contradictory results might be the variable PMA concentrations used. Concerning PMA treatment on THP-1, the conditions for the culture should be taken into consideration to obtain a homogeneous culture expressing reliable responses. In these results, the concentration of PMA and the differentiated cell density are essentially important. The differences in stimulating reagents and treatments may result in the production of different types of cytokines at different levels.

Oxidative stress

ROS production after exposure of differentiated THP-1 cells to TiO2 NPs is reported in Figure 22.

Conclusion

The highlights of the article 'Impact of the physicochemical features of TiO2

NPs on their in vitro toxicity' were:

 Rod, bigger, less agglomerated and positively charged particles are more toxic,

 Cocultured model seems more sensitive than A549 monocultures.

There was no correlation between microscopic observations of the cells and LDH, MTT results after TiO2 NPs exposure. Therefore, cell viability was determined by trypan blue method and the results are reported in Article 1.

The possible interaction between TiO2 NPs and MTT, LDH assays were examined in Chapter 5.

When the proinflammatory response was evaluated on differentiated THP-1 cells, a significant production of TNF-α was observed only for P25 treated cells.

In differentiated THP-1 cells, no significant ROS production was observed compared to the control. Cette étude collaborative a conduit à la rédaction de l'Article 2 soumis à Chemical Research in Toxicology.

Complementary Data

Acellular ROS production induced by TiO2 NPs after 30 min of UV irradiation is reported in Article 2. We also assessed the Electron Spin Resonance (ESR) intensity of the ROS signal as a function of time during 30 min irradiation of TiO2 NPs and these results are reported here as a complementary data. The DMPO-CO2• -intensity was higher for S1, S2, and P25 than S3 and S4. The most prominent production of CO2• -radicals was observed with P25, followed by S2 and S1, while S3 and S4 gave the least yields.

HO• and CO2• -radicals generation

Parameters such as crystalline structure and primary particle size affect the photocatalytic ability of TiO2 NPs. Regarding the crystalline structure, mixed phase P25 showed greater CO2• -radical intensity in the first 5 minutes and at 30 minutes slightly higher intensity was observed than for anatase S2 (21 nm) and S1 (15 nm).

This can be explained by the synergistic effect between the two phases that reduces the recombination effect, as reported earlier [START_REF] Odling | Why is Anatase a Better Photocatalyst than Rutile? The Importance of Free Hydroxyl Radicals[END_REF]. Also it is indicated that mixedphase TiO2 NPs are better photocatalysts than simple mixture of single-phase nanoparticles due to their close-contact heterophase junctions [START_REF] Likodimos | Microstructure and charge trapping assessment in highly reactive mixed phase TiO2 photocatalysts[END_REF]. Smaller and spherical shape TiO2 NPs (15 nm S1, 30 nm S2 and 21 nm P25) showed greater ROS production than bigger rod shape S3 NPs. Similar to our results, P25 showed greater ROS production than bigger (108 nm) anatase TiO2 NPs [START_REF] Tang | Photocatalytic production of hydroxyl radicals by commercial TiO 2 nanoparticles and phototoxic hazard identification[END_REF]. We also observed the surface charge impact: APTES coated, positively charged S4 NPs exhibited weaker signals whereas S2 NPs, their negatively charged counterparts, showed greater DMPO-CO2• -production. Overall, TiO2 NPs enhance photocatalytic production of ROS depending on their physicochemical properties. The potential of this compound to generate larger amounts of carbon dioxide radical anion (CO2• -) in suspension should be taken into account in preventing its potential oxidative damage.

Conclusion

The highlights of the article 'Short pre-irradiation of TiO2 nanoparticles increases cytotoxicity on human lung coculture system' were:

 Irradiated TiO2 NPs caused a greater reduction in cell viability compared to the pristine TiO2 NPs on human lung cells.

 Pristine and irradiated TiO2 NPs caused a similar pro-inflammatory response.

 No ROS production was observed either for pristine or irradiated TiO2 NPs treated cells.

30 min UV irradiation altered the average aggregate size and surface charge of NPs.

These alterations might be responsible for the greater cell loss observed when cells were exposed to pre-irradiated TiO2 NPs. The DMPO-HO• signal remained fairly unaffected by UV irradiation, however time-dependent changes in DMPO-CO2• - signal intensity of a suspension of TiO2 were observed.

When the physicochemical properties of the NPs were taken into account, the NPs with smaller size, negative surface charge and spherical shape caused more intense DMPO-CO2• -signal than bigger, rod and positively charged NPs. La formation de la corona protéique des NPs de TiO2 irradiées aux UV pendant 30 minutes a également été déterminée qualitativement / semi-quantitativement et est présentée dans ce chapitre en tant que données complémentaires.

Complemantary Data

After TiO2 NPs were exposed to UV irradiation for 30 minutes, the protein corona formation was determined. UV irradiated S2, S1 and P25 nanoparticles exhibited similar amounts of proteins in their corona while proteins were less abundantly found in the corona from S4 nanoparticles. For each nanoparticle type, a similar concentration of proteins was found in the coronas formed in 30 min UV irradiated or in pristine TiO2 NPs (no statistically significant differences). Both in pristine and UV-irradiated TiO2 NPs the corona of S2, and P25 had very similar protein profiles, S1 presented a slightly lower amount of proteins whereas S4 corona exhibited the smallest protein content. The differences were not observed between the gel profiles of pristine and UV irradiated particles. These results are in good agreement with those of protein quantification (Figure 26).

Profile of proteins adsorbed at the surface of the 30 min UV irradiated nanoparticles by gel electrophoresis

Conclusion

The highlights of the article 'Influence of the physicochemical features of TiO2 nanoparticles on the formation of a protein corona and impact on cytotoxicity' were:

 The parameters influencing the protein corona formation are mainly nanoparticle primary size and surface charge, no clear impact of the shape and agglomeration state was observed.

 Smaller NPs has slightly less important amount of proteins in their corona compared to bigger P25 nanoparticles. Protein coronas were preferentially formed on negatively charged NPs than positively charged NPs.

 No clear relationship between the protein corona and toxicity of TiO2 NPs was observed.

Exploratory studies

The consistency and accuracy of the nanomaterials toxicity test results is crucial in the design, use and legal regulations of these materials.

To date, there are contradictory reports on the toxicity of various types of nanomaterials, making it difficult to predict their biological effects [START_REF] Monteiro-Riviere | Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line[END_REF]. One of the most important factors affecting consistent toxicity results is nanomaterial interaction with the tests, leading to data artifacts and incompatible predictions of The toxicity induced by the TiO2 NPs was assesed by the LDH and MTT assays in Chapter 2. However, after cell exposure to TiO2 NPs, no correlation was found between the morphological changes of the cells and the results of these two cytotoxicity assays.

To provide insight into the bias possibilities, TiO2 NPs interaction with LDH and MTT assay components in cell free conditions were investigated in this chapter.

Chapitre 5 Etudes exploratoires

La production et l'utilisation de nanomatériaux (NMs) augmentent, et la cohérence et la fiabilité des tests de toxicité de ces matériaux sont très importantes en termes de conception, d'utilisation et de réglementation de ces matériaux.

À ce jour, il existe des rapports contradictoires sur la toxicité de divers types de nanomatériaux, ce qui rend difficile la prédiction de leurs effets biologiques [START_REF] Monteiro-Riviere | Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line[END_REF]. NPs were incubated at 37°C for 24 h. At the end of incubation, the solutions were centrifuged at 115 g for 10 min, and the supernatant was collected and was mixed with the LDH assay mixture at a ratio of 1:2 and incubated at room temperature in the dark for 30 min. The reaction was stopped by the addition of 1 N HCl (1/10 of mixture volume), and the absorbance was read at 450 nm as described earlier by Forest et al. [START_REF] Forest | Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results about the cytotoxicity of these nanomaterials[END_REF]. In cell free conditions, after adding 120 µg/mL TiO2 NPs to 0.16 U/µL LDH solution, the decrease was observed in absorbance value compared to 0.16 U/µL LDH control.

Especially S2, S3 and P25 absorbances were significantly lower than control. These results suggest that particles inactivate or absorb the LDH enzyme, preventing LDH from being measured and resulted in lower absorbances.

Our results are in agreement with those of Zaqout et al. [START_REF] Zaqout | Binding of titanium dioxide nanoparticles to lactate dehydrogenase[END_REF], they observed a decrease LDH activity induced by TiO2 NPs in cell free conditions. Similarly, other studies showed TiO2 NPs were able to adsorb or inactivate LDH protein [START_REF] Kroll | Current in vitro methods in nanoparticle risk assessment: Limitations and challenges[END_REF][START_REF] Holder | Particle-induced artifacts in the MTT and LDH viability assays[END_REF][START_REF] Duan | Interaction between nanoparticulate anatase TiO2 and lactate dehydrogenase[END_REF].

Overall, these results show that TiO2 NPs bind to LDH, and might cause false positive results in cell conditions. Particle interaction with the LDH protein needs extensive investigation, however these results show the limited utility of the LDH assay.

Titanium dioxide nanoparticle interaction with MTT (cell free condition)

TiO2 nanoparticles present in the reaction mixture can influence the readout by oxidizing the assay substrate due to its photocatalytic properties [START_REF]Titanium dioxide in nanoparticle form: ANSES defines a toxicity reference value (TRV) for chronic inhalation exposure | Anses -Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail[END_REF]. The conversion of MTT into formazan by TiO2 NPs might cause misleading cell viability results.

