Katie Buxton

Sylvie Bylent

Marta Bolsa-Ferruz

Xiaoming Yang

Gabriela Moran

Rym Boudjemaa

Ala Husseen

Albert Leo

Abdoul Sala

Mohammad Zakaria

Ammoun

Dorine Bonte

Gabriel Siracusano

Keywords: Nanoparticles, Cyclodextrins, Biodegradable polymers, intracellular bacteria, drug encapsulation, AFM-IR

Gianluca Cattinari for sharing the everyday lab-life with me

"Science means constantly walking a tightrope between blind faith and curiosity; between expertise and creativity; between bias and openness; between experience and epiphany; between ambition and passion; and between arrogance and conviction -in short, between an old today and a new tomorrow."

Heinrich Rohrer

Firstly, I would like to thank the European Community for providing the financial support to my PhD study which was performed in the frame of the European Project ITN "Cyclon Hit" (Grant no 608407). I would like to express my deep gratitude to all the members of "Cyclon Hit" group for the stimulating environment and the fruitful scientific discussions. I particularly thank all my colleagues Cyclon Hit fellows who shared with me this highly enriching as well as challenging experience.

At the end of this three years of PhD I would like to thank the whole ISMO (Institut de Sciences Moléculaires d'Orsay) and its director Dr. Bernard Bourguignon for making me feel welcomed and supported.

But, my deepest sense of gratitude goes to Dr. Ruxandra Gref, CNRS research director and my PhD supervisor, for giving me the chance to be a part of Cylon Hit project and to live these very stimulating three years. It was an honour to be directed and inspired by her passion for science. She was incredibly patient and supportive, especially during the writing of this manuscript. In particular, I thank her for sharing with me her talent in scientific writing, her ideas, enthusiasm and impressive knowledge. Heartful thanks goes also to my lab mates at ISMO. Firstly, Dr. Li Xue for sharing this thesis experience with me and for her friendship; Dr. Mario Menendez-Miranda for helping me, not only with his competence, but Antimicrobial resistance (AMR) is one of the biggest health threats that human kind is facing nowadays and that will have to face in the next decades 1 . An increasing number of drug-resistant bacterial strains are reported every year and the phenomenon is expected to further grow, leading to alarming healthcare and economic consequences. AMR is foreseen to cause 1 million deaths/year by 2050 and to generate an economic burden of 100 trillion US dollars 1 . On this basis, US as well as Europe are committed in implementing the number of social and economic plans and assets dedicated to face this threat. Europe is founding an increasing number of projects proposing innovative solutions to tackle this global crisis. This PhD research is an example of such European commitment as it has been carried on in the frame of the European Project ITN "Cyclon Hit", which aims at developing nanocarriers for the delivery of antimicrobial agents as a strategy to fight resistance mechanisms. In particular, this research project was dedicated to an especially challenging and important goal, the treatment of intracellular infections. Intracellular bacteria possess the ability to "hide" inside the cells of the host immune system, transforming them in their preferred habitat and thus escaping from the action of many antibiotics 2,3 . This project proposes the use of nanocarriers as a promising tool to efficiently entrap antibiotics, protecting them from degradation, overcoming their limitations such as low solubility, inability to cross cell membrane and unfavourable biodistribution, possibly reducing their toxic side effects and hence increasing patient's compliance.

This PhD work focused more specifically on the development, optimisation and advanced characterisation of biodegradable and biocompatible polyester-based and cyclodextrin (CD)-based nanoparticles (NPs) for antimicrobial drugs encapsulation. The main polyesters used in this study were poly(lactic acid) (PLA), poly(lactic-co-glycolic) (PLGA) and poly(ε-caprolactone) (PCL) which are all approved by FDA for pharmaceutical applications [4][5][6] . Both polyesters and CDs are among the most employed materials for the preparation of drug delivery systems with tailorable properties. The success of these two carrier systems lies in their ability to improve the physico-chemical characteristics and biodistribution of antibiotics and to disassemble in non-toxic units, which are easily cleared from the body.

A first part (Introduction) will give a literature overview on the background of this research and its related challenges. It is thus focused on intracellular bacteria and AMR and gives a summary on the structure, safety profile and applications of the two biodegradable NPs systems listed above and on the main techniques used for their characterisation as well as for their intracellular in vitro localisation.

Afterwards, the first two chapters are centered on the main results obtained in the development, optimisation and characterisation of such polymeric NPs encapsulating challenging antimicrobial drugs.

• Firstly, PLA NPs and crosslinked CD (pCD) NPs were employed for the co-encapsulation of ethionamide (ETH), a second-line anti-TB drug, and a molecule improving its activity, named Booster. This potent recently discovered drug combination 7,8 opened tremendous opportunities in tuberculosis (TB) treatment by increasing the therapeutic activity of ETH. However, this drug pair presented some limitations, mainly related to the difficulty to find a common solvent, much less a biological-compatible one, to their completely different biodistribution profiles and to the strong tendency of ETH to crystallise. These limitations greatly hampered the possibility to co-administer this drug pair in an effective way. The main pharmacological purpose and challenge of this study consisted in the co-encapsulation of these drugs to allow their administration directly in the infection site, overcoming the technological barriers of the free molecules. Once the co-encapsulation was achieved, an in-depth characterization of PLA NPs systems was conducted by using complementary advanced techniques (Chapter I -Section A). The main advantage provided by pCD NPs was the encapsulation of both drugs at a molecular level, thus totally avoiding ETH crystallization. To complement this study, a combination of spectroscopic techniques was employed to unravel the type and strength of interactions occurring between the ETH and pCD NPs (Chapter I -

Section B).

Both drug-loaded PLA and pCD NPs were tested in vitro on extracellular and intracellular bacteria by means of an automated high-content screening platform. Then their compatibility with the Microsprayer®, the device for in vivo endotracheal administration, was assessed. One of the main achievements in this work consisted in the evaluation of pCD NPs safety profile after repeated administration of high doses of such carrier. Then the antibacterial activity of loaded pCD NPs was estimated (Chapter I -Section A).

• A second study was performed to address the challenges related to the incorporation of a first-generation quinolone, pipemidic acid (PIP), in PLA/PLGA and pCD NPs with the aim of "recycling" this old antibiotic by optimising its intracellular delivery for the treatment of infections such as salmonellosis 9,10 .

The loading capabilities of polyester NPs, formulated by either nanoemulsion or nanoprecipitation methods, were compared and the role of PIP acid in NPs formation as well as its localisation and affinity for each components of the NPs were explored. In order to overcome the technological barriers related to the physical encapsulation approaches and to obtain NPs with a high PIP loading, a new self-assembling prodrug was developed. A prodrug was successfully obtained by conjugating PIP with PCL through an original catalyst-free drug-initiated polymerisation. The PCL-PIP NPs were in depth characterised by combining four advanced techniques and their degradation was studied. The NPs contained around 27 wt% PIP, highlighting the interest of developing drug conjugated by a straightforward method (Chapter II).

These formulation studies highlighted the importance of an in-depth characterisation of the complex NP structures containing one or more antibiotics susceptible to crystallize and/or to locate near the NP top layers.

Drug location deeply impact upon the release patterns and NPs stability in biological media. To the best of our knowledge, there is no experimental method yet to unravel the structure of each individual NP and to assess the homogeneity of a preparation. Thus, it was our aim to go beyond the state of art in the nanomedicine field, and we proposed in the second part of the work an original nanospectroscopic technique adapted both to unravel the structure of core-shell NPs and to localise them inside cells without the need of any labelling (Chapter III and IV). This recently developed technique combines the nanoscale spatial resolution offered by atomic force microscopy (AFM) with the chemical characterisation offered by infrared spectroscopy (IR) and it is therefore referred to as AFM-IR 11 . A general overview (Chapter III -Section A) is given at the beginning of this second part to introduce the AFM-IR technique and to explain its potential through the already developed applications 12 . Then, our studies present the intriguing information that this technique can provide when applied to NPs characterisation and detection.

• The first great opportunity offered by AFM-IR was the possibility to obtain precise chemical information on the distribution of the components of each individual PLGA NP. A wavenumber, characteristic of the specific IR absorption of each NP component, was selected as fingerprint and for the first time a chemical mapping of single NPs could be obtained, allowing unravelling their structure. The major breakthrough of this study is the first application of AFM-IR in tapping mode. Indeed, all previously published results were obtained by using the technique in contact mode, thus hampering the possibility of analysing soft or loosely-adhesive samples. The advantage of the new tapping mode was clearly shown here in the case of PLGA NPs which were crushed and/or displaced in contact mode, whereas they were very well-described thanks to tapping mode. The technique allowed to undoubtedly demonstrate for the first time the existence of PLGA NPs core-corona structure which was up to now only hypothesized. Moreover, when analysing PIP-loaded PLGA NPs, tapping AFM-IR allowed precisely localising PIP distribution and confirming the hypothesis made in our previous studies presented in Chapter II (Chapter III -Section B). Noticeably, this technique allowed visualising nano-objects as small as 10 nm mainly composed of PIP and surfactant.

Finally, tapping AFM-IR enabled obtaining the chemical mapping also in the case of PCL-PIP NPs as well as a first quantification of PIP/PCL ratio on a particle by particle basis. IR cartographies of PCL-PIP NPs displayed a well-distributed PIP loading and their analysis provided quantitative information about NPs loading in addition to the already appealing qualitative ones (Chapter III -Section C).

• AFM-IR further proved its attractivity in the label-free detection and unambiguous chemical identification of polyester NPs internalised in cells. Indeed, these NPs, totally composed of organic materials, usually require the addition of labels to be detected by the most common imaging techniques (such as confocal fluorescence microscopy and transmission electron microscopy). Thus, the NPs are indirectly detected by observing their label. Here we could apply AFM-IR to directly localise NPs by using the polymer IR specific response as a fingerprint.

Finally, the main results of this research are discussed, the main conclusions are gathered and the numerous perspectives of this work are presented.

Introduction Générale

La résistance aux antimicrobiens constitue aujourd'hui l'une des plus graves menaces pesant sur la santé publique et ainsi l'un des plus grands défis que l'humanité devra relever dans les prochaines décennies. Un nombre sans cesse croissant de bactéries résistantes à l'action de plusieurs antibiotiques est détecté chaque année et ce phénomène devrait s'amplifier, entrainant de graves conséquences sanitaires ainsi qu'économiques à l'échelle mondiale 1 . En effet, l'impact de cette résistance a été estimé à 1 million de morts par an d'ici 2050, s'accompagnant d'un poids économique énorme (environ 100 trillions de dollars américains 2 ).

C'est pour cela que les Etats Unis et l'Europe se sont engagés à mettre en oeuvre de nombreux plans au niveau économique comme social pour faire face à cette menace. Ainsi, l'Europe est en train de financer un nombre croissant de projets proposant des solutions innovantes pour affronter cette crise mondiale.

Ce travail de thèse représente un exemple de cet engagement européen du fait qu'il s'inscrit dans le cadre du Projet Européen ITN « Cyclon Hit », visant à développer des nanovecteurs pour administrer de manière optimisée les agents antimicrobiens et empêcher les mécanismes de résistance. Plus précisément, ce projet s'est consacré à un défi particulièrement ambitieux et complexe comme le traitement des infections intracellulaires. Les bactéries intracellulaires ont la capacité de « se cacher » à l'intérieur des cellules de l'hôte, en particulier celles du système immunitaire, en les transformant en leur habitat préféré et en échappant ainsi à l'action de nombreux antibiotiques 3,4 .

Ce projet propose donc l'utilisation de nanovecteurs pour encapsuler efficacement les antibiotiques, les protéger de la dégradation, surmonter leurs limitations telles que leur faible solubilité, leur incapacité à franchir les membranes cellulaires et leur biodistribution défavorable, en réduisant par là même leurs effets secondaires toxiques et en améliorant l'observance des patients aux traitements.

Ainsi, au sein de ce projet, mon travail de thèse est axé sur le développement, l'optimisation et la caractérisation avancée de nanoparticules (NPs) biodégradables et biocompatibles, composés de polyesters ou de cyclodextrines (CD), et chargées en antibiotiques. Les polyesters principaux employés dans cette étude sont l'acide polylactique (PLA), l'acide poly(lactique-co-glycolique) (PLGA) et le polycaprolactone (PCL) qui sont tous approuvés pour les utilisations pharmaceutiques par la FDA, l'agence américaine des produits alimentaires et médicamenteux [5][6][7] . De plus, ces polyesters aussi bien que les CDs sont parmi les matériaux les plus employés pour la préparation de systèmes d'administration des médicaments à propriétés modulables. La clef du succès de ces matériaux dépend surtout de leur capacité d'améliorer les caractéristiques physicochimiques et la biodistribution des antibiotiques ainsi que du fait qu'ils se désassemblent en unités nontoxiques, facilement éliminées par l'organisme.

Une première partie introductive (Introduction) consiste en une revue de la littérature touchant les domaines scientifiques à la base de cette étude et les défis majeurs qui lui sont associés.

Les bactéries intracellulaires et la résistance aux antibiotiques seront plus particulièrement abordées. La structure, le profil d'innocuité et les applications des polyesters ainsi que des CDs utilisés dans notre étude pour la formulation des NPs seront détaillés. En outre, les principales techniques utilisées pour la caractérisation de ces NPs et pour leur localisation intracellulaire in vitro seront également décrites.

Par la suite, les deux premiers chapitres seront consacrés aux principaux résultats obtenus dans le développement, l'optimisation et la caractérisation de NPs polymériques chargées de molécules antibiotiques difficiles à administrer.

• Tout d'abord, ces NPs, fabriqués en PLA ou en CD réticulées (pCDs), ont été utilisées pour la coencapsulation de l'éthionamide (ETH), un antibiotique de deuxième ligne pour le traitement de la tuberculose (TB), et du Booster, une molécule capable d'améliorer l'activité de l'ETH. L'association très puissante de ces deux molécules, découverte récemment 8,9 a ouvert des opportunités formidables dans le traitement de la TB grâce à sa capacité d'augmenter notablement l'efficacité thérapeutique de l'ETH.

Toutefois, cette association présente quelques limites, essentiellement liés à la difficulté de trouver un solvant commun, (qui plus est, compatible avec le milieu biologique), aux profils de biodistribution complètement différents des deux molécules et à la forte tendance de l'ETH à cristalliser. Ces contraintes ont freiné considérablement la possibilité de coadministrer cette association de façon efficace. Donc, l'objectif ainsi que le défi pharmacologique principal de cette étude consistait à co-encapsuler ces médicaments dans les NPs pour permettre de les administrer directement au site d'infection et « de lever les verrous technologiques » (contourner les propriétés physiques) des molécules libres. Une fois cette coencapsulation obtenue, une caractérisation approfondie des NPs en PLA a été réalisée à l'aide de L'efficacité in vitro des NPs en PLA et en pCDs chargées en antibiotiques a été évaluée sur les bactéries extra ou intracellulaires grâce à l'aide d'une plateforme automatisée de criblage à haut rendement. Ensuite, la compatibilité des NPs avec le dispositif pour l'administration in vivo par voie endotrachéale, le Microsprayer®, a été étudiée. L'un des principaux résultats de ce travail a été l'évaluation du profil d'innocuité des NPs en pCDs après administration pulmonaire répétée de fortes doses de ce vecteur. De plus, l'activité antibactérienne des NPs en pCDs chargées avec l'association des deux molécules actives a été déterminée in vivo en montrant une diminution de 3-log de la charge bactérienne pulmonaire des animaux infectés après seulement 6 administrations (Chapter I -Section A).

• Le deuxième chapitre de cette thèse est consacré à la résolution des défis liés à l'incorporation d'une autre molécule active, une quinolone de première génération, l'acide pipémidique (PIP). Le but, était de recycler un ancien antibiotique en optimisant sa distribution intracellulaire dans des infections telles que la salmonellose 10,11 .

La capacité à encapsuler le PIP des NPs en polyester, obtenus par les techniques de nanoemulsion ou de nanoprecipitation, a été étudiée. Le rôle de cet antibiotique dans la formation des NPs ainsi que sa localisation dans la structure du vecteur et son affinité pour les différentes molécules composantes les NPs ont été examinés. L'encapsulation du PIP a été confrontée à des défis liés à la cristallisation du principe actif et à sa libération incontrôlée. Afin de s'affranchir de ces « barrières technologiques »et d'obtenir des NPs avec une charge de PIP importante, une approche alternative a été développée basée sur la synthèse d'un promédicament capable de s'auto-assembler sous forme de NPs. Cette prodrogue a été obtenue en couplant le PIP avec du PCL via une réaction de polymérisation sans catalyseur initiée par le médicament.

Les NPs en PCL-PIP ont été caractérisées précisément en combinant quatre techniques de pointe et leur dégradation a été également étudiée. Les NPs contenaient environ 27 % (en poids) de PIP. Ce résultat a mis en évidence l'intérêt de développer un conjugué du médicament par une méthode simple pour en obtenir une encapsulation efficace (Chapter II).

Ces études de formulation ont mis l'accent sur l'importance d'une caractérisation approfondie de la structure complexe des NPs qui peuvent contenir à la fois plusieurs antibiotiques susceptibles de cristalliser ou de se localiser près de la surface des NPs. La localisation du médicament a une influence très importante sur son profil de libération et sur la stabilité des NPs dans les milieux biologiques. Au début de notre étude n'existait aucune méthode expérimentale permettant d'analyser la composition de chaque NPs individuellement et de déterminer l'homogénéité d'une formulation. C'était ainsi notre objectif d'aller au-delà de l'état de l'art dans le domaine de la nanomédicine et de proposer dans la deuxième partie de ce travail de thèse une technique nanospectroscopique innovante adaptée pour définir de manière approfondie la structure et la composition du noyau et de la couronne des nanostructures, mais aussi pour les détecter après internalisation dans les cellules sans avoir recours à leur marquage (Chapter III and IV). Cette technique récemment développée est capable de combiner la résolution spatiale à l'échelle nanoscopique offerte par la microscopie de force atomique (AFM) avec la caractérisation chimique apportée par la spectroscopie infrarouge (IR). Elle a donc été nommée AFM-IR 12 .

Au début de cette deuxième partie du travail, une revue générale (Chapter III -Section A) est donnée pour introduire l'AFM-IR et pour mettre en évidence son potentiel à travers la présentation des applications déjà développées 13 . Ensuite, les résultats extraordinaires de caractérisation et de détection des NPs obtenus par cette technique seront présentés (Chapter III -Section B, C).

• Le premier avantage considérable offert par l'AFM-IR a été de pouvoir acquérir des informations très précises sur la distribution des composants de chaque NP en PLGA individuellement. Une longueur d'onde, spécifique de l'absorption IR de chaque élément constituant les NPs, a été sélectionné comme leur « signature » et une cartographie chimique des NPs prise individuellement a été réalisée en permettant de décoder leur structure à l'échelle nanométrique. L'avancée majeure de cette étude a été l'application pour la première fois de l'AFM-IR en mode tapping. En effet, tous les résultats précédemment publiés ont été obtenus en utilisant cette technique en mode contact, mode qui n'offre pas la possibilité d'étudier les échantillons souples ou adhérant faiblement à la surface du support. L'avantage de ce nouveau mode a été clairement démontré dans le cas des NPs en PLGA qui étaient écrasées et/ou déplacées avec le mode contact alors qu'elles ont pu être décrites très précisément grâce au mode tapping. Cette technique a permis de prouver définitivement l'existence d'une structure coeur-couronne des NPs en PLGA, ce qui était jusqu'à présent seulement une hypothèse. En outre, lors de l'exploration des NPs en PLGA chargées en PIP, l'AFM-IR en mode tapping a rendu possible de localiser précisément la distribution du PIP à l'intérieur de NPs et ainsi de confirmer les hypothèses qu'on avait formulé lors de nos études précédentes présentées dans le chapitre II (Chapter III -Section B). L'AFM-IR a permis la visualisation de nano-objets de seulement 10 nm, principalement composés de PIP et de surfactant. Enfin, l'AFM-IR en mode tapping a permis d'obtenir également la cartographie chimique des NPs en PCL-PIP aussi bien qu'une première estimation quantitative du ratio PIP/PCL de ces NPs une par une. Les cartographies IR des NPs en PCL-PIP ont permis de quantifier la charge en PIP et de démontrer une répartition de cette charge assez homogène (Chapter III -Section C).

• Un autre avantage très attrayant de l'AFM-IR est, comme démontré dans le dernier chapitre de ce manuscrit, qu'il peut détecter sans marquage et identifier chimiquement sans ambiguïté les NPs en polyesters internalisées par les cellules. En effet, ces NPs, en étant constituées exclusivement de matériaux organiques, exigent généralement le rajout d'un marqueur pour pouvoir être détectées par les techniques les plus courantes d'imagerie (telles que la microscopie confocale à fluorescence et la microscopie électronique en transmission). Les NPs sont ainsi identifiées indirectement en visualisant leur marqueur. Dans cette étude, nous avons pu montrer que l'AFM-IR peut être employé avec succès pour repérer les NPs à l'intérieur des cellules en utilisant la réponse IR du polymère comme signature spécifique.

Pour finir, l'ensemble des résultats démontrés au cours de cette thèse seront discutés, les principales conclusions et les nombreuses perspectives ouvertes par ces études seront présentées.

Introduction 1. Intracellular bacteria and antimicrobial resistance 1.1. Intracellular bacteria

Bacteria are among the most numerous forms of life on earth and they are consistently present in the human body, both inside (especially in the gastrointestinal tract) and on its surface (skin). A considerable number of bacteria are able to live in symbiosis with humans and are innocuous, sometimes beneficial or even necessary.

However, some of them, namely pathogenic bacteria, are able to colonise, invade and harm their host thus causing diseases 1,2 . Indeed, pathogens can cause infections either in specific localised niches in the host or they can invade the entire organism, even becoming life-threatening. Some examples of such dangerous infection are tuberculosis (TB), endocarditis, pneumonia, osteomyelitis and septicaemia.

Intracellular pathogens are causative agents of some of the infections listed above which are recurrent, persistent and particularly difficult to defeat. Therefore, they represent an important public health concern.

The peculiarity of intracellular bacteria lies in their ability to thrive within the organism by hiding inside the cells of the host and surviving in active or latent form over a long period of time, despite the presence of a well-functioning immune system. Indeed, they have found a way not only to circumvent the host immune system but also to colonise and replicate in the cells belonging to it, such as the ones of the reticuloendothelial system (RES) 1 . The RES consists of phagocytic cells such as macrophages and monocytes which main role is to uptake and destroy foreign bodies trough phagocytosis and to activate the adaptive component of the immune system through presentation of bacterial antigens. Once the bacteria are engulfed by phagocytes they are enclosed in vesicles, called phagosomes, which further fuse with cells lysosomes resulting in phagolysosome formation 3 . In the phagolysosome, bacteria are usually destroyed thanks to the contribution of different factors; low pH, production of oxygen reactive species, nitric oxides, proteases etc 4,5 .

Despite the efficiency of host immune system, intracellular bacteria have hence developed a number of ingenious mechanisms to hijack the phagocytic pathway at different stages and exploit the protection offered by the cell to multiply 6 . For example, bacteria can escape from the phagosome and survive in the cytosol, or block the system at the early endosome stage such as Mycobacterium Tuberculosis (Mtb), or at the late endosome stage like Salmonella and transform these vacuoles in their preferred habitat 7 . In such way, these bacteria give rise to chronic infections difficult to defeat.

For instance, TB, a respiratory disease caused by Mtb, is ranked above HIV/AIDS as one of the leading causes of death from an infectious disease (1.8 million deaths in 2015) 8 and its treatment requires at least 6 months daily administration of an association of first-line antibiotics (isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin) or two years therapy of second-line drugs (ethionamide, aminoglycosides, fluoroquinolones, etc) in case of resistant TB [9][10][11] . Another interesting example is Salmonellosis which is caused by an opportunistic intracellular pathogen, Salmonella Typhimurium, and consists in an uncomplicated gastroenteritis in immunocompetent patients whereas can be life-threatening in immunocompromised ones.

Salmonellosis is related to a high risk of mortality and morbidity with economic losses of billions of dollars and in Sub-Saharan Africa is associated with a case of fatality of 20-25% (often relates to AIDS patients).

Multidrug resistant strains of S. Typhimurium have recently been identified and labelled as emerging diseases in Africa 12 .

Other particularly challenging facultative intracellular bacteria include Staphylococcus aureus, Listeria monocytogenes and Burkholderia pseudomallei. These pathogens, together with Mtb, stand out for their capacity of using circulating macrophages as "Trojan horses", hence spreading within the host and establishing secondary infectious foci 1 .

Antibiotics

Antibiotics are antibacterial drugs able to cause bacterial death (bactericidal drugs) or to inhibit their replication (bacteriostatic drugs). The same antibiotic molecule can be bacteriostatic at low dose and bactericidal at high dose 13 . The choice of the most suitable antibiotic and its dosage is based upon the gravity of the infection and on the specific sensitivity of the strain. Antibiotics can be classified according to different methods, mainly on the basis of their mode of action 13 :

• Inhibitors of cell wall synthesis (Beta-lactams such as penicillins, cephalosporins, monobactams and carbapenems; Glycopeptides; Fosfomycins)

• Inhibitors of protein synthesis (Aminoglycosides; Macrolides; Tetracyclines, Glycylcyclines; Phenocols; Oxazolidinones; Ansamycins)

• Inhibitors of membrane function (Lipopeptides; Polymyxins)

• Inhibitors of nucleic acid synthesis (Quinolones; Trimethoprim-sulfamethoxazole; Ryfamycins). Despite their undeniable usefulness, antibiotic efficacy is often hampered by low bioavailability, side-effects, the inability of some of them to cross tissular and cellular barriers, the onset of resistance and the ability of bacteria to organise in biofilms.

Antibiotics, in order to be effective, need to reach the infection site at a therapeutic concentration that has to be maintained stable for a sufficient amount of time. Therefore, antibiotic doses have to be high enough and administered frequently enough to make up for all the losses/degradations taking place on the way from the administration site to the target. Indeed, just as all other drugs systemically administered, antibiotics undergo a uniform distribution in the body meaning that only a small fraction of the dose actually reaches the target.

Moreover, the need to cross biological barriers and in some cases a mucus layer (lungs, gastrointestinal tract, etc) makes even harder for the antibiotic to reach the targeted bacteria. In addition, in the case of intracellular pathogens, antibiotics also have to cross the cell membrane and reach the bacteria in their subcellular hideouts.

Inability of crossing the cell membranes (polar drugs such as β-lactams and aminoglycosides), efflux phenomena (macrolides and fluoroquinolones) and inactivation due to intracellular pH or enzymes can also result in subtherapeutic concentrations and lead to inefficacy 1 . It is evident that antibiotics do not have an easy task.

Emergency of antimicrobial resistance

As outlined in the previous paragraphs, intracellular bacteria are a pharmaceutical challenge per-se and thus antibiotics are not always effective enough to defeat them. In addition to the already described difficulties, the scientific community has witnessed a progressive increase in the onset of antimicrobial resistance phenomena in a growing number of bacteria in the last 50 years.

Antimicrobial resistance (AMR) can be defined as the ability of an organism to resist the action of an antimicrobial agent to which it was previously susceptible 2 .

Bacteria can exhibit intrinsic resistance to antimicrobials due to their inherent structural (G+/G-) or functional characteristics. Indeed, all the bacteria are not sensitive to all the antibiotics. Moreover, they can acquire AMR as a result of spontaneous chromosomal genes mutations or by horizontal gene transfer (plasmids, transposons) 14 . The acquisition of resistance can be mediated by three main classes of mechanisms: i) the transport of the antibiotics through the bacterial cell wall and membrane (reduced permeability due to downregulation of porins or increased efflux phenomena), ii) the synthesis of enzymes or modification of cellular environment to inactivate the antibiotics and iii) the structural modification of the antibiotic target by genetic mutation or post-translational modification 14,15 .

When a bacterium survives to a drug, thanks to one of these mechanisms of AMR, the strain capable of surviving grows and spreads due to the lack of competition with other strains (resistant pathogen selection) 16 .

Multiple resistance arise when a bacterium already containing a drug resistance gene acquires another type of drug resistance genes 17 . This phenomena have led to the emergence of "superbugs" such as Methicillinresistant Staphylococcus aureus (MRSA) and extremely drug resistant (XDR) tuberculosis 11 which are very difficult or even impossible to treat with the existing antibiotics 18 . Moreover, we are down to using "last line" antibiotic for the treatment of drug-resistant gonorrhoea and to reintroducing antibiotics that were not used anymore because of their serious side effects. For instance, colistin, an antibiotic causing kidney failure, has been reintroduced in clinic as last-resort treatment for particular hard-to-treat gram negative bacterial infections, but colistin-resistance already emerged at the end of 2015 16 .

Although AMR is a natural adaptive response of bacteria observed since the discovery of antibiotics, it has become a serious problem in recent times due to the increased rate of resistance development and diffusion. Indeed, in the past, AMR prevailed in the healthcare environment where the antibiotic selective pressure is intensive. However, over the past decade resistant infections have arisen also in the rest of the community 16 .

The worldwide spread of resistant bacteria can be viewed as an ecological consequence of the systematic use and sometimes misuse of antibiotics. Furthermore, there is a continuous exposure to low doses of antibiotics contained in food (animal-derived products and agriculture) 1 .

Recent assessments have estimated that, without aggressive policies to stop the worrying spread of AMR, 10 million people will die every year from bacterial infections by 2050 overcoming the number of deaths due to cancer 16 . Indeed, AMR is considered as one of the biggest threats that humankind will have to face in the next decades.

Taking into account that very few new antibiotics are in the development pipeline 18 , new strategies are required to administrate already existing antimicrobial agents in an optimized manner to help fighting against this treat.

Nanomedicine is considered as one of the most promising strategies in this perspective 17 (Figure 1).

Nanomedicine

Nanomedicine is a branch of medicine that applies the knowledge and tools of nanotechnology to the prevention and treatment of disease. It involves the use of nanoscale materials, such as biocompatible nanoparticles and nanorobots, for diagnosis, delivery, sensing or actuation purposes in a living organism 19 .

During the last decades, this field gained ever-growing interest as a promising tool to overcome the drawbacks of traditional therapeutic agents (Figure 1). Indeed, drug-loaded nanoparticles are able to: i) improve physicochemical properties of drugs (low solubility, tendency to form crystals); ii) protect them towards degradation; iii) increase their bioavailability and improve their pharmacokinetic; iv) target them to the disease; v) reduce toxic side effects; vi) increase patient compliance and vii) reduce treatment duration and related costs 20 .

Figure 1 -Schematic representation of an infected cell treated with free antibiotic or with antibiotic-loaded nanoparticles (NPs).

Free drugs often poorly penetrate inside cells due to several factors such as their chemical structure (polar drugs such as β-lactams and aminoglycosides), their decreased uptake caused by resistance mechanisms (down regulation of membrane carriers), intracellular degradation and the onset of efflux phenomena. Such circumstances individually or simultaneously contribute to the inefficacy of free antibiotics. On the contrary, nanocarriers are able to protect the drugs, improve their physico-chemical properties and target them inside cells. Indeed, drug-loaded NPs are taken up inside endosomes where they degrade (hydrolytic enzymes and acidic pH) and release their payload. Then, drugs able to diffuse can escape endosomes and reach the bacteria in their preferential locations (cytosol or vacuolar niches). Interestingly, non-diffusible drugs still have the opportunity to reach the bacteria thanks to the ability of some NPs to escape endosomes or to the capacity of NPs-containing endosomes to fuse with the bacterial vacuoles 1 .

Nanoparticles as intriguing strategy to overcome drugs physicochemical limitations and tackle intracellular bacteria

Liposomes were the first nanosized delivery system introduced in 1960 and since then a broad variety of nanocarriers such as nanoparticles (NPs), nanogels, dendrimer and micelles have been prepared by using a wide variety of organic (polymers and lipids) and inorganic materials (silica and metals). Ideal NPs have to be non-toxic, biocompatible and possibly biodegradable and to have tailored well-defined size, shape and surface properties and release their payload in a controlled manner.

Drugs may be incorporated into NPs by three different methods. They can be loaded during or after the NPs formulation, absorbed to NPs surface or alternatively chemically bound to the NPs 21 .

