Keywords: Pseudomonas aeruginosa, Acinetobacter baumannii, tobramycin, colistin, lung biofilms, lipid nanoparticles

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires ABSTRACT

Pseudomonas aeruginosa (PA) and Acinetobacter baumannii (A. baumannii) are responsible for chronic lung infections often associated with the development of biofilms, i.e. bacterial aggregates trapped in a self-produced matrix of anionic polymers such as alginate or DNA. Within these biofilms, bacteria are protected from cationic antibiotics (ATB) such as tobramycin (TOB) and colistin (COL), in part through their interaction with matrix polymers, which reduce their diffusion. The main objectives of this thesis were first to develop a lung biofilm model that mimics those found in chronic lung infections of patients with cystic fibrosis (CF), one of the main lung diseases for which biofilms develop. Then use this model to evaluate a new ATB delivery system made of lipid nanoparticles developed to treat lung biofilms.

In CF patients, PA forms biofilms consisting of aggregates (5-100 μm) enclosed in their pulmonary mucus. An in vitro lung biofilm model composed of polymers present in vivo (alginate, mucins, DNA) and consisting of PA trapped in alginate beads has been developed and evaluated using COL and TOB. This model was refined by evaluating the effect of the size of the beads and the growth medium in which they were dispersed. Changing the size of the beads from 60 to 1200 µm and using a dispersion medium simulating the composition of mucus further decreased the effectiveness of TOB in this model. For high TOB concentrations, smallcolony variants (SCV) of PA, also found in the CF patient, were observed in the model, suggesting a good mimicry of the conditions in vivo. In this model, COL efficacy was less impacted than that of TOB, and prevented the appearance of SCV. Pegylated lipid nanoparticles loaded with COL and containing a lipophilic adjuvant to COL such as Farnesol (FAR) have been developed to increase its effectiveness against biofilms. Encapsulation of COL improved its efficiency by two in the lung biofilm model PA developed, but also restored the sensitivity to COL of a resistant A. baumannii and improved its efficiency in an abiotic adherent biofilm model with A. baumannii.

ii Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires RÉSUMÉ Pseudomonas aeruginosa (PA) et Acinetobacter baumannii (A. baumannii) sont responsables d'infections pulmonaires chroniques souvent associées à la formation de biofilms, i.e. des agrégats bactériens piégés dans une matrice riche en polymères anioniques tels que l'alginate ou l'ADN.

Au sein de ces biofilms, la diffusion des antibiotiques cationiques (ATB), tels que la tobramycine (TOB) et la colistine (COL), est en partie réduite due aux interactions avec les polymères de la matrice. Les principaux objectifs de cette thèse ont été dans un premier temps de développer un modèle de biofilm pulmonaire mimant ceux retrouvés dans les infections pulmonaires chroniques des patients atteints de mucoviscidose (CF), l'une des principales maladies pulmonaires dans laquelle ces biofilms se développent. Un nouveau système d'administration d'ATB, composé de nanoparticules lipidiques chargées de COL, a été par la suite développé et testé grâce à ce modèle de biofilms pulmonaires.

Chez les patients atteints de CF, PA forme des biofilms constitués d'agrégats (5-100 μm) incorporés dans leur mucus pulmonaire. Un modèle de biofilm pulmonaire in vitro composé des polymères présents in vivo (alginate, mucines, ADN) et constitué de PA piégés dans des billes d'alginate, a été développé et évalué à l'aide de la COL et de la TOB. Ce modèle a été optimisé en évaluant l'effet de la taille des billes et du milieu de croissance dans lequel elles ont été dispersées. La modification de la taille des billes de 60 à 1200 µm et l'utilisation d'un milieu de dispersion simulant la composition du mucus pulmonaire ont permis de réduire davantage l'efficacité de la TOB dans ce modèle. Pour des concentrations élevées en TOB, des variants de PA formant de très petite colonies (SCV), également trouvés chez les patients atteints de CF, ont été observés dans le modèle, ce qui suggère une bonne imitation des conditions in vivo. Dans ce modèle, l'efficacité de la COL a été moins affectée que celle de la

I. Introduction

Bacteria are responsible for both acute invasive infections and chronic localized infections.

Differences between these infections are often attributable to the mode of growth of the bacterium. Bacteria growing as isolated cells or planktonically are mainly associated to acute infections. For most of the chronic bacterial infections, bacteria grow in slime-enclosed aggregates known as biofilms which are recognized as a significant cause of antimicrobial tolerance (Bjarnsholt 2013;Boisvert et al. 2016;[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF]). These biofilm infections, such as pneumonia in cystic fibrosis (CF) patients, chronic wounds, chronic otitis media, implant-and catheter-associated infections, affect millions of people and increase their risk of death (González et al. 2018). In these biofilms, bacteria are protected from the environment and the concentration of antibiotics (ATBs) needed to eradicate a biofilm are up to a 1000-fold higher than the concentration needed to kill planktonic cells. Therefore, biofilm infections are not easily responsive to existing antimicrobial treatment (Høiby et al. 2010a).

Reduced susceptibility in biofilms is a consequence of complex physical and biological properties with multiple factors such as resistance and tolerance against antimicrobial agents through metabolic dormancy or molecular persistence programs (Bjarnsholt 2013). Besides, mismanagement of ATBs treatment used to fight biofilm accelerates the emergence of bacterial resistance to the conventional ATBs [START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF][START_REF] Ridenhour | Persistence of antibiotic resistance plasmids in bacterial biofilms[END_REF] as well as the lack of the discovery of new antimicrobial agents drove to develop another strategies to cure the biofilm infections. In this context, the therapies currently used to combat bacterial infections must be improved in order to be able to treat biofilms, for example by using new administration strategies [START_REF] Forier | Lipid and polymer nanoparticles for drug delivery to bacterial biofilms[END_REF][START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Smith | Biofilms and antibiotic therapy: Is there a role for combating bacterial resistance by the use of novel drug delivery systems?[END_REF].

For lung biofilms, promising approaches currently focus on pulmonary drug delivery systems (DDS) which, when inhaled, can improve the targeting of ATBs to lung biofilm [START_REF] Ciofu | Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients[END_REF][START_REF] Forier | Lipid and polymer nanoparticles for drug delivery to bacterial biofilms[END_REF]. This new approach consists of inhaling ATBs encapsulated in a particulate DDS such as nanoparticles (NPs) or liposomes to improve its penetration into biofilms and to control its release into the biofilm, in order to increase its local concentration and minimize its toxicity [START_REF] Sajid | Drug Delivery Systems That Eradicate and/or Prevent Biofilm Formation[END_REF]. Several excellent reviews discuss how bacteria develop biofilms and their protective mechanisms against ATBs and specific therapeutic strategies developed to combat biofilms [START_REF] Bjarnsholt | the Scandinavian Cystic Fibrosis Study Consortium: Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients[END_REF]Høiby et al. 2010a ;Høiby et al. 2010b ;[START_REF] Ciofu | Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients[END_REF][START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF][START_REF] Klinger-Strobel | Aspects of pulmonary drug delivery strategies for infections in cystic fibrosiswhere do we stand?[END_REF][START_REF] Koo | Targeting microbial biofilms: current and prospective therapeutic strategies[END_REF][START_REF] Fleming | Approaches to Dispersing Medical Biofilms[END_REF]. The main objective of this thesis was to evaluate a new DDS of lipid nanoparticles (LNP) to increase diffusion of cationic ATBs (Tobramycin (TOB) and Colistin (COL)) into lung biofilms. Our first step was to develop a Pseudomonas aeruginosa Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires (PA) lung biofilm model that mimics those found in chronic lung infections of CF patients and to validate this model by studying efficacy of cationic ATBs against PA biofilms and Acinetobacter baumannii (A. baumannii) biofilms.

In the general part of this work, biofilms structure will be developed as well as their mechanisms of regulation and tolerance towards ATBs. Secondly, in vitro and in vivo biofilm models found in literature will be presented before ending in the last part on drug delivery systems to target lung biofilms.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

II. Pulmonary biofilm a. Lung biofilms and associated pathogens

Depending on the bacterial species and the surrounding environment, bacteria produce various structured biofilms with different characteristics. In the lung, there are two main types of biofilm associated with chronic infections, where the biofilm is either produced in the mucus (Bjarnsholt et al. 2009a;Høiby et al. 2010b;Sønderholm et al. 2017a) or adhered to the wall of the endotracheal tubes (ETTs) (Boisvert et al. 2016). The first type of biofilm is characterized by unattached aggregates dispersed in the airway mucus. It is mainly observed for chronic pulmonary infections with PA associated with CF, but also with bronchiectasis (Boisvert et al. 2016;Macia et al. 2014;[START_REF] Marsh | Detection of biofilm in bronchoalveolar lavage from children with non-cystic fibrosis bronchiectasis[END_REF]. The second type is found in patients with ventilatorassociated pneumonia (VAP) and can be formed by various species often forming polyspecies biofilms. Multidrug-resistant pathogens from ESKAPE group (acronym corresponding to the names of six bacterial pathogens commonly found to carry antimicrobial resistance genes (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, PA, Enterobacter spp) play a dominant role in VAP etiology, and these organisms are frequently identified in ETTs biofilms [START_REF] Bauer | Biofilm formation in endotracheal tubes. Association between pneumonia and the persistence of pathogens[END_REF][START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF]).

1) Mucus dispersed biofilms

Bacterial biofilms developed in mucus are non-attached biofilms that are not associated with a foreign body [START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF]. Their formation is accompanied by a reduction in the motility and expression of virulence genes of bacteria. In the airways of CF patients, abnormal mucus present in excess, decreases bacterial clearance and provides a nutrient-rich environment promoting the biofilm establishment [START_REF] Balázs | Mucus obstruction and inflammation in early cystic fibrosis lung disease: Emerging role of the IL-1 signaling pathway[END_REF][START_REF] Rosa | Adapting to the Airways: Metabolic Requirements of Pseudomonas aeruginosa during the Infection of Cystic Fibrosis Patients[END_REF][START_REF] Mcdaniel | An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability[END_REF]. PA is the most common pathogen that persists as a mono-species biofilm suspended in the mucus of these patients, resulting in ineradicable chronic lung infections (Bjarnsholt et al. 2009a;Boisvert et al. 2016;[START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF]. Therefore, in CF patients, the persistence of PA in the airways mucus as a biofilm is strongly correlated with the decline in lung function and a high rate of patient morbidity and mortality [START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF]. In chronic obstructive pulmonary disease (COPD), another chronic inflammatory lung disease in which mucus is produced in excess, no direct observation of PA biofilm formation was reported [START_REF] Blasi | The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections[END_REF]Hassett . 2014;[START_REF] Martínez-Solano | Chronic Pseudomonas aeruginosa Infection in Chronic Obstructive Pulmonary Disease[END_REF].

Still, the ATB resistance patterns in COPD patients are similar to those observed for CF chronic infections and isolates from lungs of COPD patients were shown to produce more in vitro Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires biofilm than isolate collected from the blood of the same patients, suggesting the possible presence of biofilm in these patients [START_REF] Blasi | The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections[END_REF][START_REF] Rodrigo-Troyano | Pseudomonas aeruginosa resistance patterns and clinical outcomes in hospitalized exacerbations of COPD[END_REF].

In CF patient's airways, PA mature biofilms are characterized by small structured bacterial aggregates embedded in a self-produced viscous matrix of extracellular polymeric substances (EPS) composed of polysaccharides (mainly alginate, Psl, and Pel), extracellular DNA (eDNA), proteins such as CdrA, and lipids (Bjarnsholt et al. 2009a; Maunders and Welch 2017) (Figure 1). These aggregates which are enclosed in a second polymer matrix composed of the patient's mucus have a size ranging from 5 to 100 µm (Figure 1) [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]Bjarnsholt et al. 2009;[START_REF] Sønderholm | Tools for studying growth patterns and chemical dynamics of aggregated Pseudomonas aeruginosa exposed to different electron acceptors in an alginate bead model[END_REF]Sønderholm et al. 2017). The polysaccharide composition of EPS changes during the biofilm life cycle. In the early stages, two exopolysaccharides named Psl and Pel are produced and serve to initiate the formation of biofilm. Psl is a neutral polysaccharide consisting of a pentasaccharide repeat containing glucose, mannose, and rhamnose. This polysaccharide is essential for the adherence to mucin surface and epithelial cell airway and promotes cell-cell interactions [START_REF] Irie | The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms[END_REF][START_REF] Ryder | Role of polysaccharides in Pseudomonas aeruginosa biofilm development[END_REF]. Pel is a cationic polysaccharide made of N-acetylgalactosamine and Nacetylglucosamine, involved in interactions between bacteria in the biofilm and in the adhesion of bacteria to mucins [START_REF] Colvin | The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix: Polysaccharides of the P. aeruginosa biofilm matrix[END_REF][START_REF] Irie | The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms[END_REF][START_REF] Jennings | Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix[END_REF][START_REF] Ryder | Role of polysaccharides in Pseudomonas aeruginosa biofilm development[END_REF]. It is also required to initiate and maintain cell-cell interactions in biofilm environment [START_REF] Colvin | The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix: Polysaccharides of the P. aeruginosa biofilm matrix[END_REF]. For PAO1, Psl is the primary exopolysaccharide produced in the biofilm matrix, while it is Pel for PA14 [START_REF] Jennings | Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix[END_REF]. PA strains is characterized by hyperadherence and hyperaggregation of bacteria, called small colony variants (SCVs), which are frequently isolated from various biofilms, and which constitutively overexpress Pel and Psl.

In response to infection, numerous polymorphonuclear leukocytes (PMNs) surround the PA biofilm aggregates and create a chronic inflammation (Figure 1) [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]Bjarnsholt et al. 2009a;Høiby et al. 2010b). In mature biofilm, many of these PMNs are necrotic and constitute a massive source of eDNA that is incorporated into the biofilm matrix (Bjarnsholt et al. 2009a;[START_REF] Lewenza | Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa[END_REF]. PMNs that are still alive phagocytize planktonic bacteria released by biofilms and release in turn various components such as reactive oxygen species (ROS, 𝑂 2 -, 𝐻 2 𝑂 2 , 𝑂𝐻 . …), pro-inflammatory cytokines (IL-1α-IL-1β,IL-6, IL-17…), proteolytic enzymes (cathepsin G, neutrophil elastase) and antimicrobial peptides (LL-37, ...) [START_REF] Colvin | The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix: Polysaccharides of the P. aeruginosa biofilm matrix[END_REF][START_REF] Tecchio | Neutrophil-Derived Cytokines: Facts Beyond Expression[END_REF]. PMNs also degranulate to produce web-like extracellular structures known as extracellular neutrophil traps (NETs) which contain DNA that can also be incorporated into the matrix of biofilms [START_REF] Lewenza | Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa[END_REF]. Due to their high activity, PMNs consume a lot of O2, which promotes local O2 depletion and makes the bacterial aggregates surrounded by PMNs mostly anoxic (Sønderholm et al. 2017). Furthermore, all the elements Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires released by PMNs, which are in part responsible for lung damage, apply selective pressure to the colonizing PA that favors the survival of mucA gene mutants, the main mutation responsible for overproduction of alginate [START_REF] Boucher | Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection[END_REF][START_REF] Malhotra | Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials[END_REF][START_REF] Mathee | Mucoid conversion of Pseudomonas aeruginos by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung[END_REF][START_REF] Ramsey | Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis: P. aeruginosa pathogenesis[END_REF][START_REF] Ryder | Role of polysaccharides in Pseudomonas aeruginosa biofilm development[END_REF]. Indeed, alginate is a polyanionic polysaccharide composed of acid mannuronic and gluronic that act as oxygen-radical scavenger. Its overproduction protects the bacteria also from phagocytosis by PMNs and gives the colony a mucoid phenotype that is typical in ineradicable CF lung infection. As chronic infection develops in the CF airways, numerous new PA phenotypes appear and CF sputum culture can revealed the presence of mucoid cells overproducing alginate but also SCVs overproducing Pel and Psl [START_REF] Maunders | Matrix exopolysaccharides; the sticky side of biofilm formation[END_REF]. However, mucoid phenotype is rarely isolated from non-CF airway [START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF]. Both phenotypes are related to ATB resistance, persistence of infection, resistance to phagocytosis and poor lung function [START_REF] Malone | Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs[END_REF]. During later stage, biofilm is mature causing chronic infection (c), forming characteristics specific to biofilm (bacterial aggregate in self-produced matrix rich in anionic polysaccharides). Finally, some PA begin to detach and the biofilm disperses (d). (Personal creation)

The establishment of mucus dispersed biofilms can be described by several stages presented in Figure 1.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

First, planktonic PA colonized the mucus using flagella and Pili IV (Figure 1a) [START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF]. Then, in the early infection stage, planktonic bacteria begin to adapt to the mucus environment, in particular to the depletion of oxygen and the response of the host's immune system. In these stages, bacteria still produce virulence factors such as rhamnolipid, proteases and pyoverdine, and are still susceptible to ATBs treatment (Figure 1b) [START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF]. Real biofilm development begins at the stage of chronic infection, where bacteria are perfectly adapted to the mucus environment thanks to various characteristics including overproduction of alginate, loss of production of virulence factors and loss of mobility (loss of both pili and flagella). To date, it is unclear when the bacteria switch from planktonic to sessile lifestyle after airway colonization [START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF] This mature biofilm (Figure 1c) is distinguished by gradients of nutrients and oxygen that decrease from the outside to the inside of the biofilm, which creates a gradient of metabolic activity of bacteria in the same direction.

The last stage of biofilm life cycle corresponds to the dispersal phase (Figure 1d), where alterations in the mucus environment (oxygen, nutrients availability, pH) trigger the biofilm dispersion as planktonic bacteria to colonize a new region in the mucus and establish another biofilm [START_REF] Kirov | Biofilm differentiation and dispersal in mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis[END_REF][START_REF] Sousa | Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review[END_REF][START_REF] Woo | Biofilm dispersal cells of a cystic fibrosis Pseudomonas aeruginosa isolate exhibit variability in functional traits likely to contribute to persistent infection[END_REF]. This stage is associated to a coordinate release of virulent factors from the bacteria and corresponds to acute exacerbation of the chronic infection, for which planktonic bacteria can also be found in the patient's mucus.

2) Biofilms adhering to abiotic surfaces

Biofilms adhering to the surface of ETT found especially in patients with VAP are the main biofilms adhering to abiotic surfaces found in the lung [START_REF] Mcdaniel | An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability[END_REF] (Figure 2). These biofilms are likely the major microbial reservoir of VAP (Fernández-Barat and Torres 2016).

Indeed, 70% of patients with VAP have similar pathogens in their lungs as those that form the biofilm on the ETT (McDaniel et al. 2015). These biofilms can be generated within hours after intubation. Patchy biofilms are generally observed 2-7 days after ventilation initiation on the ETT surface, and 87.5% of ETT surface can be covered by biofilms after 7-10 days. After 10 days, all the ETT surface is generally covered (Boisvert et al. 2016;[START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF][START_REF] Fernández-Barat | Biofilms in ventilator-associated pneumonia[END_REF].

Compared to biofilms suspended in abnormally viscous lung mucus, these biofilms are much larger and can be up to 1000 µm thick. Consequently, steeper O2 and nutriment gradients and higher tolerance to ATBs are observed [START_REF] Roberts | Modeling Antibiotic Tolerance in Biofilms by Accounting for Nutrient Limitation[END_REF][START_REF] Stewart | Physiological heterogeneity in biofilms[END_REF][START_REF] Bjarnsholt | The in vivo biofilm[END_REF][START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF]. In addition, these biofilms have a more complex architecture than those included in mucus and are often formed by several Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires microbial species, including different bacteria, fungi and viruses [START_REF] Orazi | Pseudomonas aeruginosa Increases the Sensitivity of Biofilm-Grown Staphylococcus aureus to Membrane-Targeting Antiseptics and Antibiotics[END_REF][START_REF] Pericolini | Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro[END_REF]. Some of the pathogens which are frequently found in ETT biofilm associated to VAP are from the multidrug-resistant "ESKAPE" group such as PA, A. baumannii, Staphylococcus aureus and Klebsiella pneumoniae [START_REF] Bauer | Biofilm formation in endotracheal tubes. Association between pneumonia and the persistence of pathogens[END_REF]Boisvert et al. 2016;[START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF]. The formation begins with the adhesion of the planktonic cells on abiotic surface (such as endotracheal tube) (a). Then, the bacteria proliferate (b) and start to produce an extracellular matrix (c), leading to the formation of bacterial microcolonies. During later stage, the biofilm is mature forming characteristic structure of three-dimentionnal bacterial communities, bacterial aggregate in selfproduced matrix made of polysaccharides (d). Finally, some bacterial cells start to detach allowing the dispersion of biofilm (e) (personal creation).

As for biofilm dispersed in mucus, ETT surface adhering biofilms formation is generally described by several steps presented in Figure 2. The first step is the binding to the ETT surface, mediated in PA by the flagella involved in the swimming of bacteria and by type IV pili needed for their motility by twitching (Figure 2a). The second stage corresponds to the formation of microcolonies, in which the sessile bacteria proliferate and adhere even more to the surface of the ETT, but also to each other, while producing their own EPS (Fernández-Barat and Torres 2016; Maurice et al. 2018;[START_REF] Mcdaniel | An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability[END_REF]. In this step, stability and surface adherence is improved and bacteria division is exponential (Figure 2b and 2c) (Boisvert et al. 2016;[START_REF] Wilson | Advanced endotracheal tube biofilm stage, not duration of intubation, is related to pneumonia: The Journal of Trauma Injury Infection and Critical Care[END_REF]. Both planktonic and sessile bacteria are still present in this Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires phase [START_REF] Rasamiravaka | The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms[END_REF][START_REF] Wilson | Advanced endotracheal tube biofilm stage, not duration of intubation, is related to pneumonia: The Journal of Trauma Injury Infection and Critical Care[END_REF]. The third step concerns the maturation of the biofilm which becomes thicker and more organized (Figure 2d). Microenvironments with bacteria having different metabolic states (aerobic, micro-aerobic and anaerobic) and activity levels (high or low metabolic activity) are created [START_REF] Fernández-Barat | Biofilms in ventilator-associated pneumonia[END_REF][START_REF] Mcdaniel | An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability[END_REF][START_REF] Wilson | Advanced endotracheal tube biofilm stage, not duration of intubation, is related to pneumonia: The Journal of Trauma Injury Infection and Critical Care[END_REF]. The last step is the dispersal phase (Figure 2e), where certain bacteria are released from the biofilm to colonize new surfaces that allow its propagation (Bjarnsholt 2013;[START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF][START_REF] Fernández-Barat | Biofilms in ventilator-associated pneumonia[END_REF]Macia et al. 2014).

As with biofilms suspended in mucus, this step involved bacterial synchronization using quorum sensing (QS) system and the release of a significant amount of virulence factors [START_REF] Mcdaniel | An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability[END_REF].

The risk of development of VAP depends on many factors including the length of the intubation period that increases the risk of biofilm establishment, as well as the type of pathogens that defines the formation of early or late biofilm. For instance, VAP acquired within the first five days after intubation are more likely to be caused by ATBs sensitive bacteria such as methicillin-sensitive Staphylococcus aureus and VAP acquired more than five days after intubation caused by ATBs-resistant bacteria such as PA, A. baumanii and methicillin-resistant Staphylococcus aureus [START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF]. The ETT commonly used for patient's intubation is made of polyvinylchloride that constitutes a surface favorable to bacterial adherence and biofilm formation [START_REF] Fernández-Barat | Biofilms in ventilator-associated pneumonia[END_REF]. Several strategies can be adapted to avoid or reduce the development of biofilms on the surface of ETT. These include replacement of ETT if contamination is suspected, endotracheal aspiration of respiratory secretions, mechanical removal of ETT secretions and biofilm using a mucus razor, and use of antiseptics such as chlorhexidine [START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF][START_REF] Fernández-Barat | Biofilms in ventilator-associated pneumonia[END_REF][START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF]. More advanced strategies involve modification of the surface of the ETT, such as ETT impregnated with antiseptics or ATBs such as sulfadiazine or silver-coated ETT, which is the only one that showed benefits in clinical trials [START_REF] Diaconu | Endotracheal Tube Biofilm and its Impact on the Pathogenesis of Ventilator-Associated Pneumonia[END_REF][START_REF] Høiby | ESCMID * guideline for the diagnosis and treatment of biofilm infections 2014[END_REF][START_REF] Raad | The prevention of biofilm colonization by multidrug-resistant pathogens that cause ventilator-associated pneumonia with antimicrobial-coated endotracheal tubes[END_REF]).

b. Bacterial biofilm regulations

Several interconnected bacterial communication mechanisms have been described as being involved in regulating the evolution of biofilms [START_REF] Bjarnsholt | the Scandinavian Cystic Fibrosis Study Consortium: Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients[END_REF][START_REF] Jenal | Cyclic di-GMP: second messenger extraordinaire[END_REF][START_REF] Liu | Influence of (p)ppGpp on biofilm regulation in Pseudomonas putida KT2440[END_REF][START_REF] Rasamiravaka | The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms[END_REF][START_REF] Rutherford | Bacterial Quorum Sensing: Its Role in Virulence and Possibilities for Its Control[END_REF][START_REF] Valentini | Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria[END_REF]. Two of them, the bacterial QS system and the second intracellular messenger Bis-(3′-5′) cyclic diguanosine monophosphate (c-di-GMP) have been among the most studied.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

1) Bis-(3′-5′) cyclic diguanosine monophosphare

The c-di-GMP is one of the most important bacterial intracellular secondary messenger which regulates the biofilm life cycle [START_REF] Lin Chua | Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa[END_REF][START_REF] Valentini | Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria[END_REF]. High intracellular c-di-GMP concentration enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP concentration in biofilm cells leads to increased motility and biofilm dispersal. For example, high c-di-GMP level activate the production of Pel, Psl, and alginate in PA [START_REF] Pestrak | Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments[END_REF][START_REF] Rasamiravaka | The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms[END_REF][START_REF] Rutherford | Bacterial Quorum Sensing: Its Role in Virulence and Possibilities for Its Control[END_REF]. The concentration of c-di-GMP in bacteria is controlled by the rate of its synthesis and degradation. Its synthesis from GTP molecules is catalysed by enzymes called diguanylate cyclases and its degradation is catalysed by various phosphodiesterases (PDEs). At least five diguanylate cyclases have been described to specifically control the transition from planktonic to biofilm growth: WspR, SadC, RoeA, SiaD, and YfiN/TpbB, and several PDEs have been linked to biofilm dispersal. Mucoid or SCV phenotype of PA isolate from CF patients have high c-di-GMP concentration due to the overexpression of diguanylate cyclases [START_REF] Valentini | Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria[END_REF].

2) Quorum sensing

QS is a type of bacterial communication mechanism using diffusible molecules that synchronizes gene expression in response to population cell density to regulate population behaviors such as the production of virulence factors and biofilm maturation and dispersion [START_REF] Lee | The hierarchy quorum sensing network in Pseudomonas aeruginosa[END_REF][START_REF] Solano | Biofilm dispersion and quorum sensing[END_REF]. Biofilm formation is a cooperative group behavior that involves bacterial populations living embedded in a self-produced extracellular matrix.

Only when the population density is high enough (formation of microcolony or microaggregate), the accumulation of the signal in the extracellular environment is sufficient to activate the QS response (Fig. 3). Structurally, QS signal molecules (or autoinducers) are low molecular weight molecules that belong to a wide range of chemical classes including acyl homoserine lactones (AHLs), furanosyl borate diesters (AI2), cis-unsaturated fatty acids (diffusible signal factor), and peptides [START_REF] Solano | Biofilm dispersion and quorum sensing[END_REF]. Gram-negative bacteria communicate primarily using two Several studies have shown that PA QS systems are involved in the maturation of biofilms.

Wild-type PA forms 3D heterogeneous structured biofilms on abiotic surfaces, but LasI mutant strains which cannot produce OdDHL form homogeneous dense flat biofilms [START_REF] Davies | The Involvement of Cell-to-Cell Signals in the Development of a Bacterial Biofilm[END_REF]. In PA, LasR activates the production of enzymes involved in the synthesis of the structural Pel polysaccharide of the biofilm matrix [START_REF] Gilbert | Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR[END_REF]. Likewise, PA mutants with an inactive PQS system form thin biofilms containing reduced levels of eDNA compared to the wild type strain [START_REF] Allesen-Holm | A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms[END_REF]). The Rhl system has been shown to regulate the swarming and twitching motility of PA , which are essential for the development and assembly of PA as adherent abiotic biofilms [START_REF] Daniels | Quorum sensing and swarming migration in bacteria[END_REF][START_REF] Rasamiravaka | The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

In addition, the Rhl system control the production of rhamnolipids in PA [START_REF] Wood | Rhamnolipids from Pseudomonas aeruginosa disperse the biofilms of sulfate-reducing bacteria[END_REF].

These glycolipids composed of rhamnose with properties of surfactant and virulence factors are involved in several crucial processes of biofilm evolution. For example, they allow the formation of microcolonies, the maintenance of open channels in the biofilm allowing the transport of nutrients and wastes, and facilitate the dispersion of the biofilm in order to colonize other areas when the environment becomes less favorable. Indeed, PA mutants that produce more rhamnolipids than wild-type exhibit hyper-detaching properties [START_REF] Schooling | A role for rhamnolipid in biofilm dispersion[END_REF][START_REF] Wood | Rhamnolipids from Pseudomonas aeruginosa disperse the biofilms of sulfate-reducing bacteria[END_REF]. Lastly, Rhl system also controls the expression of the galactophilic lectins LecA and LecB, virulence factors that contribute to biofilm development in PA [START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF][START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF].

Dispersion of biofilm is often associated with the synchronized release by QS systems of virulence factors. In PA cells with low intracellular c-di-GMP concentrations, PQS and Rhl stimulate the production of pyocyanin and rhamnolipids that kill immune cells (macrophage and PMNs) and protect newly formed planktonic cells [START_REF] Lin Chua | Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa[END_REF]Lu et al. 2018).

