Thomas Stalder

Taghrid Zaiter

Wassim El Basset

Raphaël Cornu

Hélène Martin

Mona Diab

Arnaud Béduneau
email: arnaud.beduneau@univ-fcomte.fri.

WOR: water oil ratio W/O: water in oil ZnO: zinc oxide Zn: zinc

Keywords: multi-walled nanotubes medium chain triglyceride MUC5AC, mucin 5AC NPs, nanoparticles polydispersity index PLA, poly-(lactic acid) PLGA, poly-(lactide-co-glycolide) PIT, phase inversion temperature QDs, quantum dots ROS, reactive oxygen species SG, stratum granulosum SS, stratum spinosum SC, stratum corneum simulated gastric fluid SIF, simulated intestinal fluid SOR, surfactant oil ratio SWNTs, single-walled nanotubes SLN, Solid Lipid Nanoparticles SiO2, silicon dioxide TEER, transepithelial electrical resistance titanium dioxide TEM, transmission electron microscopy

List of figures

CHAPTER I: GENERAL INTRODUCTION I. Nanomaterials and nanoparticles

Definition

The definition of nanomaterials (NM) published in 2011 in the Official Journal of the European Commission (2011/696/EU) (1) has been very recently updated (2). NM are defined as natural, incidental, or manufactured materials consisting of solid particles including single, agglomerated, and aggregated particles where at least 50% of particles in the number size distribution have one or more external dimensions comprised between 1 nm and 100 nm.

Compared with the previous definition, two other conditions were added to consider the shape:

-Particles with an elongated shape, such as rods, fibers, or tubes, where two external dimensions are smaller than 1 nm and the other one is larger than 100 nm.

-Particles with a plate-like shape, where one external dimension is smaller than 1 nm and the other ones are larger than 100 nm."

The specific surface by volume greater than 60 m²/cm 3 which was another condition in the definition of 2011 but was not considered anymore. High surface area is not always directly correlated to the dimensions of particles but rather to their internal structure.

Nanoparticles (NPs) are a subclass of NM composed of three dimensions less than 100 nm (3).

Economic market

Due to their physicochemical properties, NM are used in many fields, including electronic, healthcare, aerospace, food, and textile industries. Their use is in constant increase, with an annual growth rate that should reach 13.1 % between 2021 and 2028 (4) (Figure 1).

Figure 1: estimation of various nanomaterials uses over a span of 10 years. According to Market Analysis Report (4) .

The medical segment of NM market is the most important with 29.98% of revenues in 2020.

Other applications of NPs are also continuously growing such as electronics, paints, and energy.

(Figure 2).

Figure 2: applications insights of nanomaterials in 2020. According to Market Analysis Report (4).

Considering the rapid evolution of nanofoods, the part of food industries in the global market of NM should rapidly increase.

The different types of nanoparticles

NPs are generally divided into organic, inorganic, and carbon-based NPs. Organic NPs mainly include polymeric nanospheres and nanocapsules, polymeric micelles, liposomes, nanoemulsions (NE), and dendrimers. Inorganic NPs are represented by silica NPs, metal oxide NPs, metal NPs and quantum dots. The carbon-based nanomaterials include fullerenes, graphenes and carbon nanotubes (5) (Figure 3).

Organic NPs

Polymeric NPs

In 1979, Couvreur et al. developed the first polymeric NPs based on poly-(alkylcyanoacrylates) as a drug delivery system for cancer therapy. New polymers have been used such as poly-(lactic acid) (PLA), poly-(glycolic acid) (PGA) and their copolymer poly-(lactide-co-glycolide) (PLGA). These biocompatible and biodegradable polymers are approved as pharmaceutical inactive ingredient. Their degradation does not lead to the formation of toxic metabolites and their high stability gives them essential characteristics for applications in the biomedical field. There are two types of polymeric NPs, nanospheres and nanocapsules.

Nanospheres are composed of a polymeric matrix in which drug is entrapped. Nanocapsules are composed of a core containing the drug and surrounded by a polymer shell (Figure 4) (6).

Polymeric micelles

Used as drug delivery system for cancer therapy, the polymeric micelles are the result of selfassembly of amphiphilic polymers, usually in aqueous solution (Figure 5). The inner core of these micelles includes a hydrophobic region of polymer that contains the drug, while the hydrophilic region provides stability in the aqueous environment. Depending on the route of administration, the stability of the micelles can be affected by the environmental changes such as the pH and the temperature (7).

Liposomes

The use of liposomes is focused on biomedical applications. They can be easily used for a precise active targeting. Liposomes are lipid vesicles composed of aqueous cavities enclosed by one or several lipid bilayers. They can vary in diameter, from nanometers to microns, depending on their chemical composition and the preparation methods. Hydrophilic and lipophilic drugs can be encapsulated within the inner aqueous compartments and the lipid bilayer, respectively (Figure 6) (9). Liposomes are also used for the protection and transport of nutrients. The stability of liposomes used as drug delivery system can be affected by the pH and temperature changes, causing a rapid degradation and then, the early release of the active compound (10).

Nanoemulsions

NE are lipid drug delivery systems composed of two immiscible liquids such as water and oil and stabilized by an appropriate surfactant. Many surfactants with various characteristics (ionic or nonionic) were used for their preparation. NE are classified in two categories: Oil-in-Water (O/W) and Water-in-Oil (W/O) systems. They have the ability to incorporate lipophilic and hydrophilic drugs and to enhance their oral bioavailability due to their physicochemical characteristics, especially the small droplet size (Figure 7) (11).

Dendrimers

In 1978 the branched molecules were discovered by Fritz Vogtle. Dendrimer structure begins from a central body formed by one or more atoms. From this central structure, branches of other atoms called "dendrons" grow through a variety of chemical reactions (Figure 8). In biomedical field, dendrimers are used as drug delivery system. The active molecules can be covalently bond to the ends of branches or entrapped in the hydrophobic cavities (13).

Inorganic NPs

Metallic and metallic oxide NPs

The most common metallic NPs are synthesized from aluminum (Al), cobalt (Co), gold (Au), silver (Ag), zinc (Zn), iron (Fe), copper (Cu) and titanium (Ti). The metallic oxides such as iron oxide (Fe2O3), ZnO, TiO2 are also widely used. They are prepared by chemical, photochemical, or biological methods. Conjugations with several chemical compounds such as enzymes, ligands and drug were performed for biomedical applications (15,16). Metallic nanoparticles were also developed in other fields, including electronics, cosmetics, and food industries. TiO2

NPs are commonly used as sunscreen, thickening and opacifying agents in food products.

Quantum dots

Quantum dots (QDs) are spherical crystals where the size is comprised between 2 and 10 nm.

They are composed of semiconductor metals (CdS, CdSe, CdTe, ZnS, PbS). Generally, they consist of a semiconductor core, covered with a shell such as ZnS, and a cap that allows a better solubility in aqueous buffers. QDs are used in biomedical domains, especially in cancer therapy where they have a selective binding to malignant cells and thus sparing normal ones from unwanted side effects (17).

Silica NPs

Silicon dioxide appears as the simplest model of tectosilicates. Eight crystallized forms of silica are known today. Their many applications in cosmetics, pharmaceuticals, and food industries require nevertheless a careful evaluation of their toxicity in Human (18). Silica NPs can be synthesized according to three different techniques. Inverse microemulsion, where the spherical micelles are formed by the dissolution of surfactant molecules in the presence of water. This method has been used successfully developed for nanoparticles coating and the attachment of functional groups. The high temperature decomposition of organometallic precursors is also a method used for the synthesis of silica nanoparticles. This method has been widely used in the commercial synthesis of silica nanoparticles under powder form. Sol-gel method involves the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) or inorganic salts as sodium silicate in presence of mineral acid (19).

Carbon-based nanomaterials

Carbon nanotubes are cylindrical particles with an arrangement of carbon atoms in hybrid shapes. Its internal structure is hollow, and the surface consists of one or more layers of graphene sheets. Based on the presence of layer, carbon nanotubes are divided into two categories such as single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) (Figure 9).They have a wide range of applications, especially in the biomedical field (20). Unlike carbon nanotubes, C60 fullerene is characterized by a spherical shape.

Applications of nanoparticles

The NPs currently on the market have large-scale applications in cosmetics, food industry, medicine, renewable energies, electronics, and others (Figure 10).

Cosmetics field

The use of NPs in cosmetic is to increase the stability, the solubility, and the efficiency of active compounds. Cosmetics products should not reach the bloodstream but rather act only on the superficial layers of the skin. TiO2 NPs are used as sunscreen. Their small size increases the surface area, facilitates the spreading, and reduces the whitish appearance compared with microparticles. NPs improve the quality of make-up and hair dyes (23). Cosmetic NE facilitate the skin moisturization and penetration. In addition, they offer a uniform distribution of the product on the skin, and a transparency. Kerastase  Nutritive is a NE developed for moisturizing dry hair (24). Due to their ability to incorporate both hydrophilic and lipophilic substances, liposomes are essential compounds in some anti-aging products, sunscreens, and moisturizers. Some marketed products can be cited, such as liposomes carrying ceramides, tanning agents (25). Solid lipid nanoparticles (SLN) characterized by a solid lipid core unlike NE are used in cosmetics for their stability, their ability to control the active compound release and to protect them from degradations (26). Carbon nanotubes, dendrimers, nanospheres and gold nanoparticles are used also in hair coloring, lotions, creams, hairstyling gel and hair colorant [START_REF] Kaul | Role of Nanotechnology in Cosmeceuticals: A Review of Recent Advances[END_REF]. The small size of NPs confers them very interesting properties in cosmetic products but at the same time, it could be considered as a toxicity factor. NPs could be internalized in cutaneous cells or even reach the bloodstream after penetration in the deep cutaneous layers.

Pharmaceutical field

Pharmacokinetic profile and biodistribution of active compounds can be improved by nanomedicines. NP have the ability, depending on their characteristics, to modulate the distribution, metabolism, and elimination of drugs. They allow the transport of lipophilic drugs and can control the release kinetic of active ingredients (28). Due to the small size and then their high surface area, dissolution rate of nanosized drugs can be widely improved (29). NPs protect the encapsulated active compound from enzymatic and chemical degradations (30).

Liposomes are used in drug delivery because of their ability to encapsulate hydrophilic molecules in the internal phase, as well as hydrophobic molecules in the phospholipid bilayers.

Many liposome-based products are currently on the market (Caelyx ® , Daunoxome ® , Myocet ® …). Their main advantages compared to conventional forms are to modify the pharmacokinetic and the bioavailability of drugs, usually anticancer agents and to reduce side effects. The polymeric NPs are used for the sustained release of active compounds. Metallic nanoparticles can be effectively internalized in organs and cells. The ferumoxytol (Feraheme™) was approved by FDA for the treatment of iron anemia in adult patients. Other forms such as nanoemulsions, dendrimers and inorganic nanoparticles showed a real interest for biomedical applications. Even if the nanoparticles exhibit numerous advantages as drug delivery systems, their toxicity including the empty form (without active compound) must be evaluated using tools adapted to their physicochemical properties. Their biodistribution in the body and the elimination process need also to be fully characterized for safety concerns (31).

Food industry field

Food industry uses nanotechnologies to create functional and preservative ingredients. NM allow increasing the physical stability of food dispersion by reducing the sedimentation/skimming. NE and phospholipid vesicles improve the solubility of active ingredients and flavors (32). Metal oxide nanoparticles, including zinc oxide, titanium dioxide known as E171 in Europe or silica dioxide E551, were added to some pastries as a coloring or opacifying agents. NM are also present in the packaging to increase the shelf life of food. They are usually embedded in plastic matrices to limit bacterial growth or prevent the permeation of gases or UV rays. Silver, zinc oxide and titanium dioxide nanoparticles possess antibacterial properties (34). Other applications of nanotechnologies were reported in the food sector.

Nanoporous materials were developed for water filtration and for removing unwanted flavors or allergens from food products (32). Few studies focused on the consequences of the consumption of NM-containing food in human. Due to their small size, NM could accumulate in the intestinal mucus or in Peyer's patches. A chronic exposure to NPs could alter the integrity of the epithelium and modulate the intestinal permeability (33).

Routes of human exposure to nanoparticles

The effects of NPs on the human body are related to the route of entry into the organism and to their particulate form. NPs can enter the body by ingestion, inhalation, transdermal penetration, or intravascular injection and then be randomly distributed to organs (Figure 11).

Dermal route

Human skin is characterized by a surface area of approximately 2 m². It is composed of three distinct layers, the epidermis which represents the outermost layer, the dermis, and the hypodermis. The skin forms an effective barrier against the invasion of pathogens, chemical and physical attacks, as well as the uncontrolled loss of water and solutes (35). Engineered NPs are used in many skin products as sunscreen, texture agents, colorants, and drug delivery systems. The small size of NPs could promote their interaction with skin cells. NPs could cross the barrier by paracellular pathway, crossing the stratum corneum and passing through the lipid matrix between keratinocytes or along the hair follicles (Figure 12) (36). However, most of NPs are usually located in the epidermis which represents a very efficient protective barrier.

Oral route

A large part of NPs enters in the human body by the oral route. NPs are used as anti-caking agents to obtain a more homogeneous, smoother mixture, but also to improve the assimilation of nutrients. TiO2 nanoparticles are present in most toothpaste (1-10 µg/mg of product). Their use in food products represents a significant part of oral exposure to NPs in Human (38). They are also present in environment, including natural and anthropogenic NPs. Silica nanoparticles are used as an anti-caking agent (food additives E550/551) in most food powders such as the salt. Both in vitro and in vivo studies reported that some NPs are able to cross the intestinal barrier, depending on their physicochemical characteristics and compositions (39). However, the translocation pathway stays unclear. Immunohistochemically monitoring is faced with the nanometric scale which requires a high-resolution level for imaging instruments. The translocation of ingested NPs can occur by several routes including the paracellular and transcellular routes (Figure 13) (40). The paracellular passage is classically an exchange pathway for water and electrolytes. Only small hydrophilic molecules cross the intestinal epithelium by this route in healthy individuals (41). However, NPs can increase the intestinal permeability by acting on the tight junctions and then promote the paracellular transport (42).