Therefore, we examined whether TiO2 could induce the MTT to formazan transformation under cell free conditions relevant for in vitro biological experiments.

The ability of TiO2 NPs to reduce MTT to formazan was measured in the cell free conditions. 15, 30, 60, 120 μg/mL TiO2 suspensions in DMEM were prepared. 200 µL TiO2 suspensions were incubated with 20 µg/mL MTT solution at 37°C for 3 hours.

DMEM without TiO2 NPs was used as a control. At the 10 th , 30 th , 60 th and 180 th minutes within a 3 hours period incubation, the medium was carefully aspirated and 200 μL of DMSO was added to each well. Absorbance (Acell free) measurements were made at the indicated times at 570 nm.

The absorbance of the particles in cell free conditions (Acell free) are shown in Figure 29. This is in good agreement with studies [START_REF] Ong | Widespread nanoparticle-assay interference: Implications for nanotoxicity testing[END_REF][START_REF] Lupu | The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays[END_REF][START_REF] Wang | Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO 2[END_REF] demonstrating TiO2-MTT interactions leading to the formation of purple/blue formazan. P25-MTT incubation studies showed the concentration dependent absorbance increase compared to control (without TiO2 NPs) in cell free conditions [START_REF] Lupu | The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays[END_REF], similar to our results. TiO2 may interfere with MTT viability tests by oxidizing the substrate MTT. Hence, redoxactive nanoparticles may cause false signals in assays. This leads to critical difficulties in differentiating between true cell viability and artefacts caused by NP-assay interference. It is clear from these results that NPs have the potential to interfere with the components of the assays themselves; thus, the use of these assays in cell conditions clearly requires attention.

Re-evaluation of MTT results in cellular assays

The TiO2 NPs generated formazan in cell free conditions as well as in cell conditions.

Therefore, produced formazan absorbances in cell free conditions were used to correct the cell viability results obtained in Chapter 2.

In this regard, the estimated value of the viability (Vcorrected) was recalculated by substracting the mean of the absorbance of samples in cell-free conditions (Acell-free) from the absorbance of samples observed in cell conditions (A) in accordance with Lupu et al. [START_REF] Lupu | The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays[END_REF], with the formula given below. The corrected viability percentage is shown in Figures 30 and31. In both A549 and coculture system, the corrections led to lower viability values meaning higher toxicity. The non-corrected viability values were not significantly different with respect to control for S4 but then become so after taking into account the artifactual formazan formation. These results are in agreement with [START_REF] Lupu | The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays[END_REF] showing lower cell viability after TiO2 induced formazan corrections. However, the corrected results are not fully consistent with results from the trypan blue assay reported in Article 1. This is why the correction method is not fully satisfactory.

The limit of this study is that when cells exposed to TiO2 NPs are incubated with MTT, it is difficult to determine how much of the MTT passes through the cell membrane and is reduced to formazan by cell metabolism, and how much of it interacts with TiO2 and is reduced to formazan. However, the obtained corrected cell viabilities represent the estimated values after cell exposure to TiO2 NPs.

Conclusion

In this chapter, LDH and MTT assays which are frequently used in nanoparticle toxicity assesment were evaluated regarding their potential interactions with TiO2

NPs. This study highlights the artifacts obtained with LDH and MTT assays in cell free En conclusion, les artefacts potentiels dans les essais de cytotoxicité couramment utilisés devraient être testés avant de procéder à l'évaluation de la cytotoxicité des nanoparticules afin d'éviter toute mauvaise interprétation des résultats de toxicité.

CONCLUSIONS AND PERSPECTIVES

The main objective of this thesis work focused on the effects of various physicochemical properties on the toxicity of TiO2 NPs toward human lung cells, in particular on the cytotoxicity, pro-inflammatory response, and oxidative stress mechanism. In this sense, 5 different TiO2 NPs were synthesized and thoroughly characterized with the collaboration of MINES Paris Tech and University of Torino and evaluated systematically on three human lung cell system, namely monocultures of A549 human epithelial lung cells, monocultures of macrophages differentiated from THP-1 and coculture of both cell lines.

The monocyte differentiation process represents a sensitive step for correctly proceeding in vitro toxicity experiments. In Chapter 1, the process of THP-1 differentiation into the macrophages and its optimization were discussed. THP-1 cell line has been widely used to obtain macrophages in biological and toxicological researches and PMA is extensively used to stimulate their differentiation into macrophages. However, the PMA concentration, treatment durations vary widely in the literature and there is no standardized protocol. Therefore, before conducting the toxicity assays, we demonstrated the optimal conditions required for THP-1 differentiation in our laboratory. Among the different applied protocols (different treatment durations, concentration of PMA and different cell densities) the optimal conditions required for successfull differentiation was selected as 30 ng/mL PMA for 24 hours incubation. Especially the concentration of PMA was carefully determined since it has been show that higher concentrations of PMA is associated with the upregulation of inflammatory gene expression [START_REF] Lund | The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus[END_REF][START_REF] Cooke | Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes[END_REF], as compared to lower properties. An impact on the cytotoxicity and to a lesser extent on the proinflammatory response depending on cell type was observed, namely: smaller, large agglomerated TiO2 NPs were shown to be less toxic than P25 whereas rod-shaped TiO2

NPs were found to be more toxic.

Contrarily, TiO2 NPs, whatever their physicochemical properties, did not induce significant ROS production in A549 cell system and coculture system, which is contradictory to studies from Simon-Deckers et al. (2008) and Jugan et al. (2012) [START_REF] Simon-Deckers | In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes[END_REF][START_REF] Jugan | Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells[END_REF] where ROS were systematically detected whatever the size, crystalline phase, and shape of the TiO2 NPs studied in very similar conditions to ours (same cellular model: A549 cells, exposed to various types of TiO2 NPs, for 24 h at 100 µg/mL). Direct measurement of ROS levels with high accuracy and precision is difficult due to their short lifespan and rapid reactivity with redox state regulating components [START_REF] Katerji | Approaches and methods to measure oxidative stress in clinical samples: Research applications in the cancer field[END_REF]. Indirect measurement of ROS by examining the oxidative damage these radicals cause to the lipids, proteins, and nucleic acids of the cells is a promising alternative approach. In this sense, it would have been interesting to study in more detail the oxidative stress and in particular the enzymatic antioxidants (SOD, CAT, GR) and non enzymatic antioxidants (GSH) levels, and total antioxidant capacity, protein carbonyl content and lipid peroxidation.

This study also highlighted the importance of selected cell systems since we detected clear differences in the cytotoxicity endpoints between the mono-and co-cultures.

Biological responses vary between different in vitro systems, thus toxicological evaluation of nanoparticles in only one in vitro cell system can under or overestimate their effects, as suggested by Kasurinen et al. [START_REF] Kasurinen | Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion[END_REF]. Therefore, more than one in vitro system should be used to complete evaluation of nanoparticle hazard assessment. It would have been interesting to estimate the uptake and localization of nanoparticles in two cell models by transmission electron microscopy and evaluate the physicochemical characteristics of nanoparticles impact on their cellular internalization.

In Chapter 3 and Article 2, we further investigated whether the short time UV light exposure induced changes in the physicochemical properties of five different TiO2 NPs and eventually affected their toxicity on human lung coculture system. We observed that 30-min UV irradiation altered the surface charges and agglomeration status of TiO2 NPs. Pre-irradiated TiO2 NPs (less negatively charged) induced a higher cytotoxic effect than the pristine TiO2 NPs. In cell free conditions, TiO2 NPs enhanced photocatalytic production of ROS depending on their physicochemical properties: smaller primary size, negative surface charge and spherical shape caused more intense DMPO-CO2• -signal than bigger, rod and positively charged NPs. The potential of this compound to generate larger amounts of the carbon dioxide radical anion in suspension should be taken into account in preventing its potential oxidative damage. Since TiO2 NPs in the environment are expected to be exposed to UV rays and possibly modified following photoexcitation, these results can serve as a basis for further studies as it has important implications for environmental and human health risk assessment and preventive actions to limit human exposure.

In Chapter 4 and Article 3, the protein corona formation of pristine and 30 min UV irradiated TiO2 NPs was also qualitatively/semi-quantitatively determined. The parameters influencing the protein corona formation are mainly nanoparticle primary size and surface charge, no clear impact of the shape and agglomeration state was observed. No effect of UV irradiation was observed on protein corona. When the toxicity results obtained in Chapters 2 and 3 and protein corona results were evaluated together, no clear relationship was observed between the protein corona and toxicity of TiO2 NPs. However, in this research we obtained the preliminary results, now, mass spectrometry-based proteomic analysis might be useful to identify, quantify, and characterize the protein corona protein composition [START_REF] Carrillo-Carrion | Techniques for the experimental investigation of the protein corona[END_REF].

In this thesis work, TiO2 NPs toxicity was first assessed by commonly used in vitro assay methods MTT and LDH as reported in Chapter 2. In Chapter 5 particles interaction with assay compounds by chemical reactions, and assay molecules adsorption on the particle surface were investigated in LDH and MTT method in cell free conditions. LDH assays have been shown to be affected by TiO2 NPs through the interaction of NPs with the LDH enzyme itself, causing adsorption and/or inactivation of the protein and an associated loss of activity [START_REF] Ong | Widespread nanoparticle-assay interference: Implications for nanotoxicity testing[END_REF][START_REF] Holder | Particle-induced artifacts in the MTT and LDH viability assays[END_REF], as shown in our study where TiO2 NPs-LDH interaction resulted in loss of activity. Similarly, TiO2 interfere with MTT viability tests by oxidizing the substrate MTT and TiO2 induced the MTT to formazan transformation under cell free conditions.