Poor aqueous solubility and rate of dissolution are the two critical factors that heavily impact on formulation and development of drugs, limiting their therapeutic application 22 . Indeed, recent statistical analyses have reported that around 40% of currently marketed drugs and about 90% of drugs in their developmental phase encounter solubility-related problems 23 . Failures in the development of a drug are thus tightly connected to poor biopharmaceutical properties such as solubility and permeability. Actually, on the basis of these two parameters, drugs have been divided in four main biopharmaceutical classes which are shown in Figure 2. The classification is based on drugs solubility (with a soluble drug defined as one in which highest dose intended for human use dissolves in 250 mL of water) and permeability (with a permeable drug defined as one with ≥ 90% oral bioavailability or ≥ 90% absorption) 24 .

Class II and class IV drugs represent the biggest pharmaceutical challenges when it comes to their in vivo administration 25 . Remarkably, most of the cytotoxic anticancer drugs belong to the IV class having both low aqueous solubility and permeability 26,27 . Indeed, drug discovery has evolved over the years towards highthroughput screenings putting evolutionary pressure on drug candidates. This has led to a systematic increase in drugs molecular weight, lipophilicity and decrease in their water solubility 24,27 . Challenging type II compounds (see Figure 2) are increasingly selected and their inclusion in NPs can represent an efficient strategy to make them behave as type I compounds which results in an increase of bioavailability 24 .

Furthermore, NPs can also be extremely beneficial in improving drug stability during storage, avoiding drug crystallisation or degradation, and during in vivo administration, protecting it from chemical and enzymatical degradation. Moreover, NPs not only can protect the drug from the organism, but they have also proved to be able to protect the host from drug toxicity due to non-specific interaction of the active molecule with other organs than target (i.e. Doxil, doxorubicin-loaded liposomes which reduce cardiac toxicity) 28 .

As previously pointed out, to efficiently eradicate intracellular bacteria, NPs have to be capable of carrying their drug cargo inside cells. Indeed, NPs have been shown to penetrate RES cells by different mechanisms depending on their composition, size and physicochemical properties. For instance, the modification of their surface with hydrophilic polymers such as poly(ethylene glycol) (PEG) decrease their recognition and uptake by the immune system leading to extended vascular circulation. This approach has proved its usefulness especially in cancer therapy, where NPs uptake by RES cells is not desired, and allowed NPs passive targeting to cancer cells thanks to the enhanced permeability and retention effect [29][30][31][32] . Therefore, in antimicrobial applications, PEG coating is normally avoided or considerably reduced in order to allow NPs internalisation by RES cells and hence targeting intracellular bacteria.

NPs colloidal properties are of paramount importance in establishing which endocytic pathway NPs will undergo and consequently which will be their intracellular location. Largest particles are generally taken up by phagocytosis (characteristic endocytic pathway of professional phagocytes) whereas smaller one by pinocytosis (possible in every type of cells). Pinocytosis can be further divided in clathrin-mediated, caveolaemediated as well as clathrin-and caveolae-independent endocytosis and micropinocytosis 1,33 . The mechanism of NPs internalisation determines, trough very complex trafficking processes, their intracellular fate. For instance, clathrin-mediated and caveolae-mediated internalisation determine a lysosomal or a cytoplasmic localisation, respectively 33 . Interestingly, some NPs have shown the ability to escape their endosomal/endolysosomal compartments and release their cargo in the cytoplasm 34,35 . Indeed, the subcellular location of the NPs can determine the success or the failure of the developed delivery system. As a matter of facts, in some cases NPs have shown an improved intracellular accumulation but any improvement in the antibacterial effect could be detected. This situation is most likely due to NPs being localised in a different cellular compartment than bacteria and to the inability of the released drug molecules to reach their target 1 .

Another factor which may contribute to the scene is the dormant state that some bacteria, such as Salmonella and Mtb, can assume during the infection making them insensitive to antibiotics 36,37 .

The physicochemical properties of the NPs have also a very high impact in determining the release profile of the antibiotic. Ideally, the drug should be released only once the NPs have been internalised and possibly in the vicinity of the bacteria to insure the highest probability of success. As intracellular controlled release is challenging, stimuli-responsive NPs have been designed to trigger the release at a specific moment and under a specific stimulation 34,[38][39][40][41] . Despite the decoration of NPs surface is certainly appealing and advantageous, one must keep in mind that building complex drug delivery systems normally requires multiple synthetic steps and will raise questions on their biocompatibility, long-term toxicity and genotoxicity. Thus, the number of tests, money and time necessary to market the final product will significantly increase. Therefore, a balance should be kept between nanocarrier complexity and feasibility and the necessity of surface decoration must be thoroughly assessed.

The choice of the appropriate administration route is another important parameter impacting on the efficacy of NPs against bacteria. Intravenous administration is the most adapted way to treat intracellular infections particularly when liver and spleen are involved 42,43 . Oral administration is intuitively the route of choice in the treatment of infections localised in the intestinal tract (Salmonella) 44,45 . Pulmonary administration is instead the most employed and efficient way to treat respiratory infections such as TB 1,43,46 .

A broad variety of NPs have proved their capability in reaching therapeutic drug concentration inside different cell lines thus improving antibiotic efficacy [47][48][49] . Liposomes have been the first type of carrier successfully applied in the field 39,[50][51][52][53] but they presented some drawbacks in comparison to NPs such as higher instability (drug leaking), lower capacity of encapsulate drugs and a difficulty to be sterilised 1,54 . Indeed, different types of NPs were also employed including poly(D,L-lactic-co-glycolic acid) (PLGA) NPs and polymeric NPs, mesoporous silica NPs, polymer micelles, solid lipid NPs, niosomes and dendrimers 1 . Moreover, a plethora of NPs endowed with intrinsic antibacterial activity were developed by using organic or inorganic materials (metals and metal oxides) and were used alone or in association with antibiotics 17,55,56 . Their action is generally based on the production of free radicals inducing oxidative stress and the generation of reactive oxygen species which irreversibly damage bacterial DNA.

Biocompatible and biodegradable nanocarriers

The idea of a nanocarrier, able to break-down in harmless products and be cleared from the body after delivering its drug cargo in an optimised fashion, has captured scientists' attention since the conception of Erlich's "magic bullet" at the beginning of 1900s.

To produce such NPs, a vast array of biodegradable materials, natural, synthetic and hybrid, have been studied and developed over the years 57,58 . Herein, we will focus firstly on the most employed ones, poly(lactic acid) (PLA),PLGA and poly(ε-caprolactone) (PCL) based NPs and subsequently on cyclodextrins (CDs), CDs derivatives and CD-based polymeric NPs.

PLA/PLGA and PCL nanoparticles

PLA and PLGA copolymers history, properties and degradation

Poly(glycolic acid) (PGA), PLA and their copolymer PLGA are a family of highly versatile biocompatible and biodegradable polyesters which are widely applied in the medical field.

PGA is the first polymer of this family used for biomedical applications. Indeed, it was firstly employed in 1960s in the development of biodegradable sutures marketed in 1970s under the commercial name of Dexon 57 .

However, the tension of the sutures decreased too fast due to PGA relatively fast degradation. Therefore, a PLGA copolymer was developed to lengthen the degradation time. This 10:90 PLGA was marketed under the name of Vicryl and Polyglactin 59 . From this first application, which demonstrated the good mechanical properties, the biocompatibility and the tuneable degradation rate of the material, the use of PLGA copolymers was expanded to tissue engineering and drug delivery systems 21,29 .

PLGA copolymers have therefore received the approval of the Food and Drug Administration (FDA) and several products have been marketed ranging from sutures and bone fixatives, to artificial skin and blood vessels, to drug delivery 57,59 . Lupron-Depot (leuprolide acetate) was the first parenteral (intramuscular or subcutaneous) sustained-release formulation utilising PLA polymers and it was approved in 1989.

Since then, PLGA-based NPs have been used over the years to efficiently carry active agents for the treatment of a wide range of diseases 1,21,60,61 .

Because PLGA is a copolymer, it can be employed in a broad range of formulations by varying both the lactic:glycolic ratio and the molecular weight. The different compositions of PLGA can be used to manipulate the crystallinity, hydrophobicity, the glass transition temperature (between 40 and 60°C) and the degradation rate of the copolymers 62 . Typically, a higher ratio of lactic acid results in a more hydrophobic copolymer whereas an increase in the amount of glycolic acid produces a more hydrophilic polymer matrix. Indeed, PLA present a methyl side group and it has an asymmetric carbon. Consequently, PLA exists in 2 enantiomers which display crystalline character 57 . Therefore, the use of PLA racemic mixtures is preferred over the pure enantiomer to form PLGA copolymers. In this way, disordered polymer chains can be obtained and the copolymer exhibits more amorphous features which can improve homogeneous dispersion of the drug in the polymer matrix 29 . Conversely, glycolic acid is devoid of this methyl side group and displays a highly crystalline structure (Figure 3) 31,62 . The glass transition temperature (Tg) of PLGA copolymers is above 37°C and hence they are glassy in nature and exhibit a fairly rigid chain structure. This confers them a significant mechanical strength which decreases with the decrease of the lactic content and the decrease in their molecular weight 29,31 .

The degradation of PLGA occurs by simple hydrolysis of the ester bonds which connects the lactic and glycolic units, leading first to oligomers and finally to monomers. These degradation products are both non-toxic and they are either excreted unchanged in the kidneys (only in the case of glycolic acid) or metabolised by the body and eliminated as carbon dioxide and water 29,31,62 . PLGA degradation is a bulk process which takes place by random scission wherever water is able to penetrate into the polymer structure 57 . The degradation rate depends mostly on PLA/PGA ratio, polymer molecular weight, crystallinity, size of the device and on environmental factors such as pH and presence of enzymes, among others 29 . Several studies demonstrated that the ratio of the two monomers is the parameter which affects the most PLGA degradation. However, the prediction of PLGA degradation is not trivial because of the influence of all the other parameters listed above. In general, the higher the glycolic acid percentage the faster is PLGA degradation apart from PLGA 50:50 which exhibits the fastest degradation 31 . Indeed, as already highlighted, lactic acid is more hydrophobic than glycolic acid, hence lactiderich PLGA absorb less water and hydrolyse more slowly 29 .

PCL polymer history, properties and degradation

PCL (Figure 4) was one of the first polyester polymers synthesised in the early 1930s and its biodegradable property was firstly identified in 1973 32 .

Figure 4 -PCL chemical structure

It is a hydrophobic, semi-crystalline polymer, whose crystallinity increases with the decrease of its molecular weight. PCL has a much lower Tg (-60 °C) in comparison to PGA, PLA and PLGA and this characteristic endows it with flexible mechanical properties suitable for paramedical application, wound dressing, contraceptive and dentistry 63 . The other main difference in comparison to PLA, PGA and PLGA is the much lower degradation rate. Therefore, PCL was originally used in drug delivery devices active for over 1 year and in slowly degrading suture materials (Maxon) 64 . Thanks to the development of these first devices, PCL received FDA approval. However, PCL mechanical flexibility and slow degradation rate were initially seen as a disadvantage. The polymer was too soft and flexible to be able to replace metal devices such as plates and screws and its slow degradation was perceived as a factor leading to higher toxicity risks. Consequently, PLGA copolymers were preferred over PCL for over two decades. PCL potentialities, such as superior rheological and viscoelastic properties in comparison with other resorbable-polymers, were reassessed thanks to the development of tissue engineering in 1990s and 2000s. Therefore, PCL was employed to produce a wide range of biocompatible resorbable scaffolds 64 .

Moreover, the possibility of blending PCL with a broad range of other polymers to improve its mechanical resistance and increase the degradation rate opened the way for its use in drug delivery for the development of micro and nanoparticles. Similarly to PLGA, PCL degradation occurs by random hydrolytic chain scission of the ester linkages. High molecular weight polymers take longer time to be degraded because of the necessity to cleave a higher number of ester bonds. PCL hydrolytic product, the 6-hydroxyl caproic acid, has been proved to be nontoxic and it associate to acetycl coenzyme A (Ac-CoA), enters in the citric acid cycle and is easily eliminated from the body 32,64 .

Nanoparticles preparation methods

The selection of the appropriate methodology for NPs preparation is of crucial importance to achieve specific needs and the physico-chemical characteristics of both polymer and drug must be taken into account 65 .

The preparation techniques of NPs can be mainly divided into two synthetic approaches; the self-assembly of preformed polymers and the polymerisation of monomers 66 . Generally, the methods using the first approach are preferred to avoid the presence of toxic residues which can be an unwanted consequence of methods based on monomer polymerisation. Among methods starting from preformed polymers, herein we will focus on the five most common; nanoprecipitation, salting-out, emulsification diffusion, emulsification evaporation and double emulsion solvent evaporation 65 .

Nanoprecipitation is also known as solvent diffusion or displacement and was firstly developed in 1989. The polymer and the drug are dissolved in an organic solvent, miscible with water (i.e. acetone, ethanol). This organic phase is added drop-wise under magnetic stirring to an aqueous solution which ideally has to be a nonsolvent for both the polymer and the drug. Nanoprecipitation occurs by rapid desolvation of the polymer when the two phases are mixed. Nanoparticles formation is governed by interfacial turbulences between the solvent and the non-solvent 67,68 . Local fluctuation in the polymer concentration leads to the formation of nucleation sites which further grow to form monodispersed NPs, if all operating conditions are correctly set. Ideally, the process is characterised by a high nucleation rate, strongly dependent on the supersaturation, and low growth rate 65,67 . Then the organic solvent is evaporated under stirring at room temperature or at reduced pressure. NPs with this method can be obtained with or without the need of a surfactant, such as poly(vinyl alcohol) (PVA) 68,69 . In this method, the NPs size is strongly affected by operating parameters such as polymer concentration and molecular weight, the choice of organic solvent, solvent/non-solvent ratio, stirring speed and the presence of surfactants. Nanoprecipitation method is simple, reproducible, scalable and does not require the use of organic solvents considered as highly toxic. However, it leads to a lower polymer concentration in the final solution in comparison to other techniques and it is not advantageous for hydrophilic drugs 65 .

Salting-out technique consists in forming an emulsion between an organic solvent totally miscible with water (i.e. acetone, methanol, ethanol and acetonitrile) and an aqueous solution thanks to the salting-out effect 66 . The organic solvent contains the polymer and the drug and the aqueous phase contains a colloidal stabiliser and the salting-out agent. The salting-out agent is normally an electrolyte (i.e. magnesium chloride, calcium chloride, magnesium acetate or sucrose) which hinders the mixing of the two solvents.

The obtained emulsion is then diluted with a high volume of pure water which allow the diffusion of the organic phase in the aqueous one thanks to the lowering of salting-out agent concentration below a threshold.

Similarly to nanoprecipitation, interfacial turbulences, generated because of the sudden miscibility of the two solvents, lead to NPs formation. This technique is versatile, scalable and reproducible but it requires extensive NPs washing to eliminate the salting-out agent and leads to poor encapsulation of hydrophilic drugs. Moreover, the high concentration of salts needed to prepare the NPs can be incompatible with the drug stability 65 .

Emulsification diffusion method is very similar to salting-out but avoids the high salt concentration. The technique requires a first step of mutual saturation between the organic solvent (partially miscible with water, such as ethyl acetate or methyl ethyl ketone) and water. Polymer and drug are solubilised in the organic solvent whereas the stabiliser is added in water. The two solvents are than mixed together (usually, 1:2 o/w ratio) forming an emulsion which is broken by adding water as for salting-out. The water can contain an additional amount of stabiliser (PVA or poloxamer 188). Most common disadvantages are poor encapsulation efficiency of hydrophilic drugs and the necessity to concentrate the final NPs suspension. Finally this method requires longer processing times compared with nanoprecipitation 65 .

Emulsion evaporation technique is the first method proposed for polymeric NPs preparation in 1979 65 and is one of the most frequently used together with nanoprecipitation. This time a water-immiscible and volatile organic solvent such as dichloromethane (DCM) or ethyl acetate is required. The organic phase containing drug and polymer is emulsified with an aqueous solution containing a surfactant (oil in water -o/wemulsion).

Then the emulsion is broken to even smaller drops by applying an external high sheer-stress such as sonication or homogenisation. The solvent is then evaporated under magnetic stirring or reduced pressure leading to polymer precipitation and thus to NPs formation. NPs size is affected by the force and type of sheer-stress applied, by the polymer concentration, by drug/polymer ratio by organic solvent/water ratio and by the surfactant used. This method is generally highly efficient for hydrophobic drugs encapsulation but it is not suitable for hydrophilic molecules 65 .

Double emulsion evaporation technique has been developed starting from emulsion evaporation for the need

to encapsulate hydrophilic molecules. Interestingly, this method allows the encapsulation of both hydrophobic and hydrophilic molecules separately or simultaneously. The procedure is equivalent to that of emulsion evaporation, but an additional step is added at its beginning, leading to the formation of a w/o/w emulsion. Indeed, this first additional step consists in the solubilisation of the hydrophilic drug in a first aqueous solution that will represent the inner phase of the double emulsion. This water phase is emulsified with the organic solvent containing the polymer (and/or a lipophilic drug) and a lipophilic emulsifier. This first w/o emulsion is used as "organic phase" for the standard emulsion evaporation process. This method has been successfully used to encapsulate nucleic acid and proteins. It is worth noticing that this technique can be used also to obtain o/w/o emulsions. The principal issues of the technique lie in the difficult stabilisation of the double emulsion, in the high sheer-forces used, which can damage biomolecules, and in the frequent formation of some large non-uniform NPs 65 .

Polymeric NPs biological degradation, controlled release and safety profile

Although the mechanism of degradation of polymeric devices is influenced by many variables and may therefore be complex, a generalisation of the process can be given. Initially, the water penetrates from the surface into the centre of the device triggering the hydrolysis of the polymer. As the degradation proceeds, oligomers and monomers are cleaved from the polymer matrix and begin to erode away from the device 31 . The degradation occurs first in the amorphous domains and in a second moment in the crystalline regions. As a consequence, the overall crystallinity of the device increases during time thanks to the increased mobility of the partially degraded polymer and the realignment of polymer chains 21 . At this point, heterogeneous degradation occurs in large-sized devices 21,57 . The core begins to degrade faster than the surface because the monomers in the interior are not able to diffuse away, begin to accumulate and their carboxylic end groups lower the pH catalysing further hydrolysis in the inside of the device. In the case of implants, when the outer section of the device becomes thin enough, it breaks down and releases the degradation products 57 . Such mechanism has been shown to occur both in vitro and in vivo 70,71 . This bimodal degradation mechanism takes place also in the case of microparticles where the osmotic pressure increases until the "capsule" made of the remaining non-degraded polymer burst 57,72 . Oppositely, in small microparticles and in nanoparticles the degradation occurs homogeneously with an equal degradation rate in the core and at the surface probably thanks to a faster clearance of degradation products from small particles 21,57,73 . Therefore, NPs seems advantageous over microparticles because no low-pH inner pockets are formed, thus decreasing the risk of sensitive payload damages. A difference in the NPs degradation rate is usually observed between in vitro and in vivo conditions, with the in vivo one being faster 31 . This is likely due to the fact that enzymatic degradation contribute to the hydrolytic one after in vivo administration 31,74,75 . Interestingly, the intervention of the phagocytic cells of the immune system was found to play a major role in the degradation of NPs, thus allowing the selective targeting of such cells to fight against intracellular infections or to use them as "trojan horses" to guide NPs towards a specific target 40,76,77 . The trojan horse idea has been derived by scientists from the ability of some intracellular bacteria to behave the same (see Introduction Section 1.1). Furthermore, phagocytic cells have been shown to intervene also in the last steps of polymeric microparticles and implants degradation, when their residues reach a size small enough to be phagocytated 21,32,64 .

Since the degradation process of the polymeric NPs is already influenced by a wide variety of parameters, the prediction of drug release rate is even more difficult and often impossible. However, two drug release steps can usually be observed; an initial burst release of drug associated to NPs surface and a second phase of progressive release due to drug diffusion following particle erosion 31 . The relative relevance of the two phases is due to drug characteristics, drug concentration and polymer hydrophobicity which are typical of each system. Therefore, the evaluation of drug release process must be assessed on a case by case basis and the characteristics of the polymeric nanoparticles can be tuned to reach the desired properties and, if necessary, a controlled release 31,58,62 . Interestingly, different biodegradable polymers, such as PLGA and PCL, can be combined with each other to obtain new copolymers with optimised characteristics 59,62 . Moreover, different kind of "sensors" can be coupled to the polymeric NPs to achieve controlled drug release as response to an external stimulus (increase of blood sugars, change in pH, magnetic signal, etc.) 58 . Another intriguing strategy, which can provide controlled release, is the direct conjugation of the drug molecule to the polymer through a stimulus sensitive linkage which allow the drug release in a time or location-dependent manner (i.e. colon targeting where the cleavage of the bond is ensured by bacteria) 58,78 .

One of the main advantages of polymeric NPs lies in their high biocompatibility. PLGA has shown minimal systemic toxicity and excellent biocompatibility in vitro and in vivo. An important amount of data is available thanks to the success of these materials in drug delivery over the past decades. However, this is an active area of research as the safety of newly developed systems have to be systematically proven.

NPs administration has been performed by exploiting all the possible administration ways. Herein, some examples of safe NPs delivery will be reported. Orally administered polymeric NPs have been described to be able to release their drug cargo in the gastrointestinal lumen or to cross the intestinal barrier (generally if their size is lower than 500 nm) through Payer's patches, delivering the drug to the systemic circulation. Moreover, they were demonstrated to be safe both after in vitro and in vivo evaluations 29,79 . For example, no signs of toxicity or inflammation could be detected in liver, spleen and intestine after oral administration of estradiolloaded PLGA NPs to rats 80,81 . Moreover, polymeric NPs have been reported to be non-cytotoxic on a wide range of cells such as different types of epithelial cells (Caco-2, HeLa, Hepg-2) 82 , bronchial cells 83 and alveolar cells 84 . PLGA NPs resulted safe also after pulmonary administration in mice no matter their surface charge (neutral, positive or negative) 85 . Furthermore, PLGA NPs did not present any in vitro cito-or genotoxicity on different cell types 86 .

Concerning the intravenous administration, unmodified polymeric NPs are usually taken up few minutes after injection by the mononuclear phagocytic system, mainly in Kupffer cells of the liver, and by the spleen macrophages. Indeed, to increase their blood circulation time and decrease their recognition by the host immune system, di-block copolymers, principally with PEG, were synthesised enabling reaching their target 62 .

Polymeric NPs applications

The unique properties, including versatile degradation kinetics, established safety, and biocompatibility, make these polymers ideal for a wide variety of applications in medical field. Some of the applications PLGA and PCL devices have been marketed for, are sutures, bone fixatives and regeneration, dental materials, artificial skin and blood vessels, as well as proteins, nucleic acids and small molecules delivery 32,57,59,64,87,88 .

In drug delivery domain, polymeric NPs can successfully carry drugs for a broad range of diseases such as cancer, neurodegenerative disorders, cardiovascular disease, microbial or viral infections, diabetes and osteoporosis 32,66,87 . Polymeric NPs could provide solubilisation and stabilisation of insoluble or instable drugs or improve their pharmacodynamic and pharmacokinetic properties. Furthermore they could grant a better penetration of therapeutics inside cells concealing eventual drugs toxicity and helping in the fight against multidrug resistant cancer cells or bacteria 60,89,90 . PEG-copolymers could endow NPs with long circulation abilities thus allowing their intravenous administration and passive targeting 58,60 .

Importantly, active targeting of such NPs could also be achieved, by modifying their surface with moieties able to direct them to a specific body district or target. However, the feasibility and scale up possibilities of such decorated NPs must be taken into account. The more complex the NP system is, the longer will be the process of its approval, the higher the amount of toxicity tests required to allow commercialisation and the higher will be the cost of the marketed product.

CDs were demonstrated to be a family of cyclic oligosaccharides composed of glucopyranose units bounded together by α-1,4 linkages 92 . The most common natural (native) CDs are α-CD, β-CD, and γ-CD, which consist of six, seven, and eight glucopyranose units, respectively (Figure 5).

In view of the "chair" conformation of their units, CDs display the peculiar shape of a truncated cone (or toroidal shape) with the secondary hydroxyl groups extending from the wider edge and the primary ones from the narrower, in both cases oriented towards the cone exterior. This characteristic endows the CDs molecules with a hydrophilic outer surface and with an hydrophobic central cavity 93 (Figure 5). The lipophilicity of such inner surface has been estimated to be comparable with an aqueous ethanolic solution 93,94 . This unique structure renders them capable of forming inclusion complexes (host-guest) with a wide variety of hydrophobic guests, thus acting like molecular cages. The interaction between the CD (the host) and included molecule (its guest) is maintained thanks to a set of intermolecular interactions such as hydrogen bonds, hydrophobic interactions, steric interactions and van der Waals forces [START_REF] Rekharsky | Complexation thermodynamics of cyclodextrins[END_REF] . Such interactions are dependent upon the characteristics of the guest (hydrophobicity, hydrosolubility, ionization state and steric hindrance), those of the host (native or modified CDs) and those of the solution (solvent, pH, ionic strength, temperature, presence of a cosolvent, etc.) [START_REF] Szejtli | Cyclodextrin Technology[END_REF] and they lead to a state that is more thermodynamically stable overall.

Whereas there is a general consensus about these stabilising forces, some controversy surrounds the nature of the "driving force" which lead to the host-guest complex formation and especially the role of water molecules.

According to Szejtli [START_REF] Szejtli | Cyclodextrins and their inclusion complexes[END_REF] , the main driving force is given by the displacement of the high enthalpy water molecule, which form polar-apolar unfavourable interactions with the cavity, by the entering of the guest which is a more hydrophobic molecule and therefore leading to more favourable apolar-apolar bonding. Otherwise Liu and Guo questioned this theory by stating that the water in CDs cavity do not intervene to any extent in the complexation phenomena 92,[START_REF] Liu | The driving forces in the inclusion complexation of cyclodextrins[END_REF] .

Beside the role of water molecules in the inclusion process, studies generally agree that is the most hydrophobic part of the host molecule which is preferentially included in the cavity of the CDs and the encapsulated molecules are oriented in such a way as to maximize the contact between their hydrophobic parts and the apolar CD cavity 92 . The strength of the complexation phenomena and therefore their stability are key factors for CDs applications [START_REF] Stella | Mechanisms of drug release from cyclodextrin complexes[END_REF] . Indeed, as no covalent bonds are involved in the process of complex formation, the guest-CDs complexes, upon dilution, establish rapidly an equilibrium between dissociated and associated species so that an equilibrium with free guest molecules exists at any time.

The guest-CD complex stability is determined by a thermodynamic equilibrium association constant (Ka) or dissociation constant (Kd) which are an index of changes in physico-chemical properties of a compound upon inclusion. The majority of methods for determining K values are based on titrating changes in the physicochemical properties of the guest molecules within the CDs and then analysing the concentration dependencies 93,94 . Properties that can be titrated in this way include: aqueous solubility 100-103 , phase solubility measurements [104][105][106] , chemical reactivity and photochemical stability 106-108 , molar absorptivity and other optical properties (such as circular dichroism) [109][110][111][112] , NMR chemical shift 113,114 , pH-metric and calorimetric titration 115,116 and liquid chromatography retention times. Most commonly the order of magnitude of drugs-CDs binding constant is between 10 M -1 and 2 x 10 3 M -1 (occasionally 10 4 and very rarely higher than 10 5 ) 93,117 and the molar ratios of drug-CDs complexes are tipically 1:1, 1:2 or 2:1, host-guest respectively. Besides hostguest complexes, CDs are also well known to form non-inclusion complexes and self-assembling aggregates capable of dissolving molecules through micelle-like structures 92,93,118 .

CDs derivatives

Although native CDs and their complexes are hydrophilic, their water solubility, especially in case of β-CDs (around 18 mg/mL), can be rather limited. This is attributed to the crystal state of natural CDs and to their relatively high crystal lattice energy. The random substitution of hydroxyl groups even with hydrophobic moieties such as methoxy functions dramatically improve CDs solubility. The main reason of the aforementioned CDs derivatives solubility enhancement (generally to more than 500 mg/mL) lies in the fact that chemical modification often transforms crystalline CDs into amorphous mixtures of isomeric derivatives 93 .

More generally, the aim of CDs derivatisation consist in: i) the improvement of the solubility of CDs and their complexes, ii) the improvement of the fitting and association between the CD and its guest, iii) the attachment of specific moieties to confer some catalytic, biological or therapeutic activity to CDs and iv) the immobilisation of CDs onto polymers (for example for chromatographic purposes) and v) the reduction of toxicity 119 .

The possible number of CDs derivatives is virtually unlimited as α-, β-and γ-CDs bear 18, 21 and 24 substitutable hydroxyl groups, respectively 92 . In reality, the majority of these derivatives will never find application especially because of their too high production costs and for the impossibility of scaling-up complicated multi-steps reactions 119 . Considerable efforts have been thus made over the years to optimise the synthesis in order to decrease the amount of reaction steps and to develop selective reactions to limit the postsynthetic steps (e.g. purification) to the essential 120 .

The main derivatives of pharmaceutical interest, which are industrially produced, include the hydroxypropyl derivatives (HP-β-CD and HP-γ-CD), randomly methylated βCD (RM-β-CD or RAMEB), sulfobutylether βCD sodium salt (SBE-β-CD) and the so-called branched CDs, such as maltosyl-βCD (M-β-CD) 93 .

These synthetized derivatives, both hydrophilic (e.g. HP-β-CD) and lipophilic (e.g. RM-β-CD) 121 could find successful applications in a wide variety of fields, such as analytical chemistry, pharmaceutical, cosmetic and food industry.

CD-based crosslinked polymers and nanoassemblies for drug delivery

CDs have been successfully employed to create novel nanomaterials 122 such as nanoparticles, nanosponges, nanomicelles, nanovesicles and hydrogels founding applications especially in drug delivery field 23,123 .

CD-based crosslinked polymers as well as CD-based nano-objects have emerged as an innovative approach to overcome the disadvantages related with native CDs while keeping or even improving their advantages.

Three main strategies can be identified in the preparation of cross-linked network: i) direct cross-linking of CDs (condensation with a cross-linker); ii) copolymerization of CDs with vinyl-or acrylic-comonomers, and iii) grafting of CDs to preformed polymer (i.e. PLGA, PLA, PCL, PEG etc..) 124,125 .

When taking into consideration the first strategy, cross-linked CD polymers can be prepared by using different cross-linking agents, such as aldehydes, isocyanates or epoxides 124 . Epichlorohydrin (EPI) is the first and probably the most investigated between them. It possesses two reactive functional groups which, under alkaline conditions, can react with the hydroxyl groups of CDs forming polymeric CDs (pCDs). This pCDs are normally obtained in the form of microgels. However, hydrosoluble pCDs can be obtained when stopping the reaction in the vicinity of the gelation. Interestingly, TEM investigations showed that they were under the form of 10-15 nm NPs in water 126,127 (Figure 6). pβCDs NPs synthetized by this way draw particular attention thanks to their higher water solubility in comparison to native βCDs and thanks to their ability to greatly enhance the apparent solubility of several guests 128 . This type of pCDs has also been widely employed to construct nanospheres and nanogels via host-guest interaction mediated self-assembly using hydrophobically modified hydrophilic polymers or hydrophobic polymers as guests 125 . For example, nanogels of 100-200 nm have been prepared by spontaneous selfassociation of pβCDs with dodecyl-modified dextran in aqueous solution and has been used to encapsulate hydrophilic molecules [129][130][131] .

Another intriguing CDs network is represented by polypseudorotaxanes, inclusion complexes in which CDs are threaded onto a polymer chain 125,129 .

Interestingly, CDs could also be used to decorate nano or micro particles prepared with other materials, such as mesoporous silica NPs or metal organic frameworks (MOFs) 132,133 , and to prepare hybrid organic-inorganic NPs, such as CD-MOFs using the CDs as organic ligands 134 .

Cyclodextrins biological degradation and safety profile

In solid state CDs can be stored for several years at room temperature without any detectable degradation. In aqueous solution instead, they can undergo to both non-enzymatic and enzymatic degradation. The nonenzymatic one requires quite strong acidic conditions (t1/2 15h at pH1.1 and 70°C) and it consists in the acidcatalysed hydrolysis of the α-acetal linkage, whilst they are chemically stable in basic and neutral aqueous solution. Regarding the enzymatic hydrolysis, CDs are resistant to β-amylases whereas they are slowly degraded by α-amylases, which are present mainly in pancreatic juice and saliva. The γ-CDs are more sensitive to such enzymes and therefore, after oral administration, they are almost completely digested in the gastrointestinal tract whereas α-and β-CDs are digested mostly by colon bacteria. Free CDs are generally digested faster than CDs complex 93 .