Transcellular transport occurs by endocytosis and transcytosis through enterocytes and M cells in Peyer's patches for small NPs characterized by diameter below 100 nm. (43).

Pulmonary route

Inhalation is a very commune route of exposure for NPs. Once inhaled, NM can either be exhaled or be deposited in the different regions of the respiratory tree. The upper airways include nasal cavities, mouth, pharynx, and larynx. The tracheo-bronchial tree is composed of the trachea, bronchi, and bronchioles and the pulmonary alveoli (Figure 14). The deposition of NPs depends on the diameter, the aggregation/agglomeration degree, and the density. Particles with a diameter between 10 and 100 nm are mainly deposited in the deep lung, at the level of the pulmonary alveoli. The penetration of NM through the respiratory tract is greater in impaired lung functions due for example, to chronic bronchitis (45).

Ocular route

The ocular route concerns fine particles in the air but also NPs for therapeutic purposes (Figure 15). They can be administered by periocular, intravitreal injection or corneal absorption [START_REF] Souto | Advanced Formulation Approaches for Ocular Drug Delivery: State-Of-The-Art and Recent Patents[END_REF].

Parenteral route

The parenteral route is used for the administration of NPs for biomedical applications. NPs can be intravenously, subcutaneously, intradermally, or intramuscularly injected. The intravenous route provides an instantaneous response. It is also suitable for drugs that cannot be absorbed by the gastrointestinal tract or cannot be injected into muscles or other tissues [START_REF] Parker | Pharmacoeconomics of intravenous drug administration[END_REF]. Intravenously injected NPs improve the pharmacokinetic profile of drugs by extending the plasma half-life and preserve them from chemical degradations. They can also control the biodistribution by targeting specific tissues and cells. Abraxane ® , an albumin NP was approved by the FDA in 2006 for paclitaxel-based treatments [START_REF] Kalepu | Insoluble drug delivery strategies: review of recent advances and business prospects[END_REF]. A better therapeutic response was observed in women with a metastatic breast cancer compared with the standard formulation of paclitaxel [START_REF] Chenthamara | Therapeutic efficacy of nanoparticles and routes of administration[END_REF]. Distribution of untargeted NPs after intravenous administration was observed in many organs. 60% of the injected dose of gold NPs was detected in the liver, lungs, spleen, heart, kidneys and brain after intravenous injection [START_REF] Bednarski | The influence of the route of administration of gold nanoparticles on their tissue distribution and basic biochemical parameters: In vivo studies[END_REF].

Uncoated NPs characterized by a hydrophobic surface are mainly phagocytosed by macrophages in liver, spleen, and lungs, especially when the diameter is higher than 5 nm.

Smaller NPs are eliminated from the body by renal excretion. Surface modification is an effective way to reduce clearance and enhance cellular uptake for a maximum drug accumulation in target sites [START_REF] Chenthamara | Therapeutic efficacy of nanoparticles and routes of administration[END_REF].

Regulations of nanomaterials

The articles L-523-1 to L523-3 of The French environmental Code require an annual declaration system for "substances in nanoparticle state". From January 1 st , 2013, all the manufacturers, importers, and distributors of NM need to fill a declaration in the R-nano register when quantities are greater than 100g per year. It must include the quantity, the physicochemical properties, and the application. The available information regarding the health and environmental hazards must be mentioned [START_REF]Nanomatériaux, nanoparticules[END_REF]. From January 1 st , 2020, the European Commission requested to identify the size, shape, surface structure as well as dissolution and stability parameters of nanomaterials [START_REF][END_REF]. Regulations are recommended for the use of nanoparticles in various fields, including food industry, cosmetics, and the pharmaceutical applications. Since 2014, the European INCO regulation (Information du Consommateur UE 1169/2011) has required that the mention "nano" should appear on the labels of nanoparticle-containing foods [START_REF] Aschberger | Considerations on information needs for nanomaterials in consumer products[END_REF]. In 2011, the European Food Safety Authority (EFSA) published a guide for the risk assessment of nanoscience and nanotechnology applications in the food chain. This guide details the risk assessment procedure in three steps: identification, exposure assessment and risk characterization (55). Titanium dioxide was authorized as a food additive named E171 by European regulations in foodstuffs, mainly in confectionery, bakery products and sauces. It is also used in other fields like cosmetics, paints, and medicines. In May 2021, EFSA concluded that titanium dioxide could no longer be considered safe as a food additive, due to its genotoxic potential and accumulation in the organism [START_REF]Dioxyde de titane : le E171 n'est plus considéré comme sûr en tant qu'additif alimentaire[END_REF]. EFSA panel published in 2018 a re-evaluation of the E551 as a food additive. The panel concluded the European specifications of the additive are still insufficient. This requests a better size characterization of silica particles to identify the presence and the ratio of nano silicates. Besides, it was not possible to confirm the current acceptable daily intake not specified due to the limited toxicological data. The Cosmetics Regulation made obligatory, from July 2013, the reporting of the "nanomaterials" presence in the ingredient list of cosmetic products. In February 2022, the European Commission notified the world trade organization a draft amendment to cosmetics regulation. The aim is to prohibit the use of nanomaterials in cosmetic products for which the Scientific Committee on Consumers Safety (SCCS) identified a risk for human health. This is the case for copper NPs. In France, nanoparticles used in cosmetics are subject, like other nanomaterials, to the obligation of declaration in the R-nano register [START_REF]Scientific advice on the safety of nanomaterials in cosmetics[END_REF]. Concerning the use of nanoparticles in the pharmaceutical field, it was extremely difficult to establish a single regulation given the diversity of nanomedicines, their chemical nature and their many applications [START_REF]Nanotoxicologie et réglementation des nanomédicaments[END_REF]. In general, there was a lack of studies in the evaluation of nanomedicines. Currently, new nanoparticle-based drugs are evaluated by the Food and Drug Administration (FDA), the European Medicines Agency (EMA) and other agencies using a benefit/risk analysis approach [START_REF] Desai | Challenges in Development of Nanoparticle-Based Therapeutics[END_REF].

CHAPTER II: NANOTOXICITY: BIBLIOGRAPHIC PART 1. Nanotoxicology

The fundamental aspect for understanding the toxicity of NPs is their physicochemical characterization. Their small size, surface charge and composition are critical factors governing the interactions with the biological environment. These bio-nano interactions are essential for understanding the biodistribution and toxic effects of many nanomaterials in both in vitro and in vivo models. Nanomaterials can form "aggregates" or "agglomerates" in contact with biological fluids, modulating the toxicity of the original nanomaterial (1). Due to their small size, NPs have the ability to penetrate the human body, cross the various biological barriers and reach the most sensitive organs (2). For particles with the same composition, the size change leads to different levels of cytotoxicity (3). Small NPs can be internalized in cells, causing toxicity issues (4).Changes in the surface charge result also in dramatic differences in cell internalization and biodistribution of NPs. NPs with a positively charged surface are known to have the highest toxicity. The chemical composition also influences the toxicity of NPs. In the literature, the most documented toxic NPs are metallic type NPs (5).

Toxicological model studies

To study the toxic effects of nanomaterials, cellular and animal models are usually used. The choice of model depends essentially on the study objective and the expected result. From literature, rats and mice are the most common species for in vivo toxicity studies. However, the pharmaceuticals regulation requires to include non-rodent species in safety studies. The number of in vitro toxicity studies have been multiplied in recent years due to the growing development of alternative methods to the use of animals in experiments (5). Cell types are mainly macrophages, blood cells, hepatocytes, epithelial and endothelial cells (7). An in vitro and in vivo correlation is necessary to validate the cellular model.

Oral toxicity

Ingested nanoparticles mainly come from drug delivery systems and food products. They can be also released from packaging materials. Another origin is their presence in in the air and drinking water, including natural and anthropogenic NPs. After ingestion, they are in contact with the gastrointestinal tract, especially the intestinal barrier which offers a large exposure surface. A review of the literature on the intestinal toxicity of ingested NPs was carried out in and inorganic (silica, titanium dioxide, metal and metal oxide NPs) materials [START_REF] Maroof | Scope of Nanotechnology in Drug Delivery[END_REF]. Some of them, especially inorganic NPs are present in food products due to their coloring or texturing properties. Organic NPs especially lipid and polymeric NPs are mainly used as drug delivery systems to enhance the solubility, stability, and bioavailability of drug molecules [START_REF] Dilnawaz | Polymeric Biomaterial and Lipid Based Nanoparticles for Oral Drug Delivery[END_REF]. Humans can also ingest natural and incidental anthropogenic NPs present in the environment. Silica and iron NPs are released from volcanic ash clouds while carbon nanotubes were detected during the combustion of pines [START_REF] Griffin | Natural Nanoparticles: A Particular Matter Inspired by Nature[END_REF]. After oral administration, NPs can reach the Gastrointestinal Tract (GIT), where the intestine, which offers a large exposure surface, is present. Their small dimensions and their high surface area grant those many advantages as food and pharmaceutical ingredients. At the same time, these features promote their interactions with the biological environment, including cells, mucus and bacteria. An alteration of physical and functional properties of the intestine could be reasonably assumed. However, the fate and the toxicity of NPs after oral ingestion has not been yet fully elucidated due to the growing number of nanomaterials and the lack in toxicological studies.

Are they able to modulate the intestinal permeability or reach the systemic circulation after translocation? Can they affect the microbiota and the epithelial cells, inducing digestive disorders, inflammatory responses, genotoxicity or oxidative stress? This present work faces the published toxicological data on ingested nanomaterials to identify the critical factors responsible for the intestinal nanotoxicity.

II-The multilayer intestinal barrier

The intestine is an important body organ which prevents microorganisms and xenobiotics including NPs from reaching the systemic circulation [START_REF] Lundquist | Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues[END_REF][START_REF] Segata | Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples[END_REF]. The intestine could be considered as a barrier composed of multilayers. The first one is the bacterial microbiota. It is responsible for many functions such as regulating host immunity, protecting against pathogens and preserving gut integrity [START_REF] Thursby | Introduction to the human gut microbiota[END_REF].

It also plays an important role in the collection of energy from food to synthetize vitamins and amino acids. Disruption of the bacterial microbiota can lead to many diseases as the Inflammatory Bowel Disease (IBD) and metabolic syndrome [START_REF] Barko | The Gastrointestinal Microbiome: A Review[END_REF]. The mucus represents the second layer which protects the epithelium from mechanical damages. Mucus acts as a physical barrier, hindering pathogens from reaching epithelial [START_REF] Pelaseyed | The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system[END_REF].

This hydrogel is composed of large glycoproteins, mainly mucins. Mucins are organized in two adjacent layers. The inner one is thin and strongly adherent in contrast to the outer one. The main mucins secreted in the small intestine by goblet cells are the MUC2 and MUC5AC. In the jejunum, each MUC2 binds to three other mucins to form a hexagonal mesh. The synthesis of these mucins is influenced by the intestinal microbiota as well as the luminal substances including bioactive compounds present in functional food [START_REF] Damiano | Quercetin Increases MUC2 and MUC5AC Gene Expression and Secretion in Intestinal Goblet Cell-Like LS174T via PLC/PKCα/ERK1-2 Pathway[END_REF][START_REF] Walsh | Expression of MUC2, MUC5AC, MUC5B, and MUC6 mucins in colorectal cancers and their association with the CpG island methylator phenotype[END_REF]. The intestinal epithelium represents the third layer. It is formed of a continuous polarized monolayer of cells interconnected and bound to a basement membrane by proteins, mainly integrins [START_REF] Pompili | The Charming World of the Extracellular Matrix: A Dynamic and Protective Network of the Intestinal Wall[END_REF]. It regulates solute transport and selective transporters for amino acids, electrolytes, short chain fatty acids and sugars [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF]. The epithelium is mainly composed of absorptive enterocytes, covering a large surface, and making up to 80-90 % of the epithelium and goblet cells (8 %). The apical membrane of the enterocytes is covered by microvilli that increase the intestine's surface area (Faust et al., 2014b;[START_REF] Lundquist | Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues[END_REF]. Paneth cells, enteroendocrine cells, Microfold cell (M cells) and stem cells are also present in the intestinal epithelium but at a smaller proportion [START_REF] Gehart | Tales from the crypt: new insights into intestinal stem cells[END_REF]. M cells located in the Peyer's patches are responsible for the transport of antigens from the lumen of the small intestine to the lymphoid follicles. They are involved in the mucosal immunity response [START_REF] Corr | M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis[END_REF].

Intestinal permeability is governed by two major pathways: paracellular and transcellular routes. The intercellular space is regulated by proteins [START_REF] Salvo Romero | The intestinal barrier function and its involvement in digestive disease[END_REF]. They are involved in different types of junctions such as Tight Junctions (TJs), anchoring and communicating junctions. TJs (as occludins, claudins), Junction Adhesion Molecules and tricellulin regulate the paracellular trafficking of molecules. They are arranged as multiprotein complexes that form a selective permeable seal between adjacent epithelial cells [START_REF] Lee | Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases[END_REF].