Mutation of the PQS system decreased the production of virulence factors such as pyocyanin, elastase, PA-IL lectin and rhamnolipids [START_REF] Diggle | The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR[END_REF][START_REF] Lee | The hierarchy quorum sensing network in Pseudomonas aeruginosa[END_REF][START_REF] Rybtke | Pseudomonas aeruginosa Biofilm Infections: Community Structure, Antimicrobial Tolerance and Immune Response[END_REF].

c. Biofilm tolerance to ATBs

The reduced susceptibility of bacteria in biofilms to antimicrobial agents often leads to treatment failure and is due to both resistance and tolerance of bacteria to these agents [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF]Høiby et al. 2010b). While resistance is associated with bacterial genetic change and increase in minimal inhibitory concentration (MIC), tolerance results in the ability of bacteria to survive antimicrobial treatment using their current genes and biofilms have specific phenotypic features related to ATBs tolerance [START_REF] Verderosa | Bacterial Biofilm Eradication Agents: A Current Review[END_REF].

1) Limitation of the ATBs transport through the biofilm

Various components of biofilms are involved in sequestration or reducing the diffusion of ATBs. Mainly, the matrix of bacterial biofilms contains several negatively charged polymers such as exopolysaccharides, proteins and eDNA, which form a mesh that can limit the penetration of certain antimicrobials into biofilms [START_REF] Tseng | The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin: Limited tobramycin penetration protects biofilms[END_REF]. Membrane vesicles and free soluble lipopolysaccharides (LPS) are also negatively charged components that can be abundant within biofilms and can limit diffusion of some ATBs through biofilms [START_REF] Mann | Pseudomonas biofilm matrix composition and niche biology[END_REF][START_REF] Yokota | Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin[END_REF]. The reduced penetration of ATBs into biofilms is a tolerance mechanism which is considered problematic primarily for polycationic ATBs such as Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires aminoglycosides and polymyxins due to electrostatic interactions with negatively charged biofilm matrix components [START_REF] Cao | Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model[END_REF][START_REF] Chiang | Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Davenport | Differential Protection from Tobramycin by Extracellular Polymeric Substances from Acinetobacter baumannii and Staphylococcus aureus Biofilms[END_REF][START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Hatch | Alginate Lyase Promotes Diffusion of Aminoglycosides through the Extracellular Polysaccharide of Mucoid Pseudomonas aeruginosa[END_REF][START_REF] Nichols | Inhibition of tobramycin diffusion by binding to alginate[END_REF][START_REF] Okshevsky | The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms[END_REF][START_REF] Torres | In vitro evaluation of Pseudomonas aeruginosa chronic lung infection models: Are agar and calcium-alginate beads interchangeable[END_REF][START_REF] Tseng | The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin: Limited tobramycin penetration protects biofilms[END_REF][START_REF] Walters | Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin[END_REF][START_REF] Yokota | Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin[END_REF]). Yet, formed in mucus, eDNA also come from PMNs that surround the biofilm. For instance, in CF patients, virulence factors produced by PA such as rhamnolipids and pyocyanin lyse PMNs and release eDNA that can subsequently be incorporated into the biofilms [START_REF] Alhede | Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes[END_REF][START_REF] Chiang | Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Christensen | Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model[END_REF]. In addition, during infection, PMNs release granules to form an antimicrobial matrix of extracellular fibril known as neutrophil extracellular traps, also made up of DNA. This neutrophil extracellular traps is likely a major contribution of eDNA at the site of infection [START_REF] Lewenza | Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa[END_REF].

cationic
Extracellular DNA can bind to cationic ATBs, such as aminoglycosides and antimicrobial peptides, and contributes to antimicrobial tolerance in PA biofilm by acting as an antimicrobial shield [START_REF] Chiang | Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Okshevsky | The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms[END_REF]. In addition, eDNA also induces an ATBs resistance due to its ability to bind and sequester divalent metal cations such as Zn 2+ , Mg 2+ , Mn 2+ and Ca 2+ . Indeed, the sequestration of Mg 2+ by eDNA activates PhoPQ and PmrAB two-component systems in PA. These systems promote the remodeling of lipid A and core region of the LPS of PA by adding 4-aminoarabinose at the end of the phosphate groups.

These modifications remove negative charges of the LPS and reduce the binding of ATBs such as antimicrobial peptides, COL, and aminoglycosides to the bacterial outer membrane, increasing the tolerance to these ATBs [START_REF] Hunt | Macromolecular mechanisms of sputum inhibition of tobramycin activity[END_REF][START_REF] Lewenza | Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa[END_REF][START_REF] Mulcahy | Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Okshevsky | The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms[END_REF]. To improve the efficacy of cationic ATBs against in vitro and in vivo biofilms, multiple studies have evaluated the used deoxyribonucleases (DNase) to hydrolyze the eDNA and disperse the biofilms to improve ATBs efficacy [START_REF] Alipour | Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa[END_REF][START_REF] Baelo | Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections[END_REF][START_REF] Deacon | Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: Formulation, characterisation and functionalisation with dornase alfa (DNase)[END_REF][START_REF] Nair | In vitro effects of combined iron chelation, antibiotics and matrix disruption on clinical isolates of Pseudomonas aeruginosa[END_REF][START_REF] Okshevsky | The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms[END_REF][START_REF] Tetz | Effect of DNase and Antibiotics on Biofilm Characteristics[END_REF].

ii.

Polysaccharides (Alginate,Psl,Pel)

In PA biofilms, the major polysaccharides produced, i.e. alginate, Pel, and Psl are critical in providing tolerance to various ATBs [START_REF] Billings | The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms[END_REF] [START_REF] Billings | The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms[END_REF]. In contrast, the sensitivity of biofilm of mutants unable to produce alginate or Pel was similar to that of wild type. It is important to mention that Pel and Psl are overexpressed in the SCV of PA often observed in CF patients, which correlate with an increased risk of failure of antipseudomonal treatment with ATBs [START_REF] Goltermann | Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates[END_REF].

Like eDNA, alginate is a polyanion, and one of the arguments to explain its efficiency in providing tolerance to biofilm, in particular to cationic ATBs, would be its ability to bind these ATBs by electrostatic interaction, causing a reduction in their diffusion rate or penetration into the biofilm [START_REF] Nichols | Inhibition of tobramycin diffusion by binding to alginate[END_REF][START_REF] Tseng | The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin: Limited tobramycin penetration protects biofilms[END_REF][START_REF] Walters | Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin[END_REF]. For example, PA biofilm exopolysaccharides have been shown to limit the diffusion of positively charged aminoglycosides, while neutral CIP can normally enter the biofilm. (Ciofu and Tolker-Nielsen 2019;[START_REF] Goltermann | Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates[END_REF]. Cao et al, showed on an alginate bead biofilm model that the binding of TOB to a multitude of sites led to a pronounced delay in its penetration into the biofilm [START_REF] Cao | Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model[END_REF]. This filtering of the free TOB concentration inside biofilm beads was expected increasing the survival probability of bacteria residing in the biofilm [START_REF] Bagge | Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and β-lactamase and alginate production[END_REF]Ciofu and Tolker-Nielsen 2019).

2) Host mucus can limit the transport the transport of ATBs

In CF patient, PA biofilms are mainly found embedded in the lung mucus (Bjarnsholt et al. 2009a). Lung mucus is a viscoelastic gel that covers the epithelium of the airways and plays a vital role in protecting the lungs against external factors such as bacteria [START_REF] Müller | Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin[END_REF].

Mucins (MUC5AC and MUC5B) are polyanionic glycoproteins that give mucus its mechanical flow properties. In CF, mucus viscosity and elasticity is increased and the mucin MUC5AC is overproduced [START_REF] Groneberg | Expression of MUC5AC and MUC5B mucins in normal and cystic fibrosis lung[END_REF][START_REF] Ridley | Mucins: the frontline defence of the lung[END_REF][START_REF] Witten | Molecular Characterization of Mucus Binding[END_REF]). In addition, pulmonary mucus also contains eDNA and phospholipids. As for the EPS biofilm matrix, all those polyanions can bind polycationic ATBs and provide an additional barrier to their diffusion that might allow the bacteria enough time to adapt to a more tolerant state (Bahamondez-Canas et al. 2018;Ciofu and Tolker-Nielsen 2019;[START_REF] Müller | Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin[END_REF][START_REF] Witten | Molecular Characterization of Mucus Binding[END_REF]. For example, Bos et al. showed that TOB binds to mucus with a higher affinity in the presence of eDNA, suggesting that the diffusion of TOB is significantly reduced in mixtures of biofilm/mucus [START_REF] Bos | The fate of inhaled antibiotics after deposition in cystic fibrosis: How to get drug to the bug?[END_REF]. While the effect of mucus on reducing the diffusion of aminoglycosides such as TOB has been shown by several consistent studies [START_REF] Huang | Mucin Binding Reduces Colistin Antimicrobial Activity[END_REF][START_REF] Iglesias | Activity of Antibiotics against Pseudomonas aeruginosa in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium[END_REF][START_REF] Müller | Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin[END_REF], its effect on the diffusion of polymyxins is more controversial. For instance, Huang et al. found that COL, polymyxin B (PMB), and TOB, were bound at around 80% to pig mucins in dialysis experiments [START_REF] Huang | Mucin Binding Reduces Colistin Antimicrobial Activity[END_REF].

When comparing MICs measured with or without added mucin, both polymyxins showed Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires greater than 100-fold increases in MICs for several Gram-negative bacteria. However, other studies have shown that the efficacy of COL against abiotic biofilms of PA was much less affected by the presence of mucin or human mucus than that of TOB [START_REF] Müller | Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin[END_REF].

d. Investigated therapies specific to biofilms

Biofilm dispersion is essential to allow bacteria to escape and colonize new niches when nutrients and other resources become limited and waste products accumulate. Therefore, most bacteria can switch between biofilm and planktonic growth. This coordinated dispersal event is mediated by a decrease in concentration in the bacterial intracellular second messenger cdi-GMP [START_REF] Wille | Biofilm dispersion: The key to biofilm eradication or opening Pandora's box?[END_REF]. Thus, an antibiofilm strategy has emerged in which dispersing agents are used to initiate the dispersion of the biofilm and allow to eliminate the planktonic bacteria, more vulnerable than the bacteria in the biofilm, by ATBs co-administered with these dispersing agents or by the host immune defenses [START_REF] Wille | Biofilm dispersion: The key to biofilm eradication or opening Pandora's box?[END_REF]. As reviewed by [START_REF] Fleming | Approaches to Dispersing Medical Biofilms[END_REF] and Wille and Coeny [START_REF] Wille | Biofilm dispersion: The key to biofilm eradication or opening Pandora's box?[END_REF], one strategy to induce biofilms dispersal is to use molecules or events that decrease the intrabacterial concentration of c-di-GMP, such as a change in concentration in a carbon source, oxygen depletion, administration of nitric oxide (NO) or molecules interacting with bacterial QS. A second strategy to trigger the dispersion of biofilms is the use of enzymes and molecules that act directly on the organization or chemical stability of components of the matrix.

1) Agents decreasing intracellular c-di-GMP concentration

These agents more or less directly reduce the concentration of c-di-GMP in the bacteria and lead to the production of enzymes that degrade the matrix causing biofilm dispersal. It should be noted that lowering the concentration of c-di-GMP does not always lead to dispersion of the biofilm. For example, doxorubicin reduces the concentration of c-di-GMP in PA biofilms, while it increases biofilm formation by stimulating the release of eDNA (Wille and Coenye 2020). [START_REF] Barraud | Involvement of Nitric Oxide in Biofilm Dispersal of Pseudomonas aeruginosa[END_REF]. Similarly, the NO-releasing prodrug cephalosporin-3′diazeniumdiolate, which is activated by bacterial β-lactamases showed 70% removal of the biofilm of PA strain PA01 in vitro [START_REF] Barraud | Cephalosporin-3′-diazeniumdiolates: Targeted NO-Donor Prodrugs for Dispersing Bacterial Biofilms[END_REF]. The prodrug was also active against CF isolates of PA, but the cephalosporin released was inactivated by the β -lactamases and additional ATB were needed to eradicate the biofilm [START_REF] Soren | Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa[END_REF].

Nitroxide conjugate were also used as NO-donor. Verderosa et al. synthesized CIP -nitroxide conjugates that has shown efficacy against in vitro PA biofilms [START_REF] Verderosa | Ciprofloxacin-nitroxide hybrids with potential for biofilm control[END_REF][START_REF] Verderosa | Synthesis and Evaluation of Ciprofloxacin-Nitroxide Conjugates as Anti-Biofilm Agents[END_REF]. A clinical trial using inhaled NO gas at 10 ppm in air associated to intravenous TOB combined or not to ceftazidime has been performed in CF patients. The treatment of 7 days resulted in PA biofilm clusters reduction and no adverse effects were observed [START_REF] Howlin | Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis[END_REF]. TOB is known to diffuse slowly into the lungs after intravenous administration. We can assume that the combination of NO gas with inhaled TOB instead of intravenous injection could have given more promising results.

ii. Change in nutrient concentrations

As previously described, the biofilm environment is associated with nutrient limitations which reduce the metabolism of bacteria at the center of the biofilm and increase tolerance to ATBs targeting bacterial metabolism such as TOB or CIP [START_REF] Bjarnsholt | Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent[END_REF]Ciofu and Tolker-Nielsen 2019;Høiby et al. 2010a). Interestingly, an important change in availability of nutrients, such as carbon and nitrogen sources, stimulates the passage of bacteria from biofilm to planktonic lifestyle [START_REF] Gjermansen | Characterization of starvation-induced dispersion in Pseudomonas putida biofilms[END_REF][START_REF] Sauer | Characterization of Nutrient-Induced Dispersion in Pseudomonas aeruginosa PAO1 Biofilm[END_REF]. For example, the biomass of 4-day-old PA biofilms grown on silicon tubes was reduced by 80 % by a 10-fold increase in the carbon concentration provided by succinate or glutamate in the growth medium [START_REF] Sauer | Characterization of Nutrient-Induced Dispersion in Pseudomonas aeruginosa PAO1 Biofilm[END_REF]). The increase in the concentration of glutamate is sensed by the BdlA chemotaxis sensor in PA, which activates multiple PDEs and decrease the c-di-GMP concentration (Li et al. 2014). Similarly, a strong reduction of nutriments can also induce a biofilm dispersion through c-di-GMP signaling [START_REF] Gjermansen | Characterization of starvation-induced dispersion in Pseudomonas putida biofilms[END_REF]. Based on these results, the effect of various excipients generally used to formulate drugs on the dispersal of biofilms and improving the efficacy of ATBs was tested (Bahamondez-Canas and Smyth 2018). Bahamondez-Canas and Smyth screened 190 potential excipients alone, and in combination with TOB against PA strains PA01 and PA14 grown as abiotic biofilms. Amino acids such as L-and D-alanine, Lproline, or N-acetyl-D-glucosaminitol improved significantly the antibiofilm activity of TOB.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

iii. Antibiofilm peptides Several antibiofilm peptides, natural or synthetic, have shown the ability to disperse existing biofilms [START_REF] Pletzer | Anti-biofilm peptides as a new weapon in antimicrobial warfare[END_REF]. Some of these peptides are thought to indirectly act on diguanylate cyclases and PDEs to reduce intracellular c-di-GMP levels and induce biofilm dispersion. Yet, the exact mechanism has to be identified [START_REF] Pletzer | Anti-biofilm peptides as a new weapon in antimicrobial warfare[END_REF]. Compared to other dispersing agents, most antibiofilm peptides also have intrinsic bacterial activity at concentrations greater than those required for dispersion, potentially allowing them to be used to eradicate biofilms without additional ATB treatment but simply by increasing their administered dose [START_REF] Dostert | Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application[END_REF][START_REF] Feng | The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii[END_REF][START_REF] Overhage | Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation[END_REF][START_REF] Pletzer | Anti-biofilm peptides as a new weapon in antimicrobial warfare[END_REF]. Most of these peptides are positively charged and have a wide spectrum against Gramnegative and Gram-positive bacteria [START_REF] Dostert | Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application[END_REF][START_REF] Pletzer | Anti-biofilm peptides as a new weapon in antimicrobial warfare[END_REF]. Human cathelicidin (LL-37) produced by PMNs was the first anti-biofilm peptide identified to have dispersing activity against PA and A. baumannii biofilms [START_REF] Feng | The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii[END_REF][START_REF] Overhage | Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation[END_REF]. Overhage et al. has reported that LL-37 at 0.5 mg/L (1/128 of its MIC) reduced up to 40 % the thickness of PA biofilms formed on 96-well microtiter plates [START_REF] Overhage | Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation[END_REF].

Chennupati et al. found that a high concentration of LL-37 (2.5 g/L) eradicated PA biofilms from the nasosinus mucosa of rabbits [START_REF] Chennupati | Effects of an LL-37-Derived Antimicrobial Peptide in an Animal Model of Biofilm Pseudomonas Sinusitis[END_REF]). However, these high concentrations of LL-37 have shown proinflammatory and ciliotoxic effects. All these findings encouraged the design and discovery of multiple antibiofilm peptides [START_REF] Dostert | Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application[END_REF][START_REF] Feng | The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii[END_REF].

For example, synthetic peptides derived from bactenecin have shown disruption of abiotic biofilms of PA and A. baumannii at a sub-inhibitory concentration by inducing the degradation of alarmone guanosine tetraphosphate (ppGpp), a second messenger involved in the development of the biofilm [START_REF] Dostert | Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application[END_REF][START_REF] De La Fuente-Núñez | D-Enantiomeric Peptides that Eradicate Wild-Type and Multidrug-Resistant Biofilms and Protect against Lethal Pseudomonas aeruginosa Infections[END_REF][START_REF] De La Fuente-Núñez | Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response[END_REF]. This second messenger is able to relay signal to the c-di-GMP biofilm dispersal machinery [START_REF] Díaz-Salazar | The stringent response promotes biofilm dispersal in Pseudomonas putida[END_REF][START_REF] Fontaine | Exploring the Links Between Nucleotide Signaling and Quorum Sensing Pathways in Regulating Bacterial Virulence[END_REF].

iv. Interaction with quorum sensing

The role of QS systems in biofilm maturation and dispersal offers another strategy that has been intensely studied for the development of a novel antibiofilm therapy using quorum sensing inhibitors (QSI) [START_REF] Brackman | Quorum Sensing Inhibitors as Anti-Biofilm Agents[END_REF][START_REF] Rasamiravaka | The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms[END_REF][START_REF] Solano | Biofilm dispersion and quorum sensing[END_REF]. Primarily, QSIs that target AHL-based QS systems in Gram-negative bacteria have been evaluated for their ability to disperse biofilms using in vitro and in vivo models [START_REF] Geske | Small Molecule Inhibitors of Bacterial Quorum Sensing and Biofilm Formation[END_REF][START_REF] Ishida | Inhibition of Quorum Sensing in Pseudomonas aeruginosa by N-Acyl Cyclopentylamides[END_REF]. For example, analogues of AHL, having additional aromatic fractions such as bromophenyl, have shown inhibitory activity of the Las QS system and reduced the formation of abiotic biofilm of PA PA01 [START_REF] Geske | Small Molecule Inhibitors of Bacterial Quorum Sensing and Biofilm Formation[END_REF]. Analogues in which Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires the homoserine lactone ring has been replaced by a cyclohexanone ring down-regulate the expression of LasI, resulting in reduced expression of the virulence factors pyocyanin and elastase and a change in morphology biofilm [START_REF] Smith | Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer[END_REF]. Molecules interfering with the PQS system such as benzamide-benzimidazole derivatives were also developed [START_REF] Koo | Targeting microbial biofilms: current and prospective therapeutic strategies[END_REF][START_REF] Starkey | Identification of Anti-virulence Compounds That Disrupt Quorum-Sensing Regulated Acute and Persistent Pathogenicity[END_REF]. These derivatives reduced the formation of persisters of PA strain PA14 and minimize the bacterial load when co-administered with CIP in murine lung infection model.

In signal molecule, inhibits the growth of A. baumannii once a quorum develops [START_REF] Peleg | Prokaryote-eukaryote interactions identified by using Caenorhabditis elegans[END_REF]. It was also shown that FAR interferes with the PQS synthesis of PA [START_REF] Cugini | Candida albicans-produced farnesol stimulates Pseudomonas quinolone signal production in LasR-defective Pseudomonas aeruginosa strains[END_REF]Cugini et al. 2007;[START_REF] Li | Inhibition of the pqsABCDE and pqsH in the pqs quorum sensing system and related virulence factors of the Pseudomonas aeruginosa PAO1 strain by farnesol[END_REF]McAlester et al. 2008). This interference destabilizes the biofilm of Gram-negative bacteria, making FAR attractive for antibiofilm therapy. For example, FAR co-trapped with CIP in liposomes increases the destruction of abiotic biofilms of PA by 10-fold compared to liposomes loaded with CIP alone (Bandara et al. 2016). Recently patented farnesyl-aminoglycosides conjugates that spontaneously form NP and release farnesyl and the aminoglycoside improved around 10-times the efficacy of TOB against abiotic PA14 biofilms ( [START_REF] Ho | Aminoglycoside derivatives and nano-assemblies thereof, including those with quorum sensing inhibitory function[END_REF].

QS requires the binding of a signalling molecule to a corresponding transcriptional regulator, which activates the downstream transcription of select targets. Although the increased efficacy of ATB treatment with QSI in vivo is promising, inhibitors still need access and specific targeting to site of active QS-signaling. However, QSI molecules can be sequestered or have limited diffusion through the EPS matrix and the effects may be limited to highly localized areas within the biofilm structure. Therefore, the efficacy of such inhibitors could be improved in combination with other strategies such as the use of nanoparticles to improve their delivery within the biofilm.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

2) Agents that directly target the matrix

Several enzymes, mainly bacterial hydrolases, promote the detachment of bacterial cells from the biofilm by catalyzing the degradation of the polymers of the extracellular matrix i.e.

proteins, eDNA and exopolysaccharides. Therefore, most of the biofilm dispersing enzymes discovered are proteases, DNase, and glycosidic hydrolases produced by bacteria [START_REF] Fleming | Approaches to Dispersing Medical Biofilms[END_REF][START_REF] Flemming | The biofilm matrix[END_REF]. Enzymes that were most investigated to eradicate PA pulmononary biofilms are DNase [START_REF] Whitchurch | Extracellular DNA required for bacterial biofilm formation[END_REF], such as recombinant human DNase (rhDNase) that is already used by inhalation to reduce the viscosity of the mucus in CF patients, and alginase or alginate lyase (AlgL) since alginate in one of the main polysaccharide found in mucoid PA biofilms [START_REF] Alkawash | Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms[END_REF][START_REF] Fleming | Approaches to Dispersing Medical Biofilms[END_REF]. Furthermore, AlgL reduces the amount of negatively charged alginate in the biofilm matrix, facilitating cationic ATBs, such as aminoglycosides, to penetrate through biofilms and kill the bacteria [START_REF] Alipour | Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa[END_REF][START_REF] Hatch | Alginate Lyase Promotes Diffusion of Aminoglycosides through the Extracellular Polysaccharide of Mucoid Pseudomonas aeruginosa[END_REF]. For example, coadministration of AlgL with gentamicin on in vitro biofilms of mucoid PA clinical isolates resulted in the elimination of biofilms, although treatment with gentamycin alone was not effective [START_REF] Alipour | Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa[END_REF]. Similarly, the effectiveness of aminoglycoside against in vitro PA biofilm was improved when combined with DNase or AlgL, while N-acetylcysteine was not able to improve aminoglycosides activity [START_REF] Alipour | Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa[END_REF].

Interestingly, alginate oligomers were able to disperse PA biofilms [START_REF] Powell | Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides[END_REF]. In

III. How to studying and evaluation of biofilms?

Multiple in vitro and in vivo models were developed to test the efficiency new drugs or new approaches to treat biofilms, a summary of these models is provided in this paragraph.

a. In vitro biofilms models

Different models of in vitro biofilm have been developed with increasing complexity. For example, bacteria may or may not be attached to a surface, cultured in the presence of various nutrients or media simulating in vivo conditions, co-cultured with various eukaryotic cells or other microorganisms to form poly-species biofilms (Bjarnsholt 2013;[START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF].

Depending on their complexity, these models allow either to have a high throughput system to screen for many conditions, or to have conditions that mimic as much as possible those encountered in the patient (Bjarnsholt 2013;[START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF]. These models are classified generally into two categories, static and dynamic models, which are presented in Table 1 (Coenye and Nelis 2010; [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF][START_REF] Shi | In vitro and ex vivo systems at the forefront of infection modeling and drug discovery[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

1) Static biofilm model

Static or closed biofilm models are the most widely used models, especially for high throughput screenings [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]. They are characterized by the absence of flow of growth medium on the biofilm and by the reduction in the availability of nutrients over time [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF].

In such models different parameters including nutrients availability, incubation temperature, shear stress, O2 and CO2 concentrations, as well as nitrate gradients concentrations can be monitored. Three types of static biofilm models are mostly recognized: microtiter plates including pegs lid models (i.e. Calgary device), bacteria-embedded in beads based models [START_REF] Bjarnsholt | The in vivo biofilm[END_REF][START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF], and culture-cells based model.

These different models were presented in Figure 5. -Microtiter plate and pegs lid models (Figure 5a) are intended to produce a surfaceattached biofilm simulating the adherent lung biofilm formed on ETT. The advantages of these models are easy to use, quick and cheap [START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF]. The main limitation of these models is the variability in the biofilm biomass among the wells or the pegs. In addition, oxygen and nutrients gradients were not observed as within the lung bacterial biofilm [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF]. All these challenges made this model weakly representative of the lung biofilm found on ETT.

-Cell-culture models (Figure 5b) correspond to the formation of biofilm on living cells This model can add mucus on the top of cells or used mucus producing cells such as calu-3 cells lines [START_REF] Haghi | Time-and passagedependent characteristics of a Calu-3 respiratory epithelial cell model[END_REF]).

-Bacteria-embedded biofilm models (Figure 5c) bacteria-loaded beads made from agar or alginate. This model simulates non-attached bacterial biofilm observed in chronic inflammatory lung disease (CF and bronchiectasis) [START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Pedersen | Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres[END_REF]Sønderholm et al. 2017a). Microscopic observations in such models showed dense bacterial clusters, similar in size and aspects to the biofilm aggregates found in lungs of CF patients.

Clusters are abundant at beads periphery and gradually decreased in size and abundancy from the outer to the inner of beads (Sønderholm et al. 2017a) due to the oxygen gradients, which simulate the bacterial repartition in biofilm lung environment. This agar or alginate beads-based model has a size approximately 2.4 mm [START_REF] Behrendt | Biofilm Growth and Near-Infrared Radiation-Driven Photosynthesis of the Chlorophyll d-Containing Cyanobacterium Acaryochloris marina[END_REF]Sønderholm et al. 2017a) and is used to develop a chronic lung infection in murine models, when using smallsized beads (less than 265µm).

2) Dynamic biofilm model

Dynamic or open biofilm models [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF], are based on continuous replace of growth medium which stimulates the cultures and eliminates metabolic products and dead cells [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF]. Due to the complexity of devices, the use of this model is limited to long term high throughput applications [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF]. Flow cell systems, including continuous flow cell system and flow Cell-culture models, and bioreactorbased models, including center for disease control (CDC) biofilms reactors and drip flows Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires system were the most used (Bjarnsholt 2013;[START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF][START_REF] Shi | In vitro and ex vivo systems at the forefront of infection modeling and drug discovery[END_REF][START_REF] Weiss Nielsen | Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells[END_REF]) .

These models were presented and described in Table 1 andFigure Reactor consists of four isolated rectangular channels containing a removable coupon (as shown in at the right side), with a size similar to the glass microscope slide, which promote the bacterial attachment on its surface. Drip flow reactor placed in angled way and the broth medium were pumped in channels and flows down the surface of the slide by gravity (adapted from [START_REF] Goeres | A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor[END_REF][START_REF] Radniecki | Investigating Nitrosomonas europaea Stress Biomarkers in Batch, Continuous Culture, and Biofilm Reactors[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Two types of biofilms are formed on non-irrigate surface in the lung, on ETT or respiratory airway, which makes the idea of continuous supply of fresh media not relevant to in vivo conditions. The flow cell system presents the main advantage to maintain the ATBs treatment with time. However, it may generate antimicrobial resistant mutants. Moreover, flow cell systems can produce three dimensional structured bacterial biofilms with mushroom-like structures [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]), which have never been observed in an in vivo [START_REF] Bjarnsholt | The in vivo biofilm[END_REF][START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF]. This observation raises the question of the representativeness of this model as biofilm found in CF patients.

Despite of the different characteristics of in vitro biofilm models, several missing points were observed. Firstly, the absence of implication of human body environment, such as the immune response (macrophages, PMN…) [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Roberts | The limitations of in vitro experimentation in understanding biofilms and chronic infection[END_REF].

Secondly, the limited nutrients access, especially observed with static models, which gradually decreases over time and restricts a long period time study. Also, the biofilm feedings have been regulated by the nutrients composed growth media which can variate from minimal nutrients media (Bacto R2A agar) to high nutrient media (e.g. Mueller Hinton Broth, Lysogeny broth), allowing the study of the influence of each growth media compound on the biofilm formation. In addition, lung biofilm is produced in a nutrient-rich environment (mucus and sputum) that encourages researchers to produce an artificial media simulating the sputum and mucus in its composition (Diraviam Dinesh and Diraviam Dinesh 2010). This media was used in some studies to produce bacterial lung biofilm.

In conclusion, none of the developed in vitro biofilm models adequately represents the pulmonary bacterial biofilms observed in CF patients. However, several properties obtained with these models are similar to those found in biofilms in vivo and have helped to assess the effectiveness of different anti-biofilm strategies.

