Joana Costa-Gouveia

Giuseppina Salzano

Mario Menendez- Miranda

Eik Hoffmann

Nathalie Deboosere

Didier Desmaële

Priscille Brodin
email: priscille.brodin@inserm.fr

Ruxandra Gref
email: ruxandra.gref@u-psud.fr

PLGA engineered polymeric nanocarriers

Keywords: Mycobacterium tuberculosis, tuberculosis, vancomycin, PLGA, nanoparticles, nanocarrier

Tuberculosis (TB) is a major problem of global health, responsible for 10.4 million new cases and 1.8 million deaths in 2015 according to the World Health Organization (WHO). This disease is caused by inhalation of small aerosol droplets containing Mycobacterium tuberculosis (Mtb), and lungs are usually the major site of infection.

TB can usually be treated with a daily six months course of standard, or first-line, antituberculosis drugs. If first-line drugs are misused, the onset of multidrug-resistant Mtb can occur.

The new WHO global public health strategy "End TB" aims the reduction of TB incidence 90 % by 2035. To reach these ambitious targets, new approaches are urgently needed to get a faster, less harmful and more efficient treatment for active and latent TB.

My thesis focused on the use of nanoparticles (NPs) to develop new antituberculosis therapies. Our review of the literature showed that it could be a promising approach.

Here, we investigated four potential uses of the NPs.

INTRODUCTION

Figure 11 -Children with TB sleep outside at Springfield House Open Air School in London in 1932. Like sanatoriums, these schools offered TB patients a place to receive fresh air and sunshine cure. From (1932). 4. Chapter II

ACKNOWLEDGEMENTS / REMERCIEMENTS

This thesis represents a history of personal and professional growth, build with collaboration of many people. To them, I would like to express my affection and gratitude.

I would like to thank Dr. Ruxandra Gref, Dr. Vivi Miriagou, Dr. Laurent Marsollier and Dr. Alain Baulard for having accepted to be members of my PhD jury.

I would like to thank my supervisor, Dr. Priscille Brodin, for kindly accepting me as her PhD student and providing me the great opportunity to work in an excellent laboratory with state of the art equipment.

I am very grateful to all the current and previous members of the CGIM group, Alexandre Vandeputte, Arnaud Machelart, Christophe Queval, Clara Aguilar, Eik Hoffmann, Gaspard Deloison, Isabelle Ricard, Nathalie Deboosere, Ok-Ryul Song, Raffaella Iantomasi, Romain Veyron-Churlet, Vincent Delorme and to the members of Frank Lafont's team, for their strong support and for all the great moments.

A special thanks to Nathalie Deboosere and Arnaud Machelart for writing the french part of this thesis, and for their prompt help, whenever I needed it.

I am very grateful to Rosangela Frita, for her corrections to the introduction of this thesis, for the conversations, suggestions and for being a good friend. I am also grateful to Luis Solans, Kamel Djaout, Ruben Hartkoorn and Martin Moune for all their support, encouragement and consideration. I would also like to thank all the members of the Marie Curie Initial Training Network "CycloNHit", in particular to Elisabetta Pancani, with whom I have worked more closely, developing a good friendship.

Last but not least, to my family and friends, for the unconditional support, affection and trust. For being there in all my ups and downs and for giving me the strength and peacefulness to complete this stage.

x xi

RESUME

La tuberculose (TB) est un problème de santé mondiale majeur qui est à l'origine de 10.4 millions de nouveaux cas et 1.8 millions de morts en 2015 selon l'Organisation Mondiale de la Santé (OMS). Cette maladie est causée par la bactérie Mycobacterium tuberculosis (Mtb) qui infecte principalement les poumons et qui se transmet par l'inhalation d'aérosols contaminés.

Le traitement de TB nécessite la prise quotidienne d'antibiotiques de première ligne pendant 6 mois, dont la mauvaise utilisation peut être à l'origine de l'apparition de souches Mtb multi-résistantes.

La nouvelle stratégie de santé publique de l'OMS, "End TB", vise à réduire de 90 % l'incidence de TB d'ici 2035. Pour y parvenir, il est important de définir de nouvelles approches visant à réduire la durée et la toxicité des traitements et améliorer leur efficacité vis-à-vis des bactéries actives et latentes.

L'approche abordée lors de ma thèse a consisté à utiliser des nanoparticules (NPs) pour développer de nouvelles thérapies antituberculeuses. La bibliographie sur le sujet montre que cela pourrait être une stratégie prometteuse. Nous avons par conséquent étudié quatre applications potentielles des NPs:

1-Vectorisation des médicaments pour les administrer au niveau pulmonaire.

L'éthionamide (ETH) est un antibiotique de deuxième ligne utilisé pour le traitement de TB avec des effets secondaires indésirables. L'ETH est une "pro-drogue", qui nécessite une bioactivation par une monooxygenase bactérienne, dont l'efficacité peut être ellemême augmentée par des molécules chimiques appelées "booster". Nous avons étudié l'effet de l'ETH et de booster, co-encapsulés dans des NP de poly-β-cyclodextrine (pCD) biodégradables pour le traitement de TB. Nous avons d'abord évalué leur efficacité in vitro sur la croissance extracellulaire et intracellulaire (dans les macrophages) de Mtb grâce à l'utilisation d'un système automatisé de microscopie confocale à haut contenu.

Dans les deux essais, nous avons constaté que les médicaments conservaient leur activité après encapsulation et que les NP n'étaient pas cytotoxiques. L'efficacité des NP chargées a ensuite été étudiée in vivo chez des souris infectées par voie intranasale. La suspension de NP a été délivrée sous forme d'aérosols directement dans les poumons par voie endotrachéale à l'aide d'un Microsprayer® Aerosolizer. Une réduction significative de la xii charge bactérienne dans les poumons de 3-log a été observée après 6 administrations de doses inférieures à celles thérapeutiques.

2-Amélioration de la solubilité et biodisponibilité des antibiotiques. La clofazimine (CLZ) est un antibiotique utilisé dans le traitement de la lèpre et pourrait être, au regard de son efficacité in vitro sur les souches de Mtb multi-résistantes, un candidat potentiel pour celui de TB. La CLZ est extrêmement lipophile, gênant ainsi sa solubilité. Dans notre étude, son encapsulation dans des particules de silice nanoporeuses a stabilisé l'état amorphe de la CLZ et a augmenté radicalement sa solubilité. Après encapsulation ou solubilisation dans le diméthylsulfoxyde (DMSO), la CLZ a d'autre part montré une activité antibactérienne similaire sur Mtb.

3-Libération des antibiotiques dans les compartiments intracellulaires. La

Vancomycine (VCM) est utilisée pour des applications cliniques comme alternative de la pénicilline dans le traitement de Staphylococcus aureus et pourrait être utilisée pour celui de TB. Cependant, comme beaucoup d'antimicrobiens, VCM est incapable d'atteindre les concentrations thérapeutiques optimales à l'intérieur des cellules infectées. L'encapsulation de cet antibiotique à l'intérieur de NP à base de PLGA avec la libération dépendant du pH a amélioré son efficacité contre les bactéries intracellulaires. En conclusion et au regard des options thérapeutiques limitées pour combattre les souches résistantes et de la rareté de solutions innovantes dans le pipeline de découverte de médicament, ces travaux ont montré que les NPs pouvaient constituer une approche antituberculeuse originale.

4-Activité antimycobacterial

1-Nanocarrier for pulmonary delivery of drugs. Ethionamide (ETH) is a second line antibiotic with high toxicity and several adverse side effects. ETH is a prodrug that requires bioactivation by a bacterial monooxygenase, which can be enhanced by chemical molecules named "boosters". We investigated the simultaneous delivery of ETH and boosters coencapsulated in biodegradable poly-β-cyclodextrin (pCD) based NPs by the pulmonary route for the treatment of TB. First, we evaluated the in vitro efficacy of the designed formulations on Mtb extracellular growth and intracellular growth inside macrophages using an automated confocal high-content microscopy system. And we found for both assays that the drugs maintained their activity after encapsulation and the pCD were not cytotoxic. Given these promising results, their efficacy was then tested in vivo. The NPs suspension, administered directly into mouse lungs by endotracheal way using a Microsprayer® aerosolizer, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations and using lower doses than the therapeutic ones.

2-Enhancement of the solubility and the bioavailability of antibiotics. Clofazimine

(CLZ) is an antibiotic usually used in a combination therapy for the treatment of leprosy xiv and could be a potential candidate for the treatment of TB because of its in vitro efficacy on resistant Mtb strains. CLZ is extremely lipophilic and has important solubility problem.

In our study, its encapsulation in nanoporous silica particles stabilized the amorphous state of CLZ and dramatically increased the drug solubility. On the other hand, CLZ encapsulated in nanoporous silica particles or efficiently dissolved in Dimethyl Sulfoxide (DMSO) showed a similar antibacterial activity on Mtb, validating the assessment of solubility of CLZ by encapsulation.

3-Intracellular delivery of antibiotics. Vancomycin (VCM) is used for clinical

applications for nearly 50 years as a penicillin alternative to treat penicillinase-producing strains of Staphylococcus aureus. VCM can be used for TB treatment as a repurpose.

However, like many antimicrobials, VCM is unable to reach the optimal therapeutic concentrations inside infected cells. Encapsulation of this antibiotic inside Poly (Lactic-co-Glycolic Acid) (PLGA)-based NPs with pH-triggered release enhanced its efficacy against intracellular bacteria. From [START_REF] Ma | Global tuberculosis drug development pipeline: the need and the reality[END_REF] Tuberculosis (TB) has always been present in human populations throughout known human history and it may have killed more people than any other microbial pathogens [START_REF] Daniel | The history of tuberculosis[END_REF]. Over a billion lives were lost to TB in the past 200 years (Figure 1) [START_REF] Paulson | Epidemiology: A mortal foe[END_REF].

4-Intrinsic antimycobacterial activity of

Archaeological evidence and written records describing the disease were found in several ancient civilizations from different geographical regions, showing that TB was widespread in humans since the Neolithic period and has remained so until the present day [START_REF] Daniel | The history of tuberculosis[END_REF][START_REF] Barberis | The history of tuberculosis: from the first historical records to the isolation of Koch's bacillus[END_REF][START_REF] Donoghue | Insights gained from ancient biomolecules into past and present tuberculosis-a personal perspective[END_REF].

During the 18 th and 19 th centuries, TB, also called white plague, because of the extreme anaemic pallor of those affected, reached epidemic proportions in Europe and caused millions of deaths [START_REF] Daniel | The history of tuberculosis[END_REF][START_REF] Barberis | The history of tuberculosis: from the first historical records to the isolation of Koch's bacillus[END_REF].

TB declined after the late 19 th century until the mid-20 th most probably as a result of improved socioeconomic conditions and the development of sanatoria for the isolation and treatment of infectious patients [START_REF] Daniel | The history of tuberculosis[END_REF][START_REF] Lienhardt | Global tuberculosis control: lessons learnt and future prospects[END_REF]. The development of the Bacillus Calmette Guérin (BCG) vaccine (used in humans since 1921) and the discovery of antituberculosis drugs starting in 1944 also contributed for the 1. Introduction decline of global TB incidence [START_REF] Lienhardt | Global tuberculosis control: lessons learnt and future prospects[END_REF]. These developments could have had a much greater impact on control of the disease but regrettably, as the prevalence of the disease declined in developed countries, so did the interest in global disease surveillance and research [START_REF] Zumla | Reflections on the white plague[END_REF].

TB incidence unexpectedly increased in the late 1980s. Numerous factors were responsible for this including the emergence of the Human Immunodeficiency Virus (HIV), lack of access to antituberculosis drugs, increased immigration from countries where TB is common and the rise and spread of drug-resistant strains [START_REF] Kamholz | Resurgence of tuberculosis: the perspective a dozen years later[END_REF].

In 1993, the World Health Organization (W.H.O.) declared TB a global public health emergency. Since then, it has implemented and scaled-up successive global public health strategies for TB control: the DOTS (Directly Observed Treatment Short course) in 1993, the "Stop TB" strategy in 2006 and the "End TB" strategy in 2014 [START_REF] Sotgiu | Tuberculosis-a World Health Organization Perspective[END_REF]. The first two WHO strategies successfully contributed to decrease the annual TB incidence rate but not enough to achieve the WHO target: TB elimination by 2050 globally (incidence rate of less than one TB case per million population is referred to as elimination) [START_REF] Sotgiu | Tuberculosis-a World Health Organization Perspective[END_REF]. The new WHO strategy "End TB" aims to reduce TB deaths by 95 %, to reduce TB incidence to 90 % by 2035 and to ensure that no family is burdened with catastrophic costs due to TB by 2020 [START_REF] Petersen | World TB Day 2017: Advances, Challenges and Opportunities in the "End-TB" Era[END_REF]; 1. Introduction [START_REF] Sotgiu | Tuberculosis-a World Health Organization Perspective[END_REF]). To reach these ambitious targets the annual decline in global TB incidence rates must first accelerate from 1.5 % per year in 2015 to 10 % per year by 2025 (Figure 2). To sustain progress beyond 2025 requires the development of new tools such as a new vaccine, safer, more effective and shorter treatment options for active and latent TB and rapid, affordable, diagnostic methods of all forms of drug-susceptible and drug resistant disease [START_REF] Petersen | World TB Day 2017: Advances, Challenges and Opportunities in the "End-TB" Era[END_REF][START_REF] Schito | Tuberculosis eradication versus control[END_REF][START_REF] Sotgiu | Tuberculosis-a World Health Organization Perspective[END_REF].

According to the 2016 report on TB research funding trends (T.A.G. 2016), the total funding for TB R&D in 2015 was more than 620 million dollar, with the top donors, the Bill & Melinda Gates Foundation and U.S. National Institute of Allergy and Infectious Diseases giving almost 50 percent of the total funding. This value is far from the 2.9 billion dollar total funding for malaria, in 2015 (W.H.O 2016), and the 1.9 billion dollar total funding for HIV, in 2016 (UNAIDS 2017).

For new tools to be available by 2025 more investments in research and development, as well as on political commitment to implement effective programs to control TB and TB comorbidities are required.

Clinical presentation and diagnostics 1.1.1.

Until mid-1800s many believed that TB was a hereditary disease. In 1865 a French military surgeon, Jean Antoine-Villemin, proved the contagious nature of the disease by inoculating a rabbit with "a small amount of purulent liquid from tuberculous cavity" removed at autopsy from a TB victim [START_REF] Daniel | The history of tuberculosis[END_REF]. 1. Introduction TB is transmitted mainly from person to person by small aerosol droplets of 1-5 microns in diameter, expelled by infected hosts, for example by coughing, sneezing or speaking. Those droplets may remain suspended in the air during hours and be posteriorly inhaled by another host. In consequence, TB predominantly affects the lung (85 % of the cases are pulmonary TB) (W.H.O. 2016). Nevertheless, it may access the blood stream and affect other parts of the body such as pleura, lymph nodes (scrofula), abdomen, genitourinary tract, skin, joints, meninges and bones (Pott's disease) (Figure 3), and cause extrapulmonary disease (W.H.O. 2014).

The infection with Mycobacterium tuberculosis (Mtb), the causative agent of the vast majority of TB cases, may result in extremely different outcomes (Figure 4); however, patients are categorized as having either latent TB infection or active TB disease (O'Garra, Redford et al. 2013).

In latent TB, patients remain with no signs or symptoms of the disease (O'Garra, Redford et al. 2013). It is estimated that one third of the human population is latently infected with Mtb. The diagnostic is commonly made by the classic tuberculin skin test (TST) and/or Interferon-Gamma Released Assays (IGRA). These tests measure the response of T cells to Mtb antigens and enable to evaluate if the person has been infected with Mtb [START_REF] Pai | Gamma interferon release assays for detection of Mycobacterium tuberculosis infection[END_REF][START_REF] Fogel | Tuberculosis: a disease without boundaries[END_REF][START_REF] Salgame | Latent tuberculosis infection--Revisiting and revising concepts[END_REF][START_REF] Pai | Tuberculosis[END_REF]. The TST has been used for TB diagnosis over 100 years [START_REF] Salgame | Latent tuberculosis infection--Revisiting and revising concepts[END_REF].

It is performed by intradermal injection of tuberculin Purified Protein Derivative (PPD), a mixture of antigens present in both Mtb and other mycobacteria. In a person who developed T cell-mediated immunity to these antigens, a delayed-type hypersensitivity skin reaction will occur within 48-72 hours after injection. Interpretation of TST reactions takes into account: the millimetres of induration (swelling), the person's risk of being infected with TB and of progression to disease, if infected [START_REF] Pai | Gamma interferon release assays for detection of Mycobacterium tuberculosis infection[END_REF][START_REF] Fogel | Tuberculosis: a disease without boundaries[END_REF][START_REF] Pai | Tuberculosis[END_REF]. TST has the advantage of being a simple and low cost method, for that reason it is widely used. However, it lacks specificity and sensitivity, due to its cross-reaction with BCG and Nontuberculous Mycobacteria (NTM) and false negative responses in immunosuppressed individuals [START_REF] Pai | Gamma interferon release assays for detection of Mycobacterium tuberculosis infection[END_REF][START_REF] Fogel | Tuberculosis: a disease without boundaries[END_REF][START_REF] Pai | Tuberculosis[END_REF]).

In the early 2000s IGRA was introduced as immunodiagnostic tool to solve the specificity and sensitivity issues of TST [START_REF] Pai | Tuberculosis[END_REF]. IGRA measures the interferon-γ production of whole blood isolated T-cells in vitro upon stimulation with antigens encoded by Region of Difference 1 (RD1) such as Early Secreted Antigenic Target 6 (ESAT-6) and Culture Filtrate Protein 10 (CFP-10). RD1 antigens are more specific than PPD for Mtb because they are not encoded in the genome of any BCG vaccine strains or of most NTM species [START_REF] Pai | Gamma interferon release assays for detection of Mycobacterium tuberculosis infection[END_REF][START_REF] Pai | Tuberculosis[END_REF].

However, positive TST and/or IGRA results are shared by patients who have cleared the infection but retain a strong memory T cell response, those who have developed an adaptive immune response, yet who remain infected by bacteria in a quiescent or latent state ("true" latent infection), and those with subclinical or active TB. On the contrary, individuals that cleared the infection without developing a detectable adaptive immune response or infected individuals with highly localized immune response or insufficient magnitude to be detectable systemically may present negative TST and/or IGRA negative results [START_REF] Pai | Tuberculosis[END_REF]. Thus, none of the currently available testsable to accurately differentiate between different forms of the disease.

The principle signs and symptoms of active TB include fatigue, lack of appetite, weight loss, fever, chest pain and severe coughing with bloody sputum [START_REF] Pai | Tuberculosis[END_REF]. Patients with subclinical TB don't report symptoms but are culture positive.

Introduction

The first action for the detection of pulmonary TB is generally the chest x-ray. The bacteriological confirmation is additionally performed by microscopic examination, mycobacterial culture of sputum smears and molecular tests [START_REF] Caulfield | Diagnosis of active tuberculosis disease: From microscopy to molecular techniques[END_REF][START_REF] Pai | Tuberculosis[END_REF]. Microscopic examination is able to rapidly detect Mtb using Ziehl-Neelsen staining (also called acid-fast staining). However, only half the number of TB cases are detected with this diagnostic method and it doesn't detect drug-resistance (WHO). Moreover, Ziehl-Neelsen staining cannot distinguish Mtb from other acid-fast bacillus such as Rhodococcus spp. and Nocardia spp. (Caulfield and Wengenack).

Mycobacterial culture of sputum smears has high sensitivity and is able to identify Multidrug Resistant (MDR) Mtb strains simply by using antibiotics during culture.

However, growth of Mtb on traditional solid medium requires long time, between 2 to 8 weeks, depending on the initial number of Mtb [START_REF] Caulfield | Diagnosis of active tuberculosis disease: From microscopy to molecular techniques[END_REF][START_REF] Ryu | Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms[END_REF].

The use of liquid media is significantly faster, between 10 to 14 days and is more sensitive for detecting mycobacteria than solid media. There are now automated machines such as the Bactec Mycobacterial Growth Indicator Tube 960 (Becton Dickinson), which enabled early recognition of bacterial growth by using a fluorescent sensor that detects oxygen consumption. However, liquid systems are more prone to contamination by other microorganisms and they do not allow visualization of colony morphology and pigmentation, which is useful for distinguishing colonies of Mtb and of other NTM species [START_REF] Caulfield | Diagnosis of active tuberculosis disease: From microscopy to molecular techniques[END_REF][START_REF] Ryu | Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms[END_REF].

Molecular methods have been developed over the last two decades and are being increasingly used for detection of Mtb. These tests are highly specific compared to sputum smears and are also rapid; results can be available on the same day [START_REF] Ryu | Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms[END_REF].

Besides detecting the presence of Mycobacterium tuberculosis Complex (MTBC) and some NTM, some of these methods allow to detect resistance to antituberculous drugs and identification of the exact bacterial species.

Xpert MTB/RIF (Cepheid) assay is a cartridge based sample preparation with fully automated quantitative Polymerase Chain Reaction (qPCR), targeting different sequence regions of rpoB gene with fluorescent probes. The rpoB gene encodes the β-subunit of the bacterial RNA polymerase and mutations in this gene are responsible for approximately 96 % of Rifampicin (RIF) resistance (Caulfield and Wengenack). Therefore, xpert MTB/RIF can be a useful diagnostic tool to detect both Mtb and resistance to RIF. However, GeneXpert machine is not widely available yet.

Introduction

Loop-mediated isothermal amplification (LAMP) is a constant temperature method of amplifying DNA from Mtb in a single step. Unlike Polymerase Chain Reaction (PCR), it does not need a thermal cycler and results can be read with naked eye as fluorescence or turbidity [START_REF] Dorman | Advances in the diagnosis of tuberculosis: current status and future prospects[END_REF].

Line probe assay (LPA) also uses PCR amplification of DNA that codes for resistance to a certain drug. The products are detected as colour bands on a paper strip of nitrocellulose, which contains DNA probes that activate a colorimetric indicator, when bound to target DNA [START_REF] Dorman | Advances in the diagnosis of tuberculosis: current status and future prospects[END_REF]. First-line LPA can detect resistance to first-line drugs RIF, Isoniazid (INH) and Ethambutol (EMB). Second-line LPA can detect resistance to second-line drugs such as some fluoroquinolones (ofloxacin and levofloxacin) and all second-line injectables (kanamycin, amikacin, and capreomycin) [START_REF] Group | An activist's guide to Tuberculosis Diagnostic Tools[END_REF].

Advances in several diagnostic tools have contributed for the increase of the specificity and sensitivity of TB diagnosis. Nevertheless, the development of new TB diagnostic tools must continue to increase accuracy and to decrease costs and time in order to promote timely initiation of treatment, better treatment outcomes, and, ultimately, ending the TB epidemic.

Pathogenesis of Mtb and phagocytic cells 1.1.2.

Pathogenic life cycle of Mtb initiates when small aerosol droplets are expelled by an infected host (Figure 5). Once the bacillus is transmitted to a new host and delivered to lower lungs, the immune response to Mtb is initiated. Initial bacterial recognition is made by alveolar macrophages but also neutrophils, monocytes and Dendritic Cells (DCs), through different Pattern Recognition Receptors (PRRs) expressed on the surface of these cells [START_REF] Ernst | The immunological life cycle of tuberculosis[END_REF][START_REF] O'garra | The immune response in tuberculosis[END_REF] Nitrogen Species (RNS) produced by the Inducible Nitric Oxide Synthase (iNOS), phagossome acidification, and apoptosis [START_REF] Behar | Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis[END_REF][START_REF] Kleinnijenhuis | Innate immune recognition of Mycobacterium tuberculosis[END_REF].

The fate of intracellular mycobacteria can also be influenced by autophagy, a cellular process whereby cytoplasmatic components, including organelles and intracellular pathogens, are sequestered in a double-membrane vesicle called autophagosome and delivered to the lysosome for degradation [START_REF] Kleinnijenhuis | Innate immune recognition of Mycobacterium tuberculosis[END_REF][START_REF] O'garra | The immune response in tuberculosis[END_REF]. The activation of autophagy, by IFN-γ for example, leads to phagosome maturation and an increase in its acidification and killing of Mtb [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF]).

DCs also phagocytose Mtb and then migrate to lung-draining lymph nodes to present mycobacterial antigens to naive T lymphocytes. This step is essential for the activation [START_REF] Cooper | Cell-mediated immune responses in tuberculosis[END_REF]. If the response is too slow, bacteria grow and reach a point where although a potentially protective response is being expressed, the environment is such that it is not effective [START_REF] Cooper | Cell-mediated immune responses in tuberculosis[END_REF].

For that reason, Mtb impairs antigen presentation and migration of DCs to the draining lymph-nodes and induction of protective adaptive cellular responses is delayed compared with that of other infections or immunization [START_REF] Wolf | Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo[END_REF][START_REF] Wolf | Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs[END_REF]. Initiation of adaptive immunity responses to Mtb is delayed, approximatelly 42 days after infection in humans and 11-14 days in mice [START_REF] Ernst | The immunological life cycle of tuberculosis[END_REF]). In the meantime, Mtb proliferates in the early granulomas by a continual cycle of death of infected macrophages and infection of newly arriving macrophages, by phagocytosis of infected macrophages that have recently undergone apoptosis [START_REF] Davis | The role of the granuloma in expansion and dissemination of early tuberculous infection[END_REF].

As granulomas continue to mature, macrophages form an organized aggregate whose membranes become tightly interdigitated like those of epithelial cells [START_REF] Ramakrishnan | Revisiting the role of the granuloma in tuberculosis[END_REF]. Granuloma macrophages can also fuse into multinucleated giant cells or differentiate into foam cells, which are characterized by lipid accumulation. Many other cell types are also present in the granuloma, such as neutrophils, DCs, Natural Killer (NK) cells, T cells and B cells. Bacteria are most commonly present in the central necrotic areas in which dead and dying macrophages can be seen (Figure 6). Over time, there is an increase in fibroblast proliferation around the granuloma, that together with collagen and other extracellular matrix proteins leads to the formation of a fibrous capsule that surround the lesion and prevent bacterial spread [START_REF] Guirado | Modeling the Mycobacterium tuberculosis Granulomathe Critical Battlefield in Host Immunity and Disease[END_REF].

Mtb can persist for decades within the granuloma structures in a latent state, serving as a reservoir of the bacterium and, if the host becomes immunocompromised, it may lead to disease reactivation. Eventually, bacteria starts to proliferate, leading to increasing accumulation of necrotic debris, or caseum, in the granuloma center, which eventually leads to rupture of the granuloma and bacilli escape into the airways, thus spreading the infection to new tissue and potentially new hosts.

Introduction

Granulomas are generally assumed as protective structures that prevent dissemination of infection and provide a local environment for interaction of cells of the immune system. Indeed, individuals who never showed signs of active TB have evidence of healed granulomas [START_REF] Cosma | The secret lives of the pathogenic mycobacteria[END_REF]. However, granuloma is also a place that supports Mtb expansion. Virulent Mtb bacilli evolved multiple virulence mechanisms to rapidly adapt to the hostile environment in the granuloma and manipulate it for their own advantage [START_REF] Ramakrishnan | Revisiting the role of the granuloma in tuberculosis[END_REF][START_REF] Cambier | Host evasion and exploitation schemes of Mycobacterium tuberculosis[END_REF].

Mtb evades destruction by preventing phagolysosomal fusion and persisting in the phagosome, reducing acidification of the phagosomal compartment to a pH of around 6.4, still acidic but considerably less hostile than the pH of the lysosome (pH around 4.8), thus adapting to the intracellular environment of the macrophage and creating a niche for survival [START_REF] O'garra | The immune response in tuberculosis[END_REF]Tan and Russell 2015).

Introduction

Mtb is also able to modulate host cell metabolism to favor its survival. For example, Mtb is able to induce accumulation of lipid-loaded droplets inside the cell and then use fatty acids and cholesterol as a carbon source [START_REF] Vanderven | The Minimal Unit of Infection: Mycobacterium tuberculosis in the Macrophage[END_REF].

Moreover, Mtb possess an important protein secretion system ESX-1 encoded by genes in RD1, which secretes 6 kDa early secretory antigenic target ESAT-6 and 10 kDa culture filtrate protein CFP-10, proteins that play important roles in the bacterial pathogenesis. ESX-1 is required for mycobacterium virulence and its absence attenuates the strain M. bovis BCG vaccine [START_REF] Pym | Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti[END_REF]. ESX-1 has been shown to induce phagolysosomal rupture and provide cytosolic access to Mtb, which appears to results in cell toxicity and host cell death involving necrosis [START_REF] Simeone | Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death[END_REF][START_REF] Simeone | Cytosolic access of Mycobacterium tuberculosis: critical impact of phagosomal acidification control and demonstration of occurrence in vivo[END_REF]. This releases intracellular bacteria to the extracellular environment, permitting mycobacterial growth and subsequent re-infection. ESX-1 also induces apoptosis of infected cells, most likely through its secreted effector ESAT-6, which appears to promote bacterial proliferation during granuloma formation [START_REF] Davis | The role of the granuloma in expansion and dissemination of early tuberculous infection[END_REF].

Understanding the mechanisms used by Mtb to manipulate the host and proliferate may contribute for the development of effective therapeutic and preventive options against TB.

Risk factors 1.1.3.

The diversity of clinical manifestations after exposure to Mtb depends upon different environment-, social-, pathogen-and host-specific factors (Figure 7).

The strain virulence, the concentration of the bacterial inoculum and the duration of exposure are key determinants of whether infection occurs and increases the risk of progression to active disease [START_REF] Salgame | Latent tuberculosis infection--Revisiting and revising concepts[END_REF].

Host characteristics such as immunity, genre, age and comorbidities are also related with increased risk of progression to active disease.

Introduction

The HIV infection is the strongest risk factor for TB. In 2015, HIV-infected people accounted for 12 % of all new active TB disease cases and 22 % of all TB-associated deaths (W.H.O. 2016). Indeed, TB is the most prevalent opportunistic disease among people living with HIV, contributing to nearly one third of all its mortality [START_REF] Tiberi | The cursed duet today: Tuberculosis and HIV-coinfection[END_REF].

Other medical conditions and health-risk behaviours that weaken the immune system are associated with a greater risk of developing TB, such as diabetes mellitus, silicosis, chronic renal failure needing dialysis, immune-suppressive therapy, head and neck cancer, tobacco smoking, alcohol abuse and undernutrition [START_REF] Dheda | Tuberculosis[END_REF][START_REF] Pai | Tuberculosis[END_REF].

Highest risk of infection with Mtb is seen among children (0-4 years old) and young adults, from male sex (after adolescence) and individuals with specific genetic polymorphisms [START_REF] Dheda | Tuberculosis[END_REF]. Already in classical Greece the physician Hippocrates (460-370 BC) recognized the predilection of the disease, at that time called 1. Introduction phthisis (Greek word meaning "consumption"), for young adults: "Phthisis makes its attacks chiefly between the age of eighteen and thirty five" [START_REF] Daniel | The history of tuberculosis[END_REF].

Latently infected individuals are more susceptible to develop active TB during their lifetime, which is estimated to happen in 5-10 % of the cases [START_REF] Lin | Understanding latent tuberculosis: a moving target[END_REF][START_REF] O'garra | The immune response in tuberculosis[END_REF].

Socioeconomic and environmental factors are also shown to increase the susceptibility to infection, including poverty, overcrowding and air pollution. "TB will not be eliminated anywhere until it is eliminated everywhere" Don Enarson.

When anyone not working in the fields of TB science or medicine is asked about the disease, they often recall a debilitated and pallid woman from the mid-20 th century coughing blood into a handkerchief. However, TB is not a disease of the past. It is estimated that more than 2 billion people, equally to one third of the world's population, are infected with Mtb (WHO). Fortunately, most of infections with Mtb (90-95 %) remain 1. Introduction asymptomatic (latent TB) with a 5-10 % lifetime risk of progressing to active TB [START_REF] Gideon | Latent tuberculosis: what the host "sees"?[END_REF][START_REF] Salgame | Latent tuberculosis infection--Revisiting and revising concepts[END_REF]. Even so, TB was one of the top ten causes of death worldwide in 2015, and the number one cause of death from an infectious disease, surpassing HIV and malaria (W.H.O. 2016). According to WHO, in 2015 there were 10.4 million new TB cases worldwide and 1.8 million deaths were caused by this disease, including 0.4 million HIV-associated TB deaths (W.H.O. 2016).

TB epidemiology is heterogeneously distributed worldwide, as shown on the map of estimated TB incidence (Figure 8). Most cases are estimated to occur in Asia and Africa (61 % and 26 %, respectively), with the six countries with highest number of incident cases in 2015 (India, Indonesia, China, Nigeria, Pakistan and South Africa) accounting for 60 % of the total number of cases (W.H.O. 2016).

Etiological agent of TB: Mycobacterium tuberculosis complex (MTBC) 1.1.5.

On March 24 th , 1882, Dr. Robert Koch announced the discovery, of Mtb, the etiological agent of TB disease. This important discovery is commemorated each year on March 24 th , the World TB Day [START_REF] Lakhtakia | The Legacy of Robert Koch: Surmise, search, substantiate[END_REF].