The expression of claudins 1, 4 and 5 consolidates the permeability of TJs while claudin-2 depletion enhances the barrier function of the intestine [START_REF] Suzuki | Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium[END_REF][START_REF] Weber | Dynamic properties of the tight junction barrier[END_REF]. TJs are associated with Zonula Occludens (ZO-1 ZO-2 and ZO-3) which are bound to F actin [START_REF] Rodgers | Regulation of epithelial permeability by the actin cytoskeleton[END_REF]. Thus, ZO proteins allow the anchorage of TJs to the actin cytoskeleton. The interaction loss between TJs and cytoskeleton by actin depolymerisation can lead to a drastic increase in the intestinal paracellular permeability [START_REF] Shen | Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis[END_REF].

Occludin phosphorylation by protein kinase C (PKC) can also weaken the interaction with actin, leading to a dissociation from the junctional complex [START_REF] Lee | Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases[END_REF]. The inflammatory response involving the activation of the subepithelial immune system with dendritic cells, neutrophils, and macrophages can affect the TJ integrity. Interleukin 8 (IL-8) which is a major neutrophil recruiting chemokines induces actin rearrangement, thus increasing TJ permeability [START_REF] Gerloff | Influence of simulated gastrointestinal conditions on particle-induced cytotoxicity and interleukin-8 regulation in differentiated and undifferentiated Caco-2 cells[END_REF][START_REF] Talavera | IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers[END_REF]. Other cytokines like TNF-α, IL-1β, IL-6, IL-13 and IL-17 can affect the actin arrangement and increase claudin-2 expression through the MEK/ERK and PI3K pathways [START_REF] Mankertz | TNFalpha up-regulates claudin-2 expression in epithelial HT-29/B6 cells via phosphatidylinositol-3-kinase signaling[END_REF][START_REF] Nighot | Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation[END_REF][START_REF] Shen | Tight junction pore and leak pathways: a dynamic duo[END_REF][START_REF] Suzuki | Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium[END_REF][START_REF] Talavera | IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers[END_REF]. On the contrary, anti-inflammatory cytokines secreted by regulatory T cells (T4) such as the IL-10 and TGF-β increase TJs' expression through MEK/ERK signalling and therefore reduce the intestinal permeability [START_REF] Sun | Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition[END_REF]. The anchoring junctions connect the cytoskeleton of each cell to that of neighbouring cells or to the extracellular matrix. They include adherent junction and desmosomes and play an important role in various cell processes such as differentiation, proliferation, and morphogenesis. The communicating junctions link the cytoplasm of adjacent cells by forming a channel of connexins through their membranes. They also play a crucial role in the growth and development of epithelial cells as well as in the barrier function maintenance [START_REF] Salvo Romero | The intestinal barrier function and its involvement in digestive disease[END_REF]. The transcellular pathway consists of transporting molecules across the enterocytes using different mechanisms. This can include energy-dependent selective transporters (active transport), passive or facilitated diffusion, and receptor or absorptive-mediated endocytosis.

The type of mechanism involved depends on the size and physicochemical properties of the molecules [START_REF] Turner | Intestinal mucosal barrier function in health and disease[END_REF]. Passive transport enables the diffusion of small lipophilic molecules through the enterocyte membrane. The solute carriers, termed SLC, carry drugs according to their concentration gradient without adenosine triphosphate (ATP) molecules (Liu and Liu, 2013;[START_REF] Oostendorp | The biological and clinical role of drug transporters at the intestinal barrier[END_REF]. Transporters of the ATP-binding cassette family such as the P-glycoprotein (P-gp) are involved in the efflux mechanisms. They prevent the systemic distribution of toxins and some xenobiotics located in the intestinal lumen [START_REF] Fu | Where is it and How Does it Get There -Intracellular Localization and Traffic of Pglycoprotein[END_REF].

Endocytosis is a vesicle-mediated transcellular transport. Various endocytic processes were reported such as pinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-caveolae independent endocytosis [START_REF] Panariti | The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions?[END_REF][START_REF] Talkar | Transmucosal Nanoparticles: Toxicological Overview[END_REF][START_REF] Zhang | Mechanism study of cellular uptake and tight junction opening mediated by goblet cell-specific trimethyl chitosan nanoparticles[END_REF].

III-Ingested nanoparticles 1. Nanoparticle description

NPs administered by the oral route are used as drug delivery systems, imaging agents and food additive. Many research works focused on innovative biomedical applications of NPs, especially for the diagnosis and the treatment of colorectal cancer and IBD [START_REF] Pridgen | Polymeric nanoparticle drug delivery technologies for oral delivery applications[END_REF][START_REF] Tyagi | Nanoparticles -An Overview of Classification and Applications[END_REF][START_REF] Abbas | The Potential Role of Nanoparticles as an Anticancer Therapy in the Treatment of Rectal Cancer[END_REF]. Moreover, the oral route for drug administration offers many advantages such as convenience and medication compliance, especially in the treatment of chronic or long-term diseases that require frequent administration [START_REF] Pridgen | Polymeric nanoparticle drug delivery technologies for oral delivery applications[END_REF]. Active pharmaceutical ingredients and imaging agents entrapped in NPs are protected from chemical and enzymatic degradations [START_REF] Araújo | Safety and toxicity concerns of orally delivered nanoparticles as drug carriers[END_REF][START_REF] Ojer | Toxicity evaluation of nanocarriers for the oral delivery of macromolecular drugs[END_REF]. Insulin and calcitonin were protected against proteases after encapsulation in polymeric NPs [START_REF] Lowe | Calcitonin and Insulin in Isobutylcyanoacrylate Nanocapsules: Protection Against Proteases and Effect on Intestinal Absorption in Rats[END_REF]. The encapsulation improves the solubility and the systemic absorption of drugs [START_REF] Tan | Improved Bioavailability of Poorly Soluble Drugs through Gastrointestinal Muco-Adhesion of Lipid Nanoparticles[END_REF]. A 7.3-fold increase of the curcumin bioavailability was observed with nanoemulsions of ethyl oleate [START_REF] Wan | Novel nanoemulsion based lipid nanosystems for favorable in vitro and in vivo characteristics of curcumin[END_REF]. E551 food additive composed of amorphous silica NPs is commonly used as anti-caking or thickening agents (Fruijtier-Pölloth, 2016).

Titanium dioxide (TiO2) NPs contained in E171 additive are widely present in food products due to their colouring and opacifying properties [START_REF] Younes | Safety assessment of titanium dioxide (E171) as a food additive[END_REF]. Inorganic NPs were also used in packaging materials to protect food products. Containers were designed with antimicrobial Titanium, Zinc (Zn) or other inorganic NPs to prevent the proliferation of bacteria [START_REF] Carrillo-Inungaray | Chapter 15 -Use of Nanoparticles in the Food Industry: Advances and Perspectives[END_REF]. However, their release in food products was reported [START_REF] Huang | Costbenefit analysis of capecitabine versus 5-fluorouracil/leucovorin in the treatment of colorectal cancer in the Netherlands[END_REF]. Humans are also exposed to natural nanomaterials. Volcanic activity, rock weathering and forest fires produce inorganic NPs such as silica, iron and carbon NPs (Barhoum et al., 2022). Anthropogenic NPs are incidentally released in the environment during the industrial processes and the combustion of fuels such as carbonaceous NPs (D'Anna, 2009).

Fate of ingested NPs

After ingestion, NP diffuses into the GIT. The GIT has a pH gradient varying from highly acidic in the stomach to basic with a pH of 7.5 in the colon [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. During the digestion process, NPs undergo numerous chemical modifications as oxidation, deamidation and hydrolysis [START_REF] Sood | Peroral route: an opportunity for protein and peptide drug delivery[END_REF]. In addition, gut enzymes as proteases, nucleases and lipases present in the GIT are also involved in the degradation of NPs [START_REF] Ganapathy | Protein Digestion and Absorption[END_REF].

The feeding status and meal content also need to be considered [START_REF] Lundquist | Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues[END_REF]. Therefore, potential exposure of NPs to proteins, carbohydrates and lipids present in food products in gastrointestinal fluid can also modify their physicochemical properties.

Changes in the surface properties due to adsorption at the surface of NPs as well aggregation/agglomeration or dissolution processes were reported [START_REF] Wang | Formation of Protein Corona on Nanoparticles with Digestive Enzymes in Simulated Gastrointestinal Fluids[END_REF][START_REF] Shi | Nanoparticle Digestion Simulator Reveals pH-Dependent Aggregation in the Gastrointestinal Tract[END_REF][START_REF] Mbanga | Dissolution kinetics of silver nanoparticles: Behaviour in simulated biological fluids and synthetic environmental media[END_REF]. The aggregation state, surface charge and morphology of polyvinylpyrrolidone-coated silver (Ag-PVP) NPs were modified in contact with simulated gastrointestinal fluids. The acidic pH of human gastric intestinal fluids in a fasted state termed FaSSGF, induced the release of silver (Ag) ions, subsequently forming an AgCl precipitate [START_REF] Jiang | Influence of gastrointestinal environment on free radical generation of silver nanoparticles and implications for their cytotoxicity[END_REF]. Agglomerates of TiO2 NPs were observed in simulated gastric fluid compared to water [START_REF] Jones | Human in vivo and in vitro studies on gastrointestinal absorption of titanium dioxide nanoparticles[END_REF]. Gastric digestion induced the clustering of silica (SiO2)

NPs due to the acidic pH and high electrolytes concentrations. However, NPs were reformed in the intestinal fluid, suggesting a pH-dependent agglomeration state [START_REF] Peters | Presence of Nano-Sized Silica during In Vitro Digestion of Foods Containing Silica as a Food Additive[END_REF].

The interaction of NPs with the mucus layer is mainly modulated by their physicochemical properties; especially size, surface charge and chemistry (Figure 1) [START_REF] Lamprecht | Size-dependent bioadhesion of micro-and nanoparticulate carriers to the inflamed colonic mucosa[END_REF].

The mucus mesh size of approximately 100 nm allows only the diffusion of small particles [START_REF] Fröhlich | Models for oral uptake of nanoparticles in consumer products[END_REF][START_REF] Olmsted | Diffusion of macromolecules and virus-like particles in human cervical mucus[END_REF]. For example, Ag particles characterized by a size of 200 nm were entrapped to a great extent in the mucus layer of a TC7/Caco-2 co-culture model unlike 20 nm NPs (Georgantzopoulou et al., 2016a). Similarly, SiO2 NPs with hydrodynamic diameters of 20 and 30 nm were able to reach the HT29-MTX goblet cells while NPs of 70 and 200 nm were mainly stuck to the mucus layer [START_REF] Zaiter | Size effect and mucus role on the intestinal toxicity of the E551 food additive and engineered silica nanoparticles[END_REF]. Mucins facilitate the mucoadhesion by electrostatic interactions of positively charged NPs [START_REF] Fröhlich | Models for oral uptake of nanoparticles in consumer products[END_REF]. Moreover, interactions with mucins can occur by hydrogen binding, Van der Waals interactions, hydrophobic forces or polymer chain interpenetration [START_REF] Ojer | Toxicity evaluation of nanocarriers for the oral delivery of macromolecular drugs[END_REF][START_REF] Pridgen | Polymeric nanoparticle drug delivery technologies for oral delivery applications[END_REF][START_REF] Talkar | Transmucosal Nanoparticles: Toxicological Overview[END_REF][START_REF] Woodley | Bioadhesion: new possibilities for drug administration?[END_REF]. Some NPs as polyethylene glycol (PEG)-coated NPs were characterized by mucus-penetrating properties [START_REF] Fröhlich | Models for oral uptake of nanoparticles in consumer products[END_REF][START_REF] Lai | Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus[END_REF][START_REF] Pridgen | Polymeric nanoparticle drug delivery technologies for oral delivery applications[END_REF]. PEG confers hydrophilic properties and a neutral charge to NPs, reducing the interactions with mucins. However, long PEG chains limit the mucus penetration due to steric hindrance [START_REF] Lundquist | Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues[END_REF].

Mucus-penetrating NPs reach the epithelial cell surface. Different basic internalization mechanisms of particles in cells were reported. Macro-pinocytosis is non-specific and induces the formation of vesicles with about 1 µm in diameter. It allows the internalization of large NPs or agglomerates in cells [START_REF] Sahay | Endocytosis of nanomedicines[END_REF]. Clathrin-mediated and caveolae-mediated endocytosis form vesicle at specific regions of the membrane with a diameter of less than 0.1 µm. They are the main cellular internalization pathways of nanomaterials such as PEGpolylactic acid (PLA), polylactic-co-glycolyc acid (PLGA), SiO2, chitosan and gold (Au) NPs [START_REF] Sahay | Endocytosis of nanomedicines[END_REF]. Clathrin-independent and caveolae-independent endocytosis induce the formation of small vesicles and occur continuously in the cell. They are also involved in the cell uptake of polymeric NPs such as PLGA NPs [START_REF] Palocci | Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection[END_REF]. The caveolae/lipid raft is the main internalization pathway of nanoemulsions [START_REF] Fan | Endocytosis of Corn Oil-Caseinate Emulsions In Vitro: Impacts of Droplet Sizes[END_REF]. The physicochemical properties of NPs, in particular their size and surface charge, are the most important factors involved in the cell uptake [START_REF] Sahin | A small variation in average particle size of PLGA nanoparticles prepared by nanoprecipitation leads to considerable change in nanoparticles' characteristics and efficacy of intracellular delivery[END_REF]. The size range comprised between 10 and 60 nm was considered as an optimum diameter. As for the charge, it was reported that positively charged NPs electrostatically bind to the negative cytoplasmic membrane and are then endocytosed [START_REF] Sabourian | Effect of Physico-Chemical Properties of Nanoparticles on Their Intracellular Uptake[END_REF]. However, they are not able to cross the mucus layer in a significant manner. The interaction of negatively charged NPs with the cell membrane should be low compared with cationic and neutral particles. However, a significant internalization of carboxymethyl dextran-coated NPs with a surface charge of -50 mV was observed in Caco-2 cells [START_REF] Ayala | Effect of surface charge on the colloidal stability and in vitro uptake of carboxymethyl dextran-coated iron oxide nanoparticles[END_REF].