As suggested by many researchers, to minimize the potential NPs-assay interactions and associated misinterpretation of results, choosing the in vitro test method according to the nanoparticle type and concentration is of great importance.

Therefore, the presence of NPs should be as much as possible limited in the final sample with multiple washes and/or centrifugations [START_REF] Ong | Widespread nanoparticle-assay interference: Implications for nanotoxicity testing[END_REF]. In nanoparticle in vitro toxicity assessment, each individual formulation should be tested for compatibility with all assays used. The quality control studies provide more accurate scientific data for the establishment of regulations related to safe NPs production.

Overall, in this PhD study, the impact of physicochemical properties of TiO2 nanoparticles on their toxicity was systematically investigated. The main conclusions of this thesis are:

 Pristine TiO2 NPs caused cytotoxicity on human lung cells and cytotoxicity of TiO2 NPs was dependent on their size, shape, agglomeration status and functionalization/surface charge. The effect on toxicity was higher with larger sized, less agglomerated, rod-shaped, and positively charged nanoparticles.

 Upon short-term UV irradiation, irradiated TiO2 NPs induced a higher cytotoxic effect than the pristine TiO2 NPs in relation to an altered surface charge.

 In acellular ROS production, the NPs with smaller size, negative surface charge and spherical shape caused more intense DMPO-CO2• -signal than bigger, rod and positively charged NPs.

 The parameters influencing the protein corona formation are mainly nanoparticle size and surface charge, no clear impact of the shape and agglomeration state was observed.

This research has raised many questions in need of further examination. Further experimental investigations are needed to evaluate the apoptosis by the formation of apoptotic bodies and altered expression of markers such as P53, P21, Bax, Bcl2 and cleaved caspase-3. As stated above, in oxidative stress, the production of reactive oxygen species and malondialdehyde and the activity of catalase and glutathione could be interesting to investigate. On the other hand, it has also been shown that nuclear factor-kappa B (NFκB) is activated in response to pro-inflammatory cytokines. Therefore, further researches should be undertaken in the evaluation of the involvement of NF-κB in TiO2-induced inflammation.

In a context of long or chronic exposure to TiO2 NPs, such effects can cause many pathological conditions in the lung, including cancer. In perspective, the development of non-cytotoxic TiO2 nanoparticles under the safety-by-design approach could make it possible to control the toxicity of certain nanoparticles, but at this day, no study has yet demonstrated this. The data obtained from this thesis contribute significantly to the toxicological profiles of these nanoparticles. Research on TiO2 NPs need to be combined and supported with extensive toxicological studies. Mots-clés: Nanoparticules de dioxyde de titane; cytotoxicité; réponse pro-inflammatoire; espèces réactives de l'oxygène; cellules pulmonaires humaines.

CONCLUSIONS et PERSPECTIVES

Résumé :

Avec le développement rapide des nanosciences et des nanotechnologies, les préoccupations concernant les effets potentiels des nanoparticules sur la santé se sont renforcées. Les nanoparticules de dioxyde de titane (NPs de TiO2) sont largement utilisées dans une grande variété d'applications industrielles, principalement en raison de caractéristiques remarquables telles que leur taille, leur indice de réfraction ou leurs propriétés photocatalytiques.

Bien que les recherches sur la toxicité du TiO2 soient de plus en plus nombreuses, les études examinant l'impact des propriétés physicochimiques des NPs de TiO2 sur leur toxicité sont limitées. Par conséquent, les principaux objectifs de cette thèse étaient d'évaluer : (i) la toxicité des NPs de TiO2 sur des cellules pulmonaires humaines en lien avec leurs principales propriétés physicochimiques, (ii) l'impact de l'exposition de courte durée des NPs de TiO2 à la lumière UV sur leurs propriétés physicochimiques et leur toxicité.

Pour atteindre cet objectif, quatre types de NPs de TiO2 ont été synthétisés à façon et un TiO2 commercial a été utilisé. Ces cinq types de NPs de TiO2 diffèrent par leur taille/surface spécifique, leur forme, leur agglomération et leur fonctionnalisation/charge de surface (APTES).

Dans la première étape de l'étude, l'effet des propriétés physicochimiques des NPs de TiO2 sur leur toxicité a été évalué sur 3 systèmes différents de cellules pulmonaires humaines (cellules épithéliales A549, macrophages différenciés à partir de THP-1, et cocultures de A549/THP-1 différenciées). En général, les NPs de TiO2 provoquent une diminution de la viabilité des cellules et induisent une réponse pro-inflammatoire selon le type de cellule. L'effet sur la toxicité était plus important avec des nanoparticules de plus grande taille primaire, moins agglomérées, en forme de bâtonnet et chargées positivement.

Lors de la deuxième étape de l'étude, nous avons évalué si une exposition de courte durée des NPs de TiO2 aux rayons UV indusait des changements dans leurs propriétés physicochimiques et leur toxicité.

Les NPs de TiO2 irradiées ont induit un effet cytotoxique plus élevé que les NPs de TiO2 non irradiées en lien avec une charge de surface modifiée.

Après avoir examiné la toxicité des NPs de TiO2 en fonction de leurs propriétés physicochimiques et photocatalytiques, la formation de la corona protéique, qui est un autre facteur affectant la toxicité, a été étudiée. Les paramètres influençant la formation de la "protein corona" sont principalement la taille primaire des nanoparticules et la charge de surface, aucun impact clair de la forme et de l'état d'agglomération n'a été observé. Aucun effet de l'irradiation UV n'a été observé sur la formation de la couronne protéique.

Dans l'ensemble, l'impact des propriétés physicochimiques des NPs de TiO2 sur leur toxicité a été observé et la toxicité est plus prononcée après l'irradiation UV des NPs. Une autre conclusion importante de cette étude est que le modèle de co-culture semble plus sensible aux effets indésirables des NPs de TiO2 que le modèle de monoculture.

Cette étude contribue à améliorer notre compréhension des dangers et des risques que cinq types différents de NPs de TiO2 et leurs homologues irradiées aux UV peuvent présenter pour la santé publique. Elle peut servir de base à une approche "safer by design" pour atténuer la toxicité de ces nanoparticules.

Figure 1 . 5 Figure 2 . 8 Figure 3 . 9 Figure 4 . 9 Figure 5 . 14 Figure 6 . 15 Figure 7 . 16 Figure 8 .

 152839495146157168 Figure 1. Examples of nanoparticle shapes. ............................................................................ 5 Figure 2. Crystalline forms of TiO2 [19]. ................................................................................. 8 Figure 3. Brief overview of TiO2 photocatalyst activity. ........................................................ 9 Figure 4. Main industrial applications of TiO2 (adapted from [30]). ................................... 9 Figure 5. Upper and lower respiratory system. ..................................................................... 14 Figure 6. Nanoparticle translocation in human body. .......................................................... 15 Figure 7. Cell organization of the respiratory system. .......................................................... 16 Figure 8. Scheme of the mucociliary clearance. .................................................................... 17

Figure 9 . 18 Figure 10 . 19 Figure 11 . 26 Figure 12 . 41 Figure 13 . 43 Figure 14 . 44 Figure 15 . 45 Figure 16 . 46 Figure 17 . 65 Figure 18 . 66 Figure 19 . 67 Figure 20 . 69 Figure 21 . 71 Figure 22 . 72 Figure 23 . 107 Figure 24 . 108 Figure 25 . 109 Figure 26 . 125 Figure 27 . 126 Figure 28 . 131 Figure 29 . 133 Figure 30 . 135 Figure 31 .

 9181019112612411343144415451646176518661967206921712272231072410825109261252712628131291333013531 Figure 9. Schematic illustration of the phagocytosis. ........................................................... 18 Figure 10. Protein corona formation. .................................................................................... 19 Figure 11. Interaction of NPs with cells and possible biologic effects. ................................ 26 Figure 12. 72 h DMSO treated THP-1 cell viability. DMSO solutions were prepared in culture media (RPMI) in indicated concentrations. ............................................................. 41 Figure 13. Microscopic images of THP-1 cells (7.5 x 10 6 cells/ T25 cm 2 flask) exposed to 5; 30; 50; 100 ng/mL PMA for 24 h and 48 h. Control respresents THP-1 cells without PMA treatment (20 x magnification). ............................................................................................ 43 Figure 14. THP-1 cell adhesion after treatment with the indicated PMA concentrations for 24 h and 48 h. Independent experiments were conducted three times in triplicate. Statistically different from control ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test. ............................................... 44 Figure 15. Absorbance values of 30, 50, 100 ng/mL PMA treated cells at the indicated cell densities as determined by MTT assay. ................................................................................ 45 Figure 16. Immunocytochemistry staining of CD11b (marker of macrophage activation) in THP-1-derived macrophages. The detection of protein expression was performed by fluorescence microscopy (magnification 25 X). ................................................................... 46 Figure 17. Principle of LDH release assay. ........................................................................... 65 Figure 18. LDH release 24 h after cell exposure to TiO2 NPs. Control (C-) represents unexposed cells. Lysed cells were used as positive control (C+). Values are the mean ± SEM of three independent experiments. Statistically different from control ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test. ......................................................................................................................................... 66 Figure 19. MTT reduction into purple formazan by mitochondrial dehydrogenase in viable cells. ......................................................................................................................................... 67 Figure 20. Cell viability after 24 h exposure to TiO2 NPs assessed by MTT. Control (C) represents unexposed cells. Values are the mean ± SEM of three independent experiments. Statistically different from control ( * ) P < 0.05, ( * * ) P < 0.01, ( * * * ) P < 0.0001, ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test. ........................................................................................................................ 69 Figure 21. TNF-α production after a 24 h exposure of differentiated THP-1 to TiO2 NPs. Control (C) represents unexposed cells. Values are the mean ± SEM of three independent experiments. Statistically different from control ( * ) P < 0.05 ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test. ...... 71 Figure 22. ROS production after 90 min and 24 hours exposure to the indicated concentrations of TiO2 NPs in differentiated THP-1. Control (C-) represents unexposed

  TiO2 nanoparticles in relation to their different physicochemical properties. As one of the main routes of exposure to these nanoparticles is inhalation, their toxic potential was evaluated on human lung cell lines. This thesis was carried out within the Biomaterials and Inhaled Particles Engineering Department (BioPI) of the Engineering and Health Center of the Ecole des Mines de Saint-Etienne, within the framework of the research unit U1059 Inserm Sainbiose.