Mtb belongs to a group of phylogenetically related species collectively denominated as MTBC, which in addition to Mtb includes other species such as M. africanum, M. bovis, M. canettii M. avium and M. microti [START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Galagan | Genomic insights into tuberculosis[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF]. All of these species are potential human pathogens; however, the vast majority of the cases of TB in humans are caused by Mtb and M. africanum [START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF].

Mycobacterium is a genus of the actinobacteria phylum (gram-positive bacteria with high guanine and cytosine content in DNA). Among Mycobacterium genus, Mtb belongs to the slow growth group, with a doubling time of 12-24 h under optimal conditions [START_REF] Delogu | The biology of mycobacterium tuberculosis infection[END_REF]. Mycobacterial cells are irregular rods of 0.3-0.5 µm in diameter and 1.5-4.0 µm in length [START_REF] Cook | Physiology of mycobacteria[END_REF].

A major feature of mycobacteria is the unusual cell envelope structure, which provides a strong impermeable barrier and plays an important role in the pathogen's 1. Introduction survival and virulence, It is composed of three major structural components: the plasma membrane, the cell wall core and the capsule (Kieser and Rubin 2014) (Figure 9).

The plasma membrane consists of a typical lipid bilayer with protein interactions. It is surrounded by long polymers of peptidoglycan, which comprises repeating disaccharide N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are cross-linked via short peptide chains attached to the NAM [START_REF] Kieser | How sisters grow apart: mycobacterial growth and division[END_REF]. The peptidoglycan layer confers rigidity and osmotic stability to the cell wall of mycobacteria as it does to several other gram-positive and gram-negative bacteria [START_REF] Jankute | Assembly of the Mycobacterial Cell Wall[END_REF].

A layer of arabinogalactan consisting of arabinan disaccharides and galactose monosaccharides surrounds the peptidoglycan layer. Arabinan is bound to long-chain mycolic acids, which form the characteristic hydrophobic thick lipid layer of mycobacteria. This layer constitutes approximately 50 % of mycobacterium dry weight and it is responsible for the resistance to degradation by lysosomal enzymes and by many drugs [START_REF] Kleinnijenhuis | Innate immune recognition of Mycobacterium tuberculosis[END_REF].

Glycolipids and porins are also found in the cell wall, as Lipoarabinomannan (LAM), which is anchored to the cell membrane [START_REF] Brown | Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi[END_REF]. The arabinan motifs of LAM are further decorated by capping moieties, depending on the mycobacteria species. [START_REF] Brown | Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi[END_REF].

Introduction

In the case of Mtb, LAMs are capped with mannose and are referred to as ManLAMs [START_REF] Jankute | Assembly of the Mycobacterial Cell Wall[END_REF].

Due to the high density of lipids in the cell wall, mycobacteria resist staining by ordinary methods such as a Gram stain. They are also resistant to acid and alcohol decolorization and are described as acid-fast bacilli [START_REF] Kieser | How sisters grow apart: mycobacterial growth and division[END_REF]. A different staining method must be used, namely the Ziehl-Neelsen Stain, a method in which the acid-fast mycobacterium is stained in bright red with carbol fuchsin, combined with phenol. After application of an acid decolorizing solution and a counter stain (methylene blue), non-acid-fast bacteria become blue [START_REF] Vasanthakumari | Practical Microbiology[END_REF].

Evolution and genetic diversity within the MTBC 1.1.6.

"You have to know the past to understand the present"

Dr. Carl Segan

The ancient association of TB with humanity was initially recognized by observation of the characteristic Pott's disease lesions in the spine of human fossils. However, other pathologies such as brucellosis and chronic inflammation can also cause changes in the bones that can be difficult to distinguish from TB [START_REF] Donoghue | Insights gained from ancient biomolecules into past and present tuberculosis-a personal perspective[END_REF]. Moreover, it is estimated that skeletal TB corresponds only to 3-5 % of untreated cases, so only a small fraction of ancient TB infections were detected by simple skeleton observation [START_REF] Donoghue | Insights gained from ancient biomolecules into past and present tuberculosis-a personal perspective[END_REF].

The introduction of molecular diagnostic methods to Paleopathology in the 90s has enabled the specific identification of TB in historical and archaeological specimens. [START_REF] Donoghue | Insights gained from ancient biomolecules into past and present tuberculosis-a personal perspective[END_REF]. This advance has expanded our knowledge of the probable origin of the different Mtb lineages and their geographical distribution, which opens up new challenges in the understanding of the evolution of human-pathogen relationships.

The oldest evidence of TB infection in humans comes from ancient human remains from the Neolithic era dating from 9000 years ago [START_REF] Hershkovitz | Detection and molecular characterization of 9,000year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean[END_REF].

Signs of the infection in humans were also found in Egyptian mummies dating back to c. 3000 BC [START_REF] Zink | Molecular analysis of skeletal tuberculosis in an ancient Egyptian population[END_REF]).

Introduction

Genomic studies revealed that Mtb has coexisted with human beings in the last 70.000 years [START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF]. During this period, both the pathogen and the host have adapted to each other to increase chances of survival [START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF]. From this evolution, seven different lineages of mycobacteria that constitute the MTBC have arisen (Figure 10) [START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Galagan | Genomic insights into tuberculosis[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF]. Each lineage is linked with a specific geographic region, where the bacteria are more often isolated: lineage 1 occurs around the Indian Ocean, lineage 2 is widely distributed in East Asian countries, lineage 3 occurs in Central Asia, lineage 4 has a broad distribution in Europe and America, lineage 5 and 6 are restricted to West Africa and lineage 7 is restricted to Ethiopia [START_REF] Comas | Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF].

Bacteria of the MTBC have been described as being genetically monomorphic [START_REF] Sreevatsan | Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination[END_REF][START_REF] Ramaswamy | Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis[END_REF][START_REF] Comas | Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved[END_REF][START_REF] Achtman | Insights from genomic comparisons of genetically monomorphic bacterial pathogens[END_REF][START_REF] Galagan | Genomic insights into tuberculosis[END_REF]). However, as mentioned above, infection with MTBC strains may result in a wide spectrum of disease outcomes, ranging from clearance to latent or active TB with different severity degrees. Until recently, the different outcomes of Mtb infection were totally attributed to host and environmental factors. Currently, there is increasing evidence that part of TB diversity may result from the heterogeneity of the bacteria. For instance, studies using MTBC strains from different phylogeographical lineages demonstrated that: (i) strains from the same lineage are more likely to spread among the human population from the same geographic origin [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]); (ii) different lineages of MTBC have different rates of progression from latent to active TB [START_REF] De Jong | Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in The Gambia[END_REF]); (iii) different Mtb strains lead to different macrophage and in vivo immune responses [START_REF] Portevin | Human macrophage responses to clinical isolates from the Mycobacterium tuberculosis complex discriminate between ancient and modern lineages[END_REF][START_REF] Carmona | Differential TLR recognition of Mycobacterium tuberculosis strains modulates innate immune responses with an impact on the outcome of infection[END_REF] and that (iv) the modern lineages L2 and L4 have different intracellular growth rates and induce a weaker innate proinflammatory response, when compared to ancient lineages (L1, L5, L6 and L7).

This suggests that the limited genetic diversity existent within the MTBC, impacts different aspects of TB as well as host-pathogen interactions.

Treatment and prophylaxis 1.1.7.

Before the discovery of antibiotics, treatment of TB was limited to a regimen of rest and better nutrition. In the late 19 th and 20 th , the development of sanatoriums contributed to a moderate control of the disease. In these institutions, patients were exposed to high altitude, fresh air, good nutrition, bed rest and sunbathing (Figure 11) [START_REF] Riva | From milk to rifampicin and back again: history of failures and successes in the treatment for tuberculosis[END_REF]. Here, more invasive approaches were also applied, such as lung collapse 1. Introduction therapies and surgical resection of disease lung tissue [START_REF] Riva | From milk to rifampicin and back again: history of failures and successes in the treatment for tuberculosis[END_REF]. It is doubtful whether the sanatorium treatment improved the survival of the patients, but it provided a haven for those healing from TB and it may have helped reducing the spread of the disease.

The introduction of chemotherapy against TB started in 1940s with the discovery of Streptomycin (STR), in 1943, derived from Streptomyces griseus (Figure 12). The results were impressive but soon STR resistant mutants began appearing, threatening the success of antibiotic therapy [START_REF] Kerantzas | Origins of Combination Therapy for Tuberculosis: Lessons for Future Antimicrobial Development and Application[END_REF]. Shortly afterwards, Para-Aminosalycilic acid (PAS), a synthetic antimicrobial, was introduced as an oral therapy. In 1. Introduction thought that INH could be used as monotherapy but, INH resistance subsequently appeared, demonstrating again the importance of combining different drugs for the treatment of TB [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF]. The "triple therapy", combining INH, STR and PAS, was the standard treatment during 15 years [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF].

In 1961 an extensive screening led to the discovery of EMB. This synthetic antibiotic was reported to be as effective as PAS when combined with INH even at a low dose and it was much better tolerated [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF]. For that reason, soon it replaced PAS in the standard treatment (Z Ahmad 2011).

The next major advance was the introduction of RIF in 1963, derived from Amycolatopsis mediterranei (Z Ahmad 2011). This drug exhibited a spectacular activity and was able to kill dividing and semidormant bacteria, the so called "persisters", like no other drug [START_REF] Dobbs | Chemotherapy of Tuberculosis[END_REF]. The addition of RIF to the standard regimen of STR, INH and EMB, in the 1970s, allowed to reduce the length of treatment from 18 months to 9 months and led to improved cure rates [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF]. However, it causes an increase of the hepatic Cytochrome P450 (CYP450) oxidase system, decreasing the levels of many other drugs, including many protease inhibitors, a class of antiviral drugs [START_REF] Dobbs | Chemotherapy of Tuberculosis[END_REF]. Currently, there are no sterilizing drugs that can prevent relapse as effectively as rifamycins, therefore, combining drug therapies in patients coinfected with HIV and Mtb is complex. Rifabutin, another rifamycin, is a less potent inducer of CYP450 and may be used as an alternative to RIF when drug-drug interactions or drug intolerance impedes use of RIF [START_REF] Dobbs | Chemotherapy of Tuberculosis[END_REF]. Pyrazinamide (PZA) was also added to the standard regimen allowing a shortening of treatment to 6 months due to its activity against slow-growing and non-replicating organisms [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF] According to the new WHO drug classification (2016) (Table 1) the treatment of RIFresistant or MDR-TB should include at least 5 potentially active drugs during the intensive phase: one first-line drug such as PZA and 4 second-line TB-drugs, one from of a later generation fluoroquinolone (moxifloxacin, levofloxacine or gatifloxacine; group A), an injectable aminoglycoside (amikacin, capreomycin, or kanamycin; group B) and at least 2 from group C (Ethionamide (ETH)/protionamide, cycloserine/terizidone, linezolid and Clofazimine (CLZ)). If some of these drugs cannot be used, additional drugs from group D can reinforce the regimen to bring the total to 5 drugs (2016). Group D is divided into 3 subgroups: first-line antituberculosis drugs (D1), the new drugs in confirmatory phase III trials, bedaquiline and delamanid (D2), and other agents of uncertain role in MDR-TB treatment (D3). The recommended duration is 20 months with an intensive phase (with injectable drugs) during the first 8 months (2016).

The 2016 WHO update on MDR-TB treatment guidelines suggests that partial lung resection used alongside a recommended MDR-TB regimen may be considered for the management of MDR-and XDR-TB (2016). However, weak evidence exists confirming that it increases the prognosis, compared to chemotherapy alone.

Also in 2016, WHO has recommended a shorter (9-12 months) MDR-TB treatment regimen with seven drugs, for patients with RIF-resistant or MDR-TB that is not resistant to second-line drugs (2016). The treatment includes 3 drugs (kanamycin, prothionamide, and high-dose INH) for 4-6 months, plus 4 drugs (moxifloxacin, CLZ, PZA, and EMB) given throughout the course of treatment. This new recommendation is expected to benefit the majority of MDR-TB patients; however, an inappropriate use (XDR-TB, for example) may worsen resistance.

Introduction

The treatment of XDR-TB is designed on the basis of the patient's individual results of drug susceptibility testing and on similar principles to those drawn for MDR-TB.

Not only the development of new drugs is important to eradicate TB, an effective new vaccine is also needed. Despite years of research, no vaccine currently provides reliable protection against pulmonary TB in adults, the most prevalent and contagious form of the disease [START_REF] Ottenhoff | Vaccines against tuberculosis: where are we and where do we need to go?[END_REF]. Indeed, although many components of the host immune response against MTBC are known, lack of an immune correlate of protection, have been hampering the development of a more effective vaccine [START_REF] Ottenhoff | Vaccines against tuberculosis: where are we and where do we need to go?[END_REF].

The current vaccine BCG was introduced in 1921 and remains the only available vaccine against TB [START_REF] Luca | History of BCG Vaccine[END_REF]. It was developed by Calmette and Guérin in the Institute Pasteur of Lille from a live attenuated strain of M. bovis, which lost its virulence towards humans after more than 11 years of consequent subcultures [START_REF] Luca | History of BCG Vaccine[END_REF].

The effectiveness of this vaccine is variable and limited in adulthood pulmonary TB, even though, it is among the most widely administered vaccines, with more than 4 billion doses delivered to date, and it saved a large number of deaths that would otherwise be caused by TB every year. [START_REF] Ottenhoff | Vaccines against tuberculosis: where are we and where do we need to go?[END_REF].

No universal BCG vaccination policy exists, and national vaccination schedules vary widely. In France, for example, a country with low prevalence of TB, universal mandatory BCG vaccination of children was suspended in 2007, with a shift to selective vaccination of children living in the Ile-de-France (Paris and suburbs) or French Guyana regions, and children considered by a physician as living in an environment with a high risk of exposure to TB [START_REF] Lévy-Bruhl | Recent changes in tuberculosis control and BCG vaccination policy in France[END_REF].

BCG vaccine and the existent antituberculosis drugs are clearly insufficient for worldwide TB control and alternative strategies for TB prevention and therapy are urgently needed.

Introduction

Development of new antituberculosis drugs and regimens 1.1.8.

Before a promising new drug is brought to the market it needs to pass through a series of pre-clinical and clinical trials, which is a long (10 or more years), expensive and complex process. In preclinical studies in vitro and in vivo studies are performed to study the drug's activity, physicochemical properties, pharmacokinetics and toxicity. In the TB field, mouse models are currently the most commonly used [START_REF] Cooper | Mouse model of tuberculosis[END_REF]. During clinical trials, drugs are tested on people in three steps: Phase I trials are performed in 20-100 healthy volunteers to determine safety and dosing; Phase II trials are performed in up to several hundred people with the disease to get an initial idea of efficacy and side effects; Phase III trials evaluate efficacy and monitor adverse reactions in large numbers of patients during long time (1 to 4 years); Phase IV trials are conducted to uncover more information about efficacy, safety and side-effects after being approved by U.S. Food & Drug Administration (FDA) for marketing (FDA).

Development of new antituberculosis drugs should ideally be able to overcome the problems associated with the current regimens, fulfilling a set of requirements that includes a high level of potency to reduce the treatment duration, ability to inhibit new targets, which is crucial for MDR-TB and XDR-TB treatment, good penetration into all tissues where the Mtb resides, have low toxicity profile for use in children and pregnant women, compatibility with antiretroviral therapy, absence of antagonistic activity with existing TB drugs, ease of use, and be active against different physiological states of Mtb, including latency. Alongside with these ideal criteria, other practical aspects are important such as low production costs, compound stability and low rate of spontaneous resistance emergence.

New antituberculosis drugs in development (phase III)

After the introduction of RIF in the late 1960s, the war on TB was considered winnable with existent tools and drug development for TB has stagnated for decades [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF]. Between 1970 and 2000 only fluoroquinolones were introduced for the treatment of MDR-TB [START_REF] Hofman | Emerging drugs and alternative possibilities in the treatment of tuberculosis[END_REF]. Resurgence of TB in the late 1980s renewal the interest in finding drugs to eliminate the disease. From 2000 onwards, multiple new antituberculosis drugs are in clinical development (Figure 1. Introduction 13), the majority of which are incorporated into the TB Drug Accelerator, a programme sponsored by the Bill&Melinda Gates Foundation [START_REF] Hofman | Emerging drugs and alternative possibilities in the treatment of tuberculosis[END_REF][START_REF] Pai | Tuberculosis[END_REF]. Two of these drugs, bedaquiline and delamanid, are still in confirmatory phase III trials but have been already conditionally approved in many countries and are now recommended by WHO as an addition to a background treatment for MDR-and XDR-TB, for whom an effective treatment regimen is not available (2016). Based on ongoing trials, these drugs have been proven to be effective in increasing sputum smear and culture conversion, and in improving success rates at the end of treatment, although safety concerns for these drugs, including cardiotoxicity and deaths, remains [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF][START_REF] Hofman | Emerging drugs and alternative possibilities in the treatment of tuberculosis[END_REF]. Accelerated approval of these drugs indicates the lack of viable alternatives and poor outcomes for patients with MDRand XDR-TB. Bedaquiline, previously identified as R207910 and then TMC207, is a promising new TB drug discovered in a high-throughput phenotypic screen for compounds active against M. smegmatis, and subsequently showed activity against other mycobacteria, including Mtb [START_REF] Andries | A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis[END_REF][START_REF] Chakraborty | Tuberculosis Drug Development: History and Evolution of the Mechanism-Based Paradigm[END_REF]. It is a novel diarylquinolone that works through a new mechanism, inhibiting essential ATP synthase activity of both replicating and dormant mycobacteria. The activity of bedaquiline is specific for mycobacteria, while the Minimum Inhibitory Concentration (MIC) for other prokaryotic and eukaryotic is much higher [START_REF] Andries | A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis[END_REF][START_REF] Hoagland | New agents for the treatment of drug-resistant Mycobacterium tuberculosis[END_REF]. Bedaquiline displays potent early and late bactericidal activity and excellent MICs against both drug-sensitive (0.03 µg/mL) and MDR-TB (0.12 µg/mL), as well as outstanding activity in in vivo murine models [START_REF] Andries | A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis[END_REF]. Moreover, the long effective half-life (24 h) [START_REF] Andries | A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis[END_REF]) may allow the use of bedaquiline for intermittent drug administration when combined with others drugs in the regimen against MDR-TB and XDR-TB. However, its metabolism via the CYP450 system limits its potential use as adjunct to RIF, a potent inducer of CYP450 system [START_REF] Hoagland | New agents for the treatment of drug-resistant Mycobacterium tuberculosis[END_REF].

The second promising drug, delamanid, formerly called OPC-67683, belongs to the nitroimidazoles, a class of compounds with known activity against protozoan and anaerobic bacterial infections. Delamanid is effective in killing both replicating (aerobic condition) and non-replicating (anaerobic condition) mycobacteria in vitro and in the guinea pig TB model [START_REF] Chen | Delamanid Kills Dormant Mycobacteria In Vitro and in a Guinea Pig Model of Tuberculosis[END_REF]. It inhibits the synthesis of methoxy-1. Introduction and keto-mycolic acids, crucial components of the cell wall of MTBC, thus its antibacterial activity is mycobacteria-specific. Unlike INH, delamanid has little effect on α-mycolic acid biosynthesis [START_REF] Matsumoto | OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice[END_REF]. Despite the absence of an effect 1. Introduction on α-mycolic acid, the MICs reported with delamanid with both drug-susceptible and drug-resistant Mtb, in vitro, are lower (0.006-0.024 µg/mL) than the ones reported with INH (0.05-0.78 µg/mL) [START_REF] Matsumoto | OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice[END_REF]). As a prodrug, delamanid requires activation possibly mediated via the F420-deazaflavin-dependent nitroreductase (ddn) of Mtb.

Pretomanid (PA-824) is another nitroimidazole currently in clinical development (phase III). It also exhibits bactericidal activity against both latent and active TB and it has also shown activity against both drug-sensitive and drug-resistant TB [START_REF] Stover | A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis[END_REF]. Similarly to delamanid, pretomanid inhibits the synthesis of methoxy-and keto-mycolic acids in mycobacteria cell wall and requires metabolic activation through ddn (Rv3547) [START_REF] Manjunatha | Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis[END_REF]), but MICs reported (0.015-0.25 µg/mL) are higher than delamanid and comparable to INH [START_REF] Stover | A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis[END_REF].

Pretomanid also kills non-replicating Mtb by intracellular Nitric Oxide (NO) release, which appears to be crucial for its anaerobic activity through poisoning of cytochrome c oxidase [START_REF] Singh | PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release[END_REF]. Similarly, delamanid may also induce NO production and this may be responsible for cell killing under non-replicating conditions since mycobacteria do not extensively remodel mycolic acids under anaerobiosis [START_REF] Boshoff | Is the mycobacterial cell wall a hopeless drug target for latent tuberculosis?[END_REF].

Repurposed drugs

Repurposed drugs are antimicrobials that were developed for other diseases, but which have useful antimycobacterial activity. Repurposing is attractive as a drug development strategy since much is already known about approved agents, including their pharmacokinetics and pharmacodynamics features, dosing, safety, tolerability and manufacturing. Even though further studies of drug dosing, safety, drug-drug interactions and efficacy in the context of the long multidrug regimen treatment of TB is needed, repurposing drugs accelerates their entrance in the market of TB drugs (Dheda, Barry et al. 2016). The major drawback of this strategy is the increased risk of emergence of drug resistance as a side effect of previous treatments nonrelated to TB. Several existing antibiotics are now being repurposed for treatment of TB and some of these are listed under WHO drugs (Table 1).

Introduction

Fluoroquinolones are a class of antibiotics frequently used to treat a wide variety of bacterial infections. The main targets are bacterial topoisomerase II (DNA gyrase) and DNA topoisomerase IV, two critical enzymes for bacterial viability [START_REF] Dheda | Tuberculosis[END_REF]. Several members of this class have been used as second-line drugs for the treatment of MDR-TB. Interest of moving fluoroquinolones to the first-line was renewed when it was shown that moxifloxacin instead of INH in the standard regimen could reduce the duration of therapy in a murine model of TB [START_REF] Nuermberger | Moxifloxacin-containing regimen greatly reduces time to culture conversion in murine tuberculosis[END_REF]). However, three trials have demonstrated that regimens including both moxifloxacin and gatifloxacin are not effective in shortening the treatment to 4 months [START_REF] Gillespie | Four-month moxifloxacin-based regimens for drugsensitive tuberculosis[END_REF][START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF][START_REF] Merle | A four-month gatifloxacin-containing regimen for treating tuberculosis[END_REF].

Currently, different moxifloxacin-containing regimens are in clinical trials (Figure 13) to improve the treatment of MDR-TB and patients co-infected with TB and HIV. Levofloxacin is also under a phase II clinical study to determine the highest levofloxacin area under the curve (AUC) that is associated with maximal efficacy and acceptable safety/tolerability.

CLZ is an extremely lipophilic riminophenazine antibiotic discovered in the 1950's and used since 1962 in a combination therapy for the treatment of leprosy [START_REF] Cholo | Clofazimine: current status and future prospects[END_REF]. It was initially discarded for the treatment of TB possibly because other agents brought on the market at about the same time were more effective and because of some undesirable side-effects such as pigmentation of the skin and gastrointestinal upset [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF]. CLZ demonstrated in vitro activity against drugresistant and drug-susceptible Mtb and efficacy against a mouse model of TB [START_REF] Reddy | Antituberculosis activities of clofazimine and its new analogs B4154 and B4157[END_REF]. The activity of CLZ was also observed in vitro and in vivo against Mtb persisters [START_REF] Xu | In vitro and in vivo activity of clofazimine against Mycobacterium tuberculosis persisters[END_REF]) and against latent TB [START_REF] Zhang | Streptomycin-starved Mycobacterium tuberculosis 18b, a drug discovery tool for latent tuberculosis[END_REF].

Moreover, in mouse models, CLZ demonstrated potential to shorten the duration of TB chemotherapy in combination with first-line and second-line drugs [START_REF] Grosset | Assessment of clofazimine activity in a second-line regimen for tuberculosis in mice[END_REF][START_REF] Tyagi | Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis[END_REF].

CLZ is now recommended by the WHO to treat MDR-TB as a medicine with "unclear efficacy" (group C). Although the exact mode of action is not completely understood, it appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), and subsequently after spontaneous reoxidation, liberates ROS [START_REF] Lechartier | Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis[END_REF].

Oxazolidinones are antibiotics developed for the treatment of infections caused by resistant gram-positive bacteria, which exert their antimicrobial activity by inhibiting 1. Introduction protein synthesis by binding to the bacterial 23S ribosomal RNA [START_REF] Dheda | Tuberculosis[END_REF]. The first commercially available oxazolidinone, linezolid, is now recommended by WHO to treat patients with MDR-TB (group C). Different studies suggest that linezolid containing chemotherapy for drug-resistant TB may improve treatment success, but patients must be monitored carefully because of significant side effects associated with long treatment [START_REF] Cox | Linezolid for the treatment of complicated drug-resistant tuberculosis: a systematic review and meta-analysis[END_REF][START_REF] Lee | Linezolid for treatment of chronic extensively drug-resistant tuberculosis[END_REF][START_REF] Sotgiu | Efficacy, safety and tolerability of linezolid containing regimens in treating MDR-TB and XDR-TB: systematic review and meta-analysis[END_REF][START_REF] Tang | Efficacy, safety and tolerability of linezolid for the treatment of XDR-TB: a study in China[END_REF]. Other two oxazolidinones, sutezolid (previously known as PNU-100480) and delpazolid (LCB01-0371), are currently in clinical development phase II, and one oxazolidinone, TBI-223, is in preclinical development.

β-lactams are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. This ring mimics the shape of the terminal D-alanyl-D-alanine peptide sequence that serves as the substrate for cell wall transpeptidases that form covalent bonds between different peptidoglycan chains, during periods of cell growth. The tight binding of these drugs to the transpeptidase active site inhibits the synthesis of the peptidoglycan layer of the bacterial cell wall, resulting in a weakened cell wall that is susceptible to lysis during periods of cell growth.

The most important mechanism of bacterial resistance to β-lactam antibiotics is enzymatic inactivation by β-lactamases by cleavage of the β-lactam ring. Mtb possesses a constitutively active broad-spectrum β-lactamase, BlaC, which may justify why β-lactams have received little attention as antituberculosis drugs [START_REF] Dheda | Tuberculosis[END_REF].

However, addition of clavulanate, an irreversible inhibitor of BlaC, enhances β-lactam activity [START_REF] Hugonnet | Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate[END_REF]. Carbapenems, a subclass of β-lactams, such as meropenem, and imipenem, are relatively resistant to hydrolysis by β-lactamases, and thus are active against Mtb in vitro, nonetheless their activity is enhanced by the addition of clavulanate [START_REF] Hugonnet | Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis[END_REF]. Combination of carbapenems plus clavulanate showed significantly improved survival in a murine model of TB [START_REF] Veziris | Activity of carbapenems combined with clavulanate against murine tuberculosis[END_REF], and a systematic review of carbapenem-containing regimens in individuals with MDR-XDR-TB showed to have very good safety and tolerability and treatment efficacy higher than 57 % in five studies with culture conversion rates between 60 % and 94.8 % (Sotgiu, D'Ambrosio et al. 2016). The combination of a carbapenem and penicillin, another subclass of β-lactams, such as amoxicillin might also have synergistic or additive effect in vitro [START_REF] References Gonzalo | Is there a place for beta-lactams in the treatment of multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between meropenem and amoxicillin/clavulanate[END_REF]. A recent phase 2A trial with limited number of patients examined the activity of meropenem, amoxicillin, and 1. Introduction clavulanate combination and showed early bactericidal activity in patients with pulmonary drug-susceptible TB [START_REF] Diacon | beta-Lactams against Tuberculosis--New Trick for an Old Dog?[END_REF]. However, all drugs were administered three times per day, and meropenem requires intravenous administration, which makes this combination impractical in many settings.

"Revived" old TB drugs

Many drugs developed during the early days of TB drugs development, are now rarely used because of poor efficacy and associated toxicity.

An innovative approach to make old TB drugs strong again arose from the observation that a significant number of antituberculosis antibiotics are prodrugs that must be bioactivated by mycobacterial enzymes to exert their activity, and that overproduction of the prodrug activator in recombinant mycobacteria increased the sensitivity to the prodrug. This means that the bioactivation is not fully exploited and that only part of the prodrug administered is actually exerting its antimycobacterial effect, living space for optimizing this process.

In particular, Ethionamide (ETH), an old TB drug currently recommended by WHOto treat patients with MDR-TB (group C), is a prodrug that is inactive until activation by the mycobacterial monooxygenase ethionamide activator (ethA) [START_REF] Baulard | Activation of the pro-drug ethionamide is regulated in mycobacteria[END_REF][START_REF] Debarber | Ethionamide activation and sensitivity in multidrugresistant Mycobacterium tuberculosis[END_REF], which first oxidizes ETH to a corresponding S-oxide and further catalyses the covalent addition of Nicotinamide Adenine Dinucleotide (NAD) to ETH, forming the ETH-NAD active adduct [START_REF] Vannelli | The antituberculosis drug ethionamide is activated by a flavoprotein monooxygenase[END_REF][START_REF] Laborde | Ethionamide biomimetic activation and an unprecedented mechanism for its conversion into active and non-active metabolites[END_REF]. Similar to INH, after activation, the resultant ETH adduct inhibits an enoylacyl carrier protein reductase, InhA, an essential enzyme of Mtb involved in the synthesis of mycolic acids, critical constituents of the mycobacterial cell wall. ETH activation by ethA is negatively regulated by the Ethionamide transcriptional Repressor (ethR), which limits the potency of ETH [START_REF] Baulard | Activation of the pro-drug ethionamide is regulated in mycobacteria[END_REF][START_REF] Debarber | Ethionamide activation and sensitivity in multidrugresistant Mycobacterium tuberculosis[END_REF][START_REF] Engohang-Ndong | EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator[END_REF]).

Willand N. et al. searched for compounds able to inhibit the DNA-binding function of the repressor ethR, thus boosting the bioactivation of ETH [START_REF] Willand | Synthetic EthR inhibitors boost antituberculous activity of ethionamide[END_REF].

One of the compounds selected, BDM31343, enabled to reduce the dose of ETH necessary to decrease the pulmonary bacterial load in a Mtb mice model of infection.

Introduction

Mice were treated 6 days per week, during 3 weeks, with a combination of 50 mg/Kg (body weight) BDM31343 in DMSO given intra-peritoneally twice daily and different concentrations of ETH administered in DMSO orally, once a day. After 3 weeks of treatment, the efficacy obtained with 9 or 27 mg/Kg ETH could be equalled by 3 or 9 mg/Kg of ETH in combination with 100 mg/Kg BDM31343, respectively [START_REF] Willand | Synthetic EthR inhibitors boost antituberculous activity of ethionamide[END_REF]. These results proved that by using inhibitors of ethR that boost the activation of ETH, it is possible to reduce the dose of ETH, while keeping the same efficacy.

The structure of BDM31343 was further optimized, leading to the development of BDM41906, which presents an increased activity profile, improved physicochemical and pharmacokinetic properties, and microsomal stability [START_REF] Flipo | Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors[END_REF].

Because mycobacterial enzymes that activate prodrugs are not essential for bacterial growth and pathogenesis, the genes that encode them commonly undergo mutations that cause loss of function or attenuation, as observed for the INH-, PZA-, PAS-and ETHresistant clinical isolates with mutations in katG [START_REF] Zhang | The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis[END_REF], pncA [START_REF] Scorpio | Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus[END_REF], ribD [START_REF] Mathys | Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis[END_REF][START_REF] Zheng | para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis[END_REF]) and ethA [START_REF] Baulard | Activation of the pro-drug ethionamide is regulated in mycobacteria[END_REF][START_REF] Debarber | Ethionamide activation and sensitivity in multidrugresistant Mycobacterium tuberculosis[END_REF], respectively. In the same way, mutations in ddn (Rv3547), a gene encoding an enzyme involved in the bioactivation of the new prodrugs delamanid and pretomanid, are associated with resistance to these drugs [START_REF] Manjunatha | Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis[END_REF][START_REF] Matsumoto | OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice[END_REF]).

Thus, for the case of ETH, although the use of compounds that inhibit the repressor ethR might proof very efficient to boost the efficacy of ETH against drug-sensitive and MDR bacteria, it would be ineffective against ETH-resistant Mtb strains, mutated in ethA.

However, in the process of optimizing the structure of compounds that inhibit the repressor ethR, Blondiaux N. et al. unexpectedly found molecules unable to bind to ethR but that were still highly effective in boosting ETH activity against Mtb. Because these compounds had no antibacterial activity per se, the authors hypothesized that they may trigger an alternative bioactivation pathway for ETH. Using genome wide transcriptional analysis, the authors found that while BDM41906 induced the transcription of both ethR and ethA, the new compounds, represented by SMARt-420, induced a distant pair of 1. Introduction genes with homology to ethR and ethA, which they called ethR2 and ethA2 (Figure 14).

Further, they showed that, like the analogous system, ethR2 represses the transcription of ethA2. SMARt-420 by binding to ethR2 inhibits the repression of ethA2, leading to activation of ETH. Treatment of Mtb with SMARt-420 not only increased susceptibility to

Introduction

ETH but was also highly active against all TB strains tested, including MDR strains mutated in ethA.