Figure 1: Schematic representation of the main factors affecting the intestinal distribution of NPs

Very few studies investigated the transport of NPs across the intestinal barrier. After oral administration, SiO2 NPs were found in kidneys and liver, suggesting their ability to cross the intestinal barrier [START_REF] Lee | Tissue distribution and excretion kinetics of orally administered silica nanoparticles in rats[END_REF]. A translocation of TiO2 NPs through the ileum and Peyer's patches was reported [START_REF] Brun | Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia[END_REF]. Oral administration of 75 nm TiO2 NPs in young rats for 30 days also caused hepatic damages [START_REF] Wang | Susceptibility of young and adult rats to the oral toxicity of titanium dioxide nanoparticles[END_REF]. This is in accordance with the elimination pathway of circulating NPs which involves Kupffer cells [START_REF] Moghimi | Role of Lactobacillus reuteri in Human Health and Diseases[END_REF].

However, the mechanism involved in the transepithelial transport of NPs remains unclear even if the transcytosis across the Peyer's patches seems the potential route (Jani et al., 1992).

IV. Nanotoxicology

The main toxicity endpoints reported in literature include cytotoxicity, genotoxicity, and inflammatory response on intestinal cells using both in vitro and in vivo models. Inorganic NPs were mainly responsible for intestinal damages (Table 1) while very few studies clearly demonstrated a toxicity of organic NPs (Table 2). Their influence on the microbiota and the mucus protective barrier was slightly investigated despite their critical role in the intestinal homeostasis.

Microbiota and mucus interactions

The influence of NPs on the GIT bacteria is mainly controlled by electrostatic interactions which depends on the pH and the composition of the gastrointestinal fluids [START_REF] Gangadoo | Inorganic nanoparticles as food additives and their influence on the human gut microbiota[END_REF]. Their effect is mediated through various mechanisms, such as Reactive Oxygen Species (ROS) production, release of cationic ions and disruption of cell membrane (Figure 2). Cationic

Au NPs were more toxic on both Gram-positive and Gram-negative bacteria compared with anionic NPs [START_REF] Vivian Feng | Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gramnegative and Gram-positive bacteria[END_REF]. The toxicity was explained by an accumulation at the surface of bacteria, likely causing membrane destabilization. Modifications of microbiota after oral exposure to TiO2 NPs were also reported. A significant increase in Lactobacillus reuteri was observed after 90-day oral administration [START_REF] Chen | Hepatotoxicity and the role of the gut-liver axis in rats after oral administration of titanium dioxide nanoparticles[END_REF]. This bacteria is beneficial for human health due to its ability to produce metabolic molecules and prevent the migration and expansion of opportunistic pathogens (Mu et al., 2018). In contrast, chronic ingestion of rutile TiO2 NPs-containing foods reduced the proliferation of Bifidobacterium and facilitated the invasion of opportunistic pathogens such as Escherichia-Shigella [START_REF] Li | Oral administration of rutile and anatase TiO2 nanoparticles shifts mouse gut microbiota structure[END_REF]. Toxicity of Ag NPs on commensal bacteria in human GIT was also identified due to their antibacterial properties [START_REF] Mercier-Bonin | Mucus and microbiota as emerging players in gut nanotoxicology: The example of dietary silver and titanium dioxide nanoparticles[END_REF]. Two mechanisms were hypothesized: an accumulation of Ag NPs in the bacterial cell membrane or the Ag cations release, inducing ROS production [START_REF] G1108 Ladaycia | Microbiota and nanoparticles: Description and interactions[END_REF]. SiO2 NPs in the GIT increased the abundance of Lactobacillus genus after oral ingestion. A complexation of SiO2 NPs with bacteria was reported, reducing their cytotoxicity on human epithelial cells [START_REF] Siemer | Nanosized food additives impact beneficial and pathogenic bacteria in the human gut: a simulated gastrointestinal study[END_REF]. The interactions of inorganic NPs with the microbiota seems to depend on the bacteria type, especially the composition of the cell membrane and on the features of NPs [START_REF] Vivian Feng | Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gramnegative and Gram-positive bacteria[END_REF]. Metallic NPs are well-known for their antibacterial activity while SiO2 and TiO2 inhibit or induce the proliferation of commensal bacteria, depending on the strain [START_REF] Slavin | Metal nanoparticles: understanding the mechanisms behind antibacterial activity[END_REF]. Gut microbiota dysbiosis was also observed in mice after oral exposure to polystyrene (PS) NPs having a size of 500 nm for 5 weeks. The population of Firmicutes and α-Proteobacteria was significantly affected at a concentration in NPs of 1mg/L [START_REF] Lu | Polystyrene microplastics induce gut microbiota dysbiosis and hepatic lipid metabolism disorder in mice[END_REF]. Multi-walled carbon nanotubes (MWCNTs)

administered by intratracheal instillation in doxorubicin-treated mice affected the gut microbiota through the proliferation of Helicobacteraceae and Coriobacteriaceae [START_REF] Liu | Multiwalled carbon nanotubes exacerbate doxorubicin-induced cardiotoxicity by altering gut microbiota and pulmonary and colonic macrophage phenotype in mice[END_REF]. et al., 2020). Changes in the composition of neutral and acidic mucins were also observed [START_REF] Mercier-Bonin | Exposition orale et devenir dans l'intestin des nanoparticules alimentaires : exemple de l'argent et du dioxyde de titane[END_REF].

Despite the limited number of studies, the NPs tested induced a modification of the biochemical composition of mucus. This could weaken the mucus' protective effect. Further investigations could confirm this hypothesis.

Intestinal cell damages

NPs in contact with intestinal epithelial cells can damage the brush border of enterocytes and then their absorptive properties. Scanning Electron Microscopy (SEM) revealed an affectation of the microvilli structure of Caco-2 after exposure to TiO2 NPs at a concentration of 10 µg/ml [START_REF] Koeneman | Toxicity and cellular responses of intestinal cells exposed to titanium dioxide[END_REF]. This finding was supported by other studies that showed a 42 % loss of microvilli (Faust et al., 2014a) and brush border disruption by TiO2 NPs (Faust et al., 2014b). Cytotoxicity of NPs in food products on intestinal cells was widely reported [START_REF] Fröhlich | Cytotoxicity of Nanoparticles Contained in Food on Intestinal Cells and the Gut Microbiota[END_REF][START_REF] Cornu | Ingestion of titanium dioxide nanoparticles: a definite health risk for consumers and their progeny[END_REF]. After 24 and 48 h exposure to ZnO NPs, a decrease in the cell viability of human colon cells was observed. The viability loss was concomitant with a ROS content increase, suggesting an oxidative stress-related mechanism. Direct interaction of ZnO NPs with the cell surface could trigger the intracellular signal activation, altering mitochondrial and/or endoplasmic reticulum functionalities [START_REF] De Berardis | Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells[END_REF].

Similarly, TiO2 NPs injected in the abdominal cavity of mice for 14 days significantly increased lipid peroxidation, decreased glutathione (GSH) level and altered antioxidant enzyme activity in a dose-dependent manner [START_REF] Ma | Oxidative stress in the brain of mice caused by translocated nanoparticulate TiO2 delivered to the abdominal cavity[END_REF]. A viability drop was also seen after 24 h exposure of metallic and metallic oxide NPs to human colorectal adenocarcinoma HT29 (Schneider et al., 2017a). Morphological changes of cells and granular inclusions suggests an apoptotic process. Early and late apoptotic cells were observed by flow cytometry after contact with most of NPs except for Au and cerium oxide NPs (Schneider et al., 2017a). The digestion process of NPs involving enzymes and acidic conditions was considered in viability assays. Cytotoxicity of copper oxide NPs increased by 30 % compared to native NPs when pre-treated with simulated gastrointestinal fluids. This was explained by a diameter decrease of NPs after incubation with pepsin [START_REF] Henson | Cytotoxicity of copper (II) oxide nanoparticles in rat intestinal cells: effect of simulated gastrointestinal fluids and generation of oxidative stress[END_REF]. The same effect was observed with digested TiO2

NPs after exposure to mucin-producing cells termed HT29-MTX-E12. A cytotoxicity increase was observed compared to undigested TiO2 NPs due to a reduction of the agglomeration state [START_REF] Bettencourt | Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro Digestion[END_REF]. Unlike inorganic NPs, neutral or negatively charged polymeric NPs including PLGA NPs did not show any damages on epithelial cells in use conditions. Their biodegradable feature could reduce the exposure time to cellular compounds and then limit their toxicity. Only cytotoxicity was observed with cationic polymeric NPs in Caco-2 cell models.

Chitosan NPs characterized by a size of 25 nm reduced the mitochondrial dehydrogenase activity at pH 6 while no effect was observed at pH 7.4 [START_REF] Loh | Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells[END_REF]. This was explained by the positive charge of chitosan at acidic pH. Toxicity of poly(amidoamine) dendrimers termed PAMAM on Caco-2 cells was dependent on the number of generations. While lactate dehydrogenase release was observed with generations (G) 3 and 4, no toxic effect was reported with G0-G2 (El-Sayed et al., 2002). This result suggests a synergistic effect of both size and positive surface on the cytotoxicity of dendrimers. However, the lack of a mucus layer in the Caco-2 model does not allow an extrapolation of results to in vivo conditions, especially in the case of positively charged NPs.

Genotoxicity

Nanomaterials can lead to DNA damage and mutagenic events (García-Rodríguez et al., 2018a;[START_REF] Asharani | Cytotoxicity and genotoxicity of silver nanoparticles in human cells[END_REF]. Guidelines for genotoxicity evaluation of manufactured nanomaterials were established by OECD (OECD, 2014). Ames test was excluded for nanogenotoxicity investigation while the in vitro micronucleus assay was adopted by requiring an exposure time without cyto B. Another recommendation is the need to conduct a toxicokinetic investigation prior to the genotoxicity study. The aim is to ensure that nanomaterials will reach the target cell/tissue. Ag NPs led to a DNA double-strand break in Caco-2 cells. An increase in the percentage of γH2AX-positive cells was observed in a dose and time dependent manner [START_REF] Gillois | Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products[END_REF]. Caco-2/HT29 co-culture were exposed to TiO2 nanospheres (NS), nanorods (NR) and nanowires (NW) for 48 h (García-Rodríguez et al., 2018b). NS, NR and NW were characterised using electron microscopy by sizes of 70-80, 40-70 and 8-14 nm, respectively. TiO2 NS did not modulate the gene expression of ZO-1 while it was downregulated in presence of TiO2 NR at 50 µg/ml and upregulated with NW at 150 µg/ml. Only NR induced a genotoxic damage evaluated by comet assay after 48 h exposure while no oxidative DNA damage was reported. After oral gavage of E171 food-grade TiO2 in rat, titanium accumulation was detected in the nucleus of cells located in the Peyer's Patches. However, no DNA damage including DNA strand breaks formation and oxidative DNA damages was reported after treatment for 7 days [START_REF] Bettini | Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon[END_REF]. Preneoplastic lesions were nevertheless detected in the colon after a long-term oral exposure of 100 days in rat.

Intestinal permeability modulation

NPs can modulate the intestinal permeability, especially the paracellular transport due to their toxicity on epithelial cells or by interacting with TJ network. Intestinal integrity can be assessed by measurements of the TransEpithelial Electrical Resistance (TEER) in intestinal barrier models and the paracellular flux of molecular markers such as lucifer yellow, fluorescein isothiocyanate-dextran and mannitol [START_REF] Graziani | Intestinal permeability in physiological and pathological conditions: major determinants and assessment modalities[END_REF]). An increase in the intestinal paracellular permeability could facilitate the transport in the blood compartment of pathogenic substances or modify the bioavailability of drugs and nutrients. A disruption of the intestinal barrier would facilitate the bacterial translocation, causing drastic sequelae as sepsis. Ag NPs increase the permeability of a monolayer of T84 human colorectal carcinoma cell lines in a dose and size dependent manner. A downregulation of the cytoplasmatic actin which plays a major role in the epithelial integrity maintenance and the regulation of TJs, was observed [START_REF] Baranwal | Nonredundant roles of cytoplasmic β-and γ-actin isoforms in regulation of epithelial apical junctions[END_REF][START_REF] Ku | The cytoskeleton of digestive epithelia in health and disease[END_REF]Georgantzopoulou et al., 2016a). SiO2 NPs smaller than 200 nm caused reversible relaxation of TJs in Caco-2 monolayer due to the activation of Myosin Light Chain Kinase (MLCK) enzymes. Activated MLCK enables the phosphorylation of the myosin part of the cytoskeleton, inducing TJs contraction [START_REF] Lamson | Anionic nanoparticles enable the oral delivery of proteins by enhancing intestinal permeability[END_REF]. A reversible TEER drop and a paracellular permeability increase were also observed in a Caco-2/HT29-MTX intestinal barrier models after exposure to amorphous 20 and 30 nm SiO2 NPs (Cornu et al., 2020). In the same study, no significant modulation of the permeability was observed with 200 nm SiO2 NPs due to the lack of cell internalization. In addition, no downregulation of TJs as claudin 2 and ZO-1 was observed, suggesting a TJ rearrangement after cytoskeleton disruption.

The opposite effect was observed with ZnO NPs in a Caco-2/HT29-MTX coculture model.