The question now is: Can we transfer the obtained data from laboratory workbench directly to the bedside of patient? The use of in vitro models to produce a system like-biofilm environment and evaluate the new promising therapies does not allow to study the implication of host human environment or directly understand the dynamic interactions between the host and bacterial biofilms. These perspectives can be evaluated in in vivo biofilm models.

b. In vivo models

Several animal models have been developed to produce chronic lung infections, and were mainly divided into two biofilm model types; models of biofilm embedded in mucus and models of biofilms grown on ETT of ventilated animal, listed in Table 2. -Models based on bacteria embedded in beads have been widely used to produce chronic lung infection in animals (rats, mice, guinea pigs and rhesus monkeys), in order to mimic unattached lung biofilms produced by PA in the lungs of patients with CF [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]. The size of beads is generally smaller than the in vitro models which variate (15-265 µm) [START_REF] Christophersen | Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection[END_REF][START_REF] Growcott | Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin[END_REF][START_REF] Heeckeren | Murine models of chronic Pseudomonas aeruginosa lung infection[END_REF][START_REF] Pedersen | Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres[END_REF][START_REF] Torres | In vitro evaluation of Pseudomonas aeruginosa chronic lung infection models: Are agar and calcium-alginate beads interchangeable[END_REF]. Some researchers have used single or repeated aerosol administration of PA in an attempt to reproduce the symptoms of infection caused by PA as seen in CF patients. To date, to create a PA chronic lung infection, only PA trapped into agar or alginate beads have been successful [START_REF] Cash | A Rat Model of Chronic Respiratory Infection with Pseudomonas aeruginosa[END_REF][START_REF] Growcott | Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin[END_REF]. This Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires model showed a great ability to mimic chronic lung infection, with the presence of inflammation typical of what is seen in patients with CF for up to 28 days [START_REF] Bjarnsholt | The in vivo biofilm[END_REF][START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Growcott | Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin[END_REF][START_REF] Kukavica-Ibrulj | Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies[END_REF][START_REF] Nacucchio | Short Communication. Role of Agar Beads in the Pathogenicity of Pseudomonas aemginosa in the Rat Respiratory Tract[END_REF][START_REF] Tewes | In vivo efficacy of a dry powder formulation of ciprofloxacin-copper complex in a chronic lung infection model of bioluminescent Pseudomonas aeruginosa[END_REF].

-An in vivo non-attached lung biofilm can be developed in transgenic mouse (CFTR Knockout) intratracheally inoculated with a small amount of planktonic PA [START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Kukavica-Ibrulj | Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies[END_REF]. Although the manifestations of intestinal disease are significant in these animals, there is little evidence of lung disease in CF mice maintained under normal housing conditions (Kukavica-Ibrulj and Levesque 2008). Thus, it remains unclear whether the dysfunction of CFTR directly results in an increased predisposition to pulmonary infection or whether it is due to wider sensitivity and inappropriate inflammatory responses resulting from repeated infections with PA that generate an environment that favors subsequent infections. To date, evidence of persistent infection to PA have only been achieved using the agar bead model to mimic chronic colonization in CFTR knockout mice (Kukavica-Ibrulj and Levesque 2008). Therefore, due to the high production cost of these mice and their relative fragility, the use of this model is limited.

-In vivo models of lung biofilm attached to a surface, mimicking biofilms grown on ETT in ventilated patients could be produced in ventilated animals. These models have been associated with many benefits including the possibility of keeping the animal intubated for more than 96 H allowing the study of bacterial colonization and biofilm formation on ETT [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Luna | Animal models of ventilator-associated pneumonia[END_REF][START_REF] Pulido | Does animal model on ventilatorassociated pneumonia reflect physiopathology of sepsis mechanisms in humans?[END_REF]. Hence, various types of ETT uncoated or coated with various preventive anti-biofilm agents (such as silver-sulfadiazine/chlorhexidine in polyurethane or silver hydrogel) have been evaluated ETT [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF][START_REF] Luna | Animal models of ventilator-associated pneumonia[END_REF][START_REF] Pulido | Does animal model on ventilatorassociated pneumonia reflect physiopathology of sepsis mechanisms in humans?[END_REF].

Although in vitro biofilm models are numerous, simpler to set-up, rapidly produced and relatives cheaper, in vivo biofilm models allow the production of biofilm in a more representative dynamic environment. In addition, in vivo models may offer the possibility to study the host response (local and systemic) against chronic lung infection and to evaluate anti-biofilm therapies under similar conditions of the human body [START_REF] Pulido | Does animal model on ventilatorassociated pneumonia reflect physiopathology of sepsis mechanisms in humans?[END_REF]. Regardless of legitimate ethical considerations, the use of in vivo models was early limited by legal legislation requiring that the researcher evaluates the scientific and medical benefits while considering the animal welfare [START_REF] Lebeaux | From in vitro to in vivo Models of Bacterial Biofilm-Related Infections[END_REF]. In this regard, researchers have been encouraged to optimize the conditions of in vitro models to better mimic in vivo biofilm environmental. For instance, using synthetic medium mimicking the composition of lung environment (sputum, mucus), investigating the implication of immune response and production of oxygen, nutrient At present, there is no "gold-standard" or perfect method to produce an in vitro or in vivo model fully representative to the bacterial lung biofilm. Better evaluation of anti-biofilm strategy, prior to clinical considerations, requires even more investigation to develop a more realistic in vitro biofilm models for chronic infection, so as to continue addressing this emerging challenge in biofilm development.

c. Parameters measured with biofilm models

Before being able to test the effectiveness of an antimicrobial treatment on human biofilms, it is important to be able to determine certain parameters in order to choose the appropriate therapy, such as for example the effective dose using the biofilm models. Actually, MIC and minimal bactericidal concentration (MBC) are two parameters used to determine the lowest antimicrobial concentrations required for inhibiting bacterial growth and reducing cells viability (≥99.9% of initial bacterial inoculum) respectively. These parameters are evaluated using bacteria in planktonic form and are specific to this mode of growth [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Döring | Treatment of lung infection in patients with cystic fibrosis: Current and future strategies[END_REF]. Thus, MIC and MBC are not suitable for use on biofilms, so it is essential to develop specific biofilm susceptibility tests as none of the official agencies eg. European Committee on Antimicrobial Susceptibility Testing (EUCAST) or Clinical and Laboratory Standards Institute (CLSI), have yet established standardized definitions of biofilm endpoint parameters such as MIC.

Different biofilm susceptibility parameters such as the minimum biofilm eradication concentration (MBEC), the minimum biofilm inhibitory concentration (MBIC), the biofilm bactericidal concentration (BBC), and the biofilm prevention concentration (BPC) have been suggested as a guide for the treatment of biofilm-associated infections [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Thieme | MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology[END_REF]. According to some publications, MBEC corresponds to the lowest ATB concentration reducing by 3 log10 the bacterial concentration in the biofilm compared to growth controls [START_REF] Thieme | MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology[END_REF]). However, others consider it as the BBC and describe the MBEC as the concentration allowing complete eradication of biofilm [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Thieme | MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology[END_REF].

Inhibitory effects on biofilm formation are commonly assessed by the MBIC. It is described as the minimum antimicrobial concentration that does not result in any increase over time in the average number of viable bacteria in the biofilm [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Reiter | Inhibition of biofilm maturation by linezolid in meticillin-resistant Staphylococcus epidermidis clinical isolates: comparison with other drugs[END_REF][START_REF] Thieme | MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology[END_REF]. BPC is another parameter used to assess the antibiofilm-forming property of a molecule. This is the concentration at which the antimicrobial lowers enough the cell Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires density of a planktonic culture to prevent biofilm formation [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF][START_REF] Thieme | MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology[END_REF]. BPC can be helpful in the early stages of bacterial infection of the lungs preventing the development of chronic infection [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF].

1) In vitro methods

Methods for assessing the antimicrobial efficacy of molecules obtained from in vitro models of biofilm can be divided into quantitative or qualitative methods i. Quantitative methods Among the quantitative biofilm characterization methods, some measure the total biomass, the total number of cells (live and/or dead cell), or the total organic carbon [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

These methods can be realized using direct or indirect measurements.

Direct measurements methods are based on observations using various methods such as colony forming method, flow-based cell counting or microscopic cells count.

Colony-forming unit (CFU) count method (Figure 10) is a standard quantification method widely used to determine the number of bacteria able to grow on an agar plate [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]. The CFU method offers the possibility of identifying slow-growing cells, allowing the quantification of persistent cells, such as SCV often observed in clinical isolates of PA taken from the lungs of CF patients [START_REF] Pestrak | Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments[END_REF]. Flow-based cell counting methods are more automatized methods based on counting bacteria in liquid suspension. Two main methods are used: coulter counting and flow cytometry [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]. Coulter counting is a simple method and less expensive than cytometry flux but does not differentiate between alive and dead cells. Flow cytometry is rapid and gives Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires additional information about bacteria, including metabolic activity or the presence of surface markers [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

Microscopic methods are also used for cell counting and biofilm morphology characteristics. Such observation may be improved by using fluorescence microscopy [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

For instance, intrinsic fluorescent molecules such as NADH and NAD(P)H, or fluorescent dyes and proteins are used to stain the bacteria allowing the observation of the biofilm features [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]. Alternatively, a 3D observation using a confocal laser scanning microscopy (CSLM) can be performed [START_REF] Azeredo | Critical review on biofilm methods[END_REF][START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]). However, the observation is limited to a very small portion where the light is concentrated requiring multiple time-consuming measures [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

Indirect measurements methods are based on using proxy markers, more easily measurable than direct markers to evaluate the biofilm cells number, such as dry mass, total protein content, total organic carbon, adenosine triphosphate (ATP) bioluminescence, DNA, RNA, metabolites and others.

Dry mass measurement, crystal violet (CV) staining, and fluorogenic dye Syto9 DNA staining are different methods used to estimate the overall biofilm mass. CV is a basic dye that binds to negatively charged molecules and polysaccharides of the extracellular biofilm matrix (Figure 11a). It is widely used and well optimized for visualization and indirect quantification of biofilm grown on abiotic surfaces [START_REF] Peeters | Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates[END_REF][START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]. Syto9 is a nucleic acid dye that diffuses across the cell membrane and binds to DNA of both living and dead cells, as well as to the eDNA of biofilms. Hence, this method cannot distinguish between all the components of the biofilm (matrix, dead and lives cell), which makes it unsuitable for assessing the killing of the bacteria within biofilms. In this regard, the evaluation of bacterial biofilm killing requires specific quantification method, known as the viability test, which differentiates between live and dead cells. Numerous viability tests have been used based on the study of metabolic activity of viable or living cells in biofilm, including resazurin assay (Figure 11b), fluorescein diacetate assay, 2H-Tetrazolium (XTT) assay, or based on chemical reactions occurring in living cells which produce light in proportion to the concentration of ATP [START_REF] Peeters | Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates[END_REF][START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF]. (a) Image showed biofilm formed on microtiter plate method that was evaluated using crystal violet stain. This image showed none, weak, moderate and strong biofilm producers differentiated by the concentration of crystal violet detected in each well. This method is used to estimate the viability of biofilm cells (Darwish and Asfour 2013). (b) Linear relationship between the number of bacteria and resazurin reduction. This figure showed that resazurin is not sensitive enough to the bacteria at a concentration lower than 5x10 6 , which prove that this method is not an adaptable tool to measure the cell viability (adapted from Koizumi and Picardeau 2020).

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

XTT and resazurin assays are used to measure cellular metabolic activity as an indicator of bacteria viability. These methods are based on reducing the membrane permeable reactant, XTT or resaruzin by living bacteria to colored and fluorescent dyes, respectively. Similarly, fluorescein diacetate is a cell-permeant esterase substrate that is hydrolase to fluorescent fluorescein via hydrolysis by metabolically active bacteria [START_REF] Peeters | Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates[END_REF].

Finally, the quantification of DNA, RNA and polysaccharides can be used to estimate the number of bacteria in the biofilm but it must be combined in parallel with a direct measurement method like CFU counting, because the matrix of the biofilm also contains these components which interfere with the measurement [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

ii. Qualitative biofilm characterization methods

These are generally used to study biofilm surface morphology (such as surface roughness), spatial organization, and interaction between biofilm and environment [START_REF] Wilson | Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review[END_REF].

Different qualitative can be divided into microscopic and molecular methods.

Microscopic methods: Two types of microscopy (optical and fluorescent) are mainly used to visualize the formation of the biofilm. Among fluorescence microscopy, the CLSM, which provides a 3D image of the biofilm allowing the study of its architecture and the distribution of

2) In vivo methods

The characterization methods used for observation and therapy assessment of in vivo biofilm models are fewer than the in vitro models.

In general, infected/treated desired tissues (e.g. Lung) are analyzed by several methods including viability observation using CFU determination methods or flow cytometry, and microscopic observation methods after fixing the organ (e.g. lung) to study the biofilm-formed on lung or ETT using FISH method or transmission electron microscopy or CLSM. In addition, PCR and quantitative-PCR can help to identify the genes involve in biofilm formation or bacterial mutation [START_REF] Magana | Options and Limitations in Clinical Investigation of Bacterial Biofilms[END_REF]. Unfortunately, these methods are not suitable to evaluate antimicrobial therapies that are mostly efficient at early stages of treatment. To overcome this problem and reduce the number of animals used, there are other methods to assess the treatment efficacy such as the bioluminescence or near-infrared fluorescence imaging methods based on the production of light from bacteria. This techniques allows to assess the location and the development of biofilm observed by a sensitive imaging camera [START_REF] Magana | Options and Limitations in Clinical Investigation of Bacterial Biofilms[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

IV. Drug Delivery system to target lung biofilms

Biofilm infections found in lungs are difficult to eradicate with conventional treatment [START_REF] Wu | Strategies for combating bacterial biofilm infections[END_REF]. So, the treatment of these lung biofilms requires high concentrations of ATBs that can penetrate into the biofilm and kill the bacteria [START_REF] Wenzler | Inhaled Antibiotics for Gram-Negative Respiratory Infections[END_REF][START_REF] Wu | Strategies for combating bacterial biofilm infections[END_REF].

However, these high levels of ATB can be achieved in the lungs without systemic toxicity only by using inhalation therapy [START_REF] Velino | Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis[END_REF] [START_REF] Schwarz | Colobreathe® for the Treatment of Cystic Fibrosis-Associated Pulmonary Infections[END_REF]. As shown on their chemical structures (Fig. 13), these two ATBs have five primary amino groups. These amine groups are mostly protonated at physiological lung pH (6.8-7) [START_REF] Song | Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis[END_REF]). Hence, these two ATBs are cationic and can interact with the components of mucus and biofilms, potentially decreasing their effectiveness in combating lung biofilms [START_REF] Khan | Alternative strategies for the application of aminoglycoside antibiotics against the biofilm-forming human pathogenic bacteria[END_REF][START_REF] Wenzler | Inhaled Antibiotics for Gram-Negative Respiratory Infections[END_REF]. Therefore, strategies to improve cationic ATBs diffusion through mucus and biofilms are required to improve their antibiofilm efficacy.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

New ATB delivery strategies involve the use of drug delivery DDS, which encapsulate ATB to improve its efficacy by protecting it and controlling its release at the site of infection [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Nafee | Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors[END_REF]. Different types of vectors have been studied as DDS of cationic ATBs, including nanocarriers, but other strategies such as bacteriophages or PEGylation of ATBs have also been studied to improve ATB delivery to bacteria. In this chapter, we will present various DDS formulation of ATBs and their preclinical and clinical evaluations to treat gram-negative chronic lung infection.

a. Nanotechnologies or nanoencapsulations for DDS

ATBs nanoencapsulation is considered a suitable alternative to improve the current antimicrobial therapy and constitutes a promising strategy to overcome the problem of diffusion of ATB through mucus barrier and biofilm into the lung [START_REF] Poyner | A comparative study on the pulmonary delivery of tobramycin encapsulated into liposomes and PLA microspheres following intravenous and endotracheal delivery[END_REF][START_REF] Serramitjana | Pseudomonas aeruginosa RESPIRATORY INFECTIONS IN[END_REF]. DDS based on nanotechnology can allow ATBs to interact directly with the complex structure of biofilms and treat different stages of bacterial biofilm formation as shown in Figure 14

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

The use of NPs offers several other advantages including controlled release of ATBs to achieve high dose delivered to the target infected site with less side effects (Pelgrift and Friedman 2013) and improved ATB apparent solubility [START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF][START_REF] Zhang | Development of Nanoparticles for Antimicrobial Drug Delivery[END_REF].

Additionally, multiple antimicrobial agents can be loaded into the same NP to deliver a combination therapy to the site of infection, thus preventing resistance from developing [START_REF] Pelgrift | Nanotechnology as a therapeutic tool to combat microbial resistance[END_REF][START_REF] Zhang | Development of Nanoparticles for Antimicrobial Drug Delivery[END_REF]. For instance, LIPs surface charge can be positive when using, dioctadecyl aminogylcospermine, or negative using phosphatidylserine or phosphatidylglycerol, or neutral using phosphatidylcholine and its derivatives [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF]). The membrane rigidity can vary depending on the length and saturation of the alkyl lipid chains within the phospholipids and depending on the cholesterol content, thus controlling LIPs stability and membrane permeability [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF][START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Pelgrift | Nanotechnology as a therapeutic tool to combat microbial resistance[END_REF]. In addition, the inclusion of polymers conjugated to lipids such as polyethylene glycol (PEG), polyacryl amide and polyvinyl alcohol within the membrane, may form a coat on the LIP surface that reduces LIP aggregation, prevents opsonin adsoption, and therefore stabilizing more the LIP and protecting it against phagocytosis [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Sharma | Liposomes in drug delivery: Progress and limitations[END_REF].

LIPs have the capacity to encapsulate drugs with variable degree of lipophilicity, either in phospholipid membrane, in aqueous medium (inner core), or in the bi-layer interface [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Sharma | Liposomes in drug delivery: Progress and limitations[END_REF] The encapsulation of TOB into liposomal formulation has been investigated in many studies.

For example, Beaulac et al. developed two formulations of liposomal TOB with different bilayers composition [START_REF] Beaulac | Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection[END_REF]). The first one was made of DPPC and dimyristoylphosphatidylglycerol and the second one made of DSPC and dimyristoylphosphatidylcholine. They showed that, compared to second formulation, the first formulation was effective in treating and eradicating chronic PA lung infection in rats intratracheally infected with agar beads model, which did not show any efficacy compared to free TOB. The decrease in efficacy observed with second formulation is due to DSPC which was less permeable for TOB, compared to DPPC composed in first formulation [START_REF] Beaulac | Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection[END_REF][START_REF] Forier | Lipid and polymer nanoparticles for drug delivery to bacterial biofilms[END_REF][START_REF] Omri | Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa[END_REF]. In another study, the first formulation DPPC and dimyristoylphosphatidylglycerol was administrated via pulmonary routes and demonstrated a significant improvement of the efficacy of TOB by approximately three-folds as compared to free TOB after multiple administrations in rats chronically infected with PA agar beads [START_REF] Beaulac | Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection[END_REF][START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Marier | Liposomal tobramycin against pulmonary infections of Pseudomonas aeruginosa: a pharmacokinetic and efficacy study following single and multiple intratracheal administrations in rats[END_REF][START_REF] Omri | Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa[END_REF]. This improvement in efficacy was explained by significant increase in lungs residence time [START_REF] Marier | Liposomal tobramycin against pulmonary infections of Pseudomonas aeruginosa: a pharmacokinetic and efficacy study following single and multiple intratracheal administrations in rats[END_REF]Omri et Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires al. 1994), high amounts and sustained-release of TOB proved within the lungs of both infected and uninfected rats [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Omri | Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa[END_REF]. TOB has been noted to be absent in blood and kidneys of these animals [START_REF] Omri | Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa[END_REF]. Similarly, gentamicin encapsulated in neutral or negative liposomal formulations has been developed. Neutral formulations showed a significant reduction in the MIC and MBC values and a significant prevention of in vitro PA biofilm formation using the microtiter plate model, compared to free gentamicin, while the negative liposomal formulation increased the encapsulation of gentamicin but displayed the same efficacy of free gentamicin [START_REF] Alhariri | Efficacy of neutral and negatively charged liposome-loaded gentamicin on planktonic bacteria and biofilm communities[END_REF].

Co-encapsulation in liposomes of aminoglycosides with gallium or bismuth metals has been shown to improve their efficacy against chronic PA infections. In particular, the incorporation of bismuth-ethanedithiol (Bi) into the TOB-loaded LIPs (LIP-TOB/Bi) reduced the MIC value 4-fold and 16-fold compared to TOB-Bi and free TOB, respectively [START_REF] Alipour | Attenuation of Pseudomonas aeruginosa virulence factors and biofilms by co-encapsulation of bismuth-ethanedithiol with tobramycin in liposomes[END_REF]). In addition, LIP-TOB/Bi reduced the attachment of PA to the microtiter plate surface 4

times more than free TOB-Bi [START_REF] Alipour | Attenuation of Pseudomonas aeruginosa virulence factors and biofilms by co-encapsulation of bismuth-ethanedithiol with tobramycin in liposomes[END_REF]). These results were validated in an in vivo study of chronic lung infection in rats infected with PA agar beads [START_REF] Alhariri | Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection[END_REF]. In [START_REF] Varshosaz | Development and optimization of solid lipid nanoparticles of amikacin by central composite design[END_REF]) with a drug-to-lipid ratio of 0.5. These SLN had a diameter of 149 ± 4nm and drug's EE was of 88±5% [START_REF] Varshosaz | Development and optimization of solid lipid nanoparticles of amikacin by central composite design[END_REF]. When tested in vitro, the Amikacinloaded SLN showed a reduction by two folds of MIC and MBC on PA planktonic, compared to free amikacin [START_REF] Varshosaz | Stability and antimicrobial effect of amikacin-loaded solid lipid nanoparticles[END_REF]. The administration of Amikacin-loaded SLN to rats via pulmonary route showed higher concentration of drug into lungs compared to free amikacin [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Varshosaz | Biodistribution of Amikacin Solid Lipid Nanoparticles after Pulmonary Delivery[END_REF]. These results suggested that the use of SLN-Amikacin could be used to decrease the amikacin total dose and toxicity [START_REF] Varshosaz | Biodistribution of Amikacin Solid Lipid Nanoparticles after Pulmonary Delivery[END_REF][START_REF] Varshosaz | Stability and antimicrobial effect of amikacin-loaded solid lipid nanoparticles[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

The encapsulation of CMS into SLN and NCL was envisaged to improve its efficacy in PA chronic infection in CF patients. Both formulations had size ranging between 200-400 nm, and were characterized by high EE of 79.7% and 94.8% for SLN and NCL, respectively. The in vivo distribution study of intratracheally administered lipids nanoparticles in mice showed a homogenous distribution through the lungs without migration into others organs (such as kidney, liver and spleen) for up to 48 hrs, which may prove the safety of formulations for human use [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF]Pastor et al. 2014). A study compared the efficacy of encapsulation of COL and Amikacin into negatively and positively charged NLC (coated (+) or uncoated (-) with chitosan) with size around 73-142 nm [START_REF] Vairo | In vitro and in vivo antimicrobial activity of sodium colistimethate and amikacin-loaded nanostructured lipid carriers (NLC)[END_REF]). An in vitro test of the efficacy of these formulations on different strains of PA in planktonic mode and biofilms models using pegs lids model demonstrated that COLloaded NCL decreased at least 2-fold MIC, MBEC and MBIC in almost strains compared to the free form, while Amikacin-loaded NCL showed a slight reduction of MIC (max 2 folds) with some strains and significant decreases in MBIC up to 4 fold but not in MIC and MBEC [START_REF] Vairo | In vitro and in vivo antimicrobial activity of sodium colistimethate and amikacin-loaded nanostructured lipid carriers (NLC)[END_REF]). In addition, the in vitro test of COL-loaded NCL on A. baumanii planktonically grown showed a significant reduction of MIC (4 to 8 folds) while the encapsulation of amikacin did not report a change in the MIC value [START_REF] Vairo | In vitro and in vivo antimicrobial activity of sodium colistimethate and amikacin-loaded nanostructured lipid carriers (NLC)[END_REF].

On the other hand, LNP have been used to encapsulate other molecules with antimicrobial activity against bacterial biofilm. In particular, the nano-encapsulation of QSI into SLN showed a reduction in the virulence factor pyocyanin formation by 7 folds, compared to the free form in PA planktonically grown in vitro [START_REF] Nafee | Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors[END_REF]. Furthermore, Taylor et al. have In conclusion, LNPs demonstrate potential effectiveness in treating bacterial lung infection in terms of sustaining drug release and enhancing drug distribution without migration to others organs that reduce drug toxicity. In addition to previous in vitro and in vivo studies, clinical trials of LNP would need to be conducted to determine the efficacy and safety of these formulations in human.

2) Polymeric Nanoparticles

Polymers The degradation is faster when the glycolic acid proportion increase up to 50:50 by weight [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF]. PLGA polymers with a 50:50 ratio, mainly used in nanomedicine, have been reported to have the fastest rate of degradation and release profile. [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF]).

In addition, the incorporation of an amphiphilic substance increases the porosity and water penetration within the polymers which increase its degradation rate. Hence, the release profiles iii. Dendrimers Dendrimers consists of highly ordered and branched macromolecules with tree-like structural design capable of loading both of hydrophilic and lipophilic molecules [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Velino | Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis[END_REF]. The high area-to-size ratio provided by the highly branched nature allows different molecules to be incorporate into the structure of dendrimer [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF][START_REF] Velino | Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis[END_REF]. The size of dendrimers generally ranges from 4 to 20 nm [START_REF] Kuzmov | Nanotechnology approaches for inhalation treatment of lung diseases[END_REF]. With this size, dendrimers typically diffuse rapidly into the bloodstream, reducing lung retention [START_REF] Kuzmov | Nanotechnology approaches for inhalation treatment of lung diseases[END_REF]. However, high molecular weight can increase their half-life in the lung [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF]. Many types of dendrimers

2019).

Two different studies have documented that the use of combined therapy of dendrimers with fluoroquinolones ATBs including levofloxacin, nadifloxacin and prulifloxacin enhanced significantly their efficacy against Escherichia coli planktonically grown [START_REF] Cheng | Polyamidoamine (PAMAM) dendrimers as biocompatible carriers of quinolone antimicrobials: An in vitro study[END_REF][START_REF] Wrońska | Synergistic Effects of Anionic/Cationic Dendrimers and Levofloxacin on Antibacterial Activities[END_REF]). On the contrary, it has been reported that the use of combined therapy of TOB with dendrimers (PAMAM-NH2 or PAMAM-OH) did not reduce the MIC values against PA and A. baumanii planktonically grown compared to TOB [START_REF] Winnicka | The Effect of PAMAM Dendrimers on the Antibacterial Activity of Antibiotics with Different Water Solubility[END_REF]. But until to date, there are no studies that investigate the influence of incorporating of ATBs into dendrimers for treating chronic lung infection.

Dendrimers were used to encapsulate others molecules in order to treat chronic lung infection such as cysteamine, an anti-oxydant FDA approved and known for its antibiofilm and demonstrated that the dendrimer-encapsulating cysteamine enhanced significantly its bactericidal activity against PA and displayed also an in vitro anti-mucolytic activity against porcine mucus [START_REF] Faraj | Novel cystamine-core dendrimer-formulation rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection by augmenting autophagy[END_REF].

Two studies have been documented about the efficacy of two peptides dendrimers (G3KL and TNS18) in the treatment of PA biofilm infection. In a first study conducted by Pompilio et al. the activity of dendrimers alone was compared to free TOB. Dendrimers displayed an MIC value of 8 μg/mL against PA planktonically grown while TOB MIC was 1 μg/mL. However, their antibiofilm biofilm activity measured on biofilm grown on microtitre plate was similar [START_REF] Pompilio | Peptide dendrimers as "lead compounds" for the treatment of chronic lung infections by Pseudomonas aeruginosa in cystic fibrosis patients: in vitro and in vivo studies[END_REF]. In other studies, these dendrimers have been reported to destroy the morphological structure and thickness of the PA biofilm in dose-dependent manner [START_REF] Han | Peptide dendrimers G3KL and TNS18 inhibit Pseudomonas aeruginosa biofilms[END_REF]. Therefore, G3KL and TNS18 are two peptide dendrimers that could be a promising novel antimicrobial agents for clinical treatment of PA chronic lung infection [START_REF] Han | Peptide dendrimers G3KL and TNS18 inhibit Pseudomonas aeruginosa biofilms[END_REF].