Therefore, exploring mechanisms of bioactivation is a promising strategy to give new life to old antituberculosis prodrugs, opening perspectives of dose and side effects reduction, and also reverting resistance mechanisms by inducing the activation of the prodrug through alternative pathways.

Mycobacterial resistance, tolerance and persistence to antibiotic treatment 1.1.9.

TB pathology is extremely complex and dynamic, varying between patients and within a single individual patient. The spatial and temporal heterogeneity of TB lesions imply that Mtb has to constantly adapt to different stresses created by the local microenvironment. For example, the availability of oxygen and nutrients, the pH and the presence of oxidative stress is different when Mtb is within an extracellular environment in the caseous cavity or when it is in an intracellular environment inside a macrophage, in an early granuloma or in a hypoxic granuloma. Each of these conditions induces mycobacterial metabolic and physiologic adaptations that rise distinct subpopulations with different gene expression profiles, rate of replication, and other functional characteristics [START_REF] Lenaerts | Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses[END_REF]. This heterogeneity affects the efficacy of antituberculosis chemotherapy that may not be able to act against the different mycobacterial subpopulations and most likely contributes to the long treatment duration and requirement for multi-drug combination.

For example INH, a drug that targets the biosynthesis of the cell wall, is a potent early bactericidal drug, highly active against rapidly dividing bacteria that are producing new cell wall, but it has low activity against bacteria that are maintained in a non-replicating state due to restriction of oxygen [START_REF] Paramasivan | Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic-and stationary-phase cultures of Mycobacterium tuberculosis[END_REF]. RIF, a drug that targets RNA polymerase, although not so active at early time points, it is equally active against both replicating and non-replicating bacilli [START_REF] Paramasivan | Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic-and stationary-phase cultures of Mycobacterium tuberculosis[END_REF], probably because of the constant need for RNA production, even when bacteria are not undergoing active replication. On the other side, PZA, a drug that requires low pH to be active, only works against dormant and semi-dormant organisms that reside in an acidic environment, such as the one found within granulomas and in macrophages after 1. Introduction phagosome-lysosomal fusion [START_REF] Zhang | The curious characteristics of pyrazinamide: a review[END_REF]. Thus, the same drug may have different efficacy against the same bacteria in different environments.

The introduction of antituberculosis chemotherapy subjected Mtb to another stress, which forced bacteria to develop adaptation mechanisms such as resistance and tolerance to antibiotic environment.

TB was the first infectious disease in which drug resistance was described, soon after the introduction of STR, in 1948 [START_REF] Pai | Tuberculosis[END_REF]. Resistance is the inherited ability of a microbe to grow at high concentrations of an antibiotic, irrespective of the duration of treatment, and it is quantified by the minimum inhibitory concentration (MIC) of antibiotic that is required to prevent growth. Mtb is considered drug resistant when more than 1 % of bacterial cells grow in the presence of critical drug concentrations [START_REF] Dheda | Tuberculosis[END_REF]). Mtb may be intrinsically resistant to an antibiotic, namely by cell wall impermeability, active efflux, modification of targets, enzymatic degradation or modification of antibiotics and activation of a transcriptional regulator or may acquire resistance by changes in the DNA that usually result from mutations in chromosomal genes encoding drug targets or enabling enzymes and possibly by horizontal gene transfer [START_REF] Blair | Molecular mechanisms of antibiotic resistance[END_REF][START_REF] Nasiri | New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria[END_REF] .

When drug-resistant TB results from infection with a drug-resistant strain it is called primary resistance [START_REF] References Dheda | The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis[END_REF]. For long time the impact of primary resistance was underappreciated because it was thought that the mutations that lead to resistance produced a fitness cost, and that resistant strains were less virulent or less easily transmitted. However, it is now known that, additional mutations often follow or coincide with drug resistance mutations and can compensate for the deleterious effects caused by the mutation that lead to the resistance. Molecular epidemiology studies in high-burden locations have shown high degrees of strain clustering, which is indicative of transmission (primary resistance) rather than acquisition (secondary resistance), in the majority of known MDR-TB cases and a significant part of XDR-TB cases [START_REF] References Dheda | The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis[END_REF].

When resistance is developed during the treatment course is referred as secondary or acquired resistance. Acquired resistance to antituberculosis drugs is believed to be driven by poor adherence to a long TB therapeutic regimen and programmatic failure, with doses and dosing schedules that lead to suboptimal antibiotic concentrations.

Introduction

Though, other mechanisms such as pharmacokinetic variability, induction of efflux pumps that transport the drug out of cells, and suboptimal drug penetration into TB lesions are also crucial for the development of drug resistance. Moreover, some bacterial strains have genetic hypermutable backgrounds that have greater propensity to cause acquired drug resistance [START_REF] References Dheda | The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis[END_REF].

Tolerance is the ability, inherited or not, of bacterial cells to survive a transient exposure to antibiotics at concentrations that would otherwise be lethal [START_REF] Kester | Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria[END_REF], which is often achieved by slowing down an essential bacterial process.

Dormancy is an extreme case of slow growth that leads to tolerance. Tolerance may be acquired through a genetic mutation or conferred by environmental conditions; for example, poor growth conditions have been shown to increase tolerance to different classes of antibiotics [START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF]. Unlike resistance, tolerance applies only to bactericidal antibiotics and not to bacteriostatic antibiotics, as all bacteria are expected to survive transient exposure to bacteriostatic antibiotics since they only arrest growth. Tolerance to an antibiotic is almost insensitive to the concentration and dependent only on the duration of exposure, thus MIC is not informative as a metric to evaluate tolerance. Tolerant bacteria can have the same MIC as non-tolerant bacteria. Brauner, Fridman et al., propose the use of the Minimum Duration for Killing (MDK) as a quantitative indicator of tolerance that can be extracted from a time-kill curve.

The term persistence is used to describe the ability of a subpopulation of a clonal bacterial population to survive exposure to high concentrations of an antibiotic. The "persisters" constitute the less numerous subpopulation (normally less than 1 %) and are killed at a slower rate than the susceptible cells. Persistence may be dependent on time (persisters undergo tolerance) or dependent on concentration (persisters undergo transient overexpression of a resistance factor) [START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF].

Persistence is non-heritable, when persistent subpopulation cells are isolated, regrown and re-exposed to the same antibiotic treatment, the same heterogeneous response to the drug will be observed, with the division of the population into persistent and nonpersistent subpopulations.

In the clinic, these insights may be useful for the establishment of a more effective therapy, by using inhibitors of resistance or extending the treatment duration, for example.

Introduction

Nanotechnology as new tool in TB treatment 1.2.

A bibliographic review on this subject was published on Drug discovery today.

Revue de la littérature -Résumé 1.2.1.

"Comment les nanoparticules peuvent-elles contribuer à la thérapie antituberculeuse?"

Face à la recrudescence des cas de résistance aux antibiotiques, la tuberculose (TB) est un problème de santé mondiale majeur, qui est à l'origine de 10.

How can nanoparticles contribute to antituberculosis therapy?

Authors list: Joana Costa-Gouveia 1 , José A. Aínsa 2,3,4 ,Priscille Brodin 1# ,Ainhoa Lucía 2,3,4 Ainhoa Lucía (ainhoalq@unizar.es) Tel: +34 876 55 43 45 Keywords: Tuberculosis, nanoparticles, antituberculosis drugs, tuberculosis treatment.

Teaser:

The number of effective drugs to treat TB is limited, the incidence of MDR-TB and XDR-TB is increasing, and developing novel drugs against TB is difficult. Nanoparticles (alone or carrying anti-TB drugs) are interesting alternatives with a potential for therapeutic use in treatment of TB.

Introduction

The problem of antibiotic resistance in Mycobacterium tuberculosis has been declared a global health emergency by the WHO, which may endanger the control of tuberculosis (TB). Rising cases of multi-drug resistant (MDR) TB and extensively drug resistant (XDR) TB all over the world are increasingly hindering treatment of TB, making mandatory the use of second line drugs (SLDs) [1]. Treatment of MDR-TB is expensive, prolonged (18-24 months) and complex (with at least a combination of 5 drugs that include injectable drugs) and associated with a higher incidence of adverse effects. New drugs are urgently needed, but despite several molecules are being investigated, some of them already in clinical trials, only bedaquiline and delamanid have been approved for conditional treatment of specific MDR-TB patients in the last years [2,3].

It is obvious that besides classical drug discovery strategies, new approaches are urgently needed to get a faster, more efficient and less harmful treatment. For this purpose, nanoparticles (NPs) have been shown since many years to be useful and versatile tools that can be used as antibacterial agents, and also as drug delivery systems, with numerous examples in the literature regarding various antibiotics and antibacterial agents. Nowadays, several groups across Europe including universities, small and medium-sized enterprises, research institutes and pharmaceutical companies join forces together in the frame of research consortiums to apply nanotechnology for the 1. Introduction development and implementation of novel treatments for multidrug resistant pathogens, including MDR-XDR -TB.

Use of NPs as new tools in TB treatment

NPs: characteristics and advantages for TB treatment

Very simply, we could consider a NP as a tiny object measuring less than 1000 nanometers that works as a whole unit (Fig. 1,[9]). They can be made of very different materials (biodegradable lipids, liposomes, solid-lipid NPs, polymers like poly lactic-coglycolic acid (PLGA) or polysaccharides like chitosan), and can be produced by different methods, resulting in NPs with different structures and sizes. One second, and probably the major advantage of nanosized carriers is their use as drug delivery systems owing to their unique physicochemical properties. In general, they are of small size, high surface to volume ratio, and high stability [9]. Normally they can incorporate both lipophilic and hydrophilic drugs (Fig. 1,[9]), as well as other molecules such as carbohydrates, proteins or lipids to stabilize and functionalize them [6]. In this way, NPs may improve the aqueous solubility of poorly soluble drugs, protect the drugs from degradation and allow a controlled release of the loaded drug, thereby the dose and frequency of administration can be reduced. Additionally, they can be modified to control their biodistribution allowing selective delivery to target organs or tissues [9]. For all those characteristics NPs are smart tools for the improvement of pharmacokinetics, solubility, bioavailability, toxicity or pharmacodynamics of drugs.

The development of effective and safe nanotherapeutic methods is particularly relevant in the treatment of drug-resistant tuberculosis, since very long treatments with highly toxic second-line drugs are required for those cases. The properties of NPs could diminish the dose of the drugs used to treat MDR-TB, and direct them to the infected macrophages in the lung, decreasing the toxicity and side effects, and increasing their efficacy. To this end, dedicated NPs have to be developed for each TB drug and optimized for targeting the lungs harboring extracellular as well as intracellular bacteria.

NPs with intrinsic antimycobacterial activity

Metal NPs present antimicrobial activity against a variety of microorganisms, and M. tuberculosis is not an exception (Table 1). Silver (Ag) has been used as antibacterial agent for centuries [10,11]. Its antimicrobial activity is reported to increase when in the nano-form, which may be due to the ability to enter through cell membranes [12], and nowadays silver NPs (AgNP) are widely used in consumer products such as textiles, disinfectant sprays, antibacterial ointments, 1. Introduction bandages or medical devices [13]. AgNPs are reported to have high antibacterial properties against Gram positive and Gram negative bacteria [11,12,[14][15][16][17]. Because of that, the specific antimycobacterial activity of AgNPs has been studied, and reported that AgNPs show higher activity against mycobacteria than copper oxide NPs, followed by zinc oxide NPs, with poor antimycobacterial activity for both of them

[13].
The synthesis of AgNPs can be done via chemical methods but also physical methods.

Concerning the chemical methods, sodium citrate, sodium borohydride, dimethlyformamide are typical compounds used to reduce Ag+ into metallic silver [18].

However as for organic NPs, eco-friendly pure methods based on the use of apiin, geraniol and gum kondagogu as reducing agents were also shown successful to produce AgNPs so that the synthesis process of AgNPs is no more an impediment to their All NPs need to be cleared from the body completely in a reasonable time period.

Typically after infection, NPs rapidly distribute into various organs, circulate in the blood and are excreted though the urinary system or in the feces. As metals may accumulate in various organs in the body, it is thus important to investigate the renal and fecalclearance of inorganic NPs when tested in animal models. Whether a NP can pass through the kidney is strongly dependent on their physical properties (size, charge, shape, etc.) [30]. A recent study showed that an increase in particle density correlates with a longer retention times in permeable organs for tumor targeting inorganic NPs [31]. However, irrespective of particle size and coating, acute oral exposure to AgNP is associated with predominantly fecal elimination and is not associated with accumulation in tissue or toxicity [32]. Many researchers now focus their attention to biodegradable nanoparticles.

NPs as vehicles for effective per os delivery

Oral delivery is the most common and convenient form of drug administration (Fig.

2). To be effective, the medicines to be taken per os have to resist to the acidic gastric pH, to be soluble, absorbable through the gastrointestinal (GI) mucus barrier and maintain therapeutic levels in the target organs Gelatin nanocarriers are good candidates for the oral delivery as well, due to their biocompatibility, biodegradability, low antigenicity, low cost, and ease of their parenteral use. They have been formulated to vehicle rifampicin, resulting in a better biodistribution and higher blood levels of the drug, which lead to a reduction of the CFUs in lungs and spleen with respect to free rifampicin in mice [46].

Research is still widely active to improve oral administration of the drugs: lipidic and polymeric are the most widely used nanocarriers and more recently polymer-lipid hybrid systems are being investigated [35]. These latter show a better drug encapsulation, a modulated drug release, and improved cellular uptake with respect to non-hybrid systems.

We mainly focus here on the use of NPs for drug delivery, but it is worth to mention that similar particles are investigated for the development of vaccines against TB. Oral route was used to administer guar gum NPs loaded with Ag85A [47]. The formulation was shown to protect the antigen from the gastric environment, and deliver it to the intestinal region. Moreover, in vivo data indicated that the system induced a strong mucosal as well as systemic immune response, suggesting that it can be utilized for oral delivery of vaccines.

NPs as carriers for pulmonary delivery 1. Introduction

Lungs are the primary site for M. tuberculosis infection. Upon inhalation of contaminated aerosols, M. tuberculosis is deposited in the lungs and is taken up by phagocytic cells, such as macrophages. During active TB, the bacteria replicate actively within granuloma that are multicellular complex structures that the TB drugs must reach Besides considering the characteristics of the particles, one should take into account the characteristics of the inhaler device in order to produce an appropriate aerosol.

Inhalable liquid formulations are administered using nebulizers and pressurized metered dose inhalers (pMDIs). Nebulizers are particularly useful when high pulmonary doses are required and when the patient is unable to coordinate or achieve flow rates necessary for the use of other inhalation systems [START_REF] Chan | Advances in device and formulation technologies for pulmonary drug delivery[END_REF]. PMDIs are the most popular inhaled drugdelivery systems however they are limited by small dose sizes. Inhalable dry powder formulations are administered using dry powder inhalers (DPIs). These formulations are generally more stable than inhalable liquid formulations and usually do not require refrigeration, being therefore suitable for the warm climates normally found in the developing countries where TB is prevalent [START_REF] Porter | Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs[END_REF].

In experimental animals the use of nebulizers has several limitations such as imprecision due to the difficulty of measuring the amounts of inhaled drugs and the use of pMDIs or DPIs is not possible since it requires the cooperation of the patient. There are also exposure systems such as whole-body chamber and nose-only chamber for exposing 1. Introduction single or multiple animals to the aerosol. Nose-only chamber has the advantage of minimizing dermal exposure. However, in both systems, there may be loss of materials in the devices before being inhaled. [START_REF] Wong | Inhalation exposure systems: design, methods and operation[END_REF]. To circumvent that, pulmonary delivery can be performed by intratracheal delivery using a PennCentury MicroSprayer (for inhalable liquids) or a dry powder insufflator [START_REF] Misra | Inhaled drug therapy for treatment of tuberculosis[END_REF]. In this case, a precise volume of compound is directly sprayed in the lungs.

The use of nano-and microparticles for the development of inhalable antitubercular therapies and vaccines is a promising strategy to fight TB. However, more studies are necessary to evaluate the long-term toxicity of the particles and the effect of excipients used and to discover if this kind of treatment can be used as exclusive therapy or as a combination with the conventional oral therapy.

Functionalization of NPs surface for targeting intracellular M. tuberculosis

Macrophages are a major habitat for M. tuberculosis to reside in the host for years.

Thus, to kill M. tuberculosis, one should target these cells and once internalized, the drug should reach the phagosome. NPs offer a promising platform for passively target macrophages by exploiting particle size and administration route. Additionally, there are active strategies to increase the phagocytosis of NPs by macrophages. In this case, the surface of the NP is modified by attaching targeting ligands that bind to molecules specific for the target cells. In fact, macrophages express a broad range of receptors on their surface that mediate recognition and bacterium endocytosis, and that can be targeted by nanocarriers with appropriate ligands (Table 3).

The mannose receptor is among the most commonly used for this purpose, since the receptors to this ligand are mainly expressed by macrophages for the recognition of conserved motifs on pathogens. For example, treatment of mannosylated gelatin NPs

Conclusion and future perspectives

In summary, TB treatment may benefit from the advantages of NPs for several reasons: first, there is a limited number of drugs available for XDR-MDR-TB, many of them presenting important toxicity issues; second, drug resistance is increasing and hence complicating the treatment; and third, although novel drugs have been made

available in the last decades, this seems insufficient for coping with the growing Nanotechnology-mediated drug delivery systems offer many solutions for potential therapeutic agents but they may also offer a variety of challenges for the industrial production of such medicines, mainly the development and regulatory approval costs.

However, these difficulties tend to decrease when more products enter in the market and more advanced production methods are developed, a field of study which is currently progressing [START_REF] Elahian | High-throughput bioaccumulation, biotransformation, and production of silver and selenium nanoparticles using genetically engineered Pichia pastoris[END_REF]. In addition, it would be of great interest to further develop nanocarriers totally devoid of organic solvents, surfactants and other potentially toxic compounds, not only for their synthesis, but also during the encapsulation steps. Finally, these systems are likely to improve the life quality of the patients and to reduce the overall future health care costs if they reduce the frequency and duration of the treatment, so that the cost-benefit-ratio is favorable.

The

Objectives

Current therapies often fail because of insufficient drug concentration in the infected areas and fast drug metabolism and excretion. Moreover, some drugs are limited to be used by specific administration routes due to their solubility characteristics and stability inappropriate for other administration routes.

In active collaboration with different partners from the European Union Marie Curie Initial Training Network "CycloN-Hit", this thesis aimed to develop nanoparticles (NPs) to overcome the problems mentioned above. Therefore, the main goals of this thesis were to investigate the use of nanoparticles as nanocarriers:

I. For pulmonary delivery of drugs.

II. To enhance solubility and bioavailability of antibiotics.

III. To improve antibiotic intracellular penetration.

Additionally, this thesis also aimed to:

IV. Explore the intrinsic antimycobacterial activity of NPs.

Addressing these goals will be important to develop a new concept of drug therapy to efficiently combat drug-susceptible and drug-resistant tuberculosis (TB), able to chase the pathogen from its hideout and thus paving the way for the development of new therapeutics means against TB. L'ETH est une "pro-drogue", qui nécessite une bioactivation par la bactérie Mtb (via la monooxygenase bactérienne ethA). Chez Mtb, l'expression d'ethA est inhibée par une autre enzyme ethR, responsable d'une diminution de la transformation de la prodrogue [START_REF] Baulard | Activation of the pro-drug ethionamide is regulated in mycobacteria[END_REF]. Des molécules ligands, appelées "booster", ont été récemment développées [START_REF] Willand | Synthetic EthR inhibitors boost antituberculous activity of ethionamide[END_REF] Les NP de poly-β-cyclodextrine (pCD) ont été choisies pour la suite de notre étude in vivo. To achieve the objective of drug co-administration, we simultaneously coencapsulated ETH and Booster in nanoparticles able to be administered by pulmonary route, to reach the main site of TB infection in the lungs. To do so, various encapsulation strategies and nanocarriers were considered. Drugs were incorporated in biodegradable polymeric β-cyclodextrin (pCD) nanoparticles. Advantageously, encapsulation in the CDbased nanoparticles enabled circumventing the bottlenecks inherent to the strong tendency of ETH to crystallize and poor solubility of Booster, without using any organic solvent in the encapsulation process.

CHAPTER I

Chapter I

We report for the first time the use of an automated confocal high content screening platform to study the effect of drug-loaded nanoparticles on M. tuberculosis infected cells. The drugs maintained their activity after incorporation in the nanocarriers.

Given these satisfactory results, drug-loaded nanoparticles were further investigated for their antituberculosis property in an in vivo model of infection. Nanoparticles based on CDs, which showed the best physiochemical characteristics, were selected for these studies.

The nanoparticles co-loaded with ETH and Booster were administered in vivo via pulmonary route in mice that have been priory challenged with M. tuberculosis.

Compared to untreated mice, the treatment with CD nanoparticles containing the drug combination lead to a significant decrease of the pulmonary bacterial load. Noteworthy, the results were achieved with only three administrations. This study paves the way for a future use of CD-nanoparticles for the delivery of the synergic [ETH:Booster] pair to 3. Chapter I infected lungs. Our approach could be used as a supplement to the standard treatment to content the M. tuberculosis pulmonary manifestations of the disease and to prevent its dissemination. It may be also considered useful for prevention use for healthy people in contact with TB patients, in order to contain an initial lung infection avoiding the toxicity often associated with oral drug administrations. Formulations up to an ETH concentration of 0.43 mg/mL were completely stable. In these conditions the highest achieved drug loading (DL) and encapsulation efficiency (EE)

were 11 wt % and 36 wt %, respectively. Unfortunately, raising the amount of ETH to 0.71 mg/mL in the attempt to further increase DL and EE, the DL resulted unchanged and the encapsulation efficiencies decreased to 22 % (Supplementary Fig. S2). Moreover, increasing ETH amounts in the NP preparation procedure lead to the formation of drug crystals which progressively grew in the suspension media until being seen with the naked eye, few days after NP preparation (Supplementary Fig. S3).

Supplementary Fig. S3 shows In an additional attempt to increase both DL and EE, a series of PLA and PLGA copolymers were used, with different molecular weights and compositions (P2, P3 and P4). In all cases, aggregation or drug crystallization occurred. Better results in terms of DL and EE (24 wt % and 34 wt %) were obtained with P3 and an ETH concentration of 1 mg/mL. However, crystallisation occurred after 5 days as for the other polymers.

Moreover, co-encapsulating Booster did not improve ETH loading, as it was found that Booster had stronger affinity for the polymeric NP than ETH. As an example, at the ratios [ETH:Booster] [1:0.1] and [1:1], ETH DL were lower than 2 wt %. Interestingly Booster DL and EE during coencapsulation were significantly higher than ETH ones (9 % and 29 % wt respectively, for ratio [1:1]) showing a higher affinity of the Booster for the polymeric matrix.

PLA NPs -Nanoemulsion method

To address the challenge of coencapsulating ETH and Booster, a second method based on the formation of an emulsion, stabilized with polyvinyl alcohol (PVA), a 3. Chapter I surfactant approved for pulmonary administration, was set up using P4 (see material and methods section).

An extensive study on mean diameter of PLA NPs obtained by nanoemulsion has been performed using the 3 independent methods: PCS, NTA and cryo-TEM (see main text). The results show the importance of the use of different techniques to truly characterise nanoparticulate systems. Results are reported in Supplementary Table S1.

It's interesting to point out that no significant difference in the diameter of NPs has been found between empty PLA NPs and drug-loaded PLA NPs.

Section II -Phenotypic extracellular and intracellular assays

To investigate the efficacy of drug-loaded NPs against M. tuberculosis, we used two phenotypic assays that are disease-relevant and amenable to high-throughput. The first one is a simple assay relying on the monitoring of fluorescence from a strain of M.

tuberculosis that expresses the green fluorescence protein using a microplate photometer. The second is an image-based model that allows the multi-parametric quantification of M. tuberculosis replication inside its favourite niche, the macrophage.

The dedicated protocol used for image analysis with the Columbus software is given in Supplementary S5 below. 3. Chapter I (EP). Briefly, 100 g of anhydrous β-CD was solubilized overnight in 160 mL of NaOH 33% w/w solution. Then, 81.52 g of EP was rapidly added and the reaction was stopped in the vicinity of the gelation point by addition of acetone. The β-CD NPs, recovered by ultrafiltration followed by freeze-drying, contained 70% w/w β-CD, as determined by 1 H NMR spectroscopy.

Nanoparticle preparation

Nanoprecipitation: encapsulation of ETH in PLA and PLGA NPs was carried out adapting a previously reported procedure (Bilati, Allémann et al. 2005). Briefly, 0.4 mL of DMSO solution containing 10 mg polymer (PLA or PLGA) and ETH (0.5 to 7 mg) were poured drop by drop in 7 mL of injectable water under magnetic stirring. In the case of co-encapsulation, 3 mg (ratio 10:10) or 0.3 mg of Booster (ratio 10:1) were added to the DMSO solution.

Nano-emulsion: preparation of PLA NPs was carried out as previously reported by Kumar et al. with slight modifications [START_REF] Kumar | In vitro physicochemical characterization and short term in vivo tolerability study of ethionamide loaded PLGA nanoparticles: potentially effective agent for multidrug resistant tuberculosis[END_REF]. Briefly, 10 mg of ETH were solubilized in 0.2 mL of MeOH and mixed to 1.5 mL of a dichloromethane (DCM) solution containing 20 mg of P4 in DCM. For the co-encapsulation of the synergic drugs pair, 10 mg of Booster, in case of 10:10 ratio, or 1 mg of Booster, in case of 10:1 ratio, were solubilized in the solution of PLA in DCM. Control empty NPs were prepared in the same conditions, except that the drugs were not added.

The organic phase was poured into 4 mL of an aqueous phase (injectable water) containing 0.5% w/v PVA and emulsified 20 seconds (vortex). The mixture was then sonicated (Sonopuls HD 2070, BANDELIN electronic GmbH & Co, Berlin, Germany) at 20% power for 1.5 min, stopping the procedure each 30 seconds to avoid the overheating and for 30 additional seconds to 10%. The resulting emulsion was left uncapped under magnetic stirring, checking the weight of the flask to monitor the evaporation of the organic solvents, until complete evaporation of DMC and MeOH.

Drug encapsulation in "green" pCD NPs: encapsulation of ETH and Booster in pCD NPs was carried on without using any organic solvent, by mixing for at least 4 hours the NP suspensions with the drug powders. 3. Chapter I

Nanoparticle characterization

Size measurement by PCS and Zeta Potential measurement

The average hydrodynamic diameter of the NPs was determined at 25 °C, in triplicate, with an equilibration time of 60 s, using a Malvern Zetasizer® (Nano ZS90, Malvern Instruments S.A., Worcestershire, UK). For the measurement of method 1 formulations, Samples were analyzed without performing any dilution (method 1) and with 1:10 and 1:100 dilutions in injectable water (as prescribed by ISO 22412) in the case of method 2. No significant differences were noticed in the hydrodynamic diameter of the two dilutions. All autocorrelation functions have an intercept with amplitude equal to 0.9 or higher. Mean diameters were reported as Z Average (nm) ±SE (Standard Error -with a Polydispersity Index lower than 0.1) or as number mean diameter (nm) ±SD (Standard Deviation).

The stability of the NPs prepared by nanoprecipitation and nanoemulsion was assessed at RT (25°C-storage conditions). The NPs mean diameters were monitored up to three months of storage at 25°C via DLS size measurements. (see Supplementary Fig. S1 and S5) Also the Zeta Potential (ZP) of nanoparticles was measured in KCl 1Mm by Malvern Zetasizer © . Each sample was diluted 100 times and 1,000 times in KCl 1mM. Results are reported as ZP (mV) ± SD.

Size measurement by NTA and concentration quantification

Mean hydrodynamic diameter and concentration of the NPs were measured 5 times All the samples were dosed by liquid chromatography-mass spectrometry (LC-MS-MS) and reverse phase HPLC (RP-HPLC). For LC-MS-MS analysis, a Waters Acquity I Class -Xevo TQD system using an Acquitity BEH C18 column (50x2.1mm, 1.7 µm) at 40°C was used. The mobile phase at a flow of 600 µL/min was composed by two solvents: ammonium acetate 10 mM (Solvent A, spontaneous pH) and acetonitrile (CH 3 CN) with 0,1% HCOOH (Solvent B) (UPLC gradient of B= 0% at time 0.98% from minute 2.00 to 2.50 then 0% from minute 2.60) and its flow was 600 µL/min.

Samples were prepared by diluting them 100 times in MeOH and then 100 times in a solution of MeOH containing the internal standards, then 1 µL of each sample was injected in the system.

The RP-HPLC analysis were performed after dilution with the mixture acetonitrile/water 45/55 v/v using a Agilent HPLC system (Agilent 1100 Series) equipped with a C18 column (Kinetex 5u C18, 100 A, Phenomenex, and UV detector at λ = 280 nm.

The chromatographic conditions were as follows: solvent A 0.1% Trifluoroacetic Acid (TFA) in water and solvent B 0.1% TFA in acetonitrile; 0-2 min: 0%-20% B, 2-6 min: 20%-45% B, 6-10 min: 45%-75% B, 10-15 min: 75%-0%. Flow rate: 1.0 mL/min at room temperature. 3. Chapter I In all cases, R 2 values for both ETH and Booster were higher than 0.99. From the obtained data the DL and EE were calculated as shown in equation 1 and 2, respectively.

The DL can be defined as the mass fraction of a NP that is composed of drug, while the EE can be considered as the fraction of drug effectively encapsulated into the NPs compared with the amount that was initially added during the particles preparation [START_REF] Kumar | In vitro physicochemical characterization and short term in vivo tolerability study of ethionamide loaded PLGA nanoparticles: potentially effective agent for multidrug resistant tuberculosis[END_REF][START_REF] Ankrum | Engineering cells with intracellular agent-loaded microparticles to control cell phenotype[END_REF].

( Echo 550 Series Liquid Handler (Labcyte) was used to transfer precise volumes between 5 and 500 nL from the Echo-qualified plate to the 384-well clear-bottom polystyrene assay plates (78198, Greiner Bio-One) by using sound waves. All the conditions were backfilled with DMSO until 500 nL. Prior to addition of cells, 4.5 µL of cell medium were dispensed in the Echo-dispensed wells.

NPs loaded with ETH or with the [ETH:Booster] pair were diluted in water to 0.2 mg/mL of ETH. 2-fold serial dilutions of the mother solution in a final volume of 100 µL were performed in sterile MilliQ water in a 384 deep well "diamond plate" (P-384-120SQ-C-S, Axygen) in order to obtain a dose-response curve. Posteriorly, 5 µL of the NPs were dispensed in the 384-well assay plates.

Extracellular in vitro assay

Bacteria were diluted at 2×10 6 bacteria/mL using complete 7H9 medium and 45 µL/well of bacterial suspension were added in 384-well assay plates. After 5 days incubation at 37°C, 5% CO 2 , extracellular plates were read using a fluorescence reader (Victor X3, Perkin Elmer) at excitation/emission of 485/535 nm for 0.1 seconds/well with a small emission aperture and CW-lamp energy of 50,000. The read-out, relative fluorescence units (RFU), versus the ETH concentration was then plotted using GraphPad Prism 5.0 software and the concentration required to inhibit 50% of the bacterial replication (IC 50 ) was calculated by nonlinear regression analysis using the equation for a sigmoidal dose-response curve with variable slope.

Intramacrophage in vitro assay

For intracellular assay, bacteria were mixed with RAW 264.7 macrophages to prepare a suspension at 5×10 5 cells/mL and 1×10 6 bacteria/mL (multiplicity of infection 2) in RPMI-1640 + glutamax containing 10% FBS. After 2 hours of infection at 37°C with shaking (120 rpm), infected cells were washed with RPMI-1640 + glutamax containing 3. Chapter I 10% FBS by centrifugation at 1,100 rpm for 5 minutes. The remaining extracellular bacilli from the infected cell suspension were killed by a 1 hour 50 g/mL amykacin (A2324-5G, Sigma) treatment and then washed twice with RPMI-1640 + glutamax containing 10% FBS. Finally, 45 µL/well of cellular suspension was added in the 384-well assay plate and incubated during 5 days at 37°C, 5% CO 2 . Macrophages were then stained with 5 µM Syto 60 (S11342, Molecular probes) dye for 1 hour, followed by plate sealing, imaging acquisition and data analysis.

Image acquisition

Confocal images were recorded on an automated fluorescent ultra-high-throughput microscope Opera (Perkin Elmer). This microscope is based on an inverted microscope architecture that allows imaging of cells cultivated in 96-or 384-well microplates. Images were acquired with a 20X-water immersion objective (NA 0.70). A double laser excitation (488-nm and 640-nm) and dedicated dichroic mirrors were used to record green fluorescence of mycobacteria and red fluorescence of the macrophages on two different cameras, respectively. A series of 6 pictures at the center of each well were taken and each image was then analyzed using Columbus system version 2.5.1 as previously described [START_REF] Queval | A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening[END_REF] to extract the bacterial area and the number of cells.