Figure 1 .

 1 Figure 1. Examples of nanoparticle shapes.

Figure 2 .

 2 Figure 2. Crystalline forms of TiO2 [21].

Figure 3 .

 3 Figure 3. Brief overview of TiO2 photocatalyst activity.

Figure 4 .

 4 Figure 4. Main industrial applications of TiO2 (adapted from[START_REF] Ziental | Titanium Dioxide Nanoparticles: Prospects and Applications in[END_REF]).

  The respiratory tract is a complex anatomical structure and the diameter of the upper airways is relatively large, and the lower airways and alveolar diameters are smaller. The inhaled particles deposit in the entire respiratory tract, from the nasal cavity to alveoli. Therefore, mainly the sizes and surface areas of particles, and their physical state (liquid or solid), density, shape and velocity are important factors in influencing NPs deposition [52].

Figure 5 .

 5 Figure 5. Upper and lower respiratory system.

  efficiently due to their smaller size. The retention time of nanoparticles depends on

Figure 6 .

 6 Figure 6. Nanoparticle translocation in human body.

Figure 7 .

 7 Figure 7. Cell organization of the respiratory system.

Figure 8 .

 8 Figure 8. Scheme of the mucociliary clearance. When nanoparticles come in contact with the outer membrane of a cell, it can interact with the components of the plasma membrane or extracellular matrix. They can enter the cell mainly through endocytosis. Endocytosis leads to ingestion of nanoparticles in membrane invaginations. Endocytosis is classified as phagocytosis, clathrinmediated endocytosis, caveolin-mediated endocytosis, clathrin/caveola-independent endocytosis and micropinocytosis [62].

Figure 9 .

 9 Figure 9. Schematic illustration of the phagocytosis.

  adsorption, tighter bonds, slow exchange time and high conformational change; and the soft corona, which is the second and exterior layer of proteins, characterized by weak bonds (protein-protein interactions), quick exchange time and low conformational change (Figure10)[START_REF] Forest | Electrostatic interactions favor the binding of positive nanoparticles on cells: A reductive theory[END_REF][START_REF] Monopoli | Physical-Chemical aspects of protein corona: Relevance to in vitro and in vivo biological impacts of nanoparticles[END_REF][START_REF] Foroozandeh | Merging Worlds of Nanomaterials and Biological Environment: Factors Governing Protein Corona Formation on Nanoparticles and Its Biological Consequences[END_REF].

Figure 10 .

 10 Figure 10. Protein corona composition.

1 .

 1 O2 + e -→ O2• -Superoxide radical 2. O2• + H20 → H2O• Hydroperoxyl radical 3. H20• + e -+ H + → H2O2 Hydrogen peroxide 4. H202 + e -→ OH -+ OH• Hydroxyl radical

Figure 11 .

 11 Figure 11. Interaction of NPs with cells and possible biologic effects.

  3 mg/m 3 for ultrafine (including engineered nanoscale) TiO2, as time-weighted average (TWA) concentrations for up to 10 hr/day during a 40-hour work week. These recommendations represent levels that over a working lifetime are estimated to reduce risks of lung cancer to below 1 in 1,000. The recommendations are based on using chronic inhalation studies in rats to predict lung tumor risks in humans. The hazard assessment of inhaled TiO2 NPs should be performed in consideration of the significant role of this route of exposure for both the general and occupational populations.

1 . 1 Chapter 1 1

 1111 and macrophages. In addition to commercial P25 NPs, used as a reference, four types of TiO2 NPs samples differing in size, shape, agglomeration state and surface functionalization/charge were synthesized at MINES Paris Tech. All samples were characterized before toxicity experiments regarding their size, hydrodynamic size, surface charge, shape, crystallinity, and they were tested regarding possible endotoxin contamination.The characterization and validation of accurate cell differentiation are initial steps to ensure the reliable and reproducible results before conducting in vitro toxicity experiments. Therefore, THP-1 cell differentiation into the macrophages were studied extensively and the optimal conditions necessary for THP-1 differentiation into the macrophages were determined. The THP-1 cell differentiation processes are reported in Chapter Once cell lines were well established and ready to toxicological experiments, the cell membrane integrity, cell viability, pro-inflammatory response and cellular oxidative responses were assessed after exposure to TiO2 nanoparticles and reported in Chapter 2 and in the associated article entitled "Impact of the physicochemical features of TiO2 nanoparticles on their in vitro toxicity".As TiO2 is photo-activated with either UV light, visible light or combination of both, TiO2 nanoparticles in the environment are expected to become photoactive in the UV spectrum. After the toxicity results we obtained in Chapter 2, we evaluated if a short time exposure of TiO2 NPs to UV light induced changes in their physicochemical properties and eventually affected the toxicity they induced on a human lung coculture system. These results are reported in Chapter 3 and in the associated paper entitled "Short pre-irradiation of TiO2 nanoparticles increases cytotoxicity on human lung coculture system".Then, the protein corona formation, which is another factor affecting toxicity, was compared in the five TiO2 NP types in different cell culture media. This is the topic of Chapter 4 and the article entitled "Influence of the physicochemical features of TiO2 nanoparticles on the formation of a protein corona and impact on cytotoxicity".Finally, in Chapter 5 some exploratory studies are reported on TiO2 NPs interactions with LDH and MTT assay components in cell free conditions.The findings obtained through this thesis provide a wide perspective in toxicity potentials depending on the physicochemical properties of TiO2 nanoparticles by the systematic evaluation of their adverse effects on human lung cell lines. In addition, it provides a comparative evaluation of pristine TiO2 nanoparticles and their UVirradiated counterparts. The thesis results indicate health concerns that might be due to inhalation of different properties of pristine TiO2 nanoparticles and photoactivated TiO2, and it improves our understanding of the hazards and risks that TiO2 NPs could pose to public health. It can serve as a basis for a safer by design approach to mitigate the toxicity of this material and for preventive actions to limit human exposure.Development and Validation of the In vitroBiological ModelsConsidering that the lungs are the target organs in the exposure to inhaled NPs and inhalation is the main route of the entrance of NPs in the body, in this study, the toxicity of TiO2 NPs was evaluated on in vitro human lung cell lines: widely used adenocarcinomic human alveolar basal epithelial cells (A549 cell line), differentiated THP-1 macrophages from THP-1 monocytes and coculture of both cell lines. Since primary tissue macrophages cannot be easily expanded ex vivo and are difficult to grow in vitro, THP-1 monocyte cell lines are widely used in obtaining macrophages.Due to their metabolic and morphological similarities, human monocytic THP-1 cell line can be differentiated into macrophages by Phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). However, their differentiation state may not always accurately predict the behavior of differentiated tissue macrophages depending on the experimental conditions. Thus it may lead to misinterpretation of biological responses in toxicity tests. Therefore the characterization and validation of accurate cell differentiation are important and initial steps to ensure reliable and reproducible results, before conducting toxicity experiments.Once large, round single-cell morphology and suspension THP-1 cells differentiate into macrophages, they result in a more mature phenotype with a lower rate of proliferation, higher levels of adhesion, enhanced granularity, increased expression of specific surface markers associated with macrophage differentiation such as cluster of differentiation molecule 11b (CD11b) and CD14. Therefore, the THP-1 differentiation can be verified by the simple checking of cell adhesion, morphology, and by examining the expression of macrophage specific surface markers.Since THP-1 macrophages are used as target cells in TiO2 NP toxicity in this thesis, this chapter presents the general features of THP-1 cells and the various protocols used for optimization and validation of their differentiation into macrophages, and the results of these protocols. At the end of the studies carried out in this chapter, the most suitable conditions for THP-1 differentiation were determined. The findings in this section are the fundamental results of the optimal cell differentiation conditions required to perform toxicity studies.ChapitreDéveloppement et validation des modèles biologiques in vitro Considérant que les poumons sont les organes cibles de l'exposition aux NP inhalées et que l'inhalation est la principale voie d'entrée des NP dans le corps, dans cette étude, la toxicité des NPs de TiO2 a été évaluée in vitro sur des lignées cellulaires pulmonaires humaines: cellules épithéliales basales alvéolaires issues d'un adénocarcinome humain (lignée A549, largement décrite et utilisée dans la littérature), des macrophages THP-1 différenciés à partir des monocytes de la lignée THP-1 et une coculture de ces deux lignées cellulaires. Étant donné que les macrophages tissulaires primaires ne peuvent pas être facilement multipliés ex vivo et sont difficiles à cultiver in vitro, les lignées cellulaires de monocytes THP-1 sont largement utilisées pour obtenir des macrophages. En raison de leurs similitudes métaboliques et morphologiques, la lignée cellulaire monocytaire humaine THP-1 peut être différenciée en macrophages par le Phorbol-12-myristate-13-acétate (PMA) ou la 1,25-dihydroxyvitamine D3 (VD3). Cependant, leur état de différenciation peut ne pas toujours prédire avec précision le comportement des macrophages tissulaires différenciés en fonction des conditions expérimentales. Ainsi, cela peut conduire à une mauvaise interprétation des réponses biologiques dans les tests de toxicité. Par conséquent, la caractérisation et la validation d'une différenciation cellulaire précise sont des étapes importantes et initiales pour garantir des résultats fiables et reproductibles, avant de mener des expériences de toxicité.Une fois que les cellules THP-1 en suspension sont différenciées en macrophages, elles donnent un phénotype plus mature avec un taux de prolifération plus faible, des niveaux d'adhérence plus élevés, une granularité accrue, une expression de marqueurs de surface spécifiques des macrophages accrue tels que le groupe de molécule de différenciation 11b (CD11b) et CD14. Par conséquent, la différenciation THP-1 peut être vérifiée par la simple vérification de l'adhérence cellulaire, de la morphologie et en examinant l'expression des marqueurs de surface spécifiques des macrophages.