Those studies have demonstrated the capacity of dendrimers alone, without being incorporated with antimicrobials agents, to show the antibacterial and anti-biofilm formation activity [START_REF] Calabretta | Antibacterial Activities of Poly(amidoamine) Dendrimers Terminated with Amino and Poly(ethylene glycol) Groups[END_REF][START_REF] Johansson | Inhibition and Dispersion of Pseudomonas aeruginosa Biofilms by Glycopeptide Dendrimers Targeting the Fucose-Specific Lectin LecB[END_REF][START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF]. However, the incorporating of antimicrobials agents into dendrimers may offer an additional activity or a synergistic action in targeting and treating the chronic PA lung infection.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

3) Inorganic nanoparticles

Inorganic NPs also known as metallic NPs are mainly made of gold, silver, or copper (Au, Ag, or Cu) that display significant antimicrobial activity [START_REF] Miller | Inorganic nanoparticles engineered to attack bacteria[END_REF][START_REF] Rad | The Effect of Ampicillin and Gentamicin Conjugated with Gold Nanoparticles on the Formation of Biofilms in Pseudomonas aeruginosa[END_REF][START_REF] Yeh | Nano-Based Drug Delivery or Targeting to Eradicate Bacteria for Infection Mitigation: A Review of Recent Advances[END_REF][START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF]. However, their use can be limited due to their toxicity reported against other biologicals systems such human cells [START_REF] Miller | Inorganic nanoparticles engineered to attack bacteria[END_REF]. The preparation of these particles is generally based on the reduction of a metal salt, and their characteristics may be changed depending on different reaction conditions including pH, temperature, reaction time and concentration of the reducing agent [START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF]. The reducing agent often also acts as a linker between NPs surface and the drug and require appropriate selection [START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF]. The selection of the reducing agent also affects the potentiality of loading and releasing the drug, the antimicrobial drug activity and the probability of aggregating NPs (Zaidi et al. larger size in treating planktonic PA that MIC value decreases significantly by 32 folds with decreasing the size from 100 nm to 10 nm. In addition, Ag-NPs showed a reduction of PA biofilm mass using peg lids model also depending on the size of particles. Small size of 10-nm and 20-nm could reduce significantly the biomass by 2 and 4 folds, respectively, whereas NPs with size of 100 nm did not exhibit any antibiofilm efficacy [START_REF] Habash | Potentiation of Tobramycin by Silver Nanoparticles against Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Habash | Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms[END_REF]. Moreover, the combination of Ag-NPs with ATBs showed an in vitro synergistic efficacy of ATBs against PA biofilms grown on pegs lid. Firstly, Ag-NPs with small size of 10 and 20 nm combined with aztreonam at low concentration could reduce more significantly the biomass than with 40 nm and 60 nm, while the use of ATBs alone at high concentration (up to several hundred times MIC) do not reduce the biofilm biomass with aztreonam. Combined Ag-NPs with ATBs showed a synergistic bactericidal activity against PA biofilm promoted by the penetration of small NPs in the biofilm matrix enhancing the ATBs efficacy against PA biofilm.

2017). The potential impact of inorganic

Thus, Ag-NPs could serve as a promising potential strategy for treating chronic PA infection [START_REF] Habash | Potentiation of Tobramycin by Silver Nanoparticles against Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Habash | Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms[END_REF].

Another type of metal NP recognized for its antimicrobial activity is the gold nanoparticles (Au-NPs) [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF]. Several studies investigated the efficacy of Au-NPs in treating bacterial infections. Many studies have shown that Au-NP could improve the efficacy of ATBs against PA infections in CF patients. In this regard, a previous study has documented a high interaction between Au-NPs and aminoglycosides (streptomycin, gentamycin and neomycin)

and remarkably demonstrated a binding by chelation of aminoglycosides to Au-NPs, with an initial size ranging between 15-20 nm. Au-NPs conjugates aminoglycosides showed an in vitro antibacterial activity, greater than the free form against various strains of gram-negative and gram-positive bacteria (Nirmala Grace and Pandian 2007). In another study, other aminoglycosides (ampicillin and gentamycin) were conjugated with Au-NPs and produced particles with a size of 10 nm. The results demonstrated that gentamycin NPs reduced the in vitro PA biofilm formation by two folds on microtiter plate compared to the free form, while the ampicillin showed similar anti-biofilm activity to free form [START_REF] Rad | The Effect of Ampicillin and Gentamicin Conjugated with Gold Nanoparticles on the Formation of Biofilms in Pseudomonas aeruginosa[END_REF].

Other metallic NPs were reported to have an antimicrobial activity against PA biofilms including Iron-NPs, Cu-NPs and zinc-NPs [START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF]. For example, iron-oxide-NPs have been recently assessed alone or conjugated to TOB (alginate-capped iron-oxide NP-TOB) on an in vitro PA biofilm grown on agar plate. Results showed that Iron-oxide NPs alone could inhibit the established biofilm on agar plate at a concentration two times lower than NPs capped with alginate alone or conjugated to TOB, while the others (NPs capped with PEG, and free TOB) did not exhibit any inhibition at the same concentration. This may suggest that the capping agent could influence the activity of particles against PA biofilm, particularly alginate negatively charged which could promote the diffusion of particles through biofilm barrier also formed by alginate [START_REF] Armijo | Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms[END_REF].

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Inorganic nanoparticles are therefore promising potential agents that can be used to suppress bacterial biofilm, especially by developing their use as nanocarriers to target the infection site, and their very small size that promotes their use in pulmonary delivery route. [START_REF] Seabra | Nanotoxicology of Metal Oxide Nanoparticles[END_REF][START_REF] Vallet-Regí | Nanomaterials as Promising Alternative in the Infection Treatment[END_REF][START_REF] Yeh | Nano-Based Drug Delivery or Targeting to Eradicate Bacteria for Infection Mitigation: A Review of Recent Advances[END_REF][START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF] In general, low drug loading contents is a common drawback of all formulations, which is an issue for ATBs typically administered with high dose (mg of ATBs), such as TOB dry powder: Podhaler 112 mg 4 capsules [START_REF] Shteinberg | Use of Inhaled Tobramycin in Cystic Fibrosis[END_REF], requiring large quantities of formulation including the excipients to be administered in order to achieve the desired dose. And here we question about the excipients that will be automatically given also in high quantity (is it biocompatible, toxic, accumulate in lung or degrades quickly …)

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires b. Others strategies used for antibiotic delivery 1) Bacteriophages

Bacteriophages are viruses that infect and kill specific bacteria without affecting other microbiota [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF] and are intended to be used alone or in combination to treat bacterial respiratory infection [START_REF] Bodier-Montagutelli | Inhaled phage therapy: a promising and challenging approach to treat bacterial respiratory infections[END_REF][START_REF] Domingo-Calap | Bacteriophages: Protagonists of a Post-Antibiotic Era[END_REF][START_REF] Principi | Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections[END_REF]. Many studies showed that bacteriophages could be efficient in treating PA biofilm, particularly with CF patients [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Pei | Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes[END_REF][START_REF] Sharma | Pseudomonas aeruginosa biofilm: Potential therapeutic targets[END_REF]. Some phages can lyse bacteria present in biofilm through increasing the diffusion into biofilm matrix by producing polysaccharides DNases or peptidases such as alginase that disperse the EPS matrix allowing the access to bacteria [START_REF] Donlan | Preventing biofilms of clinically relevant organisms using bacteriophage[END_REF][START_REF] Glonti | Bacteriophage-derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates of Pseudomonas aeruginosa[END_REF][START_REF] Morello | Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention[END_REF][START_REF] Pei | Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes[END_REF][START_REF] Sharma | Pseudomonas aeruginosa biofilm: Potential therapeutic targets[END_REF].

In this context, several in vitro studies have shown that bacteriophages diffuse through alginate and EPS to reach PA in biofilm [START_REF] Sharma | Pseudomonas aeruginosa biofilm: Potential therapeutic targets[END_REF][START_REF] Velino | Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis[END_REF] [START_REF] Donlan | Preventing biofilms of clinically relevant organisms using bacteriophage[END_REF][START_REF] Hanlon | Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms[END_REF]. Hanlon et al. have documented that bacteriophages-treated alginate showed a decrease in molecular weight compared to untreated alginate , which would explain the increased phages diffusion through pseudomonal biofilm by reducing alginate viscosity [START_REF] Hanlon | Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms[END_REF]. Moreover, phages can also disperse the biofilm by encoding degradation of QS molecules. An engineered T7 bacteriophage encoding AHL lactonase has been reported to inhibit the mixed strains biofilm formation, composed of PA and Escherichia coli [START_REF] Pei | Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes[END_REF].

Bacteriophages can be genetically modified in order to display peptide that help to increase the diffuse through mucus. In this respect, Leal et al. showed that modified bacteriophages could enhance the transmucosal permeability of drugs, particularly diverse peptides have been genetically engineered in phages genome to display peptides on phage surface and demonstrated that ISLPSPT peptide (having mucin-mimicking sequences) improved the in vitro diffusion of phages by 2.6 times through CF mucus model and decreased their binding affinities to mucin as compared with the control [START_REF] Leal | Mucuspenetrating phage-displayed peptides for improved transport across a mucus-like model[END_REF]. Furthermore, bacteriophages have been widely examined alone or in combination therapy to assess their efficiency in preventing or treating PA lung infection using pulmonary routes. In this aspect, Morello et al have found that the intranasal administration of novel bacteriophage (P3-CHA or PAK-P3) at high dose induced 90% of survival rate in mice infected by planktonic PA in the lungs. While, the intranasal administration of P3-CHA at same concentration as preventive treatment (given before 4 days) showed only 20% of survival rate but the increased of phages administrated dose by 10 folds cause a totally survival of mice [START_REF] Morello | Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention[END_REF] Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires [START_REF] Sharma | Pseudomonas aeruginosa biofilm: Potential therapeutic targets[END_REF]. Another in vivo study showed that the use of bacteriophage (PAK-P1) has been successful in preventing lung infection when phages were administered 24H before bacterial infection combating PA lung infection [START_REF] Debarbieux | Bacteriophages Can Treat and Prevent Pseudomonas aeruginosa Lung Infections[END_REF].

Besides, the use of two bacteriophages could reduce the risk of developing resistant colonies. Alemayehu et al. have observed a decreasing of 3-4 log CFU of PA in an in vitro PA biofilm developed on epithelial cell monolayer and in an in vivo acute lung infection, using two bacteriophages (φMR299-2 and φNH-4). In addition, phages could replicate in the presence of targeted bacteria, which has been shown as an increase by 100 folds over a 24-h period [START_REF] Alemayehu | Bacteriophages ϕMR299-2 and ϕNH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells[END_REF][START_REF] Sharma | Pseudomonas aeruginosa biofilm: Potential therapeutic targets[END_REF] .

Few studies demonstrated the efficacy of combining ATBs with phages therapy which could show a promising synergistic effect regarding eradication of biofilm. In this context, Chaudhry et al. have demonstrated a synergistic effect between phages and certain ATBs (ceftazidime, CIP and TOB) in the eradication of PA biofilm using biotic biofilm models(monolayers epithelial cells) in comparison to the use of ATBs alone while gentamicin and COL showed no synergy with bacteriophages [START_REF] Chaudhry | Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms[END_REF]). Moreover, it has been recently documented that the combination of phages with CIP at sub-MIC concentration decreased the number of cells by ∼6 log in an in vitro PA flow-cell biofilms model, as compared to the control [START_REF] Henriksen | aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination[END_REF]. Other studies have further tested the combined therapies of ATBs with phages on other species using abiotic biofilm model. For instance, it has also been reported that the combination of amoxicillin and bacteriophages significantly reduced the CFU of Klebsiella pneumonia by 5.5 log as compared with ATBs alone or phages alone, which was only reduced by 3 log and 3.5 log respectively [START_REF] Bedi | Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of Klebsiella pneumoniae B5055[END_REF]. Similarly, the combination of phage SAP-26 and rifampicin has been reported to reduce 5 log CFU of Staphylococcus aureus as compared to phage or ATBs alone that kill 3 log and 4 log respectively [START_REF] Rahman | Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin[END_REF].

The administration of bacteriophages via pulmonary routes has been examined to target and treat chronic bacterial lung infection. Indeed, the use of dry powder inhaler containing bacteriophages for pseudomonal lung treatment has been already investigated in several studies [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Golshahi | In vitro lung delivery of bacteriophages KS4-M and ΦKZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis: Lung delivery of phage powders[END_REF][START_REF] Matinkhoo | Spray-dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections[END_REF], and was found to be feasible [START_REF] Matinkhoo | Spray-dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections[END_REF]. In this context, Puapermpoonsiri et al. have developed a dry powder formulation of bacteriophages encapsulated into a biodegradable polyester microsphere, that demonstrated an in vitro biphasic release (firstly burst release of 55-63% within 30 min followed by sustained release till around 6 H) [START_REF] Puapermpoonsiri | A freeze-dried formulation of bacteriophage encapsulated in biodegradable microspheres[END_REF].

Two studies have investigated the encapsulation of bacteriophages into an inhaler powder, and speculated that bacteriophages could be effective in treating chronic pseudomonal infection in CF patient [START_REF] Golshahi | In vitro lung delivery of bacteriophages KS4-M and ΦKZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis: Lung delivery of phage powders[END_REF][START_REF] Matinkhoo | Spray-dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections[END_REF]. Indeed, Matinkhoo et al. have encapsulated bacteriophages alone (KS4-M, KS14) or mixed (KZ/D3 and KZ/D3/KS4-Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires M) into an inhaler powder with respirable size which showed an in vitro distribution of bacteriophages in the lungs [START_REF] Matinkhoo | Spray-dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections[END_REF]. Besides, Golshani et al. have formulated an inhalable powder containing two bacteriophages (KS4-M and FKZ) with fine particles, which was aerosolized via dry powder inhaler device, and demonstrated an in vitro controlled release of bacteriophages deeply diffusing into the lung airway and reaching PA infection site [START_REF] Golshahi | In vitro lung delivery of bacteriophages KS4-M and ΦKZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis: Lung delivery of phage powders[END_REF]. In a study conducted by Sahota et al., seven different aerosolized bacteriophages showed an in vitro ability to lyse a high percentage (up to 98 %) of many PA strains [START_REF] Sahota | Bacteriophage Delivery by Nebulization and Efficacy Against Phenotypically Diverse Pseudomonas aeruginosa from Cystic Fibrosis Patients[END_REF]. Thus, speculating that bacteriophages would be effective in treating chronic pseudomonal infection in CF patient.

From the above, bacteriophages have been demonstrated to display several effects including antimicrobial as well as synergistic activities and improving the delivery of molecules through mucus and biofilm matrix. In this context, the use of aerosolized bacteriophages could be regarded as future potential candidates for DDS technologies to treat PA chronic lung infection, yet further research is required towards a more profound assessment of the feasibility and safety of introducing bacteriophage therapy in combating human lung infections.

2) PEGylation of antibiotics

PEGylation is a strategy proposed to enhance the delivery of therapeutic drugs and based on conjugating the therapeutic agent, such as cationic ATB, with polyethylene glycol (PEG) [START_REF] Suk | PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[END_REF]. PEG is FDA approved and commonly used in DDS, it is recognized for being non-toxic, non-immunogenic, non-antigenic, highly water soluble (hydrophilic) [START_REF] Suk | PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[END_REF][START_REF] Veronese | PEGylation, successful approach to drug delivery[END_REF]. Several studies reported that PEGylation of drugs can decrease their clearance and improve their delivery to the desired site [START_REF] Joseph | Peptide and Protein-Based Therapeutic Agents *[END_REF]. In addition, an increase in PEG size has been reported to increase the retention time of drugs in the lungs.

For exemple, the intratracheal administration in rat lungs of PEG with small size (< 2 kDa) were cleared within 48H while the larger size (> 5 kDa) remained up to 7 days [START_REF] Gursahani | Absorption of Polyethylene Glycol (PEG) Polymers: The Effect of PEG Size on Permeability[END_REF]. PEGylation was used to modify directly ATBs and increase their efficacy against PA biofilm [START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF]. PEGylation of cationic ATBs such as polymyxins and aminoglycosides, constitutes a strategy to resolve the problem of one of the biofilm resistance mechanisms [START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF]. PEG could shield the positive charges of ATBs, thus reducing the electrostatic interaction with mucus and biofilm matrix components and promoting the diffusion through mucus and matrix biofilm components. This may allow deeper region of biofilm to be reached consequently enhancing the efficacy of the ATBs (Bahamondez-Canas et al. 2018;[START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF][START_REF] Witten | Selective permeability of mucus barriers[END_REF].

In this regard, Smyth et al. have developed a PEGylated TOB (TOB-PEG) to enhance the therapeutic aspect of TOB against PA biofilm [START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF]. TOB-PEG was formulated by a chemical conjugation, at specific site 6' amine group of TOB, between TOB and PEG.

EXPERIMENTAL WORK

Abstract

Chronic lung infections are mostly associated with biofilm-growing bacteria and P. aeruginosa (PA) is the predominant pathogen responsible for the development of biofilms in patients with chronic inflammatory disease such as cystic fibrosis. Within biofilms, embedded bacteria are more difficult to eradicate than planktonic bacteria and high antibiotic concentrations are generally required. Inhaled antibiotics are used to treat bacterial chronic infections, tobramycin (TOB) and colistin (COL) are among the first choice of inhaled treatments. However, these antibiotics (ATBs) are positively charged and their effectiveness is limited against biofilms. An alternative to get around this problem is to use inhaled drug delivery systems to improve the ATBs efficacy by increasing their penetration into the biofilm matrix. Lipid nanoparticles (LNP), which are nanocarriers capable of encapsulating and delivering both hydrophilic and lipophilic drugs, seem to be interesting candidates. In our study, the choice of fatty acid (FA) as synthetic adjuvants to LNP was evaluated as well as the addition of anti-biofilm non-ATB drugs as farnesol (FAR). The aim of this study was to develop a new delivery system by encapsulating cationic antibiotics (COL and TOB) into LNP-loaded with adjuvants such as FA (lauric acid (LA) and decanoic acid (DA)) and/or FAR, and to assess their activities against PA and A.

baumannii.

LNPs were formulated by nanoemulsification by phase inversion. Various adjuvants (LA, DA, FAR) were tested to improve the ATBs (COL) encapsulation efficiency and increase their efficacy. Size and zeta potential of these LNPs were studied to characterize the formulations.

The efficacy of LNPs was firstly evaluated on minimal inhibition concentrations (MIC) of planktonic PA and A. baumannii. In second time, the selected LNPs were evaluated on PA and A. baumannii adherent biofilms by measuring the enzymatic activity and the total biomass of biofilms. Finally, same LNPs were tested on a PA embedded in alginate beads biofilm model and their efficacies were evaluated by performing bioluminescence kinetics for 40 hrs, followed by a measurement of total bacterial concentrations (log10 CFU/ml). An inhibitory Emax model was used to describe the bacterial counts as a function of the concentrations of COL.

Results showed that Blank-LNP did not interact with cationic ATBs. Loading FA (LA or DA) into the LNP conferred negative charge to the particles, allowing the interaction with cationic ATBs by producing HIP. Then, Free COL was encapsulated into DA and/or not FAR -loaded LNP via HIP producing LNP-DA/COL, LNP-DA/FAR/COL. It was shown that less COL was present at the LNP-DA surface when it was added before the formulation process than after.

Combined therapy of adjuvants (DA and/or FAR) with COL, in encapsulated form or not, significantly increased the sensitivity of resistant strains of A. baumanii to COL. Moreover, encapsulated formulations of COL showed an in vitro effect in completely suppressing the enzymatic activity within A. baumannii biofilm which was not observed with free COL. Also,

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Introduction

Chronic lung infections are mostly associated with biofilm-growing bacteria, which are characterized by persistence of inflammation and tissue damage [START_REF] Bjarnsholt | The role of bacterial biofilms in chronic infections[END_REF]2], as well as increasing of mortality risk [START_REF] González | Chronic biofilm-based infections: skewing of the immune response[END_REF]. Biofilms are known as matrix-enclosed clusters of bacteria containing anionic polymers such as polysaccharides (alginate), eDNA and proteins [START_REF] Eze | Acinetobacter baumannii biofilms: effects of physicochemical factors, virulence, antibiotic resistance determinants, gene regulation, and future antimicrobial treatments[END_REF][START_REF] Maurice | Pseudomonas aeruginosa Biofilms: Host Response and Clinical Implications in Lung Infections[END_REF]. P. aeruginosa is the predominant pathogen responsible for the development of biofilms dispersed in the mucus of patients with chronic inflammatory disease such as cystic fibrosis (CF) and bronchiectasis [START_REF] Boisvert | Microbial Biofilms in Pulmonary and Critical Care Diseases[END_REF][START_REF] Macia | Antimicrobial susceptibility testing in biofilmgrowing bacteria[END_REF][START_REF] Pastor | Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis[END_REF]. Within biofilms, the embedded bacteria are generally more difficult to eradicate and generally require high antibiotic concentrations up to 1000-fold greater than for planktonic form [START_REF] Macia | Antimicrobial susceptibility testing in biofilmgrowing bacteria[END_REF][START_REF] Ciofu | Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics[END_REF]10].

Inhaled antibiotics have been used extensively in treating bacterial chronic infections in CF patients and ventilator associated pneumonia (VAP) patients [START_REF] Garcia-Contreras | Inhalation drug delivery devices: technology update[END_REF][START_REF] Maselli | Inhaled Antibiotic Therapy in Chronic Respiratory Diseases[END_REF][13][START_REF] Restrepo | Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: A European consensus[END_REF]. Both tobramycin (TOB)

and colistin (COL) are the first choice of inhaled treatments for chronic lung infections, especially with CF patients [START_REF] Pastor | Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis[END_REF][START_REF]Investigated therapies specific to biofilms[END_REF]. However, these antibiotics are positively charged and their effectiveness is limited against these biofilm infections. This limitation in the efficacy is due to their restricted penetration through biofilm matrix, in which antibiotics are sequestered with the anionic polymers of matrix at the periphery of bacterial biofilm [START_REF] Ciofu | Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics[END_REF].

Lipid nanoparticles (LNP) are nanocarriers capable of encapsulating and delivering both of hydrophilic and lipophilic drugs. The nano-encapsulation of antibiotics into LNP constitutes a promising drug delivery system that may prevent the interaction of antibiotics with anionic polymers matrix and overcome the problem of restricted penetration associated with biofilms [START_REF] Pastor | Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis[END_REF]. LNP can encapsulate antibiotics in mono or combined therapy with adjuvants having antimicrobial or/and antibiofilm activity in order to increase the activity of current treatment and produce a synergistic action to treat bacterial chronic lung infections.

Among these adjuvants, fatty acid (FA) are lipophilic molecules known to have an antimicrobial and antibiofilm activities [16][17][START_REF] Nicol | Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii[END_REF][START_REF] Wenderska | Palmitoyl-dl-Carnitine is a Multitarget Inhibitor of Pseudomonas aeruginosa Biofilm Development[END_REF], especially lauric acid (LA) and decanoic acid (DA) activity [16,20]. Moreover, these FAs may enhance the encapsulation of antibiotics, having opposite charge, by producing a reaction of ions pairings named hydrophobic ions pairing (HIP) [START_REF] Maha | DELIVERY SYSTEM: A REVIEW[END_REF][START_REF] Ristroph | Hydrophobic ion pairing: encapsulating small molecules, peptides, and proteins into nanocarriers[END_REF]. Additionally, Farnesol (FAR), is a sesquiterpene alcohol that was reported for its antimicrobial activity [START_REF] Gomes | Effect of Farnesol on Planktonic and Biofilm Cells of Staphylococcus epidermidis[END_REF][START_REF] Kim | Potentiation of the activity of β-lactam antibiotics by farnesol and its derivatives[END_REF] and anti-biofilm activity against P. aeruginosa [START_REF] Cugini | Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa[END_REF],

but is difficult to administer in human due high hydrophobicity. Formulating this molecule within LNP could facilitate its administration.

Consequently, the aim of this study was to develop a new delivery system by encapsulating cationic antibiotics (COL and TOB) into LNP-loaded with adjuvants such as FA and/or FAR and to assess their activity against P. aeruginosa and A. baumannii.

105

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Materials and Methods

Reagents and materials and antibiotics

Tobramycin (TOB) (Lot: LRAA5713), Colistin sulphate (COL) (Lot: SLBV1747), farnesol (FAR) (Lot: MKCC1784), decanoic acid (DA) (Lot: MKCG6788), lauric acid (LA) (Lot:

MKBP4726V), butyric acid (BA) (Lot: STBH8638), cation-adjusted Mueller Hinton broth (MHB), and seaweed alginate were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France).

Labrafac® WL 1349 (caprylic/capric acid triglycerides) was a gift from Gattefossé (Saint-Priest, France). Solutol® HS 15 also called Kolliphor® HS 15, a mixture of free polyethylene glycol 660 (30%) and various esters formed between polyethylene glycol 660 and 12-hydroxystearic acid, was donated by BASF (BASF SE, Ludwigshafen, Germany). Phospholipon ® HS90 (an hydrogenated lecithin) was a gift from Lipoid (Grasse, France)

Development of cationic antibiotics-loaded lipid nanoparticles (LNPs)

Synthesis of Blank-LNP

The preparation of LNP was based on the method previously described by Anton et al. [START_REF] Anton | Nano-emulsions and nanocapsules by the PIT method: An investigation on the role of the temperature cycling on the emulsion phase inversion[END_REF].

First, an aqueous phase formed by NaCl, MilliQ® water, and the surfactant Solutol® HS15 was mixed with an oily phase composed of triglycerides (Labrafac®) and hydrogenated Lecithin (Phospholipon®) to obtain an oil-in-water (O/W) emulsion. This O/W emulsion was heated to reach a temperature of 85°C, which corresponds to the phase inversion temperature (PIT), in order to obtain water-in-oil (W/O) emulsion. The W/O emulsion was cooled to 55°C to return to an O/W emulsion. Two more of these temperature cycles were performed, and then icy water (~4°C) was added to produce a stable suspension of LNP (Blank-LNP). Finally, the pH value of the LNP suspensions was adjusted to 7 and the formulations were stored at 4°C. The composition of Blank-LNPs is presented in Table 1.

Characteristics of LNPs

The size and ζ-potential (ZP) distributions of the LNPs were determined at 30°C by dynamic light scattering measurements and laser Doppler electrophoresis, respectively, using a Nano ZS Zetasizer (Malvern Instruments Ltd. Orsay, France). LNP suspensions were diluted (1:5 v/v) in a mix of dipotassium phosphate buffer (0.01 M; pH=6.8) and NaCl (0.01M). Size and ZP distributions were analyzed in triplicate. The mean values of ZP and the median values of the volume-weighted size were recorded.

Study of the interaction of cationic ATBs with the interface of Blank-LNP

The suspension of blank LNP was titrated with cationic ATBs (TOB and COL) by measuring their ZP for different ATB concentrations in order to study their interactions with the LNP surface. Each sample was prepared by mixing 200 µL of LNP suspension with 400 µL of dipotassium phosphate buffer (0.01 M; pH = 6.8). Then, 400 µL of a mix solution containing

ATB and NaCl at various concentrations was added to obtain the final desired ATB concentration and an ionic strength of 0.01. The TOB concentrations tested were 10 -4 M; 5.10 - 4 M; 10 -3 M; 2.10 -3 M; 3.5.10 -3 M and 5.10 -3 M. For COL, the concentrations tested were 10 -4 M; 2.5.10 -4 M; 5.10 -4 M; 7.5.10 -4 M, 10 -3 M; 2.10 -3 M; 4.10 -3 M and 5.10 -3 M. The samples were then analyzed directly using the Nano ZS Zetasizer (Malvern Instruments Ltd. Orsay, France).

Control of the surface electrical charge of LNPs

To negatively charge the surface of LNPs, various fatty acids (FA) such as lauric acid (LA), decanoic acid (DA), and butyric acid (BA) were added to the oily phase to the formulation. The FA-modified LNPs were formulated using the same method as for the Blank LNP. The effect of the amount of FA added on the size and ZP of the LNPs was evaluated using the Nano ZS Zetasizer (Malvern Instruments Ltd. Orsay, France) as described previously. The suspensions of LNP modified by the FA (LNP-LA, LNP-DA or BA-LNP) were then further investigated by measuring the effect of pH (3, 5, 6, 6.8, 8) on their ZP.

Study of the interaction of COL and TOB with the interface of LNP-LA and LNP-DA

The study of the interactions between the LNPs modified with LA or DA (LNP-LA and LNP-DA) and the TOB or the COL was carried out according to the same procedure as for the titration of blank LNP explained in 2.2.3. To further assess the interaction of COL with LNP-DA, a study of the influence of pH (3, 5, 6, 6.8 and 8) on the ZP of LNP-DA when titrated with COL was carried out.

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Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Synthesis of the formulation of COL loaded into DA-LNP

Two different methods were evaluated for incorporating COL to LNP-Das. In the first one, COL was added after the synthesis of LNP-DA, as in the titration process. In the second method (Fig. 1), the aqueous phase (composed of NaCl, MilliQ® water, and Solutol HS15) was added to the oily phase (composed of Labrafac®, Phospholipon, and DA) under stirring to obtain an O/W emulsion. The pH was then adjusted to 7 and the O/W emulsion was heated to 85°C to obtain the W/O emulsion. At this point, 1250 µL of COL solution in water (15 g/L) was added to the W/O emulsion (Figure 1). Thus, the DA/COL molar ratio was about 115 and the theoretical final COL concentration in the final suspension was 1.15 g/L (10 -3 M). The W/O emulsion was then cooled to 55°C and two more temperature cycles were performed. At the end, cold water (4°C) was added to the formulation to stabilize the LNP-DA/COL particles obtained. Farnesol, a hydrophobic acyclic sesquiterpene alcohol that has shown an antibiofilm and adjuvant property with COL was also incorporated in LNPs. Hence, additional LNP batches were prepared in which FAR was added to the oily phase before the formulation step to prepared LNP-FAR, LNP-DA/FAR, and LNP-DA/COL/FAR. FA concentration in the final suspension was around 33 g/L and the FAR/COL molar ratio was about 150.

Bacterial strain and culture media

Two bacterial strains were used during this study. A P. aeruginosa (PAO1-LUXCDEBA) strain, having a COL MIC of 1 mg/L, made bioluminescent by the chromosomal incorporation of the luxCDABE operon, which was offered by Professor Patrick Plesiat (CHU de Besançon, France). A clinical isolate of Acinetobacter baumannii AB122 (MTQH00000000), resistant to COL (MIC of 32 mg/L) [START_REF] Jaidane | Genomic analysis of in vivo acquired resistance to colistin and rifampicin in Acinetobacter baumannii[END_REF] was given by Professor Thierry Naas (CHU de Bicêtre, France).