Intramacrophage assay data analysis

Bacterial area was normalized with the negative control DMSO (0% inhibition) and the positive control INH at a concentration of 1 µg/mL (100 % inhibition) by converting it into a percentage of bacterial replication inhibition (% inhibition). % inhibition was calculated by the formula:

This formula was used to normalize the "test bacterial area" from different experiments, in order to decrease potential bias in the results due to differences in the % of infection and bacterial growth between experiments. For each compound, a plot of % 3. Chapter I inhibition versus the ETH concentration was determined with GraphPad Prism 5.0 software and the IC 50 was calculated in the same way as in the extracellular assay.

In vivo efficacy

6-week old Balb/C female mice were purchased from Janvier (Le Genest-Saint-Isle, France) and were maintained in the animal house facility of the Pasteur Institute of Lille, France (Agreement B59-350009). The project (reference 00579.01) received ethic approval.

Toxicology studies

Sixteen uninfected 8-week mice were divided in four groups and endotracheally administered with water (vehicle) or pCD NPs over a period of 2 weeks (Fig 5a).

At day 14 mice were sacrificed, lungs were harvested and soaked in 4% formaldehyde (10% formalin solution, neutral buffered, HT501128, Sigma-Aldrich) for 24 hours, before being embedded in paraffin. Tissues were sliced with microtome and 5 µm sections were stained with Hematoxylin-Eosin (H-E) for light microscopy examination by an anatomopathologist. For flow cytometry analysis, lung were harvested, cut into small pieces and incubated for 1 hour at 37°C with a mix of DNAse I (100 μg/ml, , Sigma-Aldrich) and collagenase (1.6 mg/ml, Roche) 400 U/ml. Lung cells were washed and filtered before being incubated with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μl PBS 0.2 % BSA 0.02 % NaN3 (FACS buffer) for 20 minutes at 4°C to prevent antibody binding on the Fc receptor. Various fluorescent mAb combinations in For the 1-week treatment and the 2-week treatment, administrations were on day 7, 9, 11 and on day 7,9,11,14,16,18 respectively. To assess the reproducibility of NPs administration through the MicroSprayer®, the delivered doses of NP suspensions were collected in glass vials after each spray and were accurately weighted. Then the amount of the delivered drug was quantified by HPLC as already described.

The protocol to administer the NPs in mice was adapted from a previously reported one (Bivas-Benita, Zwier et al. 2005). Briefly, mice were placed in isoflurane chamber (Aerrane®, Baxter SAS, France). Then, one mouse was placed on the back on a platform (Mouse Intubation Platform -Model MIP, Penn Century Inc., Wyndmoor, PA) with isoflurane mask and hanging on its teeth. The tongue was pulled out by a tweezer and a laryngoscope (Small Animal Laryngoscope for mouse -Model LS-2-M, Penn Century Inc., Wyndmoor, PA) was used to see the trachea and 50µL of suspensions were delivered inside the lung allowing to aerosolize 50 µL suspensions inside the lungs. At day 14 or 21, mice were sacrificed and lungs were homogenized with MM300 bead beater (Retsch) and eight ten-fold serial dilutions were plated onto 7H11 agar plates supplemented with 10% OADC. CFUs were determined after a three-week growth. Statistics were performed using Student's t-test and one-way ANOVA analysis. Same p-values for in vivo experiments were obtained with the two tests. *: p<0.1, **: p<0.01,***: p<0.001.

Supplementary Video S1 -NTA video of a NP suspension obtained by

nanoemulsion. NTA allows tracking each NP individually to determine its mean hydrodynamic diameter and the concentration of the sample. Indeed, the Nanosight instrument is able to track the Brownian motion of NPs, in order to determine its diffusion coefficient (D t ) and consequently calculate, using Stokes-Einstein equation the sphere-equivalent hydrodynamic diameter. The NP suspensions were diluted by a factor of 10 4 to fit the instrument requirements and to allow the particle to move freely Résumé article 2: "Encapsulation de la Clofazimine dans des particules de silice 4.1.

nanoporeuses pour le traitement oral de M. tuberculosis résistant aux antibiotiques"

La propagation mondiale de micro-organismes résistants aux antibiotiques constitue une menace majeure pour la santé publique mondiale. Et malgré la prise de conscience de cette menace au cours des 30 dernières années, aucun nouveau type d'antibiotiques n'a été jusqu'alors développé par l'industrie pharmaceutique, suite aux échecs coûteux de développement de nouvelles thérapies de traitement à court terme (Nathan 2004).

L'utilisation de nouveaux excipients pour reformuler des antibiotiques existants pourrait alors permettre de développer de nouvelles thérapies en améliorant leur efficacité et leur performance. Néanmoins, les dernières données in vitro ont ravivé notre intérêt pour son utilisation pour le traitement de cas de TB multi-résistantes. D'autre part, des formulations basées sur l'utilisation des nanotechnologies pour la délivrance d'antibiotiques se sont avérées efficaces in vitro et in vivo pour traiter ou prévenir d'autres maladies infectieuses (Huh and Kwon 2011;[START_REF] Semiramoth | Self-assembled squalenoylated penicillin bioconjugates: an original approach for the treatment of intracellular infections[END_REF]Abed and Couvreur 2014;[START_REF] Song | Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition[END_REF][START_REF] Wang | Rational Design of Multifunctional Dendritic Mesoporous Silica Nanoparticles to Load Curcumin and Enhance Efficacy for Breast Cancer Therapy[END_REF].

Mais le développement de produits pharmaceutiques basés sur l'utilisation de nanocarriers a été jusqu'alors entravée par des aspects réglementaires et la pénurie de connaissances relatives à l'effet biologique, la toxicité et la sécurité à long terme du matériel de transport utilisé (Crist, Grossman et al. 2013). Dans ce contexte, les particules à base de silice nanoporeuses (PSN) semblaient constituer des vecteurs attrayants pour l'administration d'antibiotiques par voie orale, de par leurs propriétés chimiques [START_REF] Mamaeva | Mesoporous silica nanoparticles in medicine--recent advances[END_REF]. Les PSN sont constituées de dioxyde de silicium amorphe (SiO2) chimiquement semblable au silicium colloidal couramment utilisé comme excipient dans l'agro-alimentaire et dans de nombreuses formulations à visée pharmaceutique (capsules, suspensions et comprimés par voie orale; préparations transdermiques, rectales et vaginales). L'Agence de protection de l'environnement (EPA) a notamment montré son absence de toxicité pour l'administration orale.

Sur la base de ces données bibliographiques intéressantes, notre étude a consisté à développer une nouvelle formulation de CLZ basée sur l'utilisation d'un nanocarrier pour améliorer la pharmacocinétique de CLZ et s'attaquer aux infections de TB multirésistantes.

Dans des études précédentes, nos collaborateurs avaient montré que l'encapsulation de molécules pharmaceutiques à l'intérieur de la structure nano-poreuse des particules stabilisait les molécules dans leur forme amorphe, en améliorant leur solubilité et leur biodisponibilité [START_REF] Xia | In vivo enhancement in bioavailability of atazanavir in the presence of proton-pump inhibitors using mesoporous materials[END_REF]Xia, Pethe et al. 2014).

Au vue de ces résultats prometteurs, le but de ce travail était d'encapsuler CLZ dans des particules de silice nanoporeuses, dans le but d'augmenter sa solubilité pour améliorer sa biodisponibilité dans des formulations destinées à une administration orale.

Une sélection de particules de silice poreuses, se différenciant par leur taille, et la taille et

the oral treatment of antibiotic resistant M. tuberculosis"

The worldwide spread of antibiotic-resistant microorganisms is no longer a prediction for the future, but it is now a major threat to worldwide public health. Despite the increasing awareness to this threat, over the last 30 years, no major new types of antibiotics have been developed by pharmaceutical companies mainly due to the persistent costly failure to discover novel antibiotics for short-term treatment therapies (Nathan 2004). The use of novel advanced excipients to reformulate existing antibiotic drugs allows the development of new therapies to enhance their efficacy and improve their performance.

Thus, the use of existing antibiotics already used in other diseases treatment, that have not acquired bacterial resistance, can offer a route to speed up the development time for new therapeutic formulations. Clofazimine (CLZ), for example, is an antibiotic discovered in the 1950s currently used for the treatment of leprosy, can be a potential candidate to the tuberculosis (TB) treatment. The CLZ efficiency was already shown in vitro against Mycobacterium tuberculosis multi-resistant strains, with extremely low frequency of drug resistance to the antibiotic (Yawalkar and Vischer 1979;[START_REF] Reddy | Antimycobacterial activities of riminophenazines[END_REF]). However, the use for TB treatment has not been considered so far, due to the variability of the therapeutic activity observed in different animal models (Van Rensburg, Anderson et al. 1997;[START_REF] Mukherjee | Programmes and principles in treatment of multidrugresistant tuberculosis[END_REF]Van Deun, Maug et al. 2010;[START_REF] Cholo | Clofazimine: current status and future prospects[END_REF].

Furthermore, oral administration in humans showed inadequate peak drug concentrations and insufficient total dose in plasma to obtain bactericidal activity against M. tuberculosis (O'Connor, O'Sullivan et al. 1995;[START_REF] Janulionis | Lack of Activity of Orally Administered Clofazimine against Intracellular Mycobacterium tuberculosis in Whole-Blood Culture[END_REF]).

Nevertheless, the promising in vitro data have rekindled our interest in its use for multi-resistant TB treatment.

Nanotechnology based formulation for the delivery of antibiotics that have proven effective in vitro and in vivo for treating or preventing infectious diseases (Huh and Kwon 2011;[START_REF] Semiramoth | Self-assembled squalenoylated penicillin bioconjugates: an original approach for the treatment of intracellular infections[END_REF]Abed and Couvreur 2014;[START_REF] Song | Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition[END_REF][START_REF] Wang | Rational Design of Multifunctional Dendritic Mesoporous Silica Nanoparticles to Load Curcumin and Enhance Efficacy for Breast Cancer Therapy[END_REF]. However, development of pharmaceutical products using nanocarriers has been hindered due to regulatory issues and limited long-term safety and toxicity data (Crist, Grossman et al. 2013). In this context, nanoporous silica particles (NSPs) have emerged as an attractive excipient with desirable properties for the delivery of oral drugs [START_REF] Mamaeva | Mesoporous silica nanoparticles in medicine--recent advances[END_REF]. NSPs are composed by amorphous SiO2 and analytically indistinguishable from colloidal SiO2, which has generally recognized as safe status from the US FDA for use its use in food and as an excipient for many dosage forms (oral capsules, suspensions and tablets; transdermal, rectal and vaginal preparations). The Environmental Protection Agency (EPA) has determined amorphous silica to show no toxicity by oral administration and therefore is exempt from requirement of tolerance status.

Based on this interesting bibliographic data, our work consisted in the development of a new formulation of CLZ using nanocarriers to improve the CLZ pharmacokinetics and fight multi-resistant TB.

In previous studies, our collaborators have shown that the encapsulation of pharmaceutical molecules inside the nanoporous structure, stabilized the molecules in their amorphous form, improving their solubility and bioavailability [START_REF] Xia | In vivo enhancement in bioavailability of atazanavir in the presence of proton-pump inhibitors using mesoporous materials[END_REF]Xia, Pethe et al. 2014).

Motivated by this finding, the aim of this work was to encapsulate CLZ into NSPs in order enhance its solubility to improve its bioavailability in oral dosage formulations. A selection of different porous silica particles were used, differing in particle size, pore size and pore volume.

Our collaborators were in charge of the physicochemical characterization of the CLZ encapsulated in the NSP, as well as to study its stability after encapsulation, its solubility, toxicity and permeability in intestinal cells models. In our study, the use of silica nanoporous allowed to stabilize the amorphous state of CLZ (for more than 6 months) and to increase up to 20 times the solubility of the antibiotic in the model gastric fluid, the rate of dissolution being a function of the morphology and texture of the NSPs used.

In our laboratory, we studied the in vitro effect of different NSPs in macrophages models infected with M. tuberculosis. The effect of the loaded NSP, empty NSP and the non-encapsulated antibiotic in the intracellular M. tuberculosis replication was evaluated by the utilization of an automated confocal high content screening (OPERA-PerkinElmer).

The results showed that the CLZ encapsulation in the NSP had considerably improved the solubility and cell permeability of the CLZ, achieving effective antimicrobial concentrations in TB-infected macrophages.

For the first time, we were able to reformulate CLZ in NSP. This work shows that the use of CLZ, a well-known marketed antibiotic for treatment of other diseases, combined with an administration mode based in nanotechnologies, can be a pertinent strategy to fight drug-resistant TB.

Materials and methods

Materials

CLZ was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). FaSSIF-V2

Powder was purchased from Biorelevant (London, UK). Buffers and consumables for cell culture were purchased from Dominique Dutscher SAS (Brumath Cedex, France), VWR (Fontenay-sous-Bois cedex, France) and Thermo Fisher (Illkirch, France). Syringe filters from VWR. Solvents and chemicals were supplied from Sigma Aldrich (Tyreso, Sweden) or VWR (Stockholm, Sweden).

Synthesis of mesoporous silica particles

Four different NSPs were used in the current investigation with different particle size and morphology and different pore sizes and surface areas. Two of the NSPs were synthesized via molecular templating methods according to previously described procedures: NFM-1 [28], SBA-15 [29]. The molecular templated synthesis route generates particles with a sharp single pore size matching that of the templating molecule. Details on the synthesis protocols are described in the supplementary material. The other two porous particles were synthesized in a sol-gel process via spraying of a silica precursor to produce spherical particles with a reasonably narrow particle size range 5-20 microns.

The porosity arises in these particles during the condensation and drying of the silica gel which yields a broader pore size compared to the templated synthesis route. The surface area, pore volume and pore size is strongly influenced by condensation conditions and can be easily varied in order to produce a variety of particles with differing textual properties.

Material characterization

Low angle X-ray diffraction measurements to investigate the pore structure of NSPs were performed on a Panalytical PRO MPD diffractometer in transmission mode using a focusing mirror and Cu-K α radiation, with samples spread on mylar films.

Nitrogen adsorption-desorption isotherm (Micromeritics Tristar II 3020-Apparatus, Norcross, GA, USA) was used to investigate the porosity of empty silica particles (before drug loading). All samples were degassed at 200 °C for 3 hours under nitrogen gas flow.

The surface area was calculated by the Brunauer-Emmett-Teller equation in the relative pressure range between 0.05 and 0.2 [30]. The pore diameters and pore volumes were calculated according to BJH Barrett-Joyner-Halenda adsorption model. 4. Chapter II Scanning electron microscopy images were obtained using a JSM-7401F scanning electron microscope (JEOL Ltd.,Tokyo, Japan) operating at 1 kV. The materials were investigated in their native state, that is, without gold coating.

Thermogravimetric analysis (TGA) was used to measure the amount of drug loaded in the silica carriers using the instrument model TGA 209 (NETZSCH) operating with a temperature ramp between 20 and 990ºC and a heating rate of 20°C/min. The plug-in gas atmosphere was dry air. The sample weights varied from 3 mg to 10 mg. As control, CLZ (pure drug crystals) was also analyzed.

Differential scanning calorimetry (DSC) (Q204, NETZSCH) was used to determine if there was crystallinity in the loaded samples which is observed as a phase transition during melting. The loaded samples (3 -4 mg) were added in pierced aluminum pan (NETZSCH), and heated from 40 to 200 or 300ºC at a rate of 10ºC min-1. An empty pan was used to calibrate the temperature scale and the enthalpy response. As control, calcined silica particles before drug loading, CLZ (pure drugs -crystals), CLZ evaporated after acetone solution and physical mixtures comprising CLZ and NSPs without loading procedure were analyzed.

Loading of Clofazimine into Mesoporous Silica

CLZ was loaded into NSPs by incipient wetness impregnation followed by solvent evaporation, since it is practical for laboratory-scale control of the loading amount. In brief, a concentrated solution of the drug (5 mg/mL) in acetone was mixed under gentle stirring at room temperature for 2 hours with NSPs previously dried at 120°C under vacuum for 3h in order to remove residual humidity in the pores. The solvent was removed by rotary evaporation (100 rpm) under reduced pressure at 40°C until complete evaporation of solvent. The powder was then dried overnight in air. The drug/silica wt% was varied from 5% to 50% in order to investigate the maximum loading capacity of each of the silica particles.

Drug release and drug dissolution experiments

The in vitro release kinetics of the pure drug and drug-loaded NSP was conducted in SGF (0.01 HCl, 0.25 % NaCl, 0.1% SDS) (pH 2) at 37ºC, with stirring rate 50 rpm. The conditions were chosen to provide the closest simulation of in vivo conditions, while using the simple pharmacopeia dissolution apparatus. The equivalent amount of drug was used in pure drug and drug-loaded NSP formulation and chosen to be 50mg since this is used in clinical applications. At each time point, aliquots of 1-2 mL were collected and filtered with a hydrophilic syringe filter (polyethersulfone: 0.45 µm) in order to avoid interference from the particles and/or drug crystals. Due to the extreme lipophilic nature of CLZ, which may alter the accuracy of the standard curve for analysis in a UV-visible spectrometer, the release profile was evaluated by HPLC-UV method (see section below).

All release experiments showed good reproducibility.

Cell and bacterial culture

Human intestinal cell lines Caco-2 was established from the parental human colonic adenocarcinoma cell line [31], and maintained as recommended. Briefly, the Caco-2 cells were cultured in DMEM (25mM glucose) supplemented with 15% heat-inactivated ( 30min, 56°C) fetal calf serum (Boehringer, Mannheim, Germany) and 1% nonessential amino acids (Life Technologies, Villebon sur Yvette, France). Cells were passaged weekly using trypsin-EDTA. The culture medium was changed daily. Caco-2 cells were maintained in a humid atmosphere at 37 °C with 10% CO2 and used between passage 51 and 57 after thawing.

Mouse macrophage RAW 264.7 (ATCC # TIB-71) were maintained at 37°C in RPMI-1640 + glutamax containing 10% fetal bovine serum (FBS) and were passed 3 three-times per week and used before passage number 7. Macrophages were harvested by using Versene (15040033, LifeTechnologies). heat-inactivated FBS (Gibco, ThermoFisher, Illkirch, France) and titrated by measuring the optical density at 600 nm.

M. tuberculosis

Cellular and transport media

For the permeation study, the Fasted State Simulated Intestinal Fluid V2 (FaSSIF-V2) transport media was chosen as the apical buffer in order to have a biorelevant medium simulating intestine fluid that was not toxic for the Caco-2 cells [32,33]. The basolateral buffer was DMEM (without phenol red) supplemented with 1% of bovine serum albumin (BSA) (Sigma Aldrich, France) to increase the recovery of lipophilic compound and allow a correlation between absorption in Caco-2 cells and absorbed fraction in human intestinal lining [34].

For each transport experiment, the FaSSIF-V2 media was freshly prepared dissolving FaSSIF-V2 powder in half of the volume of FaSSIF-V2 DMEM-maleate buffer. After complete dissolution of the powder, the rest of the FaSSIF-V2 DMEM-maleate buffer was added and the medium left to stand at room temperature for 1hour (FaSSIF-V2 final concentration: 1.79g/L). The FaSSIF-V2 DMEM-maleate was prepared as follows: NaOH (0.3475 g), maleic acid (0.5575 g) and NaCl (1.0025 g) were dissolved in 0.225 L of DMEM.

The pH was adjusted to 6. S1.

Caco-2 cell viability assay

The 3-( 4,5-dimethylythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck Millipore, Darmstadt, Germany) assay was used for determining cytotoxicity on the Caco-2 cell line. The MTT assay quantifies mitochondrial dehydrogenase activity as a measure of cell viability [35]. Caco-2 cells were seeded at a density of 2.5 ×104 cells per well in TPP® tissue culture plates 96-well plate (Dominique Dutscher, France) over a week in order to achieve the 100% of confluence and simulated the monolayer permeability. Cells were then treated with series of concentrations of CLZ (serial dilutions from a concentrated ethanolic solution) or NSP. After 6 hours of incubation, the medium was replaced with 0,05 mg/mL solution of MTT reagent in free-serum DMEM medium for 24 hours. The culture medium was further removed and replaced by 200 μL of dimethyl sulfoxide (DMSO), in order to dissolve the formazan crystals.

The absorbance of the solubilized dye was measured spectrophotometrically with a microplate reader (GENios, TECAN XFLUOR4) at 550 nm. The percentage of viable cells for each treatment was calculated from the ratio of the absorbance of each well containing the treated cells versus the average absorbance of the control wells (i.e.,

Ethanol-or growth medium-treated cells). All experiments were conducted at least in triplicate.

Caco-2 intestinal permeation

Caco-2 cells were seeded in Transwell-6 insert 24mm, TC Treated PET Membrane.

(Fisher, France) at a density of 4.5 X 105 cells/well and then incubated for 21-28 days for differentiation. Medium was changed every 2 days in both compartments. The cellular polarization and differentiation of Caco-2 monolayer was determined by measuring the transepithelial electrical resistance (TEER) using an epithelial voltmeter (EVOM2). The shorter electrode was inserted in the apical side and longer electrode in the basolateral side. Care was taken that the electrode did not touch the cell monolayer and/or the plastic. Based on the literature, a resistance reading of 50-400Ω cm2 was considered as indicative of a confluent Caco-2 monolayer with tight junctions. Caco-2 monolayers were rinsed one time with the apical and basolateral buffers before permeation experiments.

The intestinal permeation experiments through Caco-2 monolayers was designed in order to assess a possible tool to forecast in vivo performance of oral solid dosage forms.

To this end, the permeation test was performed with CLZ after dissolution in SGF. Briefly, 1.5 mM of CLZ and NLAB-Silica-CLZ formulations were incubated in SGF at 37ºC, rpm 75. The TEER change measured was transient and reversible and the sodium fluorescein transport was under 0.6% h-1cm-1 [36]. These data suggested the integrity of the cellular monolayer. Results were expressed as cumulative transport as a function of the time.

Experiments were done in triplicate.

The permeability coefficient (Papp) and accumulated insulin (%) across the Caco-2 monolayer was calculated according to Equations 1 & 2, as previously reported in literature [37].

where ∆Q/∆t (in ng/s) indicates the linear apparent rate of mass appearing on the basolateral side of the cell monolayer, Q (in ng) indicates the accumulated mass on the basolateral side of the cell monolayer, C0 (in ng/ml) is the initial concentration of drug on the apical side of the monolayer and A (in cm2) is the sur-face area of the well..

HPLC-UV method for detection and quantification of CLZ

Quantification of CLZ was made with Agilent 1100 HPLC system comprising a model G1312. A binary gradient pump with degasser (model G1322A), autosampler (model G1367A), thermostated column compartment (model G1316A) and a diode array UV/Visible detector (model G1315B). Data collection was made using Chemstation rev.

B.01.03 (Agilent, Kista, Sweden).

For the analysis of Caco-2 intestinal permeation a suitable isocratic method was developed using a 3.0×50 mm column containing 3 µm 100Å C18 functionalised silica (type L1 according to the USP classification, Nanologica AB, Södertälje, Sweden). Mobile phase A consisted of HPLC quality water with 0.05% trifluoroacetic acid (Merck, Uvasol for spectroscopy) and mobile phase B was acetonitrile (BDH Prolabo, HiPerSolv Chromanorm for HPLC, gradient grade supplied from VWR, Stockholm, Sweden). Mobile phases A and B were mixed 50/50 by the pump. The flow rate was 0.7 ml/min and the column was thermostatted at +40°C. A detection wavelength of 286 nm was used and the injection volume was 100µl. This large volume, which was needed to achieve the required detection limits, could be injected without peak distortions by careful choice of the dilution solvents. Standard solutions in the concentration range 0.01-0.5 µg/ml were prepared in methanol (BDH Prolabo, HiPerSolv Chromanorm for HPLC, gradient grade)

and diluted 800 µl + 200 µl DMEM before analysis. Samples taken from the cell experiments were likewise diluted 200 µl + 800 µl methanol during sample work-up so the final mixtures contained the same proportions of organic solvent/water. The detection limit (signal-to-noise ratio =3) was 11 nM calculated for the undiluted Caco-2 cell sample solutions.

For the analysis of CLZ in drug release and dissolution experiment a slightly different method was used. The same mobile phases were used but the column was a 4.6×50 mm column containing 3 µm 100Å C18 functionalised silica (type L1 according to the USP classification, Nanologica AB) and the flow rate was 1.5 ml/min. The injection volume was 5 µl. Standard solutions in the concentration range 1 -100 µg/ml were prepared in methanol and diluted 500 µl +500 µl 0.01 M HCl with 0.2% NaCl and 0.1% SDS. Samples taken from the dissolution experiments were likewise diluted 500 µl + 500 µl methanol during sample work-up so the final mixtures contained the same proportions of organic solvent/water.

Effect on M. tuberculosis infected RAW 264.7 macrophages

NFM-1 and CLZ-encapsulated NFM-1 were dispersed in water to concentrations of 3.9mg/mL and 4.2 mg/mL, respectively. Sixteen twofold serial dilutions of the mother solution were performed in MilliQ water in a 384 deepwell "diamond plate" (P-384-120SQ-C-S, Axygen, Thermo Fisher, Illkirch, France) in order to obtain a dose-response curve. CLZ was dissolved in DMSO (34943, Sigma-Aldrich) to 3 mg/mL and was dispensed in Echo-qualified 384-well low dead volume source plates (Labcyte, France). Volumes between 5 and 500 nL of CLZ were transferred to the assay plate using an Echo 550 Series Liquid Handler (Labcyte) and posteriorly were backfilled with DMSO until 500 nl. Prior to addition of cells, 4.5μL of cell medium were dispensed in those wells. Intramacrophage bacterial replication was assessed following procedures reported previously. [START_REF] Queval | A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening[END_REF]) Briefly, bacteria were mixed with RAW 264.7 macrophages to prepare a suspension at 5×10 5 cells/mL and 1×10 6 bacteria/mL (multiplicity of infection 2) in RPMI-1640 + GlutaMAX containing 10% FBS. After 2h of infection at 37°C with shaking (120 rpm), infected cells were washed with RPMI-1640 + GlutaMAX containing 10% FBS by centrifugation at 1100 rpm for 5 min. The remaining extracellular bacilli from the infected cells suspension were killed by a 1h 50 µg/mL amikacin (A2324-5G, Sigma) treatment and then washed twice with RPMI-1640 + GlutaMAX containing FBS. Finally, 45 μL/well of cellular suspension was added in the 384-well assay plate and incubated during 5 days at 37°C, 5% CO 2 . Macrophages were then stained with 5 μM Syto 60 (S11342, Molecular probes, Thermo Fisher, Illkirch, France) dye for 1h followed by plate sealing. [START_REF] Queval | A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening[END_REF])

Results and discussion

Nanoporous particles with different particle morphologies & pore texture were investigated for CLZ encapsulation Several types of NSPs have been studied as oral delivery carrier for CLZ. The particles in this study were all in the micron-size range, as distinct from nanometer-sized nanoporous particles, which are more commonly used in the academic literature.

Particles larger than 2-3 µm are unadsorbed in the GIT whilst they are localized in the Peyer's patches. (Florence 2005;[START_REF] Gaumet | Localization and quantification of biodegradable particles in an intestinal cell model: the influence of particle size[END_REF]) Therefore, micron-sized particles are of optimal size for drug carriers for gastrointestinal drug release with minimum risk of systemic uptake and distribution. Amorphous silica is significantly soluble only at elevated pH (above pH 10) and is therefore excreted largely unchanged in feces.

In previous studies, we have shown that different morphologies, textural and structural characteristics of the NSP have significant influence on the drug dissolution properties. [START_REF] Xia | In vivo enhancement in bioavailability of atazanavir in the presence of proton-pump inhibitors using mesoporous materials[END_REF][START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF]Xia, Pethe et al. 2014). In this study we have used spherical shaped particle (ASP277 and ASP734), rod-type (SBA-15)

and aggregated pseudo-spherical morphologies for NFM-1 (Figure 1).

The periodicity of the internal pore structure of nanoporous silica materials was investigated using low angle x-ray diffraction (Figure 2). Both NFM-1 and SBA-15 possesses a 2D hexagonal network (space group: p6mm) of cylindrical pores. Both samples showed nanoscale peaks and unit cell parameters that are consistent with previously published data. [START_REF] Atluri | Nonsurfactant supramolecular synthesis of ordered mesoporous silica[END_REF][START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF] The NFM-1 XRD (x-ray diffraction) peak showed a rather broad peak at 2.5 2 θ, which is influenced by the morphology of the particles with spherical morphology. [START_REF] Atluri | Nonsurfactant supramolecular synthesis of ordered mesoporous silica[END_REF][START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF] The pore size was determined to be 23,7 Å for NFM-1 and 62,9

Å for SBA-15 according to the Brunauer-Emmett-Telle analysis (Table 1).

The ASP277 and ASP734 particles showed a smooth curve in the low angle pattern with no distinguishing features, consistent with the expected result from a non-ordered porous structure with a nonuniform pore size. Table 1 compares the physical characteristics of the different particles measured using nitrogen sorption. Although NFM-1 and ASP734 show similar surface area (around 750 m 2 g -1 ), the pore interconnectivity is different since NFM-1 has small pore size and low pore volume, whilst ASP734 has both large average pore size and high pore volume.

Among the particles, ASP277 presents the highest average pore size (28.40 nm) with the lowest surface area (277 m 2 g -1 ), while SBA-15 presents intermediate values of pore size/volume and surface area. Together, these NSPs represent a wide range of external particle morphology, internal structure and porosity. NSPs stabilize CLZ in the amorphous form CLZ was encapsulated in the internal nanopores of the NSP by wet impregnation followed by solvent evaporation. Acetone was chosen as the solvent because of its high volatility and ability to dissolve CLZ (solubility 5 mg/mL). Interestingly, the deep red color of CLZ was visible during encapsulation in the NSP which was readily observed under a light microscope making it easy to verify drug loading in the particles (Supplementary Figure 1). The final amount of CLZ encapsulated in the NSP was determined by TGA which is a standard method commonly used for measuring adsorbed mass in these studies. [START_REF] Atluri | Nonsurfactant supramolecular synthesis of ordered mesoporous silica[END_REF][START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF] The change in mass due to decomposition of the organic pay load (i.e;, CLZ) inside the inorganic NSP was in the temperature range between 150 and 900 °C (Supplementary Figure 2). The TGA curves of CLZ-loaded NSPs were similar to the CLZ free drug suggesting that the thermal decomposition of CLZ takes place irrespective of whether or not it is encapsulated in NSP.

The physical properties of CLZ encapsulated in the NSP and its degree of crystallinity/amorphous ware investigated by differential scanning calorimetry (DSC). The results show that all the NSPs were able to completely suppress the phase change of CLZ yielding an amorphous form of CLZ encapsulated in the particles (Figure 3). The drug loading capacity while maintaining CLZ in the amorphous form was similar for all the NSP (from 11% to 17% wt%). The slight dip in the curve observed around 100 ºC is also present in the DSC curves of the empty particles without CLZ encapsulation (Supplementary Figure 3) and is attributed to surface-bound water content present in the NSP. [START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF]Xia, Pethe et al. 2014) In order to confirm that amorphous state of CLZ was due to the encapsulation in the NSP and not just from the solvent evaporation treatment, CLZ was evaporated from acetone solution without the presence of NSP. Furthermore, physical mixtures comprising CLZ and NSPs were also investigated.

In all cases, clear melting phase transition were observed, confirming that CLZ recrystallizes during evaporation from acetone if not confined in the NSP pores. The observed stabilized amorphous form of CLZ is consistent with model of suppression of crystallization (Prasad and Lele 1994;[START_REF] Rengarajan | Stabilization of the amorphous state of pharmaceuticals in nanopores[END_REF], in which drug confinement in nanopores below a critical pore diameter (d*) reduces the crystallization kinetics due to unfavourable free energy balance. Typically, molecules confined to pores below three-times their molecular size are inhibited from recrystallizing. A recent study on CLZ crystals revealed a triclinic configuration with two molecules per unit cell with dimension 10.507 x 12.852 x 9.601 Å and volume of 1204.01 Å 3 . [START_REF] Keswani | Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition[END_REF] The predicted polar surface area of CLZ is 39.99 Å 2 (drugbank). This suggests that CLZ encapsulated in pores of NFM-1 with an average pore diameter of 24 Å will be confined to an amorphous form.

Poor physicochemical stability of amorphous drugs is a major challenge in the development of pharmaceutical products. (Serajuddin 1999) Remarkably, encapsulation of CLZ in the NSP particles in this study provided a stabilized amorphous form, which was stable for over 6 months stored at room temperature (Supplementary Figure 4). This is a crucial feature of the nanoporosity of the NSP which gives a unique and distinct advantage of NSP in comparison to other methods for preparing amorphous drug formulations, such as hot melt extrusion and spray drying.

Different porosity of the NSPs affects the drug release kinetics

To evaluate if the amorphous CLZ-NSP formulations had benefit for oral drug administration, the dissolution kinetics of CLZ in SGF was investigate using USP Apparatus 2 (Paddle Apparatus). A concentration of 50mg was used as it represents a conventional tablet dose. The dissolution of CLZ in SGF represents the fate of a typical oral drugs, where drugs usually first undergo dissolution in the gastric environment. CLZ encapsulated inside NSPs resulted in significant increase in dissolution rate compared with the free CLZ (in either crystalline form as received or after solvent evaporation from acetone) (Figure 4).