Most of the pharmaceutically interesting CDs are hydrophilic and have high molecular weights. Consequently, they are poorly absorbed by the gastrointestinal tract (bioavailability below 4%) and therefore practically nontoxic after oral administration, with the only exception of RM-β-CDs. These methylated CDs derivatives display a higher lipophilicity (as compared with other derivatives and native CDs) and a higher oral bioavailability which leads often to some toxicity, probably due to their interactions with cell lipids 93,117 . The parenteral administration of CDs is also possible but more restricted. CDs are cleared rapidly from the bloodstream and are renally excreted intact. However, both native α-and β-CDs, as well as RM-β-CDs, show renal toxicity probably due to the formation of cholesterol-CDs insoluble complexes which aggregate and crystallise in the kidneys 117,135 . Moreover, an haemolytic effect on human erythrocytes is observed from some derivatives 93 . Fewer studies have also been performed on nasal, pulmonar, rectal, dermal and ocular administration [136][137][138] . The HP-β-CDs and SBE-β-CDs resulted the more versatile and therefore used for all the administration ways. The α-CDs are applied especially for parenteral route (excipient in prostaglandin infusion), γ-CDs for oral and dermal formulations and RM-β-CDs for nasal formulation 24,139 .

The availability of information regarding the toxicity of CDs supramolecular complexes, especially on humans, is limited due to their recent development. Nonetheless, it is worth mentioning that polymeric β-CDs NPs have reached clinical trials for the administration of siRNA for cancer treatment showing promising results and no toxicity to this day 140,141 .

Application fields of CDs and their derivatives

As already mentioned, CDs and their derivatives have a very broad variety of applications in almost every scientific field including chemistry, catalysis, pharmacy, medicine, foods, cosmetics, biotechnology and textile industry 142 . Herein some examples of such applications are provided.

In chemical and catalysis field CDs are used to increase molecule stability (i.e. to light or oxygen) and solubility, to produce HPLC columns allowing chiral enantiomers separation and to catalyse selective organic reactions 143,144 . In the food industry CDs are useful to prepare cholesterol-free products, stabilise volatile or unstable compounds and reduce unwanted tastes and odours 144,145 . These features are also useful for cosmetic industry applications 146 . Moreover, they demonstrated their ability in facilitating the handling and storage of toxic substances (pesticides) and in the extraction of pollutants from the environment 144 .

The most attractive applications in our case consist in the ones within pharmaceutical and biomedical fields where they are mainly employed to form inclusion complexes with drug molecules 24,125 . CDs can increase drug solubility and stability 147 , which are the main technological limitations of free drugs. Interestingly, CDs crosslinked polymeric NPs allowed increasing the apparent solubility of challenging crystalline drugs thus solving formulation issues 128,131 . Moreover, CDs are used to enhance drugs absorption 148 , mask their unpleasant smell or taste 149 , control drug release profiles 150 , decrease their toxicity 24 and improve their permeability across biological barrier 151 . Another interesting application lies in the ability of sugammadex, a γ-CDs derivative, to act as antidote against steroids curare-like agents (rocuronium, vecuronium) by forming an inactive complex with these molecules which are used as muscle relaxants in modern anesthesia 92 .

Nanoparticles characterisation techniques

The development of a drug delivery system faces several challenges in order to ideally obtain the release of the drug in a time controlled manner, in the vicinity of the target and limiting the possible drug adverse effects.

These objectives have to be achieved together with maintaining nanocarrier biocompatibility and stability until its purpose is fulfilled. Before reaching its target, the nanocarrier distributes inside the body possibly having to cross epithelial barriers. Once the target is reached, the NP still need to execute its effect and subsequently have to be degraded and/or cleared by the organism 152 .

Size, shape and surface properties are the main key factors, beside the NPs composition itself, that strongly influence NPs stability and their interactions with living systems.

The size is the first crucial factor normally assessed. Frequently, sizing techniques determine the hydrodynamic diameter of the particle rather than its dry one. The hydrodynamic diameter is basically the size of the hydrated particle and it is defined as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured 153 . A particle, dispersed in a solution, is surrounded by a dipole double hydration layer which is tightly connected to it and moves with the particle in its Brownian motion. The thickness of this layer depends on various factors, such as the electrical conductivity of the liquid (the higher the ionic strength, the thinner the dipole layer). As a consequence, if the particles surface is decorated by moieties forming a hydrated corona (PEGylated layer, surfactant, lipid layer), this corona will be accounted for as a part of the particles. This will lead to a higher diameter in comparison to that determined by the techniques which measure the dry one.

Surface properties are assessed in a first instance by measuring the surface charge of the NPs. Indeed, surface charge has a potential influence on receptor binding and physiological barrier crossing. Moreover, it governs the dispersion stability, agglomeration and aggregation 154 .

Aggregation more than agglomeration have a high impact on NPs exploitability. Indeed, whereas agglomerates are due to the association of NPs through week interactions and are thus easily redispersed to single NPs after vortexing or sonicating, aggregates are formed by tight NPs association, or even fusion, and are therefore non redispersible. Aggregation have to be cautiously avoided and its occurrence can be foreseen by estimating NPs Zeta potential. The zeta potential is the value of the potential at a distance from the surface known as the slipping plane. This is the distance within which the electrical double layer around the NP (the same that compose NPs hydrodynamic diameter) move as part of it, and from the charge point of view defines the particle. Indeed, it is important to highlight that NPs interact according to the magnitude of the zeta potential not their surface charge. Stability dividing line is generally considered to be ± 30mV for aqueous systems and correspond to the point where repulsive forces between particles dominate over attractive ones [155][156][157] . Zeta potential can be affected by changes in pH, conductivity (concentration and/or type of salt) and changes in the concentration of a formulation component (e.g. polymer, surfactant).

In addition, the shape of NPs plays and important role affecting cellular uptake, retention in tissue and degradation. NPs shape is normally visualised thanks to electron microscopies 154 .

A wide range of techniques are currently available to characterise all these aspects of NPs suspensions.

However, as none of them is perfect, NPs characterisation with at least two techniques, possibly based on different technical principles, is of key importance to insure a correct assessment of NPs size, shape and surface properties. The association of multiple technique allows combining their strong point and limiting the impact of possible instrumental error, ambiguities and results misinterpretation 152,158 .

Herein, we will focus on the basic principles, features and drawbacks of dynamic light scattering (DLS), as bulk technique, and on nanoparticle tracking analysis (NTA), tuneable resistive pulse sensing (TRPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as particle by particle techniques. All the described characterisation methods typically require NPs dilution to meet each instrument specifications.

DLS

DLS, also known as photon correlation spectroscopy, is a well-established technique to assess rapidly the mean hydrodynamic diameter, size distribution, polydispersity index (PdI) and zeta potential (ZP) of a NPs suspension. This method is the best suited for routine measurements of NPs between around 50 nm and 1 µm (although these limits are sample dependent) 152 and has been widely used thanks to its applicability to a broad spectrum of particles and dispersion media. DLS determines the hydrodynamic diameter of NPs by measuring the changes in the speckle pattern produced by particles scattering light as they undergo to Brownian motions in solution 159 .

During DLS measurements all the NPs in solution that are hit by the laser of the instrument diffract the light in all directions (Figure 7). The diffracted light coming from different particles can interfere constructively of destructively giving a speckle pattern. All the speckle patterns resulting from illumination of the sample at short time intervals is analysed by an autocorrelator which compare the light intensity in each spot over time 159 .

Changes in speckle patterns are due to fluctuations in the intensity of scattered light which in turn are due to NPs undergoing Brownian motion. However, to precisely perform these conversions, knowledge about NPs refractive index is required. NTA measures the hydrodynamic diameter of individual NPs by employing a microscope and a camera to detect and record the light diffracted by a small volume of sample. Thus, NPs are visualised as bright spots (white spots in the black field) and followed in their Brownian motions (red lines = NPs trajectories) by the instrument software. The software counts individually the bright spots and, from their trajectories, it calculates NPs size (independent from their refractive index). NTA plots NPs concentration (particles/mL) over their size. TRPS is a Coulter-type counter which measures directly NPs size and concentration on a particle by particle basis. A voltage is applied across a tuneable pore, filled of electrolyte, thus generating an ionic current. The NPs, driven by this current, traverse the pore causing a transient blockade (yellow peaks). The magnitude of this blockade is linearly proportional to NPs volume and the number of peaks corresponds to the number of particles. TRPS plots NPs concentration (particles/mL) over their diameter.

The analysis of these fluctuations by the instrument allow the generation of a correlation function which is used to calculate the diffusion coefficient (Dt) of NPs in the samples and thus derive their mean size through Stokes-Einstein equation (Equation 1):

(1)

d T k D B t  3 =
Where "T" is the sample temperature, "η" it viscosity, "kB" the Boltzmann constant and "d" the particle diameter. To obtain the diffusion coefficient, the correlation function is mathematically treated. This treatment is especially complex for polydisperse samples which are more complicate to be analysed. Different algorithms have been developed to ease the interpretation of results and minimise the errors: Cumulants analysis, Nonnegative least squares (NNLS) and CONTIN 159,160 .

The first value derived from the analysis of correlation function with the cumulants method is the z-Average diameter (Z Av) and it is correlated to a Polydispersity Index (PdI) which express the broadness of the size distribution. Traditionally in the NPs field, a sample is considered monodisperse when its PdI is at least lower than 0.2, even if a PdI lower than 0.1 is preferable 152 . These values are historically the most reported in bibliography and are defined by specific ISO regulations (ISO 13321 and ISO 22412) 152,153 . ZAv is suitable only for monodisperse samples and is highly sensitive to the presence of small portion of aggregates. This sensitivity can sometimes result as an advantage to assess monomodal NPs sample stability over time.

DLS instruments can present results through different types of size distribution (Figure 7). Intensity distribution (obtained from different patented methods according to the employed instrument, i.e. NNLS method for Malvern Zetasizer) is obtained by plotting the relative intensity of light scattered by NPs versus size. This distribution is also very sensitive to agglomerates and knowledge on solvent refractive index (RI) and viscosity are needed. The intensity distribution can be converted to volume distribution or number distribution using the Mie theory 153 . Such conversions are necessary to compare DLS results with that of other techniques but they require a knowledge of the RI of the NPs, which is very difficult to be obtained.

Beside NPs size, DLS is also able to assess NPs Zeta Potential (ZP). Indeed, the existence of electrical charges on NPs surface lead to the exhibition of a certain effects under the influence of an applied electric field. Thus, to determine NPs ZP, an electric field is applied to NPs dispersion and the velocity of particles moving towards the electrodes is measured normally using a laser interferometric technique. The electrophoretic mobility of NPs is then calculated, and from this, their ZP 154 .

The main drawbacks of DLS lie in the necessity of using deconvolution algorithms to analyse the results, on its limited capacity to accurately describe polydisperse samples, in the need of knowing the RI and viscosity of the sample and in its high sensitivity to the presence of larger particles in a sample 159,161 . Indeed, as the amount of scattered light varies strongly with the NPs size, a small number of large NPs scatters more light than a big number of small NPs. This phenomenon can significantly bias the results artificially shifting the intensity peak towards bigger diameters and thus leading to an overestimation of NPs size 161 .

NTA

NTA determines the hydrodynamic diameter of NPs in a similar fashion as DLS. Indeed, NTA uses both light scattering and Brownian motion to obtain NPs size distribution. However, while DLS is a bulk method, NTA is a particle-by-particle technique that offers the ability to directly visualise, size and count nanoparticles 162 .

An incident laser beam is passed in the sample chamber through a prism edged glass and illuminates a small volume of the sample (20 µL) (Figure 7). A pump is connected to the sample chamber to insure a slow renewal of NPs suspension and hence the analysis of "new" NPs. NPs are visualised as bright spots thanks to a microscope and their Brownian movements are registered thanks to a camera (Figure 7). NPs movements in x and y directions are analysed by the software and the NPs diffusion coefficient is determined. Also NTA uses Stokes-Einstein equation (Equation 1) to determine the hydrodynamic NPs diameter but, in contrast to DLS, there is no necessity of mathematical data deconvolution. Furthermore, the instrument is able to provide number distribution of NPs without the need of knowing their RI. This represents one of the major advantages of the technique over DLS. Moreover, the intensity of the individual bright spots corresponding to each NP is not an indication of their diameter. The diameter is calculated from the trajectories of the particles. Therefore, NTA does not suffer from the intensity weighting problems which strongly affect DLS leading to NPs size overestimation.

Additionally, as the volume of solution analysed by the instrument is known and the NPs are counted during the measurement, the concentration of the sample in particles/mL can be calculated 161 (Figure 7).

Another very interesting feature of the instrument is the possibility to introduce a filter to the sample so that only fluorescent NPs (wavelength dependent from the selected laser source) are detected. This peculiarity allows the detection and the measurement of NPs sizes in complex solutions containing other scattering moieties besides NPs, such as biological samples (i.e. cell culture media) 161 .

The detection limits of NTA depend on the sample and on the intensity of the diffracted light. For materials with high refractive index (i.e. colloidal gold and silver) is possible to accurately measure sizes down to 10-15 nm. For very week scattering materials (i.e. polymers, liposomes) instead, the smallest detected particle is around 40 nm. The upper limit is found around 1-2 µm when the particles move too slow that the tracking of their trajectory became inaccurate. Concentrations in the range of 10 7 to 10 10 particles/mL are needed to insure a correct assessment of NPs concentration. Despite all its advantageous features, also NTA have some limitations. The analysis requires a high dilution of the samples so that the NPs are distant enough to be detected by the software as different objects. This necessitates to increase measurement times in comparison to DLS to obtain statistically significant data.

TRPS

TRPS is a Coulter-type counter technique which is recently gaining more interest in NPs sizing. This technique provides direct measure of NPs size, concentration and surface charge on a particle by particle basis.

Historically, resistive pulse sensor or coulter counters found their main application in automated cell counting and sizing and for many years the only possible application of this technique in particles field was restricted to microparticles 159 . Thanks to the recent advances in nanofabrication of pores its applications has been extended to NPs and the instrument demonstrated to be able to efficiently resolve polydisperse (multimodal) samples of close mean diameter 159 .

A voltage is applied across a tuneable pore, filled of electrolyte, thus generating an ionic current. The NPs, driven by this current, traverse the pore with a speed dependent on their ZP, causing a transient blockage in the ionic current (Figure 7). The magnitude of this blockade is linearly proportional to NPs volume (proportional to the cubic of NPs diameter according to Equation 2) allowing size determination with high resolution.

(2)

∆𝑅 𝑅 = 𝑑 3 𝐷 2 𝐿
R is the pore resistance and D its diameter, d represents the particle diameter and L is the pore length for the simple case of a spherical particle and a cylindrical pore 159 . For more complex situations, additional parameters have to be taken into account 163 .

The instrument is also capable of measuring NPs concentration by multiplying the NPs count rate by the fluid flow rate which is dependent to the pressure applied with the instrument. Additionally, NPs ZP can be measured on the base of the resistive pulse duration which allow the assessment of NPs electrophoretic mobility and the calculation of the ZP using Smoluchowski equation 164 .

The main advantage over the traditional coulter counters is that the pore size can be adjusted in real-time depending on the NPs diameter by radially stretching the membrane. The pore is formed by mechanical puncture of a micron sized pore into an elastic membrane and its tuneability offers the possibility to increase the measurement sensitivity and resolution 165 . However, in contrast with the other techniques presented here, a calibration of the pore size with a standard NPs suspension is needed prior to each sample measurement thus increasing the measurement time.

As for every other technique, also TRPS display some drawbacks. An inherent disadvantage of the technique is the issue of pore blockages due to particles too large to fit through the pore, to particles attracted by the pore surface and to the presence of small aggregates or bubbles. Such blockages can result in time-consuming cleaning processes and in the necessity of repeating the sample measurements several times. Moreover, some tiny NPs can be undetected because they do not create a significant resistive pulse event 159 . Additionally, the choice of the appropriate electrolyte is crucial because it has to be conductive enough to allow the measurement but it does not have to impact the sample stability (i.e. causing particles aggregation/agglomeration).

SEM and TEM

Electron microscopy (EM) is a technique which uses beams of accelerated electrons as illuminating source and electrostatic or electromagnetic lenses, to generate images. This technique has a much higher resolution (lower than 1 nm) in comparison to optical microscopy because of the much shorter wavelength of electrons (up to 100,000 times shorter) in comparison to visible light photons 154 . In EM, measurements are performed under ultra-high vacuum because the molecules which compose air can scatter electrons and therefore make the measurement impossible. It is important to highlight that EM techniques generally measure the sample in dried or frozen state thus leading to measure the dried mean diameter of NPs instead of the hydrodynamic one.

The main and most used types of EM are SEM and TEM which principally differ for the portion of electrons that is collected by the detector after contact with the sample. SEM is a surface technique and the specimen is scanned with a focused electron beam. When the electrons come in contact with the sample, part of their energy is lost through different mechanisms. This energy loss is converted in the emission of elastic scattering electrons, namely backscattered electrons, inelastic scattering of electrons, called low-energy secondary electrons, light emission (cathodoluminescence) or X-ray emission.

All of them bring information about the properties of the sample surface allowing the reconstruction of sample topography and providing some information about sample composition. Among these emissions, the detection of the secondary electrons is the most common mode, achieving a resolving power of around 1 nm 154 . SEM measurement often requires a quite complex sample preparation. Firstly, the sample needs to be dried which may cause shrinkage or flattening of NPs thus altering their size 154 . Secondly, a coating of an ultrathin electrically conductive material is often required for non-conductive samples that tend to acquire charge and deflect insufficiently the electron beam leading to artefacts. Moreover, the vacuum together with the drying process may cause the formation of crystals or thin film of other dissolved species present in the sample and thus obscure some details. Conductive coating can be avoided by using low voltage mode of SEM operation.

TEM is the most used EM technique for imaging nanomaterials. A high-voltage electron beam, usually

produced by an electron gun, is transmitted through a very thin sample layer. Electrons are in part transmitted and in part scattered by the sample. The image is then focused onto an imaging device (fluorescent screen, photographic film, etc.) 154 . TEM allows imaging internal features of the sample and not only the sample surface as in the case of SEM.

Moreover, it provides an even higher resolution than SEM and can be coupled to a wide variety of analytical techniques to acquire additional information.

The main drawback of TEM lies in the need of extremely thin specimen sections (around 100 nm) to achieve electron transparency and often, for organic polymers, the use of staining with heavy atoms to achieve sufficient contrast. Negative stain TEM brings along the potential of artefacts 159 . Furthermore, the high energy electron beam can damage biological and polymer based samples 166 . The development of cryogenic preparation methods adapted to hydrated samples (i.e. high pressure freezing, freeze fractures, cryoultramicrotomy..) have further increased the sample preparation time but they have also considerably improved the ability of TEM to analyse objects, while maintaining optimum conditions that preserve the initial structure 167 . SEM and TEM microscopies are valuable techniques to acquire information on NPs shape. However, when it comes to statistical analysis of NPs size distribution, they are efficient (timely and costly) only if the process can be automated to measure a representative number of particles. The process automatization is anyhow possible only for very well dispersed materials, which is often not the case for NPs due to the formation of aggregates or agglomerates during the sample preparation for EM 159 .

Nanoparticles detection in cells

Shedding lights on the interaction between NPs and living systems is of outmost importance in drug delivery applications. Multiple parameters (i.e. administration route, dose frequency and concentration, etc.) have to be carefully adjusted in order to increase the probability of reaching an active concentration at the target site. Indeed, once NPs are administered, they distribute inside the body possibly having to cross multiple barriers before being able to reach their target. Moreover, once the target is finally reached, NPs interact specifically with the cells basing on their physico-chemical properties.

Composition, size, shape and surface properties of NPs are the key factors which mainly determine their ability to cross tissue barriers and their cell binding, uptake and intracellular fate. An inadequate knowledge of NPscells interaction can determine the failure of the system. Thus, advanced imaging methodologies are employed to investigate the fate of engineered NPs when they come into contact with cells.

Fluorescence microscopy together with electron microscopy represent the most widely used techniques in the intracellular NPs detection field.

Fluorescence microscopy has proved over the years to be an essential tool in countless biomedical applications 168 . In particular, confocal laser scanning microscopy enables for 3D multicolour imaging in living cells, but its resolution is restricted by diffraction limit to around 200 nm depending on the wavelength and on the numerical aperture of the employed microscope. Thus, single NPs are not detectable. To overcome such limitation, a variety of super resolution techniques have been developed over the years 169,170 . Among them, structured illumination microscopy (SIM), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM) and photoactivation localization microscopy (PALM) are the most used to image cellular structures with sub-diffraction resolution and were successfully adapted also to NPs detection 171 . The main advantage of such techniques lies in the possibility of separately labelling NPs and cell membrane or subcellular compartments thus allowing unveiling NPs precise localisation within cells, together with their uptake mechanism 171 . SIM is the less resolutive of the listed techniques (lateral resolution ̴ 100 nm and axial resolution ̴ 200 nm) and the data processing is quite complex, but it has the advantage of using standard organic dyes. STED displays a higher lateral resolution ( ̴ 30-50 nm) and is able to acquire images at high speed but it still has a limited axial resolution ( ̴ 30-600 nm), requires the use of specific organic dyes and a high label density 172 . Finally, PALM and STORM provide the highest resolution (both lateral and axial ̴ 10-40 nm) but the image acquisition is quite slow, the data processing complex and they both need the use of very particular fluorescent dyes. PALM makes use of photo-activable proteins while STORM uses the photoswitching of pairs of cyanine dyes or blinking cyanine dyes. All these dyes are prone to cross talk and requires high labelling density 172 . The use of labelling agents has greatly facilitated the investigation of complex biological interactions. However, in the case of NPs, the fluorescent dyes, either physically encapsulated or covalently grafted 173,174 , can lead to several inconveniences 168 . Indeed, the physical encapsulation of fluorescent probes is prone to the leakage or even to the transfer of the fluorophore to cell membrane [175][176][177][178] whereas the covalent linkage is more reliable, but it can lead to changes in the size, shape or surface properties of the particles. These changes are well known to alter the interaction (uptake and intracellular fate) of labelled-NPs with cells and tissues in comparison to plane NPs [179][180][181][182][183] . Of note, finding fluorescent dyes compatible with encapsulation or conjugation to NPs together with meeting the specific criteria for super-resolution techniques can be quite challenging thus complicating or even hampering the development of suitable labelled NPs. Furthermore, NPs detection in biological media can be hampered by cells autofluorescence, particles scattering or photobleaching 184 .

The other main imaging technique, extensively used to localise NPs in subcellular compartments, is TEM 168 . This technique possesses an impressively high spatial resolution (down to 0.1 nm) allowing discriminating subcellular compartments with very high definition. Indeed, this method was the first one to enable visualising molecular events such as NPs engulfment inside cells and to clearly image NPs precise subcellular localisation 171 . Moreover, TEM is sufficiently sensitive to differentiate isolated nanoparticles from the corresponding aggregates 185 . Despite the clear potential advantages of using such technique, a number of limitations remains. Indeed, biological samples need complex preparation including chemical fixation, dehydration and embedding in a polymer resin or freezing process (cryo-TEM) to allow stabilising the sample enough for ultra-sectioning 185 . Among these sample preparation processes, cryo-TEM is particularly convenient as it allows to reduce the presence of artefacts in comparison to chemical fixation. Nonetheless the long sample preparation and prolonged image analysis greatly limit the analytical throughput of this technique.

In addition, TEM is only suitable for electron-dense NPs thus obliging to label organic particles (such as PLA/PLGA/PCL NPs) by encapsulating electron-dense material that can provide sufficient contrast 186,187 .

It is of outmost importance to highlight that both fluorescence and TEM microscopies rely on the use of labelling agents or material modification in order to detect the particles internalised by cells. As a consequence, they provide an indirect observation of the object of interest through the signal of the label. Indeed, the moiety detected and followed by fluorescent or electron microscopy throughout the experiments is the molecular probe and not the particles itself. Thus, at any moment, the integrity of the NPs cannot be proven and the dye, separated from the particle, could be followed instead of the NPs.

To overcome such problematics, there has been a tremendous interest, during the last 20 years, in developing innovative techniques, or combining the existing ones, to provide direct simultaneous imaging and chemical identification of NPs avoiding the use of labelling. This improvement could be particularly useful for the detection of polymeric organic NPs, such as the ones made of PLA, PLGA or PCL, which do not possess any contrast property.

For example, nano-scale secondary ion mass spectrometry (NanoSIMS) 188,189 in correlation with TEM 190 has recently emerged for its ability to offer high imaging resolution (less than 100 nm) 190 together with chemical characterisation. However, this technique is destructive, expensive, demand complex and time-consuming experimental procedure, and an isotopic labelling of the element of interest is still necessary.

The first technique able to detect unlabelled polymeric particles in cells was Raman microspectroscopy, which couples Raman spectroscopy and optical microscopy, offering the possibility to perform imaging together with chemical analysis 191 . Using the Raman signal, described as a modulation of the scattered light by molecular vibrations, the subcellular architecture as well as intracellular dynamics of polymeric NPs could be scrutinised 192 . Some studies have also been performed on the intracellular degradation of such NPs and on the subcellular drug delivery 191,192 . Indeed, an important feature of this technique is the possibility to perform the measurements in liquid media and even on living cells in contrast for example with TEM 193 . However, also Raman microspectroscopy displays some limitations. In fact, its signal is usually overwhelmed by the large autofluorescence of the cells and irreversible photo-damages are induced to the sample when trying to circumvent this issue by increasing the acquisition time or the incident power 194 .

Moreover Raman microscopy resolution is limited by light diffraction to around 300 nm -1 µm, depending on the excitation source 168,195 . Thus, particle agglomerates rather than individualised NPs are detected 191,192,195 . Therefore, given the small size of the polymeric NPs (usually less than 200 nm), part of information is lost 168 .

Chapter I SECTION A: Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles (Paper 1)

SECTION B: Efficient loading of ethionamide in cyclodextrin-based carriers offers enhanced solubility and inhibition of drug crystallization (Paper 2)

SECTION A:

Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles (Paper 1)

General objectives

Tuberculosis (TB) is one of the most life-threatening infections caused by an intracellular pathogen which is able to colonize and multiply inside alveolar macrophages in host lungs.

In an attempt to develop an adapted treatment of TB, we report in this study the design of an engineered Despite their high drug loading (around 36% and 26% for ETH and Booster, respectively), PLA NPs could not totally avoid ETH crystallization during storage.

On the other hand, pCD NPs displayed a lower drug loading but they could incorporate [ETH:Booster] at a molecular level (hampering ETH crystallization) by a "green" method totally solvent-free.

The efficacy of the formulations was evaluated in TB-infected macrophages using an automated confocal highcontent screening platform, showing that the drugs maintained their activity after incorporation in the NPs.

Given these positive results, the successful passage of the developed formulations through a Microsprayer®, a device for the endotracheal administration in rodents, was carefully assessed. The pCD NPs could be reproducibly passed through the device without any loss of material and were thus selected for in vivo studies.

The pCD NPs suspensions could be administered directly into mouse lungs via the endotracheal route. We first show, using histology and flow cytometry studies, that repeated administrations of pCD NPs had no toxic effect on lung homeostasis. Then, in a mice model of M. tuberculosis infection, we obtained a dramatic 3-log decrease of the pulmonary bacterial load after 6 treatments as compared to untreated mice.

This study paves the way for a future use of the CD-based NPs for the pulmonary delivery of the potent 72 The first attempt of formulation has been performed using P1 (1.43 mg/mL polymer in suspension). The encapsulation of Ethionamide (ETH) alone was studied by trying to encapsulate increasing concentrations of this drug (from 0.07 to 0.71 mg/mL). The results are shown in the Supplementary Fig. S2. 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 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.

Chapter I -SECTION A -Supplementary Information 91

Supplementary Table S1 -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.

Formulation

pCD NPs -Stability and release properties

The drug-loaded particles were stable upon storage and drugs were released only upon dilution of the NPs suspensions. Indeed, the drug molecules inside pCD NPs are in equilibrium with the ones in the suspension medium. Both ETH and Booster, poorly soluble in aqueous solution, strongly bound to pCD NPs. For instance, if the NPs are diluted ten times, one can calculate that around 90% of Booster will still be associated to the NPs.

The mechanism of drug release from pCD NPs, based on a partition mechanisms, has been thoroughly described in our previous publications [1][2][3] .

94

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. In Supplementary Table S2 we present the results of 3 independent in vivo experiments after 2 weeks of treatment as described in the manuscript. In the 3 experiments we observed a significant decrease in the pulmonary bacterial load after treatment with ETH and [ETH:Booster] encapsulated in pCD NPs. The Booster named BDM41906 (5,5,5-

Supplementary

Trifluoro-1-[4-(3-thiazol-2-yl-1,2,4-oxadiazol-5-yl)piperidin-1-yl]
pentan-1-one) was synthesized as previously described 4 .

β-CD was kindly supplied by Roquette, Lestrem, France. pCD NPs of around 10 nm were produced as previously described 5,6 by crosslinking β-CD under strongly alkaline conditions with epichlorohydrin (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 7 . 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 coencapsulation, 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 8 . 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.

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 andS5).

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 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. 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 8,9 .

Cryogenic transmission electron microscopy (cryo-TEM

(

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). 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 (781091, 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% CO2, 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 (IC50) 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 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 amikacin (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% CO2. 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 10 to extract the bacterial area and the number of cells.

Intramacrophage assay data analysis

The intracellular 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 as shown in the equation 3. 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 % inhibition versus the ETH concentration was determined with GraphPad Prism 5.0 software and the IC50 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 received ethical approval by French Committee on Animal Experimentation and all experiments were performed in accordance with relevant guidelines and regulations.

Lung histology

Infected or uninfected 8-week-old mice were divided in groups and endotracheally administered with water (vehicle) or pCD NPs (Fig 5a, Fig. 6).

At the determined end-point mice were euthanized, 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 for anatomopathology.

Flow cytometry

Harvested lungs were 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 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), APC-coupled RB6-8C5 (anti-GR1), phycoérythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APCcoupled 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.

Efficacy studies

8-week-old 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). 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. Body weight were recorded after each treatment (Fig. 5b). 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 weighed. 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 11 . 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 euthanized 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. Represented data are mean values ± standard deviation of one representative experiment from three independent experiments. Results for each independent experiment were summarized in Supplementary Table S2. 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 (Dt) 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 according to their Brownian motion. More than 2000 NPs are followed in each experiment.

SECTION B:

Efficient loading of ethionamide in cyclodextrin-based carriers offers enhanced solubility and inhibition of drug crystallization (Paper 2)

General objectives

The characterisation methods employed in Chapter I -Section A for PLA NPs were not adapted to describe the characteristics of pCD NPs with the exception of cryo-TEM. Indeed, pCD NPs have mean diameters of only around 10-15 nm and a high water content. Therefore, a complementary study has been performed here to unravel the type and strength of interactions occurring between ETH and pCD NPs, hereby exceptionally named pβCyD to distinguish them from CD which stand for circular dichroism.

We explored here the possibility of incorporating ETH in a molecular state in two different CyD polymeric carriers by using a combination of spectroscopic techniques. As previously described, ETH suffers from poor solubility in water and strong tendency to crystallize which could be overcome thanks to the incorporation in pβCyD carriers by a green approach. The binding of ETH to the two polymeric βCyD carriers was investigated in neutral aqueous medium by means of circular dichroism (CD), UV-vis absorption titrations, solubility phase diagrams and fluorescence measurements and compared with the corresponding βCyD monomer.

The first investigated polymer, obtained by crosslinking CyDs with epichlorohydrin (EPI), has relatively high molecular weight (200 kDa) and exists under the form of 10-15 nm NPs (pβCyD NPs), as reported in Chapter IA. These NPs could be highly concentrated in aqueous media (up to more than 200 mg/mL) highlighting the interest of using them for drug encapsulation instead of monomeric βCyD, which otherwise display a very limited aqueous solubility (around 17 mg/mL). pβCyD NPs increased by tenfold the apparent solubility of ETH (in agreement with the results in Chapter IA) up to 25 mM. Moreover, the formulation was stable up to six months of storage and ETH crystallisation could be totally prevented.

Of note, we evidenced that ETH, once included in βCyD NPs, experiences a chiral environment resulting in an induced CD signal of the drug. Interestingly, ETH was found to be located not only in βCyD cavities, but also in confined microdomains inside the crosslinked NPs. Next, the binding constant of ETH with pβCyD NPs and monomeric βCyD were determined by means of optical time-resolved spectroscopic techniques affording information on the affinity of this drug for the polymers and the model monomer.