1. 1 . Optimal PMA exposure time and cell density determination after exposure to various PMA concentrations 1 . 1 . 1 .

 1111 Protocol 1 (72 hours PMA exposure + 5 days resting) 5 x 10 6 THP-1 cells were treated with 0, 50, 100, 150, 200 ng/mL PMA for 72 hours. At the end of incubation PMA containing-medium was discarded and cells rested in PMA-free culture medium for 5 days. Medium being refreshed every 2 days for a period of 5 days resting. Cell adhesion was evaluated by optical microscopy (Leica ICC50 HD, Leica Microsystems) at the end of a total 8 days. Adherent cells were detached from culture flask enzymatically and the morphological changes in terms of cell granularity were determined by an increase in side scatter (SSC) on flow cytometry which allows the discrimination between differentiated THP-1 and THP-1 monocytes. The cell size (FSC) and granularity (SSC) of cells and viability were analyzed by flow cytometry using a BD FACSCalibur instrument (Becton Dickinson Biosciences, Le Pont-de-Claix, France) equipped with an air-cooled argon laser (488-nm emission; 20 mW).

Figure 12 .

 12 Figure 12. 72 h DMSO treated THP-1 cell viability. DMSO solutions were prepared in culture media (RPMI) in indicated concentrations.

Figure 13 .

 13 Figure 13. Microscopic images of THP-1 cells (7.5 x 10 6 cells/ T25 cm 2 flask) exposed to 5; 30; 50; 100 ng/mL PMA for 24 h and 48 h. Control respresents THP-1 cells without PMA treatment (20 x magnification).

Figure 14 .

 14 Figure 14. THP-1 cell adhesion after treatment with the indicated PMA concentrations for 24 h and 48 h. Independent experiments were conducted three times in triplicate. Statistically different from control ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

Figure 15 .

 15 Figure 15. Absorbance values of 30, 50, 100 ng/mL PMA treated cells at the indicated cell densities as determined by MTT assay.

2 x 10 5

 5 cells/well were differentiated in 8 Well Chamber Slide (corresponding to 1 x 10 5 cells per well in 96-well plate) for 24 hours with 0, 30, 50, 100 ng/mL PMA. After, cells were fixed in 4% paraformaldehyde for 10 min. At the end of fixation, cells were permeabilized in 0.2% Triton X-100 for 10 min and incubated with 2% BSA for 30 min. Cells were incubated for 60 min with a primary antibody directed against human CD11b coupled to FITC (fluorescein isothiocyanate) (2% BSA-FPS). After the cells were washed with PBS and stanined with DAPI dye. Cells were examined under the fluorescence microscope IX81 (Olympus, Tokyo, Japan) equipped with the Cell^P imaging software (Soft Imaging System GmbH, Munster, Germany).

Figure 16 .

 16 Figure 16. Immunocytochemistry staining of CD11b (marker of macrophage activation) in THP-1-derived macrophages. The detection of protein expression was performed by fluorescence microscopy (magnification 25 X).

1. 1 .

 1 Cell membrane integrity-Lactate dehydrogenase release Lactate dehydrogenase (LDH) is released by the cells to the extracellular medium when the cell membrane ruptures. LDH converts lactate to pyruvate, generating NADH from NAD+. The NADH interacts with the iodonitrotetrazolium chloride (INT)and subsequently INT is converted into a red color formazan (Figure17). The amount of colored product formed is directly proportional to the LDH activity in the sample.

Figure 17 .

 17 Figure 17. Principle of LDH release assay.

Figure 18 .

 18 Figure 18. LDH release 24 h after cell exposure to TiO2 NPs. Control (C-) represents unexposed cells. Lysed cells were used as positive control (C+). Values are the mean ± SEM of three independent experiments. Statistically different from control ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

Figure 19 .

 19 Figure 19. MTT reduction into purple formazan by mitochondrial dehydrogenase in viable cells.

68 10 5

 685 Figure 20.

Figure 20 .

 20 Figure 20. Cell viability after 24 h exposure to TiO2 NPs assessed by MTT. Control (C) represents unexposed cells. Values are the mean ± SEM of three independent experiments. Statistically different from control ( * ) P < 0.05, ( * * ) P < 0.01, ( * * * ) P < 0.0001, ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

Figure 21 .

 21 Figure 21. TNF-α production after a 24 h exposure of differentiated THP-1 to TiO2 NPs. Control (C) represents unexposed cells. Values are the mean ± SEM of three independent experiments. Statistically different from control ( * ) P < 0.05 ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

Figure 22 .

 22 Figure 22. ROS production after 90 min and 24 hours exposure to the indicated concentrations of TiO2 NPs in differentiated THP-1. Control (C-) represents unexposed cells, positive control (C+) cells incubated with H2O2. Statistically different from control ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

 3 Une

 3 photocatalytique. Compte tenu du passage du TiO2 à un état photoactif sous la lumière du soleil, les travailleurs et la population en général sont exposés au TiO2 photoactivé par inhalation. À ce jour, la toxicité des NPs de TiO2 a fait l'objet de recherches approfondies alors que des données limitées sont disponibles concernant la toxicité des NPs de TiO2 qui ont été pré-exposées à la lumière UV, et leur impact sur l'homme reste inconnu.

1. 1 .

 1 Electron spin resonance method for detection of HO • and CO2• -radicals from UV-irradiated TiO2 nanoparticles Electron Spin Resonance is a spectroscopic technique that detects species with unpaired electrons. This method involves the addition of a spin trap compound such as DMPO that reacts with any reactive radicals e.g., hydroxyl, carboxyl radical (carbon dioxide radical anion (CO2• -), etc. present, and gives rise to a radical adduct than can be subsequently identified. Typical spectra of the spin adduct DMPO-HO• and DMPO-CO2• -are given in the Figure 23 b. To detect the DMPO-radical adducts in UV pre-irradiated samples, ESR spectra were recorded on aliquot (50 µL) withdrawn at the 5 th , 10 th , 20 th and 30 th min of UV irradiation (Figure 23 a). ESR spectra of negative control (NC) were recorded only at the 30 th min of UV irradiation.

Figure 23 .

 23 Figure 23. Detection of DMPO-HO• and the DMPO-CO2• -from UV-irradiated TiO2 samples by ESR method (a), example of ESR spectrum of the DMPO-HO• and the DMPO-CO2• -radical adducts (b).

Figures 24 and 25

 25 Figures 24 and 25 show the ESR spectra of DMPO-HO• and DMPO-CO2• -adducts obtained on irradiation of TiO2 NPs suspension.

Figure 24 .

 24 Figure 24. Generation of HO radicals from 5, 10, 20, and 30 min UV irradiated TiO2 NPs and 30 min UV irradiated negative control (NC-without TiO2 NPs).

Figure 25 .

 25 Figure 25. Generation of CO2• -radicals from 5, 10, 20, and 30 min UV irradiated TiO2 NPs and 30 min UV irradiated negative control (NC-without TiO2 particles).

111 LesChapter 4 Chapter 4 4

 111444 photocatalytic properties in Chapter 2 and Chapter 3, protein corona formation around TiO2 NPs, which is another factor affecting toxicity, has been investigated in this chapter.

1. 1 .

 1 Figure 26 reports the mean concentrations of proteins eluted from the corona of the 30 min UV irradiated nanoparticles (NPs-UV) and pristine TiO2 NPs incubated in cDMEM.

Figure 26 .

 26 Figure 26. Concentrations of proteins eluted from the protein corona of pristine and 30 min UV iradiated NPs after incubation in cDMEM. Control is cDMEM. Results are means of 3 independent experiments, standard deviation is also indicated. Statistically different from P25 (****) P < 0.0001. Statistical differences between S2 and S4 ( § § § §) P < 0.0001. Statistical analyses were conducted using oneway Anova analyses followed by Tukey's multiple comparison test.