Experiments of this study were conducted using two growth media for bacterial culture: the cation-adjusted Mueller-Hinton Broth "MHB" (Sigma-Aldrich, France), and an Artificial Sputum Medium "ASM" prepared according to the method defined by Sriramulu Diraviam Dinesh (Sriramulu Diraviam Dinesh 2010) with some modifications. A liter of ASM was prepared first by dissolving separately in 150 mL of MiliQ® water, 5 g of mucin from pig stomach mucosa (Sigma-Aldrich, France), 4 g of low molecular-weight salmon sperm DNA (Sigma-Aldrich, France), 5 g of casein hydrolysate (Fluka, Sigma-Aldrich, France), 5 g of NaCl (Sigma-Aldrich, France), 2.2 g of KCl (Sigma-Aldrich, France), 1.81 g of Trizma base (Sigma-Aldrich, France), and 5.9 mg of diethylene triamine pentaacetic acid (DTPA) (Sigma-Aldrich, France). The pH was then adjusted at 7.0 and the medium was autoclaved at 110 o C for 15 minutes. After cooling, 5 ml of egg yolk emulsion (Sigma-Aldrich, France) was added under sterile conditions and then the ASM media was stored at 4 °C for one month.

MIC determination

Bacterial suspensions (PAO1 or AB122) were grown at 37 °C overnight in MHB, and then their optical densities at 600 nm (OD600 nm) were adjusted to 0.6. The suspensions were then diluted 100 times in MHB and 100 μL were dispensed in 96-well plates. Next, 100 μL of COL solution or COL-LNP suspensions were added to obtain final COL concentrations ranging from 64 to 0.125 μg/mL for PAO1 and from 256 to 0.5 μg/mL for AB122. The plates were incubated at 37 °C in an orbital shaker for 18 h-24h and the OD600 was read using the Infinite® M200 PRO plate reader (Tecan, France). Negative Controls made of pure broth were used to verify the absence of contamination. Assays was performed in triplicates.

Development of in vitro biofilm models

Bacterial inoculum

Before each experiment, the bacterial strain (PAO1 or AB122) was isolated on MH agar incubated for 24 hrs at 37 °C. Then, 10 ml of the growth medium (MHB or ASM) which will be used for the rest of the experiments, were inoculated with an isolated colony and incubated overnight at 37 ° C in an orbital shaker. After incubation, the obtained bacterial suspension 109 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires was washed twice with PBS by centrifugation, and dispersed in the growth medium (MHB or ASM) in order to obtain OD600=0.6.

In vitro efficacy test on adherent bacterial biofilms (PAO1 and AB122)

Development of attached bacterial biofilm

Bacterial biofilm was prepared by the method described by Tewes et al. [START_REF] Tewes | Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In vivo -Like Characteristics[END_REF] with slight modifications. Bacterial suspensions in MHB having an OD600=0.6 were diluted 10 times in MHB. Then, 100 μL was distributed in a white, high binding, 96-well microplate (Greiner Bio-One, ref. 655074) and incubated at 37 °C for 48 hrs. The supernatants were then removed and the wells were washed three times with 150 μL of PBS. Next, 200 μL of free COL or formulated as LNP dispersed in MHB with concentrations ranging from 256 to 0.5 mg/L for AB122, and from 16 to 0.03 mg/L for PAO1, were introduced into the wells. The microplates were again incubated for 24 hrs at 37°C, the supernatants were then removed and the wells were washed three times with 150 μL of PBS. Finally, the enzymatic activity in the biofilms was measured using the fluorescein diacetate (FDA) test, and the total mass of the biofilms was determined using the crystal violet (CV) staining method as described below.

Determination of biofilm enzymatic activity by FDA test

The test was adapted from a method previously described by Tewes et al. [START_REF] Tewes | Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In vivo -Like Characteristics[END_REF]. A fresh FDA working solution was prepared by diluting a stock solution (10 mg/mL in acetone) 2000fold in PBS buffer (pH 7.4-0.05 M). In each well of the microplate, 200 μL of the FDA working solution was added and the plate was incubated at 37 °C in the Infinite M200 PRO plate reader (Infinite M200 PRO, Tecan, France). Fluorescence of fluorescein produced by the FDA hydrolysis was recorded every 2.5 min for 15 min (λex: 494 nm and λem: 518 nm). The rate of the linear portion of the fluorescence intensity kinetics measured for each well was normalized to the rate obtained in the absence of COL. Results were expressed as a mean ± standard deviation, n = 6. Data were analyzed using a non-parametric one-way ANOVA test (GraphPad Prism software (version 8.0.1)). P˂0.05 values were considered as statistically significant.

Evaluation of biofilm mass using crystal violet staining

For each assay, a CV working solution (0.05%) was freshly prepared by diluting a stock solution (10 mg/mL in a 1/1 v/v water/ethanol mix) 20 fold in water. After the FDA test, each well of the microplates were rinsed 2 times with PBS andfixed with 150 μL of methanol for 20-30 min. Then, biofilms were stained with 150 μL of CV working solution for 30 min. After rinsing 4-5 times with deionized water, the microplates were allowed to air-dry overnight. The next day, the CV content was solubilized in 200 μL of 30% (v/v) acetic acid in water. After adequate dilution, the CV absorbance was measured at 550 nm using a plate reader (Infinite M200 PRO, 110 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires Tecan, France). A calibration curve of CV in 30% acetic acid in water ranging from 20 to 0.1 μg/mL was used to calculate the amount of CV (µg) per well. Results were expressed as a mean ± standard deviation, n = 6. Data were analyzed using a non-parametric one-way ANOVA test (GraphPad Prism software (version 8.0.1)). P˂0.05 values were considered as statistically significant.

Effect of COL-LNP in an in vitro non-attached PAO1 biofilm model

Preparation of alginate beads loaded with P. aeruginosa strain PAO1 Firstly, a suspension of PAO1 was prepared by mixing 9 mL of autoclaved seaweed alginate (2% w/v) with 1 ml of the P. aeruginosa suspension prepared as described in 2.3.1.1 in order to obtain a concentration of 5.10 7 CFU/mL. This suspension was filled in an Eppendorf repetitive pipette (Multipette® plus), which was placed 1-2 cm above the surface of a sterilized Tris-HCl, CaCl2 solution (CaCl2 in Tris-HCl buffer solution 0.05 M, pH=7) under magnetic stirrer. Droplets of 2 μl were released to produce spherical uniform beads which were hardened for one hour in Tris-HCl, CaCl2. Then, the beads were washed two times with NaCl 0.9% by centrifugation.

Bioluminescence kinetics of PAO1 loaded in alginate beads.

In order to describe the dynamics of the effect of free and encapsulated COL against P. aeruginosa loaded in alginate beads, the kinetics of bioluminescence of PAO1 were measured in the presence of various COL concentrations. PAO1-loaded alginate beads freshly prepared were dispersed and then diluted into ASM to obtain a final bioluminescence signal of 20000-30000 RLU estimated by a plate reader (Infinite M200 PRO, Tecan, France). Then 100 μL of beads suspension was dispensed in a non-binding (to avoid adherent biofilm formation), white, 96-well microplates (Greiner bio-one, France), followed by 100 μL of free or formulated COL prepared in ASM. Final COL concentrations ranged from 0.25 to 200 times the COL MIC (MICCOL =1 mg/L). The plate was covered with a transparent, gas permeable moisture barrier membrane and then incubated at 37°C in a plate reader (Infinite M200 PRO, Tecan, France).

Luminescence was recorded every 30 min for 40 hrs. Negative controls without bacteria were used to verify the absence of contamination and positive controls without COL were utilized to assess the effect of the medium on bacterial growth.

Viable cells count after killing kinetics

After 40 hours of exposure to free or formulate COL, P. aeruginosa counts were performed using a modified version of the method proposed by Sønderholm et al. [29]. P. aeruginosa were released from alginate beads by dissolving them in one mL of a solution of NaHCO3 111 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires (0.05M) and citric acid (0.02 M). Then, the solubilized beads were serially diluted in 0.9% NaCl and 0.1 mL was plated on Mueller-Hinton agar. The plates were incubated at 37°C and formed colonies (numbers of CFU) were counted after 24, 48 and 72 hrs.

Data analysis and modelling

RStudio software version "1.2.1335" and package "ggplot2 version 3.10 were used to plot the smoothed average luminescence values against time using a Generalized Additive Model (GAM) as the smoothing method. The shaded area around the mean value curve is the 95% confidence level interval for the mean values predicted from the GAM smoothing model.

The antibacterial effect of TOB and COLI in the different conditions tested (effect of the bead size and dispersion media) was investigated by modelling the bacterial survival after 40 hours of exposure to ATB versus their concentrations using RStudio software version "1.2.1335" and the package Rstan version "2.19.3". An inhibitory Emax model (Equation 1) was used to describe the average decrease in bacterial concentrations CFU in relation with the ATB concentrations (C) expressed in the number of time the MIC:

𝐶𝐹𝑈 ̂= 𝐶𝐹𝑈 0 - (𝐶𝐹𝑈 0 -𝐶𝐹𝑈 ∞ )×𝐶 𝛾 𝐸𝐶 50
𝛾 + 𝐶 𝛾 (Equation 1)

In equation 1, CFU0 refers to the mean concentration in P. aeruginosa (CFU/ml) measured in the absence of ATB. CFU∞ refers to the lowest mean P. aeruginosa CFU value when the ATB concentration tends to infinity. EC50 is the ATB concentration needed to achieve 50% of CFU0 and  is the slope factor (Hill coefficient) that measures the sensitivity of the bacterial survival within the ATB concentration range. The predicted mean CFU concentration (𝐶𝐹𝑈 ̂)

were evaluated for each ATB concentration based on the hypothesis that individual CFU values were normally distributed around (𝐶𝐹𝑈 ̂) with a standard deviation . Parameters were evaluated using the Rstan version 2.3 by performing Bayesian data analysis and using the following weakly informative prior distributions: the log of CFU0 is uniformly distributed between 5 and 10; EC50 is normally distributed between 0 and 100; and  between 0 and 10.

Simulations were run for six chains with 1000 burn-in steps followed by 1000 Markov chain Monte Carlo (MCMC) steps.

112 The blank-LNP had a size of 65 ± 1.6 nm and a ZP of -1.1 ± 0.7 at pH 6.8 (Fig. 2). The addition of TOB at concentrations of 0.1 mM to 5 mM had no influence on the size and the ZP value of the particles, suggesting the lack of interactions between the LNP and TOB (Fig. 2A-B). The addition of COL in the same concentration range had no effect on particle size (Fig. 2C). However, a change in the ZP values, which increased from -1.65 mV to +1.61 mV, was obtained with COL (Fig. 2D). This switch in ZP value sign from negative to positive suggests the interaction of the positively charged COL with the LNP surface. Indeed, while the TOB is hydrophilic, the COL is amphiphilic and can be anchored to the surface of the LNP via its alkyl chain moiety (Fig. 5). The adsorption of COL to the surface of particles has also been shown using liposomes, for which the ZP went from -48 to + 5.2 mV after incorporation of COL [START_REF] Li | Colistin-entrapped liposomes driven by the electrostatic interaction: Mechanism of drug loading and in vivo characterization[END_REF].

Control of the surface electrical charge of LNPs.

To improve the interaction of the cationic COL and TOB with the LNP surface, various FAs, including medium carbon chain FA such as lauric acid (LA), capric or decanoic acid (DA), and the small carbon chain FA butyric acid (BA) were added to LNP formulation to negatively charge their surface. The influence of the FA concentration on the LNP size and ZP is presented in Fig. 3. The data obtained with BA were not presented as no influence of BA on LNP ZP was observed, suggesting the absence of its adsorption to the surface of LNPs.

114 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires For LA and DA, an increase in their concentration up to 20 g/L induced a sharp decrease in their mean ZP value from -1.1 mV to around -15.9 ± 2.7 mV and -16.0 ± 1.5 mV for LNP-LA and LNP-DA, respectively. In the concentration range of 0 to 20 g/L, both FAs first slightly reduced the size of the LNPs, which then increased back to the same size values as those obtained without FA (around 65 nm), for a FA concentration of 20 g/L (Fig. 3 A andC). This reduction in size can be attributed to the surfactant properties of the two FAs which adsorbed to the surface of the LNPs, reducing their ZP value and size, as also observed by Umerska et al. [START_REF] Umerska | Antibacterial action of lipid nanocapsules containing fatty acids or monoglycerides as cosurfactants[END_REF].

Increasing the concentration of the two FAs in the range of 20 to 40 g/L had less effect on decreasing the ZP value of LNPs than in the range of 0 to 20 g/L. Indeed, for a concentration of 40 g/L, the mean ZP was -22.9 ± 4.4 mV and -17.3 ± 2.3 mV for LA and DA, respectively.

However, above 20 g/L, the increase in LA concentration strongly increased the LNP size. For instance, at 40 g/L LA, the mean of the LNPs median size was 109 ± 16 nm. These results showed that a FA concentration of around 20 g/L leads to the formation of LNPs with the most negative ZP value allowing interacting with cationic ATBs, but also with a small particle size (around 65 nm) allowing its diffusion through the mesh network of polymers forming the biofilm and mucus. Based on these results, the following part of the study was performed using the LNP-DA formulation only. To further characterize the LNP-DAs, the influence of pH on their ZP was evaluated and compared with the profile obtained for the blank LNP (Fig. 4). The ZP value of the white LNPs was almost independent of pH in the tested range (pH [START_REF] González | Chronic biofilm-based infections: skewing of the immune response[END_REF][START_REF] Eze | Acinetobacter baumannii biofilms: effects of physicochemical factors, virulence, antibiotic resistance determinants, gene regulation, and future antimicrobial treatments[END_REF][START_REF] Maurice | Pseudomonas aeruginosa Biofilms: Host Response and Clinical Implications in Lung Infections[END_REF][START_REF] Boisvert | Microbial Biofilms in Pulmonary and Critical Care Diseases[END_REF][START_REF] Macia | Antimicrobial susceptibility testing in biofilmgrowing bacteria[END_REF][START_REF] Pastor | Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis[END_REF], showing the absence on their surface of ionizable groups having a pKa value between 3-8. Indeed, the only component that contains an ionizable group in blank LNP is phospholipon® HS90, a hydrogenated lecithin having a phosphate group with pKa∼1.5 [START_REF] Wu | Chitosan coatings on lecithin stabilized emulsions inhibit mycotoxin production by Fusarium pathogens[END_REF]. At the contrary, the ZP of LNP-DA was strongly dependent of pH. At pH 3, ZP of LNP-DA was positive (4.5 ± 0.5 mV) and decreased almost linearly to reach a minimal value of around -14 mV for pH 6.8.

This ZP-pH profile shows that the carboxyl group of DA, having a pKa∼4.9 is present at the LNP surface [START_REF] Barratt | Quantitative structure-activity relationships (QSARs) for skin corrosivity of organic acids, bases and phenols: Principal components and neural network analysis of extended datasets[END_REF][START_REF] Wellen | Surface pKa of octanoic, nonanoic, and decanoic fatty acids at the air-water interface: Applications to atmospheric aerosol chemistry[END_REF]. The pH 6.8 was the one allowing the highest negative pH to be obtained.

Hence, this pH was chosen as the optimal pH for COL and TOB adsorption as it allows to negatively charge the LNP-DA but is low enough compare to pKa values of the amine groups of TOB and COL to maintain these ATB positively charged. Indeed, using the pKa values found in the literature for TOB and COL, the percentage of protonated, positively charged form of each ATBs, can be estimated as a function of the pH (Fig. 5). At pH 6.8, 100% of COL molecules are positively charged and bear 5 positive charges (Fig. 6), and, on average, TOB molecules carry 4 positive charges (Fig. 6). In addition, this pH is very close to the physiological pH value found for the airway surface liquid 6.9 ± 0.1 [35], which will be of benefit for pulmonary delivery of the LNPs as a liquid aerosol. Compared to the blank LNP (Fig. 2) for which TOB did not change their ZP value, the ZP value of the LNP-DA increased hyperbolically with the concentration of TOB in the range of 0.1 mM to 5 mM indicating a saturation phenomenon. Their ZP value went from -13 ± 1.7 mV without TOB to a plateau around -3.3 ± 0.8 mV for 5 mM of TOB (Fig. 7B). Similar behavior was obtained with COL, but the increase in ZP was greater and allowed to reach a plateau and to reverse the ZP value of LNPs at + 2.6 ± 0.6 mV already from a concentration of 4 mM.

The adsorption of TOB and COL to the surface of the LNP also had an impact on their size (Fig. 7 A,C). For both ATBs, the size of the LNPs increased following a biphasic profile. For ATB concentrations allowing to obtain a ZP value lower than -5 mV (for less than 1 mM of COL and less than 2 mM of TOB), no effect on the particle size was observed that stayed around 65 ± 5 nm. From ATB concentrations that produced a LNP ZP value above than -5 mV, a sharp increase in the particle size was observed. For example, for TOB concentration of 3.5 mM, ZP was of -4.8 ± 0, 6 mV and the size increased to 93 ± 25 nm and the mean of the LNP median size increased further to 169 ± 43 nm for TOB concentration of 5 mM (Fig 7 A andB). For the same COL concentration, the mean of the LNP median size was 209 ± 225 nm (Fig. 7C). This increase in size could be due to an aggregation of the negatively charged LNP induced by the polycationic ATBs. Other reports exist on the charge reversal induced by multivalent ions [START_REF] Szilagyi | Destabilization of Colloidal Suspensions by Multivalent Ions and Polyelectrolytes: From Screening to Overcharging[END_REF].

For example, aggregation of negatively charged latex particles in the presence of positively charged linear oligoamines having 1 to 6 amine groups were observed [START_REF] Szilagyi | Charging and aggregation of negatively charged colloidal latex particles in the presence of multivalent Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires oligoamine cations[END_REF]. Low-valent oligoamines behave like inert salts and destabilize suspensions by screening their charge. At low concentrations, higher valence oligoamines triggered particle aggregation by charge neutralization. This aggregation was also shown with negatively charged particles that were more efficiently aggregated with multivalent metal cations than monovalent ones [START_REF] Oncsik | Aggregation of Negatively Charged Colloidal Particles in the Presence of Multivalent Cations[END_REF].

According to the literature, the pKa value of all amine groups of COL is approximately 10 in aqueous solution (Fig. 6) [START_REF] Liu | Colistin nanoparticle assembly by coacervate complexation with polyanionic peptides for treating drug-resistant gram-negative bacteria[END_REF]. Hence, at the pH 6.8 more than 99.9% of amine groups are protonated and the five amines of the COL molecules are positively charged (Fig 5 and6). For TOB, due to its lower pKa value, only 4 amines are positively charged at pH 6.8 and TOB is less effective in aggregating negatively charged particles (Fig. 7).

To further assess the interaction of LNP-DA with cationic ATB, the ZP-pH profiles of LNP-DA were measured in the presence of various COL concentrations (Fig. 8). At low COL concentration of 0.1 mM, no change was observed on the ZP-pH profile compare to the blank LNP. For 1 mM the ZP-pH profile was shifted toward positive values, but ZP value at pH 6.8 was still lower than 5 mV. For 5 mM of COL, all ZP values were positively charged, whatever the pH value. Compare to the different pH tested, pH 6.8 was those that induced the strongest shift in ZP value, allowing to find conditions for which COL could be adsorbed at the LNP surface but still keeping the surface charge relatively low for enhancing LNP diffusion through the biofilms and mucus. Indeed, some studies have found than highly charged particles have low penetration in those systems [START_REF] Ibaraki | The effects of surface properties of liposomes on their activity against Pseudomonas aeruginosa PAO-1 biofilm[END_REF][START_REF] Peulen | Diffusion of Nanoparticles in a Biofilm[END_REF]. Based on this result, the concentration of 1mM of COL (1.15 mg/mL) was selected to formulate the LNP. This concentration allowed to obtain stable LNP, with concentration 1000-times higher than the MIC value of COL against most of the gram-negative bacteria (generally around 1 µg/mL).

Synthesis of the formulation of COL loaded into DA-LNP. The addition of COL before or after the formulation process involving 3 heat cycles (Fig. 1) had no influence on the size of the LNP-DA that was around 60 ± 5 nm for all conditions tested (Fig. 9A). However, the incorporation of COL in the aqueous phase before the formulation process reduced the ZP value of the LNP to -10.4 ± 1.91 mV, i.e. closer to the value obtained for the blank LNP-DA (-13.1 ± 1.1 mV) than when COL was added after formulation as for the titration experiments (-7.4 ± 0.6 mV) (Fig. 9B). This result suggest that less COL is present at the LNP-DA surface when it was added before the formulation process than after. The 3 cycles of heat (55-85°C) HIP can be produced between COL and LNP-DA by the electrostatic interaction between NH3 + of COL and COO -of DA, so to achieve this interaction, it was important to keep the maximal functional groups of both of COL and FA in the ionic form.

Many studies have assessed the formation of HIP to form different nanocarriers. For instance, the polymyxin B was precipitated with oleic acid sodium salt to form water insoluble HIP that aggregated as nanoparticles [START_REF] Lu | Hydrophobic Ion Pairing of Peptide Antibiotics for Processing into Controlled Release Nanocarrier Formulations[END_REF]. COL was encapsulated into negatively charged poly(lactic acid) NPs by forming HIP with the PLA [START_REF] Tang | An innovative method for preparation of hydrophobic ion-pairing colistin entrapped poly(lactic acid) nanoparticles: Loading and release mechanism study[END_REF]. Furthermore, TOB encapsulation into solid lipid nanoparticle was allowed by forming HIP with negatively charged hexadecylphosphate [START_REF] Cavalli | Solid lipid nanoparticles (SLN) as ocular delivery system for tobramycin[END_REF],

and dioctylsulfosuccinate allowed the effective loading of TOB into PLGA-NPs [START_REF] Hill | Formulation of Antimicrobial Tobramycin Loaded PLGA Nanoparticles via Complexation with AOT[END_REF].

Farnesol (FAR) is a hydrophobic sesquiterpene alcohol (Log P= 4.84) that has been reported to prevent the formation of bacterial biofilms [START_REF] Gomes | Effect of Farnesol on Planktonic and Biofilm Cells of Staphylococcus epidermidis[END_REF][START_REF] Kim | Potentiation of the activity of β-lactam antibiotics by farnesol and its derivatives[END_REF][START_REF] Jabra-Rizk | Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility[END_REF], and to potentiate the activity of antibiotics [START_REF] Kim | Potentiation of the activity of β-lactam antibiotics by farnesol and its derivatives[END_REF][START_REF] Bandara | Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro[END_REF][START_REF] Brehm-Stecher | Sensitization of Staphylococcus aureus and Escherichia coli to antibiotics by the sesquiterpenoids nerolidol, farnesol, bisabolol, and apritone[END_REF][START_REF] Kostoulias | Impact of a Cross-Kingdom Signaling Molecule of Candida albicans on Acinetobacter baumannii Physiology[END_REF]. Indeed, FAR used in combination with ciprofloxacin in liposome showed an enhanced effectiveness in eradicating P. aeruginosa biofilm compared to monotherapy of ciprofloxacin [START_REF] Bandara | Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro[END_REF]. FAR also affected A. baumannii membrane integrity and mobility, and synergized with colistin in killing MDR A. baumannii [START_REF] Kostoulias | Impact of a Cross-Kingdom Signaling Molecule of Candida albicans on Acinetobacter baumannii Physiology[END_REF]. Therefore, FAR seems valuable for adjuvant therapy of MDR gram-negative bacteria, including colistin-resistant isolates. However, its high lipophilicity and low aqueous solubility make its administration difficult, which probably limited its development. One option to solve this issue consists in loading FAR within LNP. Hence, FAR was encapsulated in LNP at a concentration of 33 g/L in the total LNP suspension. These LNPs had properties similar to the blank particles, with a mean average size of 68 ± 3 nm and a mean ZP of -2 ±1 mV.

A study that have evaluated the diffusion of various nanoparticles showed that relative selfdiffusion coefficients decreased exponentially with the square of the radius of the nanoparticle [START_REF] Peulen | Diffusion of Nanoparticles in a Biofilm[END_REF]. Therefore, in order to ameliorate the size of different LNP, an additional step was introduced to reduce the size of NPs. Specifically, after mixing the lipid phase containing DA with or without FAR to the aqueous phase, the pH of the mixing solution was adjusted at 6.8 before the formulation process. When adjusting the pH before the formulation step the size of the LNPs was reduced (Fig. 10), while ZP remained unchanged (Data not showed). LNPs size of different formulations decreased from 59 ± 1.2 nm to 37 ± 0. LNP suspension containing COL, supplemented or not with the lipophilic adjuvant molecule FAR, that seemed the most suitable for antibiofilm properties were formulated by incorporating the COL in the aqueous phase which pH was adjusted to 6.8 before the formulation process.

Indeed, this formulation process allowed producing LNPs having the smallest possible size, in order to increase their diffusion through the polymer networks that are the biofilms and the mucus. The efficacy of the various formulations developed were then test against planktonic bacteria and the two types of biofilms that can be found in the lungs.

122 None of the two gram-negative species tested, P. aeruginosa (strain PAO1-Lux, susceptible to COL, (MIC= 1 mg/L)) and A. baumannii (AB122, resistant to COL (MIC~ 32-16 mg/L)), saw their growth, measured by turbidity after 24 hours, slowed down by one of the formulations without COL (LNP-DA, LNP-FAR, LNP-DA/FAR) (results not shown).

COL formulated as LNP or supplemented with LNP-DA, LNP-FAR, or LNPDA/FAR had the same efficacy than free COL against planktonic PAO1, and in all condition tested, the MIC in equivalent COL concentration was of 1 mg/L (Fig 11A). However, the effect of COL formulated as LNP or supplemented with LNP-DA or LNPDA / FAR was significantly lower than that of free COL (p<0.0001) (Fig 11B). Indeed, the MIC value of free COL (MICCOL) against planktonic AB122 was around 16-32 µg/ml and decreased to 1-4 µg/ml and 0.5-2 µg/ml in the presence of 500 mg/L of LNP-DA or 100 mg/L of LNP-FAR, respectively. Mixing DA and FAR in the same LNP as LNP-DA/FAR with a 60/100 mass ratio between DA and FAR did not improve the efficacy compare to 100 mg/L LNP-FAR alone. However, combining the three molecules in the same LNP as LNP-DA/FAR/COL decreased again the MICCOL against AB122 to 0.5-2 Additionally, free LNP-FAR also reduced COL MIC from 4 to 1 µg/ml with a susceptible strain of A. baumannii (AB121 -data not shown), decreased from 16 µg/ml to 1 µg/ml the polymyxin B MIC against AB122 (Data not shown). These results suggest that FAR could potentiate the activity of both polymyxin (B and E) against A. baumannii, especially with the resistant strain, and that LNP-FAR could be used to enhance the activity of antimicrobial treatment against these bacteria.

In vitro evaluation of the efficacy of free and LNP-COL against adherent biofilms

The efficacy of the two COL formulations, LNP-DA/COL and LNP-DA/FAR/COL, were assessed against 48-hours-old adherent biofilms formed on 96-well plate by PAO1 and AB122.

The efficacy of treatment was first evaluated by measuring the total enzymatic activity within the biofilms (i.e. the metabolic activity), determined by calculating the rate of increase in the fluorescence produced by FDA hydrolysis. The enzymatic activity within PAO1 and AB122 biofilms decreased progressively with an increase in the concentrations of free COL or LNP-DA/COL or LNP-DA/FAR/COL (Fig. 12 A and B). For AB122, 256 mg/L of free COL reduced the enzymatic activity only by 50% compared to the control. In comparison, 4 mg/L of COL as LNP-DA/COL and LNP-DA/FAR/COL reduced by 70% the enzymatic activity within the biofilm and 32 mg/L of COL as DA/COL and LNP-DA/FAR/COL almost completely suppressed it (Fig. 12B). Enzymatic activity within PAO1 biofilm was more susceptible to free COL than biofilms formed by AB122.

Determination of the amount of CV retained by the biofilm mass, which allows indirect estimation of the total biofilm mass shows a decrease of biomass of PAO1 adherent biofilm when COL concentrations increase, both as a free drug and as an LPN (Fig. 12C). However, main difference between free COL and formulations was observed for a COL concentration of 1 (µg/mL) and not for higher concentrations (Fig. 12C). Concerning AB122 adherent biofilm, the decrease of biomass is less spectacular and only a decrease of 50% of biomass was observed for formulations compared to free COL for higher concentrations (128 and 256 µg/mL). These high concentrations in COL can only be obtained after inhalation as it was observed in sheep after pulmonary administration of COL prodrug (CMS: colistin methanesulfonate) at a dose of 8mg/kg or direct COL at a dose of 3mg/kg [START_REF] Landersdorfer | Substantial Targeting Advantage Achieved by Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model[END_REF]. In the absence of COL, the bioluminescence kinetics for PAO1 within alginate beads maintained the same value of bioluminescence at time zero (remains stable) until 7.5 hrs, beyond this time the bioluminescence intensity elevated gradually to reach a peak value at hrs followed by an augmentation in this rate to achieve a stationary level as obtained with COL (Fig. 13).

At a concentration 2 times the MIC of encapsulated COL, LNP-DA/FAR/COL displayed a bioluminescence kinetics approximately similar to the one obtained with the smaller concentration (< 2 times MIC) except the initial decrease was until 10 hrs instead 7.5 hrs.

Moreover, the addition of LNP-DA/COL at a concentration of 2 the times MIC decreased the bacterial growth rate until 10 hrs with a bioluminescence intensity slightly higher than that with LNP-DA/FAR/COL, then after 10 hrs the growth rate increased progressively to reach a peak higher than the peak obtained with COL at 17.5 hrs. This rate then diminished progressively to reach a bioluminescence intensity lower than obtained with COL or LNP-DA/FAR/COL (Fig. 13).

At a concentration 5 times MIC, the addition of free COL displayed a profile of bacterial growth rate similar to the one obtained with LNP-DA/FAR/COL at a concentration of 2 times the MIC, bioluminescence intensity decreased slightly until 10 hrs then the growth rate increased progressively to reach a peak at 17.5 hrs followed by a progressive decrease to reach a steady state. The addition of encapsulated COL at a concentration of 5 times the MIC, substantially decreased the growth rate as compared to free COL. The decrease in the growth rate was greater and faster with LNP-DA/COL that reached a bioluminescence value at 5 hrs approximately 2 times lower than that obtained with LNP-DA/FAR/COL at 7.5 hrs. After reaching the lower intensity bioluminescence, the growth rate of PAO1 increased progressively in the presence of two forms of encapsulated COL with a bioluminescence intensity lower with LNP-DA/COL than LNP-DA/FAR/COL until 20 hrs. After that, the growth rate of the two encapsulated forms was similar and continued to increase to reach a steady state at 25 hrs higher than that with CO (Fig. 13).