To complete the study, a fluorescent pβCyD polymer labelled with fluorescein (FITC-pβCyD) was prepared in view of future in vitro experiments on carriers uptake and intracellular trafficking. Spectroscopic studies were performed for this polymer too, both in presence and absence of ETH. These studies highlighted the presence of interactions between FITC moieties and CyD cavities belonging to the same polymer (intramolecular) and to a different polymer chains (intermolecular). The spectral behavior of the compound was found to be dependent of ionization state of FITC and therefore of pH solution. Interestingly, the study evidenced that the labelling of pβCyD NPs did not hamper ETH encapsulation even if the binding constant was found to be slightly lower probably due to lower number of available CyD cavities since a small amount of them was occupied by FITC moieties. Of interest, ETH complexation improved the fluorescent signal of FITC-pβCyD paving the way towards the use of this polymer to study intracellular trafficking of CyD carriers. This interdisciplinary work was conducted in collaboration with two partners of Cyclon Hit Project: ISOF of Bologna and CycloLab of Budapest. E. Pancani contributed to the experiments on ETH solubility and on the long-term stability of pβCyD NPs, to results analysis and to the writing of the paper.

For supplementary Information see Annexes Section at the end of the manuscript.

Nanoparticles with high payloads of pipemidic acid, a poorly soluble crystalline drug: drug-initiated polymerization and self-assembly approach (Paper 3)

General objectives

The design of nanocarriers to efficiently load poorly soluble drugs with a high tendency to crystallize is a very difficult task. In this study we describe the challenges related to the incorporation of a typical example of such drugs, pipemidic acid (PIP), a pyridopyrimidine quinolone. Due to its physico-chemical characteristics, PIP administration is very challenging and limits its therapeutic applications. Nanotechnology could offer an opportunity to deliver this "old" drug in an optimized fashion.

PLA/PLGA and pCD NPs are among the most employed drug delivery systems and were investigated here for PIP encapsulation. A series of PLA/PLGA formulations were prepared using the common nanoemulsion and nanoprecipitation methods and adjusting several formulation parameters with the aim of obtaining high PIP payload. However, PIP readily formed crystals during NP formation and presumably adsorbed at the NPs' surface, resulting in burst release upon NP dilution no matter the selected parameters. As a consequence of the poor affinity of PIP for the biodegradable NPs, drug loadings were low (< 10 wt%). In addition, PIP did not show any affinity for pCD NPs and the apparent solubility of this drug was not improved at all.

In this context, we used an alternative approach to PIP incorporation in NPs, consisting in coupling the drug to the chain end of another biodegradable polymer, PCL. Drug-initiated polymerization was successfully used to produce in a single step a PIP-PCL conjugate without the need of catalyst. PCL-PIP was thoroughly characterized by 1 Email address: ruxandra.gref@u-psud.fr (Ruxandra Gref).

Nanoemulsion method -PIP release upon dilution

The release of PIP from NPs was thoroughly studied after progressive dilution in water and PIP content was lost within less than 5 minutes. Interestingly, there was a correlation between the amount of burst-released drug and the dilution factor. For example, the DL of NPs made using polymer P33 (see Main text -method section 4.1) decreased from 8.7% to 4.1%, 2.4% and 1.7 wt% after 0, 10, 20 and 50 times dilution, respectively.

Nanoprecipitation method supplementary information

The P19 NPs were formulated in presence and in absence of PIP. Interestingly, the size measurement of the obtained particles highlighted a 50-60 nm difference in the mean size of empty and loaded NPs. For example, in the case of a final P19 concentration of 5 mg/mL, the empty NPs displayed a size of 170 ± 1.3 nm while the loaded ones of only 117 ± 1.6 nm (PIP/P19 ratio =1:10). The same behavior was evidenced also when increasing P19 final concentration to 10 mg/mL (or higher) (empty NPs= 212 ± 2.3 nm, PIP NPs= 150 ± 2 nm). The same tendency was evidenced also when formulating the NPs by using other polymers.

Chapter III

Advanced nanoparticles characterization by nanoscale infrared spectroscopy (AFM-IR)

SECTION A: Introduction on AFM-IR technique

SECTION B: Tapping AFM-IR: a powerful new tool to unravel the structure of individual drug-loaded polymeric nanoparticles (Paper 4)

SECTION C: Tapping AFM-IR: a versatile method to determine the drug content of each polymeric nanoparticle

SECTION A:

Introduction to AFM-IR technique 1. Atomic force microscopy

The invention of atomic force microscopy (AFM) in early 1980s has been a breakthrough in the history of nanotechnology allowing investigating the matter at the atomic scale. New impressive opportunities were opened in chemistry and physics as well as in biology and medicine. Its inventors, G. Binnig and H. Rohrer, were rewarded with the Nobel Prize for Physics in 1986.

AFM is a type of scanning probe microscopy (SPM) able to investigate surfaces with a very high signal-tonoise ratio and with an impressive resolution, in the order of fractions of nanometers.

The AFM consists principally of a cantilever, a sample holder, a laser and a position sensitive photo detector.

The cantilever and/or the sample stage are usually connected to piezoelectric elements that allow adjusting the cantilever -sample relative position in the x, y and z dimensions. The detector receives the laser beam reflecting off the cantilever and therefore provides a feedback on its deflection (Figure 1). A sharp tip is placed at the free-end of the cantilever which is typically made of silicon 1 .

The tip of the cantilever is used to approach and scan the sample surface measuring its morphology at the atomic scale. When the tip enters in contact with the sample surface, attractive or repulsive forces are established between them, resulting in the cantilever deformation and thus in a change on its deflection.

Therefore, an atomic scale phenomenon can be translated into a macroscale phenomenon (the cantilever deflection). This deflection is detected thanks to the laser diode reflected by the extremity of the cantilever into the photodiode detector (Figure 1). A feedback loop allows the system to react to the deflection changes, correct the cantilever z-position consequently maintaining it at a constant force or height over the sample. Of note, AFM resolution is limited only by the radius of the tip apex, routinely achieving resolutions lower than 20 nm and in some specific configurations down to atomic scale 2 .

AFM imaging modes

AFM can operate in different acquisition modes depending on the sample and on the desired application.

Regarding the imaging applications of AFM, its acquisition modes can be mainly classified in two types: static (contact or non-contact) or dynamic modes (tapping mode, non-contact). This subdivision is performed on the base of the cantilever fixed or oscillating status 3 . Here, contact and tapping mode will be briefly illustrated.

In contact mode the tip scans the sample surface by remaining in close contact to it. The sample surface profile is generated by operating in constant force mode. Indeed, the sample is scanned by exercising a constant force between the tip and the sample. The force is kept constant thanks to a feedback loop able to correct the piezoelectric positioning (with the application of the required voltage) adapting it to the surface of the sample.

In this way, the desired deflection of the cantilever is maintained. The voltage applied by the feedback to the piezoelectric element is recorded as a function of the lateral position on the sample and it is thus used to generate an image of the sample surface (topography). The main drawback of this acquisition mode is due to the shear forces resulting from the contact between the tip and the sample during the scanning. Indeed, such forces can damage soft samples and/or distort the generated image.

Consequently, the contact mode is limited to non-soft surfaces and to samples strong interacting with the support which cannot be moved by the tip scanning.

To address this issue and extend the use of AFM to delicate samples, a second mode has been developed. As a matter of fact, in this mode, namely tapping mode, the cantilever oscillates at or near its natural resonance with an amplitude that should be higher than the curvature radius of the tip. Usually, the amplitude is in the range of 20-100 nm when the tip is not in the contact mode, and the tip impact the sample for a minimal amount of time. This reduces the interactions between the tip and the sample to the minimum required to correctly describe the sample surface and therefore also minimise the probability of damaging or altering the sample.

The oscillation of the tip is generated by a force applied at the base of the cantilever by the piezoelectric element. The intermittent impacts between the tip and the sample cause an energy loss which results in a change of the cantilever oscillation amplitude, according to the surface topography. In presence of convexity, the oscillation amplitude of the cantilever decreases as a result of the reduction of the vibrating space.

Otherwise, in presence of a depression, the amplitude increases. These amplitude changes are detected and the piezoelectric element is used to adjust the cantilever z-position to maintain a constant amplitude. Such amplitude changes are function of the lateral position of the cantilever and are used to reconstruct the topography of the sample. This last mode has the advantage of being suitable for delicate soft samples overcoming friction and adhesion that are typical of the contact mode 3 .

AFM applications

The unique versatility of AFM to image, probe and manipulate materials and its high resolution guarantee the possibility to efficiently image a wide range of samples. Several AFM techniques have been developed to discriminate materials on the basis of various properties such as, for example, friction/adhesion, elasticity, flexibility, damping, optical, electronic and magnetic properties 2 . Indeed, AFM has evolved from simple imaging technique to a multifunctional toolbox by converting the AFM tip into a "lab on a tip" through specific surface modification 4 . Hence, multiparametric and multifrequency imaging technique could be developed 5 .

Of note, the possibility to operate in liquid environment and at ambient temperature opened the possibility to use AFM techniques in the biological field, observing and manipulating biomolecular machinery in its native environment 4 . Time-lapse AFM, for example, could be used to observe dynamic processes at atomic, molecular and macroscopic scale in real-time accessing directly to the structure-function relationship of cellular nanomachinery 4,5 . Some applications of this mode include the study of the activity of RNA polymerase 6 , of the opening and the closure of the pores of a bacterial surface layer 7 , of microtubules formation 8 and of conformational changes of channel-forming proteins 8,9 . AFM also offers the possibility of using single molecule modification (functionalising the tip with specific chemical groups or ligands 5 ) to access directly to inter-and intramolecular interactions thus unravelling receptor-ligand interactions 4 and protein folding and unfolding mechanisms 10,11 (for example, the misfolding of proteins involved in neurodegenerative disease such as Alzheimer and Parkinson 12 ). Another example of single-molecule analysis is the study of the molecular interactions occurring between a drug, or a drug delivery system, and its target opening fascinating avenues in pharmacology domain 4,13,14 .

The AFM tip can be modified not only with single molecules (i.e. drugs and proteins), but also with single cells, viruses or bacteria to study their interactions with other cells at a molecular level. Thus, cells interactions in tumour metastasis and bacterial or viral attack towards cells could be deeply examined 4 . Indeed, spaceresolved AFM enabled mapping the nanoscale distribution of single cells adhesion proteins 15 and antibiotic binding site 16 giving insights on pathogen-host and drug-pathogen interactions. Interestingly, thanks to AFM it could be establish that different cell types display peculiar elastic properties 17 , enabling for example the distinction of cancer cells from healthy ones even when they have similar shape 18,19 .

In recent years, correlative imaging techniques involving AFM have been developed and have drawn increasing attention 5 . Complementary techniques including optical or fluorescent microscopy, Förster resonance energy transfer, total internal reflection fluorescence and super-resolution were successfully coupled to AFM thus combining their advantages and enabling the access to additional information 5 . As an example, specific receptors, channels and transporters could be localised on cells surface thanks to their fluorescent labelling and then their nanometric structure could be unravel thanks to AFM.

However, despite their countless applications and their undeniable advantages, the AFM and its related techniques briefly illustrated above were not able to provide unambiguous chemical analysis and identification of samples.

Infrared spectroscopy

Infrared (IR) spectroscopy is undoubtedly one of the most important analytical techniques available to scientists. Infrared spectrometers have been commercialised since 1940s but the most significant advances in the field have come with the introduction of Fourier-transform spectrometer which dramatically improved the quality of the spectra and greatly minimized the time needed to obtain the data 20 .

Principles of the technique

The IR spectroscopy is a technique based on the vibrations of atoms in a molecule. Indeed, a molecule is made up of atoms linked by chemical bonds which can be seen as spherical masses connected with a spring which vibrate at a specific natural frequency. This frequency depends on the characteristics of both the atoms and their linkage and thus every molecule has its own peculiar vibrational frequency which can be used as a fingerprint to recognise it. Indeed, when energy in the form of IR radiation is applied to a molecule, the atoms start to vibrate. When the frequency of the IR radiation is equivalent to the one of the natural vibration of the molecule, absorption takes place and a loss of energy is observed in the radiation spectrum.

Hence, IR spectroscopy measures the absorption (or transmission) of IR radiation by a sample as a function of wavelength (or frequency). Indeed, the IR radiation is an electromagnetic radiation in the wavelength region of 780 nm and 1000 µm, between the visible and the microwave ranges.

The IR spectral region is normally divided into three main sub-regions: the near-infrared (NIR), the midinfrared (MIR) and the far-infrared (FIR), in function of their distance from visible light 21 . NIR wavelengths range goes from 0.78 to 2.5 µm (corresponding to 1300-4000 cm -1 ), MIR from 2.5 to 50 µm (corresponding to 4000-200 cm -1) and FIR from 50 to 1000 µm (corresponding to 200-10 cm -1 ).

The wavenumber (ν) is defined as the number of waves in a length of 1 centimetre and it is related to the wavelength (λ) by:

(1)

  / 1 =
This unit has the advantage of being linear with the energy of IR absorption.

The absorption of IR radiation results in the transition of a molecule from its ground state to a vibrational state, typically the lowest available. To be absorbed, the incoming IR radiation must have the same frequency as one of the fundamental modes of the molecule of interest. A single vibrational transition is associated to a number of rotational energy states. The contribution of vibration and rotational states participate in giving to each IR absorption band its overall shape.

There are two predominant vibration modes, the first involving the change in bond length, thus a change in the interatomic distances, and the second involving a change in bond angle. They are called stretching and bending modes, respectively. Both stretching and bending vibrations can be symmetrical or asymmetrical. Additionally, bending vibrations can also be in plane or out of plane. More energy is required to stretch a bond than to bend it, therefore stretching will always occur at higher frequencies than bending. In order for a molecule to show IR absorption and thus be considered as IR-active, it must possess a particular feature which is the change of the dipolar moment of one or more of its bonds during the vibration.

The interesting aspect of IR spectroscopy consist in the fact that functional groups absorb IR radiations in a spectral region (or range of frequencies) which is characteristic of the group, as a "signature". This feature allows to employ IR spectroscopy to identify an unknown compound, to elucidate samples chemical structure and even to quantitatively analyse several known species.

The MIR region is particularly interesting for the analysis of the vibration of chemical bonds in organic compounds as most of the fundamental transitions of their functional groups can be found from 4000 to 600 cm -1 . In a general way, the stretching vibration of single bonded atoms (X-H stretching region: mainly O-H, C-H, N-H) occur in the range 4000-2500 cm -1 , the stretching of triply bonded (e.g. C  C, C  N) in the range 2500-2000 cm -1 and the one of double bonded (e.g. C=O, C=C, C=N) in the range 2000-1400 cm -1 . The frequency region below 1300 cm -1 in known as "fingerprint" region because it is generally composed of a series of sharp peaks, which are peculiar of a particular compound rather than of functional groups.

In is important to highlight that the chemical environment of functional groups is fundamental and that the presence of electron donating or withdrawing groups, aromatic rings, the formation hydrogen bonds, etc. can produce changes in the frequency range of the characteristic group absorption band. Therefore, IR spectroscopy is able to provide a direct measurement of the influence of the environment on each specific molecule.

Tables of the characteristic absorption frequencies exist, together with dedicated books, to assist in the correct assignment of the bands in a spectrum 21,22 .

Brief instrumentation overview

Early IR spectrophotometers employed monochromators containing a prism to disperse the IR radiation into its spectral components.

The revolution in IR spectrometers occurred with the development of instruments based on interferometry and exploiting the mathematical process of Fourier-transformation. Fourier-transform infrared (FTIR) spectrometers dramatically improved the overall efficiency of the technique and are widely used nowadays.

FTIR instruments grant a very high repeatability of data.

FTIR spectrophotometers can be used with two types of configuration depending on sample's nature; transmission and attenuated total reflexion (ATR).

Transmission spectroscopy is the measurement of the IR radiation transmitted through a sample according to Beer-Lambert law:

(2)

l C I I T A  =         - = - = 0 log log .
The absorbance (A) is the logarithm of the reciprocal of the transmittance (T) with T being the ratio of the intensity of the light transmitted by the sample (I) to the intensity of the light entering the sample (I0).

Moreover, the absorbance of the sample is directly proportional to sample thickness and concentration and it can be calculated by multiplying sample concentration (C) by the pathlength (l) and by the molar absorptivity (ε, constant of proportionality) 20 .

The transmission mode requires the deposition of the sample on an IR-transparent support (typically zinc selenide -ZnSe -windows). The advantage of transmission lies in the fact that the IR radiation pass through all the sample layer enabling a good bulk qualitative information. Quantitative analysis can be performed by mixing precise amounts of sample with KBr powder (which is IR-transparent in the MID-IR), produce a thin film/disc of a known thickness by using a press and determine the molar absorptivity (ε) of the sample with a calibration curve.

However, transmission is hardly applicable for highly absorbing or opaque powders, for soft polymers or powders and for samples containing particles of sizes which may scatter light resulting in an attenuation of the IR beam not associated with sample absorption.

To tackle these inconveniences, the ATR mode can be a solution. In the ATR approach, the sample is placed on a crystal transparent in the IR, made of a material with no water solubility and with a high refractive index (like ZnSe covered with a diamond layer). The IR beam is directed into the crystal at an angle greater than the critical one and it undergoes a total reflection thanks to the difference of refractive index at the interface crystalsample 21 .

It has been shown that, although the internal reflection of the IR beam is total, an evanescent wave is created at the interface with the sample. This evanescent wave, extending beyond the prism into the sample, loses a part of its energy at the specific absorption wavelengths of the sample. The resulting attenuated IR beam is measured, plotted in function of the wavelength and an absorption spectrum is thus produced.

ATR advantageously requires little or no sample preparation and it can be used on a wide range of sample types. However, it primarily provides information on samples surface or on the sample fraction in closest contact with the prism as the penetration of the IR beam into the sample is very small (typically between 0.5 and 2 µm depending on the prism material and on the wavelength) 23 . Therefore, ATR does not provide bulk information unless the sample is very homogenous and does not interact with the prism. Moreover, a high and homogenous contact between the prism and the samples is required to obtain good quality spectra. The ATR is thus not suitable to measure hard materials which cannot be crushed in powder to achieve sufficient contact with the prism.

IR spectroscopy life-science applications and coupling to other analytical techniques

One of the advantages of IR spectroscopy consists in its versatility and in the ability of obtaining spectra rapidly and reproducibly from a wide variety of samples in liquid, solid or gaseous state.

This technique has thus found application in almost all the fields of science. In particular, IR spectroscopy can play an important role in identifying molecules of interest in different domains such as organic and inorganic chemistry, polymer science and biology 20 . Indeed, this technique has proved its significance in life science for the study of biological samples including in particular lipids 24 , proteins [25][26][27] and nucleic acids 20,28 . Moreover, in the last decades, the number of studies involving more complex systems have raised. IR spectroscopy can be used for diagnostic purposes on sample such as diseased tissues and microbial cells [29][30][31][32] .

For all these applications, FTIR can be used as a stand-alone non-destructive analytical method but it has also been successfully coupled to several other analytical techniques to combine their advantages and obtain complementary information 21 .

For example, FTIR spectroscopy has been combined to separation methods such as gas or liquid chromatography where it is used as a detector to identify the eluted molecules [33][34][35][36] .

Moreover, FTIR has also been coupled to destructive techniques such as thermal analysis methods (e.g. thermogravimetric analysis -TGA) to measure and characterise the gaseous products which evolve from sample heating (evolved gas analysis -EGA) [37][38][39] .

One major advance, which opened the way to the analysis of small samples with important applications in polymer chemistry and life-science, is the coupling between IR spectroscopy and microscopy, namely IR microspectroscopy. Despite numerous improvements, the achieved resolution of commercially available microspectrometers was limited to the range from λ to 3λ, thus depending mainly on the illuminating wavelength (λ) and secondly on the instrument configuration (i.e. numerical aperture of the objective) 2,40-42 .

The resolution of microspectrometers could be increased to approach 1 µm by coupling to Synchrotron beamlines 43 or thanks to the recent advances in optics and array detectors 44,45 . However, nowadays no IR microspectrometer could reach a sub-micrometer resolution which clearly constrains the applications of this technique.

Atomic Force Microscopy in InfraRed (AFM-IR)

The intriguing potentialities together with the major limitations of AFM and IR spectroscopy, as stand-alone techniques were described in the previous paragraphs. While the first is able to provide high resolution images (up to fractions of a nanometre), it lacks in providing chemical characterisation. Vice versa, the second offers the unambiguous chemical identification of compounds, but lacks in resolution (limited by the diffraction).

The original idea of atomic force microscopy in IR (AFM-IR) of Dazzi et al was born in 2005 46 from the ambition to combine the advantages of both techniques and bring the power of IR spectroscopy below the diffraction barrier.

AFM-IR is a technique which couples an IR pulsed laser with an AFM to allow the detection of photo-thermal effects. Importantly, the absorption spectra measured with AFM-IR has been proved to well correlate with traditional FTIR spectra collected in transmission mode 42 .

First setting: bottom-up IR illumination (nanoIR)

The first setting of AFM-IR instrument (nanoIR), shown schematically in Figure 2, has been developed in 2005 by Dazzi et al. 46 , patented in 2007 together with the american company Anasys Instruments and a benchtop version of the instrument was commercialised in 2010 47 .

In this configuration, the sample is placed on a IR transparent prism (ZnSe) and AFM is used in contact mode.

The sample is illuminated bottom-up by the IR beam (tuneable optical parametric oscillator, OPO, as IR source) in total reflexion, thus an evanescent wave is produced with a depth in the sample proportional to the wavelength (same as ATR-FTIR explained in section 2.2) 46 .

Figure 12 -Schematic representation of the nanoIR initial configuration

Once the sample is irradiated by IR light, it absorbs energy at specific wavelengths and produces a photothermal response. Indeed, when IR photons are absorbed by the sample, a part of the energy of the excited state is converted into heat when the samples molecules relax back to the ground state. This heat is converted into a local temperature increase which results in a thermal expansion of the absorbing region of the sample.

This thermal expansion is detected by the AFM tip in contact with the surface as a shock, inducing the oscillation of the cantilever on its eigenmodes. The cantilever deflections, which follow a ringdown mode, are recorded by the AFM detection system. In contrast with other previous techniques 48 , the AFM tip was used here to detect surface deformation rather than temperature increase thus providing sub-wavelength resolution.

The group of Dazzi demonstrated that the amplitude of cantilever oscillation is directly proportional to the absorbance of the sample 42,49 . Moreover, they demonstrated from the theoretical point of view that the resolution of the technique is given by the size of cantilever tip apex.

Two kinds of acquisition are available with the system. The position of AFM tip can be fixed, while the laser is subsequently tuned at different wavenumbers. The amplitude of cantilever oscillation is measured as a function of the wavenumber, hence obtaining an IR absorption spectrum of a precise location (local spectrum)

in the sample (Figure 3a). Alternatively, the tip can scan the sample surface, while the laser wavenumber is set at a fixed value. In this case, the chemical cartography of a sample region is obtained, together with the sample topography. The amplitude of cantilever deflection is measured at each point and plotted against the xy tip position on the sample surface, thus showing the absorbing region of the sample at the selected wavenumber (Figure 3b). This is possible as the IR absorption is characteristic of each molecule and functional groups as extensively explained above (Section 2.1). A complete study of the maximum resolutions of this instrumental setting has been performed 50 . The lateral resolution has been confirmed dependent only to the curvature radius of the cantilever tip and consisted of around 10-20nm. On the other hand, the minimal thickness to obtain a significant deflection amplitude and thus a significant signal/noise ratio (vertical sensitivity) was found to be in the range of 50 and 100 nm.

Such resolution limits have been further improved mainly by increasing the system sensitivity using two strategies; the resonance enhanced mode (and consequently the change of laser source) and the lightening rod effect.

Both strategies have been investigated and proved efficient by the group of Dr. Belkin at the University of Texas 51,52 . The resonance enhanced mode consisted in tuning the repetition rate of the laser to match one of the contact mode resonances of the AFM cantilever. In this way, the cantilever changes from a decaying transient oscillation (ringdown) into a continuous wave oscillation (Figure 4) 2 . This continuous oscillation mode, namely resonance enhanced mode, allowed a much more efficient detection of the sample expansion and hence an increase in the instrument sensitivity. The laser source has been replaced in order to maintain the cantilever continuous oscillation. Indeed, the OPO has a repetition rate fixed at 1 KHz which is insufficient to control the resonant contact mode of the cantilever (resonant at around 100 KHz).

Therefore, a quantum cascade laser (QCL), whose repetition rate can reach 500 KHz, has been used instead of the OPO. Moreover, a feedback loop has been added to the instrument to adjust the repetition rate of the laser to the one of the cantilever at any moment. This first modification of the instrumental setting allowed improving the instrument resolution (especially in the z direction) 51 .

The second strategy studied by Belkin's group was the lightening rod effect which consists in the exaltation of the electrical field between the cantilever tip and the support by using metal coatings.

In order to explain this second strategy, we have to introduce the newest setting of nanoIR instrument.

New setting: top-down IR illumination (nanoIR-2)

The major innovation of nanoIR-2 configuration is the change in the way of irradiating of the sample 2 . The IR irradiation is not anymore performed through the prism (from bottom-top) but the laser is instead focused from top-down in proximity of the cantilever tip (Figure 3). The advantage of this configuration lies in the fact that the prism is not anymore necessary and thus the deposition of the sample can be performed on a wider range of substrates.

Moreover, the feedback loop and the QCL source where used in order to take advantage of the improved resolution allowed by the resonance enhanced mode. This new setting was the one used by Belkin's group to prove the efficacy of lightening rod effect in combination with the enhanced resonance mode 52 .

The lightening rod effect is a complex phenomenon. It is basically obtained thanks to the use of a metalcoated (gold-coated) tip and sample substrate. These coatings allowed the focalisation and intensification of the local electric field, generated by the incident IR radiation, at the apex of the cantilever tip, thus increasing the technique sensitivity.

Coupling the two effects has enabled obtaining spectra on single monolayer of maximum 2 nm thickness and chemical imaging on samples as thin as 5 nm 2,52 .

Despite all the undeniable advantages offered by AFM-IR, it cannot be neglected the fact that, as for every other technique, also this one comes with some limitations. After all, there is no such thing as the perfect technique in science. The coupling between the IR and AFM caused the loss of some of the characteristic advantages offered by AFM. As already discussed above, one of the most appealing feature of AFM is the possibility to study samples in liquid media and to examine even living organisms, such as cells and bacteria.

These features are partially lost in AFM-IR as the sensitivity of the technique is dramatically deteriorated when used in aqueous environment 53 . Indeed, water is a strong IR absorber and scatter along the majority of MIR range. Moreover, in the regions where water absorption is not elevated per-se (for example, around 1500 cm - 1 ), water remains usually the major compound in the investigated samples and its signal overwhelms all the other signals.

Thus, water hamper the measurement by masking the IR signal coming from the sample. Moreover, the performances of the technique in water are highly decreased because of the strong mechanical damping of cantilever oscillations. Therefore, samples analysed by AFM-IR were dehydrated previously to their analysis.

When performing the experiments under these controlled conditions, AFM-IR has proved its invaluable usefulness in the last ten years in a countless number of applications (see section 3.4).

However, the possibility of studying samples in their native aqueous environment rather than in air, thus avoiding their dehydration process which can lead to sample modifications/deformations, is very appealing.

Some pioneering attempts of AFM-IR nanospectroscopy in water have been performed in 2008 54 on cells and more recently in 2017 53 on poly(methyl methacrylate) layers with quite promising results. However, these results are still preliminary and the literature on the subject is restricted to few examples. Further studies are needed to acquire more data and confirm such promising results

Comparison of AFM-IR with related techniques

In the last 20 years, IR-based techniques were developed in the attempt to provide subwavelength images.

Despite some very exciting results, the usefulness of these technique is essentially limited to UV-VIS and the applications to IR range still have to address significant challenges.

The most notable ones are scattering scanning near field optical microscopy (s-SNOM) 55 and tip-enhanced Raman Spectroscopy (TERS) [56][57][58] .

The fundamental difference between AFM-IR and both techniques consist in the fact that the first measures the absorbed light, whereas the other two measure the light scattered by the sample. Therefore, both s-SNOM and TERS ideal samples are strong light scatterers, feature which is especially important in the case of TERS.

Indeed, the main drawback of TERS lies in the necessity to increase its sensitivity by using special fieldenhancing tips to reach satisfying amounts of scattered light. The key limitation of such tips is the difficulty to obtain a sufficient and reproducible enhancement factor.

While AFM-IR measures directly the absorption of the sample, s-SNOM measure the amount of scattered light from the sample which depends not only from the sample absorption but also from the complex optical properties of the tip, the sample and the substrate 2 . As a consequence, a complex modelling of radiation/tip/sample interaction together with interferometric detection are needed in order to limit the band distortions and peak shifts. In addition, s-SNOM bands are sensitive to sample thickness and to substrateinduced band shifts.

Moreover, AFM-IR possess another unique characteristic feature over s-SNOM and TERS. S-SNOM and TERS allow gaining information only in the top layers of the samples as they enable probing only few manometers around AFM tip. In contrast, AFM-IR is able to investigate the sample to a maximum depth of 1-2 µm 2 , potentially allowing reaching invaluable information of complex systems, such as particles or molecules embedded in a matrix or in a cell.

AFM-IR applications

Thanks to its unique features, AFM-IR has found an important number of applications in a wide range of fields allowing the discrimination of sample details "invisible" to bulk techniques or confirming their results at the nanometer level 2 .

The technique was successfully applied in resolving and describing the features of complex polymer blends 59,60 , co-polymer and/or multi-layer films [61][62][63] , polymer nanostructures 64 , polymer-drug blends 65 and several biomedical and pharmaceutical materials [66][67][68] . Additionally, the instrument could also provide information about sample stiffness variation together with the topography and the IR mapping 42,59 . Moreover, AFM-IR technique allowed to distinguish amorphous and crystalline domains and establish the molecular orientation of polymer chains in individual electrospun nanofibers 42,69 .

Another key application of this technique is the study of the degradations of medical devices such as tubing, blood bags and catheters. The aging-effects of polymer and additives, which compose these devices and that are often in contact with body fluids, are a major concern in the medical industry as they represent a potential health risk for patients 70,71 . Interestingly, AFM-IR enabled the detection of very low quantities of lubricant ( 4nm thickness) leaked from polyurethane catheters over time 72 .

Furthermore, AFM-IR significantly contributed to the better understandings of research problems in lifescience. Some of the main applications in this field consisted in the chemical subcellular imaging and spectroscopy of bacteria, viruses and mammalian cells 73,74 . For example, the production and localization of biopolyester or lipid vesicles inside different species of bacteria could be thoroughly characterized [75][76][77][78] . Other investigations involved the study of the interaction between nanodiamonds and cells 79 and the structure and disposition of light-harvesting lipids in plants 80 . Moreover, thanks to this technique, studies were conducted about bone proteins and minerals 81 , microdroplets 82 and the unravelling of protein secondary structure 83,84 including studies of the connection between β-amyloid conformation and neurodegenerative diseases 85 .

Correlative studies between AFM-IR and luminescence microscopy were also performed to localize the distribution of a hormone inside cells. The correlation between the two techniques was possible thanks to the synthesis by click-chemistry of a double-labelled version of the hormone (by using an organometallic molecule for IR detection together with a luminescent probe for UV-VIS detection) which enabled the detection of the molecule by the two systems 86 . Unique results were obtained on tissue samples such as skin and hair sections.

The ratio between IR mapping or spectral bands, characteristic of two different sample components, enabled the determination of the relative concentration of the component of interest in the tissue and to study its distribution 87,88 .

Finally, the AFM-IR versatility is proven as a broad range of other diverse research areas could benefit from its use. Just to name a few, the technique could be applied to the study of semiconductors 89 , solar cells 90,91 , plasmonic resonators 92,93 and cultural heritage 94 .

In all research applications, until today, the AFM-IR technique has been used in contact mode, independently from the employed instrument setting (nanoIR or nanoIR-2). Recent advances on nanoIR-2 introduced the possibility to acquire IR mapping and spectra in tapping mode instead of contact mode. This innovation is particularly meaningful as it extends the use of nanoIR to the study of soft materials or non-surface-adhesive samples, whose analysis was practically impossible until now. In the next section (section B) of this chapter the development and application of tapping AFM-IR are reported for the first time. In our case tapping 152 AFM-IR proved to be an invaluable asset to enable imaging and characterization of single NPs which in contrast were crushed or displaced in contact mode and therefore practically impossible to study.