Figure 27

 27 Figure 27 illustrates the protein profile of the corona of the NPs. The picture is representative of the three experiments performed.

Figure 27 .

 27 Figure 27. Electrophoresis gels for pristine and UV irradiated S1, S2, S4 and P25 NPs incubated in cDMEM. As controls, the complete biological media were also run (last lane).



  30 minutes of UV radiation did not cause a remarkable change in the protein corona of the NPs compared to pristine TiO2 NPs. Therefore, no effect of UV irradiation was observed on protein corona. Les points clés de l'article 'Influence of the physicochemical features of TiO2 nanoparticles on the formation of a protein corona and impact on cytotoxicity' sont les suivants :  les paramètres influençant la formation de la corona protéique sont principalement la taille primaire des NPs et la charge de surface, aucun impact clair de la forme et de l'état d'agglomération n'a été observé. Les petites NPs ont une quantité de protéines légèrement moins importante dans leur couronne que les grandes NPs de P25. Les coronas protéiques sont formées de préférence sur des NPs chargées négativement plutôt que sur des NPs chargées positivement. Aucune relation claire entre la corona protéique et la toxicité des NPs de TiO2 n'a été observée. 30 minutes d'irradiation UV n'ont pas provoqué de changement remarquable dans la corona protéique des NPs par rapport aux NPs de TiO2 non irradiées. Par conséquent, aucun effet de l'irradiation UV n'a été observé sur la corona protéique. 129 Chapter 5

  toxicity. Possible interactions include: (a) different optical properties of nanomaterials that can interfere with visible light absorption or fluorescence detection systems, (b) particles can interfere with assay compounds by chemical reactions, and (c) assay molecules can adsorp on the particle surface[START_REF] Ong | Widespread nanoparticle-assay interference: Implications for nanotoxicity testing[END_REF][START_REF] Monteiro-Riviere | Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line[END_REF].Widely used tests LDH and MTT for toxicology screening have been reported to be affected by different NPs. Through MTT assays, particle absorbance adds to the MTT absorbance and cause light absorption artifact, and particles generate formazan adding to the assay absorbance therefore leading to false results. Regarding LDH assay, particles adsorb LDH or inactivate the LDH enzyme.

L

  'un des facteurs les plus importants qui affectent la cohérence des résultats de toxicité est que les nanomatériaux interagissent avec les tests utilisés et entraînent des artefacts et des estimations de toxicité incohérentes. Les interactions possibles des particules sont : (a) les différentes propriétés optiques des nanomatériaux peuvent interférer avec les systèmes d'absorption de la lumière visible ou de détection de la fluorescence, (b) les particules peuvent interférer avec les composés de tests par des réactions chimiques, et (c) les molécules de tests peuvent s'adsorber sur la surface des particules [182,192]. Les tests LDH et MTT largement utilisés pour le dépistage toxicologique ont été signalés comme étant affectés par différentes NPs. Dans le test MTT, l'absorbance des particules s'ajoute à l'absorbance du MTT et provoque un artefact d'absorption de la lumière, et les particules génèrent du formazan qui s'ajoute à l'absorbance du test, ce qui entraîne des résultats erronés. En ce qui concerne le dosage de la LDH, les particules adsorbent la LDH ou inactivent l'enzyme LDH. Grâce au dosage de la LDH, en raison de la présence de particules dans le milieu de dosage pendant l'analyse spectrophotométrique, il se peut qu'il n'y ait pas d'artefact d'absorption de la lumière. La toxicité induite par les NPs de TiO2 a été évaluée par les tests LDH et MTT au chapitre 2. Cependant, après l'exposition des cellules aux NPs de TiO2, aucune corrélation n'a été trouvée entre les changements morphologiques des cellules et les résultats de ces deux essais de cytotoxicité. Pour donner un aperçu des possibilités de biais, l'interaction des NPs de TiO2 avec les composants des tests LDH et MTT dans les conditions acellulaires a été étudiée dans ce chapitre. We hypothesized that TiO2 NPs may either absorb or inactivate the LDH protein with both mechanisms leading to decreased absorbance in the LDH assay. Particle interference was tested by incubating particle suspensions with LDH protein of known concentration and measuring the LDH absorbance using an LDH kit. Within this kit a LDH solution was provided as a positive control solution. This LDH was extracted from bovine heart and prepared in DMEM at 0.16 U/µL concentration. A solution of 0.16 U/µL LDH, and a mixture of 0.16 U/µL LDH and 120 µg/mL TiO2

Figure 28 .

 28 Figure 28. Absorbance at 450 nm of TiO2 NPs (120 µg/mL) and LDH (0.16 U/µL) mixture and LDH (0.16 U/µL) alone (cell free condition). Absorbances are the mean ± SD of three independent experiments. Statistically different from 0.16 U/µL LDH standard ( * * ) P < 0.01, ( * * * ) P < 0.001, ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test, compared with the value of LDH standard.

Figure 29 .

 29 Figure 29. Absorbance measured during incubation of TiO2 NPs with MTT. DMEM was used as a control. The absorbances were measured during 3 hours incubation at 10; 30; 60; 180 min.

Figure 30 .

 30 Figure 30. Corrected cell viability of A549. Control (C) represents unexposed cells.Values are the mean ± SEM. Statistically different from control ( * ) P < 0.05, ( * * ) P < 0.01, ( * * * ) P < 0.0001, ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

Figure 31 .

 31 Figure 31. Corrected cell viability of cocultured cells. Control (C) represents unexposed cells. Values are the mean ± SEM. Statistically different from control ( * ) P < 0.05, ( * * ) P < 0.01, ( * * * ) P < 0.0001, ( * * * * ) P < 0.0001. Statistical analyses were conducted using one-way Anova analyses, followed by Dunnett's test.

  conditions. Particularly, we observed nanoparticle concentration dependent interference with MTT assay. In addition, TiO2 NPs could prevent LDH from being measured by either inactivation or adsorption of LDH. In conclusion, potential artifacts in commonly used cytotoxicity assays should be tested prior performing cytotoxicity evaluation of nanoparticles to avoid misinterpretation of toxicity results. Dans ce chapitre, les tests LDH et MTT qui sont fréquemment utilisés dans l'évaluation de la toxicité des nanoparticules ont été évalués en ce qui concerne leurs potentielles interactions avec les NPs de TiO2. Cette étude met en évidence les artefacts obtenus avec les tests LDH et MTT dans des conditions sans cellule. En particulier, nous avons observé une interférence dépendante de la concentration des nanoparticules avec le test MTT. En outre, les NPs de TiO2 pourraient empêcher la mesure de la LDH par inactivation ou adsorption de la LDH.

  concentrations of PMA. Chapter 1 shows that the differentiation conditions must be carefully assessed within the context of the experimental question. After ensuring the suitable cell culture conditions, biological responses against TiO2 nanoparticles were assessed in the following chapters. The physicochemical features of TiO2 NPs mainly determine their toxicological characteristic and establish the keystone of their fate in the biological system. To date, toxicological evaluations of TiO2 nanoparticles have been extensively studied however what property makes a TiO2 more or less safe has not yet fully been addressed. In this sense, Chapter 2 and Article 1 provide comprehensive conception on the TiO2 nanoparticles toxicity on human lung cells depending on their physicochemical