At a concentration 10 times the MIC, the addition of free COL progressively decreased the bacterial growth rate to reach a bioluminescence intensity value lower than 2.5 𝐿𝑜𝑔 10 at 10 hrs as compared to the initial value. following that, the growth rate increased progressively to reach a steady state at 30 hrs. On the other hand, the encapsulated COL decreased the bacterial growth rate more important and more rapid than with COL to reach a bioluminescence intensity value at 7.5 hrs lower by 4 𝐿𝑜𝑔 10 as compared to the initial value. After that, the bacterial growth increased in a rate more importantly with LNP-DA/COL than with LNP-DA/FAR/COL (Fig. 13).

At a concentration 25 times MIC, the addition of free COL or encapsulated COL rapidly decreased the growth rate of PAO1 to reach a very low value of bioluminescence intensity (e.g. the absence of viable cells) that was faster with the encapsulated form at 2.5 hrs in comparison with the free form of COL at 7.5 hrs. At a concentration higher than 25 times the MIC, free or encapsulated COL had similar efficacy against PAO1 with alginate beads. After 40 hrs of exposure to various concentrations of ATB, bacterial survival was estimated by measuring the colonies formed on agar plates after the solubilization of alginate beads (Fig. 14). The PAO1 survival curve for various concentrations of free COL shown in Figure 14A showed that the bacterial concentrations were similar (CFU0 = 8.7 ± 0.17 By comparing in each graph, the parameters corresponding to each treatment, the CFU0 Based on our preliminary results, the encapsulated COL was more effective in treating the in vitro biofilms of PA and A baumannii. This enhancement could be elucidated firstly by the nanoencapsulation of COL and secondly, by the adjuvant effect of DA and FAR with COL. To our knowledge, there are no studies in the literature that attempt these combinations in the treatment of bacterial biofilm. Many studies reported about the efficacy of FA alone in treating biofilm infection [START_REF] Kumar | Fatty Acids as Antibiofilm and Antivirulence Agents[END_REF], such as long chain FA showed antibiofilm activity against PA biofilm [17,[START_REF] Wenderska | Palmitoyl-dl-Carnitine is a Multitarget Inhibitor of Pseudomonas aeruginosa Biofilm Development[END_REF] and mono-unsaturated chain of FA against A. baumanii biofilm [START_REF] Nicol | Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii[END_REF]. DA is a medium carbon chain FA reported for its antimicrobial activity [16,20]. DA was reported to inhibit the biofilm formation of C. albicans [START_REF] Murzyn | Capric acid secreted by S. boulardii inhibits C. albicans filamentous growth, adhesion and biofilm formation[END_REF]. Concurrently, the poly-unsaturated FA has been reported

to influence the membrane permeability, biofilm formation and surface motility of A. baumannii [START_REF] Eder | Exogenous polyunsaturated fatty acids (PUFAs) alter phospholipid composition, membrane permeability, biofilm formation and motility in Acinetobacter baumannii[END_REF]. Also, Nicol et al. have reported that the mono-unsaturated palmitoleic alone and myristoleic acids decrease the A. baumannii biofilm formation by dispersing the biofilm and reducing the motility [START_REF] Nicol | Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii[END_REF]. Yet, no previous study investigated DA in treating PA and A.

baumanii biofilm.

FAR is a fungal QS molecule [START_REF] Bandara | Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro[END_REF], reported to have antimicrobial activity against certain bacteria [START_REF] Gomes | Effect of Farnesol on Planktonic and Biofilm Cells of Staphylococcus epidermidis[END_REF][START_REF] Kim | Potentiation of the activity of β-lactam antibiotics by farnesol and its derivatives[END_REF]. FAR is known for its anti-biofilm activity against PA biofilm by inhibiting QS molecules, pyocyanin production, and swarming motility in PA biofilm [START_REF] Cugini | Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa[END_REF]59,[START_REF] Mcalester | Signal-mediated interactions between Pseudomonas aeruginosa and Candida albicans[END_REF]. It has been stated that FAR inhibit significantly the biofilm formation of Streptococcus mutans and S.

epidermidis [START_REF] Jeon | Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms[END_REF][START_REF] Pammi | Farnesol Decreases Biofilms of Staphylococcus epidermidis and Exhibits Synergy with Nafcillin and Vancomycin[END_REF]. Few studies demonstrated that the use of FAR in combined therapy may enhance the activity of ATBs against bacterial biofilm. For instance, using the combination of FAR and ciprofloxacin in liposomal formulation showed an enhanced effectiveness in eradicating PA biofilm compared to monotherapy of ciprofloxacin [START_REF] Bandara | Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro[END_REF]. Additionally, FAR can penetrate the biofilm matrix increasing the permeability of bacterial cells within biofilm to ATBs and may help in reducing the adherent bacteria [START_REF] Nowacka | Farnesol-Containing Macromolecular Systems for Antibiofilm Strategies[END_REF], such as vancomycin against It has been documented that FAR disrupts the bacterial membrane integrity and alters bacterial morphology and modifies the virulence properties including biofilm formation and twitching motility of A. baumannii biofilm [65].

Some strategies combined several ATBs together or with another molecules having antibacterial/anti-biofilm activities which can produce a synergistic activity enhancing or producing a total eradication of pulmonary biofilm [START_REF] Bandara | Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro[END_REF]. In this context, we tried to find a strong combination to completely eradicate the biofilms infection with minimum ATBs concentration.

For this purpose, FAR was used in our formulation in a combined therapy with DA and COL.

FAR was also incorporated into LNP loaded with DA and COL to produce LNP-DA/FAR/COL.

In addition, the two encapsulated forms of COL (LNP-DA/COL and LNP-DA/FAR/COL) showed an enhancement in the in vitro efficacy of COL in treating our developed PA biofilm model. In addition, this enhancement was also demonstrated on an adherent biofilm model of PA and A. baumanni mimicking the attached lung biofilms. Thus, suggesting that the encapsulation of COL into LNP formulations (LNP-DA/COL and LNP-DA/FAR/COL) may help to increase the diffusion of COL through biofilm, and consequently to treat chronic lung biofilm.

In literature there are no studies demonstrating these combinations in treating bacterial biofilm.

Several studies illustrated the efficacy of saturated and un-saturated FA alone in treating biofilm infection of PA and A. baumanii [17][START_REF] Nicol | Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii[END_REF][START_REF] Wenderska | Palmitoyl-dl-Carnitine is a Multitarget Inhibitor of Pseudomonas aeruginosa Biofilm Development[END_REF][START_REF] Kumar | Fatty Acids as Antibiofilm and Antivirulence Agents[END_REF]. DA have antibacterial efficacy [16,20] and was reported to inhibit the biofilm formation of C. albicans [START_REF] Murzyn | Capric acid secreted by S. boulardii inhibits C. albicans filamentous growth, adhesion and biofilm formation[END_REF], yet there is limited investigation on it. The combination of linoleic acid and TOB synergistically inhibited PA biofilm formation of PA by inhibiting QS systems [START_REF] Chanda | Combined effect of linolenic acid and tobramycin on Pseudomonas aeruginosa biofilm formation and quorum sensing[END_REF].

In this aspect, DA and FAR may be considered as potential agents for adjunctive therapy in treating the biofilm infection and their use may decrease the COL concentration in combined therapy and thereby reducing COL toxicity and may lead to a promising formulation against gram negative lung infection.

GENERAL DISCUSSION

& PERSPECTIVES

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

GENERAL DISCUSSION & PERSPECTIVES

Chronic lung infections are often associated with bacterial biofilms. Biofilm is a bacterial community enclosed in a negatively charged polymeric matrix rich in polysaccharides (such as alginate), eDNA and proteins. Two types of lung biofilms are mainly encountered, the biofilms dispersed in mucus that colonize the airways of CF and bronchiectasis patients, and biofilms adhering to abiotic surface seen in patients with VAP (Boisvert et al. 2016;Sønderholm et al. 2017a). Such biofilms are tolerant to antimicrobial treatments and are difficult to eradicate with conventional treatments [START_REF] Ciofu | Antibiotic treatment of biofilm infections[END_REF]Høiby et al. 2010b;[START_REF] Wu | Strategies for combating bacterial biofilm infections[END_REF]. Cationic ATBs including aminoglycosides and polymyxins are less effective against these lung biofilms. The tolerance of lung biofilms to treatment with cationic ATBs is in part attributed to the restriction of their penetration to reach and kill the embedded bacteria (Ciofu and Tolker-Nielsen 2019).

This restriction is due to the electrostatic interactions between biofilm matrix and ATBs that slow down the diffusion of ATBs trough the bacterial biofilms. This thesis aims to overcome the problem of restricted penetration of ATBs by developing a delivery system which may prevent the electrostatic interaction and target the delivery of drug to treat lung biofilm infections.

To achieve this goal, two main steps have been developed in this thesis:

(1) Optimization of an in vitro biofilm model mimicking the lung biofilm infections that may serve to rapidly evaluate the developed delivery system

(2) Developing a nanocarrier system that diffuses through the biofilm and delivers the cationic ATB to eradicate the bacteria. This delivery system is assessed by an optimized model to verify its effectiveness

The first part of this thesis focuses on the selection and development of an in vitro biofilm model that offers an in vivo pulmonary biofilm imitation taking several parameters into considerations. We tried to optimize an in vitro model of mucus dispersed PA biofilm, which was conducted in two articles. Firstly, we demonstrated that calcium-alginate beads were more effective than agar beads in shielding PA from cationic ATBs (TOB) [START_REF] Torres | In vitro evaluation of Pseudomonas aeruginosa chronic lung infection models: Are agar and calcium-alginate beads interchangeable[END_REF].

Accordingly, calcium-alginate, negatively charged, was selected to be used as a matrix component for the beads. Then, we demonstrated that increasing the size of the beads or dispersing these beads into an artificial mucus medium further decreases the efficacy of TOB.

Therefore, the biofilm model selected to evaluate the ATB nanocarrier system consisted of PA loaded in large calcium-alginate beads dispersed in an artificial mucus medium. This model is referred to as bacteria embedded-based model ( to assess the in vivo efficacy of antimicrobial treatment against chronic lung infection [START_REF] Christophersen | Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection[END_REF][START_REF] Growcott | Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin[END_REF][START_REF] Heeckeren | Murine models of chronic Pseudomonas aeruginosa lung infection[END_REF][START_REF] Pedersen | Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres[END_REF]. While other researchers have used this model with 2.4 mm sized beads to study the structure morphology of bacterial biofilm in vitro [START_REF] Behrendt | Biofilm Growth and Near-Infrared Radiation-Driven Photosynthesis of the Chlorophyll d-Containing Cyanobacterium Acaryochloris marina[END_REF]Sønderholm et al. 2017a) [START_REF] Pedersen | Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis[END_REF]Sønderholm et al. 2017b). Yet, the model developed in this thesis was never used to assess the in vitro efficacy of antimicrobial treatment.

On the other hand, Sriramulu et al. attempted to grow PA in microcolonies in an artificial mucus medium to create mucus dispersed biofilm, but did not succeed to mimic the desired model [START_REF] Sriramulu | Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung[END_REF]. To date, there is no study that has investigated the impact of mucus on bacteria embedded model, as it was performed in our study. Our developed model has brought together all these studies by producing PA-loaded large alginate beads dispersed in artificial mucus media and was used to assess the in vitro effectiveness of antimicrobial agents.

Is this model representative of the mucus dispersed biofilms or not?

In general, the previous developed in vitro biofilm models attempted to represent as many characteristics as possible of human biofilms while remaining easy to produce, which makes these models complicated to develop. Our optimized model simulated many characteristics found in the in vivo conditions including the negatively charged alginate matrix; acting as first barrier, and the mucus media; acting as a second barrier. Several advantages are associated with our developed model, such as its simplicity and flexibility, rapid assessment of the antimicrobial therapy, a more homogeneous biofilm due to the presence of homogeneous alginate beads, and the produced bacterial clusters are similar to the aggregates observed in vivo in the CF patients as demonstrated by Sønderholm et al. [START_REF] Bjarnsholt | The in vivo biofilm[END_REF]Sønderholm et al. 2017b). The features of this model better reflect unattached bacterial lung biofilms, but several limitations are present. In particular, the implication of the human body environment is absent, especially the immune response, and the limitation of nutrients which limited the study on a long time interval. Additionally, the biofilm matrix is not only composed of alginate but also eDNA, proteins and other constituents make the use of the alginate matrix limited and not globally representative of the matrix in vivo. Furthermore, the mucus in CF airway is more viscous than the one used in our model, which makes the viscosity condition lacking in our model. Still, the present selected model can be optimized by introducing other features such as including the other matrix components in the alginate beads or increasing the viscosity of artificial mucus media to be comparable as observed in the CF airway that requires a specific rheological study. But is this simple to establish without complicating the model?

Two barriers were involved in our model (biofilm matrix and mucus) but what about the third barrier constituted by the PMN cells within the mucus? Actually, PMN accumulated around the bacterial aggregates can regulate the biofilm formation by O2 and nutrients consumption, which increases the antibiotic tolerance in the biofilm [START_REF] Kolpen | Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis[END_REF][START_REF] Sønderholm | Tools for studying growth patterns and chemical dynamics of aggregated Pseudomonas aeruginosa exposed to different electron acceptors in an alginate bead model[END_REF].

For instance, our developed model could also be supplemented by PMN cells to study the interaction between PMN and our developed model. Researchers have studied the in vitro interaction between PA biofilm and PMN [START_REF] Shi | In vitro and ex vivo systems at the forefront of infection modeling and drug discovery[END_REF]. In this regard, it has been reported

than PMNs can enhance mucoid PA biofilms formation in hanging-peg model [START_REF] Tan | Polymorphonuclear Leukocytes or Hydrogen Peroxide Enhance Biofilm Development of Mucoid Pseudomonas aeruginosa[END_REF].

So, the addition of PMN may allow investigating the effect of three barriers presented in biofilms against the anti-biofilm treatment. This step is actually under development in our laboratory using THP-1 cells differentiated into macrophages.

The question now is up to which point we may still continue to develop the in vitro biofilm model. It is indeed critical to bear in mind that what we are developing is a model that can simulate and predict as maximum as possible what occurs in vivo. In this regard, even if we add more characteristics, some features may still be lacking and establishing them in previously developed models could make them more complex meanwhile our aim is to develop a model that brings maximum information with easy production. The results obtained with the in vitro experiments were usually used mostly to perform animal experiments. The behavior of human lung biofilm is totally different even with the presence of many characteristics, and it is essentially missing the implication of host human environment or directly understanding the dynamic interactions between the host and bacterial biofilms. In this thesis, we adapted the developed model of large beads trapped PA dispersed in artificial mucus as an in vitro model for rapid evaluation of the efficacy of anti-biofilm strategies before performing in vivo experiments.

The second part of thesis is based on the development of deliverance system of cationic ATBs that may protect the drug from the electrostatic interactions and target its delivery to the biofilm. The development of ATBs delivery system was illustrated in the third article of this thesis.

Treatment of lung biofilms requires an efficient ATB concentration at the site of infection, which can be reached by the use of inhaled therapy (Restrepo et al. 2015;[START_REF] Wenzler | Inhaled Antibiotics for Gram-Negative Respiratory Infections[END_REF].

Inhaled ATBs have been used extensively in the treatment of chronic lung infections (Garcia-Contreras et al. 2015;Maselli et al. 2017;Quon et al. 2014;Restrepo et al. 2015), but have failed to totally eradicate them. Recently, the challenge of fighting these lung biofilms is to improve current inhalation therapies using DDS targeting infection sites [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Nafee | Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors[END_REF]. Different types of vectors could be used as DDS including the encapsulation into nano or micro carriers or other strategies such as bacteriophages, chemical modification and PEGylation (Bahamondez-Canas and Smyth 2018). Such therapeutic strategies may enhance the penetration of the drug into the biofilms and expand its release to increase the drugs activity and minimize its toxicity [START_REF] Sajid | Drug Delivery Systems That Eradicate and/or Prevent Biofilm Formation[END_REF]. In our study, we chose a nanoformulation strategy to enhance the efficacy of cationic ATBs activity against biofilm infections. This strategy is advantageous because it improves the diffusion through the biofilm and the mucus matrix to a greater extent than microencapsulation [START_REF] Suganya | Microencapsulation and Nanoencapsulation: A Review[END_REF].

The nanoencapsulation requires adequate nanocarriers allowing high drug loading, sustained and controlled release of drug, high mucus penetration and maintained activity of encapsulated drug [START_REF] Nafee | Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors[END_REF]. In this sense, we selected the lipid nanoparticles (LNP) as a nanocarrier system, among the other nanocarrier systems (LIPs, polymeric NPs). LNP were chosen for their excipient's biocompatibility and low toxicity, and for the simplicity of Theoretically, COL should be strongly incorporated into LNP-DA thanks to the formation of HIP, especially since COL is amphiphilic and already has a hydrophobic part (alkyl chain)

allowing it to interact with LNP. To verify the success of COL encapsulation in LNP, it is critical to assess the drug loading, which was not presented in the article 3 due to the lack of appropriate method for separating free COL and associated with LNP. Indeed, measuring the amount of drug successfully entrapped into LNP requires methods allowing the rapid separation of LNPs containing encapsulated drug from their surrounding aqueous medium than contains free drug. Then the COL concentration can be measured by the LC-MS/MS method routinely used in our laboratory. Different methods are generally employed for the separation of free and encapsulated drugs, including ultracentrifugation, centrifugal filtration, equilibrium dialysis-based methods and cationic exchange chromatography methods [START_REF] Gioria | Are existing standard methods suitable for the evaluation of nanomedicines: some case studies[END_REF][START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF]. It should be noted that large size NPs can be separated by simple filtration, while small-sized NPs (such as our generated formulations) are more challenging to be separated [START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF].

Ultracentrifugation method was demonstrated to be fast and simple to characterize free and encapsulated drug proportions [START_REF] Gioria | Are existing standard methods suitable for the evaluation of nanomedicines: some case studies[END_REF][START_REF] Mehn | Analytical ultracentrifugation for analysis of doxorubicin loaded liposomes[END_REF][START_REF] Mittal | Analytical Ultracentrifugation of Model Nanoparticles: Comparison of Different Analysis Methods[END_REF][START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF]. For example, the determination of free and encapsulated COL into LIPs using ultracentrifugation methods demonstrated that only ~1% of LIPs remained in the supernatant [START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF]. In comparison to LIPs, LNP are softer and fragile and can merge upon the centrifugation force but which may cause the release of drug.

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Equilibrium dialysis-based methods could be used to separate free drugs from encapsulated drugs using a semipermeable membrane with an appropriate molecular weight cutoff. This allows the passage of free drugs across the membrane, from a donor to an acceptor compartment while the encapsulated drug remains in the donor side [START_REF] Marques | Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers[END_REF][START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF]. Nevertheless, the longtime needed to equilibrate may cause the release of hydrophilic ATBs from the NPs during dialysis, and thus may not reflect the exact proportion of free drug [START_REF] Marques | Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers[END_REF][START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF].

Ultrafiltration method uses a device where NPs-encapsulated ATBs are loaded into the centrifuge ultrafiltration compartment and the filtration is performed under centrifugation [START_REF] Liu | Controlled Release of Biologically Active Silver from Nanosilver Surfaces[END_REF][START_REF] Tran | Synergistic Therapeutic Strategy of Dual Drug-loaded Lipid Polymer Hybrid Nanoparticles for Breast Cancer Treatment[END_REF][START_REF] Wallace | Drug release from nanomedicines: Selection of appropriate encapsulation and release methodology[END_REF]. We tried this method, proposed by Wallace et al., and our results showed encapsulation efficiency more than 98% with different formulations (LNP-DA/COL and LNP/DA/FAR/COL). One of the advantages of this method as compared to ultracentrifugation is the low centrifugal force used which may cause less particle deformation. However, LNPs can be easily deformed and can also pass through the filter at the same time with free ATBs, but in this case the encapsulation efficiency should be lower than the results obtained. Additionally, another limitation of this method is the risk of clogging the filter with LNPs, which may explain the high encapsulation efficiency obtained in our study.

Ion exchange chromatography method is a useful method for separation of free charged ATBs from encapsulated ATBs. We also tried this method using the Oasis MCX cation exchange cartridge (Oasis Sample Extraction Products), which is a water-wettable (pKa <1) cation exchange polymer. This cartridge must retain free COL molecules, while those associated with DAs and trapped on LNP are eluted. COL is positively charged at neutral pH and LNP are negatively charged, in this regard, separating free COL from encapsulated COL is possible if LNPs stay stable during the exchange process. However, COL is an amphiphilic molecule localized on the LNP surface and thus can also be extracted from LNP if the interaction with SO3 -, are stronger than with COO -of DA. This could thus make this method inappropriate for determination of free and encapsulated proportion. Our findings showed that the encapsulation efficiency of COL into LNP is 20-30% in all produced formulations. This reduction of encapsulation efficiency, compared to the first method, could be explained by the extraction of encapsulated COL from LNP.

Finally, we used the ZP measure to indirectly assess the COL interaction with LNPs. The main idea of this method is measuring the ZP values to interpret the efficiency of COL interaction with LNP. Basically, if COL is adsorbed on the surface of LNPs, the ZP value becomes less negative. In the presence of FA (FA-loaded LNP), if COL forms HIPs with FA and is incorporated into the core of LNPs, the ZP value of LNPs should be more negative than if COL remains adsorbed at their interfaces. However, COL is an amphiphilic molecule which can interact with the surface of LNP. Our results demonstrated that FA incorporated into LNPs makes them negatively charged. Formulating COL with LNPs, by incorporating COL into the Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires liquid phase before the formulation process, makes LNP ZP values less negative by only 20 to 30%. However, the addition of the same amount of COL after the formulation process reduces the negative charge of LNPs by about 50%. Thus, in accordance with an increase of LNP negative charges, COL may be partially on the surface of LPNs but also partially encapsulated in the LNP. This indirect method of measuring ZP values cannot be used as an alternative to the direct method but it can be used as a supplementary technique to the direct method.

Generally, direct methods are considered more accurate than indirect methods but the use of both, if feasible, may be more efficient to determine the encapsulation efficiency. In our scenario, the only way to determine encapsulation efficiency was to use both direct and indirect methods. In our present study, direct methods (Ultrafiltration and cation exchange method)

showed discrepancy in encapsulation efficiency results, not allowing us to conclude, but ZP measurement showed an interaction of COL at the surface of LNP.

The key objective of this study is first to enhance the encapsulation of cationic ATBs (COL) into LNP and prevent the electrostatic interaction with the matrix components by using an adjuvant (DA) serving as counter-ion in HIP formation, and secondly to benefit from the activity of the adjuvants used in formulation (including DA and FAR). In this aspect, DA and FAR may be considered as potential agents for adjunctive therapy in treating the biofilm related infections and their use may decrease the COL concentration needed and thereby reducing COL toxicity. Both molecules used as adjuvants (DA and FAR) alone did not reveal any efficacy against planktonic PA and A. baumannii. Contrarily, their use in combined therapy with COL significantly increased the sensitivity of planktonic A. baumanii to COL. Additionally, encapsulated formulations of COL (LNP-DA/COL and LNP-DA/FAR/COL) showed an in vitro efficacy in treating PA and A. baumannii biofilms. Since DA and FAR were not tested alone against the bacterial biofilms, it would be necessary to verify if they have any anti-biofilm activity alone. Hence, further in vivo studies are needed to confirm these findings.

In our laboratory, we have developed in vivo models of PA chronic lung infection (Data not yet published), where the efficacy of our developed formulations could be tested on this model. These formulations can be delivered directly to the lungs of rodents (rats and mice) by using nose-only inhalation system (TSE®) (Figure 19) to assess the efficacy and toxicological safety of formulations. This nose-only inhalation system is in an ABSL-2 lab located in the animal housing facility (PREBIOS). Canas et al. 2018;[START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF]. Moreover, it has been reported that the PEGylation of TOB via chemical conjugation significantly augmented its efficiency against PA biofilm grown in standard media and in CF-like mucus media (Bahamondez-Canas et al. 2018;[START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF].

In fact, we tried to directly PEGylate the TOB in our laboratory, but we did not succeed to produce this chemical conjugation, and this requires further testing and would actually be a future project of interest.

  TOB, et aucune SCV a été observé. Des nanoparticules lipidiques PEGylées chargées de COL et contenant un adjuvant lipophile à la COL tel que le Farnésol ont été développées pour augmenter son efficacité contre les biofilms. L'encapsulation de la COL a amélioré son efficacité par un facteur deux dans le modèle de biofilm pulmonaire à PA précédemment développée. Ces nanoparticules ont permis également de restaurer la sensibilité à la COL d'une souche résistant d'A. baumannii et ont amélioré son efficacité dans un modèle de biofilm d'A. baumannii formé sur une surface abiotique. Mots clés : Pseudomonas aeruginosa, Acinetobacter baumannii, tobramycine, colistine, biofilms pulmonaires, nanoparticules lipidiques.

1 . 2 . 1 . 2 . 4 . 6 .Figure 1 . 6 Figure 2 . 8 Figure 3 . 11 Figure 4 13 Figure 5 .Figure 6 . 26 Figure 7 . 26 Figure 8 . 27 Figure 9 . 27 Figure 10 . 32 Figure 11 . 34 Figure 12 . 36 Figure 13 . 37 Figure 14 . 38 Figure 15 . 39 Figure 16 . 40 Figure 17 . 52 Figure 18 . 57 Figure 19 .

 1212461628311413562672682792710321134123613371438153916401752185719 Figure 1. Schematic representation of different steps of formation of mucus dispersed biofilms ....................................................................................................................... 6 Figure 2. Schematic representation of different steps of formation of the biofilm adherent to abiotic surface ......................................................................................... 8 Figure 3. Scheme presented the characteristics of PA transition from planktonic to biofilm lifestyle, corresponding to PA microevolution during the CF airways infection ................................................................................................................................. 11 Figure 4.................................................................................................................... 13 Figure 5. Scheme representative for static biofilm models linked to lung infections 24 Figure 6. Scheme represent the principe of the medium flow through dynamic biofilm models. ..................................................................................................................... 26 Figure 7. Schematic drawing of flow cell system ..................................................... 26 Figure 8. Schematic diagram of Center for Disease Control (CDC). ........................ 27 Figure 9. Schematic diagram of Drip flow reactor. ................................................... 27 Figure 10. Schematic representation of the quantification of bacterial colonies (CFU) from a biofilm. ........................................................................................................... 32 Figure 11. Schematic representation of indirect measurements methods. .............. 34 Figure 12. Schematic representation of microscopic methods................................. 36 Figure 13. The chemical structure of TOB and COL. ............................................... 37 Figure 14. Schematic representation of interaction of nanoparticles in DDS with different biofilm stages. ............................................................................................. 38 Figure 15. Schematic representation of NP as a drug carrier .................................. 39 Figure 16. Schematic representation of different types of LIP. ................................ 40 Figure 17. Different types of NPs used as drug delivery system. ............................. 52 Figure 18. Scheme of Research work ...................................................................... 57 Figure 19. Inhalation Nose only system from (TSE®) ............................................ 142

Figure 1 .

 1 Figure 1. Schematic representation of different steps of formation of mucus dispersed biofilms. The formation begins with the mucus colonization by planktonic PA (a) leading to acute infection (b).During later stage, biofilm is mature causing chronic infection (c), forming characteristics specific to biofilm (bacterial aggregate in self-produced matrix rich in anionic polysaccharides). Finally, some PA begin to detach and the biofilm disperses(d). (Personal creation)

Figure 2 .

 2 Figure 2. Schematic representation of different steps of formation of the biofilm adherent to abiotic surface. The formation begins with the adhesion of the planktonic cells on abiotic surface (such as endotracheal tube)(a). Then, the bacteria proliferate(b) and start to produce an extracellular matrix (c), leading to the formation of bacterial microcolonies. During later stage, the biofilm is mature forming characteristic structure of three-dimentionnal bacterial communities, bacterial aggregate in selfproduced matrix made of polysaccharides(d). Finally, some bacterial cells start to detach allowing the dispersion of biofilm (e) (personal creation).

AHLs, N-( 3 -

 3 Oxododecanoyl)-L-homoserine lactone and Nbutyryl-L-homoserine lactone (C4-HSL or BHL), which can diffuse through bacterial membranes (Figure3). PA also produces another membrane permeable signal molecule, the 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the PA quinolone signal or PQS. Four main interconnected QS systems have been identified in PA, namely: Las, Rhl, and PQS and IQS (Lee and Zhang 2015) (Figure3). Las and Rhl use the AHLs as signaling molecules. The AHL synthases LasI and RhlI are enzymes that produce OdDHL and BHL, Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires respectively[START_REF] Girard | Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa[END_REF][START_REF] Li | Quorum sensing: How bacteria can coordinate activity and synchronize their response to external signals?: QS to Regulate Gene Expression in Bacteria[END_REF]. When AHLs reach critical concentration, they bind to the cytoplasmic regulation proteins (LasR and RhlR), which then act as transcriptional regulators of genes of various virulence factors, genes involved in biofilm evolution, and also activate transcription of AHL synthases, providing a signal amplification mechanism via an auto-induction loop[START_REF] Lee | The hierarchy quorum sensing network in Pseudomonas aeruginosa[END_REF][START_REF] Li | Quorum sensing: How bacteria can coordinate activity and synchronize their response to external signals?: QS to Regulate Gene Expression in Bacteria[END_REF][START_REF] Solano | Biofilm dispersion and quorum sensing[END_REF].

Figure 3 .