Moreover, tapping mode allowed us to study drug distribution within each single nanoparticle and have access to invaluable information inaccessible to any other characterization technique. 160 10-20 nm.

These findings have particular relevance in the NPs domain because this approach could be extended to other NPs systems. To the best of our knowledge, this technique is the first one able to provide such precise information about NPs components localisation on a particle by particle basis and with such high resolution.

This innovative study was performed in collaboration with the group of Dr A. Dazzi of the LCP of Orsay, which developed the AFM-IR, and with the society "Anasys Instrument" from Santa Barbara (USA) which commercialise the instrument.

ABSTRACT

AFM-IR is a nanospectroscopic technique which combines the high-resolution of AFM and the possibility to unambiguously identify studied components by infrared (IR) spectroscopy. This well-established photothermal technique has been used in contact mode in various fields of applications. However, its use for the investigation of soft non-adhesive samples remains limited. We present here for the first time the setup of AFM-IR in tapping mode. After describing its theoretical basis, applications were developed in nanotechnology for the investigation of non-adhesive polymeric nanoparticles (NP). The design of optimized drug-loaded NPs with sizes lower than 200 nm necessitates a deep understanding of their morphology and composition, with special emphasis on drug location and corona presence. Up to now, the preferential locations of NP components were hypothesized according to experimental results, but it was not possible to verify these suppositions by directly visualizing NP composition. Here we show that tapping AFM-IR allows not only to visualize the location of the NP hydrophilic shells, but also to locate the drug. Pipemidic acid, a highly crystalline antibiotic, was incorporated at only 1 wt%. In spite of the challenge, the location of the drug was clearly identified using its specific IR fingerprint. The drug preferential location in the NP corona was established. Moreover, small NPs of 8-20 nm could be studied, highlighting the good resolution of the method.

The following paper is under preparation for submission in Analyst.

162 These studies pave the way towards the use of tapping AFM-IR as exquisite tool to control the quality of NP formulations based on individual NP detection and component quantification.

Key words: nanoparticle; corona; tapping AFM-IR; drug identification; chemical characterization

Infrared (IR) spectroscopy is one of the most powerful and widely employed analytical techniques able to directly probe the molecular vibrations of a broad variety of samples. IR spectra can indeed be advantageously used to specifically identify chemical species.

To achieve spatially localized measurements, IR spectroscopy was historically firstly coupled to optical microscopy. However, microspectroscopic techniques are limited to a spatial resolution in the micrometer range. Therefore, to bring the power of IR spectroscopy below the diffraction barrier, a classical atomic microscope (AFM) was coupled to an IR laser about ten years ago 1 . The resulting hybrid technique, called AFM-IR, combines the nanoscale spatial resolution provided by AFM together with the chemical characterization offered by IR spectroscopy. Indeed, the AFM cantilever is used as a detector to locally sense the photo-thermal expansion of the sample resulting from IR absorption.

The signal detected by AFM-IR was proved to be proportional to the absorption coefficient of the sample 1,2 and the technique was able to investigate matter with a resolution of around 10 nm [3][4][5] .

Since its development, this technology has been employed to characterize a wide collection of samples in different domains 4 and has rapidly emerged has a powerful and versatile tool.

Nevertheless, AFM-IR was used so far exclusively in contact mode thus impairing its application to samples with particular geometries and to soft or loosely adhesive samples. Indeed, the contact between AFM tip and sample surface is essential to detect the photo-thermal expansion.

The will to overcome such limitation and extend the technique application to those challenging samples motivated the development of an AFM-IR able to perform the IR acquisition in tapping mode. The tapping AFM-IR setup is presented in this study for the first time and the key physical phenomena involved in the detection of IR absorption during non-linear interaction are described.

The second main breakthrough of this study was the first-time application of the newly developed technique to nanotechnology domain and, in particular, to sensitive weekly surface-adhesive polymeric biodegradable NPs.

Nanotechnology provides the opportunity to organize matter at the nanometer scale, elaborating intriguing reservoirs able to release their drug cargo in a controlled manner at targeted sites in the body [6][7][8] . It revolutionized drug delivery by allowing transcytosis of drugs across biological barriers to reach (intracellular) targets. Nowadays, poly(lactic acid) (PLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) polymers are the 163 most employed biomaterials to elaborate drug nanocarriers. Approved by the Food and Drug Administration (FDA), they were demonstrated to be biocompatible and biodegradable. Several drug-loaded nano(micro)particles are already on the market 9,10 and intensive research is focused on developing novel optimized nanocarriers.

The design of drug-loaded nanoparticles (NPs) with sizes lower than 200 nm necessitates a deep understanding of their morphology and structure with a special emphasis on drug location and physical state. Indeed, a drug located near the NP surface would be released in a "burst" manner whereas controlled release can be achieved with drugs incorporated in the intermingled polymeric chains forming the cores [11][12][13] . Besides, a deep knowledge on the NP corona chemical composition and structure is also of main interest as the NPs' surface is the theatre of many complex interactions with the living systems 14 . This information is generally obtained by using surface analysis techniques such as X-ray photoelectron spectroscopy (XPS) or secondary ion mass spectrometry (SIMS) or by indirect methods such as measuring the electrophoretic mobilities, the hydrophobicity and/or mobility of polymer chains forming the corona. However, direct methods give only bulk information but cannot assess if the samples are homogeneous.

In this context, there is a tremendous interest to develop a method to both visualize individual NPs and characterize their chemical composition, enabling detecting the locations of both drugs and coronas.

In this study, we show that this arduous challenge can be addressed by using the developed tapping AFM-IR.

We will demonstrate that the soft exploration provided by the technique allowed perfectly imaging looselyadhesive and spherical PLA/PLGA NPs, without distorting or displacing them. Simultaneously to the topographic information, tapping AFM-IR allowed the localization of each constituent of the NPs, by detecting their specific IR response, thus unravelling their chemical composition.

The presence of a hydrophilic surfactant corona around NPs core was here directly demonstrated for the first time. The high-resolution of the technique was verified as the morphology and the chemical composition of NPs/micelles as small as 10 nm could be investigated. Moreover, to test the method up to its limits a drug of interest was incorporated at only 1% with respect to NP weight. Despite the challenge, the location of the drug could still be identified.

164

RESULTS AND DISCUSSION

Tapping AFM-IR

AFM-IR description

The AFM-IR technique combines an infrared source with an Atomic Force Microscope. The infrared source is typically a tunable pulsed infrared laser which can be tuned through the mid IR range, but this technique has also been demonstrated using sources extending into the visible range 15 . The infrared light is focused to a spot coincident with the AFM tip. In the original AFM-IR technique 3,1 the infrared laser was pulsed at a low repetition rate, typically 1 kHz, with a fast pulse in the few ns range. When the sample absorbs the infrared light, it heats and rapidly expands causing an impulse to the AFM probe which causes the AFM cantilever to oscillate at its contact resonant frequencies. The amplitude of the contact resonant frequencies is proportional to the absorption in the sample and so by tuning the laser a IR spectrum can be collected which correlates well with traditional FTIR spectra.

A later improvement to the AFM-IR technique made use of faster pulsing laser sources such as Quantum Cascade Lasers (QCLs) in which the repetition rate can be varied over a broad range. This technique is typically referred to as resonance enhanced AFM-IR 16 . In this technique the repetition rate of the laser is tuned to match a contact resonance of the AFM cantilever. As opposed to the traditional technique in which a single laser pulse is used to induce a ringdown in the cantilever, the repeated laser pulses tuned to the contact resonance will drive a larger oscillation amplitude into the cantilever. This improves the sensitivity of the technique allowing for the measurement of thinner samples down to as thin as a single monolayer.

Tapping AFM-IR setup

In both techniques mentioned above, in order to detect the IR absorption of the sample the AFM must be in contact with the surface of the sample. This is typically not a problem for collecting point spectra in which the AFM tip is held at a fixed location on the sample surface and the wavelength of the IR source is tuned over its range. This is true as long as the sample is not particularly soft or mobile. For collecting IR images, in which the IR source wavelength is fixed and the AFM tip is scanned across the sample surface, imaging in the contact mode can be more problematic. For soft or mobile samples, the tip can deform or displace the sample leading to inaccurate results. This problem was also faced in the early days of Atomic Force Microscopy and led to the development of the tapping mode, in which the tip is not in continuous contact with the sample surface but taps, making intermittent contact with the sample surface. This allows highly reproducible imaging of a broader range of samples even if they are very soft or loosely adhered. Tapping mode is typically performed by driving the AFM cantilever at its fundamental free resonance, bringing the AFM tip down close enough to the sample 165 surface that the oscillation amplitude is limited by contact with the sample surface and using a feedback loop to map the sample topography by maintaining the amplitude as constant 17 .

Tapping AFM-IR (Figure 1) was developed using principles from heterodyne or multifrequency AFM in which multiple flexural eigenmodes of the AFM cantilever are excited or measured to enhance the sensitivity of the probe sample interaction and measure mechanical properties of the sample [18][19][20] . Tapping AFM-IR is typically performed in a similar fashion as standard tapping mode by tapping at the fundamental free resonance but in addition the laser is pulsed at the tip-sample location at higher repetition rates. Similar to the resonance enhanced AFM-IR, the laser pulsing, if at an absorption of the sample, will induce heating and an oscillating expansion of the sample surface. The highest signal would be achieved by pulsing the laser directly at the second cantilever resonant frequency. In this case the oscillation amplitude would be enhanced by the quality factor of the AFM cantilever at this mode. The challenge with this operating mode is that there is a small but non zero absorption of the IR illumination in the AFM cantilever as well as a non local contribution from the heating of the sample due to the large spot size of the IR illumination. This results in a large background signal obscuring the local measurements of the sample chemistry at the tip location. In order to achieve local measurements a heterodyne approach is used in which the repetition rate of the laser is set to the difference frequency of the fundamental and second flexural eigenmodes of the AFM cantilever. The nonlinearity of the force interaction in the tapping mode mixes the tapping oscillation with the sample expansion at the difference frequency generating amplitude at the second mode of the cantilever. An alternate approach to this measurement is to perform the tapping oscillation at the second mode of the cantilever and sense the IR absorption using the fundamental mode. In either case the signal is then dominated by the local signal at the 166 tip sample contact and the spatial resolution is only limited by the AFM tip radius. Measurements using this technique have demonstrated spatial resolution of better than 5 nm.

Tapping AFM-IR theory

Tapping AFM-IR is based on heterodyne detection like the acoustic AFM techniques developed decades ago 20,21 . In our case, the photothermal effect is detected by the second tapping mode of the cantilever. The transduction of the photothermal expansion into the second tapping mode is predominantly due to the nonlinear contact between the tip and the surface 19 . In the following theory we will focus on the case of tapping at the fundamental free resonance and measuring at the 2 nd mode but the theory is applicable to the alternate operational modes. The motion equation of the second mode can be written as: (1) where z is the motion of the 2 nd tapping mode, 2 the pulsation of the 2 nd mode,  the damping, m* the associated mass of the 2 nd mode and Fts the interaction force between the tip and the surface.

A Fourier Transform can directly give the amplitude of the 2 nd mode z.

(

The interaction force Fts can be expressed like the first non-linear term of the of the Hertzian contact expression 19 :

F ts (t) = -2c ts (Õ(t )A 1 cos(w 1 t +j 1 ))* d(t -nT 1 )

å é ë ù û u 0 (t) (3)
where ts is the non-linear term of elasticity, A1 the driving amplitude of the 1 rst mode, 1 the phase of the 1 rst mode, T1 is the period of the 1rst mode, u0 the photothermal expansion and Õ the box distribution corresponding to the time of contact  of 1 rst mode.

The Fourier transform of the interaction force leads to the following expression:

where a0 is the maximum of the thermal expansion, tp the duration of the laser pulse and p corresponds to the repetition rate of the laser.

Replacing expression (4) into the expression (2) and reducing the amplitude to his modulus we finally find the expression of the amplitude of the 2 nd mode:

(

) 5
where Q2 is the quality factor of the 2 nd tapping mode.

167

The amplitude of the 2 nd mode is proportional to the driving amplitude A1 and also to the thermal expansion a0. This last parameter is probably the most important because it demonstrates that spectra obtained by the tapping AFM-IR are directly equivalent to those from the FTIR measurement like the spectra obtained by AFM-IR technique. Even if the detection is linked to non-linear interaction between the tip and the surface, the source of 2 nd mode excitation a0 is proportional to the absorbance 3 making the spectra of this technique perfectly reliable for infrared spectroscopy analysis.

Contact versus tapping AFM-IR

AFM-IR is a well-established photothermal technique 2 which has been used for more than a decade to perform IR spectroscopy and imaging at the nanometer scale on a large variety of samples 4 . The measurement of the thermal expansion was achieved until now in AFM contact mode, dramatically limiting the types of samples which could be studied. Indeed, the contact mode on soft materials will have limited resolution and particulate samples can be readily displaced by the AFM probe. PLA/PLGA NPs are typical examples of non-adhesive samples, challenging to be analyzed by AFM-IR in contact mode. Hence, they were used here to compare the usefulness of AFM-IR in both modes to unravel their structure.

Therefore, PLA NPs were prepared by a nanoemulsion method, as previously described 22 and characterized by two complementary techniques, dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA)

(Table S1 -Supplementary Information). Their mean hydrodynamic diameters were 170 ± 45 nm and they displayed a polydispersity index lower than 0.1, indicating a narrow size distribution. The NPs were comparatively imaged by AFM-IR in contact and tapping modes Figure 2). When using applied forces in the equivalent range, as expected, the NPs appear crushed in the contact mode whereas they can be well imaged in the tapping one (Fig. 2 A and B). Moreover, in contact mode, the AFM tip displaced the NPs, making the observations very challenging.

In contrast, the tapping mode enabled to precisely image the PLA NPs unravelling their typical 3D spherical shape, without any deformation caused by the tip (Figure 2B).

169

In addition to the topography profiles, AFM-IR in tapping mode allowed the acquisition of precious information about NPs chemical structure (Figure 2B andC). Indeed, the NPs, made of PLA, are stabilized by a surfactant, polyvinyl alcohol (PVA), which confer them colloidal stability. The IR specific absorption of each component were used as fingerprints to detect their location within each NP. It was hypothesized that PLA and PVA assemble in a core-shell structure during NP formation [23][24][25] . Indeed, it was demonstrated that during NPs formation, PVA chains remain entangled in the PLA/PLGA core in such a way that they cannot be removed, even by extensive washing 24 . The parameters that affected the amount of residual PVA include the concentration of the surfactant and the type of organic solvent used in the preparation procedure 23 . The residual PVA incorporated at the NPs' surface reached up to 13% w/w 26 and influenced the properties of the NPs such as size distribution, Zeta potential, surface hydrophobicity, drug loading and cell interaction 27 .

In the case of the PLA NPs studied here, the thickness of the hydrated PVA shell was determined to be of around 40 nm according to a set of complementary experiments including cryo -transmission electron microscopy (cryo-TEM), nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) 22 .

However, despite the numerous experimental indirect evidences that point towards the arrangement of PLA/PLGA and PVA in a core-shell structure, this peculiar conformation could not be visualized and therefore

undoubtedly proved yet.

In the light of these considerations, AFM-IR in tapping mode was used here in an attempt to unravel the NPs structure.

To map the NPs' components distribution, two specific IR regions were selected as fingerprints for PLA and PVA (see IR spectra in Figure S1 -Supplementary Information). In the case of PLA, multiple characteristic bands were visible and the very intense ester carbonyl one at 1760 cm -1 was selected. In the case of PVA, the choice of a peculiar band was limited by the presence of few IR peaks most of which overlapping those of PLA. Fortunately, a small C-H bending band at 1415 cm -1 stood out and could therefore be employed to univocally assess the PVA presence.

These two bands were thus employed to acquire IR chemical maps of PLA NPs and to track the distribution of their two components (Figure 2).

The IR absorption of PLA and PVA is represented through a color code in the three 3D topographies of Figure

2.

The green corresponds to no IR absorption whereas the red and the blue to PLA and PVA IR absorption, respectively. By comparing panel A and B, it is clear that the IR acquisition of PLA obtained by tapping AFM-IR resulted much more accurate and uniform in comparison to contact mode and that the absorbing region fitted well with the NPs' position in the topography. Thanks to the tapping mode, the IR signal of PLA could be clearly detected within the entire NP volume.

From the NP characterization point of view, the most important finding made available thanks to the new tapping mode was the possibility to identify their PVA shell (Figure 2B andC) thus proving for the first time the core-shell structure of the PLA/PVA NPs.

Interestingly, the detected PVA corona (Figure 2C, blue signal) had a thickness of around 15-20 nm and was found to be principally located at the NP borders, especially in the regions where the NP were in contact with the support. Of note, the obtained PVA thickness was about half the one previously obtained for these NPs in their hydrated state (40 nm 22 ). This could be due to the drying process and the hypothesized mechanism is illustrated in Figure 2D.

Indeed, during drying, PVA chains are progressively dehydrated and thus flattened on the NP surface or deployed on the AFM substrate. Therefore, the thickness of the corona, which is most likely uniform in the hydrated state, is altered and deformed in dried conditions as schematized in Figure 2D. The PVA chains prone to accumulate in the confined space between the NP and their substrate are thus more easily detectable.

Indeed, located closer to the gold substrate, PVA chains are strongly affected by the local electric field intensification due to the tip-surface interaction which locally increase the IR sensitivity 28 . On the contrary, PVA chains at the top of the NPs are flattened during drying and thus less detected.

It can be concluded that tapping AFM-IR was adapted to the investigation of non-adhesive and soft core-shell NPs allowing imaging their shell, despite the PVA low signal (Figure S1, Supplementary Information).

The only drawback of this new acquisition mode, noticed during the IR mapping acquisition, lies in the fact that the power of the IR laser needed to be increased by a factor of 3 as compared to the traditional contact mode configuration. This makes the tapping AFM-IR more "aggressive" towards the samples, leading to the necessity to pay particular attention to avoid sample overheating during IR cartography acquisition. However, by carefully controlling the laser power, NPs could be imaged without any damage or deformation.

Application of tapping AFM-IR to the characterization of drug-loaded NPs

The design of a successful drug delivery system requires not only a deep knowledge of the core-shell structure, but also of the drug location within the carrier which highly influence the drug release profile. Indeed, drugs located in the NPs' top layers are prone to be released in an uncontrolled "burst" manner, whereas those intermingled with the polymeric chains in the cores are presumed to be released in a controlled fashion [11][12][13] .

Besides, NP suspensions might contain drug aggregates, micelles or nanocrystals difficult to be separated. In this context, the assessment of drug distribution on a one-by-one NP basis appears as one of the most fascinating and ambitious goals in the drug delivery field. It was our aim to test here the ability of AFM-IR in the tapping mode to reach this challenging objective.

In many cases, drugs are incorporated in low amounts (less than 10 wt%) in NPs, rendering their detection by AFM-IR even more complicated. This was the case with pipemidic acid (PIP), a first generation quinolone, which attracted interest for encapsulation in NPs with the aim of restore its clinical use 29 . Indeed, 171 nanoencapsulation of such "old" drugs could offer a tremendous opportunity to deliver these drugs in an optimized manner conferring them "a new life" [30][31][32][33] .

In this optic, our group previously reported a comprehensive study on PIP incorporation in PLA/PLGA NPs 34 .

The very low aqueous solubility of PIP 35 and its high tendency to crystallize made its incorporation in NPs particularly arduous, as it was already reported for other crystalline drugs such as busulfan or tamoxifen 11,13,36,37 .

A series of PLA/PLGA copolymers and formulation techniques were previously tested and a PLGA polymer with a 50:50 lactic:glycolic ratio and a relatively low molecular weight (see Method section) was reported to be relatively effective in PIP encapsulation 34 . Therefore, the PIP-PLGA NPs were chosen here for further studies using AFM-IR.

Characteristic IR bands of each NPs' component in their bulk form were selected by recording their IR spectra in the 2000-1000 cm -1 range. The IR spectra of PLGA, PVA and PIP are illustrated in Figure 3. PLGA showed characteristic bands at 1760 cm -1 , 1425 cm -1 and 1398 cm -1 similar but not identical to the ones evidenced for PLA. Among these bands, all suitable for AFM-IR investigation, the one at 1425 cm -1 was selected. Indeed, the ester carbonyl band at 1760 cm -1 was so intense, that it saturated the chemical map, not allowing to easily map the chemistry.

The selection of a PVA specific absorption band was more challenging. Indeed, the C-H bending bands in the region 1500-1300 cm -1 were shifted in PLGA as compared to PLA (Figure 3 and Figure S1), thus overlapping with the PVA signal at 1420 cm -1 . As no exclusive IR band could be attributed to PVA, the 1415 cm -1 wavenumber, where PVA absorbs stronger than PLGA, was selected as fingerprint for PVA detection, .

Similarly, the carbonyl signal at 1640 cm -1 was selected to detect the presence of PIP, as no other component of the NPs absorbs in this region.

Unloaded PLGA NPs

Firstly, the empty PLGA NPs (mean diameter of 140 ± 40 nm -Table S1, Supplementary Information) were investigated to verify if the selected 50:50 PLGA co-polymer granted the NPs the same core-shell structure already proved for PLA NPs. Despite the absence of an IR band exclusively attributable to PVA, the tapping AFM-IR revealed interesting information about the chemical distribution of PLGA and PVA polymers within single NPs. The chemical mapping and spectra recorded on such particles are presented in Figure 4.

The chemical map at 1425 cm -1 (Figure 4B), where the signal of PLGA is preponderant over PVA, shows a strong absorption only in the NP core, as the signal is clearly included in the black dotted line delimiting the NP topographical borders. In contrast, it is interesting to notice that, when taking into consideration the mapping at 1415 cm .1 (Figure 4D) where PVA signal is prevalent, an absorption can be notice not only in the NP core but also on the substrate surface around it. Indeed, a thin layer of approximately 15 nm (comparable to the one already evidenced in the case of PLA NPs) was detected outside the dotted line delimiting the NP core. The comparison of these two mappings lead to the conclusion that the PVA is surely located around the NPs, as already reported for PLA NPs, but its presence or absence in the particle core and on their top could not be surely ascertained. Indeed, while around NPs an absorption signal could be detected exclusively at 1415 cm -1 and could thus be attributed to PVA, in the inner core of the particles an absorption at both wavenumbers (1415 and 1425 cm -1 ) was clearly present. Therefore, the respective contribution of PLGA and PVA to such absorptions could not be clarified. To complete the study, a map at 1760 cm -1 was recorded and was found to exhibit the same PLGA distribution as in the 1425 cm -1 map (Supplementary Information, Figure S2) thus validating once more the selection of 1425 cm -1 as PLGA fingerprint.

However, for some NPs, a lack of intensity with a moon croissant shape was revealed in the chemical maps (Figure 4B andD). The IR signal at the upper part of the big NP in the center of the region of interest (ROI)

is not complete and do not match well the NP topography. The phase image (Figure 4C) corresponding to these maps can clearly explain this effect. Given the important size of this NP (around 160 nm), the tip contact change considerably during the IR mapping acquisition thus leading to an excessive and sudden phase change.

As a consequence, the synchronicity of the 1 rst mode does not match the laser pulse, making the 2 nd excitation mode down to zero. In our experimental settings, the AFM set point of the driving tapping mode (1 rst ) was regulated to produce a soft contact (80% of free amplitude) thus avoiding an excessive pressure on the NPs. In fact, a possible strategy to minimize these phase problems could be to increase the contact between the tip and the sample by decreasing the set point, hence reducing the phase effect.

Besides allowing the chemical characterization of relatively big PLGA NPs (of around 160 nm), the tapping AFM-IR proved its sensitivity and high-resolution by enabling the additional observation of smaller objects.

These small NPs or micelles, with sizes of around 10-20 nm, could be well imaged and chemically mapped without displacing or crushing them. Indeed, successive topography images of the ROI were identical.

Of interest, these small objects displayed a larger absorption at 1415 cm -1 (main signal of PVA) than at 1425 cm -1 (main signal of PLGA). This clearly shows that the smallest objects have a higher PVA content. This characteristic could indicate a PVA-PIP micelle-like structures of these objects.

In addition to chemical mapping, AFM-IR is able to record IR spectra at specific locations within the sample.

For instance, Figure 4E illustrate the spectra obtained in the middle of one NP and on its corona. The IR spectrum in the center of the NP shows non-ambiguously the 3 main bands characteristic of PLGA at 1763 cm -1 , 1425 cm -1 , 1398 cm -1 . The specific PVA band at 1420 cm -1 could be clearly observed in the NP corona.

These IR spectra are consistent with the FTIR ones recorded on the bulk NP components (Figure 3) and with the hypothesis made so far. These spectra enabled confirming once more the identity of the two NPs component and their respective location.

Drug-loaded PLGA NPs

Once the presence of a PVA corona around the PLGA NPs was confirmed, PIP-loaded NPs were prepared and characterized by tapping AFM-IR aiming at unravelling PIP location. Whatever the preparation method (nanoemulsion or nanoprecipitation) 34 , the final loadings after dilutions in water (necessary for AFM investigations) were of only 1 wt%, underlining the low affinity of PLGA for PIP.

Given this challenging background, it was interesting to investigate drug location by tapping AFM-IR and test if this technique would be sensitive enough to detect PIP at such low loadings.

The PIP NPs obtained by nanoemulsion and nanoprecipitation techniques were characterized separately to assess the influence of NPs preparation on the components distribution.

Nanoemulsion technique

Firstly, the PIP-loaded PLGA NPs prepared by nanoemulsion (mean diameter = 140 ± 40, Table S1, Supplementary Information) were investigated and showed similar core-shell features as the unloaded ones (Figure 5B andC) exhibiting a PVA coating of around 15-20 nm thick.

In addition, a cartography at 1640 cm -1 (Figure 5D) was performed to detect the antibiotic location.

This map enabled the acquisition of intriguing information indicating clearly that PIP is exclusively localized around the PLGA NPs forming a shell with a thickness of around 15-20 nm corresponding well to the location of the PVA corona (Figure 5C andD). These results suggest a co-localization of PIP and 175 PVA in the NPs' coronas. Of note, this preferential location was hypothesized in our previous studies, based on the fact that PIP behaved as a surfactant during NPs preparation 34 . Tapping AFM-IR enabled here to clearly visualize the location of both drug and surfactant in the shells thus validating the previous hypothesis.

Moreover, the total lack of PIP signal in the NPs' core confirmed the low affinity of the drug for the PLGA polymeric matrix thus explaining the extremely low drug loading and "burst" release from the shell previously reported 34 . Interestingly, no significant PIP IR absorption could be evidenced on the top of the NPs as already reported in the case of PVA for unloaded PLA NPs. Presumably, as suggested in the case of PVA (Figure 2D), the slow drying of the corona flattens the PVA shell together with the associated PIP which eventually accommodated in the confined interstice between the NPs and their support (Supplementary information -Figure S3), hence conferring the peculiar IR absorption pattern visible in Figure 5D. Indeed, PIP forms a corona around the NPs and small particles of around 10 nm could be detected. Of note, due to the smaller size of the NP prepared by nanoprecipitation as compared to nanoemulsion (126 ± 26 nm and 150 ± 40 nm, respectivelysee Table S1, Supplementary Information) the phase shift was also sensibly smaller (Figure 6C and Supplementary Figure S4)

The average spectrum obtained on the NPs core (pink spectrum) reveals exclusively the presence of PLGA characteristic bands at 1762 cm -1 , 1425 cm -1 and 1396 cm -1 (Figure 6E). Thus, the absence of PIP in NPs core is confirmed. The average spectrum of the corona shows a mix of PIP and PVA spectra as we can easily detect the PIP band at 1635 cm -1 , the PVA one at 1420 cm -1 and the additional band at 1550 cm -1 , which presence was already highlighted in the corona of PLGA NPs obtained by nanoemulsion. It is worth pointing out that the carbonyl band of PIP is found at 1635 cm -1 in the case of nanoprecipitation and at 178 1645 cm -1 in the case of nanoemulsion. This shift is probably due a different environment experienced by the antibiotic when interacting with the PVA.

In a nushell, tapping AFM-IR enabled for the first time the visualization of the PVA corona around PLA/PLGA NPs core and its presence was detected in loaded and un-loaded NPs prepared either by nanoemulsion or by nanoprecipitation. Moreover, a preferential localization of PIP in the PVA corona was detected despite the low drug loading (around 1 wt%) of the analyzed NPs.

Conclusion

The development of AFM-IR in tapping mode is reported in this study for the first time and the key physical phenomena involved in the detection of IR absorption were described. The amplitude of the signal detected by the AFM as a consequence of sample photothermal response was demonstrated to be proportional to sample absorbance despite the non-linear interactions occurring between the tip and the sample.

The development of this new mode allowed extending AFM-IR application to loosely-adhesive or sensitive sample, such as polymeric NPs, which were very difficult or impossible to analyze in contact mode.

Thus, tapping AFM-IR was used here to unravel the structure of NPs possessing a hydrophilic PVA shell and a PLGA core. A crystalline drug, PIP was incorporated at a loading of only 1 wt%. In spite of this challenge, the location of the drug could be clearly identified based on its specific IR fingerprint. The drugs' preferential location in the hydrophilic coronas was assessed here for the first time. Moreover, objects (NPs or micelles)

as small as 10-20 nm were detected and successfully imaged. These studies pave the way towards an in-depth characterization of both NP composition and drug incorporation.

With further investigations, tapping AFM-IR could be an exquisite tool to control the quality and the composition of NPs on a particle by particle basis and could give access to information inaccessible with other techniques. PLA ester terminated (MW:10-18 kDa), PVA (88% hydrolysed) and pipemidic acid (PIP) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Injectable water was purchased from Cooper (Melun, France), while dichloromethane (DCM -Analar Normapur), methanol (MeOH) and acetone from Prolabo (Fontenay-sous-bois, France). The solvents were all analytical grade.

METHODS

Chemicals

Nanoparticles synthesis and characterization

Nanoemulsion method -Biodegradable PLGA NPs were prepared by an oil-in-water emulsion method using PVA as a surfactant. Briefly, 20 mg of PLGA were dissolved into 1.5 mL of DCM and the organic phase was poured into 4 mL of injectable water containing 0.5% w/v PVA and vortexed for 20 s. For drug-loaded NPs, 4 mg of PIP together with 0.2 mL of MeOH (which helped solubilising the PIP) were added to the PLGA organic phase, previously to the phase mixing. The emulsion was sonicated with a sonicator probe (Sonopuls HD 2070, BANDELIN electronic GmbH & Co, Berlin, Germany) at 20 % power for 1.5 min in ice bath to avoid overheating. The emulsion was sonicated again at 10 % power for 30 s. Organic solvent was evaporated at room temperature under gentle magnetic stirring.

Nanoprecipitation method -50 mg of PLGA were solubilised in 2.5 mL of acetone. The polymer solution was added to 5 mg of PIP powder. As PIP is poorly soluble in acetone, 0.4 mL MeOH were added to completely solubilize the drug. Then, the organic phase was added drop-wise into 5 mL of 0.5% PVA aqueous (injectable water) solution under continuous vigorous magnetic stirring leading to the instantaneous precipitation of the polymer under the form of NPs. The organic solvent was evaporated at room temperature under gentle magnetic stirring. Duration of solvent evaporation was controlled.

The average hydrodynamic diameter of the NPs was determined at 25 °C

AFM-IR

The AFM-IR data reported in this study were obtained using a NanoIR2s (Anasys Instrument) coupled with a multichip QCL source (MIRcat-QT, DAYLIGHT solutions; peak powers up to 1W; average powers up to 0.5W and wavelength repeatability < 0.1 cm-1) covering the range from 1325 cm -1 to 1960 cm -1 of the mid-IR region) with a tunable repetition rate (up to 500 kHz). An Au-coated silicon probe (tapping AFM-IR cantilever, Anasys -spring constant 0.03 N/m) was employed to avoid effects linked to the IR absorption of the silicon. Topography and IR maps were treated using MountainsMap 7.3 software and the spectra were filtered using a Stavitsky-Golay algorithm (order 3 polynome using 5 points).