After 24 h incubation, a significant TEER increase was observed [START_REF] Mittag | Effects of Zinc Oxide Nanoparticles on Model Systems of the Intestinal Barrier[END_REF]. Zn increased the expression of ZO-1 in a Caco-2 model due the activation of PI3K/AKT/mTOR signaling pathway [START_REF] Shao | Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells[END_REF]. Oral exposure to Single-Walled Carbon Nanotubes (SWCNTs) increased the intestinal permeability in mice at a daily dose of 2.5mg/kg for 7 days (Chen et al., 2018). Intestinal permeability modulation of organic nanomaterials is usually attributed to the additives contained in the formulation rather than the nanoparticle structure.

The manufacturing of lipid-drug delivery systems such as nanoemulsions and solid lipid NPs requires surfactants to decrease the interfacial tension between the immiscible phases and to guarantee their long-term storage [START_REF] Chen | Lipid excipients and delivery systems for pharmaceutical development: a regulatory perspective[END_REF]. Due to their amphiphilic properties, they insert into the membrane of epithelial cells, increasing their fluidity [START_REF] Danielsen | Intestinal surfactant permeation enhancers and their interaction with enterocyte cell membranes in a mucosal explant system[END_REF][START_REF] Dimitrijevic | Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers[END_REF]. Direct interaction of surfactants with TJs was reported. Structural separation of TJs was observed in a Caco-2 model in the presence of sucrose monoester fatty acids [START_REF] Mine | Surfactants enhance the tight-junction permeability of food allergens in human intestinal epithelial Caco-2 cells[END_REF]. Modulation of the efflux pump was also observed in presence of surfactants [START_REF] Nieto Montesinos | Delivery of P-glycoprotein substrates using chemosensitizers and nanotechnology for selective and efficient therapeutic outcomes[END_REF]. Nanoemulsions increased the apical-tobasolateral flux of paeonol across a Caco-2 monolayer by a factor of 1.7 compared with the solution and decreased the efflux ratio in the same range. This effect was attributed to the P-gp inhibition by the Cremophor EL35 surfactant (S. Chen et al., 2018). Amphiphilic polymers used in the preparation of micelles and NPs such as Pluronic block copolymers were also able to inhibit the multidrug resistance mechanism in the intestinal epithelium [START_REF] Fang | Pluronic P85-coated poly(butylcyanoacrylate) nanoparticles overcome phenytoin resistance in Pglycoprotein overexpressing rats with lithium-pilocarpine-induced chronic temporal lobe epilepsy[END_REF][START_REF] Kabanov | Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery[END_REF]. Mixed micelles of Pluronic P85 and F68 reversed the efflux activity of the intestinal multidrug resistance protein 2 by affecting the structure and the function of mitochondria [START_REF] Chen | Pluronic P85/F68 Micelles of Baicalein Could Interfere with Mitochondria to Overcome MRP2-Mediated Efflux and Offer Improved Anti-Parkinsonian Activity[END_REF]. Pluronic 85 was able able to inhibit the Pgp drug efflux due to an ATP depletion associated to a membrane fluidization [START_REF] Batrakova | Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization[END_REF].

Inflammatory response

Inflammatory reactions after oral administration of NPs were widely reported in literature [START_REF] Martirosyan | Food Nanoparticles and Intestinal Inflammation: A Real Risk?[END_REF]. Ag NPs having a size of 20 nm exposed to Caco-2/TC7 cells at a concentration of 30 mg/L increased the expression of IL-8 proinflammatory cytokines while no effect was observed with 200 nm NPs (Georgantzopoulou et al., 2016a ;[START_REF] Őrfi | The immune system of the gut and potential adverse effects of oral nanocarriers on its function[END_REF], suggesting a size effect. This finding was confirmed by an in vivo study on female rats that showed a pro-inflammatory response in the intestinal mucosa after oral gavage of Ag NPs for 13 days [START_REF] Orr | Alteration in the mRNA expression of genes associated with gastrointestinal permeability and ileal TNF-α secretion due to the exposure of silver nanoparticles in Sprague-Dawley rats[END_REF]. However, no size-effect was observed with TiO2 NPs after 10day gavage in mice. Both 66 nm NPs and 260 nm microparticles induced in the same manner, the secretion of pro-inflammatory cytokines such as TNF-α and IFN-γ (Carolina Maciel Nogueira et al., 2012). The differences observed between nanomaterials could be explained by their composition. The small size of Ag NPs increases their surface area, facilitating the release of Ag ions responsible for the inflammatory response. By contrast, TiO2 NPs directly interact with the immune cells, especially macrophages which are able to phagocytose both small and large NPs [START_REF] Becker | Microparticles and their impact on intestinal immunity[END_REF][START_REF] Huang | Titanium dioxide nanoparticles prime a specific activation state of macrophages[END_REF]. Crystal structure of TiO2 NPs also needs to be considered. Unlike rutile NPs, anatase TiO2 NPs at a concentration of 50 µg/ml increased the expression of IL-8 and IL-1β in Caco-2 and THP-1-derived macrophages, respectively [START_REF] Tada-Oikawa | Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells[END_REF]. Concomitant production of IL-8, H2O2/OH•, and intracellular GSH by LoVo human colon cells was observed after 24 h exposure to ZnO NPs [START_REF] De Berardis | Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells[END_REF]. Inflammatory response in this case was correlated to oxidative stress. Another indirect mechanism involving the microbiota was reported. Oral administration of single wall carbon nanotubes at 2.5 mg/kg per day for 1 week induced the proliferation of proinflammatory bacteria named Alistipes uncultured bacterium and Lachnospiraceae bacterium A4. They were responsible for the inflammatory cell infiltrations and the production of IL-1β, IL-6 and TNFα in the duodenum and colon (H. Chen et al., 2018). General population of lymphocytes was characterized by flow cytometry after oral ingestion of SiO2 NPs in rats for 3 months.

Reductions by 33 % and 13 % in the number of leukocyte and T helper cells were observed. A 27 % decrease of the CD4/CD8 ratio was observed. This change in the immune cell populations is characteristic of an immunosuppression mechanism [START_REF] Shumakova | Toxicological assessment of nanostructured silica. III. Microecological, hematological indices, state of cellular immunity[END_REF]. The immune response in the gut was affected by Ag NPs after 13-day exposure. A decrease in the expression of immunomodulatory genes such as FOXP3, GPR43, TLR 2 and 4 was identified [START_REF] Williams | Effects of subchronic exposure of silver nanoparticles on intestinal microbiota and gut-associated immune responses in the ileum of Sprague-Dawley rats[END_REF]. E171 TiO2 additive reduced Treg cells activity after 100 day-oral exposure in rat while TiO2 nanotubes affected the immune response by inhibiting MAPK and NF-κB inflammatory signalling pathways [START_REF] Bettini | Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon[END_REF][START_REF] Neacsu | Attenuation of the macrophage inflammatory activity by TiO2 nanotubes via inhibition of MAPK and NF-κB pathways[END_REF]. 0.5 µm PS NPs exposed to Zebrafish for 14 days increased both the mRNA and the protein levels of IL1α, IL1β

and interferons in the gut [START_REF] Jin | Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish[END_REF]). An Inflammatory response was also reported in mice after oral administration of SWCNTs at a dose of 2.5mg/kg for one week. An increase of the expression of IL-1β, IL6 and TNF-α was observed in duodenum and colon (Chen et al., 2018).

Intratracheal instillation in mice of MWCNTs increased the M1-like polarization of macrophages in colon. This effect associated to a gut microbiota dysbiosis exacerbated the cardiotoxicity of doxorubicin [START_REF] Liu | Multiwalled carbon nanotubes exacerbate doxorubicin-induced cardiotoxicity by altering gut microbiota and pulmonary and colonic macrophage phenotype in mice[END_REF]. This combined toxicity of the loaded active ingredient and the MWCNTs emphasizes the importance to consider the intrinsic toxicity of nanomaterials, especially when they are used as drug delivery systems. Harmonized guidelines should be established for a better prediction of the intestinal nanotoxicity. For example, the presence of a mucus layer in in vitro models would prevent an overestimation of the nanotoxicity. Toxicokinetic investigations and consideration of digestion process would guarantee the relevancy of toxicological endpoints.

IV-

Conclusion

Dermal toxicity

Nanoparticles are used in cosmetic and dermatology products. The environment is also a source of human exposure to nanoparticles. Their toxicity after contact with the skin is still questioned mainly due to the lack of relevant in vitro models mimicking accurately the skin barrier. While the nanotoxicity by direct exposure to cutaneous cells was widely demonstrated, their ability to reach the viable epidermis stays unclear. The present review focused on the in vitro models and the toxicological effects of nanoparticles in contact with the skin barrier.

CHAPTER III: Nanotoxicity: Experimental Part 1. SILICATES NANOPARTICLES

Contexts and objectives

Silica particles are widely used in food products to prevent the powder agglomeration. Despite their presence in the market for a long time, doubts regarding their oral toxicity remain as mentioned by the EFSA in its report on the re-evaluation of the food additive E551. This could be explained by the insufficient toxicological data but also the variability in the size distribution of additives between manufacturers. No specifications have been yet established, allowing the presence of nano silica. Due to their small size, they could interact with the intestinal cells and then alter the intestinal barrier. Thus, an investigation on the intestinal toxicity of silica particles was performed using in vitro models of the intestinal barrier. The influence of the mucus, digestion process and the size was evaluated using E551 additive and engineered silica NPs.

Methodology

The effects of silica NPs, native and digested E551 additives on the intestinal barrier were evaluated in vitro on Caco-2 and HT29-MTX cell lines in mono and coculture cultivated in Transwell ® . This support mimics the in vivo configuration of the intestine. It is composed of an apical compartment that represents the intestinal lumen and a basolateral compartment corresponding to the blood vessels. These two compartments are separated by a membrane on which the cells forming the intestinal barrier are seeded. The Transwell ® is used to study the intestinal permeability of drugs or the passage of toxins (Figure 16). E551 additive was incubated in gastric and intestinal simulated fluids to accurately mimic gastrointestinal digestion before exposure. The engineered silica NPs, native and digested E551 were then incubated with the cells for 7 days. Cytotoxicity, ROS production, cytokine expression and transepithelial electrical resistance modulation were investigated.

Figure16: schematic representation of a Transwell ® .

Main Finding/Conclusions

This study showed that:

 Digestion did not affect the physicochemical properties of the E551 additive  The mucus layer produced by a single culture of HT29-MTX acted as an effective protective barrier against both small and large silica particles.

 Small nanoparticles affected the transepithelial electrical resistance of the Caco-2/HT29-MTX co-culture despite the presence of a mucus layer.

 70 nm was considered as a threshold diameter above which silica particles do not cause intestinal toxicity

Methodology

In our study, the effect of the process temperature on the nanoemulsification of medium and long chain triglycerides (MCT & LCT) was investigated. The impact on the size and the stability of NE of the lipid composition, the molecular weight, and the viscosity of triglycerides as well as the surfactant-to-oil ratio was also studied. 1

Main Finding/Conclusions

This study showed that:

 A heating at 90°C was required to obtain nanodroplets of LCT while nanoemulsions of MCT were obtained at both 90°C and 37°C

 Due to a low molar volume, the NE of glyceryl trioctanoate (GT) were destabilized by Ostwald ripening unlike lipid mixtures of GT with glyceryl tridecanoate (Labrafac ® ) or LCT  Mixtures of LCT and MCT allowed designing stable and fine NEs at 37°C.

 The surfactant-to-oil ratio influences the size of NEs.

Scientific article

Cytotoxicity of NE

NE preparation

NEs were formulated by the PIT method. Non-ionic surfactant (Kolliphor ® HS15) was provided by Sigma-Aldrich (Saint Quentin Fallavier, France). Labrafac ® WL 1349, a mixture of glyceryl trioctanoate (56%) and glyceryl tridecanoate (43%), was obtained from Gattefossé (Saint Priest, France). Oil and Kolliphor ® were added in a 20 ml vial. Approximately 15 ml of purified water were added into another 20ml vial. Both vials containing water and oil mixture were closed using a septum and heated in a water bath at 90°C for 15 minutes. After heating, 6.2 ml of water was transferred into the lipid phase and mixed under magnetic stirring for 5 minutes at 90°C.

Formulation was then removed from the water bath and magnetically stirred at room temperature.

Samples were sterilized by filtration across 0.22 µm membrane and transferred into 2ml sterile vials under a laminar flow hood. Vials were closed with an elastomer cap and crimp with an aluminium seal. Samples were stored at 4°C.

The quantities of Kolliphor ® and Labrafac ® were adjusted according to the desired size (Table 1). Hydrodynamic diameters, polydispersity index (PDI) and Zeta potentials of all the NEs were tested by Dynamic Light Scattering and laser doppler electrophoresis using the Zetasizer® Nano ZS90 (Malvern, UK). The stability of nanoemulsions during the incubation times with cells was 0 assessed by measuring the size during 2 hours at 37°C. Three independent measurements were performed for each time point. As demonstrated in the previous study, the surfactant-to-oil ratio (SOR) controls the final size of NEs (Table 2). Immediately after preparation, the hydrodynamic diameters were of 21.8 nm and 227.6 nm for SOR of 3.5 and 0.5, respectively. The polydispersity index was below 0.1 suggesting a monomodal distribution except for the NE200 with a value of 0.32. No size variation was observed during the 2h incubation at 37°C, demonstrating the stability of NEs.