In the case of CLZ-encapsulated in ASP734 a dramatic enhancement in dissolution (area under the curve) of approximately of 20-fold was observed in comparison to the CLZ as free drug. The dissolution kinetics were significantly different depending on the NSP particle used. This is due to the intrinsic properties of the nanoporous particles (pore size, surface area and geometry) which influence the release kinetics of the drug, and offers the potential for tuneable drug release profile from different NSP compared to other methods to stabilize the amorphous form. Our previous work on the subject [START_REF] Xia | In vivo enhancement in bioavailability of atazanavir in the presence of proton-pump inhibitors using mesoporous materials[END_REF][START_REF] Kjellman | Influence of microporosity in SBA-15 on the release properties of anticancer drug dasatinib[END_REF] showed that pore size and the presence of microporosity could drastically affect the release kinetics. The release profile of the CLZloaded NSPs suggest that, in this case also, the surface area plays an important role in the dissolution kinetics. For instance, ASP734 (medium pore size, high surface area) resulted in the fastest dissolution kinetics followed by SBA-15 (medium pore size, medium surface profile. Slower release kinetics from particles with narrow pores (of the order of a few nm's) can be rationalized from slower diffusion kinetics within the pores. In contrast, the slower diffusion kinetics of drug molecules from the particles with larger pores has been observed previously and may result, rather counter-intuitively, from formation of ultrafine crystals in the pores due to the local concentration exceeding the crystallization saturation concentration. Formation of crystals will shift the equilibrium conditions and hinder the dissolution rate.

NSPs enhance CLZ permeability through human Caco-2 intestinal cells

In order to simulate drug uptake via oral administration, aliquots from the dissolution of CLZ in SGF were incubated with human intestinal Caco-2 monolayer and the concentration of drug transported through the monolayer was investigated. Caco-2 cell monolayers are routinely used in drug development protocols to assess intestinal drug permeability and are accepted as a surrogate for human intestinal permeability measurements by the FDA. [START_REF] Bergstrom | Early pharmaceutical profiling to predict oral drug absorption: current status and unmet needs[END_REF]) Intestinal in vitro permeation studies are usually performed in saline solution such as Hanks' balanced salt solutions despite the fact that the physicochemical and biochemical properties are very different from the intestinal fluid. [START_REF] Lind | Development of simulated intestinal fluids containing nutrients as transport media in the Caco-2 cell culture model: assessment of cell viability, monolayer integrity and transport of a poorly aqueous soluble drug and a substrate of efflux mechanisms[END_REF][START_REF] Antoine | Biorelevant media resistant co-culture model mimicking permeability of human intestine[END_REF] Here, in order to improve the in vitro/in vivo correlation, we used the FaSSIF-V2 biomimetic fluid, for which, the composition has been recently revised to predict in-vitro dissolutions of oral dosage forms [START_REF] Markopoulos | Biorelevant media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness[END_REF], [START_REF] Porter | Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs[END_REF]) and does not affect the integrity of Caco-2 monolayer, in contrast to Fed State Simulated Intestinal Fluid. [START_REF] Ingels | Simulated intestinal fluid as transport medium in the Caco-2 cell culture model[END_REF] The addition of 1% BSA in the basolateral compartment was found to be important to prevent adsorption of the lipophilic CLZ on the plastic of the Transwell in order to measure the drug concentration. The parameters chosen for the residence time and drug concentration were aligned to obtain the best correlation between absorption of lipophilic compounds in Caco-2 cells and absorbed fraction in humans. [START_REF] Fossati | Use of simulated intestinal fluid for Caco-2 permeability assay of lipophilic drugs[END_REF] All the experiments were performed in a CLZ concentration range with good cell viability according to cell cytotoxicity test (Supplementary Figure 5). It is important to note that the silica particles do not induce cytotoxicity on the intestinal cells. Moreover, change in the TEER was transient and reversible and the sodium fluorescein transport showed that no cellular damage occurred. As shown in Figure 5, CLZ-loaded NSP formulations dramatically increased the intestinal permeation in comparison to the CLZ as free drug, which was below the detection limit (10 -10 g/ml). The data indicates a slightly higher permeability is achieved from the CLZ-loaded in ASP734 which showed more rapid dissolution kinetics in comparison to the slower release profile from CLZ-loaded in NFM-1. However, this difference is not statistically significant in the experimental setting used. Over a time period of 2 -3h the release profile from the NFM-1 shows a linear permeation profile with a similar equilibrium concentration being reached compared to the ASP734 particles (Figure 4). The concentration of CLZ in the basolateral component was in the range of 0.01-10 μg/ml with an average concentration of 0.1-1 μg/ml detected after 180 mins through dissolution from NFM-1.

Reformulation of CLZ in NFM-1 enhances solubility to reach its antimicrobial concentration.

The in vitro dissolution kinetics and subsequent Caco-2 permeability measurements provide strong support that in vivo CLZ oral bioavailability can be enhanced by encapsulation in NSP. In order to evaluate if NSP-CLZ formulation lead to enhanced antibacterial effect, we investigated the antimicrobial activity of CLZ on M. tuberculosis infected RAW macrophages. The NFM-1 particles were chosen for formulation of CLZ since they showed a good reproducibility in the dissolution and permeation studies and, as we have reported previously, shown to provide enhanced bioavailability and pHindependent solubility of lipophilic drugs. TB-infected macrophages were exposed to a 10 5 fold dilution series of CLZ dissolved from NFM-1 particles. Since CLZ as the free drug is virtually insoluble in aqueous conditions it was not possible to conduct a control experiment with dissolved CLZ in aqueous solution. Instead CLZ was dissolved in DMSO in which it is fully soluble. As such, this control served as a comparison to a fully soluble, 100% bioavailability, of CLZ. As shown in Figure 6 (bottom), there is a clear antibacterial effect of CLZ on M. tuberculosis with a marked antibacterial effect at concentrations in the range 10 -7 g/mL to 10 -6 g/mL. The dose response curves for CLZ dissolved in DMSO and that released from NFM-1 particles showed very similar concentration dependency. The antibacterial concentration of CLZ achieved by dissolution from NFM-1 in aqueous conditions matches the MIC obtained from a fully dissolved concentration in solvent. The MIC observed is in this study is in very good agreement with very recent results from a multi-lab study using a 7H9 broth microdilution method to establish MICs of several firstline and second-line TB drugs. [START_REF] Kaniga | A Multi-Laboratory, Multi-Country Study to Determine Minimal Inhibitory Concentration Quality Control Ranges for PhenotypicDrug-Susceptibility Testing of Selected First-Line Anti-TB Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine and Linezolid[END_REF] The MIC of CLZ was determined to be in the range: 0.03-0.25 μg/ml. It is important to recall that the concentration of CLZ measured in the basolateral medium in the permeation studies was of the order of 0.1-1 μg/mL from NFM-1. Therefore, the enhanced permeation of CLZ by encapsulation in NSP leads to a concentration range up to an order of magnitude higher than the CLZ MIC. This significant result indicates that the formulation of CLZ in NSP can lead to an enhanced permeation of CLZ to achieve the MIC after a single dose.

As a control, in order to verify that the observed bacterial inhibition of CLZ was due to antibacterial action and not simply because of cell toxicity, the macrophage cell viability was measured. In Figure 6 (top), the number of viable cells was counted and visualized for each group of treatment. Notably, the unloaded NFM-1 particles and CLZ encapsulated in NFM-1 did not present cytotoxicity at any concentrations tested,  CLZ released into aqueous media from nanoporous silica particles showed the same antibacterial effect as CLZ dissolved in organic solvents, while CLZ alone is insoluble in water.

 CLZ's permeation through the GIT was enhanced to levels of concentration range above the MIC on Mycobacterium tuberculosis.

Résumé article 3: "L'utilisation de nanocarriers à base de PLGA améliore 5.1.

l'efficacité de la Vancomycine contre Mycobacterium tuberculosis intracellulaire"

Face à la nécessité de définir de nouvelles stratégies pour améliorer l'efficacité des traitements contre les souches multirésistantes de TB, l'utilisation d'antibiotiques approuvés pour le traitement d'autres maladies est une alternative intéressante pour améliorer le développement de nouveaux traitements antituberculeux (Dheda, Barry et al. 2016).

La Vancomycine (VCM), un antibiotique de référence utilisé depuis des décennies comme alternative de la pénicilline pour le traitement des souches de Staphylococcus aureus Methicillin-résistantes (MRSA) (Chua and Howden 2009), avait montré une activité bactéricide sur des cultures axéniques de bactéries Mtb (Collins and Uttley 1988;[START_REF] Provvedi | Global transcriptional response to vancomycin in Mycobacterium tuberculosis[END_REF][START_REF] Dinesh | Involvement of efflux pumps in the resistance to peptidoglycan synthesis inhibitors in Mycobacterium tuberculosis[END_REF][START_REF] Schoonmaker | Nonclassical transpeptidases of Mycobacterium tuberculosis alter cell size, morphology, the cytosolic matrix, protein localization, virulence, and resistance to beta-lactams[END_REF][START_REF] Soetaert | Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria[END_REF][START_REF] Rens | Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria[END_REF]. Ces données nous ont permis d'envisager son utilisation comme nouvelle stratégie pour le traitement de TB, sachant également qu'aucune donnée concernant l'efficacité de cet antibiotique vis-à-vis de Mtb intracellulaires n'était jusqu'alors disponible.

Au vue des échecs cliniques liés à la faible pénétration intracellulaire de cet antibiotique précédemment décrits [START_REF] Lamer | Analysis of vancomycin entry into pulmonary lining fluid by bronchoalveolar lavage in critically ill patients[END_REF]Kollef 2007;[START_REF] Lehar | Novel antibody-antibiotic conjugate eliminates intracellular S. aureus[END_REF], VCM serait ainsi incapable d'accéder aux macrophages infectés pour atteindre les concentrations thérapeutiques optimales dans les niches intracellulaires de Mtb. En revanche, les cellules phagocytaires sont bien connues pour leur capacité à internaliser les nanoparticules (NP) contenant des médicaments (Pelgrift and Friedman 2013;[START_REF] Ladaviere | Toward an optimized treatment of intracellular bacterial infections: input of nanoparticulate drug delivery systems[END_REF][START_REF] Costa-Gouveia | How can nanoparticles contribute to antituberculosis therapy?[END_REF][START_REF] Costa-Gouveia | Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles[END_REF]. Ainsi, l'utilisation de nanocarriers capables de pénétrer dans des cellules infectées pour y libérer un antibiotique actif et atteindre ainsi les agents pathogènes, est une stratégie prometteuse pour surmonter la faible capacité de VCM à entrer dans les cellules.

Dans le cadre de notre étude, plusieurs critères ont été préalablement fixés, de façon à choisir un nanocarrier adapté pour favoriser un effet intracellulaire de VCM. Ces nanoparticules devaient ainsi: i) avoir un diamètre moyen inférieur à un micron pour être 5. Chapter III efficacement phagocytés par les macrophages infectés ; ii) permettre d'encapsuler plus de 10 % de VCM par NP ; iii) libérer l'antibiotique de manière contrôlée dans les compartiments intracellulaires ; iv) être préparée par une méthode simple avec une utilisation minimale d'excipients ; et v) être stable durant le stockage. Dans d'autres études, VCM avait été incorporée dans des liposomes et délivrée dans les macrophages [START_REF] Ma | Nano-hydroxyapatite/chitosan/konjac glucomannan scaffolds loaded with cationic liposomal vancomycin: preparation, in vitro release and activity against Staphylococcus aureus biofilms[END_REF][START_REF] Pumerantz | Preparation of liposomal vancomycin and intracellular killing of meticillin-resistant Staphylococcus aureus (MRSA)[END_REF][START_REF] Sande | Liposomal encapsulation of vancomycin improves killing of methicillin-resistant Staphylococcus aureus in a murine infection model[END_REF][START_REF] Surewaard | Identification and treatment of the Staphylococcus aureus reservoir in vivo[END_REF], mais avec de faibles efficacités d'encapsulation (< 20 % VCM) . Des nanoparticules à base de polymères d'acide poly(lactique-co-glycolique) (PLGA) ou de chitosan ont également été utilisées, mais une faible quantité de VCM avait pu être encapsulée (4-6 % poids) à cause des propriétés hydrophobes de l'antibiotique [START_REF] Hu | Nanoparticle biointerfacing by platelet membrane cloaking[END_REF]. Plus récemment (Pei, Mohamed et al. 2017) However, like many antimicrobials, VCM is unable to access infected cells to reach the optimal therapeutic concentrations within the intracellular replicative niches. In contrast, phagocyte cells are well known for their ability to internalize drug-loaded NPs.

Thus, the use of engineered nanocarriers able to readily penetrate infected cells to release their active drug cargo in the vicinity of intracellular pathogens appears as a promising strategy to overcome the poor ability of VCM to bypass membranes.

Here we show the potential of engineered nanoparticles (NPs) loaded with VCM to kill Mtb H37Rv strain in mouse macrophage RAW 264.7 cell line. A thorough optimization of the NP preparation procedure enabled the formation of NPs of around 320 nm including large aqueous compartments with entrapped VCM. Drug loadings reached 14.5 wt%, overpassing the ones reported so far in the literature. Despite their simple preparation method, easy to be scaled-up, the NPs possessed desired features for intracellular drug delivery, including a pH-triggered release.

Noteworthy, it was shown that the NPs were efficiently taken up by infected cells.

For instance, after 5 h incubation with the NPs, 99 % of the macrophages contained particles. Confocal microscopy enabled to ascertain that the NPs were inside the cells and were not just adsorbed on their surface. NPs acted as "Trojan horses" massively penetrating inside the macrophages and possibly delivering their drug cargo because of the lower pH in the endosomes. As a result, the activity of entrapped VCM was around 5 times higher than that of free VCM.

With further studies, we believe that these VCM-loaded NPs could find potential applications for TB treatment.

Introduction

Tuberculosis (TB), which is mainly caused by Mycobacterium tuberculosis (Mtb), is still killing 1.8 million people annually in the world (1). Although the TB death rate has decreased, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections is highly worrisome. As in some regions such as Eastern Europe, the percentage of XDR-TB cases increased, there is a growing concern to discover new potent drugs to fight MDR-and XDR-TB.

Repurposing "old" drugs used for other diseases is an attractive drug development strategy since much is already known about approved agents, accelerating their entrance in the market of TB drugs (2).

Vancomycin (VCM) is a glycopeptide antibiotic molecule able to form a hydrogen bond with the terminal D-alanyl-D-alanine moieties during peptidoglycan biosynthesis, thereby preventing bacterial cell wall backbone synthesis. It has been the treatment of choice for serious infections caused by methicillin-resistant Staphylococcus Aureus (MRSA) for decades (3). Due to its efficiency in treating MDR infectious agents and the lack of other therapeutic options for the treatment of MRSA, its use has been reserved to serious infections not treatable with any other antibiotics in order to prevent development of resistant strains.

Currently, although VCM remains the gold-standard treatment of MRSA, new alternative antimicrobials are available and VCM-resistant S. aureus strains are emerging (4,5).

Repurposing VCM for the treatment of TB is considered nowadays a valid and interesting strategy supported by studies showing the activity of VCM against axenic Mtb cultures (6)(7)(8)(9)(10)(11), but this antibiotic was never tested against intracellular Mtb. Some factors affect VCM clinical activity, such as poor bioavailability, protein binding (≤50 %) (12) and poor intracellular penetration. Indeed, like many antimicrobials, VCM is unable to access infected cells to reach the optimal therapeutic concentrations within the intracellular replicative niches of Mtb.

Clinical failures with this drug were mainly attributed to its poor intracellular penetration (13)(14)(15)(16). In contrast, phagocyte cells are well known for their ability to internalize drugloaded NPs (17)(18)(19)(20). Thus, the use of engineered nanocarriers able to readily penetrate infected cells to release their active drug cargo in the vicinity of intracellular pathogens could be a promising strategy to overcome the poor ability of VCM to bypass membranes.

An efficient VCM nanocarrier should: i) have mean diameter lower than one micron to be efficiently taken up by infected macrophages; ii) possess loadings higher than 10 wt% and good entrapment efficiencies; iii) release the drug in a controlled manner in the intracellular compartments; iv) be prepared by a straightforward method with a minimal use of excipients and v) be stable upon storage. Indeed, as VCM is a relatively expensive drug, entrapment efficiencies should be increased to reduce production costs. Similarly, VCM loadings should be maximized to avoid the use of large quantities of excipients which could cause adverse side effects. Ideally, materials with known safety profiles should be used.

VCM was incorporated in liposomes for its delivery to macrophages (21)(22)(23)(24). However the entrapment efficiencies were low (less than 20%) (21)(22)(23). Polymeric poly (lactic-coglycolic acid) (PLGA) NPs or chitosan NPs have also been used to encapsulate VCM, but the loadings (amount of VCM/NP) was low (4-6 wt%) in reason of the high hydrophilicity of the drug (25,26). Of note, VCM was loaded in NPs of around 200 nm made of a poly(ethylene glycol) -poly(L-histidine)-PLGA (PEG-PLH-PLGA) copolymer designed to shield nontarget interactions at pH 7.4 but bind avidly to bacteria in acidic conditions (27). More recently (28), VCM was entrapped in composite NPs made of PLGA, a diblock poly(ethylene glycol) (PEG)-PLGA copolymer, Eudragit and a chitosan derivative.

Interestingly, the NPs with sizes in the range of 500-1000 nm and containing up to 9.5 wt% VCM were able to release the drug in a pH-dependent manner. However, the preparation method is sophisticated and requires the use of several ingredients added in precise amounts.

Here we show a simple method enabling to load up to 14.5 wt% VCM in PLGA NPs.

By optimizing the preparation procedure, the release of the drug could be controlled and was found to be pH-dependent, with almost no release at neutral pH and fast delivery at pH 5.5 and not. Noteworthy, the pH-controlled release could be achieved by using only PLGA copolymers, avoiding the use of pH-responsive ones. The NPs were taken up by infected macrophages. The VCM loaded NPs were tested in vitro and demonstrated their ability to reduce the Mtb payloads in infected macrophages.

Results

Biodegradable PLGA NPs

PLGA NPs containing VCM have been successfully developed by adapting a double emulsion solvent evaporation method (w 1 /o/w 2 ) schematized in Figure 1. A concentrated VCM aqueous solution was poured into a dichloromethane (DCM) organic phase containing PLGA. After sonication, a w 1 /o emulsion was formed. A second aqueous phase containing a salt (NaCl) and a surfactant (PVA) was added to this emulsion, followed by a second sonication step. At this point, a w 1 /o/w 2 was created, where VCM was dispersed in aqueous droplets located inside the organic phase droplets. After solvent removal under gentle stirring, NPs were formed with characteristics gathered in Table 1. The morphology of the drug-loaded NPs was investigated by cryo-TEM, a method with the advantage of gaining insights into the inner structure of the NPs. Of note, in all VCM-PLGA NP samples, the inner aqueous compartments could be clearly observed (Figure 1).

The structure of the VCM-PLGA NPs is typical of a w 1 /o/w 2 emulsion, after polymer solidification. The size of the inner aqueous inclusions (light regions as compared to the dark PLGA matrix) is relatively large, making that they are separated from the outer NP surfaces by only a 10-40 nm thick polymer layer.

According to cryo-TEM investigations, the NPs had mean diameters in the range of 200-400 nm. This was in agreement with the DLS data showing mean diameters of 310-320 nm and polydispersities of around 0.2 (Table 1). An extensive study has been carried on to optimize the preparation procedure to maximise the VCM loading. Drug loading was determined by two methods: i) a direct one, consisting in dissolving the NPs and quantifying the extracted drug and ii) dosing the non-incorporated drug in the supernatants after NP centrifugation. Both methods were consistent with less than 5% differences.

The best strategy to efficiently incorporate VCM was to use a salt, NaCl, in the emulsification process (Figure 1). The addition of only 1% NaCl had a tremendous benefit upon VCM incorporation, while it did not modify the mean diameter of the NPs (Table 1).

Drug loadings (DL) increased from 9.6 to 14.5 wt% without and with 1% NaCl, respectively. Moreover, loading efficiencies increased from 24 to 36.6 % when NaCl was added.

The NP formulation was stored at 4°C for 3 months. Their mean diameter remained unchanged (less than 5% variation) indicating a good stability. Of note, less than 4% of the incorporated VCM was released over this time (Table 1).

Drug release studies were performed after NP dilution in PBS or in the cell culture media (Figure 2). VCM release was less than 10 % in PBS or cell culture media, at pH 7.4 (red and blue curves; Figure 2). In contrast, around 60% of the entrapped VCM was released within 30 min after incubation at pH 5.5.

Intracellular accumulation of PLGA-Rhodamine NPs 5. Chapter III

The ability of empty PLGA NPs to be taken up by infected cells was investigated by automated confocal fluorescence microscopy. RAW264.7 murine macrophages, previously infected with Mtb H37Rv-GFP were incubated with PLGA-Rho for different times (0.5, 2 and 24 hours) and stained with DAPI. This allowed gaining information about the location of both NPs (red signal) and Mtb (green signal) in cells (blue signal). A continuous accumulation of the NPs in the cells was observed in the first 24 hours of incubation (Figures 3A and3B). After 0.5 hours, only 7% of the cells contained NPs. This amount was dramatically increased after 2 and 24 h incubation, reaching 73% and 99% of cells including NPs, respectively. These data highlight the efficacy of the PLGA NPs to be internalized.

In order to ascertain that the NPs were indeed located inside the cells and not only attached to their surface or present in the suspension media, a complementary study was undertaken by confocal microscopy allowing to observe with high resolution each individual macrophage. Z-stack images of infected cells incubated with PLGA-Rho and labelled with nuclei and F-actin specific stains were performed. A typical orthogonal view is presented in Figure 3C, clearly showing that several PLGA-Rho NPs were clearly located inside the cell cytoplasm and were not just surrounding the cell or attached to their membranes. It was also possible to observe that several NPs inside the cell were larger (micron-size particles) than the size previously determined by PCS (320 nm), probably due to clustering effects.

Efficacy of the PLGA containing VCM NPs against intracellular Mtb replication

After confirmation of the ability of PLGA NPs to enter in RAW264.7 cells, we investigated the activity of these VCM-loaded NPs against intracellular Mtb replication. Briefly, compound dilutions and bacteria were prepared, cells were infected for 2 hours and extracellular bacteria were removed by successive washings and 1 hour incubation with amikacin. After 5 days incubation with the compounds at 37°C, the amount of bacteria was quantified using two different methods: confocal fluorescence microscopy and Colony-Forming Unit (CFU) counting (Figure 4A).

For the image-based assay, the analysis of the intracellular bacteria area was used as a correlate of intracellular growth. For both methods, dose-response curves were generated after normalization on water-negative and INH-positive control, (Figures 4B and Fig. 4C).

As expected, in both methods, free VCM doesn't reach complete bacterial replication inhibition. On the contrary, PLGA-encapsulated VCM reached MIC at 40 µg/mL, indicating that the intracellular drug release was critical to the antibacterial activity in Mtb infected macrophages. In terms of IC 50 , PLGA-encapsulated VCM showed 4 to 5 times improved efficacy comparing with the free VCM in both methods (IC 50 decreased from 5.7 to 1.3 µg/mL in the first method and from 9.7 to 5.1 µg/mL in the second method), clearly demonstrating that the encapsulation of VCM is beneficial for its intracellular activity.

Discussion

PLGA NPs of around 320 nm containing VCM have been successfully developed using a double emulsion solvent. In contrast to previously reported studies where up to four ingredients were added to efficiently load VCM in NPs (28) the VCM DL reached 14.5 wt% use just one polymer (PLGA) and by adding a salt (NaCl). The salt plays a key role in favouring the precipitation of PLGA, thus efficiently trapping the inner droplets containing VCM by avoiding their leakage during the evaporation step. CryoTEM images clearly showed NP structures with large cavities, presumably loaded with VCM. The stability of the VCM-loaded NPs (no release of the incorporated drug upon storage) and simplicity of their preparation method could be assets for applications and scale-up. The NP formulation prepared in the presence of NaCl appears as particularly promising, overcoming the DL and encapsulation efficiencies reported so far for VCM incorporation in NPs or liposomes.

Noteworthy, VCM was rapidly released at pH 5.5 and not at pH 7.4 (Figure 2). This interesting and unexpected result might be due to the peculiar structure of the VCM-PLGA NPs (cryoTEM image, Figure 1). They typically possess a thin PLGA shell delimiting aqueous internal compartments presumably containing the VCM payload. When the pH decreases, the solubility of the entrapped VCM increases (29) leading to the fast release of the entrapped drug. As such, 60% of the entrapped VCM was released within the first for a pH triggered release if the NPs are taken up in endosomes with a slightly acidic pH (around 4.8) (31).

For a long time, the use of VCM has been restricted to the treatment of critically ill patients, with drug-resistant infectious agents, particularly MRSA, in the tentative to avoid the development of resistant strains to this important antibiotic. Nowadays, the lack of effective alternatives for the treatment of TB makes the repurpose of known drugs such as VCM an attractive strategy. The Mtb strain used here was indeed sensitive to VCM, with a MIC of 40 µg/mL, in agreement with previously reported data (9)(10)(11).

Mtb is a facultative intracellular pathogen, able to survive and multiply inside phagocytes such as macrophages by subverting the effector functions of these important innate immune cells. Mtb can persist in the host in a dormant state and there is a risk of reactivation, even decades after infection (30). For a good efficacy, drugs must reach both extra-and intracellular Mtb.

Encapsulation of VCM in the developed PLGA NPs resulted in 5 times activity improvement compared to free VCM against intracellular Mtb growth. This supports the fact that VCM has low membrane permeability as previously reported (13)(14)(15)(16). It can be hypothesized that the observed intracellular antibacterial activity was driven by VCM released in the cells rather than the drug released prior to macrophage uptake. Possibly, VCM was readily released from the internalized NPs if these were located in compartments with low pH such as endosomes. The presumed intracellular release of the drug could be responsible of the antibacterial activity. Indeed, VCM-PLGA NPs are readily engulfed in infected cells and after 24 h incubation, practically all the macrophages contained NPs.

In a nutshell, PLGA NPs acted as "Trojan" horses to deliver VCM inside the macrophages. These data the ability of PLGA to entrap high drug payloads. Further studies will be dedicated to the encapsulations of synergic VCM/drug combinations to further improve the capacity of the NPs to kill intracellular Mtb. The combination of VCM and mycobacterial lipids synthesis inhibitors are among potential synergic combinations (10)(11).

Hopefully, this and subsequent work will contribute to the development of new effective anti-TB therapies and to improve current treatment for many other infection diseases caused by gram-positive bacteria.

Materials and methods

Chemicals

Rho-PLA conjugate synthesis

Rho-PLA was synthesised according to a previously reported method (32). Briefly, Rho was reacted with piperazine to allow its grafting to the terminal carboxylic group of PLA through an amide bond. To do so, 11.3 mL of a 2M solution of AlMe 3 in toluene were added to 17.5 mL of a 0.22 mg/mL solution of piperazine in DCM. After completion of the gas evolution, the reaction mixture was refluxed for 24 h at 50°C in the presence of 0.5 mg/mL Rho under N 2 atmosphere. After quenching with a 1 M solution of aqueous HCl, the resulting Rho-piperazine amide was extracted, dried and purified by silica gel chromatography. Then, 5 mg Rho-piperazine amide was added to 50 mg of PLA acidterminated in 0.5 mL DCM in the presence of 1 mg of DMAP as catalyst and 2 mg EDC as coupling agent. The obtained Rho-PLA was then extracted, purified by precipitation and dried. It was used in blends (1 wt%) with PLGA to form the NPs.

Preparation of PLGA NPs containing VCM

VCM-loaded PLGA NPs were prepared by an optimized w/o/w solvent evaporation method. Briefly, 0.2 mL of an aqueous internal phase containing VCM at 150 mg/mL was emulsified for 15 s in 1 mL of DCM (containing 75 mg of PLGA) using a probe sonicator at 40% of power (Sonopuls HD 2070, BANDELIN electronic GmbH & Co, Berlin, Germany). To this primary emulsion, 5 mL of 0.5% wt PVA solution containing or not NaCl (1 % in weight) were poured and sonicated for 30 sec at 40% of power to create a w/o/w emulsion. This emulsion was left at room temperature under gentle stirring overnight to allow the organic solvent to evaporate. Control plain NPs (without VCM) were prepared similarly. All formulations were prepared in triplicate.

NP size measurement and cryoTEM analysis.

The average hydrodynamic diameter of the NPs was determined at 25 °C with an equilibration time of 60 s using a Malvern Zetasizer® (Nano ZS90, Malvern Instruments S.A., Worcestershire, UK). Dilutions with injectable water were done according to ISO 22412 and experiments were performed in triplicate. Mean diameters were reported as Z Average (nm) ± SE (Standard Error -with a PdI lower than 0.1). The NPs mean diameters were monitored up to three months of storage at 25 °C. For cryoTEM analysis, 5 µL of NPs suspensions were deposited onto a 200 mesh copper grid and flash-frozen in liquid ethane cooled down at liquid nitrogen temperature. Cryo-TEM images were acquired on a JEOL 2200FS energy-filtered (20 eV) field emission gun electron microscope operating at 200 kV.

VCM drug loading

PLGA NPs were formulated with 6 mg/mL of initial VCM. Drug encapsulation was determined by quantifying both: i) the amount of non-encapsulated drug present in the supernatant obtained after centrifugation at 10,000 rpm for 10 min and ii) the drug extracted from the NPs by dissolving them in DMSO. All the samples were dosed by reverse-phase HPLC (RP-HPLC). The RP-HPLC analysis were performed after dilution with deionized and filtered water using a Agilent HPLC system (Agilent 1100 Series) equipped with a C18 column (Kinetex 5u C18, 100 A, Phenomenex), and UV detector at λ = 280 nm.

The chromatographic conditions were as follows: solvent A 0.1% Trifluoroacetic Acid (TFA) in water and solvent B 0.1% TFA in acetonitrile; 0-2 min: 0%-20% B, 2-6 min: 20%-45% B, 6-10 min: 45%-75% B, 10-15 min: 75%-0%. Flow rate: 1.0 mL/min at room temperature.

In all cases, R2 values for VCM was higher than 0.99.

The DL is defined as the mass fraction of a NP that is composed of drug, while the EE can be considered as the fraction of drug effectively encapsulated into the NPs compared with the amount that was used to prepare the NPs.

The DL and EE were calculated as shown in equations 1 and 2, respectively.

(1)

Release studies

To estimate VCM release, PLGA VCM NPs 1% NaCl were diluted 10 times in serum containing media, PBS pH 7.4 and PBS pH 5.5 and incubated at 37 °C with constant agitation. At different time points, the suspension was centrifuged at 10,000 rpm for 10 min at room temperature (RT), and the supernatant was analysed by HPLC.

Bacteria and macrophages

A recombinant strain of Mtb H37Rv constitutively expressing the green fluorescent protein (H37Rv-GFP) was cultured at 37°C for 16 days in Middlebrook 7H9 medium (Difco) supplemented with 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC, Difco), 0.5% glycerol (Sigma-Aldrich), 0.05% Tween 80 (Sigma-Aldrich) and 50 µg/mL hygromycin B (Invitrogen). Mycobacteria were washed three times with Dulbecco's Phosphate-Buffered Saline (DPBS free from MgCl 2 and CaCl 2 , Gibco) and resuspended in RPMI-1640 Glutamax medium (Difco) containing 10% heat-inactivated Fetal Bovine Serum (FBS, Life Technologies). Clumped mycobacteria were pelleted by centrifugation at 700 rpm for 2 min and homogeneous supernatants were used for infection. Bacterial titre was determined by measuring the optical density at 600 nm and by measuring EGFP fluorescence on a Victor Multilabel Counter (Perkin Elmer). The 5. Chapter III bacterial suspension was diluted at the required titre in RPMI 1640 supplemented with 10% FBS (RPMI-FBS) prior to infection. RAW264.7 murine macrophages (ATCC TIB-71) were maintained at 37°C in RPMI-FBS.

Macrophages were harvested by using Versene (LifeTechnologies).

Assay plate preparation

VCM dissolved in H 2 O to 5 mg/mL was diluted to 0.4 mg/mL and VCM-loaded PLGA NPs were diluted in water to 0.4 mg/mL of VCM.

Eight 2-fold serial dilutions of the mother solution in a final volume of 100 µL were performed in sterile MilliQ water in a 384 deep well "diamond plate" (Axygen) in order to obtain a dose-response curve. Posteriorly, 5 µL of each concentration of the NPs or VCM were dispensed in 384-well clear-bottom polystyrene assay plates (Greiner Bio-One).

A standard anti-TB drug, such as isoniazid (INH, Sigma-Aldrich) was used as positive control. INH was dissolved in DMSO (Sigma-Aldrich) to 10 mg/mL and diluted to 0.1 mg/mL in H 2 O prior to be added in 384-well assay plates to reach the final concentration of 10 µg/mL. Water was used as negative control.