 3 Figure 3. Scheme presented the characteristics of PA transition from planktonic to biofilm lifestyle, corresponding to PA microevolution during the CF airways infection. In planktonic lifestyle corresponding to the early stage of infection, PA is presented in non-mucoid phenotype and full equipped with cell-associated virulence factors including flagella and pili type IV which are serving for bacterial swimming and twitching motility, type 3 secretion systems (T3SS), and secreted virulence factors including proteases, pyoverdine, and rhamnolipid and exhibit ATBs sensitivity. In biofilm lifestyle corresponding to the chronic stage of infection, PA adapted the CF environment and exhibits a variation in characteristics including the loss of motility and overproduction of alginate, reduced of virulence factors especially the implicated in establishment for initial infection, and PA are tolerant/resistant to ATBs (expression of efflux pumps, restricted penetration…), and trigger the QS system.

  ATBs such as TOB and COL are among the most widely used for treating lung infections by inhalation in CF patients. Additionally, in these patients, the thick mucus in which the PA biofilms are formed is also composed of negatively charged polymers such as mucins which can further reduce the diffusion of positively charged ATBs (Bahamondez-Canas et al. 2018; Müller et al. 2018; Torres et al. 2019).

Figure 4 .

 4 Figure 4. Scheme representative of biofilm colony formed on infected tissues within viscous mucuslayer, characteristics of a chronic infection in the CF airway, illustrating the restriction of the penetration of cationic ATBs (TOB and COL) due to their interactions with components negatively charged in mucus (eDNA and mucins) and biofilms (anionic polymers including alginate, DNA). Biofilms and mucus constitute barriers for cationic ATBs diffusion and prevent their reaching the target bacterial colonies.[START_REF] Armijo | Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms[END_REF]

  vitro PA biofilms treated with an alginate oligomer (OligoG CF-5/20) were dispersed in a dosedependent manner. Furthermore, a significant biofilm reduction (up to 2.5 log reduction in colony-forming units) was observed in an in vivo lung infection model. The combined treatment of 5% w/v of OligoG CF-5/20 and COL reduced 128-fold the in vitro MBEC (Minimum Biofilm Eradication Concentration) against PA biofilms. Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Figure 5 .

 5 Figure 5. Scheme representative for static biofilm models linked to lung infections. (a) Biofilms were developed on abiotic surface of 96-well microtiter plate, which can be conventional where biofilm is attached to the surface of microplate, or can be pegs lid model (also known as Calgary device) where biofilm is grown on pegs attached to the surface of lid (Brown et al. 2019). (b) Cell-culture models. The bacterial biofilm is developed on a monolayer of living lung. Particularly, the monolayer of epithelial cells is cultured on a Transwell® inserts to mimic the lining in lung airway. Bacteria are incorporated in the culture media placed on the apical compartment and a culture media is placed in the basolateral compartments (TecMonterrey 2018) (c) Bacteria-embedded in beads based models. Bacteria are loaded into agar or alginate beads, dispersed in a growth or culture media (personal creation).

(

  [START_REF] Coenye | In vitro and in vivo model systems to study microbial biofilm formation[END_REF][START_REF] Moreau-Marquis | Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells[END_REF], such as bacterial biofilm formed on airways cells (e.g. monolayers of human airway epithelial cells). This type of model allows the study of interaction between the biofilm forming bacteria and the host cells and can reflect the phenomena occurring during bacterial colonization in the lungs[START_REF] Moreau-Marquis | Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells[END_REF].

  6 to 9.

Figure 6 .

 6 Figure 6. Scheme represent the principe of the medium flow through dynamic biofilm models.Dynamic biofilm systems based on a continuous sypplying of nutrients using an inlet port and eliminating of spent culture using an outlet port. (a) Flow cell system, with medium inlet and outlet ports at the same level (b) CDC biofilm reactor with inlet port placed at a level higher than the outlet ports (c) Drip flow reactor with inlet and outlet ports and an air exchange port.[START_REF] Franklin | New Technologies for Studying Biofilms[END_REF].

Figure 7 .Figure 8 .

 78 Figure 7. Schematic drawing of flow cell system. The assembled system composed basically of medium and waste bottles, a peristaltic pump, bubbles traps, and the flow cells (Biofilm is grown on the flow cell, particularly in the transparent chamber). Bacterial cells are supplied with nutrients and oxygen from a medium flask (medium inlet port) via a peristaltism pumps and the spent culture is eliminated in the waste container (outlet port). The bubble trapping device restrict the air bubble from tube to prevent the disruption of biofilm.(adapted from Weiss Nielsen et al. 2011).

Figure 9 .

 9 Figure 9. Schematic diagram of Drip flow reactor is shown on the left of the figure and a scheme of the reactor is shown on the right. The assembled system is similar to the other dynamic systems.Reactor consists of four isolated rectangular channels containing a removable coupon (as shown in at the right side), with a size similar to the glass microscope slide, which promote the bacterial attachment on its surface. Drip flow reactor placed in angled way and the broth medium were pumped in channels and flows down the surface of the slide by gravity (adapted from[START_REF] Goeres | A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor[END_REF][START_REF] Radniecki | Investigating Nitrosomonas europaea Stress Biomarkers in Batch, Continuous Culture, and Biofilm Reactors[END_REF].

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires gradients and other conditions could help to optimized the in vitro model and enhance the fitting of in vivo observation.

Figure 10 .

 10 Figure 10. Schematic representation of the quantification of bacterial colonies (CFU) from a biofilm. An example of biofilm formed on the glass slide of drip flow reactor was collected and suspended in a growth media than plated for colony counting (adapted from Wilson et al. 2018).

Figure 11 .

 11 Figure 11. Schematic representation of indirect measurements methods(a) Image showed biofilm formed on microtiter plate method that was evaluated using crystal violet stain. This image showed none, weak, moderate and strong biofilm producers differentiated by the concentration of crystal violet detected in each well. This method is used to estimate the viability of biofilm cells(Darwish and Asfour 2013).(b) Linear relationship between the number of bacteria and resazurin reduction. This figure showed that resazurin is not sensitive enough to the bacteria at a concentration lower than 5x106 , which prove that this method is not an adaptable tool to measure the cell viability (adapted from Koizumi and Picardeau 2020).

Formulations d 'Figure 12 .

 d12 Figure 12. Schematic representation of microscopic methods. a) Image of PA biofilm using the confocal microscopy (CLSM). At the top of the picture is a one-day old biofilm of PA (PA strain expressing green fluorescent protein (GFP) are used in this biofilm). At the bottom of the picture, the biofilm was treated with ciprofloxacin showing a reduction of living bacteria in the biofilm. This method is useful in estimating the efficacity of treatment of anti-biofilm infection (adapted from Khandekar et al. 2018). (b) In vitro (1) and in vivo (2) aggregate PA. A CLSM image of GFP-PA (green) embedded into alginate beads grown in vitro for 24H, was shown in Fig b-1. Also, a CLSM image of in vivo PA aggregate found in CF lungs was showed in Fig. b-2, where PA are visualized with PNA-FISH probe. The in vitro bacterial aggregate showed a similarity in aspects and in size compared to the in vivo bacterial aggregates (Sønderholm et al. 2017a).

Figure 13 .

 13 Figure 13. The chemical structure of TOB and COL. (a) Chemical structure of TOB with the corresponding individual pka values of each amino group presented in its structure (Alkhzem et al. 2020; Pagano et al. 2011; Walter et al. 1999). (b) Chemical structure of COL with the individual pka values of each amino group and which are almost equal to 10 (Liu et al. 2018; Mutasim Elimam et al. 2015).

  (Dos Santos Ramos et al. 2018), either interact directly with planktonic cells (Figure 14b) during bacterial adhesion or dispersal phase, or interfere with EPS biofilm matrix enabling the entry of NPs (e.g. drug-loaded NPs) inside the biofilm (Figure 14c) (Dos Santos Ramos et al. 2018; Fulaz et al. 2019).

Figure 14 .

 14 Figure 14. Schematic representation of interaction of nanoparticles in DDS with different biofilm stages. (a) Different stage of biofilm formation. Nanoparticles can treat biofilm infection by interacting with planktonic cells (b) and EPS matrix (c). (Dos Santos Ramos et al. 2018).

Figure 15 .

 15 Figure 15. Schematic representation of NP as a drug carrier.NPs act as a drug carrier that can either absorb the drug on its surface(a) or transport it in its core(b). NPs allow drug penetration into the biofilm, usually entrapped in biofilm matrix such as cationic ATBs, to reach and kill them and disrupt the biofilm. In addition, NP controls the release of the drug. (adapted from[START_REF] Qayyum | Nanoparticles vs. biofilms: a battle against another paradigm of antibiotic resistance[END_REF] Nano-DDS may confer many other characteristics adapted to ATB delivery compared to free form, such as protection against degradation, targeting desired infection site, controlled release, enhanced uptake by cells for intracellular bacteria[START_REF] Andrade | Nanotechnology and pulmonary delivery to overcome resistance in infectious diseases[END_REF].Different types of NPs have been used to vectorize ATB, in particular liposomes, lipid NPs, metallic NPs or dendrimers. This chapter summarizes the definition, characteristics and mechanisms of action of some of these NPs formulation and discusses their advantages as well as disadvantages (listed in Table3) and their application in treatment of gram-negative chronic lung infection.

Figure 16 .

 16 Figure 16. Schematic representation of different types of LIP. Three types of LIP are known depending of the number of phospholipid bilayers and their size. LIP can be unilamellar consisting of a single layer, which can be either small (SUV) size ranging from 25 to 50 nm or large (LUV) with size ranging from 50-500 nm , and can be multilamellar (LMV) consisting of multiple layers. The LMV size can can vary from 500 to 10000 nm, depending on the number of layers.

  . For example, hydrophilic drugs such as gentamicin and ceftriaxone are trapped into the aqueous phase of LIP, whereas lipophilic drugs are trapped inside the lipid bilayer. Amphiphilic drugs such as COL are trapped between the two regions of the lipid phase of LIP[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF][START_REF] De Leeuw | Liposomes in dermatology today[END_REF][START_REF] Rukavina | Current Trends in Development of Liposomes for Targeting Bacterial Biofilms[END_REF]. The trapping efficiency into LIPs depends on the physicochemical properties of LIPs and the preparation methods used[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF]. Additionally, it has been demonstrated that the use of lipids and ATBs with opposite charges has provided a high trapping efficiency[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF]. For example, ticarcillin, a carboxypenicillin that is negatively charged at pH=7, showed a high entrapment into a cationic LIP[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF].Aerosolized LIPs have been extensively studied in treating chronic lung infections. Some of which are currently investigated in clinical trials and others have been approved for clinical use[START_REF] Sharma | Liposomes in drug delivery: Progress and limitations[END_REF]. Nebulized LIPs are stable and can sustain the release of ATBs to maintain a prolonged lung exposure and therapeutic effect against chronic lung infection[START_REF] Okusanya | Pharmacokinetic and Pharmacodynamic Evaluation of Liposomal Amikacin for Inhalation in Cystic Fibrosis Patients with Chronic Pseudomonal Infection[END_REF]. LIPs can be loaded with multiple antimicrobials with various physicochemical properties and could be more beneficial compared to free ATB monotherapy. In addition, many studies have shown that the encapsulation of ATBs into LIPs may increase the bacterial killing efficacy compared to the free form[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF]. For instance, ATBs-loaded LIP including aminoglycoside (such as TOB and amikacin), PMB, CIP, clarithromycin, cefepime and ceftazidime, and meropenem have improved the efficacy of antimicrobial treatment against PA[START_REF] Alipour | Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa[END_REF]. It has been documented that controlling liposomal properties such as surface charge and PEG modification may contribute to the bacterial biofilm destruction, retention and permeability. In particular, the negatively charged LIP demonstrated higher bacterial biofilm permeability and lower biofilm retention than the positive LIP. TheFormulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonairesPEGylated form of both types of LIP (positive and negative) increased their permeability and decreased their retention in bacterial biofilm in)(Ibaraki et al. 2020). Aminoglycosides encapsulated into LIPs showed an efficacy in treatment of PA chronic lung infections. The use of saturated lipid dipalmitoylphosphatidylcholine (DPPC) and cholesterol or distearoylphosphatidylcholine (DSPC) and Cholestrol for the formulation of liposomal TOB, amikacin and gentamycin have shown an enhanced efficacy in the treatment of PA biofilms[START_REF] Forier | Lipid and polymer nanoparticles for drug delivery to bacterial biofilms[END_REF]. A liposomal amikacin for pulmonary inhalation was developed byMeers et al., commercially known as Arikace TM , formed by DPPC:Chol and characterized by high stability and drug loading, and showed an in vivo biphasic released in rats lung[START_REF] Meers | Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections[END_REF]. Arikace TM showed an in vitro improvement in diffusion through mucus and biofilm environment, as compared to free amikacin, in an in vitro flow-cell model and demonstrated more efficiency in reducing cell counts by two orders of magnitude as compared to the free form in rats intratracheally infected with PA agar beads[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Meers | Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections[END_REF]. The use of Arikace TM , which is administrated by eFlow® nebulizer, is in phase III of clinical trials[START_REF] Ehsan | Management of Pseudomonas aeruginosa infection in cystic fibrosis patients using inhaled antibiotics with a focus on nebulized liposomal amikacin[END_REF]. Arikace TM has been reported to prolonged half-life in lung compared to LIP-free Amikacin allowing its use once daily[START_REF] Ehsan | Management of Pseudomonas aeruginosa infection in cystic fibrosis patients using inhaled antibiotics with a focus on nebulized liposomal amikacin[END_REF][START_REF] Li | Characterization of Nebulized Liposomal Amikacin (Arikace TM ) as a Function of Droplet Size[END_REF], and to improve lung function and reduce the sputum bacterial density in PA chronic lung infections with CF patients[START_REF] Okusanya | Pharmacokinetic and Pharmacodynamic Evaluation of Liposomal Amikacin for Inhalation in Cystic Fibrosis Patients with Chronic Pseudomonal Infection[END_REF] Sønderholm et al. 2017a). In this respect, Okusanya et al. have suggested a possible efficient and single dosage of 500 mg of liposomal amikacin administered daily to treat chronic PA infection with CF patients[START_REF] Ehsan | Management of Pseudomonas aeruginosa infection in cystic fibrosis patients using inhaled antibiotics with a focus on nebulized liposomal amikacin[END_REF][START_REF] Okusanya | Pharmacokinetic and Pharmacodynamic Evaluation of Liposomal Amikacin for Inhalation in Cystic Fibrosis Patients with Chronic Pseudomonal Infection[END_REF]).

  this study, LIP-TOB/Bi showed a significant reduction by 1.6 log10 CFU/lung in PA counts compared to free TOB-Bi[START_REF] Alhariri | Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection[END_REF]. In another study, LIP-TOB/Bi showed a reduction in BEC by 4 to 32 folds[START_REF] Alipour | Bismuth-ethanedithiol incorporated in a liposome-loaded tobramycin formulation modulates the alginate levels in mucoid Pseudomonas aeruginosa: Liposomal tobramycin-bismuth[END_REF]) and prevented the PA biofilm formation at high TOB concentration[START_REF] Halwani | Bismuth-thiol incorporation enhances biological activities of liposomal tobramycin against bacterial biofilm and quorum sensing molecules production by Pseudomonas aeruginosa[END_REF] on peg lids biofilm model. Furthermore, LIP-TOB/Bi has been reported to prevent the bacterial adherence to A459 monolayers of human lung cells and prevent biofilm formation[START_REF] Alhariri | Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection[END_REF] Halwani et al. 2008a).Simultaneously, the incorporation of gallium in gentamicin-encapsulated LIP has displayed a complete eradication of PA biofilms developed on peg lids at very low concentration compared to free gentamycin and liposomal gentamycin. The PA counts was reduced to 2-4 log10 CFU/ml in the presence of gallium-gentamicin-encapsulated LIP as compared to control with 7-8 log10 CFU/L(Halwani et al. 2008b).Studies using LIPs to encapsulate polymyxins are limited. PMB has been encapsulated in LIPs to maintain and control the release of PMB, and improve the efficacy of the antibiofilm treatment while reducing its toxicity[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Sharma | Liposomes in drug delivery: Progress and limitations[END_REF][START_REF] Singh | Nanoparticles for Control of Biofilms of Acinetobacter Species[END_REF]. It has been reported that liposomal PMB may improve the ATB activity against PA resistant cell while reducing its side effects[START_REF] Huh | Nanoantibiotics": A new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era[END_REF][START_REF] Pelgrift | Nanotechnology as a therapeutic tool to combat microbial resistance[END_REF][START_REF] Zhang | Development of Nanoparticles for Antimicrobial Drug Delivery[END_REF]. It has been postulated that liposomal PMB can act by deforming the PA membrane and allows fusion between the LIPs and the bacterial membrane[START_REF] Pelgrift | Nanotechnology as a therapeutic tool to combat microbial resistance[END_REF][START_REF] Zhang | Development of Nanoparticles for Antimicrobial Drug Delivery[END_REF].Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonairesii. Lipid nanoparticles (SLN and NLC) Lipid nanoparticles (LNPs) are colloidal nanocarrier systems, which have been introduced since 1990 in nanotechnology arsenal as a substitute carrier systems to LIP, emulsions and polymeric NPs[START_REF] Santos Ramos | Nanotechnology-based drug delivery systems for control of microbial biofilms: a review[END_REF][START_REF] Nafee | Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors[END_REF][START_REF] Naseri | Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application[END_REF][START_REF] Varshosaz | Development and optimization of solid lipid nanoparticles of amikacin by central composite design[END_REF]. There are two main types of LNPs including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), which are widely studied to treat chronic PA lung infections. It has been documented that LNPs are more stable in in vitro and in vivo studies and have prolonged released profiles compared to LIPs, with lower potential toxicity compared to other nanoformulations[START_REF] Naseri | Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application[END_REF][START_REF] Serramitjana | Pseudomonas aeruginosa RESPIRATORY INFECTIONS IN[END_REF]. Typically, LNPs have a spherical morphology with an average size between 40 nm and 1000 nm in diameter, and are composed of solid lipid phase dispersed in aqueous solution of surfactant[START_REF] Fang | Nanostructured Lipid Carriers (NLCs) for Drug Delivery and Targeting[END_REF][START_REF] Naseri | Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application[END_REF][START_REF] Serramitjana | Pseudomonas aeruginosa RESPIRATORY INFECTIONS IN[END_REF]. Generally, the size is affected by the type and concentration of the surfactant[START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF]. Several other criteria can influence the character of LNP including the preparation method, type of lipid, type and concentration of surfactant and temperature[START_REF] Varshosaz | Development and optimization of solid lipid nanoparticles of amikacin by central composite design[END_REF].SLNs are formulated using 0.1-30% (w/w) lipids, which are solid at body and room temperature, such as triglycerides, fatty acids, steroids, and wax, and surfactants such as poloxamers, phosphatidylcholine, in concentrations of about 0.5-5% (w/w) (Dos Santos Ramos et al. 2018; Martin et al. 2014; Nafee et al. 2014; Naseri et al. 2015; Serramitjana and Ciordia 2017; Varshosaz et al. 2010; Weber et al. 2014; Zhang et al. 2010) SLNs can contain one or more solid lipid components in the core which confer rigidity to the SLN and provide less mobility to the drug and thus allow their controlled release (Dos Santos Ramos et al. 2018). The lipid core can crystallize during the SLN storage which changes their properties. The use of liquid lipids in the core of LNP can reduce the ability of crystallization (Salvi and Pawar 2019; Serramitjana and Ciordia 2017). NLC are a modified generation of SLN, which differ by the composition of the lipid matrix to obtain a more solid and less crystalline core matrix than the SLN core (Salvi and Pawar 2019; Serramitjana and Ciordia 2017). NLC are formulated by replacing a fraction of solids lipids matrix with liquid lipids so that the core matrix contains a combination both of solid and liquid at room and body temperatures (Fang et al.; Naseri et al. 2015; Salvi and Pawar 2019; Serramitjana and Ciordia 2017). NLC's core is less organized in lipid matrix, and composed of different types of lipids, providing more space for active molecules (Serramitjana and Ciordia 2017). There are three structural types of NLC: (i) NLC Type I or imperfect type, where a fraction of solid lipid are replaced by liquid lipid that causes formation of imperfect crystal matrix, and gives more availability for loading drug than SLN; (ii) NLC Type II, known as amorphous type, with no crystalline structure which makes loading drugs more important than Type I with preventing the expulsion of loaded drugs; and (iii) NLC Type III or multiple type, Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonairessimilar to w/o/w emulsions, appropriate for drugs with a high solubility in liquid than solid lipid and associated with improving drug loading capacity and stability[START_REF] Naseri | Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF]. LNP are used as nanocarriers which can encapsulate and deliver both of hydrophilic and lipophilic drugs via many routes, especially pulmonary routes[START_REF] Naseri | Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application[END_REF] Pastor et al. 2014;[START_REF] Weber | Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) for pulmonary application: A review of the state of the art[END_REF]. Many inhaled LNP formulations were tested in mono-or combined therapy to treat the chronic lungs infection, which will be discussed below. In general, SLN and LNP are used to improve the drug efficacy against PA infection in CF patients. It should be noted that SLN are commonly used to target bacterial biofilm-forming more than the mature biofilm[START_REF] Martin | Strategies for Antimicrobial Drug Delivery to Biofilm[END_REF].LNPs have been used to encapsulate the antimicrobial agents in order to increase their activity in treating bacterial chronic lung infection. It has been reported that the encapsulation of aminoglycosides into LNPs enhanced their efficacy against PA clinical isolates (Sans-[START_REF] Sans-Serramitjana | Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients[END_REF]. In this context, many studies have been conducted to develop and test the efficacy of aminoglycosides (TOB and Amikacin) encapsulated into lipid NPs. For example, TOB has been loaded into NLC which produced a spherical NPs with a diameter of 250 nm and negatively charged -24 mV. Results showed that TOB-loaded NLC has displayed high encapsulation efficiency (EE) (≥93%) and biphasic released profile (initial rapid release than modulate and progressive release for three days)(Moreno-Sastre et al. 2016b). In an in vitro study, TOB-loaded NLC was safe against alveolar epithelial cell line (A549) and the encapsulation did not affect the activity of TOB against planktonic PA. In addition, a large distribution of NLC into lungs has been demonstrated after TOB-loaded NLC administration to mice via intratracheal route(Moreno-Sastre et al. 2016b). In another study, TOB has been loaded into TOB-loaded SLN and TOB-loaded NLC, which produced a negatively charged spherical NPs (-20 to -23 mV) with a diameter of 254 and 302 nm respectively. In vitro susceptibility of both formulations on PA strains grown planktonically and as biofilm using pegs lid model showed a slight reduction of MIC and MBEC values (1-2 log) against susceptible stains but no change against resistant isolates as compared to free TOB(Sans-Serramitjana et al. 2017a). A formulation of amikacin loaded into SLN has been developed byVarshosaz et al.

Furthermore, another study

  carried out by Sans-Serramitjana et al. demonstrated that both formulations have similar antimicrobial activity (same MIC values) against planktonic bacteria as free COL. In contrast, COL-loaded NCL demonstrated more efficiency in the eradication of PA biofilm, with an MBEC value (4-16 folds) lower than free COL in an in vitro pegs lid model (Sans-Serramitjana et al. 2016) and a faster killing of bacterial biofilm in an in vitro biofilm model formed on µ-Slide 8 glass bottom wells (Sans-Serramitjana et al. 2017b), while the BPC values were similar with free and COL-loaded NCL on the pegs lids models (Sans-Serramitjana et al. 2016). In addition, COL-loaded NCL could provide the ability to reach both of the outer and inner (deeper) layers of biofilm, which may explain why COL-loaded NCL has been more effective and much faster than free form in in vitro eradicating of PA biofilm (Sans-Serramitjana et al. 2017b). Additionally, a stability study of nanoencapsulated COL has been carried out over one year according to the International Conference of Harmonisation guidelines by evaluating the antimicrobial activity against PA and the physicochemical properties. Data demonstrated that COL-loaded SLN lost its antimicrobial activity after three months while COLloaded NCL which was more stable than COL-loaded SLN, maintained its activity throughout the study (Moreno-Sastre et al. 2016a).

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires developed as SLN loaded with free fatty acids including stearic acid, lauric acid (LA) and oleic acid, and with antimicrobial lipids usually found in human tissues known for their bactericidal activity[START_REF] Santos Ramos | Nanotechnology-based drug delivery systems for control of microbial biofilms: a review[END_REF][START_REF] Taylor | Multi-scale strategy to eradicate Pseudomonas aeruginosa on surfaces using solid lipid nanoparticles loaded with free fatty acids[END_REF]. These negatively charged SLN formulations have a diameter of 200 nm[START_REF] Santos Ramos | Nanotechnology-based drug delivery systems for control of microbial biofilms: a review[END_REF]. SLN trapped LA and oleic acid have been revealed to destabilize the adhesion of PA by rupturing the bacterial cell membrane and preventing the biofilm formation, in addition to preventing the bacterial resistance and reducing the adhesion of dead bacterial cell[START_REF] Taylor | Multi-scale strategy to eradicate Pseudomonas aeruginosa on surfaces using solid lipid nanoparticles loaded with free fatty acids[END_REF].

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires are used as nanocarriers including polyamidoamine dendrimers (PAMAM), which is mostly used, polypropylenimine, polyetherhydroxylamine, and polyether dendrimers(Velino et al.

  mucolytic activity. Two different studies have reported an improvement of the activity of cysteamine encapsulated into dendrimers against PA infection. Firstly, Brockman et al. have encapsulated cysteamine into PAMAM and demonstrated an in vitro significant decreasing in infection and bacterial growth in CF epithelial cells as compared to cysteamine or PAMAM alone (Brockman et al. 2017; Menjoge et al. 2010)(Menjoge et al. 2010). Later, Faraj et al.

  NPs in treating the planktonic bacterial infection and preventing or eradicating bacterial biofilm have been widely investigated. It has been documented that the metal NPs can induce the production of ROS and H2O2 leading disrupting the biofilm establishment. Among the metals NPs, Ag is the most promising one in bacterial treatment[START_REF] Vallet-Regí | Nanomaterials as Promising Alternative in the Infection Treatment[END_REF].Ag-NPs have been proved to have an antimicrobial efficacy against multi-drug resistant bacteria in CF patient, particularly against PA resistant strains[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF]. It has been reported that pretreatment of planktonic bacteria with Ag NPs could inhibit the formation of biofilm[START_REF] Park | Biofilm-inactivating activity of silver nanoparticles: A comparison with silver ions[END_REF]. Ag-NPs have antimicrobial activity presented by many mechanisms including releasing of Ag+ ions, which in turn generates ROS that cause cell damage[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Singh | Anti-biofilm effects of gold and silver nanoparticles synthesized by the Rhodiola rosea rhizome extracts[END_REF]. Other mechanisms such as disruption of cell membrane, DNA damage and enzyme inhibition have been observed[START_REF] Park | Biofilm-inactivating activity of silver nanoparticles: A comparison with silver ions[END_REF][START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF]. Several studies have investigated the efficacy of Ag-NPs alone, combined or incorporated with ATBs on bacteria in planktonic and biofilm modes. Several ATBs were tested in combination with Ag-NPs and showed a synergistic activity against bacterial infection. In this regard, synergistic effects of Ag NPs with ATBs and other agents have also been explored; for example, Ag NPs in combination with penicillin G, amoxicillin, erythromycin, and vancomycin resulted in enhanced antimicrobial effects against various Gram-negative and Gam-positive bacteria[START_REF] Fayaz | Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria[END_REF]. Al-Obaid et al. have developed a formulation of inhaled CIP encapsulated into Ag-based NPs made of silica coated with Ag NPs and embedded in chitosan. This formulation was significantly more effective in inhibiting the development of PA biofilm on the microtiter plate by 3-4 times compared to free CIP, and showed a significant decrease in cell count after 12 hrs of treatment of PA biofilm (Al-Obaidi et al. 2018). In another study, Habash et al. have studied the efficacy of citrate-capped Ag-NPs, alone or combined with TOB or aztreonam on PA (Habash et al. 2014). Firstly, it was demonstrated that the efficacy of Ag-NPs depends on its size, where the smaller size was more effective than the Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Figure 17 .

 17 Figure 17. Different types of NPs used as drug delivery system. NPs: nanoparticles -LIP: liposomes -SLN: solid lipid nanoparticule -NLC: nanostructured lipid carriers -PLGA: poly-lactic-co-glycolic acid -PLA: poly-lactic acid -PEG: polyethylene glycol.(Adapted from Joseph et al. 2016; Krishnamurthy and Chan 2020; Sarkar et al. 2017;[START_REF] Yeh | Nano-Based Drug Delivery or Targeting to Eradicate Bacteria for Infection Mitigation: A Review of Recent Advances[END_REF][START_REF] Yingchoncharoen | Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and What Is Yet to Come[END_REF]

Figure 18 . 59 Formulations d '

 1859d Figure 18. Scheme of Research work

Figure 1 .

 1 Figure 1. Schema describing the protocol used to formulate the LNPs loaded with COL (COL: Colistin, DA: Decanoic acid, FAR: Farnesol, adjuvants: DA and FAR, HIP: Hydrophobic ions pairing)

FormulationsFigure 2 .

 2 Figure 2. Interaction of blank LNP with COL and TOB. Variation of the size (A and C) and ZP (B and D) as a function of TOB (top panels) and COL (bottom panels) concentrations. Samples were prepared with an adjusted pH at 6.8. Each point is a mean value ± SD of 3 measurements of the same sample.

Figure 3 .

 3 Figure 3. Characterization of different LNP loaded with LA (top panels) and DA (Bottom panels). Variation of LNP median volume-weighted size versus A) LA and C) DA concentration in the total formulation. Variation of the LNP mean ZP versus B) LA and D) DA concentration in the total formulation. Size and ZP distributions were recorded in triplicates. Each displayed measurement is a mean value ± SD. Samples were prepared with an adjusted pH at 6.8. (n=3; each color represents some different formulations; each formulation was measured 3-9 replicates).