Cytotoxicity study of NE on intestinal cells

NE were incubated with Caco-2 and HT29-MTX at different concentrations for 2 hours. MTS assay was then performed to evaluate the viability of cells. The relationship between the cell viability and the Kolliphor ® concentration was showed in Figure 17 to consider the well-known intrinsic toxicity of surfactant. At 0.01 g kolliphor ® /ml, the viability of Caco-2 cell-lines was of 76 % and 44% after exposure to NE20 and NE200, respectively (Figure 17A). This suggests that the cytotoxicity of NEs was not directly correlated to the concentration in Kolliphor ® . The same finding was observed with HT29-MTX (Figure 17B). A viability drop was observed from a concentration in Kolliphor ® of 0.086 and 0.025 g/ml for NE20 and NE200. For both cell-lines, at the same Kolliphor ® concentration, NE200 seem much more cytotoxic than NE20. 1

Figure 17: Influence of the concentration in Kolliphor ® on the viability of Caco-2 (A) and HT29-MTX (B).

In Figure 18, the viability results were expressed as a function of the NE concentration (oil and Kolliphor ® ). For all the NEs, a viability drop was observed from a concentration of 0.05 and 0.11g/ml for Caco-2 and HT29-MTX, respectively. The HT29-MTX were less sensitive to NEs compared with Caco-2, likely due to the protective mucus barrier. Unlike previous results, no size influence was observed between the different NEs. Cytotoxicity was only dependent on the concentration in NE that includes the oil and the Kolliphor ® amounts. NE200 were prepared with 2.3 fold less Kolliphor ® compared with NE20. Thus, a high amount of NE200 was necessary to reach the same surfactant concentration as the small NE. This explains the differences observed between the different NEs in Figure 17. The lack of size influence could be due to the ability of NEs to be internalized in intestinal cells at the same levels, even for the large ones. This suggests an internalization process different between NEs and silica NPs. Surprisingly, despite high concentrations in Kolliphor ® , small NEs were not more toxic than the large ones. This could be explained by the adsorption of Kolliphor ® at the surface of droplets, characterized by a large surface area. Nanotechnology represents all sciences and techniques aimed for the producing and the using of objects at the nanometer scale and more precisely between 0.1 and 100 nm, for at least, one of their dimensions. The challenges for the researchers are to industrialize the manufacture of nanoobjects to exploit their characteristics. The nanotechnology revolution has quickly resulted in the emergence of a very large number of nanoproducts with multiple applications on the market (1). Some of them so-called nanomedicines are used as drug delivery systems, to carry drugs until the action site. For example, a nanocapsule with optimized physicochemical properties were designed to cross both the mucus layer and the intestinal epithelium to finally reach the blood compartment (2). Biodegradable nano-objects such as NE, liposomes, polymeric micelles and nanoparticles were designed for the transport of anticancer agents (3). Inorganic NM are used as food additives in confectionery, pastry, and many culinary preparations such as the E171 additive. Colloidal silica particles contained in E551 additive prevent the agglomeration of food powders such as sugar and salt (4). However, their use in food and pharmaceutical products requests a deep investigation on their potential effect along the gastrointestinal tract, especially the intestinal barrier. It is also important to understand the mechanisms involved and to determine if these particles are able to cross biological barriers and then be disseminated in the body (5).

The numerous applications of silica particles in food products (6) and the emergency of oral lipid-based formulation such as nanoemulsions in therapy and nutrition request to fully characterize their effect on the intestinal barrier (7). This barrier is necessary for the absorption of vital nutrient and the protection of the organism against foreign bodies. Its alteration would be a source of several pathologies such as infection, local or systemic inflammation (8).

Technological advances over the 40 past years in the field of cell culture facilitated in vitro investigations on intestinal disorders and physiological mechanisms of the intestinal barrier, thus reducing the number of in vivo experiments. Despite properties very close to in vivo, the use of primary cell cultures is faced with availability issues and their short lifespan, limiting longexposure treatments. Cell-lines represent a good alternative due to their homogeneity in terms of phenotype and genotype, their low cost and easy access (9). The first cell model of the intestinal barrier was the Caco-2 model. This model is defined as the gold standard for predictive studies of permeability and transport of molecules through the intestinal epithelium. This cell-line was isolated from human colorectal adenocarcinoma and established by Fogh &Trempe in 1975 (10).

This lineage is well characterized as an enterocyte model regarding its morphological and functional characteristics expressed after differentiation. After reaching confluence, they spontaneously differentiate during approximately 21 days of culture. Once differentiated, they form a polarized cell monolayer, an intercellular junction complex, and a brush border with microvilli on the apical side (11). They express enzymes and membrane transporters (12).

However, this cell line has some limitations, including the formation of a heterogeneous monolayer related to culture time and number of passages (13). However, Caco-2 model should be not considered as a relevant model for toxicity studies due to the lack of a mucus layer.

Considering that the mucus could act as a protective barrier, an overestimation of the intestinal toxicity is usually observed. A model composed of mucus-secreting HT29-MTX cells associated with Caco-2 cells was developed to overcome this limitation. The HT-29 cell line is another intestinal cell-line established by Fogh & Trempe. It is derived from colon adenocarcinoma and has been used as an intestinal model for bioavailability and mechanistic studies (14). The HT-29 line is considered a pluripotent gut line because the changes in the culture medium can lead to different differentiation pathways. Unlike the Caco-2 cell line, HT-29 differentiation is not spontaneous but rather depends on nutritional and culture conditions (15). The major difference between HT-29 and Caco-2 cell lines is that under appropriate culture conditions, HT-29 differentiate into goblet cells and then produce mucus. They moderately express tight junction (16). The stable clone HT29-MTX is a sub-population of HT-29 cells resistant to high concentrations of methotrexate (MTX). This clone is thus capable of spontaneously differentiating into mucus-producing cells (17). The mucus produced by HT29-MTX is a waterinsoluble gel composed mainly of mucins such as MUC2 and MUC 5AC that form a protective layer in the intestine. The association of HT29-MTX with Caco-2 cell lines allows mimicking more accurately the human intestinal barrier by considering the mucus layer (18). In addition, the ratio between the number of Caco-2 and HT29-MTX cells can be adapted for a better correlation to the different segments of the intestine.

Oral toxicity of silica nanoparticles 1.Methodology

Regarding the first study, a re-evaluation of the E551 food additive was carried out according to the European Food Safety Authority (EFSA) recommendation. Structurally, this additive is composed of agglomerates and aggregates of primary NPs strongly bond between them. There are no available current specifications concerning their physical properties in terms of size distribution and polydispersity (19). To cover the full-size range of NPs potentially present in E551, four sizes of engineered silica NPs were evaluated in addition to the food additive. Besides, most in vitro tests using human cells do not consider the potential changes that NPs may undergo in physiological condition, for example after digestion. NPs can agglomerate, especially in acidic mediums, react with components of food bowl or digestive enzymes (20). The modification of physiochemical properties could then influence the interaction of silica particles with the gastrointestinal tract and therefore their toxicity (21). To consider the impact of digestion process, a digestion protocol was established according to the standardized INFOGEST protocol (22).

Several studies have clearly shown the reproducibility of the standardized INFOGEST protocol (23). It was considered as an alternative method to the animal experiment to mimic the static in vitro digestion (21). The composition of gastric and intestinal fluids come from the US Pharmacopeia. After E551 digestion, single and coculture of Caco-2 and HT29-MTX were exposed to engineered silica NPs, native and digested E551 at 1mg.ml -1 for 7 days. The concentration choice agreed with the recommendations of the EFSA panel. The scientific committee noted that the highest exposure doses in the various available toxicity studies were always below no observed adverse effects (NOAEL). This explains why the panel could not guarantee the acceptable daily intake "not specified". Thus, concentrations of silica NPs above daily dietary intake, estimated between 0.3 and 0.8 mg/kg, were evaluated to identify a toxic concentration threshold. Besides, in the absence of a long-term study with nano-silicon dioxide, the scientific panel of EFSA could not extrapolate the toxic effects from the chronic available studies (24). For this reason, a long-term exposure in accordance with the lifespan of in vitro models (7 days) was performed.

Results

Transmission electron microscopy and BET specific surface area results confirmed the classification of E551 as nanomaterials. E551 is composed of primary aggregates particles strongly bonded between them, with a size of 14 nm (25). After successive digestions in simulated gastric and simulated intestinal fluids (SGF, SIF), the physicochemical characteristics of E551 did not change in a significant way. The size of the digested additive stayed around of 200 nm. Our results are in agreement with those of Desai et al., where the transmission electronic microscopy images showed that the mesoporous silica NPs structure was intact after incubation with SGF and SIF at pH 1.2 and 6.8 respectively (26). However, studies reported in literature

were not in accordance with our findings. Sakai-Kato et al. observed an agglomerated state of amorphous silica NPs after incubation with simulated intestinal fluids [START_REF] Kaul | Role of Nanotechnology in Cosmeceuticals: A Review of Recent Advances[END_REF]. McCraken et al,

showed that the incubation of negatively charged silica NPs in SGF led to an agglomeration (28). This was explained by the modification of the surface charge at low pH and the presence of ions and enzymes in the simulated gastric fluid (29). Incubation in SIF by a pH of 6.8 could nevertheless reverse the agglomeration state of silica particles. Thermogravimetric analysis showed the absence of organic compounds at the surface of digested particles, likely due to washing steps after digestion. Besides, the isoelectric point was the same before and after the digestion process, indicating the lack of surface modification.

After full physicochemical characterization of native, digested E551 food additives and engineered silica NPs, single and coculture of Caco-2 and HT29-MTX were exposed to NPs.

After 7 day-exposure, the cellular viability decreased in a size dependent manner in contact with Caco-2 single culture. 70 nm was identified as a cutoff size, from which viability of cells was preserved. Unlike large NPs, 20 nm and 30 nm nanoparticles induced a viability loss in Caco-2 single culture (30). This was in accordance with literature that showed the size-dependent cytotoxicity of silica NPs on different intestinal cell-lines. Small silica nanoparticles of 12 and 40 nm induced a viability decrease of Caco-2 (31), human hepatoma cell line (HepG2) (32), human colon cancer epithelial cell line (HCT116) (33,34).This result could be explained by an accumulation over the time of NP20 and NP30 in Caco-2 cell line. A time dependent disruption of actin filaments of Caco-2 was observed with NPs of 30 nm. Our findings were similar to those of Cornu et al where Caco-2 were exposed to 30 nm silica NPs during 2h and at a dose of 10 mg.ml -1 (35). The actin cytoskeleton remained intact with larger NPs and food additives,

Perspectives

It would be interesting to investigate the mechanism involved in the proliferative effect of silica NPs in contact with HT29-MTX single culture. Previous studies showed that silica NPs can stimulate the proliferation of human gastric carcinoma cell line GXF251L. The level of a nuclear proliferation marker Ki-67was enhanced in response to high concentrations of silica NPs. The author suggested the involvement of MAPK/EGFR pathway in the proliferation at different levels. Moreover, we could investigate more deeply the involved mechanism in the actin dependent disruption after Caco-2 exposure to NP30. Several mechanisms can be at the origin of the actin disruption. A direct physical interaction between NPs and actin filaments could be suggested. Cytoskeleton could be also affected in an indirect way due to the ROS or cytokine productions (37). To understand the differences observed between the HT29-MTX single culture and the co-culture, mucus characterization could be performed. Biochemical composition, thickness and mesh could be studied. To assess the safety of E551 food additive in humans, it is necessary to confirm our results with in vivo models. It would be possible to evaluate the distribution, the excretion, and the toxic effects on the GIT but also in other organs such as the liver in the case of translocation. Long-term exposure could be also performed (38).

An assessment of nanomaterials impact on our health and the understanding of the involved toxicity mechanisms are recommended. This would help to define an appropriate regulatory and health framework for a safety use of NPs systems.

Oral toxicity of lipid nanoparticles

Methodology

A wide range of pharmaceutical nanoforms was developed as delivery systems of active substances. Among them, NEs have been designed for the administration of pharmaceutical active ingredients and lipid supplementation (39). Oil-in-water NEs allow the delivery of active ingredients with a poor-water solubility (40). The solubility of the active substance is a great challenge in biopharmaceuticals, especially for the drug adsorption. Thanks to the solubility improvement and protection against gastrointestinal enzymatic degradation, NEs increase the bioavailability of active substances orally administered (41). Due to their size in the nanometer range, NE can also facilitate the transport of drug though the intestinal epithelium by interacting with intestinal cells. However, this mechanism could also cause toxicity by altering the intestinal barrier integrity.

Methods such as high shear/pressure homogenization, ultrasonic or micro-fluidizer are used to generate nanoscale droplets. The desired size of NEs can be controlled by the amount of external energy applied. However, an excess applied energy can destabilize the NEs due to a local heating and affect the stability of the incorporated drug. 99% of the applied external energy is usually dissipated in heat. In contrast, the low-energy methods play on the tremendous reduction of the tension at the interface oil/water using surfactants and co-surfactants (42). Some of them require heating to shift the Hydrophilic Lipophilic Balance (HLB) of surfactants, inducing a phase inversion of the emulsion (43). Unlike phase inversion temperature (PIT) method, the spontaneous emulsification does not require any intense heating. The process depends mainly on the interfacial tension, viscosity, structures and concentrations of surfactants and co-surfactants (44). Low-energy processes often require a high concentration in surfactant. Due to their amphiphilic properties, surfactants can insert into the cell membrane and reach the cytoplasmic compartment, affecting the viability and biological functions of cells. Thus, beside the nanometric size of droplets, the presence of a high amount of surfactant in NE preparations could cause an intestinal toxicity. In this second part, an evaluation of different critical parameters on the features of nanoemulsions was performed. This investigation aimed to prepare NE with different physicochemical properties and compositions to identify in a second step, the toxicity factors such as the size and the surfactant concentration. This was initiated through a viability assessment of Caco-2 and HT29-MTX exposed to different NEs.