L

  'objectif principal de ce travail de thèse était d'étudier les effets de diverses propriétés physicochimiques sur la toxicité des NPs de TiO2 envers des cellules pulmonaires humaines, en particulier sur la cytotoxicité, la réponse proinflammatoire et le stress oxydatif. Pour ce faire, 5 types différents de NPs de TiO2 ont été synthétisés et soigneusement caractérisés avec la collaboration de MINES Paris Tech et de l'Université de Turin et ont été évalués systématiquement sur 3 systèmes différents de cellules pulmonaires humaines (cellules épithéliales A549, macrophages différenciés à partir de THP-1, et cocultures de A549/THP-1 différenciées).La différenciation des monocytes représente une étape importante pour garantir des résultats fiables et reproductibles, avant de mener des expériences de toxicité in vitro.Au Chapitre 1, la différenciation des THP-1 en macrophages et son optimisation ont été abordés, la lignée cellulaire monocytaire humaine THP-1 a été très utilisée pour obtenir des macrophages dans les recherches biologiques et toxicologiques et le PMA est fréquemment utilisé pour stimuler leur différenciation en macrophages.Cependant, la concentration en PMA utilisée, ainsi que les durées de traitement varient considérablement dans la littérature et il n'existe pas de protocole standardisé.Par conséquent, avant de faire les essais de toxicité, nous avons déterminé les conditions optimales requises pour la différenciation des THP-1 dans notre laboratoire. Parmi les différents protocoles appliqués (différentes durées de traitement, concentrations de PMA et différentes densités cellulaires), les conditions optimales requises pour une différenciation efficace ont été sélectionnées comme suit : 30 ng/mL de PMA pour une incubation de 24 heures. La concentration de PMA a été déterminée avec soin, car il a été démontré que des concentrations plus élevées de PMA sont associées à une régulation positive de l'expression des gènes inflammatoires[START_REF] Lund | The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus[END_REF][START_REF] Cooke | Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes[END_REF], par rapport à des concentrations plus faibles de PMA. Le Chapitre 1 montre que les conditions de différenciation doivent être soigneusement évaluées dans le contexte de la question expérimentale. Après s'être assuré des conditions de culture cellulaire appropriées, des réponses biologiques envers des NPs de TiO2 ont été évaluées dans les chapitres suivants.Les caractéristiques physicochimiques desNPs de TiO2 déterminent principalement leur profile toxicologique et constituent la base de leur devenir dans le système biologique. À ce jour, les évaluations toxicologiques des NPs de TiO2 ont été largement menées, mais la question de savoir quelle propriété rend un TiO2 plus ou moins sûr n'a pas encore été entièrement traitée. En ce sens, le Chapitre 2 et l'Article 1 présentent une étude complète de la toxicité des NPs de TiO2 sur des cellules pulmonaires humaines en fonction de leurs propriétés physicochimiques. En général, les NPs de TiO2 provoquent une diminution de la viabilité des cellules et induisent une réponse pro-inflammatoire selon le type de cellule. L'effet sur la toxicité était plus important avec des nanoparticules de plus grande taille primaire, moins agglomérées, en forme de bâtonnet et chargées positivement. Au contraire, les NPs de TiO2, quelles que soient leurs propriétés physicochimiques, n'ont pas induit de production significative de ROS dans le système cellulaire A549 et le système de coculture, ce qui est contradictoire avec les études de Simon-Deckers et al. (2008) et Jugan et al.(2012)[START_REF] Simon-Deckers | In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes[END_REF][START_REF] Jugan | Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells[END_REF] où des ROS ont été systématiquement détectés quelles que soient la taille, la phase cristalline, et la forme des NPs de TiO2 étudiés dans des conditions très similaires des nôtres (même modèle de cellules : A549 exposées à différents types de NPs de TiO2, pendant 24 h à 100 µg/mL). La mesure directe des niveaux de ROS avec une grande précision et exactitude est difficile en raison de leur courte durée de vie et de leur réactivité rapide avec les composants régulant l'état redox[START_REF] Katerji | Approaches and methods to measure oxidative stress in clinical samples: Research applications in the cancer field[END_REF]. La mesure indirecte des ROS en examinant les dégradations oxydatives que ces radicaux provoquent sur les lipides, les protéines et les acides nucléiques des cellules est une approche alternative prometteuse. En ce sens, il aurait été intéressant d'étudier plus en détail le stress oxydatif et en particulier les antioxydants enzymatiques (SOD, CAT, GR), les antioxydants non enzymatiques (GSH), la capacité antioxydante totale, la teneur en carbonyle des protéines et la peroxydation des lipides. Cette étude a également mis en évidence l'importance de certains systèmes cellulaires puisque nous avons détecté des différences nettes dans les paramètres de cytotoxicité entre les monocultures et les co-cultures. Les réponses biologiques varient entre les différents systèmes in vitro, de sorte que l'évaluation toxicologique des nanoparticules dans un seul système cellulaire in vitro peut sous-estimer ou surestimer leurs effets, comme le suggèrent Kasurinen et al. [202]. Par conséquent, plus d'un système in vitro devrait être utilisé pour réaliser l'évaluation du danger des nanoparticules. Il aurait été intéressant d'estimer l'absorption et la localisation des nanoparticules dans deux modèles cellulaires par microscopie électronique à transmission et d'évaluer les caractéristiques physicochimiques de l'impact des nanoparticules sur leur internalisation cellulaire. Dans le Chapitre 3 et l'Article 2, nous avons étudié si l'exposition de courte durée à la lumière UV provoquait des changements dans les propriétés physicochimiques de cinq types différents de NPs de TiO2 et affectait finalement leur toxicité sur le système de coculture de cellules pulmonaires humaines. Nous avons observé qu'une irradiation UV de 30 minutes modifiait les charges de surface et le degré d'agglomération des NPs de TiO2. Les NPs de TiO2 pré-irradiées (moins chargées négativement) ont provoqué un effet cytotoxique plus important que les NPs de TiO2 non irradiées. En milieu acellulaire, les NPs de TiO2 induisaient une production de ROS variable en fonction de leurs propriétés physicochimiques : une taille primaire plus petite, une charge de surface négative et une forme sphérique provoquaient un signal DMPO-CO2• -plus intense que celui observé pour les NPs plus grandes, en bâtonnets et chargées positivement. Le potentiel de ce composé à générer de plus grandes quantités de l'anion radical du CO2• -en suspension doit être pris en compte dans la prévention de son risque de lésions oxydatives. Étant donné que les NPs de TiO2 présentes dans l'environnement devraient être exposés aux radiations UV et éventuellement modifiées à la suite d'une photoexcitation, ces résultats peuvent servir de base à des études ultérieures car ils ont des implications importantes pour l'évaluation des risques pour l'environnement et la santé humaine et pour les actions préventives visant à limiter l'exposition humaine. Au Chapitre 4 et à l'Article 3, la formation de la couronne protéique des NPs de TiO2 non irradiées et irradiées aux UV pendant 30 minutes a également été déterminée de qualitative/semi-quantitative. Les paramètres influençant la formation de la couronne protéique sont principalement la taille primaire des nanoparticules et la charge de surface, aucun impact clair de la forme et de l'état d'agglomération n'a été observé. Aucun effet de l'irradiation UV n'a été observé sur la couronne protéique. Lorsque les résultats de toxicité obtenus dans les chapitres 2 et 3 et les résultats de la couronne protéique ont été évalués ensemble, aucune relation claire n'a été observée entre la couronne protéique et la toxicité des NPs de TiO2. Cependant, dans cette recherche, nous avons obtenu les résultats préliminaires, une analyse protéomique basée sur la spectrométrie de masse pourrait être utile pour identifier, quantifier et caractériser la composition de la couronne protéique [203]. Dans ce travail de thèse, la toxicité des NPs de TiO2 a d'abord été évaluée par des tests classiques (MTT et LDH) comme indiqué au chapitre 2. Au Chapitre 5, l'interaction des particules avec les composés de ces tests soit par réaction chimique soit par adsorption des réactifs du test à la surface des particules a été étudiée pour les méthodes LDH et MTT dans des conditions sans cellules. Il a été démontré que les dosages de la LDH sont affectées par les NPs de TiO2 par interaction des NPs avec l'enzyme LDH elle-même, provoquant l'adsorption et/ou l'inactivation de la protéine et une diminution d'activité associée [182,204], comme le montre notre étude où l'interaction NPs de TiO2-LDH a entraîné une diminution d'activité. De même, le TiO2 interfère avec les tests de viabilité du MTT en oxydant le substrat MTT et le TiO2 a induit la transformation du MTT en formazan dans des conditions sans cellule. Comme le suggèrent de nombreux chercheurs, pour réduire les interactions potentielles entre les tests de NPs et les erreurs d'interprétation des résultats qui en découlent, il est très important de choisir la méthode de test in vitro en fonction du type et de la concentration de nanoparticules. Par conséquent, la présence de NPs doit être limitée autant que possible dans l'échantillon final par des lavages et/ou des centrifugations multiples [182]. Dans l'évaluation de la toxicité in vitro des nanoparticules, chaque formulation individuelle doit être testée pour vérifier sa compatibilité avec tous les tests utilisés. Les études de contrôle de la qualité fournissent des données scientifiques plus précises pour l'établissement de réglementations relatives à la production sûre de NPs. Dans l'ensemble, dans cette étude de doctorat, l'impact des propriétés physicochimiques des NPs de TiO2 sur leur toxicité a été systématiquement étudié. Les principales conclusions de cette thèse sont les suivantes :  Les NPs de TiO2 ont causé une cytotoxicité sur des cellules pulmonaires humaines et la cytotoxicité des NPs de TiO2 était dépendante de leur taille, forme, état d'agglomération et fonctionnalisation/charge de surface. L'effet sur la toxicité était plus important avec des nanoparticules de plus grande taille primaire, moins agglomérées, en forme de bâtonnet et chargées positivement. Lors d'une irradiation UV de courte durée, les NPs de TiO2 irradiées ont induit un effet cytotoxique plus important que les NPs de TiO2 non irradiées en lien avec une charge de surface modifiée. Dans la production de ROS acellulaire, les NPs de taille plus petite, de charge de surface négative et de forme sphérique ont provoqué un signal DMPO-CO2• -plus intense que les NPs plus grandes, en forme de bâtonnet et chargées positivement. Les paramètres influençant la formation de la couronne protéique sont principalement la taille primaire des nanoparticules et la charge de surface, aucun impact clair de la forme et de l'état d'agglomération n'a été observé. Cette recherche a soulevé de nombreuses questions nécessitant des recherches plus avancées. Des recherches expérimentales supplémentaires sont nécessaires pour évaluer l'apoptose par la formation de structures apoptotiques et la modification de l'expression de marqueurs comme P53, P21, Bax, Bcl2 et la caspase-3. Comme indiqué ci-dessus, en cas de stress oxydatif, la production d'espèces réactives de l'oxygène et de malondialdéhyde et l'activité de la catalase et du glutathion pourraient être intéressantes à étudier. D'autre part, il a également été démontré que le facteur nucléaire-kappa B (NFκB) est activé en réponse à des cytokines pro-inflammatoires. Par conséquent, des recherches approfondies devraient être entreprises pour évaluer l'implication du NF-κB dans l'inflammation induite par le TiO2. Dans un contexte d'exposition longue ou chronique aux NPs de TiO2, de tels effets peuvent provoquer de nombreux problèmes pathologiques dans les poumons, y compris le cancer. En perspective, le développement de nanoparticules de TiO2 non cytotoxiques dans le cadre de l'approche "safety-by-design" pourrait permettre de contrôler la toxicité de certaines nanoparticules, mais à ce jour, aucune étude ne l'a encore démontré. Les données obtenues dans le cadre de cette thèse contribuent de manière significative à l'établissement des profils toxicologiques de ces nanoparticules. La recherche sur les NPs de TiO2 doit être combinée et soutenue par des études toxicologiques approfondies. IN VITRO DE NANOPARTICULES DE DIOXYDE DE TITANE SUR DES CELLULES PULMONAIRES HUMAINES -IMPACT DES CARACTERISTIQUES PHYSICOCHIMIQUES Spécialité: Génie des Procédés

Table 5 .