FTIR spectrocopy

Transmission-FTIR spectra of NPs components were obtained by using a Brüker Vertex 70 FTIR spectrometer (MCT detector, cooled down by liquid nitrogen). Each NPs component was solubilized in a suitable solvent, deposited on 20 mm ZnSe windows and dried with nitrogen flow. Spectra were recorded in the range of 2000-1000 cm -1 with an accumulation of 100 scans and a resolution of 4 cm -1 . The ester carbonyl band at 1732 cm -1 (C=O stretching vibration) was assigned to PCL and chosen as its signature whereas the tertiary amide band at 1630 cm -1 (C=O stretching vibration) was selected for PIP. Indeed, as shown in Chapter II, each PIP molecule is conjugated to a chain of about 10 PCL units through an amide III bond. These selected bands were employed to register IR cartographies on a series of PCL-PIP NPs. An example of the obtained results is shown in Figure 2. The first interesting observation which can be formulated by looking at the topography images (Figure 2) is that PCL-PIP NPs do not look spherical like PLGA ones (see Chapter II). Indeed, by taking the bigger NP as example, the topography maximum height corresponds to 100 nm whereas its width is of around 500 nm. Thus, the NP appears flattened on the support surface. One may argue that this flattening effect could be a consequence of tapping AFM-IR scan and that this new tapping mode is not as adapted as previously claimed to sensitive samples. However, it is interesting to point out that SEM investigations revealed the same morphological features (Chapter II). Given the similar shapes observed by the two techniques, two hypotheses can be formulated. Firstly, a non-spherical shape of these NPs could be assumed. To ascertain this hypothesis and complete the study, Cryo-TEM experiments (without drying) will be carried out in the future.

The second and most likely hypothesis is that the flattening effect observed with the two techniques is caused by NPs deposition and drying process on the two supports (gold for AFM-IR and silicon wafer for SEM). In Similarly, a consistent difference is evidenced in the case of MP which is around 170 °C for PLA/PLGA and around 60°C for PCL. These differences can explain the sensitivity of PCL NPs to the drying process and surface deposition which conversely do not impact on PLA/PLGA NPs.

It is important to highlight that the low PCL melting point could potentially be an obstacle in the characterisation of PCL-PIP NPs, hampering the possibility of performing multiple acquisitions on the same ROI at different wavenumbers in order to acquire the desired information. Indeed, the first IR mapping of NPs could be sufficient to damage them preventing further investigation.

However, by comparing the two topographies of Figure 2, where the one in panel C was obtained after a first IR mapping at 1732 cm -1 , it can be observed that they are almost identical and that no alteration is visible.

Indeed, the laser power of AFM-IR could be conveniently tuned to enable the acquisition of multiple chemical mappings of the same NPs without distorting or melting them.

The most interesting results of this investigation are visible in the IR mappings of Of note, it is important to highlight that, although PVA was used as surfactant in NPs formulation, its IR signal could not be detected and no PVA corona was evidenced during these investigations, contrary to the results presented in Chapter III Section B on PLA/PLGA NPs. Two hypotheses can be formulated to explain this discrepancy. Firstly, there was a difficulty to find an IR wavenumber suitable as PVA fingerprint, because its bands were overlapped by PCL-PIP absorption. Secondly, when trying to use the same strategy employed in

Chapter IIIB for PLGA NPs and record the cartography at 1415 cm -1 (where PCL-PIP contribution is less important) no PVA accumulation could be detected neither around NPs nor in other locations of the sample.

This can be explained by a lower affinity of PVA for PIP-PCL. This hypothesis could be further confirmed by a quantification of the PVA content in the NPs by colorimetric methods set up in our group 1 . Alternatively, it will be interesting to perform further studies by replacing PVA with other surfactants, such as Pluronic, approved by the FDA and which use is already reported for the efficient formulation of PCL NPs [2][3][4] .

One of the most ambitious goals in the drug delivery domain is the assessment not only of the drug localisation but also of its amount. It would be revolutionary for the development and the study of nanoparticulate systems to be able to determine their loading on a particle by particle basis.

Thus, this study reports the first steps towards the determination of the amounts of each component in individual NPs. To do so, we took advantage of the unique information provided by the tapping AFM-IR.

The IR maps of PCL-PIP NPs were employed to calculate a ratio between each compound of interest.

Indeed, these NPs were formulated by employing a PCL-PIP conjugate synthesised by a drug-initiated polymerization reaction (Chapter II). In this reaction the caprolactone monomer was used as a solvent for the reaction. It should be kept in mind that trace of water in the reaction medium and even some impurities can also initiate the polymerisation reaction, leading to the formation of poly(e-caprolactone) homopolymers. This has been well described in studies dealing with the acid-initiated caprolactone polymerisation, highlighting the obtention of polymers with different chain ends but similar molecular weight which were very difficult to be separated by chromatographic methods 5 . Very probably, this could be the case here, with the synthesis of a majoritary PCL-PIP component and some trace PCL homopolymer. In other words, NPs are prepared of blends of PCL-PIP (major component) and PCL (minor compound). Therefore, knowing the lack of affinity between PCL and PIP it will be of high interest to investigate by AFM-IR if each NP has the same PIP content, or if there is a different segregation of the blocks.

It should be kept in mind that the global composition of PCL-PIP was accurately determined by NMR indicating a molar ratio of one PIP molecule per 10 caprolactone units. By analysing several NPs, the global composition determined by NMR should be found also by AFM-IR.

In order to calculate the molar ratio of PCL and PIP through the recorded IR maps, the relative force of the absorption peaks of interest must be determined by measuring the absorption coefficient "α" which varies in function of the wavenumber.

The measure of α has been performed in collaboration with Dr. Emmanuel Dartois (Institut d'Astrophysique Spatiale, Paris-Sud University) by using an IR microscope (Thermo Scientific Nicolet iN10). A thin layer (around 1 µm) of the bulk compound PCL-PIP has been created with the help of a compression cell made of synthetic diamond. This experiment was delicate because of the elasticity of PCL-PIP copolymer which was difficult to be compressed to form a smooth, flat layer, essential to allow a precise evaluation of both the layer thickness "l" and α.

Once a flat surface was obtained, α could be determined from the principal interference fringe distance of IR light created by the optical index changes between the diamond support and the polymer which are measured 191 by the IR microscope according to the Beer-Lambert law:

(1)

l e I I T  - = = 0
where T is the transmittance, "I" the intensity of the light transmitted by the sample and "I0" the intensity of the light entering the sample. "I0" is known whereas T and I were directly measured from the sample. The unknown parameters of the equation are hence "l" and α. "l" could be measured as follows:

(2)

   RI l 2 1
where "RI" is the refractive index of the polymer, that in this study has been approximated to the one of PCL (=1.439) 6 , and "Δν" is the principal interference fringe distance, measured directly by the instrument. With the measured l, α could be derived for each wavenumber from the inversion of equation 1:

( The values of α for the two wavenumbers at which the NPs cartographies were recorded (1732 cm -1 for PCL and 1630 cm -1 for PIP) where derived from Figure 3 and a 1.9 PCL/PIP ratio resulted between them. This means that in the studied material, the PCL part produces an IR signal 1.9 times more intense than the PIP moiety.

This factor has been taken into account in the further investigations to determine for each NP its molar PIP:PCL ratio, which should be globally close to 1.

Subsequently, for each NP, the cartography at the two wavenumbers of interest have been individually analysed by using MountainsMap 7.3 software and the IR signal at each wavenumber has been integrated over the IR signal area. Thus, the NPs integrated signal, expressed in arbitrary units (A.U.) per nm 2 (A.U./nm 2 ), was obtained, as shown in Figure 4. Then, the ratio between NPs IR integrated signal at 1630 cm -1 and the one at 1732 cm -1 , corrected by the α factor ratio, has been calculated for 10 different NPs. Moreover, when on the same NP a different laser power was employed to acquire the two cartographies, their signal was normalised by taking the relative power into account.

The PIP/PCL ratio mean value corresponded to: r(PIP/PCL) = 1 ± 0.4.

The ratio calculated from the IR cartographies was very close to the theoretical one (=1) thus proving that tapping AFM-IR could provide a relative quantification of the drug/polymer ratio on a particle by particle basis. However, it can be noticed that the standard deviation of the measurements is quite important. Indeed, these studies needs to be completed on a higher number of NPs to have better statistics. Another possible explanation of the variability of r between the particles consist in the formation of crystalline microdomains of PCL and/or PIP embedded in an amorphous matrix. The AFM tip encounters domains more or less rigid which make difficult to control the contact between the AFM tip and the sample. Tapping AFM-IR signal, which is measured on the 2 nd tapping mode (Chapter III Section A), is highly dependent to the tip-sample contact, hence when the last is not constant, it interferes with the correct detection of IR-induced sample thermal expansion. With further studies the data acquisition could be improved by adjusting the parameters (i.e, the setpoint) with the purpose of minimising the contact variations.

It is worth to highlight that this hypothesis is corroborated by the spectra in Figure 2E. Indeed, it can be observed that the maximum of the amide C=O band (used as PIP fingerprint) is not constant at 1630 cm -1 but varies of few nanometres depending on the considered spot. This is also most likely explained by the presence of crystalline domains in the different regions of the NPs. This complicates the data treatment as the cartography has been acquired at 1630 cm -1 which is not the maximum of the band for the entire NPs, thus introducing an error which further increase the measurement standard deviation.

In addition to these factors, the variability between the measurements was also increased by the fact that the integration of NPs surface was complicated by their irregular form.

Despite the method reported here certainly needs to be further developed, the tapping AFM-IR was demonstrated here to enable reaching, for the first time, the challenging goal of determining the drug/polymer molar ratio on individual NPs. These findings represent the first step towards particle by particle quantification opening tremendous opportunities in the nanomedicine field.

Conclusions and perspectives

In this section it was demonstrated that the newly developed tapping AFM-IR represents a powerful and versatile tool to improve our knowledge of NPs structure.

The successful incorporation of PIP thanks to its conjugation to PCL and its localisation inside the formed NPs was here undoubtedly confirmed. Moreover, the investigation suggested the absence of PVA corona around these NPs which could explain their lower stability in comparison to PLGA NPs. Moreover, a first strategy was presented here to allow the determination of drug/polymer molar ratio on a particle by particle basis. With further study the current approach could be improved and a more precise quantification could be achieved.

The drug quantification on a particle by particle basis could represent a revolution in the drug delivery domain allowing the determination of NPs homogeneity and the eventual relationship between the colloidal properties of the particles, (such as their size) and their drug loading.

Tapping AFM-IR stands out as an invaluable tool in NPs development and its use could be extended in the future to other kind of NPs helping in unravelling their nanoscale structure.

High resolution label-free detection of biocompatible polymeric nanoparticles in cells (Paper 5)

General objectives

The design of efficient drug nanocarriers necessitates a deep understanding of their interaction with targeted cells. Indeed, size, shape and surface properties of NPs play a key role in establishing their internalization pathway as well as their intracellular fate.

The detection of NPs with sizes lower than 200 nm, such as PLGA NPs, inside cells is usually enabled by high-resolution imaging techniques which require appropriate labelling. Unfortunately, the addition of a dye, either physically entrapped or covalently linked, to NPs often alter their physicochemical properties and biological fate. Such techniques enable only indirect detection of NPs; indeed, they detect the dye, rather than the NPs themselves. Moreover, no high-resolution method allows the precise detection, together with the identification of the chemical composition of single NPs in cells, which is of outmost interest to gain insights on their fate.

Here we address these challenges by using AFM-IR in contact mode. AFM-IR was reported here for the first time as a reliable, non-destructive and direct method able to locate precisely each PLA NPs within cells, by using the specific IR response of their constitutive polymer. This response was employed as a fingerprint to enable the unambiguous chemical identification of the nanoparticles.

Nanoparticles (NPs) characterisation

The NPs size distribution was evaluated by using two techniques: i) dynamic light scattering (DLS) and ii) NP tracking analysis (NTA). The results are reported in Supplementary Table S1. NTA has been previously shown to be the most adapted technique to estimate the hydrodynamic diameter of PLA/PLGA NPs 1 . Indeed, NTA has the advantage of tracking the Brownian motion of each individual NP, in order to determine its diffusion coefficient (Dt) and consequently calculate, using Stokes-Einstein equation (equation 1), the sphere-equivalent hydrodynamic diameter (d):

d T k D B t  3 = (1)
The sample temperature "T" and viscosity "η" are known and kB is the Boltzmann constant 2 .

It is thus not necessary to know the refractive index (RI) of the material that compose the NPs. On the other hand, the hydrodynamic diameter calculated by DLS is dependent to the RI of the NPs. As a consequence, DLS can often lead to an overestimation of NPs size due to the fact that the largest NPs scatter more light than the smaller ones thus shifting the measure to higher values 2,3 and explaining the difference in size between number distribution determined by DLS and NTA. However, given the DLS sensitivity to detect large objects such as NPs aggregates, this technique remain the ideal method to study the NPs suspension stability 1 . 1. Main characteristics of fluorescently labelled and un-labelled NPs. NP mean diameter was determined by both DLS and NTA. For DLS measurements data are reported as Z average and also by number distribution to allow the comparison with NTA results. Concentration results correspond to the concentration (particles/mL) in the NP suspensions, measured after diluting 10,000 times the samples to allow the NPs to move freely according to their Brownian motion without impeding each other. Pharmacy is the science and the technique of preparing and dispensing drugs. Indeed, drugs are a fundamental component of both the traditional and the modern medicine.

Supplementary Table

Formulation

One of the major examples of serendipity in science, and more precisely in pharmaceutical field, was the discovery of antimicrobial agents at the beginning of the 20 th century (Fleming 1928) which led to a true revolution in humans' everyday life. Their introduction made far-reaching changes in the treatment of bacterial infections 1 . Diseases which were previously deadly became harmless. Several natural and synthetic molecules have been introduced in the clinic since then and until the 1970s. Indeed, this period of time is generally named the "golden era" of antibiotics. However, the rate of discovery of new antimicrobial agents has dramatically decreased since 1980s. The perception of infectious diseases as a "yesterday problem" have caused a decline in investment in the R&D of antibiotics and pharmaceutical companies have focused on other research areas with a higher commercial return 2 .

However, in the last decades, the onset of AMR phenomenon is jeopardizing again human health. This does not only have a strong impact on the treatment of infectious diseases but it also undermines the safety of medical interventions such as cancer chemotherapy, organ transplantation, invasive or non-invasive surgery procedures and patients intubation which are de-risked by effective antibiotic prophylaxis and treatments 2 .

In this context intracellular infections represent one of the biggest dangers.

To tackle this global crisis, the development of NPs, which can protect antibiotics and increase their efficacy, represent a promising and clever therapeutic strategy. Moreover, NPs gained ever-growing interest as a tool to overcome the drawbacks of traditional therapeutic agents such as low solubility, poor bioavailability and unfavorable physico-chemical properties. The recent discovery of the family of boosters of ethionamide (ETH), a 2 nd line anti-TB drug with poor therapeutic index, has opened new opportunities for a more efficient treatment of TB 5,6 . Herein, we focused on one of the molecules of this family, BDM41906, namely Booster.

The aim of this study consisted in the co-encapsulation of [ETH:Booster] in polymeric NPs to allow their administration directly in mouse lungs, the major site of Mtb infection, by means of a Microsprayer® device.

Indeed, these two molecules as free drugs have important limitations such as a completely different biological distribution and bioavailability and the impossibility to be solubilised in a solvent compatible with pulmonary administration which could allow a more efficient treatment of this respiratory disease.

The efficient co-incorporation of these molecules had to overcome several technological barriers. Indeed, the formulation process was complicated by the difficulty to find a common solvent for the two molecules and by the strong tendency of ETH to crystallize.

After a thorough formulation study, polymeric PLA NPs containing both drugs have been successfully

prepared by an optimised nanoemulsion technique. They displayed reproducible sizes, a low polydispersity and the crystallization of ETH during storage has been overcome. However, slight temperature changes during storage could still induce crystallisation. Of note, despite monodisperse NPs could be obtained by nanoprecipitation without the use of surfactant, the presence of these stabilizing molecules was necessary to insure a better drug encapsulation andavoid the rapid formation of ETH crystals during storage.

The average mean diameter of the obtained NPs has been thoroughly characterized using three complementary techniques: DLS, NTA and cryo-TEM. This work highlighted the importance of NPs characterisation with multiple techniques to obtain an overall picture of their physico-chemical features. This enables avoiding the misinterpretations that could be induced by taking into consideration only a single technique 7,8 . Indeed, in this specific case, NTA appeared to be the most adapted method to perform a realistic estimation of NPs diameter in their hydrated state. By comparing and analysing the results obtained with the three techniques, it was concluded that DLS led to an overestimation of NPs hydrodynamic diameter due to its intrinsic sensibility to the presence of a small number of large NPs. This population, although minor, was sufficient to bias the results shifting the NPs mean diameter towards bigger values (i.e. from a mean diameter value of 178 nm to 235 nm, obtained by NTA and DLS, respectively). On the other hand, cryo-TEM allowed obtaining valuable information about NPs morphology, but the experimental conditions inherently lead to flatten the surfactant corona onto the NPs surface, thus leading to a measurement of their dried diameter rather than their hydrodynamic (hydrated) one. Consequently, the mean diameter (around 100 nm) obtained by statistical analysis of TEM images was sensibly lower than the one obtained by both NTA and DLS. Interestingly, DLS sensitivity to big objects resulted advantageous when evaluating NPs suspension stability during storage allowing the immediate detection of NPs aggregates. The developed drug-loaded NPs were found to be stable during storage for at least three months. High encapsulation efficiencies (76±5 wt% and 51±8 wt%) were obtained, together with important loadings (36±4 wt% and 26±3 wt%) for ETH and Booster respectively.

Despite the positive features showed by drug loaded PLA NPs, the formulation was not totally satisfying.

Indeed, the formation of tiny ETH crystals was still observed during storage.

Therefore, to overcome the technological challenge of completely avoiding ETH crystallization, a different approach was attempted by encapsulating this drug at a molecular level using polymeric β-CDs (pCD) NPs.

Indeed, thanks to their peculiar truncated-cone structure, CDs are able to act as molecular containers by accommodating drug molecules inside their hydrophobic cavity. Noticeably, pCD NPs combine the advantages offered by native βCDs together with the higher aqueous solubility offered by their polymeric form and they have thus been employed to increase the apparent water solubility of several drugs [9][10][11] .

The pCD NPs enabled an efficient one-step incorporation of both ETH and Booster by "green" procedure.

Both drugs could be loaded into pCD NPs by simple incubation of drug powders with a water suspension of NPs. In this way, ETH apparent aqueous solubility could be increased of around 10 times without the need of any organic solvent. In particular, pCD NPs at a concentration of 200 mg/mL were able to incorporate up to 5 mg/mL of both ETH and Booster totally preventing ETH crystallisation. Of note, a water solution containing 40-50% DMSO was needed to solubilise an equivalent amount of ETH.

In the specific case of ETH, pCD NPs resulted more advantageous in comparison to PLA ones thanks to their capability of incorporate drugs in a molecular state thus totally hampering the drug crystallisation.

Interestingly, the two drugs could be hosted by pCD NPs without impeding each other.

Of the three methods used in this study (Chapter I -Section A) to deeply characterize PLA NPs size, only TEM (TEM micrograph after freeze fracture) was adapted also for gaining insights in the characteristics of pCD NPs. Indeed, these NPs were too small (5-10 nm) to be described by the other techniques.

Therefore, a complementary study (Chapter I -Section B) has been performed simultaneously to elucidate the strength of the interactions between ETH and pCD NPs and the localisation of ETH molecules inside NPs structure. This interdisciplinary study has been conducted in collaboration with two partners of Cyclon Hit consortium: ISOF from Bologna and CYCLOLAB from Budapest.

ETH-pCD NPs interactions were investigated by means of solubility phase diagrams, circular dichroism and UV-VIS absorption titrations. Native βCD were employed as a reference. Spectroscopic titrations of ETH with both pCD NPs and native CDs evidenced small changes in the absorption spectra of ETH most likely due to the alcohol-like environment experienced by the drug in the presence of the carriers. This confirmed the presence of an interaction between the CDs and the drug.

Further information about the interactions existing between this drug and the carrier were provided by circular dichroism spectroscopy (CircD). Since ETH is not chiral, it is not endowed by CircD signal. However, after addiction of the carriers, a quite intense CircD signal, characterized by a negative peak at 280 nm (in correspondence of the absorption maximum) and a positive band at 320 nm (with an intense shoulder at 380 nm), appeared. This signal was similar in intensity and shape for both native CDs and pCD NPs but not identical. Particularly, the relative intensities between the main bands were not the same. These findings suggested a different complexation environment depending on the carrier and not involving exclusively the CDs cavity. Indeed, if the only interactions occurring between ETH and the carriers were host-guest type the CircD spectra would be the same for CDs in their monomeric or nanoparticulate form. The spectral differences are justified by assuming that, in the case of pCD NPs, ETH can locate both in the hydrophobic alcoholic CD cavities and in other confined spaces inside the tiny NPs formed by the crosslinked polymer.

Moreover, CircD spectra allowed the calculation of the equilibrium binding constant which resulted slightly higher for the complexes formed by ETH and pCD NPs assuming in both cases a 1:1 complexation stoichiometry.

Additionally, phase solubility studies were performed to support the conclusions of spectroscopic studies regarding the affinity between ETH and the two carriers. The apparent solubility of ETH increased linearly with growing monomeric CD concentration up to a limit of 15mg/m. Indeed, the low aqueous solubility of monomeric βCD was the limiting factor in this study. In contrast, since pCD NPs solubility is considerably higher, a continuous linear increase of ETH apparent solubility was observed. The binding constant (log(K1:1/M -1 ) around 2) calculated from phase solubility diagram were in agreement with the ones derived from the spectroscopic study.

Interestingly, phase solubility studies with pCD NPs confirmed an increase of around 10 times in ETH apparent solubility (against the 2-fold improvement provided by monomeric βCD). Of importance, [ETH]-loaded pCD NPs were stable more than 6 months thanks to the ability of CDs to encapsulate drugs at a molecular level so that it cannot self-assemble in crystals anymore. Taking into consideration the overall findings of the current study, the use of pCD NPs resulted highly advantageous over monomeric βCD for ETH complexation and the interactions occurring between the drug and its host were unraveled.

In vitro tests were performed on both PLA and pCDs loaded formulations in collaboration with the Pasteur Institut of Lille (Chapter I -Section A) on extracellular as well as on intracellular Mtb. The efficacy of the two drugs was not impaired by their encapsulation.

For the first time, an automated confocal high-content screening platform was employed for the evaluation of NPs efficacy on Mtb infected cells. Moreover, it is worth highlighting that the automated confocal microscope allowed monitoring the viability of the macrophages during the experiments. Importantly, no sign of cytotoxicity was evidenced in vitro.

Given these satisfactory results, in vivo tests on Mtb-infected mice were performed on both formulations.

An important limiting step of the in vivo study was the assessment of NPs compatibility with the Microsprayer®, the device employed to endotracheally administer the solutions to mice. Indeed, pulmonary administration in animals is more challenging than in humans. Whereas in humans, the administration of the medication is possible by self-dispensation and without any stress or pain, in animals, it has to deal with important technical limitations. Most of methods available for pulmonary delivery in animals do not allow a precise dose deposition and/or are extremely invasive for the animal. In this frame, Microsprayer® has proved to be the most efficient and reliable device allowing precise bilateral drug deposition together with a limited animal suffering. Unfortunately, not all the suspensions are compatible with this system.

The two developed NPs formulations were repeatedly passed through the device and the reproducibility of their delivery was assessed. PLA NPs were not uniformly dispensed by the Microsprayer® leading to high variability of ETH content in each spray. Thus, the study of their in vivo efficacy was compromised.

On the contrary, pCD NPs could be efficiently and reproducibly administered using the device. In fact, 98 ± 0.7 wt % of the NPs suspension was recovered after passage through the Microsprayer® and the amount of ETH in each spray was highly reproducible (99 ± 4%). The safety and the efficacy of pCD NPs were assessed on healthy and infected mice, respectively.

Of main importance. pCD NPs were demonstrated to be non-toxic after repeated pulmonary administrations (6 administrations in total). Even if a mild increase in the number of macrophages and CD4 T-cell infiltrates was highlighted, both the histological studies and flow cytometry did not evidence any major modification of the composition of the immune cell subsets. This is a remarkable finding of this project as, up to now, no study in the literature deals with the repeated administration of high doses (500 mg/Kg/day) of polymeric NPs in the lungs. The demonstrated safety of pCD NPs opens the way for the pulmonary delivery of other drugs to treat respiratory diseases.

Remarkably, when pCD NPs containing the [ETH:Booster] combination were administered to infected animals, they led to a dramatic 3-log decrease in lungs CFU in comparison to untreated animals after only 6 administration. This effect was clearly visible in the histological sections of animal lungs. Predictably, when the same treatment schedule was administered orally no antibacterial effect could be observed.

It is worth highlighting that the treatment developed in this study enabled reaching with 6 endotracheal doses of [ETH:Booster] loaded NPs the same anti-microbial effect of 18 oral doses of ETH. The reduction of the number of administration has the potential to decrease the systemic side effect of ETH thus increasing the patients' compliance.

With further studies, this green strategy could be used as a supplement to the standard treatment helping to contain TB pulmonary manifestations and it could be extended to the encapsulation of other more recent and more powerful booster molecules 12 , or of other anti-TB drug combinations. It is worth noting that these studies proved that pCD NPs are an excellent platform for co-entrapment of drugs, hence their use could be extended also to the encapsulation of tandem drugs for the treatment of other diseases.

The PLGA and pCD NPs were then challenged for encapsulation of a second drug of interest (Chapter II).

The study aimed to improve the action of pipemidic acid (PIP) against intracellular infections such as the ones caused by the enteropathogen Salmonella Typhimurium (S.Typhimurium). The choice of this drug has been made with the idea of giving a "new life" to this old antibiotic by encapsulating it in NPs and delivering it in an optimised manner 13,14 . Indeed, PIP belong to the first generation quinolones which are no longer used in therapy because of their unfavourable physico-chemical profiles, lower tissue penetration and narrower antibacterial spectrum in comparison to the following generations of quinolones 15 . PIP was selected over parent drug molecules, belonging to the same generation, for its reported better activity against some intracellular bacteria such as Pseudomonas aeruginosa, E. coli and S.Typhimurium 16,17 . Similarly to ETH, PIP is poorly soluble in water and it has a strong tendency to crystallise. Therefore, also PIP encapsulation was particularly challenging.

To address this issue, NPs were prepared by using a series of PLA and PLGA copolymers with different characteristics (lactic:glycolic ratios, molecular weights and terminal groups). The two more common formulation methods, nanoemulsion and nanoprecipitation, were compared and multiple preparation parameters (such as PIP wt%, solvents ratio, polymer concentration, etc) were adjusted in the attempt to optimise PIP loading.

All the employed polymers could successfully form monodisperse NPs with both formulation methods. In particular, when varying the kind of polymer, nanoprecipitation technique produced NPs with a wider size range than nanoemulsion (Z average determined by DLS; 110-270 nm vs 200-270 nm, respectively) but the PdI of the single formulations remained lower than 0.1 (indicating their narrow distribution). These findings suggested that nanoprecipitation method was influenced by the polymer change at a higher degree than nanoemulsion. However, even by adjusting multiple formulation parameters and by testing several different polymers, PIP was still poorly entrapped, the presence of drug crystals was often detected by optical microscopy and the drug was burst-released upon the dilution of the NPs suspensions. The maximum drug loading achieved was higher in the case of nanoprecipitation (11% for polymer P33) although PIP crystals were formed in the NPs suspension. Indeed, neither of the two formulation methods could grant a high PIP loading together with the prevention of its crystallisation. In two only cases the prevention of crystals formation was effective: i) by preparing NPs with nanoemulsion technique replacing the sonication with the homogenisation or ii) by nanoprecipitation using the polymer P19 until a maximum PIP/P19 ratio of 1:10.

However, NPs formed in the first way were considerably larger in size (around 400 nm), polydisperse and PIP burst-release was observed upon dilution. NPs formed the second way, instead, displayed a very low drug loading (around 1 wt%).

In addition, both formulation techniques presented burst-release problems after dilution in water and an interesting correlation was found between the amount of burst-released drug and the dilution factor. This phenomenon, whilst not wanted, gave some suggestions about the reasons of the unsatisfactory results obtained for PIP encapsulation. Indeed, these findings implied a preferential localisation of PIP on the surface of NPs, as already reported for other crystalline drugs [18][19][20] , and a low affinity between PIP and the polymer employed.

Most likely, PIP has stronger affinity for PVA, the surfactant that stabilizes the NPs, than for the polymeric matrices.

The location of PIP on NPs surface and its affinity for PVA was firstly hypothesized when nanoemulsion was employed and it was further corroborated when formulating the NPs by nanoprecipitation. Indeed, when using nanoprecipitation technique, blank NPs had considerably higher sizes than the loaded ones prepared in the same experimental conditions. This indicate that, possibly, PIP plays a role as a co-surfactant, reducing the size of the NPs. Moreover, as previously mentioned, in some circumstances nanoprecipitation granted the absence of PIP crystals in spite of the fact that the drug loading was really low. Noticeably, in that case, the concentration of PIP in the suspension media was 1.5 mg/mL, 5 times more than its water solubility (around 0.3 mg/mL). The most likely explanation is the formation of PIP/PVA micelles, which is in line with previous studies showing that the surfactant used to produce NPs by nanoprecipitation can also form micelles in the suspension media 21,22 . These findings highlighted once more the affinity between PIP and PVA which probably helped solubilizing the drug. In a nutshell, PIP and PVA played a key role in the NPs formation.

Indeed, the study of the contribution of each component in the frame of nanoprecipitation method was very fascinating. When the solvent containing the polymer and PIP is added dropwise to the surfactant aqueous solution, the solvent diffuses and mix with the aqueous phase. Nanoparticles formation is governed by interfacial turbulences between the solvent and the non-solvent when the supersaturation of the polymer is reached. Local fluctuation in the polymer concentration leads to the formation of nucleation sites which further grow by sticking other polymer molecules from the surrounding solution to form monodispersed NPs.

When the growing NPs encounter a surfactant molecule, its growth is arrested and the surfactant molecule is often entangled within the polymeric matrix of the NP. The results of this study suggest that PIP act as a cosurfactant with PVA cooperatively blocking the growth process. Thus, a higher number of smaller NPs is formed, explaining the size difference between loaded and blank particles when all the experimental parameters were identical. This co-surfactant action also supports the idea of PIP localisation at NPs surface.

Given the unsatisfactory results obtained with PLA/PLGA NPs and the persistent presence of PIP crystals, the same approach as for ETH in the previous study was attempted. PIP incorporation at a molecular level was investigated by using pCD NPs. Despite some promising results were reported in the literature 23 and despite the fact that pCD NPs already proved their abilities in increasing the aqueous solubility of other crystalline drugs, PIP did not show any affinity for these NPs and the loadings were lower than 1 wt%.

Some interesting considerations can be made by comparing the results obtain with ETH and PIP during this PhD research. Despite their similar physico-chemical behaviour, these two drugs showed very different affinities for the two NPs systems. In the case of ETH, even though PLA NPs granted a higher drug loading (around 36%) they could not totally hamper ETH crystallisation. Thus, pCD NPs resulted the most effective carrier in spite of their lower loading (2.5 wt%) thanks to their ability to stabilise ETH at a molecular level. In the case of PIP instead, pCD NPs were totally ineffective in increasing the apparent solubility of this drug which remained lower than 1 mg/mL (drug loading lower than 1 wt%). Moreover, PIP did not show a satisfactory affinity also for PLGA and the loadings remained much lower than the ones obtained for ETH.

These is no such thing as a universal drug carrier, thus pCD NPs which resulted very effective in one case, completely failed in the other.

These findings point out how the physico-chemical properties of drugs can heavily impact on their interaction with the carriers. Therefore, there's a necessity of adapting the formulation strategy to the peculiarities of each drug molecule (even when the physico-chemical properties of two drugs seem similar). Consequently, the work of medical researchers and formulation scientists can be very intriguing but also very challenging.

In light of the reported results, in the specific case of PIP, the physical encapsulation was not successful with any of the two studied NPs platforms. For this reason, new strategies were employed in collaboration with Dr Desmaële (Institut Galien of Paris). Firstly, PIP structure was chemically modified through O-methylation in an attempt to avoid PIP crystallisation and to increase the drug affinity for the polymeric NPs. The derivative completely avoided PIP crystallisation, however its affinity for the NPs and hence the drug loading were very low (less than 1 wt%).