Figure 4 .

 4 Figure 4. Study of the influence of pH on the ZP value of LNP-DA and Blank LNP. For each pH values, ZP value was recorded in triplicate. Each displayed measure is a mean value ± SD.

Figure 5 :

 5 Figure 5: Diagram showing the percent ionization of cationic ATBs (COL and TOB) and FA (DA and COL as function of pH. pH value variate from approximately 1 to 14. The percent of ionized form was determined using the pka values of each ionic groups of different molecules reported in literature.

Figure 6 : 8 117Figure 7 .

 687 Figure 6: The chemical structure of TOB and COL. (a) Chemical structure of TOB with the corresponding individual pka values of each amino group presented in its structure [36-38]. (b) Chemical structure of COL with the individual pka values of each amino group and which are almost equal to 10 [39,40].

Figure 8 .

 8 Figure 8. Study of the influence of pH on the ZP value of LNP-DA supplemented with various COL concentrations. For each pH values, ZP value was recorded in triplicate. Each displayed measure is a mean value ± SD.

Figure 9 .

 9 Figure 9. Effect of COL formulation process with LNP-DA containing of 20 g/L of DA on A) LNP median particle size and B) LNP mean ZP value. COL was incorporated either before LNP-DA/COL or after LNP-DA + COL the formulation process at a final concentration of 1 mM in the total suspension. Each displayed measure is a mean value ± SD. Samples were prepared with adjusted pH at 6.8. (*p<0.05, ** p<0.01)

  involved in the formulation gives energy to the cationiques pour traiter les biofilms bactériens pulmonaires system that could favor the formation hydrophobic ion pairs (HIP) between COL and DA and their extraction in the oil core of the LNPs. LC-MSMS assays of COL from both type of formulations gave similar concentrations values around 1.15 mg/mL, showing the absence of COL degradation (results not shown).

Figure 10 :

 10 Figure 10: The influence of pH adjustment at 6.8 before formulation on different LNPs size (LNP-DA, LNP-DA/COL and LNP-DA/FAR/COL). Size distribution was recorded in triplicate. Each displayed measure is a mean ± SD.

FormulationsFigure 11 :

 11 Figure 11: Comparison of MIC between free COL and various LNP-COL, measured against A) PAO1 and B) AB122. For LNP-DA+COL, DA concentration was 500 mg/L. For LNP-FAR+COL, FAR concentration was 100 mg/L. For LNP-DA/FAR+COL, DA/FAR mass concentration ratio was 60/100. For LNP formulated with COL, the ratios between molecules evolved along with the concentration of COL. Error bars represent the means ± SD (n=4-21). Efficacy of treatment was statistically evaluated using the one-way ANOVA test (****P<0.0001).

124

  Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Figure 12 :Figure 13 :

 1213 Figure 12: In vitro evaluation of the efficacy of LNP-DA/COL and LNP-DA/FAR/COL against adherent gram-negative biofilms formed by PAO1 (panels A and C) and AB122 (panels C and D). Antibiofilm activity was measured by evaluation of the total enzyme activity (FDA test, A and B) and by the amount of CV retained by the biofilm mass (C and D). Biofilms were grown on 96-well plate for 48 hours and treated for 24H. The positive control presents the bacterial biofilm in absence of antibiotics or active ingredients.

12. 5 5 126

 55 hrs, then it progressively decreased to reach a stationary level (steady state) beyond 20 hrs. The additional of free COL at a low concentration, e.g. 0.25 times MIC, did not change the bacterial growth rate as compared to control. The addition of free COL at a concentration lower or equal to 2 times the MIC (0.5≤ [COL] ≤2) did not affect the value of bioluminescence intensity until 7.5 hrs as compared to the control. Beyond 7.5 hrs, the bacterial growth increased progressively to achieve a stationary level value higher than that obtained with the control.Contrary, the addition of encapsulated COL (LNP-DA/COL and LNP-DA/FAR/COL) at a concentration lower than 2 times the MIC slightly decreased the bacterial growth rate until 7.Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Figure 14 :

 14 Figure 14: Evolution of PAO1 concentration after 40 hrs of exposition to various concentrations of free (A) or formulated COL as LNP-DA/COL (B) and LNP-DA/FAR/COL(C) in the ASM, expressed in the number of times the MIC (1 mg/L). PA concentration is expressed as 𝑳𝒐𝒈 𝟏𝟎 CFU/mL. Gray band around lines corresponds to 95% confidence level interval of the GAM smoothing model's expected mean values (n=2-13). Colony concentrations are fitted following the equation of inhibitory Emax model (Eq. 1), where CFU(t) represents bacterial survival within various ATBs concentrations. The limit of quantification was fixed at a (𝑳𝒐𝒈𝟏𝟎 𝑪𝑭𝑼/𝒎𝑳) value of 2.3.

  (Log10 CFU/mL)) at concentrations less than or equal to 10 times the MIC. Increasing the COL concentration from 10 to 25 times the MIC suddenly decreased the bacterial concentration (Log10 CFU/mL) from 8.70 ± 0.17 (Log10 CFU/mL) to reach a value under the limit of quantification; and this value remains under the LOQ beyond the concentration of 25 times MIC.The bacterial survival within various encapsulated COL (LNP-DA/COL or LNP-DA/FAR/COL) is shown in Fig.14Band Fig.14Crespectively. The addition of both formulations at a concentration lower or equal to 5 MIC exhibited similar bacterial concentration, corresponding to the CFU0 value. This bacterial concentration decreased with the addition of formulated COL at a concentration of 10 times the MIC, which produced a reduction of more than 50% of the initial bacterial concentration. The addition of both formulated COL forms at a concentration greater than 10 times MIC displayed a bacterial concentration under LOQ.

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

  formulation process. Firstly, the assay of encapsulating COL into Blank-LNP failed due to the cationic ATBs (such as TOB and COL) being very polar molecules which makes them difficult to be encapsulated in lipophilic nanocarriers. Accordingly, we decided to improve the interactions of cationic ATBs with LNPs by hydrophobic ions pairs (HIP) formation[START_REF] Li | Driving forces for drug loading in drug carriers[END_REF] Ristroph and Prud'homme 2019). The LNPs were loaded with FA like DA, an amphiphilic molecule having a carboxylic group (hydrophilic) which imparts negative charges to LNP, which attracts the positively charged ATB and allows its association with LNPs. So, cationic ATBs (especially COL) were incorporated into LNP loaded with FA (especially DA) and producing LNP-DA/COL. Did the nanoencapsulation of FA-loaded LNP enhance the encapsulation of cationic ATBs?

Formulations d '

 d antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Figure 19 .

 19 Figure 19. Inhalation Nose only system from (TSE®)

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

	; Goltermann and Tolker-Nielsen 2017;
	Murakami et al. 2017). Indeed, Goltermann et al. demonstrates that overproduction of these
	polysaccharides in PA aggregates increases the tolerance of bacteria to TOB but also to
	ciprofloxacin (CIP) (Goltermann and Tolker-Nielsen 2017) . Similarly, Cotton et al. showed that
	treating abiotic biofilms of PA with alginate lyase increased the efficacy of 1.0 μg/mL
	gentamicin and 0.25 μg/mL CIP by 30% and 100%, respectively (Cotton et al. 2009). As
	diffusion of CIP, a relatively small and uncharged molecule, should not be limited by the matrix
	polysaccharides, these may indicate that the presence of these components altered the
	susceptibility of the bacteria in aggregates to ATBs by other mechanisms (Ciofu and Tolker-
	Nielsen 2019). In another study, Billings et al. showed that biofilm made by a PAO1 mutant
	incapable of producing Psl was 4 times more sensitive to COL than wild-type PAO1 biofilms

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires et

  al. 2006). Thus, NO-donor molecules have been evaluated in vitro and in vivo for PA biofilm

	dispersal. For example, NO-donor sodium nitroprusside applied at 500 nM for 24h reduced the
	biomass of in vitro abiotic biofilms of PA by 80%. Addition of TOB at 100 µM (0.2 µg/L) caused
	almost-complete removal of the biofilms
	i. Nitric oxide
	NO is the most studied agent for PA biofilm dispersion (Barraud et al. 2014; Fleming and
	Rumbaugh 2017; Wille and Coenye 2020). At concentrations non-toxic to humans, NO
	activates several PDEs in PA, resulting in degradation of c-di-GMP and increased expression
	of enzymes such as the endonuclease EndA, which degrade the matrix (Barraud et al. 2009;
	Barraud et al. 2006; Wille and Coenye 2020). It also increases the motility of PA cells (Barraud

Table 1 .

 1 In vitro biofilm models linked to gram-negative chronic lung infections

	Types of models	Methods	Examples	Description	Advantages	Disadvantages	Ref
		Models adsorbed on abiotic inert surfaces	Microtiter plate / Pegs lid models	-biofilm attached to the bottom of a microtiter plate wells -Peg lid model: based on biofilm formation on the pegs of	-Feasibility and reproducibility -Produce biofilm similar to attached biofilm formed on ETT	-Biological variation between wells -No differentiation between components matrix, living and dead cells -Absence of human body involvement (immune cells	(Ciofu et al. 2017; Coenye and Nelis 2010; Harrison et al. 2010; Lebeaux et al. 2013;
				a lid of a well plate.		response, interaction between	Roberts et al.
						pathogen and host cells)	2015)
		Models		Based on a biofilm			(Coenye and
	Static biofilm models	adsorbed on biotic surfaces	Cell-culture models	formed on living cells (e.g. epithelial cells of the human respiratory tract)	--Interaction between pathogen and host cells	-Absence of human body involvement (immune cells response)	Nelis 2010; Moreau-Marquis
				cultured in monolayers.			et al. 2010)
					-Simple and flexible and		(Coenye and
					produce bacterial		Nelis 2010;
		Non-attached biofilm model	Bacteria-embedded in beads based models	Biofilm produced by trapping bacteria into agar or alginate beads dispersed in growth medium	clusters similar to aggregate observed in vivo in CF patients -Nutrients and oxygen gradients -Heterogeneous growth	-Absence of human body involvement (immune cells response, interaction between pathogen and host cells)	Pedersen et al. 1990; Sønderholm et al. 2017a; Torres et
							al. 2019)
	Dynamic biofilm models	Flow system	Continuous flow cell system	Biofilm grown on a flat walled transparent chamber supplied by a continuous flow of nutrients and oxygen.	High structured biofilm -Allows a continuous supply of ATBs, which allows the detection of viable cells and	-Time consuming and low reproducibility -High biofilm variation in the same chamber in an in vivo lung biofilm -Mushroom structure not observed	(Bjarnsholt 2013; Ciofu et al. 2017; Coenye and Nelis 2010; Lebeaux et

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires 2014; Roberts et al. 2015; Shi et al. Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Table 2 .

 2 In vivo models linked to PA chronic lung infections

	Type of
	biofilm-related	Types of models	Description	Animals	Route of administration	Ref
	infection
						(Bielen et
						al. 2017;
						Bragonzi et
		Bacteria-embedded in beads-based models	Bacteria embedded into agar or seaweed alginate microsphere beads	Rats, mice, guinea pigs, rhesus monkeys	Intratracheal instillation of beads	al. 2005; Christopher sen et al. 2012; Coenye and
	CF lung infections					Nelis 2010; Lebeaux et al. 2013)
		CF mouse model infected with chronic lung infection	Transgenic murine animal was inoculated intratracheally with agar beads or PA mixed with alginate	Transgenic mice (CFTR-)	Intratracheal instillation of PA embedded into agar beads or PA mixed with alginate (2%)	(Bragonzi 2010; Hoffmann et al. 2007; Hoffmann et al. 2005; Lebeaux et al. 2013)
						(Bielen et
					Oropharyngea	al. 2017;
	Endotrach eal tubes	Lung biofilm attached to a surface in ventilated animal	Inoculation of high bacterial concentration in mechanical ventilated animal, or insertion of catheters/ETT colonized with PA	Pigs (mostly used), dogs, rats and mice	l /orotracheal instillation of planktonic PA or intratracheal insertion of catheters/ETT colonized with	Groneberg et al. 2002; Kaneko et al. 2001; Lebeaux et al. 2013; Li Bassi et al. 2014;
					PA	Nagata et
						al. 2004)

  . Inhaled ATBs have been used extensively in treating bacterial chronic infections in CF patients and in others lower respiratory tract infections such as COPD and VAP bacterial infection(Garcia-Contreras et al. 2015; Maselli et al. 2017; Quon et al. 2014; Restrepo et al. 2015). Due to their antimicrobial efficacy and apparent low permeability across the blood-pulmonary barrier[START_REF] Marchand | Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 3. Tobramycin[END_REF]

[START_REF] Marchand | Aerosol Therapy with Colistin Methanesulfonate: a Biopharmaceutical Issue Illustrated in Rats[END_REF]

, the two most commonly used ATBs by inhalation to control chronic lung infections caused by Gram-negative bacteria are TOB and COL (administered as a prodrug called colistimethate sodium (CMS))

[START_REF] Bos | The fate of inhaled antibiotics after deposition in cystic fibrosis: How to get drug to the bug?[END_REF][START_REF] Hamed | Tobramycin inhalation powder for the treatment of pulmonary Pseudomonas aeruginosa infection in patients with cystic fibrosis: a review based on clinical evidence[END_REF][START_REF] Heirali | The effects of inhaled aztreonam on the cystic fibrosis lung microbiome[END_REF]

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires and

  residence time of PLGA NPs can be tailored by modifying polymers or adding an additive[START_REF] Klinger-Strobel | Aspects of pulmonary drug delivery strategies for infections in cystic fibrosiswhere do we stand?[END_REF][START_REF] Ungaro | Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers[END_REF]. In this context, Ungaro et al. have developed a formulation of TOB loaded into PLGA NPs with different hydrophilic polymers (polaxamers, chitosan, alginate or polyvinyl alcohol)[START_REF] Klinger-Strobel | Aspects of pulmonary drug delivery strategies for infections in cystic fibrosiswhere do we stand?[END_REF][START_REF] Ungaro | Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers[END_REF]. This nanocarrier was trapped into microcarriers made of lactose for pulmonary delivery routes. They showed that the use of alginate allows effective trapping and release of TOB over one month.

	The in vivo biodistribution analysis in rat lungs after intra-tracheal delivery demonstrated that
	only polyvinyl alcohol -modified PLGA NPs penetrated deeply into lung while chitosan-modified
	PLGA NPs were found frequently in the upper lung airway (Ungaro et al. 2012).
	ii. Chitosan

Chitosan is a hydrophilic polymer used to formulate polymeric NP formulation or as an excipient in PLGA formulation. Chitosan is a natural, non-toxic and cationic linear polysaccharide bio-polymer obtained by deacetylation of chitin. It is composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)

[START_REF] Danhier | PLGA-based nanoparticles: An overview of biomedical applications[END_REF][START_REF] Forier | Lipid and polymer nanoparticles for drug delivery to bacterial biofilms[END_REF]

. Due to its mucoadhesive, biodegradable, and antimicrobial properties, and its easy modification, chitosan was widely used in drug encapsulation for pulmonary delivery

[START_REF] Danhier | PLGA-based nanoparticles: An overview of biomedical applications[END_REF][START_REF] Zaidi | Nano-therapeutics: A revolution in infection control in post antibiotic era[END_REF][START_REF] Zeng | Recent advances of chitosan nanoparticles as drug carriers[END_REF]

).

Aminoglycosides such as TOB were encapsulated into chitosan NPs in order to treat PA lung infection. In a study conducted by Decon et al., TOB was encapsulated into chitosanalginate NPs of 505 nm. The encapsulated TOB displayed similar MIC than free TOB against planktonic PA, but increased the survival rate from 40% (obtained with free form) to 80% (obtained with encapsulated form) by administrating the encapsulated TOB 96 hrs prior to PA infection on an in vivo model of PA infection (in Galleria mellonella)

[START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Deacon | Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: Formulation, characterisation and functionalisation with dornase alfa (DNase)[END_REF]

.

Table 3 .

 3 Advantages and disadvantages of the NPs formulations

	Types of Formulations	Benefits of administration	Disadvantages	References
		via pulmonary routes
	Liposomes	• Encapsulate both		• High cost production	(Akbarzadeh et al. 2013;
		hydrophilic and lipophilic	• Rapid uptake by	Anwekar et al. 2011;
		drugs			reticuloendothelial system	Çağdaş et al. 2014; Dos
		• Increase drug efficiency	reducing circulation half-life	Santos Ramos et al. 2018;
		and therapeutic index	(short half-life)	Huh and Kwon 2011; Madni
		• Increase drug stability by	• Low water solubility	et al. 2014; Mehta 2016;
		encapsulation		• Possibility of phospholipids	Shah et al.)
		• Biodegradable and	oxidation and hydrolysis
		biocompatible		• Leakage and fusion of
		• non-toxic and non-	encapsulated drug during
		immunogenic lipids	lyophilization and process jet
		• Solubilise poorly soluble	milling
		drugs			• Relative instability during
		• Good safety profile	storage and nebulisation
					• Rapid clearance
	Lipid nanoparticles (NCL and	• Better biopharmaceutical	• Variability of drug release	(Cipolla et al. 2014;
	SLN)	properties		profile due to degradation of	Ghasemiyeh and
		• Biodegradability		some lipid composed matrix	Mohammadi-Samani 2018;
		• Better bioavailability	• Potential toxic effects of burst	Mehta 2016; Pastor et al.
		• Good tolerability in the airway • Mucoadhesion and prolonged drug retention	drug release • Macrophage drug clearance • Small particles size makes them not appropriate for	2014; Sans-Serramitjana et al. 2016; Serramitjana and Ciordia 2017(Hidalgo et al. 2015))
		time in lungs		deeply lung delivery that can
		• Avoiding the first pass	overcome by encapsulating
		effect	and	reducing	into lipid microparticles or
		administered dose	coating with PEG

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

In vitro evaluation of Pseudomonas aeruginosa chronic lung infection models: Are agar and calcium-alginate beads interchangeable?

  alginate de calcium. À une concentration de la TOB équivalent à 10 fois la CMI, la luminescence émise par PA dans les billes de l'agar était réduite de 95 % après 40 h, tandis qu'elle revenait à la même valeur initiale pour PA piégés dans des billes de l'alginate. La réduction de l'efficacité de la TOB était encore plus importante lorsque les billes d'alginate étaient dispersées dans un milieu artificiel simulant le mucus. Ces résultats montrent que les modèles de billes de l'agar et de l'alginate de calcium peuvent être interchangeables uniquement pour l'ATB non chargé, comme la CIP, mais pas pour l'ATB cationique, comme la TOB. Les expériences in vitro réalisées dans le cadre de cette étude pourraient être un moyen

	Résumé
	Les modèles d'animaux d'infection pulmonaire chronique à Pseudomonas aeruginosa (PA)
	sont des outils (bénéfiques) utilisés pour améliorer le traitement d'antibiotique (ATB). Deux
	modèles principaux basés sur l'instillation pulmonaire de PA incorporée dans de billes de l'agar
	ou des de l'alginate de calcium sont actuellement utilisés. Cependant, ces deux polymères qui
	ont été utilisés pour préparer des billes ont des propriétés différentes ; par exemple, l'agar est
	un polysaccharide neutre alors que l'alginate est anionique. Nous avons émis l'hypothèse que
	l'effet d'un ATB sur PA incorporée dans des billes de l'agar ou de l'alginate de calcium dépend
	de leurs propriétés physico-chimiques, notamment de sa charge et de sa concentration.

Pour tester cette hypothèse, les PA ont été incorporées dans des billes de l'agar ou de l'alginate de calcium, et sont dispersées dans un milieu de croissance contenant soit de la tobramycine (TOB), sélectionnée parmi les ATB cationique (sélectionnée comme un ATB cationique), soit de la ciprofloxacine (CIP) sélectionnée comme un ATB zwitterionique neutre.

In vitro, des courbes de bactéricide évaluant l'efficacité des ATB en fonction de temps ont été réalisées grâce à la mesure de la luminescence émise par PA (en mesurant la lumière émise PA bioluminescente) pendant 42 h en présence de d'ATB à différents concentrations (0-100 fois la CMI). En présence de la CIP, les courbes de bactéricide obtenues avec du PA piégé dans des billes de l'agar ou de l'alginate de calcium étaient comparables, quelle que soit la concentration de la CIP utilisée. En présence de la TOB, une nette différence a été observée entre les courbes de bactéricide obtenues avec du PA piégé dans des billes de l'agar ou de l'alginate de calcium. Bien que PA piégés dans des billes de l'agar ont montrés la même sensibilité que celle des PA planctoniques, PA ne répond pas (ne sont pas sensible) à la TOB pour des concentrations allant jusqu'à une fois la CMI lorsqu'elles sont piégés dans des billes de l'rapide pour évaluer l'effet de chaque modèle sur un ATB donné avant de procéder à des expérimentations animales.

ARTICLE 3

Antibacterial and Antibiofilm Effects of colistin-loaded lipid nanoparticles treating gram negative chronic lung infection 99 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires Antibacterial and Antibiofilm Effects of colistin-loaded lipid nanoparticles treating gram negative chronic lung infection Rana Awad 1,2 , Sandrine Marchand 1,2,3 , William Couet 1,2,3 Mohamad Nasser 4 , Fréderic Tewes 1,2

	1 INSERM U1070, Université de Poitiers, France

2 Faculté de Médecine et de Pharmacie, Université de Poitiers, France 3 Service de Toxicologie et de Pharmacocinétique, Centre Hospitalier Universitaire (CHU) de Poitiers, France 100 Formulations d'

antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires Résumé Les

  infections pulmonaires chroniques sont principalement associées à des bactéries produisant des biofilms et P. aeruginosa (PA) est le pathogène prédominant responsable du développement de ces biofilms chez les patients atteints de maladie inflammatoire chronique telle que la mucoviscidose. Les bactéries au sein des biofilms sont plus difficiles à éradiquer que des bactéries planctoniques et des concentrations élevées d'antibiotiques sont généralement nécessaires. Les antibiotiques inhalés sont utilisés pour traiter les infections bactériennes chroniques, la tobramycine (TOB) et la colistine (COL) sont parmi les premiers choix de traitements inhalés. Cependant, ces antibiotiques (ATBs) sont chargés positivement et leur efficacité est limitée contre les biofilms. Ce problème peut être contourné en utilisant des systèmes particulaires administrable par inhalation afin d'améliorer l'efficacité des ATBs en augmentant leur pénétration dans la matrice de biofilm. Les nanoparticules lipidiques

	cinétiques de bioluminescence pendant 40 heures, suivie par un comptage des concentrations
	bactériennes (log10 CFU/ml). Un modèle d'inhibition Emax a été utilisé pour décrire les
	concentrations bactériennes en fonction des concentrations de COLI.
	Les résultats ont montré que les particules blanches (Blank-LNP) n'ont pas interagit avec les
	ATBs cationiques. L'incorporation de FA (LA ou DA) dans la LNP a conféré une charge
	négative aux particules, ce qui a permis l'interaction avec les ATBs cationiques en produisant

(LNP), qui sont des nanotransporteurs capables d'encapsuler et de délivrer des médicaments à la fois hydrophiles et lipophiles, semblent être des candidats intéressants. Dans notre étude, le choix des acides gras (FA) comme adjuvants de synthèse aux LNP a été évalué ainsi que l'ajout de molécule non ATB anti-biofilm, comme le farnesol (FAR). L'objectif de cette étude a été de développer un nouveau système d'administration en encapsulant des antibiotiques cationiques (COL et TOB) dans des LNPs chargés d'adjuvants tels que l'acide laurique (LA) ou l'acide décanoïque (DA) et / ou le FAR, et d'évaluer leurs activités sur PA et A. baumannii. Les LNPs ont été formulées par nanoémulsification par inversion de phase. Divers adjuvants (LA, DA, FAR) ont été testés pour améliorer l'efficacité d'encapsulation des ATBs et augmenter leur efficacité. La taille et le potentiel zêta de ces LNPs ont été étudiés pour caractériser les formulations. L'efficacité des LNPs a d'abord été évaluée sur les concentrations minimales d'inhibition (CMI) des PA et A. baumannii planctoniques. Dans un second temps, les LNPs sélectionnées ont été évaluées sur des biofilms adhérents de PA et d'AB en mesurant l'activité enzymatique et la biomasse totale des biofilms. Enfin, les mêmes LNPs ont été testés sur un modèle de biofilm PA intégré dans des billes d'alginate et leur efficacité a été évaluée par des des appariements d'ions hydrophobes (HIP). Ensuite, la COL a été encapsulée dans la LNP chargée avec du DA en présence ou non de FAR, ce qui conduit à la réalisation de deux formulations différentes de COL encapsulée (LNP-DA/COL, le LNP-DA/FAR/COL). Les 101 Formulations d'

antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires résultats

  ont montré que la COL était moins présente à la surface de LNP-DA lorsqu'elle était ajoutée avant le processus de formulation qu'après ce processus. En terme d'efficacité, une thérapie basée sur la combinaison des adjuvants (DA et/ou FAR) avec la COL, sous forme encapsulée ou non, a montré une augmentation significative de la sensibilité des souches résistantes d'A. baumannii à la COL. Les formulations de COL ont de plus montré in vitro, une suppression complète de l'activité enzymatique d'un biofilm adhérent d'A. baumannii, ce qui n'a pas été observée avec la COL libre. Enfin, la COL encapsulée a réduit la concentration efficace 50 (EC50) d'un facteur 2 par rapport à la COL libre sur la croissance d'un modèle in vitro de biofilm pulmonaire non adhérent à PA. Ceci suggère que l'encapsulation de la COL dans les deux types de formulations a augmenté son efficacité contre les biofilms bactériens en comparaison avec la COL libre et que l'effet synergique des adjuvants (DA, FAR) a montré un intérêt potentiel dans ces formulations. Ces résultats sont prometteurs et suggèrent que l'encapsulation de la COL pourrait améliorer l'activité antibactérienne et anti-biofilm de la COL contre les infections pulmonaires à Gram négatif.

Mots clés: Pseudomonas aeruginosa, Acinetobacter baumannii, biofilm, antibiotiques cationiques, acide décanoïque, farnesol, nanoparticules lipidiques 102 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Table 4 . Composition of Blank-LNP

 4

	Compound	Reagents	Mass (mg)
		MiliQ® water	3000
	Aqueous	NaCl	90
	phase	Solutol® HS15	850
		Icy MiliQ® water	10000
		Labrafac® lipophile WL1349 1000
	Oily phase
		Phospholipon® HS90

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

A V B 123 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires µg

  /ml, but this MIC was obtained for much a lower DA/FAR mass ratio of 28/17, i.e. 28 mg/L of DA and only 17 g/L of FAR.

  COL displayed an CFU∞ value of 0.6 ± 0.2 (Log10 CFU/mL), whereas the formulated COL displayed a value of 0.1± 0.1 (Log10 CFU/mL) with LNP-DA/COL and 0.2 ± 0.2 (Log10 CFU/mL) with LNP-DA/FAR/COL. Although the difference between treatments with free COL and LNP-DA/COL was more important as compared to others, we couldn't compare the CFU∞ values since they were under LOQ. The difference between diverse treatments was better

	observed by comparing the EC50 values, where free COL displayed a value of 20 ± 3 mg/L
	higher than those obtained with encapsulated COL that displayed 10 ± 0.5 mg/L with LNP-
	DA/COL and LNP-DA/FAR/COL. There was no difference between EC50 values obtained with
	both encapsulated COL forms

value was approximately similar with free or formulated COL (approximately 8.70 Log10 CFU/mL). On another aspect, the CFU∞ values variate with different treatments, in which free 128 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

  Staphylococcus aureus biofilm[START_REF] Bhattacharyya | Sol-gel silica controlled release thin films for the inhibition of methicillin-resistant Staphylococcus aureus[END_REF]. Jabra-Rizk et al. demonstrated that FAR increased the in vitro susceptibility of Staphylococcus aureus biofilm against gentamicin[START_REF] Jabra-Rizk | Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility[END_REF].

129 Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

  table 1 in general introduction). Several studies have utilized models related to this model using bead sizes ranging from 15 to 265 µm

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

PRASE, Université Libanaise, Hadath-Beirut

LIST OF TABLES

 Table 1. In vitro biofilm models linked to gram-negative chronic lung infections ...... Table 2. In vivo models linked to PA chronic lung infections ....................................

Formulations d'antibiotiques cationiques pour traiter les biofilms bactériens pulmonaires

Data reported that the MIC value of TOB-PEG was approximately 10 times higher than that of free TOB (MICTOB-PEG=13.9 µmol/L ˃ MICTOB=1.4 µmol/L) on planktonic PA, while it was 3.2 times more effective in removing in vitro PA biofilm on microtiter plate [START_REF] Du | Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms[END_REF]. In another study, the Smyth group synthesized TOB-PEG with three different conjugation methods by chemical conjugation of monomethyl ether succinate (mPEG-COOH) to TOB, producing three TOB-PEG displaying a difference in activity against in vitro PA biofilm. The most important antimicrobial activity against pseudomonal biofilm was found with TOB-PEG formulated using the conjugation method previously developed in proteins PEGylation developed by Hermanson et al., which was 4 times more effective than TOB and 2 times than the classic method used to PEGylate the TOB. This selected TOB-PEG showed MIC80 value higher by 8.6 times than free TOB on planktonic PA (8.6 µM vs. 1 µM), while MIC80 was approximately 5.6 times lower than free TOB on PA biofilm grown on microtiter plate. In addition, compared to free TOB, TOB-PEG was significantly more effective than free TOB in minimizing the proliferation of PA culture growing in CF-like mucus barrier model by 2.8-times, 4.2-times, and 4.9-times after 5H,7H and 9H (Bahamondez-Canas et al. 2018). PEGylation of ATB appears to be a promising potential approach to overcome the problem of bacterial resistance in chronic PA infection and requires further study in the future.