Intracellular bacterial growth assay

RAW264.7 macrophages suspension at 5×10 5 cells/mL were infected with H37Rv-GFP at a MOI of 2 in RPMI-FBS under mild stirring (120 rpm). At 2 h post-infection, infected cells were washed with RPMI-FBS and treated with 50 g/mL amikacin (Sigma-Aldrich) for 1 h, to kill the remaining extracellular bacilli. Cells were washed twice with RPMI-FBS and 45 µL of cell suspension was added per well, in 384-well assay plates. After 2 h, supernatants were removed and fresh RPMI-FBS was added. Plates were incubated for 5 days at 37°C with 5% CO 2 . RAW264.7 cells were stained with 5 µM Syto 60 Invitrogen) for 1 h at 37°C before imaging.

After image-acquisition, 5 selected wells, corresponding to different concentrations of VCM (40, 10, 2.5, 0.6 and 0.2 µg/mL), and containing infected cells, were washed and lysed with DPBS-0.1% Triton X-100 buffer for 1 min. The lysates were tested for CFU determination.

CFU determination.

5-fold serial dilutions were performed in DPBS and plated onto Middlebrook 7H11 (Difco) agar plates, supplemented with 0.5% glycerol, 10% OADC and 50 µg/mL hygromycin B. After 3-weeks incubation at 37°C with 5% CO 2 , CFUs were calculated.

Study of intracellular PLGA-Rho NPs accumulation

RAW264.7 macrophages suspension were seeded in 384-well plates at a density of 2×10 4 cells per well. After overnight incubation at 37°C with 5% CO 2 cells were, infected with H37Rv-GFP at a MOI of 2. At 2 h post-infection, extracellular bacilli were removed by washing three times with D-PBS and 50 g/mL amikacin treatment for 1 h. Cells were washed twice with RPMI-FBS. 100 µg/mL PLGA-Rho were added by well and incubated for 0.5 h, 2 h or 24 h. Cells were washed, fixed using 10% neutral buffered formalin solution (Sigma-Aldrich) for 30 min. Fixed cells were washed twice with DPBS and then nucleus were stained with 2.5 µg/ml DAPI for 10 min. Cells were finally washed twice with DPBS before imaging.

In parallel, RAW264.7 macrophages were seeded in 12-well plates, containing coverslips, infected and treated with 100 µg/mL PLGA-Rho NPs for 2 h, as described above. Cells were fixed formalin solution for 24h and permeabilized using DPBS-0.1% Triton X-100 for 5 min at room temperature. Cells were then stained with 5 µg/mL DAPI and Alexa Fluor 660 phalloidin dye (ThermoFisher) for 30 min at room temperature. After three washing with DPBS, coverslips were placed in mounting medium before image acquisition.

Image acquisition and Image-based analysis

For intracellular assays performed in 384-well plates, image acquisitions were performed on an automated fluorescent confocal microscope (Opera, PerkinElmer), using a 20Xwater objective (NA 0.70). The confocal microscope was equipped with 405, 488, 561 and 640 nm excitation lasers. The emitted fluorescence was captured using 3 cameras associated with a set of filters covering a detection wavelength ranging from 450 to 690 nm. A series of 6 images was taken by well and each one was analysed using the imageanalysis software Columbus system (version 2.5.1, PerkinElmer). For the quantification of Mtb intracellular growth, cells (nuclei and cytoplasm) were detected by an intensity detection algorithm applied on the SYTO60 channel. A spot detection algorithm based on the GFP channel was applied for the detection of Mtb-GFP in cells and the bacterial intensity and area in pixels were measured. The intracellular bacterial growth was quantified by the total intracellular bacterial area (pixel) per well. For the study of intracellular PLGA-Rho NPs accumulation, cells were detected by an intensity detection algorithm applied on the DAPI (blue signal) channel. Spot detection algorithms based on the GFP (green signal) and PLGA-Rho (red signal) channels were used for the detection of Mtb-GFP and NPs, respectively, in cells. Images were analysed to determine the percentage of infected cells containing NPs in the cytoplasm area.

For the intracellular assays performed in 12-well plates to study the intracellular PLGA-Rho NPs accumulation, confocal images were acquired using a confocal microscope Zeiss LSM880 equipped with a 63x objective (NA 1.4), and Zen imaging software (Zeiss). Image analysis and generation of maximum projections of z-stacks was carried out by Fiji software (33).

Results analysis for intracellular bacterial replication assay

The read-outs, intracellular bacterial area and CFU, were normalized in comparison with the values obtained for the negative control (water value corresponding to 0% inhibition) and the positive control (INH value corresponding to 100% inhibition) and could be converted into a percentage of bacterial replication inhibition (% inhibition). % inhibition was calculated by the formula:

A plot of % inhibition versus the VCM concentration was determined using GraphPad Prism 5.0 software. The concentration required to inhibit 50% of the bacterial replication (IC 50 ) was calculated by nonlinear regression analysis using the equation for a sigmoidal dose-response curve with variable slope. As part of the CycloNHit consortium, different compounds and nanoparticles (NPs) synthesised by our partners were tested during this PhD, both against extracellular and intracellular bacteria using the fluorescent assays mentioned in the previous chapters. Cyclodextrins 6.1.

Cyclodextrins (CDs) were by far the most representative, with 89 CDs tested.

Due to the relatively high number of compounds tested simultaneously, a high throughput screening (HTS) platform available at the BioImaging Center of Lille was initially used. This platform contains different liquid dispensers, and plate handlers that allow a more precise and faster distribution of the compounds. In particular, the Echo liquid handler 550 (LabCyte) uses the technology ADE (Acoustic Droplet Ejection) that uses sound energy to transfer 2.5 nL droplets from source plates to destination plates, without using tips. The transfer is more accurate and precise than traditional dispensers, reduces the compound consumption and the final solvent concentration, which is a great advantage for cell assays with compounds dissolved in dimethyl sulfoxide (DMSO)).

However, for the dispensing of NPs I noticed many inconsistencies in the results, leading me to prefer doing all the serial dilutions of NPs manually.

From the total CDs tested 24 were neutral, 21 cationic, 32 anionic and 12 polymeric.

The structure of each one is confidential thus they are here named with code identification. From the 89 CDs tested, only 5 (6 % of the total) presented an IC50 bellow 1×10 -4 M, against extracellular Mtb. (Figure 15, first column). All of them were cationic.

The compound with the best extracellular efficacy was 3K with an IC50 of 1×10 -5 M, followed by 2G with 2×10 -5 M, 1G and 3G with 3×10 -5 M and 1K with 7×10 -5 M. Compound

Silver Nanoparticles 6.2.

One core-shell silver NP was also tested against extracellular Mtb. Silver NPs are well known for their antimicrobial activity. However, the tested particle presented no effect on bacterial growth at concentrations bellow 30 mM (data not shown).

Nanoporous Metal-Organic Frameworks 6.3.

Nanoporous Metal-Organic Frameworks (NanoMOFs) were also tested. Studies performed by Laleh Majlessi's group at Institut Pasteur Paris revealed that NanoMOFs induce significantly the autophagic marker light chain 3 (LC3) expression, with no significant toxicity found at the tested conditions. Results were obtained from flow cytometric analysis of the autophagy bio-marker LC3-II in bone marrow-derived dendritic cells (BM-DC) from C57BL/6 mice treated with NanoMOFs for 16 h. We confirmed this LC3 increased expression by fluorescence microscopy in non-infected RAW 264.7 macrophages after 16 h incubation with NanoMOF (same condition as our partner).

Using the LC3 marker, but also the polyubiquitin-binding protein P62/SQSTM1, another autophagy marker (Figure 16). We observed that incubation with NanoMOFs increase the number of LC3 positive cells and P62 positive cells, from 6 to 40 % and form 2 to 50 %, respectively (Figure 17 A). We could also see that LC3 and P62 spots colocalize (see arrows, Figure 16). Figure 17 B shows an example image of how the LC3 spots detection was performed.

These results lead us to hipothesize that NanoMOFs could induce autophagy, an immune defense mechanism that has been associated with increased Mtb killing [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF][START_REF] Lam | Role of apoptosis and autophagy in tuberculosis[END_REF]. Moreover, induction of autophagy has been claimed as a promising host-directed chemotherapeutic intervention in TB [START_REF] Gupta | Targeted pulmonary delivery of inducers of host macrophage autophagy as a potential host-directed chemotherapy of tuberculosis[END_REF]. Consequently, we decided to test the effect of NanoMOFs against intracellular Mtb replication in our intracellular in vitro assay.

In this case, the incubation of NanoMOFs with the cells occurred during 5 days, the time necessary to obtain significant difference between the compounds active and nonactive in terms of Mtb replication. The increase of incubation time resulted in dosedependent cytotoxicity, which increased Mtb replication, both in the infected and noninfected cells (Figure 18). Identification of the mechanisms and pathways of NanoMOF-induced cytotoxicity is out of scope in this thesis. However, we believe that autophagic cell death may be involved. Mycobacterium tuberculosis, the main etiological agent of TB, is one of the most successful human pathogens in history Mtb is an ancient and obligate human pathogen.

Contrarily to zoonotic infections, human infection and disease is essential for the evolutionary survival of Mtb, which, therefore, evolved complex strategies for persistence in the human host. Instead of classical virulence factors such as toxins and enzymes to poison host cells and to compete with other bacteria, Mtb initiates infection in the lower lung, which harbours few commensals and thus, can deal just with its host.

Mtb is able to survive and multiply inside host macrophages by subverting the effector functions of these important innate immune cells and can persist in the host in a dormant state for decades prior to progressing to active disease.

TB is a complex and dynamic disease that includes a wide spectrum of pathologies.

To treat this disease, multiple antituberculosis drugs with unpleasant side-effects must be taken for long periods, which discourage patients' adherence to treatment. Currently, the co-infection with human immunodeficiency virus (HIV) and the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections have further complicated TB treatment. Therefore, it is crucial to find faster, less harmful and moreefficient alternative therapies for both drug-susceptible TB (DS-TB) and drug-resistant TB (DR-TB), in order to improve the quality of life of TB patients and to tackle the TB burden.

Despite the increasing awareness to this threat, no major new types of antibiotics have been developed by pharmaceutical companies, over the last 50 years, mainly due to the high cost and time consumed to develop new drugs and low profitability expected. In recent years, only bedaquiline and delamanid have been conditionally approved. Even if only limited clinical trial data exist for these drugs and adverse events have been noted, they are already used when no other treatment options exist.

Combining the decline in efficacy of known active agents with the difficulty associated with the development of new drugs, we have reached a point where a "postantibiotic" era is a real possibility.

Considering this, it is worthwhile to investigate the development of new regimens using old TB drugs and to repurpose drugs used to treat other infectious diseases, for antituberculosis treatment. Many of these drugs present limited antituberculosis activity due to their solubility characteristics, low intracellular permeability, instability, low bioavailability, fast metabolism and excretion, etc. The use of nanoparticles (NPs) to reformulate existing antibiotic drugs may be a solution to overpass the unfavourable characteristics of some old drugs and enhance their efficacy, thus speeding up the entrance of alternatives to the market.

In this thesis, different antibiotics reformulated using NPs were evaluated for their ability to inhibit extracellular and/or intracellular Mtb replication.

The strain used in this thesis, Mtb H37Rv, is the most studied strain of Mtb in research laboratories and is the standard for TB studies. The drawbacks of using a virulent Mtb strain include the danger of manipulating a biosafety level three (BSL3) pathogen, which demands specific research facilities and extensive personal training, and the low growth rates of these strains, which slows down investigation progress. Among alternative models, the most popular are the attenuated M. bovis BCG and the avirulent M. smegmatis and M. marinum [START_REF] Shiloh | To catch a killer. What can mycobacterial models teach us about Mycobacterium tuberculosis pathogenesis?[END_REF]. M. bovis belongs to the MTBC, has similar growth rates compared to Mtb, yet is a biosafety level two (BSL2) pathogen. M. smegmatis exhibits much faster growth rates, but it is evolutionary different and lacks pathogenicity. M. marinum is a BSL2 pathogen with relatively fast growth and close evolutionary proximity to the MTBC and TB-like pathogenesis in ectotherms [START_REF] Cosma | Zebrafish and frog models of Mycobacterium marinum infection[END_REF]. Nevertheless, M. marinum has a lower temperature for optimal growth than Mtb. Therefore, Mtb is the most phenotypically accurate choice.

Antimycobacterial activity in axenic liquid cultures, here called extracellular activity, is a crucial characteristic that is frequently used in antitubercular drug discovery. Colony Forming Units (CFU) enumeration on specialized mycobacterial agar medium continues to be the "gold standard" for evaluating the effectiveness of antituberculosis compounds.

However, this technic is laborious, involves long incubation periods (3 to 4 weeks) and is prone to contamination. Several alternative in vitro assays are currently available for the quantification of bacteria replication. Among them is the resazurin reduction assay, the measurement of ATP levels using the BacTitre-Glo™ microbial cell viability assay, the measurement of oxygen consumption using the BD BACTEC™ or the MGIT™ and the spectrophotometric measurement of optical density (OD) [START_REF] Blanco-Ruano | Antimicrobial susceptibility testing for Mycobacterium sp[END_REF]. However, none of these methodologies is suitable for assessing the drug activity inside macrophages. In contrast, reporter strains that express either a fluorescent protein or a luciferase can be used for drug testing both in extracellular and intracellular assays.

Here, Mtb H37Rv strain constitutively expressing the green fluorescent protein (H37Rv-GFP) was used as a reporter for the replication assays. The amount of bacteria was quantified by acquisition of the GFP-fluorescence signal on a multimode reader for the extracellular assay.

Despite the relevance of extracellular antimycobacterial activity, successful chemotherapy against Mtb must also eradicate the bacterium within the context of its host cell, a key subpopulation of bacteria that are less readily eliminated by therapy.

However, several drugs with high extracellular activity show insignificant intracellular activity.

The reasons for this include low drug concentration achieved inside the phagosome where the bacteria reside, due to low membrane permeability, host metabolism and/or activities of molecular pumps [START_REF] Hartkoorn | Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein[END_REF]. Moreover, the host environment may have impact on drug action. During infection, the host cell imposes multiple pressures on intracellular M. tuberculosis, such as acidic stress, restricted nutrient availability, microbicidal peptides, and toxic reactive nitrogen and oxygen species [START_REF] Warner | The survival kit of Mycobacterium tuberculosis[END_REF][START_REF] Liu | Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo[END_REF]. Thus intracellular survival requires metabolic and physiological adaptations relative to extracellular growth.

Transcriptome analyses revealed that M. tuberculosis's response inside macrophages differs from that observed in standard broth culture, suggesting that, inside phagosome, M. tuberculosis switches its carbon source from glucose and glycerol to fatty acids, activate DNA and cell envelope repair and induce the production of secreted siderophores to facilitate the acquisition of iron, among other adaptations [START_REF] Schnappinger | Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment[END_REF]. Host-derived stresses negatively affect M. tuberculosis drug susceptibility [START_REF] Liu | Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo[END_REF] [START_REF] Aljayyoussi | Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration[END_REF].

Thus, to improve the efficiency of drug discovery process and to reduce identification of false positive hits that have poor translation to in vivo models, it is important to test also the efficacy of the drugs against intracellular M. tuberculosis, an environment that better reflects the common niche of this pathogen.

Usually, studies of M. tuberculosis growth within host cells in vitro rely on time consuming and labor intensive techniques, such as determination of CFUs or RLU titration, that are complicated by the long incubation periods required for forming a colony on solid media from a single M. tuberculosis [START_REF] Delorme | Testing chemical and genetic Modulators in Mycobacterium tuberculosis infected cells using phenotypic assays[END_REF]. The readouts from these methods measure the intracellular bacterial load but don't rigorously control the host cell viability in vitro. However, some infection conditions, such as the multiplicity of infection (MOI), can lead to intense replication resulting in severe host cell death. This condition results in low intracellular CFU counts and leads to the wrong conclusion of low M. tuberculosis replication, whereas in reality, much replication occurred.

Here, a phenotypic cell-based assay, developed in 2009 by Priscille Brodin's team [START_REF] Christophe | High content screening identifies decaprenylphosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors[END_REF][START_REF] Brodin | High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling[END_REF][START_REF] Queval | A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening[END_REF], was used for the screening of drugs and NPs that interfere with the replication of M. tuberculosis within macrophages. Advantageously, with this method it is also possible to follow the accumulation of NPs inside cells, (see chapter III) and to obtain additional information, such as the cytotoxicity of the studied compounds, (see chapter I, II and IV).

However, discrimination between bacteriostatic and bactericidal effects can only be achieved by determining CFUs.

Christophe et al. have demonstrated that the total pixels of green areas per field correspond to the quantity of the recombinant strain M. tuberculosis H37Rv-GFP and showed a linear relationship between the total green area and the actual number of bacteria by CFU counting [START_REF] Christophe | High content screening identifies decaprenylphosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors[END_REF]). More recently, the team reported the same validation study showing the correlation between mycobacteria fluorescence and the CFUs [START_REF] Queval | STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection[END_REF].

Similar high content imaging methods have now been successfully implemented in several laboratories for drug discovery purpose and there is more and more consensus that this technique is as reliable as CFUs for the quantification of bacterial load [START_REF] Stanley | Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth[END_REF][START_REF] Sorrentino | Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages[END_REF][START_REF] Manning | A high content microscopy assay to determine drug activity against intracellular Mycobacterium tuberculosis[END_REF].

Nonetheless, it is still often that, during manuscript revision, reviewers ask validation of this phenotypic assay and to compare the results obtained with different parameters.

The correlation between bacterial area versus bacterial area × fluorescence pixel intensity versus volume was done using a reference compound (rifampicin) (Supplementary Figure 3). The IC50 values were very similar and in the range of the values previously published for the three imaging measurements. Therefore, we consider that bacteria area is a valid parameter for the quantification of M. tuberculosis and determination of the IC50 of an antimycobacterial compound. Moreover, the comparison between the results obtained with the image-based method with the classical CFU counting method resulted in similar dose-response curves, confirming once again the reliability of this image-based method. Additionally, it was also demonstrated here that the 1 hour amikacin treatment step, present in the protocol since 2009, does not impact on the extracellular bacteria viability (Supplementary Figure 4). We concluded that the critical point to eliminate the extracellular bacteria is the repetition of washings while the 1 h incubation with amikacin could be avoided.

The present thesis showed that NPs may play different roles in different antituberculosis therapies.

In the chapter I of this thesis, biodegradable polymeric β-cyclodextrins (pCD), PLA and PLGA were studied as pulmonary delivery carrier for Ethionamide (ETH), an old second line antituberculosis drug, simultaneously with its booster BDM41906, here called Booster. The aim was to circumvent the bottlenecks related to the limited water solubility of the booster, the strong ETH tendency to crystalize and the difficulty to find a common solvent for ETH and Booster compatible with pulmonary administration.

In vitro studies on M. tuberculosis H37Rv in axenic conditions as well as in infected RAW 264.7 macrophages showed that the treatment with the developed NPs containing [ETH:Booster] pair was as potent as that with free [ETH:Booster] pair solubilized in organic solvents. Among tested formulations, the pCD NPs displayed the best physicochemical characteristics and were thus selected for in vivo studies.

The mouse model was chosen in this study because it is currently the most commonly used [START_REF] Cooper | Mouse model of tuberculosis[END_REF] and considered by almost every scientist in the TB field as an essential model for investigating novel TB drug regimen prior to clinical trials.

No major modification of the composition of lungs immune cell subsets was observed after administration of the vehicle (water) or NPs formulations to uninfected mice. These results suggest that the endotracheal administration of particles in the lungs using a Microsprayer device does not cause any major irritation effects.

Then developed pCD NPs loaded with [ETH:Booster] were administered via the endotracheal route to the lungs of M. tuberculosis-infected mice. The formulation was able to reduce the mycobacterial load using subtherapeutic doses in a short course of infection model. The recommended dosage of ETH is 15 -20 mg/kg daily. Here a lower dose was tested, 10 mg/kg, 3 times per week, due to limitations on: a) frequency of administrations using a Microsprayer that mice can tolerate; b) encapsulation efficiency. However, even using a lower dose and lower frequency, a significant decrease of the pulmonary bacterial load was observed as compared to untreated mice, using just 3 doses of the [ETH:Booster] pair within a 1 week administration. Increasing the treatment to 6 doses within 2 weeks resulted in an impressive decreased by 3-logs in the mycobacterial load in the lungs, as compared to the non-treated animals. In the future, it would therefore be interesting to investigate whether a longer course of treatment could lead to a greater efficacy. It would also be interesting to investigate the effect of this treatment in a mouse model of chronic TB.

Noteworthy, when the formulation containing [ETH:Booster] was given by oral gavage using the same regimen the mycobacterial load in the lungs was similar as the one from non-treated controls, clearly demonstrating the benefit of using the pulmonary route for ETH and Booster co-loaded nanoparticles delivery.

Our observations are in line with previous studies that reported that pulmonary administration increase the drug concentration in the lungs, with less systemic side effects and less administrations required to reach the same antituberculosis effect as oral route [START_REF] Pandey | Poly (DL-lactide-co-glycolide) nanoparticle-based inhalable sustained drug delivery system for experimental tuberculosis[END_REF][START_REF] Ahmad | Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis[END_REF][START_REF] Bivas-Benita | Pulmonary DNA vaccination: concepts, possibilities and perspectives[END_REF]. By oral route, the stability and bioavailability of the compounds may be decreased due to numerous gastrointestinal limitations such as acidic pH in the stomach, high degree of enzymatic degradation, reduced passage of the intestinal barrier, hepatic first-pass effect and transit time between the place of administration and the place of infection [START_REF] Morales | Challenges and Future Prospects for the Delivery of Biologics: Oral Mucosal, Pulmonary, and Transdermal Routes[END_REF].

Because the present approach drastically reduces the dose and the frequency of the treatment, it has the potential to diminish the systemic side effects of ETH. In humans, the instrument used for pulmonary delivery is usually self-administered without stress or pain, thus the effect of this formulation may potentially be greater because the administrations frequency can be higher.

In the future, this strategy could be used as a supplement to the standard treatment to contain the M. tuberculosis pulmonary manifestations and to prevent its dissemination. Therefore, this study opens new therapeutic perspectives for TB patients.

Moreover, given the fact that the current regimen for TB consists in a cocktail of four drugs, the approach used here could be extended to the encapsulation of more than two drugs, simplifying the treatment, decreasing the systemic side effects and increasing the patients' compliance to limit drug misuse.

In the chapter II, several types of Nanoporous Silica Particles (NSP), with different pore size, surface area and geometry, were studied as oral delivery carrier for Clofazimine (CLZ). CLZ is an extremely lipophilic antibiotic used today in a combination therapy for the treatment of leprosy. Due to its poor solubility in the gastro intestinal tract (GIT), the intestinal adsorption of CLZ is low, resulting in unfavourable oral pharmacokinetics. Nonetheless the promising in vitro data against MDR/XDR strains make CLZ a candidate for the treatment of human TB [START_REF] Cholo | Clofazimine: current status and future prospects[END_REF].

Encapsulation in the NSP particles stabilized the amorphous form of CLZ, which resulted in a significant increase in the dissolution rate in simulated gastric fluid and a dramatic increase in the intestinal permeation in comparison to the CLZ as free drug.

These results provided strong support that, in vivo, CLZ oral bioavailability can be enhanced by encapsulation in NSP.

When incubated with M. tuberculosis infected macrophages, CLZ released from NSP formulation in aqueous conditions matched the same antibacterial effect obtained from a fully dissolved concentration in organic solvent.

The concentration of CLZ dissolved from NSP, after permeation through Caco-2 monolayer was up to an order of magnitude higher than the MIC, indicating that the formulation of CLZ in NSP can lead to an enhanced permeation of CLZ to achieve the MIC after a single dose. Moreover, the NSP carrier, already generally recognized as safe for oral administration, was demonstrated here to show no cell cytotoxicity. Encapsulation of CLZ in NSP presents a new and simple refurbishment of a wellknown marketed antibiotic with bioavailability issues, for a new indication in the treatment of antibiotic-resistant M. tuberculosis infections.

In the chapter III of this thesis, vancomycin (VCM), the gold-standard treatment of methicillin-resistant staphylococcus aureus (MRSA), was encapsulated in PLGA engineered polymeric NPs. Though already demonstrated active against extracellular M. tuberculosis, this antibiotic has long been restricted to the treatment of critically ill patients, with drug-resistant infectious agents, particularly MRSA, in the tentative to avoid the development of resistant strains to this important antibiotic. Nowadays, the new options available for the treatment of MRSA and the lack of effective alternatives for the treatment of TB, makes the repurpose of VCM a valid strategy. Some factors affect VCM clinical activity, namely its poor intracellular penetration [START_REF] Lamer | Analysis of vancomycin entry into pulmonary lining fluid by bronchoalveolar lavage in critically ill patients[END_REF]Kollef 2007;[START_REF] Lehar | Novel antibody-antibiotic conjugate eliminates intracellular S. aureus[END_REF] . In contrast, phagocyte cells are well known for their ability to internalize drug-loaded NPs (Pelgrift and Friedman 2013;[START_REF] Ladaviere | Toward an optimized treatment of intracellular bacterial infections: input of nanoparticulate drug delivery systems[END_REF][START_REF] Costa-Gouveia | How can nanoparticles contribute to antituberculosis therapy?[END_REF][START_REF] Costa-Gouveia | Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles[END_REF]). Thus, encapsulation in PLGA NPs acted as "Trojan" horses to deliver VCM inside the macrophages.

Interestingly, the release of the drug could be controlled and was found to be pHdependent, with almost no release at neutral pH and fast delivery at pH 5.5. This behaviour is particularly interesting for the selective release of VCM in acidic compartments, such as when the NPs are taken up in endosomes with a slightly acidic pH (around 4.8) (Tan and Russell 2015).

Encapsulation of VCM in the developed PLGA NPs resulted in 5 times activity improvement compared to free VCM against intracellular M. tuberculosis growth. This supports the fact that VCM has low membrane permeability as previously reported [START_REF] Lamer | Analysis of vancomycin entry into pulmonary lining fluid by bronchoalveolar lavage in critically ill patients[END_REF][START_REF] Cruciani | Penetration of vancomycin into human lung tissue[END_REF]Kollef 2007;[START_REF] Lehar | Novel antibody-antibiotic conjugate eliminates intracellular S. aureus[END_REF]. It can be hypothesized that the observed intracellular antibacterial activity was driven by VCM released in the cells, from internalized NPs located in low pH endosomes, rather than the drug released prior to macrophage uptake. Indeed, NPs were readily taken up by infected cells and after 24 h incubation, practically all the macrophages contained NPs.

Besides more efficient, this "smart" drug delivery of VCM might also be less toxic than conventional drug delivery.

Over 100 years have passed since Paul Ehrlich postulate of creating "magic bullets" for a highly targeted medical treatment. Today, nanotechnology brings this idea closer to reality.

Another trend in the field of antituberculosis drugs is the search for synergic drug combinations, which can increase treatment efficacy and decrease drug dosage to avoid toxicity. Further studies will be dedicated to the encapsulations of synergic VCM/drug combinations to further improve the capacity of the NPs to kill intracellular Mtb. The combination of VCM and mycobacterial lipids synthesis inhibitors are among potential synergic combinations [START_REF] Soetaert | Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria[END_REF][START_REF] Rens | Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria[END_REF]).

In the chapter IV, the intrinsic antimycobacterial activity of different compounds and NPs was investigated. From 89 Cyclodextrins (CDs) tested, 5 were active against extracellular activity, with IC50 between 10 and 70 µM. However, no significant activity was observed against intracellular Mtb and all the compounds presented cytotoxicity.

One silver NP was also tested against extracellular Mtb without success. Moreover, NanoMOFs called our attention because of the observed induction of the autophagy marker LC3 expression, without signs of cytotoxicity, upon 16 h incubation with NanoMOFs. This led us to investigate if NanoMOFs could induce autophagy in host cells and if that plays a beneficial role against Mtb intracellular replication. However, after the 5 days of incubation necessary for the intracellular Mtb replication assay, NanoMOFs presented a cytotoxic effect. These results show the importance of varying incubation times while accessing toxicity of any NP.

Altogether, this thesis show that NPs engineered with distinctive compositions, sizes, shapes, and surface chemistries to enable a wide range of biological applications. In particular, the use of NPs as drug carriers may: i) allow alternative administration routes

  Intrinsèque des NPs. Différentes NPs (89 polymeric βcyclodextrines (pCD), 1 NP mésoporeuses hybrides (NanoMOF) et 1 NP en argent) ont été évaluées in vitro. Aucune n'a présenté d'activité antituberculeuse intrinsèque prometteuse.

Figure 1 - 3 Figure 2 - 4 Figure 3 - 5 Figure 4 - 6 Figure 5 - 10 Figure 6 - 12 Figure 7 - 14 Figure 8 - 15 Figure 9 -

 132435465106127148159 Figure 1 -Number of deaths caused by different infectious diseases during the last 200 years. From (Paulson 2013). ........................................................................................... 3 Figure 2-Desired decline in global TB incidence rates to reach the WHO 2035 targets. From (WHO 2017). ................................................................................................................ 4 Figure 3 -The remains of Nesparehan, a priest of Amun, in the 21 st Dynasty of the Ancient Egypt, typical of Pott's disease. a) big right psoas abcess; b) angular curvature of the spine. From (Said 2014) .................................................................................................. 5 Figure 4-TB clinical presentations. From (Pai, Behr et al. 2016) ................................... 6 Figure 5 -Pathogenic life cycle of Mtb. From (Cambier, Falkow et al. 2014) ............. 10 Figure 6 -Structure and cellular constituents of the tuberculous granuloma. From (Ramakrishnan 2012) .......................................................................................................... 12 Figure 7 -Risk factors for Mtb. ..................................................................................... 14 Figure 8-Estimated worldwide TB incidence rates in 2012, as determined by the WHO. From (WHO 2016). .................................................................................................... 15 Figure 9-Diagram of the basic components of the mycobacterial cell wall. Adapted from (Brown, Wolf et al. 2015). .......................................................................................... 17

Figure 10 - 19 Figure 11 - 20 Figure 12 -

 1019112012 Figure 10 -Map summarizing phylogeographic and dating analyses of MTBC. Major splits are annotated with the median value (in kya) of the dating of the relevant node. . 19 Figure 11 -Children with TB sleep outside at Springfield House Open Air School in London in 1932. Like sanatoriums, these schools offered TB patients a place to receive fresh air and sunshine cure. From (1932). .......................................................................... 20 Figure 12 -History of drug discovery and development of treatment regimens for TB.

21 Figure 13 -Figure 14 - 34 Figure 15 -Figure 16 -Figure 17 -Figure 18 -

 2113143415161718 Figure 13 -Global TB new drug pipeline and ongoing clinical development research. ............................................................................................................................................. 28

Figure 1 -

 1 Figure 1 -Number of deaths caused by different infectious diseases during the last 200 years. From (Paulson 2013).

Figure 2 -

 2 Figure 2-Desired decline in global TB incidence rates to reach the WHO 2035 targets. From (WHO 2017).

Figure 3 -

 3 Figure 3 -The remains of Nesparehan, a priest of Amun, in the 21 st Dynasty of the Ancient Egypt, typical of Pott's disease. a) big right psoas abcess; b) angular curvature of the spine. From[START_REF] Said | Orthopaedics in the dawn of civilisation, practices in ancient Egypt[END_REF]

Figure 4 -

 4 Figure 4-TB clinical presentations. From[START_REF] Pai | Tuberculosis[END_REF]

  . Of the PRRs, Toll-Like Receptor 2 (TLR2) recognizes the largest number of identified mycobacterial Pathogen-Associated Molecular Patterns (PAMPs), including lipoproteins, phosphatidylinositol mannans and lipomannan (Ernst 2012). This triggers various intracellular signaling cascades that leads to the production of proinflamatory cytokines such as Interleucin 1β (IL-1β), Tumor Necrosis Factor (TNF) and IL-12. This response leads to the recruitment of other immune cells. Neutrophils and monocytes are the first ones arriving, phagocytose additional 1. Introduction bacteria, secrete more cytokines and chemokines, and begin to organize the early granuloma, the hallmark of this disease. Mtb recognition leads also to different microbicidal mechanisms, such as the production of toxic effector molecules like Reactive Oxygen Species (ROS) or Reactive

Figure 5 -

 5 Figure 5 -Pathogenic life cycle of Mtb. From[START_REF] Cambier | Host evasion and exploitation schemes of Mycobacterium tuberculosis[END_REF]

Figure 6 -

 6 Figure 6 -Structure and cellular constituents of the tuberculous granuloma. From (Ramakrishnan 2012)

Figure 7 -

 7 Figure 7 -Risk factors for Mtb.

Figure 8 -

 8 Figure 8-Estimated worldwide TB incidence rates in 2012, as determined by the WHO. From (WHO 2016).

Figure 9 -

 9 Figure 9-Diagram of the basic components of the mycobacterial cell wall. Adapted from[START_REF] Brown | Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi[END_REF].

Figure 10 -

 10 Figure 10 -Map summarizing phylogeographic and dating analyses of MTBC. Major splits are annotated with the median value (in kya) of the dating of the relevant node.