In this context, the idea was to covalently link PIP to a biodegradable polymer to form a prodrug able to selfassemble in NPs. An original drug-initiated polymerization technique was employed to produce such conjugate in a single step and without the use of catalysts. PCL was preferred over PLGA as it has been previously

showed that it can polymerize in a controlled manner without catalysts forming monodisperse polymers with controlled and adjustable molecular weight 24 . A PIP-PCL conjugate was successfully formed with a drug:polymer molar ratio of 1:10. The structure of this prodrug was thoroughly characterised by 1 H and 13 C NMR, IR and mass spectrometry leading to the conclusion that the polymerisation occurred on the piperazine free amino group of PIP.

The conjugate was shown to be able to self-assemble in round-shaped (SEM determination) NPs with the nanoemulsion method. NPs could be successfully obtained by employing 100% of the PCL-PIP conjugate or by mixing it at 50:50 ratio with PLGA. Furthermore, different surfactant amounts (0.5% and 1%) were tested.

The maximum drug loading was obtained for 100% PCL-PIP NPs which contained around 27 wt% PIP. All NPs where characterised with four complementary and advanced techniques: DLS, NTA, TRPS and SEM.

NTA and TRPS are both interesting particle by particle methods even if based on completely different principles. These two techniques are able to determine at the same time the particles mean diameter and their concentration. NTA and TRPS were in good agreement regarding the size of all measured PCL-PIP NPs (i.e.

130 ± 37 and 126 ± 52 nm, respectively).

DLS and SEM led both to an overestimation of NPs size for different reasons. As previously explained, DLS is particularly sensitive to the presence of small amounts of big particles hence shifting the size distribution towards higher values (around 200 nm). Regarding NPs sizes estimated by SEM, the overestimation (values of around 300 nm) was most likely caused by the sample preparation. Indeed, PCL-PIP appeared to be flattened on the support surface. This effect, which was not evidenced in the case of PLGA NPs, can be ascribable to different physico-chemical properties of PCL-PIP conjugate, such as glass transition and melting temperatures.

These NPs resulted to be stable for around 10 days and further experiment could be performed to increase their long-term stability. However, the bioconjugate did not degrade in the presence of enzymes because of the tertiary amide conjugation.

Further studies are focused on the synthesis of novel PCL-PIP conjugates able to be degraded to release PIP.

It would be then very captivating to establish if such PCL-PIP NPs would be able to optimise the delivery and increase the efficacy of PIP towards intracellular bacteria. Moreover, if this approach will be proved effective it could be easily extended to other more active molecules of quinolones family or to other drugs.

The formulation studies performed within this work provided fairly clear indications about the importance of the interactions between each component of NPs to obtain a successful carrier. A deep understanding of the morphology and structure of the NPs, on their corona and core composition and on the location of the drug is crucial to explain the obtained results and to move forward in the formulation process. It has been shown how the localisation of the drug at the surface of NPs can induce a burst release whereas controlled release could be obtained if the drug was entangled in the NPs polymer matrix. However, these conclusions have been drawn from experimental results or have been obtained using surface analysis (such as XPS or SIMS) or indirect methods (such as electrophoretic mobilities, hydrophobicity and mobility of polymer chains). No technique able to visualise and thus prove the truthfulness of these conclusion was available until now for PLA/PLGA NPs and for all the nanocarriers which do not possess any particular contrast properties (such as a fluorescent component or electron density). Therefore, at least formally, these findings can still be considered as hypothesis. Moreover, the available techniques can give only bulk information but cannot assess if the samples are homogeneous.

Similarly, the surfactant corona around PLA/PLGA NPs, which is considered a certitude in drug-delivery domain, has never been visualised and therefore its presence was never undoubtedly proved.

In the light of these consideration, formulation scientists have always been fascinated and interested in the development of a method able to visualise the individual NPs and provide at the same time their chemical visualising for the first time the PVA corona surrounding the PLA core. Thus, the existence of the NPs coreshell structure was here finally undoubtedly proved. Interestingly, the detected PVA corona had a thickness of around 15-20 nm. Of note, the comparison of NTA (hydrodynamic diameter) and cryo-TEM (dry diameter) results reported in the Chapter IA on the same unloaded PLA NPs suggested a thickness of this corona of around 40 nm. Therefore, a discrepancy between the two sets of experiments is evident. Nonetheless, this difference can easily be explained by the fact that while NTA values were obtained on hydrated NPs, the AFM-IR measurements were performed on dried NPs. Thus, the PVA chains were progressively dehydrated during the drying process (Figure 2D Chapter IIIB) and flattened on NPs surface, considerably decreasing the corona thickness.

Given these very promising results, a more ambitious goal was set in the study. Indeed, as previously pointed out, the design of a successful drug delivery system requires a deep knowledge of the drug location within the carrier, which highly influence the drug release profile. This was especially evident in our studies on PIPloaded NPs. Thus, these particles were chosen for tapping AFM-IR investigations. In particular, the PIP NPs formulated with PLGA polymer P19 were selected for their low drug loading (less than 1wt%) together with their ability of avoiding PIP crystallisation which were thoroughly discussed above. Tapping AFM-IR provided the fascinating perspective of confirming by direct visualisation the hypothesis formulated during the NPs development, such as the PIP localisation on NPs surface and its affinity for PVA.

Once more, a IR signature for each component was selected thanks to FTIR spectra. PLGA (P19) was detected at 1425 cm -1 (C-H stretching) while the band at 1760 cm -1 , previously employed for PLA localisation, was discarded because it saturated the chemical mapping. The selection of a PVA specific absorption band was more challenging. Indeed, the PVA signal at 1420 cm -1 was overlapped by PLGA bands. As no exclusive IR signal could be attributed to PVA, the 1415 cm -1 was chosen as wavenumber for PVA detection because it was the only region where the PVA was a stronger absorber than PLGA. PIP displayed multiple interesting peaks but only a few were not overlapping with PLGA and PVA. Hence, its carboxylate band at 1639 cm -1 was selected.

A complete set of chemical mappings and spectra were recorded for empty PLGA NPs and for PIP-loaded ones, formulated by both nanoemulsion and nanoprecipitation methods. Of note, objects, NPs or micelles, as small as 10 nm could be detected and chemically mapped thanks to tapping AFM-IR.

Despite the absence of a wavenumber exclusively attributable to PVA, a surfactant corona could still be visualised around the PLGA core in both loaded and unloaded NPs. Fascinatingly, regardless of the difficult circumstances, the tapping AFM-IR managed to further confirm PVA identity by recording IR spectra directly on the thin NPs corona.

Moreover, the principal purpose of the study was achieved, as PIP could be clearly localised within NPs structure. Indeed, the tapping mode allowed the study of drug distribution within each single NP and gave access to invaluable information, inaccessible to any other characterization technique. The hypothesis formulated in Chapter II could be here clearly confirmed: PIP was detected around NPs and it was colocalised with the PVA corona. These findings indeed validated the hypothesis that the drug was located at NPs surface explaining the burst-release upon dilution. Of note, the PIP spectral changes, detected when the drug was localised in NPs corona, in comparison to pure PIP, further reinforced the idea of a strong affinity Drug-loaded pCD NPs were proved to be compatible with the Microsprayer ® and could therefore be endotracheally aerosolised in mouse lungs. Remarkably, empty pCD NPs showed no major toxicity after repeated pulmonary administrations of high doses opening the way for further applications. Impressively, a 1000 times decrease in the lung bacterial load of infected animals could be obtained with only 6 doses of ETH:Booster loaded pCD NPs in comparison with non-treated animals.

2. A complementary study was performed on ETH loaded pCD NPs to unravel the type and strength of interactions occurring in these systems. The ability of pCD NPs to increase ETH apparent solubility of about 10 times was confirmed. Moreover, the spectroscopy studies evidenced that ETH in NPs is located not only in CD cavities but also in confined domains between CDs, inside the crosslinked NPs. ETH was incorporated in a molecular state, thus avoiding its crystallisation even over long storage time. These NPs could be highly concentrated in aqueous media (up to more than 200 mg/mL) emphasizing the interest of using them for drug encapsulation instead of monomeric βCDs, which otherwise display a very limited aqueous solubility (<18 mg/mL).

3. The ability of PLA/PLGA and pCD NPs to achieve the encapsulation of PIP, a poorly soluble firstgeneration quinolone with a strong tendency to crystallize, was investigated. PIP was unfortunately poorly entrapped in PLA/PLGA NPs formulated by either nanoemulsion or nanoprecipitation. Drug crystallisation remained a major issue and PIP burst-release was evidenced upon dilution thus leading to the conclusion that PIP was mostly localised on NPs surface. Moreover, PIP did not show any affinity for pCD NPs. In light of these results, the drug was directly conjugated to the polymers to obtain stable NPs with high PIP payloads. An original drug-initiated and catalyst-free polymerization was developed. The PCL-PIP conjugate was thoroughly characterised and it was shown to self-assemble in NPs. PCL-PIP NPs displayed a monodisperse size distribution and contained around 27 wt% PIP.

However, as PIP was conjugated to PCL through a tertiary amide bond, particularly difficult to hydrolyse, the complete enzymatic degradation of the conjugate could not be achieved. Further studies are now focused on the synthesis of novel PCL-PIP conjugates able to release PIP. Main applications will include the delivery of PIP to infected cells.

4. In an attempt to go beyond the state of art in the NPs characterisation field, an original nanospectrocopic technique was adapted to unravel the structure of core-shell PLGA NPs. This method, namely AFM-IR, combines the high resolution provided by atomic force microscopy (AFM) with chemical characterisation offered by IR spectroscopy. A region of the IR spectrum specific of each NPs component was chosen as its specific fingerprint and it was used to detect and image its chemical distribution with the AFM-IR.

An additional innovation of this study is the first-time use of the recently developed tapping mode of AFM-IR. The tapping mode allowed the investigation of soft or non-adhesive samples such as PLGA NPs which could be well-described without crushing or pushing them as it happened when the contact mode was employed. One of the main achievements of this study was the visualisation of the surfactant corona around NPs. Moreover, the localisation of a drug of interest, PIP, within the NPs, could be assessed thus confirming the surface localisation of this drug and its affinity with the PVA corona which where hypothesised in the second chapter of this work. It is remarkable to highlight that PIP was incorporated at only 1% with respect to NPs weight but, despite the challenge of such low loading, the location of the drug could still be identified. Additionally, tapping AFM-IR was very useful to undoubtedly confirm the homogeneous localisation of PIP inside PCL-PIP NPs and to move the first steps towards a particle by particle drug quantification. Indeed, a first strategy confirming the PIP/PCL theoretical ratio by means of IR cartographies on individual NPs was successfully presented here. With further studies, the current approach could be improved and a more precise quantification could be achieved opening the way to a revolution in drug delivery domain. Furthermore, the soft exploration by tapping AFM-IR allowed detecting NPs as small as 10-20 nm.

These findings have particular relevance in the NPs field because, to the best of our knowledge, this technique is the first one able to provide such precise information about NPs components localisation on a particle by particle basis and with such high resolution.

5. In addition to the achievements in the domain of NPs characterisation, here the AFM-IR was proved to be a reliable, non-destructive and direct method able to locate PLA NPs of around 170 nm within cells with a high lateral resolution, by taking advantage of the specific IR response of their constitutive PLA polymer.

This response was used as a fingerprint to enable the unambiguous chemical identification of the NPs, avoiding the need of labelling. Indeed, the most common techniques usually employed to intracellularly detect NPs require the modification of plain NPs with a dye. Such labelling can lead to several inconveniences and it is the label, not the object itself, which is detected by the instrument. The AFM-IR instead, detect directly the object of interest thus avoiding misinterpretations.

In this context, with further studies, AFM-IR could become a powerful tool to unravel the fate of individual unlabelled NP inside cells. Moreover, the identification of the NPs chemical composition with highresolution could enable determining drug release and NP degradation. AFM-IR could find a plethora of applications in the field of drug delivery and its use could be extended to other kinds of NPs.

Conclusion

245

In conclusion, during this PhD work, two promising approaches for the treatment of intracellular diseases were developed by means polymeric NPs. The potential of such polymeric carriers for the efficient entrapment of challenging drugs was illustrated. Moreover, the application of an original IR nanospectroscopic technique to NPs characterisation enabled the acquisition of invaluable and unprecedented information about their structure, the localisation of drug and its amount on a particle by particle basis. This technique was also proved to be a reliable, direct and label-free method for NPs intracellular detection. Moreover, it was sensitive enough to detect nanostructures of only 10-20 nm and drug molecules despite loadings of less than 1 wt%.

In a nutshell, these studies pave the way towards an in deep investigation of NPs and we believe that the AFM-IR methodology developed here could be an invaluable tool in the near future in the development of complex NPs containing one or more synergic drugs. Abstract: The treatment of intracellular infections is very challenging given the ability of bacteria to "hide" inside the cells of the host, especially the ones of the immune system, thus hampering the action of many antimicrobial agents. The battle against these bacteria has been further exacerbated by the increasing diffusion of antimicrobial resistant strains. In this frame, nanoparticles (NPs) are a very promising strategy to overcome the limitations of free antimicrobial agents by administering them in an optimized manner. This PhD work, performed as part of the European Project ITN Cyclon Hit, aimed at the development and advanced characterisation of antibiotic-loaded biodegradable and biocompatible NPs made of poly (lactic acid) (PLA), poly (lactic-co-glycolic) (PLGA) and polycaprolactone (PCL) or of polymerised cyclodextrins (pCDs). The first two chapters are dedicated to the encapsulation of powerful but challenging drugs in polymeric NPs. Firstly, these carriers were employed for the simultaneous delivery of a potent drug combination recently discovered, ethionamide (ETH) and its booster, for tuberculosis therapy. Secondly, they were used to address the challenges related to the incorporation of a first-generation quinolone, pipemidic acid (PIP), with the aim of optimising its intracellular delivery in infections such as salmonellosis. The efficient co-incorporation of ETH and booster had to overcome several technological barriers. These drugs presented solubility, crystallisation and bioavailability-related problems which were overcome thanks to the developed NPs. Our engineered PLA and pCD NPs were both able to efficiently co-encapsulate the two molecules. Among the in depthcharacterised formulations, pCDs NPs displayed the best physico-chemical properties and were shown to host the drugs both in the CD cavities and in confined spaces inside NPs crosslinked polymer. The pCD NPs were administered in vivo by endotracheal route directly to the infection site.

Empty NPs were shown non-toxic after repeated pulmonary administration of high doses. Moreover, loaded pCD NPs led to a 3-log decrease in the pulmonary bacterial load of infected animals after only 6 administrations. Similarly, the incorporation of PIP faced challenges mainly related to PIP crystallization and burst release. Unfortunately, PIP displayed poor affinity for all the studied polymeric materials and its physical encapsulation was unsuccessful. Thus, an alternative approach was developed by coupling PIP to PCL by using an original catalyst-free drug-initiated reaction. The PCL-PIP conjugate self-assembled in NPs with up to 27 wt% PIP which were thoroughly characterised. However, the conjugate couldn't be enzymatically degraded. With the design of novel PCL-PIP conjugates, this self-assembly approach could represent a promising strategy. The deep understanding of the structure and composition of complex core-corona nanocarriers containing one or two active molecules is crucial for their optimisation. The last two chapters are devoted to the innovative application of AFM-IR, an original nanospectroscopic method combining atomic force microscopy (AFM) with infrared (IR) spectroscopy, to the chemical analysis of PLGA NPs or to their label-free detection after cell internalisation. AFM-IR is able to provide chemical characterisation at the nanometer scale (resolution ~10nm). One main breakthrough here is the application of the recently developed tapping mode allowing the investigation of single polymeric NPs. The specific IR signal of NPs constituents was used to unravel the chemical composition of their core and corona as well as to precisely locate the drug. Moreover, the AFM-IR in contact mode enabled for the first time the label-free localisation and unambiguous chemical identification of NPs inside cells using the polymer IR specific response as a fingerprint. This work paves the way for countless application of this technique in the field of drug delivery.

I

  would also like to thank the members of my thesis committee: firstly, Dr. Vivi Miriagou, researcher at the Hellenic Pasteur Institute of Athens, and Dr. Laurent Marsollier, INSERM research director at Université d'Angers, for granting me the honour of evaluating my work as "rapporteurs"; furthermore, Dr. Priscille Brodin, INSERM research director at the Pasteur Institute of Lille, Dr. Alexandre Dazzi, professor at Université Paris-Sud, Dr. Didier Letouneur, CNRS research director at Université Paris-Diderot and Dr. Marie-Françoise Bernet-Camard, assistant professor at Université Paris-Sud, for having accepted to evaluate my thesis. Je tiens à remercier particulièrement Dr. Marie-Françoise Bernet-Camard pour avoir aussi été ma coencadrante et m'avoir formée à la culture cellulaire. Merci pour ta disponibilité, ton support, tes conseils et pour nos échanges professionnels et personnels. I would like to express my gratitude to all the collaborators which enriched my work with their significant contribution. The team of Dr. Priscille Bordin: Ms. Joana Costa-Gouveia, Dr. Arnaud Machelart, Mr. Samuel Jouny and Ms. Nathalie Deboosere for the in vitro and in vivo studies on Mycobacterium Tuberculosis. The team of Pr. Alexandre Dazzi: Dr. Ariane Deniset-Besseau and Mr. Jérémie Mathurin for teaching me how to use AFM-IR, for their help with IR data treatment, for their experience in the nanospectroscopy field and for their very strong cooperation and support. I deeply thank Dr. Didier Desmaële for his great competence in organic chemistry and for the very fruitful scientific advices. Many thanks to the CPBM team, especially to Ms. Sandrine Lécart for forming me at the use of confocal microscopy. I also thank Mr. Michel Terray and Mr. Laurent Lachmanski of Malvern Instruments for the helpful discussions.

  techniques complémentaires et avancées (Chapter I -Section A). Le principal avantage offert par les NPs en pCDs résidait dans leur capacité d'incorporer les deux médicaments au niveau moléculaire et d'éviter ainsi totalement la cristallisation de l'ETH. En complément à cette étude, une combinaison de techniques spectroscopiques a été employée pour analyser le type et la force des interactions qui existent entre l'ETH et les NP en pCDs (Chapter I -Section B).

Figure 2 -

 2 Figure 2 -Biopharmacutical Classification System (BCS) of drugs established by FDA.The classification is based on drugs solubility (with a soluble drug defined as one in which highest dose intended for human use dissolves in 250 mL of water) and permeability (with a permeable drug defined as one with ≥ 90% oral bioavailability or ≥ 90% absorption)24 .

Figure 3 -

 3 Figure 3 -Chemical structure of PGA, PLA and PLGA

Figure 5 -

 5 Figure 5 -Structure, configuration and main characteristics of native CDs.

Figure 6 -

 6 Figure 6 -Polymerisation of βCD with epichlorohydrin leading to the formation of cross-linked polymeric CDs (pCDs) which in water assemble under the form of NPs.

Figure 7 -

 7 Figure 7 -Schematic representation of the three main characterisation techniques (DLS, NTA and TRPS)measuring hydrodynamic diameter of NPs. DLS is a bulk technique able to rapidly assess NPs hydrodynamic diameter by detecting the light scattered in every direction by the all the particles in the sample. The diffracted light is typically detected at 90 or 173° and mathematically analysed by an autocorrelator. The instrument provides an intensity distribution in function of the particles size from which it can calculate a volume and a number distribution by using Mie theory. However, to precisely perform these conversions, knowledge about NPs refractive index is required. NTA measures the hydrodynamic diameter of individual NPs by employing a microscope and a camera to detect and record the light diffracted by a small volume of sample. Thus, NPs are visualised as bright spots (white spots in the black field) and followed in their Brownian motions (red lines = NPs trajectories) by the instrument software. The software counts individually the bright spots and, from their trajectories, it calculates NPs size (independent from their refractive index). NTA plots NPs concentration (particles/mL) over their size. TRPS is a Coulter-type counter which measures directly NPs size and concentration on a particle by particle basis. A voltage is applied across a tuneable pore, filled of electrolyte, thus generating an ionic current. The NPs, driven by this current, traverse the pore causing a transient blockade (yellow peaks). The magnitude of this blockade is linearly proportional to NPs volume and the number of peaks corresponds to the number of particles. TRPS plots NPs concentration (particles/mL) over their diameter.

  nanoparticulate drug carrier able to host a powerful drug combination. The drugs of interest in this research are ethionamide (ETH), a second line anti-TB drug with low therapeutic index, and its Booster (BDM41906), belonging to a family of molecules able to boost ETH bioactivation, thus having the potentiality of improving the current TB clinical outcome. Indeed, the clinical use of ETH is limited by its severe side effects and by the high doses need to obtain a therapeutic effect.The administration of the [ETH:Booster] pair is very challenging, as the two molecules possess completely different physico-chemical properties and biodistributions and are not soluble in biological-compatible solvents. Moreover, ETH administration is further hampered by its strong tendency to crystallize in aqueous media and the necessity to use large amounts of organic solvents to solubilize it.In this context, we have investigated the co-encapsulation of the [ETH:Booster] pair in nanoparticles made of biocompatible and biodegradable polymers such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) or polymerized cyclodextrins (pCDs). PLA NPs coencapsulating [ETH:Booster] were successfully formulated by nanoemulsion and characterized with advanced complementary physico-chemical methods.

[

  ETH:Booster] pair. With further developments, our approach could be used as a supplement to the standard treatment to contain the M. tuberculosis pulmonary manifestations of the disease and to prevent its dissemination, especially for MDR-XDR cases. Graphical abstract -Endotracheal delivery of polymeric β-cyclodextrin nanoparticles loaded with ethionamide and one of its boosters (BDM41906) into mouse lungs infected by M. Tuberculosis. This multidisciplinary study was performed in collaboration mainly with the team of Dr. P. Brodin at the Pasteur Institute of Lille, partner of the European project "Cyclon Hit" which founded this PhD research. E. Pancani prepared, optimised and characterised the NPs used in the study, helped in dosing NPs drugs content by HPLC and assessed the NPs passage though the Microsprayer. Moreover, she actively participated in close cooperation with the Pasteur Institute of Lille in conceiving the in vitro and in vivo experiments, helped in the interpretation of the obtained results and contributed in writing the paper. Chapter I -SECTION A -Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles 73 The following paper and its supplementary information are published in Scientific Reports DOI: 10.1038/s41598-017-05453-3 Online version available at : https://www.nature.com/articles/s41598-017-05453-3#Sec26

N

  P s e m p ty R T N P s e m p ty 4 °C N P s [E T H :B o o s te r] 1 :1 R T N P s [E T H :B o o s te r ] 1 :1 4 °C N P s E T H 0 .4 3 m g /m L R T N P s E T H 0 .4 3 m g /m L 4 °C Supplementary Figure S10 -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.

  e m p ty N P s E T H N P s [E T H :B o o s te r ] [1 :1 ] N P s [E T H :B o o s te r ] [1 :0 .1 ] N P s B o o s te r Supplementary Figure S4 -Stability of NPs obtained by nanoemulsion over a period of 1 month. No crystallization was observed during storage and no significant size or polydispersity changes (DLS) were found.

  Supplementary Figure S6-Effect of empty NPs on intra-and extra-cellular assay. (a) % of inhibition of bacterial RFU obtained with empty PLA made by the nanoemulsion method (solid circle) and with empty pCD (solid triangle). (b)% of inhibition of intracellular bacterial area (black points) and number of cells (grey points) obtained with empty PLA made by the nanoemulsion method (solid circle) and with empty pCD (solid triangle).

) 5 µl

 5 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 (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 further dilution in DMSO and/or with acetonitrile/water 45/55 v/v.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 Acquity 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 (CH3CN) 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.

  The amount of encapsulated drug has been calculated with a direct or indirect estimation, depending on the sample fraction considered]Bacteria and macrophages M. tuberculosis 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 )

 3 % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (1 -Test bacterial area-INH bacterial area DMSO bacterial area-INH bacterial area ) × 100

  H and13 C NMR, IR and mass spectrometry to unambiguously describe its structure. The newly synthesized PCL-PIP was employed to prepare self-assembled NPs which were comprehensively characterized by a set of complementary techniques: dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS) and scanning electron microscopy (SEM). Noteworthy, the PIP content in the NPs was as high as 27 wt%. Unfortunately, due to the presence of a tertiary amide bond, PLC-PIP couldn't be entirely enzymatically degraded and hence the antibacterial activity of PIP couldn't be restored. With further studies, PIP-initiated polymerization of biodegradable esters could be a promising strategy to produce NPs with high PIP content. The novel methodology established here could be applied to a variety of other active molecules. This study was performed in collaboration with Dr D. Desmaële of the Institut Galien, for the synthesis of PCL-PIP conjugate and with Dr M.F. Bernet-Camard of the EA4043 (UBaPS) for the studies on Salmonella Typhimurium. E. Pancani prepared and characterised (size, zeta potential, concentration, HPLC dosage) all the NPs formulated in the study. Moreover, she analysed the results, interpreted the data and wrote the manuscript with Dr R. Gref. Original Article -Acta Pharmaceutica Sinica B Nanoparticles with high payloads of pipemidic acid, a poorly soluble crystalline drug: druginitiated polymerization and self-assembly approach Elisabetta Pancani a , Mario Menendez-Miranda a , Alexandra Pastor a,b , François Brisset c , Marie-Françoise Bernet-Camard d , Didier Desmaële b , Ruxandra Gref a,* a Institut de Sciences Moléculaires d'Orsay (ISMO), CNRS UMR 8214, Univ. of Paris-Sud, Université Paris-Saclay, 91405 Orsay, France b Institut Galien, UMR8612 Univ. of Paris-Sud, Université Paris-Saclay, 92290 Châtenay-Malabry, France c Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), CNRS UMR 8182, Univ. of Paris-Sud, Université Paris-Saclay, 91405 Orsay, France d EA4043 "Unité Bactéries Pathogènes et Santé" (UBaPS), Univ. of Paris-Sud, Université Paris-Saclay, 92290 Châtenay-Malabry, France Received 3 February 2018; received in revised form 28 February 2018 ; accepted 16 March 2018 *Corresponding author. Tel.: +33 1 69 15 82 34.

Figure S1 .

 S1 Figure S1. Optical image of PIP crystals in PLA/PLGA NPs. Typical example of PIP crystals formed in NPs suspension observed with 10 times and 40 times (zoomed panel) magnification in an inverted optical microscope over a Malassez chamber.

Figure S2 .

 S2 Figure S2. PLGA PIP-loaded NPs observed by SEM. a: SEM micrograph. b: Histogram of the size distribution of the NPs showing a mean diameter d = 164 ± 27 nm. N (d) refers to the total counts and the scale bar corresponds to 500 nm.

Figure S3. 13 C

 13 Figure S3. 13 C NMR spectrum of PCL-PIP in DMSO-d6.

Figure S4 -

 S4 Figure S4 -IR spectrum of PCL-PIP.

Figure S5 .

 S5 Figure S5. IR spectra of PCL, PIP, PCL-PIP conjugate and PCL+PIP physical mixture.

Figure S56 - 1 H

 1 Figure S56 -1 H NMR of decarboxylated caprolactone adduct (4) (see Fig. 2 main manuscript).

Figure 11 -

 11 Figure 11 -Schematic representation of an AFM setting

Figure 13 -

 13 Figure 13 -Acquisition modes of AFM-IR. a) First mode = Local IR absorption spectrum obtained from measuring the amplitude of AFM cantilever oscillation as a function of wavenumber. b) Second mode = AFM topography of an eukaryotic cell and IR cartography of the amide I band of the same cell (1660 cm -1 ). Partially adapted with permission from Anasys Instrument.

Figure 14 -

 14 Figure 14 -Comparison of the cantilever ring-down oscillation obtained in the case of conventional AFM with the cantilever continuous wave oscillation obtained when working in resonance enhanced mode. Adapted with permission from Anasys Instrument.

Figure 15 -

 15 Figure 15 -NanoIR-2 configuration

E.

  Pancani prepared and characterised the NPs used for the study, contributed to design and perform the AFM-IR experiments, to interpret the obtained results and to paper writing. Tapping AFM-IR: a powerful new tool to unravel the structure of individual drug-loaded polymeric nanoparticles Jérémie Mathurin 1# Elisabetta Pancani 2# , Ariane Deniset-Besseau 1 , Kevin Kjoller 3 , Craig.B Prater 3* , Ruxandra Gref 2* , Alexandre Dazzi 1* . 1 Laboratoire de Chimie Physique (LCP), CNRS UMR 8000, Univ. of Paris-Sud, Université Paris-Saclay, 91405 Orsay, France 2 Institut de Sciences Moléculaires d'Orsay (ISMO), CNRS UMR 8214, Univ. of Paris-Sud, Université Paris-Saclay, 91405 Orsay, France 3 Anasys Instruments, Santa Barbara, CA, United States. # equally contributing first authors * senior corresponding authors

Figure 16 -

 16 Figure 16 -Setup of the NanoIR2s (Anansys Instrument) in tapping mode.

Figure 17 -

 17 Figure 17 -Comparison of contact AFM-IR (A) and tapping AFM-IR (B, C) chemical map on PLA NPs and illustration of NPs drying process (D). The NP topography variations are represented by the 3D. The color code represents the absorption distribution of the ester carbonyl band of PLA at 1760 cm -1 (red corresponds to a strong absorption and green to no absorption) and of the C-H bending band of PVA at 1415 cm -1 (blue corresponds to a strong absorption and green to no absorption). Schematic representation of the drying process of a PLA NP on an AFM support (D).

Figure 18 -

 18 Figure 18 -IR spectra of bulk PLGA, PVA and PIP. In blue: spectrum of PLGA characterized by the ester carbonyl band at 1760 cm -1 and C-H bending of CH2 and CH3 around 1500-1300 cm -1 . In black: spectrum of PVA showing in the 1500-1300 cm -1 range the C-H bending but with different positions compared to the PLGA. In red: spectrum of PIP showing characteristic carbonyl bands at 1640 cm -1 (carboxylate C=O asymmetrical stretching), 1615 cm -1 , N-H bending at 1570 cm -1 and C=O (carboxylate) symmetrical stretching at 1360 cm -1 .

Figure 19 -

 19 Figure 19 -AFM-IR study of empty PLGA NPs. A) Topography image of the PLGA NPs. B) Tapping AFM-IR map at 1425 cm -1 corresponding to a strong absorption of PLGA and very weak absorption of PVA. C) Phase image of the NPs revealing the significant shift produced by the topography effect. D) Tapping AFM-IR map at 1415 cm -1 corresponding to a weak absorption of PLGA and strong absorption of PVA. This map shows clearly that the PVA is localized around the NP. E) Infrared spectra obtained by tapping AFM-IR at 2 positions: on the NP (orange arrow corresponding to orange spectrum) and on the NP border (pink arrowpink spectrum).

Figure 20 -

 20 Figure 20 -AFM-IR study of PIP-loaded PLGA NPs prepared by nanoemulsion. A) Topography of PIP-loaded nanoemulsion PLGA NPs. B) Tapping map at 1425 cm -1 corresponding to the strong absorption of PLGA. C) Tapping AFM-IR map at 1415 cm -1 corresponding to a weak absorption of PLGA and strong absorption of PVA. The presence of a PVA absorbing region outside the NPs core borders is here clearly visible. D) IR Map at 1640 cm -1 showing the spatial distribution of the PIP colocalized with PVA in the corona around the PLGA NPs core. E) Average IR spectra of PIP corona (cyan arrowcyan spectrum) and PLGA particles (pink arrowpink spectrum).

Figure 21 -

 21 Figure 21 -AFM-IR study of PIP-loaded PLGA NPs prepared by nanoprecipitation. A) Topography of PIP-loaded nanoprecipitation PLGA NPs. B) Map at 1425 cm -1 corresponds to a strong absorption of PLGA. C) Phase image associated to the topography showing a small edge effect on each NPs. D) Map at 1640 cm -1 shows the distribution of the PIP around the nanoparticles. E) Average infrared spectra of PIP corona (cyan arrowcyan spectrum) and PLGA nanoparticles (pink arrowpink spectrum).

PLGA 50 :

 50 50 acid terminated (MW=5-20 KDa, 10P019) was kindly donated by PCAS (Expansorb, Aramon, France).

Figure S22 -

 S22 Figure S22 -IR spectra IR of bulk PLA and PVA in the range 1500-1300 cm -1 . In green: spectrum of PLA characterized by C-H bending of CH2 and CH3. In black: spectrum of PVA showing its C-H bending bands which have different positions compared to the PLA. Indeed, the PVA band at 1420 cm -1 stand out from the PLA spectrum and could thus be used as a fingerprint to undoubtedly detect PVA localization.