 5 Percentage values of differentiated THP-1 cell viability monitored by flow cytometry after treatment with PMA (50, 100, 150 and 200 ng/mL PMA) for 72 h + 5 days resting (without PMA). ........................................................................................................... 41

	viii

Table 2 .

 2 Workplace measurements in TiO2 depending on the activity types[START_REF] Methner | Nanoparticle emission assessment technique (NEAT) for the identification and measurement of potential inhalation exposure to engineered nanomaterials-part b: Results from 12 field studies[END_REF][START_REF] Plitzko | Workplace exposure to engineered nanoparticles[END_REF][START_REF] Ding | Airborne engineered nanomaterials in the workplace-a review of release and worker exposure during nanomaterial production and handling processes[END_REF].

	Background
	p/

cm 3 nm During activity p/cm 3 Sampling size range Mean size a Agglomeration Production (flame pyrolysis)

		7000-20000	21000	5 nm to >32	2 µm	Agglomerated
				µm
	Spraying	33500	144800	10-1000	N/A	Agglomerated
				nm
	Cleaning	7000-20000	22000	5 nm to >32	0.5 -1	Agglomerated
				µm	µm
	(p: particle, a: Mean particle size was taken from the mode sizes in number size distributions
		or estimated from TEM/SEM images).

Table 3 .

 3 In vitro studies in literature releated to NPs size impact on its toxicity.

	NPs	Size (nm)	Cell lines	Evaluation techniques	Main conclusions	Ref
			Osteoblasts,	Alkaline
	TiO2	14-196	Human fetal hepatocyte (L-02),	phosphatase and	Size-dependent cytotoxicity, 100 nm	[98]
			Human embryonic	zymography	critical size
			kidney (HEK 293)	evaluation
			The adrenal
	QDs	2.2, 5.2	phaeochromocytom a (PC12), Murine	MTT assays	Smaller NPs more toxic	[99]
			microglia (N9)
					5 nm, cytotoxic; 15 nm
				Colony forming	non-cytotoxic
	Au	5, 15	Mouse embryo (Balb/3 T3)	efficiency, Trypan Blue	Autophagosomes	[100]
				assays	observed after AuNPs
					5 nm exposed cells
					Au NPs, increased
					toxicity for smaller NPs
	Au Ag	3-38 3-25	Mouse monocyte macrophage cell (J774 A1)	Sizing and counting of cells	Smaller Au NPs up-inflammatory genes regulate the expressions of pro-	[101]
					Ag NPs, no size-
					dependent toxicity
	SiO2	32, 83	Human colorectal adenocarcinoma cells (Caco-2)	WST-1 assays, comet assays

Table 4 .

 4 Protocols for determining optimal conditions for THP-1 differentiation

	1 cells were cultured in Roswell Park Memorial Institute Medium
	(RPMI-1640, Gibco, Life Technologies, France) containing 10% FBS and 1%
	penicillin-streptomycin.
	Optimal conditions required for accurate THP-1 differentiation into macrophages
	were investigated by applying different protocols as summarized in Table 4. First of
	all, PMA was chosen as differentiation agent. Stock solution of PMA (1 mg/mL) was

prepared in dimethyl sulfoxide (DMSO). Fresh dilutions of PMA were prepared in culture medium at 5, 30, 50, 100, 150, and 200 ng/mL to determine the minimum PMA concentration required for the macrophage differentiation. These concentrations of PMA were applied to different density of cells at different exposure times. Afterwards, cell adhesion, morphology, and expression of macrophage surface marker CD11b were evaluated by various protocols (Table

4

).

a After removing the PMA-containing medium then incubating the cells in fresh culture medium, ICC Immunocytochemistry

Table 4 .

 4 Continue Protocols for determining optimal conditions for THP-1 differentiation After removing the PMA-containing medium then incubating the cells in fresh culture medium, ICC Immunocytochemistry

		Protocol	Cell density	PMA concentrati on	PMA exposure time	Method	Parame ter	Ref.
			6.5 x 10 4
	Cell density determination	Protocol 3	cells, well (96 well 10 x 10 4 cells, 13 x 10 4 cells per	30, 50, 100 ng/mL PMA	24 hours	MTT assay	Metaboli c Activity	-
			plate)
	Concentration determination	Protocol 4	2 x 10 5 Slide cells per 8 Well Chamber	30, 50, 100 ng/mL PMA	24 hours	ICC	CD11b expressio n	[144, 153,1 63]

a

Table 5 .

 5 Percentage values of differentiated THP-1 cell viability monitored by flow

	cytometry after treatment with PMA (50, 100, 150 and 200 ng/mL PMA)
	for 72 h + 5 days resting (without PMA).
		50 ng/mL	100 ng/mL	150 ng/mL	200 ng/mL
		PMA	PMA	PMA	PMA
	Cell Viability	59.6%	60.4%	60.1%	75.1%

La principale voie d'exposition aux NPs de TiO2 est l'inhalation. Elle concerne essentiellement le travailleur, le consommateur moyen étant lui plus probablement concerné par la voie alimentaire. Cependant, les atmosphères environnementales et professionnelles ne sont pas bien caractérisées pour les NPs, ce qui peut expliquer le manque d'études épidémiologiques sur la relation entre l'exposition humaine aux NPs et leurs effets négatifs. Malgré cela, pour montrer les associations entre l'exposition aux particules fines et ultrafines présentes dans l'atmosphère et les effets indésirables sur la santé, leur toxicité potentielle a été étudiée sur des modèles animaux et cellulaires. Deux études sur l'inhalation chronique ont signalé un risque de cancer du poumon élevé après exposition à du TiO2 fin [5] ou ultrafin [6] chez le rat. Par conséquent, le Centre international de recherche sur le cancer (CIRC) a classé le TiO2 comme cancérogène possible pour l'homme (groupe 2B) [7]. Cette classification s'applique à toutes les formes de TiO2 sans tenir compte du critère de taille et donc des formes nanométriques. Cependant, compte tenu de leurs spécificités, la structure nanométrique pourrait susciter davantage de préoccupations en matière de santé et de sécurité.En raison des effets adverses observés pour certaines formes de nano TiO2, son usage dans les produits alimentaires sous la forme de l'additif E171 a été suspendue pour un

Article 2. Short pre-irradiation of TiO2 nanoparticles increases cytotoxicity on human lung coculture system

With the growing development of nanoscience and nanotechnology, concerns on the potential health effect of nanoparticles have increased. Titanium dioxide nanoparticles (TiO2 NPs) are widely used in a wide variety of industrial applications, mainly because of their unique properties such as their small size, high refractive index, or photocatalytic properties.

Although there are growing researches on TiO2 toxicity, studies examining the impact of TiO2 NPs physicochemical properties on their toxicity are limited. Therefore, the main aims of this thesis were: (i) to evaluate the toxicity of TiO2 NPs in human lung cells as a function of their main physicochemical properties, (ii) to evaluate if a short time exposure to UV light induced changes in the physicochemical properties of TiO2 NPs and eventually affected their toxicity.

For these purposes, 4 custom made and one commercial TiO2 NPs which differ in size/SSA, shape, agglomeration, and surface functionalization/charge (APTES) were used.

In the first stage of the study, the influence of the physicochemical properties of TiO2 NPs on their toxicity was evaluated on 3 different human lung cell systems (A549 epithelial cells, macrophages differentiated from THP-1, and A549/differentiated THP-1 cocultures). In general, TiO2 NPs caused a decrease in cell viability and induced a pro-inflammatory response depending on the cell type. The effect on toxicity was higher with larger sized, less agglomerated, rod-shaped, and positively charged nanoparticles.

In the second stage of the study, we evaluated if a short time exposure of TiO2 NPs to UV light induced changes in their physicochemical properties and their toxicity. Irradiated TiO2 NPs induced a higher cytotoxic effect than the pristine TiO2 NPs in relation to an altered surface charge.

After examining the toxicity of TiO2 NPs depending on their physicochemical and photocatalytic properties, protein corona composition, which is another factor affecting toxicity, was investigated. The parameters influencing the protein corona composition are mainly nanoparticle primary size and surface charge, no clear impact of the shape and agglomeration state was observed. No effect of UV irradiation was observed on the formation of the protein corona.

Overall, the impact of the TiO2 NPs physicochemical properties on their toxicity was observed and the toxicity was more pronounced after UV irradiation of NPs. Besides another main finding of this study was that the co-culture model seemed more sensitive to the adverse effects of TiO2 NPs than the monoculture model. This study improves our understanding of the hazards and risks that five different types of pristine TiO2 NPs and their UV irradiated counterparts could pose to public health. It can serve as a basis for a safer by design approach to mitigate the toxicity of this material.