Results

Nanoemulsification at 37°C of Medium Chain Triglycerides (MCT) led to the formation of 25 nm lipid droplets. This process temperature is slightly above the melting point of the Kolliphor ® but does not lead to the phase inversion. Larger sizes, above 100 nm were obtained with Long Chain Triglycerides (LCT) at the same temperature. This agreed with Chang et al demonstrated that the oil phase composition has a major effect on the size of NEs. Only very fine nanoemulsions were obtained with LCT using the PIT method. The molar volume (MV) of triglycerides was considered as a critical factor for the spontaneous nanoemulsification. The MV of LCT could explain their limited diffusion in the aqueous phase (45). Addition of MCT with LCT at ratio 1:1 reduced the average MV of the oil mixture, allowing the spontaneous nanoemulsification at 37°C. The concentration in surfactant also played a major role in the nanoemulsification process by affecting the size and the polydispersity of droplets. High surfactant concentrations facilitated the production of fine NEs. At least two-fold more surfactants was requested to obtain droplets in the nanometer range. Despite the surfactant presence, NEs of glyceryl trioctanoate (GT) were not stable. Ostwald ripening was observed after a few days of storage. GT mixed with glyceryl tridecanoate (Labrafac ® ) and LCT oils such as glyceryl trioleate stabilized the nanoemulsions during at least 3 months. The mixture allowed increasing the VM value of the lipid core of NE, limiting the diffusion of oils in the water bulk phase and then the Ostwald ripening. By predicting the water solubility of triglycerides, VM could be a very interesting indicator for both the spontaneous emulsification and the Oswald ripening-mediated destabilization.

In vitro study

NEs have been developed as promising nano-object for the administration of pharmaceutical active substances (39).The major interest in this field focuses on O/W NEs for the delivery of hydrophobic active ingredients with a poor water solubility and to increase their oral bioavailability (40,41). In our study, we demonstrated that all NEs affected the viability of Caco-2 from a concentration of 0.05 g/ml without size influence. The same result was observed with HT29 MTX but at a higher concentration in NEs, 0.11 g/ml, suggesting a protective effect of mucus. Surprisingly, the Kolliphor ® concentration that is inversely proportional to the size of NEs did not play a major role in the cytotoxicity of NEs. Despite high Kolliphor ® concentrations in nanoemulsions of 20 nm and 30nm, no cytotoxicity difference was observed compared with larger NE. Majority of surfactant could be adsorbed at the surface of droplets characterized by a large surface area, limiting the interaction with cells. The lack of size influence could be explained by the ability of NEs to be internalized into the cells, even for lipid droplets of 200 nm.

Thus, rather than the Kolliphor ® concentration and the size, only the NE concentration affected the viability of intestinal cells. This finding suggests nanotoxicity factors depend on the nature of NPs. While the size governs the toxicity of silica NPs, only the exposed dose influenced the cytotoxicity of nanoemulsions.

Perspectives

This toxicity study needs to be completed by evaluating the influence of the digestion process on the physicochemical properties of NEs. The presence of lipase in the intestinal fluid could affect their structure. The potential release of surfactants from the surface of nanodroplets to gastrointestinal fluids could also exacerbate the toxicity of NEs. Free surfactants would interact then with the biological environment, especially the intestinal cells. Influence of NEs on the permeability of the intestinal barrier should be also investigated. Surfactants are well known to interact with the multidrug resistance mechanism, especially the P-gp. An alteration of the P-gp would enhance the bioavailability of some xenobiotics and cause adverse effects, especially when NEs are used as drug delivery systems. Expression and functions of tight junctions such as ZO-1 and claudins could be also affected. The influence of NEs on the gut microbiota and the mucus layer needs also to be evaluated to identify a potential intestinal toxicity.

General conclusion

Despite the wide presence of nanomaterials in numerous manufactured products, their potential health hazard is still unclear due to the lack of available toxicological data and harmonized protocols. In addition, the exposure doses and times in the toxicity studies are not often correlated with the use conditions, especially for the daily products. In this present thesis work, we have demonstrated that the mucus needs to be considered in nanotoxicity studies due to its protective role. A size effect was identified in the toxicity mechanism of silica NPs. From a hydrodynamic diameter of 70 nm, silica particles were unable to be internalized in intestinal cells and to interact with the cytoskeleton. Our study also demonstrated that the exposure time is a critical parameter, especially for the lowly biodegradable particles. Small silica NPs accumulate into the cells, until reaching a toxic dose. The safety of the Emprove  E551 Food additive was demonstrated after subacute exposure to intestinal cells. This was explained by the large mean size of silica particles, close to 200 nm, even after the digestion process. Due to the lack of specifications on the dimensions of particles, nanosilicates could be nevertheless present in E551 additives provided by other manufacturers, affecting their toxicological profile. In contrast to silica NPs, no size effect was reported in the cytotoxicity of nanoemulsions. They provoked a cell viability drop in a concentration dependent manner for all the tested NE. This demonstrates that the mechanisms involved in the nanotoxicity is dependent on the composition of nanoparticles and consequently, toxicity factors cannot be extrapolated to all NPs. production after 7-day exposure. However, the single culture of goblet cells was not affected due to the protective barrier of mucus. Silica NPs with a size of at least 70 nm and additive E551 were not toxic, even in the lack of mucus. Unlike NP20 and NP30, they did not significantly modulate the transepithelial electrical resistance of the co-culture. Regarding nanoemulsions, a cytotoxicity in a dose-dependent manner was observed in Caco-2 and HT29-MTX cultures, without any size effect. The lipid composition and the presence of surfactant could facilitate the cell internalization of droplets, even the largest ones. This study demonstrates that the nanotoxicity factors vary according to the composition of NPs. Unlike nanoemulsions, the toxicological profile of silica particles is strongly dependent on the hydrodynamic diameter. Mucus also needs to be considered in toxicity studies for a better prediction.

Annexes

Figure 1 :

 1 Figure 1: Estimation of various nanomaterials uses over a span of 10 years. According to Market Analysis Report.

Figure 2 :

 2 Figure 2: Applications insights of nanomaterials in 2020. According to Market Analysis Report.

Figure 3 :

 3 Figure 3: Classification of NPs. According to Shah et al 2020.

Figure 4 :

 4 Figure 4: The types of polymeric nanoparticles used as drug delivery. According to Christoforidis et al 2012.

Figure 5 :

 5 Figure 5: Schematic representation of polymeric micelles. According to Ghezzi et al 2021.

Figure 6 :

 6 Figure 6: Schematic representation of liposome. According to lembo et al 2010.

Figure 7 :

 7 Figure 7: Schematic representation of nanoemulsion types. According to Che Marzuki et al 2019.

Figure 8 :

 8 Figure 8: Schematic representation of dendrimers structure. According to Santos et al 2013.

Figure 9 :

 9 Figure 9: Schematic representation of carbon nanotubes. According to Abazari et al 2020.

Figure 10 :

 10 Figure 10: Global nanotechnology market. According to Talebian et al 2021.

Figure 11 :

 11 Figure 11: Main exposure routes of human body to nanoparticles. According to Naseer et al 2018.

Figure 12 :

 12 Figure 12: Schematic represent the different NPs penetration pathways through skin barrier. According to Nafisi et al 2018.

Figure 13 :

 13 Figure 13: Schematic represent the different NPs penetration pathways through gastrointestinal tract. According to Bellmann et al 2015.

Figure 14 :

 14 Figure 14: Deposition of nanoparticles in respiratory tract depending on their size. According to Geiser et al 2010.

Figure 15 :

 15 Figure 15: Schematic represent the different NPs penetration route through eye. According to Souto et al 2019.

Figure 16 :

 16 Figure 16: Schematic representation of a Transwell ® .

Figure 17 :

 17 Figure 17: Influence of the concentration in Kolliphor ® on the viability of Caco-2 (A) and HT29-MTX (B).

Figure 18 :

 18 Figure 18: Influence of the concentration in NEs on the viability of Caco-2 (A) and HT29-MTX (B).

Figure 3 :

 3 Figure 3: classification of NPs. According to Shah et al 2020 (5).

Figure 4 :

 4 Figure 4: the types of polymeric nanoparticles used as drug delivery. According to Christoforidis et al 2012 (6).

Figure 5 :

 5 Figure 5: schematic representation of polymeric micelles. According to Ghezzi et al 2021 (8).

Figure 6 :

 6 Figure 6: schematic representation of liposome. According to lembo et al 2010 (9).

Figure 7 :

 7 Figure 7: schematic representation of nanoemulsion types. According to Che Marzuki et al 2019 (12).

Figure 8 :

 8 Figure 8: schematic representation of dendrimers structure. According to Santos et al 2013 (14).

Figure 9 :

 9 Figure 9: schematic representation of carbon nanotubes. According to Abazari et al 2020 (21).

Figure 10 :

 10 Figure 10: Global nanotechnology market. According to Talebian et al 2021 (22).

Figure 11 :

 11 Figure 11: main exposure routes of human body to nanoparticles. According to Naseer et al 2018 (34).

Figure 12 :

 12 Figure 12: schematic represent the different NPs penetration pathways through skin barrier. According to Nafisi et al 2018 (37).

Figure 13 :

 13 Figure 13: schematic represent the different NPs penetration pathways through gastrointestinal tract. According to Bellmann et al 2015 (44).

Figure 14 :

 14 Figure 14: deposition of nanoparticles in respiratory tract depending on their size. According to Geiser et al 2010 (46).

Figure 15 :

 15 Figure 15: schematic represent the different NPs penetration route through eye. According to Souto et al 2019 (47).

1 . 2 .

 12 this part. Toxicokinetic and toxicological endpoints including cytotoxicity, genotoxicity, oxidative stress, and inflammatory response were considered. Introduction ..........................................................................................................Microbiota and mucus interactions ......................................................................... Intestinal cell damages ............................................................................................. 3. Genotoxicity ..........................................................................................................definition issued by the European Commission (EC) in 2011, nanomaterials were described as solid particles including single, agglomerated, and aggregated particles where at least 50 % of particles in the number size distribution have one or more external dimensions between 1 nm and 100 nm (Commission Européenne, 2011). Two other conditions have been added in the new definition published in 2022 by the EC (Commission Européenne, 2022). This includes the elongated particles characterized by two dimensions smaller than 1 nm and another one larger than 100 nm such as the rods, fibers, and tubes. Platelet particle with one dimension below 1 nm and two others above 100 nm are also defined as nanomaterials. According to the ISO/TS 80004-2:2015 guidelines, nanoparticles (NPs) are characterized by three external dimensions in the nanoscale comprised between 1 and 100 nm and approximately in the same range ("ISO/TS 80004-1:2015, Nanotechnologies ). NPs represent a sub-class of nanomaterials. They are composed of organic (lipid and polymeric NPs, dendrimers, micelles)

Figure 2 :

 2 Figure 2: Schematic representation of interactions between nanomaterials and bacteria.Figure is reproduced with permission from reference[START_REF] G1108 Ladaycia | Microbiota and nanoparticles: Description and interactions[END_REF]

  Physicochemical properties of nanomaterials are critical factors mediating the distribution of nanomaterials in the intestine. A small size usually below 100 nm, neutral or negatively charged NPs facilitate the penetration across the mucus layer, acting as a protective barrier for the intestinal epithelium. The positive NPs, potentially more toxic for cells are stuck to the mucus layer and are unable to reach the epithelial cells in healthy models. Physical and chemical modifications of nanomaterials after contact with gastrointestinal fluids also need to be considered in toxicity studies. Changes in the surface charge and agglomeration state were reported, impacting their toxicological profile. Some nanomaterials are able to interact with the different layers of the intestinal barrier including microbiota, mucus layer and epithelial monolayer. Gut microbiota dysbiosis and changes in the secretion and the composition of mucus were observed. Intestinal permeability modulation can be directly correlated to epithelial cell damages. Another mechanism is attributed to the destabilization of the actin cytoskeleton of epithelial cells and the rearrangement of TJs. Besides the physicochemical properties of nanomaterials, the composition is another critical factor in the intestinal nanotoxicity. Inorganic materials are usually more toxic than polymeric and lipid-based NPs. Mainly surfactants used as additives for the preparation of organic particles were responsible for the cytotoxicity and the intestinal permeability modulation. Two toxicity pathways are suggested for the inorganic nanomaterials. The high surface area of metallic NPs such as Ag particles facilitates the release of ions, affecting the biological functions of cells. The second could be attributed to the lower biodegradation rate of inorganic NPs such as SiO2 and TiO2 NPs compared with organic materials. The contact time with the cellular compounds is then prolonged and a chronic exposure would result in their accumulation in the cells. Microbiota was affected by some NPs through the proliferation of pathogenic and pro-inflammatory bacteria. The antibacterial features of some metallic properties reduced the population of commensal microorganisms.Unlike to epithelium damages, the inflammation response triggered by nanomaterials was less dependent on their physicochemical properties. Despite the numerous investigations, consistency issues were usually observed in the toxicological data reported in literature.

  Lipid nanoparticles are composed of a solid (SLN) or liquid (NE) lipid core surrounded by an external corona of surfactants including phospholipids and PEGylated lipids. They exhibit numerous advantages as drug delivery systems. Hydrophobic drugs can be solubilized in the lipid core of NPs. The small size of droplets and the presence of surfactants improve the transport of drugs across the biological barriers. High and low energy methods can be used to produce nanoemulsions. High shearing rates are required to reduce the size of oil droplets in the nanometer scale. Another approach consists in the lowering of the tension at the oil/water interface using high quantities of surfactants and co-surfactants. A spontaneous nanoemulsification can then occur. Phase Inversion Temperature (PIT) is an alternative method based on the modification of the hydrophilic-lipophilic balance (HLB) of PEGylated surfactants with the temperature. A hydrophilic surfactant at low temperature becomes hydrophobic during the heating, leading to the inversion of the emulsion from O/W to W/O. This present work evaluates the critical parameters for the preparation of small and stable nanoemulsions containing medium and long chain triglycerides.