Figure S23 -

 S23 Figure S23 -AFM-IR study of empty PLGA NPs (complementary to Figure 4, Main text). A) Topography image of the PLGA NPs. B) Tapping AFM-IR map at 1760 cm -1 corresponding to a very strong absorption of the carbonyl ester group (C=O stretching) of PLGA.

Figure S24 -

 S24 Figure S24 -Schematic representation of the deposition and drying process of a PIP-loaded PLGA NP on an AFM support. During the drying process, PVA chains are progressively dehydrated and thus flattened on the NP surface or deployed on the AFM support. The thickness of the corona is therefore sensibly reduced

Figure S25 - 5 ,

 S255 Figure S25 -AFM-IR study of PIP-loaded PLGA NPs prepared by nanoemulsion (complementary to Figure 5, Main text). A) Topography of PIP-loaded nanoemulsion PLGA NPs. B) Phase image associated to the topography showing the croissant moon shape effect which explains the lack of intensity in IR cartographies.

Figure 27 -

 27 Figure 27 -AFM-IR study of PCL-PIP NPs. a) Topography image of PCL-PIP NPs. b) Tapping AFM-IR map at 1732 cm -1corresponding to the ester carbonyl band of caprolactone units (laser power 1.85%) c) Topography image of PCL-PIP NPs. This image shows clearly that the two topographies (a and c) are identical and that the first IR acquisition did not damage the NPs. Indeed, the parameters of the instruments were carefully set in order to avoid overheating of PCL which possess a low melting point (around 60°C) thus degrading very easily. d) Tapping AFM-IR mapping at 1630 cm -1 corresponding to the absorption of PIP (laser power 1.85%). This map reveals the colocalization between PCL and PIP thus confirming that PIP is successfully located inside NPs. e) spectra obtained by tapping AFM-IR in multiple locations inside the NPs. All the spectra confirm that PCL and PIP are colocalized. Topography colour grading: 0-100 nm. IR mapping colour grading = 0-2 Arbitrary Units (A.U.), from green to pink.

  fact, in the case of both techniques, NPs flattening was observed only for PCL-PIP NPs and not for PLGA ones, which undergo to the same sample preparation procedures. Consequently, the phenomenon must be connected to the physico-chemical properties of PCL. Indeed, PCL displays a much lower glass transition temperature (Tg) and melting point (MP) when compared to both PLA and PLGA polymers. PCL has a Tg of about -60 °C whereas the one of PLA and PLGA is in the range of 40-60 °C (Introduction -Section 3.1).

Figure 2 (

 2 2B and 2D) which clearly show that PCL and PIP absorption signals are perfectly overlapping. Herein, PIP was clearly localised inside NPs and co-localised with PCL. These images confirm unequivocally that the PIP covalent conjugation with the PCL polymer was a successful strategy enabling to finally obtain drug-loaded NPs.

Figure 28 -

 28 Figure 28 -Absorption coefficient (Alpha=α) as function of the wavenumber.

Figure 29 -

 29 Figure 29 -Graphical representation of IR signal integration (A.U./nm 2 ) of a single NP at a certain wavenumber. The IR intensity in z (A.U.) is integrated over the IR signal area (nm 2 ).

A

  correlative study between confocal fluorescence microscopy and AFM-IR was conducted to prove the reliability of the technique. To do so, fluorescent rhodamine (Rho)-NPs were prepared. Rho-NPs internalisation in cells was confirmed by using confocal fluorescence microscopy. Then, the exact same cell was subsequently analysed by confocal microscopy and by AFM-IR. The images of NPs obtained by the two techniques were in excellent agreement. These results were further confirmed on non-labelled PLA NPs. Thus, AFM-IR allowed ascertaining the chemical composition of nanoparticles of around 170 nm inside a cell without the need of any labelling and with a high lateral resolution.The novel methodology established here could be applied to a variety of NPs containing one or several active molecules. Indeed, combinatorial drug therapies emerge among the most promising strategies to treat complex pathologies such as cancer and severe infections. Thus, advanced imaging methodologies are needed to investigate the fate of engineered NPs containing drug combinations inside cells. In this context, with further studies, AFM-IR could become a powerful tool to unravel the fate of each individual unlabelled nanoparticle inside cells. This study was performed in collaboration with the group of Dr A. Dazzi of LCP in Orsay and with the help of Dr. M.F. Bernet-Camard of the EA4043 (UBaPS) for the in vitro experiments. E. Pancani performed the NPs preparation and characterisation, the in vitro experiments and fluorescence confocal imaging. Moreover, she contributed in performing the experiments for the correlative imaging, in performing data processing and in paper writing. The following paper is the pre-peer reviewed version of the article: E. Pancani, J. Mathurin, S. Bilent, M.-F. Bernet-Camard, A. Dazzi, A. Deniset-Besseau, R. Gref, Part. Part. Syst. Charact. 2018, 1700457, which has been published in final form at 10.1002/ppsc.201700457. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

  Medical researchers and formulation scientists are committed to the hunt for efficient drug delivery systems which design and improvement are areas of intensive ongoing research. This PhD work has focused particularly on the development and advanced characterisation of two polymeric carriers; polyester (PLA, PLGA, PCL)-based and CD-based NPs.As already pointed out in the introduction to this work, these two drug delivery platforms stand out among the others for their ability to encapsulate challenging drugs overcoming their limitations as free molecules, for their tailorable properties, for their advantageous biodegradable and biocompatible characteristics and for the well-established safety profile. The first two chapters of this work allowed a comparison between pCD NPs and PLGA NPs, by studying the encapsulation abilities of these two systems towards different challenging drugs, namely the therapeutic [ETH:booster] pair and the first-generation quinolone, PIP.The first objective of this research consisted in improving the efficacy of [ETH: booster] pair for the combination therapy of TB. Indeed, TB is one of the most threatening diseases caused by an intracellular pathogen resulting in 1.8 million deaths annually3 . The progressive discovery and spread of MDR TB and XDR TB strains have reached the level of a global emergency4 . TB, being a chronic disease, necessitates an intensive and long antimicrobial therapy which causes frequent side-effects encountering low patient compliance. Given this alarming situation, growing efforts have been perpetrated in optimising the use and the efficiency of existing treatments and in developing new approaches.

  , the ability of PLA/PLGA and pCD NPs to encapsulate different antibiotics have been investigated. The obtained drug-loaded systems have been comprehensively characterised by using complementary techniques at the forefront in the field. Moreover, in an attempt to go beyond the state of art, a new technique, namely the AFM-IR, has been applied for the first time to unravel the structure of individual PLGA NPs and to detect them in eukaryotic cells.1. PLA and pCD NPs co-loaded with ETH and Booster were successfully prepared and deeply characterised with the purpose of obtaining a combined therapy in the treatment of TB. PLA NPs allowed achieving a high drug loading of both drugs, but they were not able to totally avoid ETH crystallisation. On the other hand, pCD NPs, despite their lower loading capability, enabled preventing the formation of ETH crystals.Noticeably, ETH and Booster maintained their in vitro antimicrobial activity on extracellular and intracellular Mtb after encapsulation in both PLA and pCD NPs.

Université

  Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Mise au point et caractérisation avancé de nanoparticules chargées en antibiotique dirigées contre des bactéries intracellulaires Mots clés : Nanoparticules, Cyclodextrines, Polymères biodégradables, Bactéries intracellulaires, encapsulation médicament, AFM-IR Résumé : Le traitement des infections intracellulaires est compliqué par la capacité des bactéries à «se cacher» à l'intérieur des cellules de l'hôte, en particulier celles du système immunitaire, entravant ainsi l'action de nombreux agents antimicrobiens. La diffusion croissante de souches résistantes est très inquiétante. Dans ce cadre, les nanoparticules (NPs) constituent une stratégie prometteuse pour administrer de manière optimisée des agents antimicrobiens. Ce travail de thèse, réalisé dans le cadre du projet européen ITN Cyclon Hit, visait à développer et caractériser des NPs biodégradables et biocompatibles chargées en antibiotiques, composés d'acide polylactique (PLA), d'acide poly (lactique-co-glycolique) (PLGA) et de polycaprolactone (PCL) ou de cyclodextrines polymérisées (pCD). Les deux premiers chapitres sont consacrés aux verrous technologiques liés à l'encapsulation de certains médicaments puissants dans les NPs polymériques. Tout d'abord, ces vecteurs ont été utilisés pour la délivrance simultanée d'une combinaison de molécules actives récemment découverte, l'éthionamide (ETH) et son Booster, pour le traitement de la tuberculose. Deuxièmement, ils ont été employés pour relever les défis liés à l'incorporation d'une quinolone de première génération, l'acide pipémidique (PIP), dans le but d'optimiser sa distribution intracellulaire dans des infections telles que la salmonellose. La co-incorporation efficace de l'ETH et du booster a dû surmonter de nombreuses difficultés liées à des problèmes de solubilité, de cristallisation et de biodisponibilité. Nos NPs en PLA et en pCD ont montré leur capacité de coencapsuler efficacement les deux molécules et tout particulièrement celles en pCD. Elles incorporent les médicaments à la fois dans les cavités des CD et dans des microdomaines hydrophobes. Les NPs en pCD, non toxiques après administration pulmonaire répétée de fortes doses, ont été administrés in vivo par voie endotrachéale directement au site d'infection. Elles ont permis une diminution de 3-log de la charge bactérienne pulmonaire des animaux infectés après seulement 6 administrations. De même, l'incorporation de PIP a été confrontée à des défis liés à la cristallisation de PIP et à sa libération incontrôlée. Malheureusement, le PIP présentait une faible affinité pour tous les matériaux polymériques étudiés et son encapsulation physique était infructueuse. Ainsi, une approche alternative a été développée en couplant le PIP au PCL via une réaction sans catalyseur initiée par le médicament. Le conjugué PCL-PIP se auto-assemble en forme de NPs avec une charge en PIP de 27%. Cependant, le PCL-PIP n'a pas pu être dégradé in vitro, mais l'approche de synthèse de conjugués est séduisante pour obtenir de particules stables et avec un contenu important en PIP. La compréhension approfondie de la structure et de la composition du noyau et de la couronne des nanostructures contenant une ou deux molécules actives est cruciale pour leur optimisation. Les deux derniers chapitres sont donc consacrés à l'application innovante de l'AFM-IR, une méthode nanospectroscopique originale combinant la microscopie à force atomique (AFM) avec la spectroscopie infrarouge (IR), à l'analyse chimique des NPs en PLGA ou à leur détection sans marquage après internalisation dans les cellules. L'AFM-IR est capable de fournir une caractérisation chimique à l'échelle nanométrique (résolution ~10 nm). Une avancée majeure du travail est l'application du mode tapping permettant l'investigation individuelle de chaque NP. Le signal IR spécifique des composants des NPs a été utilisé pour appréhender la composition chimique de leur coeur et couronne ainsi que pour localiser précisément le médicament. De plus, l'AFM-IR en mode contact a permis pour la première fois la localisation sans marquage et l'identification chimique des NP à l'intérieur des cellules. Ce travail ouvre la voie à d'innombrables applications de cette technique dans le domaine de la nanomedecine.

Table S2-Comparison of 3 independent in vivo experiments.

	Log10 CFU in lungs mean ± SD	Control	pCD [ETH]	pCD [ETH:booster]
	at 21 days post infection
	Exp 1	9.15 +/-0,44	7.83 +/-0.59 *	5.77 +/-0.27 ***
	Exp 2	7.51 +/-0.16	6.25 +/-0.28 ***	5.87 +/-0.48 ***
	Exp 3	7.22 +/-0.23	6.04 +/-0.72 *	5.12 +/-0.28 ***

*:p<0.1. ***:p<0.001 Section III -

Detailed Materials and methods

	Chemicals
	PLGA 75:25 acid terminated (P1) (MW: 37-84 KDa, 10P002), PLGA 50:50 ester terminated (P2) (MW: 70-
	100 KDa, 10P016) and PLGA 50:50 acid terminated (P3) (MW: 5-20 KDa, 10P019) were kindly provided by
	PCAS (Expansorb, Aramon, France). PLA ester terminated (P4) (MW =10-18 kDa), PVA (87-90%
	hydrolyzed), DMSO-99.5%, ETH and amikacin were all purchased from Sigma-Aldrich. Injectable water was
	purchased from Cooper (Melun, France). Dichloromethane (DCM-Analar Normapur stabilized with about
	0.002% of 2-methyl-2-butene) and MeOH were purchased from Prolabo (Fontenay-sous-Bois, France). KCl
	(99%) was purchased from Alfa Aesar (Karlsruhe, Germany).

& co-senior authors

Chapter IV -High resolution label-free detection of biocompatible polymeric nanoparticles in cells

Acknowledgements

Acknowledgements

ACKNOWLEDGEMENTS

Financial support for this work was provided by the European Community through the Marie Curie ITN CycloN Hit Grant no 608407. We thank the C'nano IDF DIM for founding this research through the "Nanoimagerie" grant. This work is supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (Labex NanoSaclay, reference: ANR-10-LABX-0035). We are grateful to Dr K. Salim (PCAS, Longjumeau, France) for kindly donating the PLGA polymer samples.

Label-free imaging of PLA NPs in THP-1 cells

Figure 2. Visualisation of internalised PLA NPs with nanoIR. AFM topography of a THP-1 macrophage (Topography colour grading = 0-1.4 µm) (a). IR map at 1770 cm -1 showing internalised PLA NPs (indicated by black arrows) (IR mapping colour grading = 0-70 A.U.) (b). The scale bar represents 10 µm.

Abbreviations

Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles

Joana Costa-Gouveia 1# , Elisabetta Pancani 2# , Samuel Jouny 1 , Arnaud Machelart 1 , Vincent Delorme 1 , Giuseppina Salzano 2 , Raffaella Iantomasi 1 , Catherine Piveteau 3 , Christophe J Queval 1 , Ok-Ryul Song 1 , Marion Flipo 3 , Benoit Deprez 3& , Jean-Paul Saint André 4 , José Hureaux 4& , Laleh Majlessi 5& , Nicolas Willand 3& , Alain Baulard 1& Gref, Ruxandra, (ruxandra.gref@u-psud.fr), Brodin, Priscille (priscille.brodin@inserm.fr)

Supplementary Information

Section I -Nanoparticles development

Section II -Phenotypic extracellular and intracellular assays and in vivo results

Section III -Detailed materials and methods

Section I -Nanoparticles development PLA/PLGA NPs -Nanoprecipitation method

Several PLA and PLGA polymers were employed to prepare the nanoparticles by nanoprecipitation (P1, P2, P3, P4, see materials and method section). In all cases the formulated NPs were remarkably stable upon storage up to 5 months, with less than 5% size variation. The size variation over a period of 1 month is shown in the Supplementary Fig. S1.

Interestingly no difference in the size evolution of NPs was shown between the formulation stored at room temperature or in the refrigerator (4°C).

Supplementary Figure S8 -Stability of nanoparticles obtained by nanoprecipitation method over a period of 1 month (measured by DLS). The graph shows size stability of nanoparticles (charged or not) over 1 month. RT: storage at room temperature, 4°C: storage in the refrigerator. 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 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.

Section II -Phenotypic extracellular and intracellular assays and in vivo results

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.

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.

Chapter II

Nanoparticles with high payloads of pipemidic acid, a poorly soluble crystalline drug: drug-initiated polymerization and self-assembly approach (Paper 3)

Graphical abstract

Drug-initiated ring-opening polymerization of ε-caprolactone leads to the formation of a polymer which selfassembles under the form of nanoparticles. Drug content reaches 27% (w/w).

The following paper and its supplementary information are published in Acta

General objectives

The studies reported in the previous chapters highlight the importance of an in-depth characterisation of NPs.

Complementary techniques should be employed with the aim of obtaining a picture as clear as possible of NPs structure and on its component localisation. However, we have to deal with the limitations of the existing techniques which imply that some conclusions can only be indirectly assessed by assembling the information provided by each technique, such as building a puzzle. Moreover, the existing techniques give only bulk information but cannot assess if the samples are homogeneous.

In an attempt to go beyond the state of art in the NPs characterisation and to confirm the conclusions drawn by combining the results of the other characterisation techniques, an original nanospectrocopic technique was here applied. This method, namely AFM-IR, combines the high resolution provided by atomic force microscopy (AFM) with chemical characterisation offered by IR spectroscopy. AFM-IR is able to track the location of each sample component by using its specific IR spectra as a fingerprint.

A comprehensive overview of AFM-IR and its originating techniques has been given in the section A of this chapter (Chapter III) placing emphasis on its advantages and versatility.

The study presented here aimed at employing AFM-IR to unravelling the structure of empty and drug-loaded PLGA NPs. However, the results obtained by trying to map the NPs chemical composition by AFM-IR in contact mode were inadequate despite the fact that some promising images could be collected. Indeed, PLGA NPs poorly adhered to ZnSe and CaF2 windows notwithstanding the efforts we deployed in improving NPs adherence. The adhesion on gold surface was slightly improved but was still not strong enough to avoid crushing and pushing the NPs with AFM-IR tip.

These adhesiveness issues could be addressed thanks to the recently developed tapping mode of AFM-IR which was applied here for the first time. Indeed, the tapping mode allowed extending the use of the technique to soft and non-adhesive samples such as PLGA NPs.

A comparison of the two modes on NPs imaging clearly proved the superiority of tapping mode and a technical and theoretical explication of this new mode were provided here.

Thanks to this innovation, invaluable information could be collected on PLGA NPs structure. A peculiar band of each NP component was selected and used as a fingerprint for its localisation within NPs structure. The surfactant corona surrounding NPs could be imaged here for the first time. Moreover, when applying the technique on PIP-loaded PLGA NPs, developed in Chapter II, the surface localisation of PIP could be confirmed, thus explaining the drug burst-release. A clear colocalization of PIP and NPs surfactant could be evidenced. It is worth noting that PIP was incorporated at only 1 % with respect to NP weight but AFM-IR could still identify it. Furthermore, the soft exploration by tapping AFM-IR allowed detecting NPs as small as Again, tapping AFM IR proved to be able to efficiently map not only large NPs but also small objects of about only 8 nm thus emphasizing the high-resolution of the technique (Figure 5B). As in the case of unloaded NPs, the smallest object had the highest PVA content. In all cases, the cores were devoid of PIP.

Furthermore, IR spectra were recorded on NPs cores and coronas (Figure 5E)

The pink averaged spectra recorded on the biggest PLGA NP core (showed the two specific bands of PLGA at 1425 cm -1 and 1398 cm -1 .

On the other hand, the average spectra of the NPs corona (Figure 5E blue line) reveal the presence of the PIP which produce an absorption band at 1645 cm -1 . Interestingly, the second band of the doublet characteristic of the pure antibiotic and visible at around 1615 cm -1 in FTIR spectra of bulk PIP (Figure 3) is not detected anymore in NPs. This is most likely due to different molecular environment experienced by PIP when located in NPs corona and to PIP very low concentration. Indeed, this is witnessed by the appearance of a unique absorption band at 1550 cm -1 , which do not correspond to PIP FTIR spectra, and by the presence of a broad band in the 1425 cm -1 region which do not resemble PLGA ones and which could hence be generated by a shift of the PVA band typically found at 1415 cm -1 as a consequence of its interaction with PIP. These bands most likely belong to a complex mixture of both compounds enforcing the hypothesis of a strong affinity of PIP for PVA.

Nanoprecipitation technique

In the case of the nanoprecipitation, the distribution of PIP represented by the mapping at 1640 cm -1 (Figure 6D) is similar to the one in the NPs prepared by the nanoemulsion. In both cases, the antibiotic appears to be located only around the NPs.

AUTHOR CONTRIBUTIONS

A

ADDITIONAL INFORMATION

Supplementary information accompanies this paper.

Competing financial interests: The authors declare no competing financial interests.

ACS Nano

Tapping AFM-IR: a powerful new tool to unravel the structure of single drug-loaded polymeric nanoparticle

Jérémie Mathurin 1# Elisabetta Pancani

SECTION C:

Tapping AFM-IR: a versatile method to determine the drug content of each polymeric nanoparticle

In the previous section of this chapter, the capability of the recently developed AFM-IR tapping mode to efficiently image and chemically characterise PLGA NPs was demonstrated, addressing the challenges related to the non-adhesive character of these NPs on the AFM supports. The superiority of the NPs 3D topography obtained by tapping AFM-IR was striking when compared with the one acquired by contact mode.

The technique provided the fascinating opportunity to prove the existence of a PVA corona and thus the coreshell structure of both empty and drug-loaded NPs. Moreover, the preferential PIP surface localisation, hypothesised in Chapter II to explain the drug burst-release upon water dilution, was confirmed thanks to tapping AFM-IR. Indeed, PIP was clearly shown to co-localise with PVA in the NPs corona but was not detected in the PLGA core. This finding, together with the PIP spectral changes, occurring when the drug was localised in the corona (in comparison to free-PIP), further corroborated the conclusion that this drug has a strong affinity for PVA and practically no affinity for PLGA.

The possibility to access to NPs components precise localisation on a particle by particle basis is invaluable in the drug delivery domain.

Given the very satisfactory results obtained on PLGA NPs, in this last section the abilities of tapping AFM-IR were further tested in collaboration with the team of Pr. Alexandre Dazzi. Thus, the features of the PCL-PIP NPs, developed and characterised in Chapter II, were explored here at the nanoscale level. The first objective of the study consisted in proving PIP localisation in the particle core. To do so, the IR spectrum of the bulk PCL-PIP conjugate was employed to identify the specific fingerprints of PCL and PIP (Figure 1).

Chapter IV

High resolution label-free detection of biocompatible polymeric nanoparticles in cells (Paper 5)

High resolution label-free detection of biocompatible polymeric nanoparticles in cells

Elisabetta Pancani

Supplementary information

composition and the incontrovertible localisation of each NP component.

The main ground-breaking novelty of this PhD lies in demonstration of the possibility to address all these challenges and obtain the desired information by applying an original IR nanospectroscopic technique to NPs characterisation (Chapters IIIB and C). These studies have been performed in collaboration mainly with the LCP of Orsay, where the technique was developed, and with the society "Anasys Instrument" which commercialise the instrument. This technology, combining the spatial resolution offered by atomic force microscopy (AFM) together with the chemical characterisation provided by IR spectroscopy, is called AFM-IR and is employed here for the first time to unravel the structure of PLA/PLGA NPs.

A literature overview about the technique is given in the Chapter IIIA. Briefly, the nanoIR-2 setting of AFM-IR, used in this work, consists of a tuneable IR laser focused on the sample near an AFM tip. When the sample absorbs the IR radiation at specific wavelengths characteristic of the sample functional groups, the absorbing region of the sample undergo a thermal expansion which is detected by the AFM tip. Two types of acquisition are possible. In the first mode, the position of AFM is fixed while the laser is tuned subsequently at different wavenumbers allowing recording the absorption spectrum of that precise spot (local spectrum). In the second mode instead, the laser is set at a specific wavenumber and the AFM tip is moved on the sample surface. As a consequence, the chemical cartography of the scanned region is obtained, together with its topography.

This technique already proved its usefulness in several fields of science but until now it has been used exclusively in contact mode. Thus, the characterisation of soft and surface loosely-adhesive samples was impossible or was complicated by multiple sample preparation steps to improve the adhesiveness of the sample to the support. A typical example of such problematic material is indeed PLA/PLGA NPs which were almost impossible to be imaged in contact mode as they were either crushed or displaced during acquisition.

The will to overcome such limitation and extend the technique application to challenging samples motivated the development of an AFM-IR able to perform the IR acquisition in tapping mode.

The tapping AFM-IR setup, developed by Anansys Instrument, was presented in this study (Chapter IIIB) for the first time and the key physical phenomena involved in the detection of IR absorption during non-linear cantilever-sample interaction were described.

The abilities of the newly developed technique were then tested on unloaded PLA NPs. As expected, the superiority of the tapping mode over traditional contact was striking when investigating these particles. The tapping mode could perfectly describe the 3D typical profile of NPs together with simultaneously providing precious chemical information about their structure (Figure 2A,B,C -Chapter IIIB).

Indeed, to achieve the unique goal of unravelling PLA NPs chemical features, a characteristic IR band was chosen as a fingerprint for each NP component (PLA and PVA) and it was used to detect specifically their localisation and distribution within the sample. Therefore, FTIR spectra of bulk materials between 2000 and 1000 cm -1 were recorded. The strong ester carbonyl band at 1760 cm -1 (C=O stretching) was selected as signature for PLA presence, whereas a small C-H bending band at 1415 cm -1 could be univocally attributed to PVA and used as its signature.

Fascinatingly, the chemical mappings recorded in tapping mode at the selected wavenumbers allowed directly between this drug and PVA.

Therefore, tapping AFM-IR demonstrated to be an exquisite tool able to visualise individual NPs and to unravel their chemical composition detecting the locations of both drug and corona without distorting or displacing them.

In an attempt to challenge this technique even more, another very ambitious and fascinating goal was set in this project and some preliminary results were reported in Chapter IIIC. The aim was the assessment not only of drug localisation but also of its amount. Indeed, it would be revolutionary for the development and the study of nanoparticulate systems to be able to determine their loading on a particle by particle basis.

The PCL-PIP NPs were selected as sample of choice to move the first steps towards the determination of drug/polymer ratio. Indeed, in PCL-PIP conjugate, the drug and the polymer are advantageously covalently linked and the global composition of the copolymer is known thanks to NMR experiments (10 PCL units for each PIP molecule). Therefore, it would be of high interest to investigate by tapping AFM-IR if each NP has the same PIP/PCL ratio (theoretical ratio: 1 PIP molecule for every PCL chain).

Firstly, the efficient encapsulation of PIP in NPs was investigated by recording the IR mappings at the specific wavelength of PCL (ester carbonyl at 1732 cm -1 ) and PIP (tertiary amide carbonyl of the PIP-PCL linkage at 1630 cm -1 ). The obtained cartographies showed clearly that PIP was perfectly colocalised with PCL ester signal, confirming unequivocally its localisation inside NPs. Thus, PIP covalent conjugation with the PCL polymer was a successful strategy enabling to finally obtain PIP-loaded NPs.

Interestingly, these NPs displayed a less spherical shape in comparison to PLGA ones and a quite flattened profile. These results are in good agreement with the ones evidenced by SEM images. Indeed, the flattening effect observed with the two techniques was most likely caused by the NPs deposition and drying process on the supports. As the phenomenon was limited only to PCL-PIP NPs, it must be connected to the physicochemical properties of PCL (glass transition temperature and meting point) which make it more prone to deformation during sample drying.

Secondly, the IR mappings obtained on 10 different NPs were analysed to estimate PIP/PCL ratio. To do so, the absorption coefficient (α) of the bands of interest (1732 cm -1 and 1630 cm -1 ) was measured by using an IR microscope and the PCL portion of the conjugate was found to produce an IR signal 1.9 times more intense than the PIP moiety. Then, the signal of each IR cartography at the two wavenumbers were integrated over the IR signal area (Chapter IIIB). The ratio between the integration at 1630 cm -1 and the one at 1732 cm -1 was calculated (after correction by α factor = 1.9).

The PIP/PCL ratio mean value corresponded to 1 ± 0.4 and was thus very close to the theoretical one. However, a high variability was found between the particles (high SD of the measurement). This variability was probably so high for two main reasons. Firstly, the presence of PCL chains devoid of PIP end group was hypothesised.

Indeed, the presence of small traces of water during the PCL-PIP conjugation could have initiated the polymerisation reaction in place of PIP. Therefore, some traces of PCL homopolymer could be present in the final conjugate, thus explaining the interparticle variability. The other possible explanation consists in the presence inside particles of crystalline microdomains of PCL and/or PIP embedded in an amorphous matrix.

Such more or less rigid domains could make difficult to control the contact between the AFM tip and the sample during the images acquisition interfering with the correct detection of IR thermal expansion.

In a nutshell, despite the method reported here certainly needs to be further developed, the tapping AFM-IR was demonstrated to enable reaching, for the first time, the challenging goal of determining the drug/polymer molar ratio within individual NPs.

The estimation of the amount of drug on a particle by particle basis could lead to very remarkable advances in nanomedicine. The homogeneity of the particles drug loading as well as its dependence on formulation parameters could be studied for each single NP. This would open to the possibility of a better understanding of formulation processes and to the chance of facilitating NP design.

Of note, this approach could be extended to a wide range of NPs, other than polymeric ones (hybrid organicinorganic, liposomes, lipid nanoparticles, etc).

Once NPs have been successfully prepared and characterised, their interactions with the living systems represents a critical step in their development and in the determination of their effectiveness. Scientist must possess a deep understanding of such interactions to be able to improve and optimise nanocarriers efficacy.

Therefore, different imaging techniques are nowadays available to track the NPs fate inside cells and tissues.

The most commonly employed techniques are fluorescence microscopy and transmission electron microscopy.

Despite their differences, they have a common point. Both microscopies require NPs labelling with a fluorescent dye or an electron dense material, respectively, to enable NPs detection. Indeed, organic particles, such as PLA/PLGA ones, do not possess any contrast properties to be distinguished from the surrounding.

However, the modification of NPs with a dye is known to possibly cause changes in their size, shape or surface properties which can alter their interactions with cells and tissues [25][26][27] . In fact, NPs surface is the theatre of all the complex interactions with the living media 28 . Moreover, dyes which are not covalently linked to the NP could leak out or even be transferred to cell membranes.

The key limitation of the use of labelling lies in the fact that what is observed and followed during experiments is the label and not the object of interest. Therefore, such techniques provide only an indirect measurement and at any time they cannot insure that the NP is still intact.

In this context, there is a tremendous interest to develop a label-free method for NPs detection in living systems.

Some progresses have been performed in this direction with the development of Raman microspectroscopy.

Although Raman could detect unlabelled polymeric particles, its resolution is limited by light diffraction to 300 nm-1 µm depending on the excitation source. This enables the detection of particle agglomerates rather than individualised NPs.

The bottleneck represented by the detection of individual unlabelled PLA/PLGA NPs could be overcome thanks to AFM-IR. Here (Chapter IV), AFM-IR was proved to be a reliable, non-destructive and direct method able to locate NPs of around 170 nm within cells with a high lateral resolution, by taking advantage of the specific IR response of their constitutive PLA polymer. The very intense ester carbonyl band of PLA NPs could be used as a fingerprint for their detection as it clearly stands out against the carbonyl stretching band of cellular constituents.

A correlative study between confocal fluorescence microscopy and AFM-IR was conducted to prove the reliability of the technique. Firstly, fluorescent NPs of 170 nm were developed by using a small percentage of rhodamine-labelled PLA (Rh-PLA) polymer. Rhodamine was covalently linked to PLA to avoid any leakage.

Then, Rh-PLA NPs were incubated with macrophages and their successful internalisation in the cells was proved by confocal microscopy.

Subsequently, the exact same region of interest (ROI) was sequentially studied by confocal fluorescence microscopy and by AFM-IR. The IR map at 1770 cm -1 , corresponding to the PLA ester, clearly indicated the presence of PLA NPs into the cell and the results were in excellent agreement with the confocal images. Thus, it was proved that the objects detected by AFM-IR were indeed NPs. One of the major advantage of this technique was to further zoom on a particular ROI in a cell to acquire more accurate information detecting even single NPs. Moreover, despite the fact that NPs were embedded in the cell, their chemical composition could be still assessed from the local spectra acquisitions. The IR spectra obtained with a resolution of 1 cm -1 were in complete accordance with those of the PLA NPs. Multiple spectra could be recorded on the same NP to assess its chemical homogeneity. Similar results were obtained when Rh-PLA NPs were replaced with unlabelled ones.

In a nutshell, AFM-IR could offer a high level of resolution, together with the possibility of chemical identification, that to the best of our knowledge no other technique is able to provide.

AFM-IR can offer a considerable amount of interesting perspectives. The spectral range of the IR source could be extended to enlarge the applications of the technique and to increase the probability to find a fingerprint band for a wider range of molecules. A future challenge would also be to couple the AFM-IR with fluorescence confocal microscopy allowing identifying the intracellular compartments where the NPs are located.

Alternatively, a comprehensive study of the specific IR absorption of each type of subcellular compartment could be performed with the aim to find a fingerprint band for each one of them, hence enabling to locate NPs by using exclusively AFM-IR. Moreover, this technique could be applied not only to NPs localisation but also to the study of their degradation and to the tracking of one or more drugs, following their release from the carrier and unravelling their intracellular fate. This intriguing tool could be used to design nanocarriers capable of releasing their drug cargo at intracellular targets.

Conclusion Annexes

Supplementary Information of Chapter 1 -SECTION B