  1948, researchers from the Britain Medical Research Council demonstrated that combined treatment with STR and PAS was superior to each drug alone and prevented the emergence of drug resistance (Z Ahmad 2011; Kerantzas and Jacobs 2017). These results provided the first evidence of the necessity for multiple drugs in treating TB to prevent development of drug resistance. INH, which remains the most potent drug against actively dividing tubercle bacilli, was discovered and brought into clinical use in 1952. In addition to its potency, INH is remarkably selective for mycobacteria (Chakraborty and Rhee 2015). It was initially

Figure 12 -

 12 Figure 12 -History of drug discovery and development of treatment regimens for TB. From (Ma, Lienhardt et al. 2010).

Figure 13 -

 13 Figure 13 -Global TB new drug pipeline and ongoing clinical development research.

Figure 14 -

 14 Figure 14 -Alternative activation of ETH with the use of SMARt-420. A: In susceptible Mtb, transcription of the enzyme ethA is only partially repressed by the transcription factor ethR, allowing some production of ethA and conversion of ETH to the NAD adduct, the active moiety that inhibits cell-wall synthesis; B: In ETH-resistant Mtb loss-offunction mutations in the gene encoding ethA results in no activation of the drug; C: Normally, ethA2 is completely repressed by ethR2. Adding SMARt-420, blocks the binding of ethR2 to DNA and allows high-level expression of ethA2, thus representing an alternative mechanism for the activation of ETH. From (Rubin 2017).

  4 millions de nouveaux cas et 1.8 millions de morts en 2015 selon l'Organisation Mondiale de la Santé (OMS). Cette maladie est causée par la bactérie Mycobacterium tuberculosis (Mtb) qui infecte principalement les poumons et qui se transmet par l'inhalation d'aérosols contaminés. Cette mycobactérie est capable de survivre et de se répliquer à l'intérieur des cellules phagocytaires, comme les macrophages pulmonaires. A l'heure actuelle, le traitement de TB nécessite la prise quotidienne de plusieurs antibiotiques, dits de première ligne, qui sont l'isoniazide (INH), l'éthambutol (EMB), la rifampicine (RIF) et la pyrazinamide (PZA), pendant 6 mois. Une mauvaise utilisation de ces antibiotiques, notamment liée à leurs effets secondaires indésirables, est en partie à l'origine de l'apparition de souches Mtb multi-résistantes (MDR) et nécessite alors l'utilisation d'antibiotiques de seconde ligne pendant 18 à 24 mois. La combinaison de plusieurs antibiotiques est alors associée à des effets secondaires sévères. De nouveaux médicaments sont nécessaires, mais, en dépit du fait que plusieurs molécules soient en cours d'étude, et certaines d'entre elles en essais cliniques, seules la Bedaquiline et Delamanid ont été approuvées pour le traitement conditionnel de patients atteints de TB résistantes (OMS, 2014 ; OMS, 2013). Dans l'attente de nouvelles molécules, il serait intéressant d'améliorer l'efficacité des traitements existants. Une stratégie prometteuse consiste à cibler directement Mtb au niveau des macrophages pulmonaires en administrant les composés antituberculeux directement au site d'infection par aérosolisation. De plus, il est indispensable de définir de nouvelles 1. Introduction approches pour réduire la durée et la toxicité des traitements et améliorer leur efficacité vis-à-vis des bactéries actives et latentes. Ainsi, l'encapsulation de composés antituberculeux dans des nanoparticules (NP) pourrait constituer une approche prometteuse pour le traitement de TB. Les NP sont déjà largement utilisées dans les domaines de l'agro-alimentaire et de la médecine, dont le domaine des maladies infectieuses. Dans ce contexte, notre travail avait pour principal objectif de présenter une bibliographie exhaustive sur l'utilisation des NP pour le traitement de TB, en croisant toutes les données disponibles de la littérature, afin d'avoir une vue globale sur cette application avant d'envisager de nouveaux développements. Ainsi, dans cette revue bibliographique, nous avons recensé plusieurs groupes à travers l'Europe (comprenant des universités, des entreprises, des instituts de recherche et des industries pharmaceutiques) qui ont unis leurs forces dans le cadre d'un consortium international pour utiliser les nanotechnologies pour le développement et la mise en oeuvre de traitements originaux contre les cas de TB multi-résistante. Ces données montrent essentiellement deux domaines d'application possibles des NP dans le cadre des traitements antituberculeux reposant sur: (i) leurs propriétés antimycobactériennes intrinsèques ; et (ii) leur utilisation comme vecteurs ou « nanocarriers » pour les médicaments antituberculeux connus, de façon à réduire les doses et les effets secondaires associés et permettre leur administration par voie orale ou pulmonaire. Des résultats prometteurs ont été obtenus dans des modèles d'infection in vitro et in vivo sur l'animal, et ont besoin à présent d'être évaluer en tant que médicament -candidat à l'échelle clinique. Ce travail bibliographique a été un point de départ extrêmement important pour dégager des pistes de nouvelles approches thérapeutiques antituberculeuses et nous a ainsi conduits à étudier quatre applications potentielles des nanoparticules: utilisées (i) comme nanocarriers pour les antibiotiques pour les administrer au niveau pulmonaire; (ii) pour améliorer la solubilité et la biodisponibilité des antibiotiques; (iii) pour la libération des antibiotiques dans les compartiments intracellulaires; et (iv) leurs propriétés antimycobactériennes intrinsèques. 1

# Affiliations: 1 :France 2 :

 12 University of Lille, CNRS, INSERM, University Hospital Center of Lille, Pasteur Institute of Lille, U1019 -UMR 8204 -CIIL -Center for Infection and Immunity of Lille, 59000 Lille, Departamento de Microbiología (Facultad de Medicina), and BIFI, Universidad de Zaragoza, Zaragoza (Spain); 3: Instituto de Investigación Sanitaria Aragón (IIS-Aragón), Zaragoza (Spain); 4: CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid (Spain) #Corresponding authors: Priscille Brodin (priscille.brodin@inserm.fr) Tel :+33 6 79 42 78 53

Figure 1 .

 1 Figure 1. Composition and properties of nanoparticles. (a) Composition of the different types of nanoparticles that have been tested for activity against M. tuberculosis, such as those encapsulating first or second-line anti-TB drugs, alone or in combination, as well as other potentially useful particles such as antimicrobial peptides, other therapeutic agents or small DNA fragments. (b) The main nanoparticle properties that can influence the uptake, distribution and efficacy of nanoparticle-based therapies are: nature of the polymer, size, surface charge, surface hydropathy, and targeting ligands. Source: Adapted, with permission, from [9].

  [34]. In this sense, formulations of TB drugs with NPs are being developed to increase their bioavailability, protect the drugs from degradation and release them in a time-and location-controlled manner. 1. Introduction Orally administered NPs must cross the GI mucus barrier, which in vivo has a considerable hydrophobic character due to the presence of lipids. Due to their nature and characteristics, lipid and hydrophobic polymer based NPs are the two dominant types of nanocarriers used for oral drug delivery[35]. But in addition to hydrophobicity/hydrophilicity, other characteristics like mucoadhesion, NP surface charge, or particle size are also important parameters to optimize for efficient oral drug delivery[36] and to improve absorption of the nanosystems in the GI tract.Several NPs have been already reported to be potent carriers for oral administration of antitubercular drugs and the resulting formulations were tested in animal models of TB infection (Table2). As one example, polymeric PLG (poly (lactide-co-glycolide)) loaded with first line drugs [RIF, INH, PZA and EMB], drug levels were maintained 4-5 days in plasma, and 7-9 days in the organs. For this combination of drugs in PLG NPs, 5 oral doses given every 10 days was sufficient

Figure 2 .

 2 Figure 2. Drug delivery systems for antituberculosis nanoparticles. Whereas pulmonary delivery (a) drives nanoparticles directly to the infected organ, the lungs, it is still challenging and requires use of nebulizers or inhalers. Delivery via oral administration (b) is simpler but it may encounter difficulties due to stability at acidic pH, absorption in the gastrointestinal tract, etc.

[ 48 ]

 48 . Most systemically administered drugs present poor pulmonary distribution and need to be administered in high doses to treat pulmonary TB[49].Stemming from the successful results obtained for the administration of antibiotics via the pulmonary route for the treatment of pulmonary infections in cystic fibrosis patients since several decades, pulmonary administration of antitubercular drugs has started to be investigated[START_REF] Porter | Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs[END_REF]. Inhalable antibiotics can be easily self-administered using appropriate devices (Fig.2). The use of this route of administration allows maximal drug delivery to the target site of infection, decrease in drug interactions in the gastrointestinal tract and escaping the hepatic first-pass metabolism [49,51,52]. Drugs delivered via pulmonary administration may also produce systemic effects. Lungs have a large surface area covered by a thin epithelium and high perfusion rates, allowing the transport of drug molecules into the systemic circulation [51]. As a consequence, drugs administered in the lungs could be detected in various other organs (liver, spleen and kidney) for up to 24 hours after nebulization [53]. In particular, chitosan NPs was studied for its ability to increase lung retention and residence time of antibacterial compounds [54]. Encapsulation of isoniazid in chitosan NPs improve the efficacy of the parent drug against an in vitro model of M. avium infection [55] and a mice model of M. tuberculosis infection [53]. Another important feature of NPs is that they can increase the selectivity for phagocytic cells, where M. tuberculosis usually resides. For instance, higher quantities of rifampicin-loaded solid lipid NPs were delivered of rifampicin into alveolar macrophages [56]. In addition, NPs may act as adjuvants enhancing the immune response. Conjugation of Ag85B antigen with pluronic-stabilized poly(propylene sulphide) NPs proved to improve antigen-specific CD4+ and CD8+ T cell activation when administered in combination with the adjuvant CpG via the pulmonary route [57]. They observed that NP-Ag85B and CpG administered via the pulmonary route led to enhanced immune responses and increased the protection of mice against TB, comparing with soluble 1. Introduction Ag85B with CpG and to formulation delivered via the intradermal route. These findings highlight the potential of NP-based formulations delivered through the pulmonary route for the development of novel TB vaccines.To explore the advantages of lung delivery, inhaled drugs have to be deposited in the deep respiratory airways, where M. tuberculosis is normally found. Many parameters influence this deposition, in particular the size, shape, porosity, density, electrostatic charge and aerodynamic diameter of the particles administered[START_REF] Costa | The formulation of nanomedicines for treating tuberculosis[END_REF]. For this reason, it is important to study the particles characteristics to evaluate their potential for pulmonary delivery[START_REF] Abdulla | Rehydrated lyophilized rifampicin-loaded mPEG-DSPE formulations for nebulization[END_REF]. Particles larger than 5 µm undergo deposition in the oropharyngeal region by inertial impaction and they are consequently swallowed[START_REF] Markopoulos | In-vitro simulation of luminal conditions for evaluation of performance of oral drug products: Choosing the appropriate test media[END_REF][START_REF] Andrade | Nanotechnology and pulmonary delivery to overcome resistance in infectious diseases[END_REF]. The optimal particle size for deposition in lower airways after inhalation is commonly accepted to be in the range of 1-3 µm, while particles smaller than 1 µm are believed to be exhaled[START_REF] Markopoulos | In-vitro simulation of luminal conditions for evaluation of performance of oral drug products: Choosing the appropriate test media[END_REF][START_REF] Andrade | Nanotechnology and pulmonary delivery to overcome resistance in infectious diseases[END_REF].However, studies in the area of environmental toxicology showed that particles smaller than 1 µm are also likely to be deposited in deep lungs by diffusion [61]. To render small NPs suitable for deep lung delivery in inhalable dry powder formulations [62], NPs loaded drugs could be further encapsulated in larger microparticles of size ranging from 1 to 5 µm. Alternatively, microparticles in solution may disaggregate in NPs [63].

  lead to lower CFUs in the lungs of M. tuberculosis infected mice in comparison to treatment of non-mannosylated gelatin particles and free drug [67]. As another example, a higher uptake in alveolar macrophages was found for mannosylated cationic nanostructured lipid carriers in comparison with non-mannosylated particles [68]. The coupling of loaded-NPs with other sugars, such as lactose, was also reported to enhance 1. Introduction the lung uptake of the drug and particles, in comparison with non-coupled particles and free drug [69]. Surface coating with polyethylene glycol (PEG) is also part of the active targeting strategies. In this case, it inhibits the recognition and phagocytosis by macrophages, prolonging the blood circulation of the drug-loaded NP and decreasing the requirement of repetitive doses [70]. Recently, mycobacterial cell wall mycolic acids were exploited as targeting ligands for drug-encapsulating poly lactic-co-glycolic acid polymer (PLGA) NPs [71]. The inclusion of mycolic acids in the nanocarrier resulted on a significant increase in the uptake of NPs and a higher frequency of fusion of NPs-containing phagolysosomes with mycobacterium-containing phagolysosomes [71].

  long duration of the TB treatment makes drug toxicity more relevant than for other short-term microbial infections, hence NPs could help in reducing doses, targeting specifically infected tissues and organs (mostly the lungs in pulmonary TB, the most frequent form of M. tuberculosis disease), and reducing toxicity issues. The cases described in this review have shown a wide diversity of NPs, containing or not additional drugs, having important antimycobacterial activity. These are a solid starting point, a promising guarantee, for thinking of a new future when treating TB, MDR-TB and/or XDR-TB with NPs could come true. If we ask ourselves, how can nanoparticles contribute to antituberculosis therapy, just think of the case of amphotericin B and you will get the answer: hope. 1. Introduction The European community has recently committed to support this area of research through the financing of several research consortia like CycloN-Hit, FORMAMP and NAREB. This very likely that exciting results will come out in the next coming years. échouent souvent à cause de concentration insuffisante de médicament au niveau des sites d'infection et de leur métabolisme et excrétion rapide. De plus, les voies d'administration de certains antibiotiques sont limitées et spécifiques, en raison de leurs caractéristiques de solubilité et de stabilité. En collaboration avec les différents partenaires du réseau de formation innovante "CycloN-Hit" constitué dans le cadre du programme européen d'Actions Marie Curie, cette thèse visait à développer des nanoparticules (NP) pour surmonter les problèmes précédemment mentionnés. Le but principal de cette thèse était d'étudier l'utilisation de nanoparticules comme nanocarriers pour administrer les antibiotiques directement au niveau des poumons, et améliorer la solubilité et la biodisponibilité, ainsi que la pénétration intracellulaire des antibiotiques. Cette thèse avait également pour objectif d'explorer l'activité anti-mycobacterienne intrinsèque des NP. Atteindre ces buts était important pour développer un nouveau concept de thérapie antibiotique contre TB capable d'atteindre l'agent pathogène dans sa niche de réplication et à combattre ainsi efficacement les cas de tuberculose (TB) sensibles et résistants aux antibiotiques.

  (ETH) est le principal antibiotique de seconde ligne utilisé pour le traitement antituberculeux, mais il est à l'origine d'effets secondaires sévères.

  . Ces boosters sont capables d'inhiber l'action d'ethR et ainsi d'augmenter l'expression d'ethA et la métabolisation de la prodrogue[START_REF] Blondiaux | Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420[END_REF].L'utilisation de ces molécules permet d'une part de diminuer la dose utilisée de l'ETH, réduisant ainsi la toxicité du traitement, et améliore d'autre part fortement son efficacité vis-à-vis des souches résistantes (Bivas-Benita,[START_REF] Bivas-Benita | Pulmonary DNA vaccination: concepts, possibilities and perspectives[END_REF]. Leur utilisation offre ainsi de nouvelles perspectives pour combattre les cas de TB résistants aux médicaments existants.Pour augmenter l'efficacité du traitement, une autre stratégie consiste à cibler Mtb principalement localisé dans les macrophages pulmonaires et à favoriser l'entrée les composés antituberculeux sous forme d'aérosols directement dans les poumons. Au vue des données bibliographiques, l'encapsulation de composés antituberculeux dans des nanoparticules (NP) constitue une approche prometteuse[START_REF] Costa | The formulation of nanomedicines for treating tuberculosis[END_REF].Dans cette étude, nous avons évalué l'effet de l'ETH et du booster BDM41906(Flipo, Desroses et al. 2012) ([ETH:Booster]) co-encapsulés dans des NP et administrés directement dans les poumons, pour le traitement de TB. Différents types de NP ont été préalablement sélectionnés sur la base des données bibliographiques: (i) les NP à base de copolymères d'acide poly(lactique-coglycolique) (PLGA) biodégradables et d'acide poly(lactique) (PLA), pour lesquelles une biocompatibilité et la biodégradabilité étaient bien documentées ; et (ii) les NP de poly-βcyclodextrine (pCD), présentaient également une biocompatibilté chez l'homme bien établie et l'avantage d'être bien tolérée in vivo grâce notamment au procédé 3. Chapter I d'encapsulation utilisé (dite « verte »), ne nécessitant pas l'utilisation de solvants organiques[START_REF] Gref | New self-assembled nanogels based on host-guest interactions: characterization and drug loading[END_REF][START_REF] Zuckerman | Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA[END_REF].Nos collaborateurs ont co-encapsulé les 2 composés dans ces différentes NP et caractérisés celles-ci d'un point de vue de la taille et de l'efficacité de l'incorporation de [ETH:Booster]. Cette encapsulation nous a permis d'une part de nous aguerrir des obstacles majeurs liés à la cristallisation de l'ETH et la faible solubilité du booster dans l'eau. De façon intéressante, les NP de poly-β-cyclodextrine (pCD) permettent notamment l'incorporation en une seule étape de [ETH:Booster]. Pour étudier l'efficacité de différentes formulations de [ETH:Booster] coencapsulés contre Mtb, nous avons réalisé au sein de notre laboratoire 2 essais phénotypiques, qui consistaient à comparer l'effet de NP chargées, des NP vides et du couple [ETH:Booster] non encapsulés, sur la croissance (i) extracellulaire et (ii) intracellulaire de Mycobacterium tuberculosis (dans des macrophages murins infectés par Mtb), grâce à l'utilisation d'un système de microscopie confocale à haut contenu automatisé (OPERA-PerkinElmer). Dans les deux essais, nous avons constaté que les médicaments conservaient leur activité après encapsulation et que les NP n'étaient pas cytotoxiques.

Figure

  Figure de résumé: Administration pulmonaire de nanoparticules de poly-β-cyclodextrines chargées avec l'ethionamide et un de ses boosters (BDM41906) dans des macrophages infectés avec Mtb.

  Fig. S1.

  the decreasing of DL and EE (P1 and ETH concentration of 0.71 mg/mL) during the first 8 days after NPs formulation. The formation of crystals was observed after 5 days and corresponds to a further decrease in NPs DL and EE. ETH crystallisation in P1 NPs was avoided only at ETH concentrations below 0.71 mg/mL, which is insufficient for in vivo applications. Attempts to concentrate the NPs suspensions failed because of a strong aggregation in the absence of protective surfactants. -DL% and EE% decrease during storage. DL and EE decreased during first days of storage leading to the formation of crystals at the bottom of the flask (represented by the yellow star in the figure) and to a decrease of both DL and EE.

  No crystallization was observed during storage and no significant size or polydispersity changes (DLS) were found.

  for 60 seconds at 25°C by NTA (NanoSight LM10, Malvern Instruments S.A., Worcestershire, UK, equipped with a 565 nm filter and a NanoSight syringe pump). NPs prepared by nanoprecipitation were diluted 2,000 times and the ones made by nanoemulsion 10000 times. Results are reported as Mean diameter ± SD.Cryogenic transmission electron microscopy (cryo-TEM)5 µl of NPs suspensions (diluted 4 times in water) were deposited onto a 200 mesh holey copper grid (Ted Pella Inc.) and flash-frozen in liquid ethane cooled down at liquid nitrogen temperature. Cryo-TEM images were acquired on a JEOL 2200FS energy-filtered 3. Chapter I (20eV) field emission gun electron microscope operating at 200 kV using a Gatan ssCCD 2048 × 2048 pixels. Several thousands of images were automatically acquired for each NP formulation to obtain representative results.Drug sample preparation for drug(s) dosageTo determine the amounts of ETH and Booster incorporated in PLGA NPs, 500 µL of NP suspensions were centrifuged at 17,000g for 15 minutes. Then, 200 µL of the supernatant were withdrawn to assess the quantity of non-encapsulated drug (indirect estimation). The NPs pellet was dissolved in 250 µL DMSO and dosed (direct estimation of the encapsulated drug). For the pCD NPs, aliquots of pCD NPs containing drug(s) were centrifuged at 5000 rpm for 10 min to remove unloaded drug(s). The supernatant containing the pCD NPs was then diluted with DMSO (1:1 volume ratio) and dosed by HPLC after dilution in DMSO and/or with acetonitrile/water 45/55 v/v.

  H37Rv strain constitutively expressing the green fluorescent protein (H37Rv-GFP) was used as a reporter for the replication assay. Bacteria were cultured at 37 °C for 16 days in complete 7H9 medium containing 0.5 % glycerol (50405, Euromedex), 10% Middelbrook oleic acid-albumin-dextrose-catalase (OADC) (211886, Becton Dickinson), 0.05% Tween 80 (2002A, Euromedex) and 50 µg/mL hygromycin B (10687010, Invitrogen). In the day of the experiment, M. tuberculosis were washed with D-PBS Ca-Mg-(14190169, LifeTechnologies) 3 times with centrifugation at 5,000 RPM for 5 minutes and centrifuged at 700 RPM for 2 minutes to remove clumped bacteria. Bacteria were resuspended in RPMI-1640 + glutamax (61870044, LifeTechnologies) containing 10 % heat inactivated fetal bovine serum (FBS) (10270106, Gibco) and titrated by measuring the optical density at 600 nm. Mouse macrophage RAW 264.7 (ATCC # TIB-71) were maintained at 37°C in RPMI-1640 + glutamax containing 10% FBS and were passed 3 times per week and used before passage number 7. Macrophages were harvested by using Versene (15040033, LifeTechnologies). 3. Chapter I Assay plate preparation ETH, BDM41906 and INH were diluted in DMSO (34943, Sigma-Aldrich) to 10 mg/mL and were dispensed in Echo-qualified 384-well low dead volume source plates (Labcyte).

FACS

  buffer were used to stain 3-5x106 cells. Acquisitions were done on FACScantoII cytofluorometer (Becton Dickinson) with the following mAbs from BD Biosciences: Fluorescein (FITC)-coupled HL3 (anti-CD11c), FITC-coupled 145-2C11 (anti-CD3), APCcoupled RB6-8C5 (anti-GR1), phycoérythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APC-coupled BM8 (anti-F4/80). APC-eF780-coupled M1/70 (antiCD11b) was purchased from eBiosciences and fixable viability dye eFluor 506 (eBiosciences) was used to gate viable cells. 3. Chapter I Efficacy studies 8-week mice (4 mice per group) were inoculated with M. tuberculosis H37Rv via the intranasal route to 10 6 bacteria in the lungs at day 0. On day 7, 9 and 11 after infection, NPs or INH were administered to mice using a Microsprayer® (MicroSprayer® Aerosolizer -Model IA-1C-M and FMJ-250 High Pressure Syringe, Penn Century Inc., Wyndmoor, PA).

  Ainsi l'utilisation d'antibiotiques déjà utilisés pour le traitement d'autres maladies, et pour lesquels aucune résistance bactérienne n'a été observée, pourrait constituer une stratégie pertinente pour accélérer le développement de nouvelles formulations thérapeutiques, et notamment pour la lutte contre la tuberculose multi-résistante et latente.Par exemple, la Clofazimine (CLZ), un antibiotique découvert au cours des années 1950 et utilisé dans le traitement de la lèpre, pourrait être un candidat potentiel pour le traitement de la tuberculose (TB). L'efficacité de CLZ a déjà été montrée in vitro sur des souches de Mycobacterium tuberculosis multi-résistantes, avec une fréquence extrêmement basse de résistance à cet antibiotique(Yawalkar and Vischer 1979;[START_REF] Reddy | Antimycobacterial activities of riminophenazines[END_REF]. Cependant, son utilisation pour le traitement de TB n'a pas été envisagée jusqu'à présent, en raison de la variabilité de son activité thérapeutique observée dans différents modèles animaux(Van Rensburg, Anderson et al. 1997;[START_REF] Mukherjee | Programmes and principles in treatment of multidrugresistant tuberculosis[END_REF] Van Deun, Maug et al. 2010;[START_REF] Cholo | Clofazimine: current status and future prospects[END_REF]. De plus, son administration par voie orale chez l'homme a également montré des concentrations d'antibiotique inadaptées et une dose totale dans le plasma insuffisante pour obtenir une activité bactéricide contre M. tuberculosis[START_REF] O'connor | The pharmacology, metabolism, and chemistry of clofazimine[END_REF][START_REF] Janulionis | Lack of Activity of Orally Administered Clofazimine against Intracellular Mycobacterium tuberculosis in Whole-Blood Culture[END_REF]).

  le volume des pores, ont été étudiées. Nos collaborateurs se sont chargés de la caractérisation physico-chimique de CLZ encapsulée dans les différentes PSN, ainsi que d'étudier sa stabilité après encapsulation, sa solubilité dans un milieu mimant le fluide gastrique, ainsi que sa toxicité et sa perméabilité dans des cellules intestinales modèles. Dans notre étude, l'utilisation de particules de silice nanoporeuses a permis de stabiliser l'état amorphe de CLZ (pendant plus de 6 mois) et d'augmenter jusqu'à 20 fois la solubilité de l'antibiotique dans le liquide gastrique modèle, la vitesse de dissolution étant fonction de la morphologie et de la texture des PSN utilisées. Dans notre laboratoire, nous avons étudié l'effet in vitro de différentes PSN sur des macrophages modèles infectés par Mtb. L'effet des PSN chargées, des PSN vides et de l'antibiotique non encapsulé sur la croissance intracellulaire de Mtb ont été évalués par utilisation d'un système de microscopie confocale à haut contenu automatisé (OPERA-PerkinElmer). Les résultats ont montré de façon intéressante que l'encapsulation de CLZ dans des PSN avait amélioré considérablement la solubilité de CLZ et sa perméabilité cellulaire, en atteignant des concentrations antimicrobiennes efficaces sur Mtb dans des macrophages infectés. Pour la première fois, nous avons réussi avec succès à reformuler CLZ dans des PSN. Cette étude montre que l'utilisation de CLZ, un antibiotique bien connu et commercialisé pour le traitement d'autres maladies, combinée à un mode d'administration basé sur l'utilisation de nanotechnologies, peut constituer une stratégie pertinente et originale pour s'attaquer aux infections de TB résistantes aux antibiotiques.

5 Affiliations: 1 :

 51 mesoporous silica particles for the oral treatment of antibiotic resistant M. tuberculosis infections Authors list: Sabrina Valetti**, 1,2, Xin Xia 1 , Joana Costa-Gouveia 3 , Priscille Brodin 3 , Marie Françoise Bernet-Camard 4 , Margareta Andersson 1 & Adam Feiler* 1,Nanologica AB, SE-151 36 Södertälje, Sweden 2: Biofilms -Research Center for Biointerfaces, Department of Biomedical Sciences, Faculty of Health & Sciences, Malmö University, Sweden 3: Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur deLille, U1019 -UMR 8204 -CIIL -Center for Infection & Immunity of Lille, F-59000 Lille, France 4: EA4043 Unité BactériesPathogènes et Santé (UBaPS), Univ. Paris-Sud, Université Paris-Saclay, 92296, Châtenay-Malabry Cedex, France 5: Surface and Corrosion Science, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden *Author for correspondence: adam@nanologica.com **Author for correspondence: sabrina.valetti@outlook.com Keywords: amorphous • Caco-2 cells • clofazimine • drug carrier • HPLC • intramacrophage • in vitro assay • nanoporous silica particles • oral drug delivery • tuberculosisIntroductionThe worldwide spread of antibiotic-resistant microorganisms is no longer a prediction for the future, but it is now a major threat to worldwide public health.(WHO 2015)Currently, three of the top ten causes of death worldwide are from infectious diseases.In 2013, there were around 480 000 new cases of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) identified in 100 countries.[START_REF] Who | Fact sheet N°194 -Antimicrobial resistance[END_REF] The antibiotic therapy is the pillar of the modern medicine, enabling routine medical and surgical procedures to be effective and successful with the protection of antimicrobial treatment.(Nathan and Cars 2014) However, unconstrained overprescription, misuse and poor patient adherence to the treatment regimen has caused the systematic and uncontrolled evolution of drug-resistant bacteria. Despite the increasing awareness to this threat, over the last 30 years, no major new types of antibiotics have been developed by pharmaceutical companies mainly due to the persistent, costly failure to discover novel antibiotics for short-term treatment therapies.(Nathan 2004) The cost for development of antibiotic therapy, which necessarily needs to be produced in high volumes and sold at a low price, and for a short treatment regimen does not induce commercial incentive for pharmaceutical companies.A solution that can synergistically improve and expedite the pharmaceutical development of antibiotics is to use novel advanced excipients to reformulate existing antibiotic drugs to enhance their efficacy and improve their performance. Refurbishment of clinically tested pharmaceutical drugs that have not acquired bacterial resistance offers a route to dramatically speed up the development time for new therapeutic formulations. In this context, clofazimine (CLZ), has great potential as a candidate for the treatment of human tuberculosis (TB).(Cholo, Steel et al. 2012) CLZ is an extremely lipophilic riminophenazine antibiotic, used today in a combination therapy for the treatment of leprosy, marketed under the trade name Lamprene. CLZ was discovered in the 1950s and originally investigated as an anti-TB drug. It is impressively active in vitro against MDR/XDR strains with extremely low frequency of drug resistance and good safety profile.(Yawalkar and Vischer 1979; Browne, Harman et al. 1981; Reddy, O'Sullivan et al. 1999; van Ingen, Simons et al. 2010) However, its use to treat human TB has, so far, been hampered mainly because of its inconsistent therapeutic activity in various animal models.(Van Rensburg, Anderson et al. 1997; Mukherjee, Rich et al. 2004; Van Deun, Maug et al. 2010; Cholo, Steel et al. 2012) Oral administration in humans showed inadequate peak drug concentrations and insufficient total dose in plasma to obtain bactericidal activity against M. tuberculosis. (O'Connor, O'Sullivan et al. 1995; Janulionis, Sofer et al. 2004) The unfavorable oral pharmacokinetics of CLZ is well-known in leprosy treatment, where a study of daily administration of dosages of 100, 300 or 400 mg CLZ resulted in average plasma levels of 0.7, 1.0 and 1.41 mg/L, respectively.(Yawalkar and Vischer 1979) The intestinal adsorption of CLZ is low due to its poor solubility in the gastro intestinal tract (GIT) and the majority of the unabsorbed CLZ is largely excreted unchanged in the feces.(Cholo, Steel et al. 2012) Nonetheless the promising in vitro data and focus on improved pharmaceutical formulations to offer a defense against the acute need to tackle TB infection has rekindled interest in novel drug delivery carriers that can improve the CLZ pharmacokinetic profile for its use in MDR-and XDR-TB. There are a few examples of nanotechnology-based formulation for the delivery of antibiotics that have proven effective in vitro and in vivo for treating or preventing infectious diseases.(Huh and Kwon 2011; Semiramoth, Di Meo et al. 2012; Abed and Couvreur 2014) However, despite enormous expectation surrounding the nanomedicine field, rapid translation from research into clinically approved products is hindered due to regulatory issues and limited long-term safety and toxicity data.(Bawa 2013; Crist, Grossman et al. 2013). The anticipated rapid adoption of nanotechnology-based formulation has suffered from the paucity of knowledge relating to the biological effect of carrier materials with unknown, but potentially adverse side effects, from the carrier material at the nanometer size range. In this context, mesoporous silica-based particles (NSPs) have emerged as an attractive excipient with desirable properties for the delivery of oral drugs.(Mamaeva, Sahlgren et al. 2013) NSPs comprise pure amorphous silicon dioxide (SiO 2 ), and analytically indistinguishable from colloidal silicon dioxide, which has Generally Regarded As Safe status from the US FDA (Food and Drug Authority) for use its use in food and as an excipient for many dosage forms (oral capsules, suspensions, and tablets; transdermal, rectal, and vaginal preparations). The Environmental Protection Agency has determined amorphous silica to show no toxicity by oral administration and therefore is exempt from requirement of tolerance status.(Agency September 1991) Our previous studies in vitro and in vivo have demonstrated how the encapsulation and confinement of pharmaceutical drug molecules inside the internal nanoporous structure of the NSP stabilize the molecule in its amorphous form, leading to enhanced solubility and bioavailability.(Xia, Zhou et al. 2012; Kjellman, Xia et al. 2014; Xia, Pethe et al. 2014) Motivated by this finding, the aim of this work was to encapsulate CLZ into NSPs in order enhance its solubility to improve its bioavailability in oral dosage formulations. A selection of different porous silica particles were used, differing in particle size, pore size and pore volume. A full physicochemical characterization of the CLZ encapsulated in the NSP, in vitro dissolution in simulated gastric fluid (SGF) and intestinal permeation as well as the antimicrobial activity in TB-infected macrophages were performed in order to investigate the potential benefit in refurbishment CLZ in oral TB treatment.