Figure 18 :

 18 Figure 18: Influence of the concentration in NEs on the viability of Caco-2 (A) and HT29-MTX (B).

  confirming the lack of cell uptake. The interactions of silica NPs with HT29-MTX exhibited a different pattern compared with Caco-2. The continuous mucus layer spread on HT29-MTX single culture prevented the cell internalization of silica NPs, even the smallest ones. No toxic effect including cytotoxicity, actin disruption, ROS production and inflammatory response was reported. Surprisingly, a proliferative effect of HT29-MTX cells was identified in presence of silica particles. Mucus layer secreted by HT29-MTX served as an effective barrier, preventing both small and large NPs to reach the cells. A rapid accumulation of engineered silica NPs in mucus layer was observed, due to the strong interaction of NPs with the mucus gel(36). In the lack of a mucus layer on HT29-MTX, a size-dependent cell uptake was noticed, similarly to Caco-2 cell line, proving the role of mucus Despite the mucus on the co-culture, a transepithelial electrical resistance drop was noticed with the 20 and 30 nm nanoparticles. The low ratio of HT29-MTX in the coculture model could change the mucus layer properties in comparison with the one spread on the HT29-MTX single culture. No damage of the co-culture was reported with E551 additive and engineered NPs of 70 and 200 nm. The safety of E551 food additives could be then guaranteed if it contains only silica NPs with a size of a least 70 nm.

Evaluation

  in vitro du profil toxicologique des nanoparticules de silice et nanoémulsions Mots clés : nanoparticules de silice, nanoémulsions, toxicité, barrière intestinale, mucus Résumé : La quantité de nanoparticules manufacturées sur le marché est en constante augmentation du fait de leurs propriétés uniques. Elles sont utilisées comme excipients en cosmétique, additifs alimentaires, et systèmes de délivrance de médicaments. Leur taille nanométrique est responsable de leur forte interaction avec l'environnement biologique. Ce travail a pour but d'évaluer la toxicité intestinale de deux types de nanoparticules (NPs) : des nanoparticules de silice potentiellement présentes dans l'additif E551 et des nanoémulsions utilisées pour la délivrance de lipides et substances actives. L'évaluation toxicologique a été réalisée à partir de modèles in vitro, à savoir des monocultures et une co-culture de cellules intestinales (Caco-2 et HT29-MTX). Avant l'incubation, l'additif a été digéré en présence de fluides gastriques et intestinaux simulés afin de mimer fidèlement les conditions in vivo. Les plus petites NPs de silice caractérisées par des diamètres de 20 et 30 nm ont altéré la viabilité des entérocytes et induit la production de ROS après 7 jours d'exposition.Cependant, ces dernières n'ont pas causé de cytotoxicité sur la monoculture de cellules caliciformes en raison du rôle protecteur du mucus. Les NPs de silice avec une taille supérieure à 70 nm et l'additif E551 n'ont présenté aucune toxicité, même en l'absence de mucus. Contrairement aux NP20 et NP30, elles n'ont pas modulé significativement la résistance électrique transépithéliale de la co-culture. Concernant les nanoémulsions, une cytotoxicité dose-dépendante a été observée dans les cultures de Caco-2 et HT29-MTX, sans influence de la taille. La composition en lipides ainsi que la présence de tensioactif pourraient faciliter l'internalisation cellulaire des gouttelettes, même les plus grossières. Cette étude démontre que les facteurs de nanotoxicité varient selon la composition des NPs. Contrairement aux nanoémulsions, le profil toxicologique des particules de silice est fortement dépendant du diamètre hydrodynamique. Le mucus doit également être pris en compte dans les études de toxicité pour une meilleure prédiction.In vitro investigation of the toxicological profilKeywords: silica nanoparticles, nanoemulsions, tox Abstract : The quantity of manufactured nanoparticles (NPs) on the market is constantly increasing due to their unique properties. They are used as cosmetic ingredients, food additives and as drug delivery systems. Their size in the manometer range confers them a strong ability to interact with the biological environment. This work aims to evaluate the intestinal toxicity of two types of nanoparticles (NPs): silica NPs potentially present in E551 food additive and nanoemulsions used as drug/lipid delivery system. The toxicological evaluation was carried out using in vitro models consisting in single cultures and co-culture of intestinal cells (Caco-2 and HT29-MTX). Prior to the incubations, E551 additive was digested with gastric and intestinal simulated fluids, to accurately mimic the in vivo conditions. The smallest silica NPs characterized by hydrodynamic diameters of 20 and 30 nm, altered the viability of enterocytes and induced ROS Université Bourgogne Franche-Comté 32, avenue de l'Observatoire 25000 Besançon e of silicate nanoparticles and nanoemulsions icity, intestinal barrier, mucus

Table 1 : Intestinal toxicity of inorganic NPs

 1

	NPs	Size (nm)	Concentration	Exposure	models	Observations	References
		Surface charge (mV)	of NPs		Time
					In vitro models
	Ag	20 and 200 nm	0.1 -100 mg/mL Short time Coculture of Human	Increase in IL-8 in a dose-and (Georgantzopoulou
		-12.8 and -13.9 mV			exposure: 24 colon colorectal	size-dependent manner with a lack et al., 2016b)
					h	adenocarcinoma Caco-	of cytotoxicity and oxidative
						2/TC7 and HT29-MTX	stress.
						cells
	Bare-Ag (Ag-B)	23 nm	0.1 -2.0 µg/mL	Short time	Human colon colorectal	Decrease in cellular viability in a	(Chen et al., 2016)
		-7.7 mV			exposure: 24	adenocarcinoma Caco-2	dose and coating dependent
	Citrate-coated Ag (Ag-CIT)	24 nm -9.6 mV			h Long time exposure:	cell	manner Increase in IL-8 release in a dose and time dependent manner.
	poly (N-vinyl-2-	30 nm			21 days
	pyrrolidone)-	-8.4 mV
	coated Ag NP
	(Ag-PVP)
	TiO2	265 nm	62.5, 250 and	12 h and 24 h Human intestinal	Change of cells morphology	(Setyawati et al.,
		-14.1 mv	1000 µM			carcinoma epithelial cell		2015;	García-
	Pure anastase	70 -80 nm				lines, SW480 / Normal		Rodríguez et al.,
	crystal-structure nanospheres of		12.5 350 μg/mL	and	24 and 48 h	human intestinal mucosa epithelial cell line	Decrease in cellular viability in a dose and time dependent manner	2018c)
	TiO2					NCM460 and coculture	Increase in DNA damage.
						of Human colon
						colorectal
	Pure rutile	40 -70 nm				adenocarcinoma Caco-2 and HT29-MTX cells
	crystal-structure
	nanorods of TiO2

Table 2 : Intestinal toxicity of organic NPs.

 2

	NPs	Size (nm)	Concentration	Exposure	Intestinal models	Observations	Reference
		Surface charge (mV)	of NPs			Time
						In vitro models
	Chitosan (CS)	290 nm	1 mg/mL		1 h	Human colon colorectal	No significant cytotoxicity of CS	(Slütter et al., 2009)
	loaded with a	43.3 mV					adenocarcinoma Caco-2	NPs was observed
	model antigen						cells
	ovalbumin
	(OVA)
	Chitosan	135.2 nm	0.125, 0.25,		3 h		No cytotoxicity was observed
	conjugated with a	6.3 mV	0.375 and 0.5
	goblet cell-target		mg/mL
	peptide, (CSK)
	peptide.
	Poly	/	0.1 1 and 10 mM 210 min	Human colon colorectal	G2, G3, G4 induced a significant	(El-Sayed et al., 2002)
	(amidoamine) PA						adenocarcinoma Caco-2	leakage of Lactate Dehydrogenase
	MAM						cells	in a dose dependent manner.
	Generation
	number:
	G0, G1, G2
	G3, G4
	PLGA	175 nm	12.5	-	200 8 h	Human colon colorectal	(Chaves et al., 2018)
			µg/mL				adenocarcinoma Caco-2
							cells
		211 nm	15.63	-250		Human colon colorectal
			µg/mL				adenocarcinoma Caco-2	No cytotoxicity was observed.
							cells and HT29 MTX
						In vivo models
	Chitosan	253.2 nm	5 mg/kg/day		7 days	Male Wistar rats	No cytotoxicity was observed	(Sonaje et al., 2011;
		28.2 mV						Liu et al., 2013)

  Disruption of epithelial cell microvilli was also observed in mice exposed to Ag NPs for 21 days at a dose of 20 mg/kg (van der[START_REF] Van Der Zande | Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure[END_REF]. An alteration of actin filaments reported

for TiO2 and Ag NPs

[START_REF] Déciga-Alcaraz | Irreversible disruption of the cytoskeleton as induced by non-cytotoxic exposure to titanium dioxide nanoparticles in lung epithelial cells[END_REF][START_REF] Xu | Silver nanoparticles (AgNPs) cause degeneration of cytoskeleton and disrupt synaptic machinery of cultured cortical neurons[END_REF]

could be hypothesized, considering that brush border is supported by a bundle of cytoskeleton compounds (Costa de

[START_REF] Costa De Beauregard | Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells[END_REF]

.

Table 1 : The composition of blank nanoemulsions

 1

	Batch	Products	Theoretical amount Surfactant to oil ratio
				(SOR)
		Kolliphor ®	0.98g
	NE20	Labrafac ®	0.28g	3.5
		Osmosed water	6.24g
		Kolliphor ®	0.84g
	NE30	Labrafac ®	0.42g	2
		Osmosed water	6.24g
		Kolliphor ®	0.63g
	NE70	Labrafac ®	0.63g	1
		Osmosed water	6.24g
		Kolliphor ®	0.42g
	NE200	Labrafac ®	0.84g	0.5
		Osmosed water	6.24g

Table 2 : Hydrodynamic diameter and Zeta potential of engineered blank NEs

 2

		NE20	NE30	NE70	NE200
	Surfactant -to-oil ratio	3.5	2	1	0.5
	Dynamic light scattering
	Hydrodynamic diameter (nm)
	T0	21.8±0.9	31.8±0.2	76.5±0.6	227.6±2.4
	T2h	21.4±0.1	32.4±0.6	75.8±1.5	213.4±3.6
	Polydispersity index
	T0	0.04±0.01	0.057±0.05	0.049±0.03	0.32±0.03
	T2h	0.04±0.02	0.039±0.03	0.075±0.03	0.23±0.03
	Laser Doppler
	electrophoresis
	Zeta Potential (mV)
	T0	-6.7	-1.9	-3	-3.1
	T2h	-4.2	-3.3	-2.7	-3.2

Acknowledgement

This work was achieved in the department of Pharmaceutical Engineering of the PEPITE EA4267, University of Franche-Comté; Besancon; France.

Allow me to thank the members of the jury: Dr. BOLAND Sonja and Pr. SAPIN-MINET Anne for accepting to be part of my thesis jury as reporters, Dr. MARTIN Hélène, and Pr. DAHER Ahmad for agreeing to examine this work.

I would like to thank Dr. Raphael Cornu for his support and for his presence during the period of my thesis. I would also like to express my immense gratitude for your guidance, your scientific and moral help. Thank you for attending as a guest member.

I would like to thank my thesis director Dr. Arnaud Béduneau for all his advices and encouragement throughout my thesis, and to his scientific honesty. His teachings have always been of high quality, they have brought me a lot and will remain an example for me. I also thank him for the long hours of work on this manuscript. I also express my thanks to my co-director, Pr. Mona Diab-Assaf. Thank you for your guidance and help. I would like to thank Pr. Céline Demougeot for trusting and welcoming me to the laboratory. I would also like to thank Dr. Hélène Martin and Dr. Georges Moarbess for agreeing to follow this work during the thesis monitoring committees. I would also like to thank Pr. Yann Pellequer and Claire Chrétien for their help and advice. I thank Pr. Nadine Millot and her team for their participation in the physicochemical characterisation of E551 food additive. I thank my colleagues Wassim El basset and Thomas Antoine for the great moments we shared in the laboratory. I dedicate this work to my adorable mum Insaf and my dear dad Haidar, to my loving brothers Acknowledgement T. Zaiter is supported by a fellowship from the "Centre Islamique d'Orientation et de l'Enseignement Supérieur (CIOES)". Some figures were designed using BioRender.com.

Abstract

The gastrointestinal tract represents one of primary routes of entry for many nanomaterials.

Their size in the nanometer range and their high surface area confer them very interesting properties as food additives. They are used as texturizing, opacifying or anticaking agents. Food packaging contains nanomaterials with antimicrobial properties. Humans are also orally exposed to nanoparticles (NPs) present in the air or drinking water. Ingested NPs can then reach the intestinal lumen and interact with the gastrointestinal fluids, microbiota, mucus layer and the epithelial barrier, allowing a potential translocation. The toxicological profile of ingested NPs is still unclear due to the variety of NPs in terms of composition and physicochemical properties as well as the limited number of investigations. Their unique properties related to their small size could however affect the intestinal ecosystem but also the physical and functional properties of the intestinal barrier. This review focuses on the fate of ingested organic and inorganic NPs in the intestinal lumen and their toxicity on the microbiota and epithelial cells.

Keywords : Nanoparticles, Intestinal cells, Microbiota, Intestinal permeability, Cytotoxicity, Genotoxicity

Conflicts of interest

There are no conflicts to declare.