  H37Rv strain constitutively expressing the Green Fluorescent Protein (GFP) was used as a reporter for the replication assay. Bacteria were cultured at 37°C for 16 days in complete 7H9 medium containing 0.5% glycerol (50405, Euromedex, Souffelweyersheim, France), 10% middelbrook OADC enrichment (211886, Becton Dickinson, Le Pont-de-Claix, France), 0.05% Tween 80 (2002A, Euromedex) and 50 μg/mL hygromycin B (10687010, Invitrogen, ThermoFisher, Illkirch, France). On the day of the experiment, M. tuberculosis were washed with Dulbecco's phosphate-buffered saline Ca-Mg-(14190169, LifeTechnologies) three-times with centrifugation at 5000 rpm for 5 min and centrifuged at 700 rpm for 2 min to remove clumped bacteria. Bacteria were resuspended in RPMI-1640 + GlutaMAX (61870044, Life Technologies) containing 10%

  5 and made up to a volume of 0.25 L with DMEM. The FaSSIF-V2 DMEM-maleate buffer was sterilized with Vacuum Filter/Storage Systems, Sterile, Corning® (VWR, Fontenay-sous-Bois cedex, France) and kept in the fridge until used. The final composition of the biorelevant transport media are given in Supplementary Table

After 1

 1 hour (average gastric emptying time in fasted state), aliquots(16.67 µL) were diluted to 2.5 mL with the FaSSIF-V2 transport media and then this solution was transferred in the apical compartment of the Transwell. At the same time, 2.8 mL of DMEM (without phenol red) with 1% of BSA was put in the basolateral side. The permeation experiments were performed from apical to basolateral direction. At each endpoint, aliquots (200 uL) from the basolateral side was taken and replaced with the same amount of fresh basolateral buffer in order to keep the volume constant. Aliquots were extracted in MeOH (800 uL) and then centrifuged at 14000 rpm for 20 min. 4. Chapter II Recovery test was performed in order to investigate the best procedure for CLZ extraction in presence of BSA. Samples were stored at -18ºC until HPLC analysis. At the end of the experiment, the integrity of the Caco-2 monolayer was evaluated by measuring the TEER and the transport of sodium fluorescein (1 mg/ml). When a TEER change was recorded more than the 20% of the initial value, the TEER recovery was tested after overnight incubation in cell culture medium. Sodium fluorescein concentration in the basolateral side was measured by UV spectrophotometry at 490 nm.

Figure 1 -

 1 Figure 1 -Scanning electron microscopy images of the mesoporous silica particles: a) NFM-1, b) SBA-15, c) ASP277 and d) ASP734

Figure 2 -

 2 Figure 2 -Low angle X-ray diffraction patterns of a) NFM-1; b) SBA-15; c) typical pattern for ASP277 and ASP734.

Figure 3 -

 3 Figure 3 -Solid-state characterization by differential scanning calorimetry of CLZ (crystals -pure drug), CLZ after acetone evaporation, CLZ with NSP as physical mixture (pm) and CLZ-loaded NSPs. In the physical mixtures, CLZ was solubilized in CLZ: Clofazimine; NSP: Nanoporous silica particle.acetone and mixed with NSP (with similar % wt to the respective CLZ-loaded NSP sample) without loading procedure.

Figure 4 -

 4 Figure 4 -Dissolution release profile in simulated gastric fluid of clofazimine and clofazimineloaded nanoporous silica particles. Experiment was made in triplicate. Figure shows the typical release profile. CLZ: Clofazimine.

Figure 5 -

 5 Figure 5 -Intestinal permeability test. Accumulative amount of clofazimine transported through Caco-2 cell monolayer from the apical to the basolateral side as a function of time. The insert shows extended permeation study for 360 min. Results are expressed as mean ± standard deviation (n = 3). In the graph, the 0% of accumulative clofazimine transported is referred to the limit of detection of the HPLC technique (<0.237%). CLZ: Clofazimine.

Figure 6 -

 6 Figure 6 -Number of viable macrophage cells (top) and bacterial area (bottom) after treatment with CLZ dissolved in DMSO (black circle), empty NMF-1 (gray triangle) or CLZ encapsulated in NFM-1 (purple square) in the intramacrophage in vitro assay. Experiment was done in triplicate. CLZ: Clofazimine.

  , VCM a été encapsulée dans différentes NP à base de PLGA, d'un polymère de polyéthylène glycol -PLGA ou d'un dérivé de chitosan. De façon intéressante, les NP de 500-1000 nm, et contenant jusqu'à 9.5 % VCM (poids), ainsi générées, étaient capables de libérer l'antibiotique de manière dépendante de la valeur de pH. En revanche, la méthode de préparation était complexe et exigeait l'utilisation de quantités précises de plusieurs ingrédients.Dans notre étude, nous proposons d'encapsuler VCM dans des nanoparticules (NP) à base PLGA, dans le but de stabiliser VCM et de délivrer l'antibiotique directement dans les macrophages infectés.Nos collaborateurs ont caractérisé les nanoparticules ainsi obtenues et montré par cryomicroscopie électronique de transmission (cryo-TEM) de grandes inclusions dans les NP contenant vraisemblablement l'antibiotique. La préparation des NP a permis de charger jusqu'à 14.5 % (poids) de VCM par NP. Ces NP ont pu être conservées plus de trois mois sans diminution significative de la quantité d'antibiotique encapsulée. Et de façon intéressante, il a été montré que VCM était libérée à pH 5.5, mais pas à pH neutre, ni dans les milieux de culture de cellule.Au sein de notre laboratoire, nous avons étudié l'effet in vitro de VCM encapsulée ou non sur la croissance intracellulaire de Mtb dans des macrophages murins infectés.L'utilisation de la microscopie confocale (système de microscopie confocale à haut contenu automatisé OPERA-PerkinElmer) nous a permis de montrer que les NP étaient efficacement phagocytées par les macrophages infectés. 73 et 99 % des cellules contenaient des NP après une incubation de 2 et 24 heures, respectivement. Et, alors que VCM présente une faible stabilité dans les milieux biologiques, le traitement avec VCM encapsulée dans ce type de NP a été 5 fois plus efficace sur les bactéries Mtb intracellulaires qu'un traitement avec l'antibiotique libre. L'encapsulation de cet antibiotique à l'intérieur de NP à base de PLGA a donc amélioré 5 fois l'efficacité de l'antibiotique.En conclusion, l'encapsulation de VCM dans des NPs à base de PLGA pourrait constituer une nouvelle application dans le traitement de TB.Ces nouvelles données montrent également la capacité des NP à base de PLGA à encapsuler de quantité importante d'antibiotique et nous permet d'envisager de poursuivre une autre étude qui visera à encapsuler des combinaisons synergiques de VCM et d'autres antibiotiques, et notamment avec des inhibiteurs de la synthèse des lipides mycobactériens qui ont déjà montré un effet synergique potentiel[START_REF] Soetaert | Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria[END_REF][START_REF] Rens | Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria[END_REF].Ce travail devrait contribuer au développement de nouvelles thérapies antituberculeuses efficaces et améliorer le traitement actuel pour d'autres maladies infectieuses causées par des bactéries Gram positives. against intracellular Mycobacterium tuberculosis " Tuberculosis (TB), which is mainly caused by Mycobacterium tuberculosis (Mtb), is still killing 1.8 million people annually. Although the TB death rate has decreased, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections is highly worrisome. Nowadays, the lack of effective alternatives for the treatment of TB makes the repurpose of known drugs such as vancomycin (VCM) an attractive strategy.Indeed, recent studies highlight the potential of VCM, alone or in combination with other drugs, to treat MDR-TB.

  Poly (D,L-lactide-co-glycolide) (PLGA 50:50 acid terminated (MW: 5-20 KDa, 10P019) was kindly provided by PCAS (Expansorb, Aramon, France). Vancomycin hydrochloride (VCM) was kindly provided by the Centre International de Recherche en Infectiologie (Lyon, France). Poly (vinyl alcohol) (PVA) (88% hydrolyzed), Rhodamine B (Rho), 4-Dimethylaminopyridine (DMAP), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), piperazine, trimethyl aluminium (AlMe 3 ), sodium chloride, dichloromethane, trifluoracetic acid, hydrochloric acid, potassium chloride, sodium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate and endotoxin-free water were all obtained from Sigma-Aldrich. Dichloromethane (DCM-Analar Normapur) was from Prolabo. Injectable water was purchased from Cooper (Melun, France). The solvents were analytical grade.

Figure 1 .

 1 Figure 1. Schematic representation of the preparation of VCM-loaded PLGA NPs using a double emulsion (w1/o/w2) method. CryoTEM images of PLGA NPs loaded with VCM (14.5 wt%).

Figure 2 .

 2 Figure 2. Release of VCM from PLGA NPs containing 14.5 wt% VCM, prepared in the presence of NaCl. The NPs were diluted 10 times in PBS at pH 7.4 (blue), PBS at pH 5.5 (green) and in cell culture total media (containing serum) (red). All the samples were incubated at 37°C degree under gentle stirring.

Figure 3 .

 3 Figure 3. PLGA-Rho NPs inside RAW264.7 macrophages infected with Mtb H37Rv-GFP. (A) Representative images of cells containing PLGA-Rho NPs (red) and Mtb H37Rv-GFP (green), at the indicated time points, obtained by image acquisition using an automated confocal microscope. (B) % of cells containing PLGA-Rho NPs after RAW264.7 macrophages infection with Mtb H37Rv-GFP calculated by image analysis using the software Columbus. Data are presented as mean ± SEM. (C) Orthogonal view of an infected RAW264.7 cell imaged by confocal microscopy. F-actin was labelled by phalloidin (gray), and nuclei were stained by DAPI (blue). Shown is one z-stack in xy, xz and yz dimension. Scale bar = 5 μm.

Figure 4 .

 4 Figure 4. Intracellular antitubercular activity. (A) Scheme representing the steps performed to analyse activity of VCM (grey triangle) and PLGA VCM (black square) against Mtb H37Rv-GFP inside RAW264.7 macrophages. (B) % of inhibition on intracellular bacterial area detected by automated confocal microscopy and measured by the image-analysis software Columbus. (C) % of inhibition on intracellular bacterial growth measured by CFU counting.

  1K and 3K presented also a slight inhibition of intracellular Mtb replication (Figure15, second column). Compound 2G and 3G didn't present any clear effect against intracellular Mtb replication, while compound 1G had a negative effect, by stimulating intracellular replication. All the compounds presented cytotoxicity (Figure15, third column).

Figure 15 -

 15 Figure 15 -Extracellular and intracellular activity of the compounds with best antitubercular activity tested. First column: % of inhibition on bacterial RFU; Second column: % inhibition of intracellular bacteria area; Third column: Number of cells in the intracellular assay.

Figure 16 -Figure 17 -

 1617 Figure 16 -Fluorescence microscopy. RAW 264.7 cells were plated 24 h before addition of NPs. After incubation with the NPs during 16 h, cells were fixed, stained with DAPI (blue) and immunostained with LC3 (red) and P62 (yellow). Arrows indicate colocalization in LC3 and P62 spots. Scale bars: 20 µm.

Figure 18 -

 18 Figure 18 -Dose-response induction of cytotoxicity in non-infected cells (NI) and in cells infected with Mtb H37Rv-GFP (green) with MOI 2. Cells were stained with Syto 60 (red). INH was used as positive control and H2O was used as negative control.

7 .

 7 DISCUSSION & PERSPECTIVES/DISCUSSION & PROSPECTSThe development of the world's first antibiotic, penicillin, in 1943, was one of the most important advances in the history of medical science. Antibiotics have revolutionised modern medicine by making previously incurable infections treatable and protecting patients undergoing common surgical procedures from bacterial infections. As a result, countless lives have been saved over the last 70 years.However, despite the development of modern medicine, tuberculosis (TB) is now the world's most deadly infectious disease and is on the top 10 causes of death worldwide.

Table 1 -

 1 WHO recommended antituberculosis drugs and actions.............................. 24

	xix

  .

	(XDR) is defined as MDR with additional resistance to at least one fluoroquinolone
	(ofloxacin, levofloxacin, gatifloxacin, moxifloxacin) and a second line injectable drug
	(amikacin, capreomycin, and kanamycin) (W.H.O. 2016).
	The treatment of MDR-or XDR-TB is complex, expensive, lengthy, and uses drugs
	with uncertain efficacy and characterized by high toxicity, which correlates with lower
	patient adherence and increases the probability of treatment failure and development of
	even more resistance to antibiotics. Treatment success rate is only 52 % for MDR TB and
	28 % for XDR TB (W.H.O. 2016).
	Currently, the standard therapy for new-cases of drug-susceptible TB has a high
	success rate of 83 % (W.H.O. 2016) but require taking antibiotics daily during long time. It
	consists of a combination of the first-line drugs INH and RIF for at least 6 months and PZA
	and EMB during the first 2 months (Pai, Behr et al. 2016).
	Despite the existence of an effective treatment, drug resistance has continued to
	grow. TB resistant to at least INH and RIF is termed MDR. Extensively Drug-Resistant
	1. Introduction

Table 1 -WHO recommended antituberculosis drugs and actions. Drug Mechanisms of action Activating enzyme/ Gene target Group

 1

	Isoniazide (P)	Inhibit production of the cell wall component mycolic acid	katG, inhA	First-line D1 (high dose)
	Rifampicin	Inhibit RNA synthesis	rpoB	First-line
	Ethambutol	Inhibit production of the cell wall component arabinogalactan	embB	First-line D1
	Pyrazinamide (P) Disrupt plasma membrane	pncA, rpsA	First-line D1
	Rifabutin	Inhibit RNA synthesis	rpoB	Alternativ e to RIF
	Levofloxacin	Inhibit DNA gyrase and topoisomerase IV	gyrA/B	A
	Moxifloxacin	Inhibit DNA gyrase and topoisomerase IV	gyrA/B	A
	Gatifloxacin	Inhibit DNA gyrase and topoisomerase IV	gyrA/B	A
	Kanamycin	Inhibit protein synthesis	rrs	B
	Amikacin	Inhibit protein synthesis	rrs	B
	Capreomycin	Inhibit protein synthesis	rrs tlyA	B
	Streptomycin	Disrupt protein synthesis	rpsL, rrs, gidB	B
	Ethionamide (P)	Inhibit production of the cell wall component mycolic acid	ethA, inhA	C
	Linezolid R	Disrupt protein synthesis	23S rRNA, rplC	C
	Clofazimine	Interfere with redox	Rv0678	C
	Cycloserine	Inhibit production of the cell wall component peptidoglican	cycA, alrA	C
	Bedaquiline (TMC207)	Inhibits ATP synthase	atpE	D2
	Delamanid (P)	Inhibit production of the cell wall component mycolic acid	ddn	D2
	p-aminosalicylic acid	Interfer in the process of folate synthesis	thyA, folC	D3
	Imipenem plus cilastatin			D3
	Meropenem			D3
	Amoxicilin plus clavulanate			D3
	Thiocetazone			D3
	(P): Pro-drug
	Known chemical classes are colour coded:

fluoroquinolone, rifamycin, , oxazolidinone nitroimidazole, diarylquinoline, aminoglycoside, cyclic peptide, pyridine, aminosalicylate, riminophenazine.

Table 1 . Nanocarriers with antimycobacterial activity.

 1

	Material (+load)	Features	Refs
	AgNPs	In vitro antimycobacterial activity	[13, 20, 21]
	AgNPs + cationic peptides	Antimycobacterial activity in vitro and in infected	[22]
		macrophages
	AgNPs	Selection of resistant mutants	[23]
	GaNPs	More efficacy in vitro and in vivo than free gallium	[26]
		In vivo targeting NPs to the macrophages
	AuNPs	In vitro antimycobacterial activity	[27]
	FeNPs + SM	In vitro antimycobacterial activity	[28]
	PAA covered with Fe oxide	Efflux inhibition. Sinergy with RIF, INH and norfloxacin	[29]
	EWNS	In vitro activity	[33]

Abbreviations: AgNPs: silver nanoparticles; GaNPs: gallium nanoparticles; AuNPs: gold nanoparticles; FeNPs: iron nanoparticles; SM: streptomycin; PAA: polyacrylic acid; RIF: rifampicin; INH: isoniazid; EWNS: engineered water nano-structures.

  AuNPs), and lower than the bimetallic ones (Au-Ag) despite the mechanism of action of each metal is still under investigation. Similarly, polyacrylic acid (PAA)-coated iron oxide NPs produces an inhibition in the efflux of drugs in M. smegmatis, resulting in synergistic effect of these NPs with rifampicin, isoniazid and norfloxacin[29]. Remarkably, this only happens when using the stabilized, coated NPs, and not the PAA or iron NPs alone. So a new use of metallic coated NPs as efflux pumps inhibitors can be considered.

	Other strategies like combining the effect of biogenic-AgNPs and antimicrobial
	cationic peptides are being investigated [22], and are revealed as a promising
	antimycobacterial strategy.
	Certain types of NPs, like any other antibiotic, may carry the risk of developing
	resistance. After exposure to AgNPs, Mycobacterium smegmatis populations resistant to
	AgNPs and AgNO3 were obtained, and they were cross-resistant to antibiotics as well,
	although no cross-resistance was observed to other inorganic compounds like CuSO4 and
	Although gold (Au) has been reported to be less active against mycobacteria than
	silver, thiol conjugated gold NPs show high activity against M. smegmatis [27].

development

[19]

. A study showed that AgNPs synthesized by synthetic agents present greater bacterial inhibition

[20]

, although AgNPs synthesized from medicinal plantsderived reducing agents also presents antimycobacterial activity

[21]

. In both studies it is reported that the antimycobacterial activity of AgNPs is higher than that of gold

NPs 1. Introduction (ZnSO4 [23]

. This suggests that general resistance mechanisms such as efflux pumps could be mediating resistance to these materials, which needs to be properly addressed.

Besides its in vitro antibacterial properties, AgNPs have been reported to suppress innate responses of monocyte-derived macrophages in response to M. tuberculosis infection

[24]

. This ability of NPs to modulate pathogen-induced immune responses must be further taken into account when one wants to fully understand the impact of NPs in in vivo models of infection. Gallium (Ga) is a metal very similar to iron (Fe), which is essential for the metabolism and growth of M. tuberculosis. Substituting Ga for Fe in the active site of enzymes may render them non-functional, and this is a potential approach for novel antituberculous therapy. Gallium has been reported to present antimicrobial activity against mycobacteria

[25]

, showing efficacy in murine tuberculosis models where M. tuberculosis CFUs in the lungs, liver, and spleen of mice decreased when they were treated with gallium nitrate. Nanoformulating the gallium, (GaNP), a sustained release of the metal was obtained, achieving a prolonged action. GaNPs were more efficient than free gallium, and the particles could be targeted to the macrophages, where inhibited growth of M. tuberculosis for up to 15 days following single drug loading

[26]

.

Covering iron NPs (FeNPs) with a polysaccharide (chitosan) to load in its surface an antibiotic like streptomycin, produces a new type of magnetic nanocomposite with various properties as a potential tool to both diagnose and treat tuberculosis infections 1. Introduction [28].

Table 2 . Nanocarriers as antimycobacterial drug delivery systems.

 2 Another example worth to mention is the use of PLGA for the encapsulation of levofloxacin with promising results in terms of sustained release and levels of the drug in the organs[43]. Results in mice reveal 21-fold increase in the bioavailability with respect to the intramuscularly injected streptomycin. In this case, 8 weekly doses are comparable to 24 injections of free streptomycin. Same drug has been loaded in another

			Drug	Nanocarrier	Achievement	Refs
			RIF/INH/PZA/EMB PLG	Extend drug levels in plasma and lungs Decrease doses in mice and guinea pigs	[38-40]
			RIF/INH/PZA	WGA coated PLG	Improve absorption Decrease doses in guinea pigs	[35] [41]
			ECO/MOXI	WGA coated PLG	Decrease doses in mice	[42]
	delivery	LEVO	PLGA	Sustained release and increase level of drugs in the organs	[43]
	Oral	SM	PLG	Oral delivery of an injectable drug Increase bioavailability in mice	[44]
			SM	chitosan	Oral delivery of an injectable drug with same efficacy	[45]
			RIF	gelatin	Better biodistribution and higher blood levels in mice	[46]
	Pulmonar	delivery	INH/RIF RIF	chitosan solid-lipid NPs	Improve in vitro and in vivo efficacy Higher RIF levels into alveolvar macrophages vs epithelial cells	[53-55] [56]

Abbreviations: RIF: rifampicin; INH: isoniazid; PZA: pyrazinamide; EMB: ethambutol; PLG: poly (lactide-coglycolide); WGA: Wheat germ agglutinin; ECO: econazol; MOXI: moxifloxacin; LEVO: levofloxacin; PLGA: poly lactic-co-glycolic acid; SM: streptomycin. and as efficient in clearing TB infection as 46 daily doses of free drug in mice

[37,38]

or guinea pigs

[START_REF] Johnson | Oral therapy using nanoparticle-encapsulated antituberculosis drugs in guinea pigs infected with Mycobacterium tuberculosis[END_REF]

. These PLG NPs were able to sterilize the lungs, liver, spleen and also in the meninges

[40]

and this was attributed to improved pharmacokinetics and bioavailability of the drugs upon encapsulation. Further functionalization of these PLG NPs with lectin contributed to improve the relative bioavailability of the drugs. In particular, wheat germ agglutinin (WGA) was used to model NPs in order to improve the comparable with 45 doses of oral free drugs

[41]

. The same type of material has been used to encapsulate other drugs like econazol and moxifloxacin with similar therapeutic 1. Introduction results after oral delivery in mice

[42]

.

type of carrier, the polysaccharide chitosan. These particles led to a reduction of one log in growth of bacilli, being as effective as injected streptomycin in aqueous solution at the same concentration

[45]

.

Table 3 : Functionalized nanocarriers for antimycobacterial drug delivery

 3 Introductionincidence of the disease and the increasing drug resistance of the microorganisms causing these diseases.When searching for alternative therapeutic approaches to treat TB, we should consider the use of NPs as a promising and worthwhile option, given the lessons we have learnt with fungal infections and amphotericin B liposomal formulations. Indeed, from the nineties, nanoformulations of amphotericin B (a powerful antifungal drug, but having severe toxic side effects) are available[START_REF] Voltan | Fungal diseases: could nanostructured drug delivery systems be a novel paradigm for therapy?[END_REF]. These are based on liposomal NPs, and indeed have been successful in retaining drug activity while reducing considerably their toxic effects.

	Coating	Drug	Nanocarrier	Achievement	Refs
	Mannose	INH	gelatin	Reduce bacteria in TB infected mice Reduce hepatotoxicity of the drug	[67]
	Mannose	RIF	Cationic lipids	Higher uptake efficiency by alveolar macrophages	[68]
	Lactose	RIF	PLGA	Increase lung uptake	[69]
	PEG	MOXI	PLGA	Prolonged blood circulation	[70]
	Mycolic acids	INH	PLGA	Uptake increase and fusion with lysosomes containing mycobacteria	[71]

Abbreviations: INH: isoniazid; RIF: rifampicin; PLGA: poly lactic-co-glycolic acid; PEG: poly ethylene glycol; MOXI: moxifloxacin. 1.

Supplementary Table S2 -Main characteristics of empty and drug-loaded NPs made of P4 by the nanoemulsion method

  . NP mean hydrodynamic diameters were determined by PCS and NTA. Nanoparticles dry diameter was determined by cryo-TEM and nanoparticle concentration was obtained by NTA. Concentration results given were obtained after 10,000 times dilution of each sample as prescribed to fit the instrument requirements and to allow the particle to move freely according to their Brownian motions.NPs formulated with the nanoprecipitation method were remarkably stable upon storage more than 5 months, with less than 10 % size variation and a PdI still lower than 0.1. The size variation over a period of 1 month is shown in the Supplementary Fig.S4

				Mean diameter		Concentration
	Formulation	D PCS Z Average (nm ± SE)	PdI PCS	D PCS (nm ± SE) Number	D NTA (nm± SD)	Cryo-TEM	NTA (particle/mL)
	NPs	254 ± 4	0.054	220 ± 10	176 ± 45	110 ± 50	1.01x10	9
	NPs [ETH]	267 ± 3	0.074	226 ± 10	176 ± 48	100 ± 40	0.88x10	9
	NPs [ETH:Booster] [1:0.1]	258 ± 3	0.058	223 ± 10	180 ± 51	100 ± 50	1.03x10	9
	NPs [ETH:Booster] [1:1]	274 ± 4	0.090	235 ± 11	178 ± 52	100 ± 50	0.89x10	9
	NPs [Booster]	277 ± 5	0.075	238 ± 11	171 ± 49	120 ± 55	0.91x10
							3. Chapter I

Table 1 -Surface areas calculated by Brunauer-Emmett-Teller (BET), pore diameters and pore volumes according to Barrett-Joyner-Halenda adsorption model.

 1

	Sample	Average pore width (nm)	BET Surface Area (m 2 g -1 )	Pore volume (cm 3 /g)
	NFM-1	2.37 small	770.34 high	0.4294 low
	SBA-15	6.29 medium	600.23 medium	0.763 medium
	ASP277	28.40 high	277.27 low	1.563 high
	ASP734	14.29 medium	733.89 high	1.914 high

Table 1 . Main characteristics of VCM-loaded PLGA NPs. Effect of NaCl on the incorporation of VCM.

 1 Stability upon storage at 4°C.

Table 1 . Main characteristics of VCM-loaded PLGA NPs. Effect of NaCl on the incorporation of VCM. Stability upon storage at 4°C. NaCl (wt%) DL (wt% ± SD) Loading efficiency (% ± SD) Mean diameter (nm) ± SD PdI ± SD VCM release 3 month storage 4⁰C (%± SD)

 1

	0%	9.6 ± 0.9	24.0 ± 2.4	320 ± 30	0.190 ± 0.05	4.1 ± 1.4
	1%	14.5 ± 0.5	36.6 ± 1.6	310 ± 59	0.226 ± 0.08	2.0 ± 0.9

Nous avons étudié l'efficacité de ces NP chargées de[ETH:Booster] in vivo chez des souris infectées avec Mtb par voie intranasale. La suspension de NP a été délivrée sous forme d'aérosols directement dans les poumons par voie endotrachéale à l'aide d'un Microsprayer® Aerosolizer. Une réduction significative de la charge bactérienne dans les poumons de 3 log a été observée après 6 administrations de doses inférieures à celles thérapeutiques.En conclusion, nous avons proposé une approche thérapeutique originale combinant l'utilisation de molécules boosters et de nanotechnologies pour améliorer les traitements existants de la tuberculose.

Chapter I

Chapter II

Chapter II area). Small (NFM-1) or large (ASP277) pore sizes resulted in slower kinetic release

Chapter III

7.Discussion & Prospects

7.Discussion & Prospects that wouldn't otherwise be possible; ii) change the physicochemical properties of the drugs, such as solubility, stability and consequent bioavailability and iii) allow controlled release of drugs under specific environmental conditions. Though much work is still needed to fully understand the long-term effects of human exposure to NPs, the information that we already possess suggest that NPs may open new perspectives in antituberculosis therapy and improve the life quality of the patients.

Acknowledgements / Remerciements .......................................................................... ix Résumé .......................................................................................................................... xi Abstract........................................................................................................................ xiii Acknowledgements The Universidad de Zaragoza is a partner in the European funded consortium NAREB (Nanotherapeutics against resistant emerging bacterial pathogens) (FP7 grant 604237). Pasteur Institute of Lille is a partner in the European ITN Network "CycloN Hit" (FP7-PEOPLE-2013 n° 608407) and the ANR-TB-NANO (ANR-14-CE08-0017). P. Brodin is recipient of ERC-STG INTRACELLTB Grant n° 260901. Both teams acknowledge support from the More Medicines for Tuberculosis (MM4TB) consortium (Grant n°260872). We thank Ruxandra Gref and Arnaud Machelart for critical reading of the manuscript. 3. Chapter I according to their Brownian motion. More than 2000 NPs are followed in each experiment. Acknowledgments We gratefully acknowledge Ok-Ryul Song, Isabelle Ricard, Alexandre Vandeputte, Rosangela Frita, Xue Li, Alexandra Pastor, Sabrina Brown, Grant Kiely and Alain Baulard for technical assistance and helpful discussions. We acknowledge the PICT-IBiSA and 5. Chapter III Frank Lafont from BiCEL for providing access to microscopy equipment. We are grateful to Sylvain Trepout for cryoTEM experiments. Financial support for this work was provided by the European Community (CycloN Hit n° 608407, ERC-STG INTRACELLTB n° 260901, MM4TB n°260872), the Agence Nationale de la Recherche (ANR-10-EQPX-04-01, ANR-14-CE08-0017, ANR-16-CE35-0009), the Feder (12001407 (D-AL) Equipex Imaginex BioMed) and the Région Nord Pas de Calais (convention n° 12000080). This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (Labex NanoSaclay, ANR-10-LABX-0035).

Abstract

Therapeutic approaches using nanoparticles are being successfully used in foods and in several fields of medicine, including infectious diseases. Regarding TB treatment, nanoparticles may be a useful strategy for two distinct applications: (i) for their intrinsic antimycobacterial activity; (ii) as vehicles for known antitubercular drugs to allow reduction of dose and drug-associated side effects, and administration via user-friendly administration routes such as pulmonary or oral ones. Promising results were obtained in vitro and in animal M. tuberculosis models and need now to be translated into clinical drug candidates. Such a prospect may provide an opportunity to the current limited therapeutic options for drug resistant TB and to the scarcity of novel antituberculosis drugs in the drug discovery pipeline.

Conflicts of interest

The authors declare that they have no competing financial interest. 3. Chapter I Supplementary Figure S5-Columbus script used for image analysis. Two colour-images were analysed. The red recorded on the channel "Exp1Cam3" was used for the nucleus and cytoplasm detection. The green recorded on the channel "Exp1Cam2" was used to detect the bacteria. ROI:

Region of interest.

In Supplementary Fig. S6 we point out that the % of inhibition in the presence of empty PLA and empty pCD is negligible (close to zero) independently from the concentration of the NPs, both in the intra-and extracellular assay. These results show that the antibacterial effect observed is due to the presence of ETH and Booster released from the nanoparticles since the unloaded nanoparticles were not able to impair bacterial replication.

Define Results

Calculate Morphology Properties (2)

Output Properties : Bacteria

Find Spots

Output Population : Spots

Calculate Intensity Properties

Output Properties : Intensity Spot Exp1Cam2 France). KCl (99%) was purchased from Alfa Aesar (Karlsruhe, Germany).

Select

The Booster named BDM41906 (5,5,2,piperidin-1-yl] pentan-1-one) was synthesized as previously described [START_REF] Flipo | Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1, 2, 4-oxadiazole EthR inhibitors[END_REF]).

β-CD was kindly supplied by Roquette, Lestrem, France. pCD NPs of around 10 nm were produced as previously described [START_REF] Gref | New self-assembled nanogels based on host-guest interactions: characterization and drug loading[END_REF][START_REF] Othman | A comprehensive study of the spontaneous formation of nanoassemblies in water by a "lock-and-key" interaction between two associative polymers[END_REF]) by crosslinking β-CD under strongly alkaline conditions with epichlorohydrin

9.Supplementary information

For the 3D analysis, pictures of 65 sequential z-stacks interspaced by 0.5 µm were recorded and analysed using the Imaris software. This allowed quantification of the bacterial volume (Supplementary Figure 2). Bacterial area or bacterial area × mean fluorescence intensity or bacterial volume versus the rifampicin concentration (Log10. Scale) was plotted with GraphPad Prism 5.0 software and the IC50 was calculated by nonlinear regression analysis using the equation for a sigmoidal dose-response curve with variable slope (Supplementary Figure 3).

The IC50 obtained with the analysis of bacterial area, bacterial area × mean fluorescence intensity and bacterial volume was, respectively, 3.8×10 -7 , 2.5×10 -7 , 4.1×10 -7 g/mL. These IC50 values are very similar and in the range of the values previously published. Therefore, we consider that the three imaging measurements are valid for the quantification of M. tuberculosis and determination of the IC50 of an antimycobacterial compound.

It is clear that the 3D-analysis is currently time-consuming due to the limitation of the Imaris software. Indeed the quantification of the bacterial load in the 3D-pictures have to be done field by field, whereas that on the 2D-images can be done on multiple plates, thus for drug discovery purposes we consider that analysis of 2D-images is a better option.

Supplemetary

9.Supplementary information

Amikacin Treatment: Is it necessary? 9.2.

The combination of judicious centrifugations and amikacin treatment was introduced in the protocol in 2009 due to a reviewer request to allow optimal removal of extracellular bacteria. The correct elimination of extracellular bacteria was confirmed when we subcultured the supernatant of the last wash observing the absence of all mycobacteria that were not internalized during the infection step (Christophe et al., 2010). However, we are fully aware that the amikacin alone is not sufficient for killing the extracellular bacteria. To answer to this question, we incubated a suspension of 2×10 6 bacteria/mL with 50 µg/mL amikacin during 1h in the same conditions as the assay (37°C, 120 rpm) and ten-fold serial dilutions were plated onto 7H11 agar plates supplemented with 10 % OADC. We did not observe any differences in the CFU of treated and nontreated bacteria (Supplementary Figure 3).

We conclude that the critical point to eliminate the extracellular bacteria is the repetition of washings, 3 washings in total, and that the 1 h incubation with amikacin could be avoided. Nevertheless, we kept this step in the procedure since it is generally considered as an important step for the standardization of HTS/HCS phenotypic assays related to M. tuberculosis infected cells, which was published in Mycobacteria Protocols book [START_REF] Delorme | Testing chemical and genetic Modulators in Mycobacterium tuberculosis infected cells using phenotypic assays[END_REF].

Supplemetary Figure 4 -Effect of 1 h treatment of 2×10 6 H37RV-GFP/mL with 50 µg/mL amikacin.