Soutenance mémoire
Alteration of bacteria’s efflux pump activity
In this regard, recently-reported advances could be mentioned. Khameneh et al. developed piperine-containing nanoliposomes as a vector for gentamicin. The liposomal formulation was specifically developed to fight methicillin-resistant Staphylococcus aureus (MRSA), an antibiotic-resistant bacteria which is widely recognized as a nosocomial pathogen [20]. The encapsulation of gentamicin in classical nanoliposomes or piperine-containing nanoliposomes resulted in a dramatic decrease of minimum inhibitory concentration (MIC) values of 16- and 32 folds, respectively. Similarly, minimum bactericidal concentration (MBC) values were also reduced 4- and 8- folds for encapsulated gentamicin in classical nanoliposomes or piperine-containing nanoliposomes, respectively. These hopeful results were attributed to the piperine inhibiting effect on the bacterial efflux pump. This argument was confirmed using ethidium bromide (EtBr) fluorescence assay. The fluorescence of this compound occurs only when it is bound to nucleic acid. Accordingly, bacterial suspension was incubated with EtBr for 30 min in the presence of: i) bare nanoliposomes (without piperine), ii) piperine-containing nanoliposomes or iii) piperine in its free form. After centrifugation and washing of bacteria, the loss of fluorescence was checked in order to investigate the efflux of EtBr outside bacterial cells. Consistently, a gradual decrease of fluorescence during the assay period was observed in the first case, i.e. in the absence of piperine. However, in the presence of piperine the fluorescence was significantly enhanced indicating a significant inhibition of the efflux pump [20]. Therefore, the enhanced antibacterial activity of gentamicin encapsulated in piperine-containing nanoliposomes is likely to be the consequence of an increase in its intracellular concentration. It is of note that piperine in its free form was less effective in inhibiting the efflux pump than the liposomal one, as demonstrated by the EtBr fluorescence assay.
Antibiofilm activity
Nitric oxide (NO)-releasing NP were found to prevent the formation of bacterial biofilms and to eradicate already formed biofilms. Some examples of recent breakthroughs in this domain are presented hereafter. Jardeleza et al. encapsulated isosorbide mononitrate (ISMN), as NO donor into different liposomal formulations with the purpose to enhance the antibiofilm activity against Staphylococcus aureus’s biofilms [21]. NO-releasing multilamellar vesicles (MLV) efficiently eliminated S. aureus’s biofilms in vitro. A five min-exposure to 60 mg/mL ISMNloaded MLV induced an almost complete eradication of the biofilms. Paradoxically, the authors observed that at low concentrations NO-releasing MLV enhanced the formation of biofilms, which is in accordance with previously obtained results [22]. Duong et al. developed nanoparticulate NO-core cross-linked star polymers as new therapeutics able to combating biofilms that are frequently formed during long exposure of the body to medical devices and catheters [23]. These systems were found to release NO in a controlled and slowed-down manner in bacterial cultures and showed great efficacy in preventing both cell attachment and biofilm formation in P. aeruginosa over time. This study unveiled, in part, the inherent mechanisms of NO’s antibiofilm activity. Accordingly, NOreleasing NP inhibit the switch of planktonic cells in contact with a surface to the biofilm form by continuously stimulating phosphodiesterase activity. Thus, NO-releasing NP maintained low intracellular concentrations of cyclic di-guanosine monophosphate (c diGMP) in the growing bacterial population, thereby confining growth to an unattached freeswimming mode [23]. The dual delivery of two antibiotics via their co-encapsulation in nanoliposomes is another proposed strategy to bypass resistance mediated by biofilm formation. For instance, Moghadas-Sharif proposed vancomycin/rifampin-co-loaded nanoliposomes as a new therapeutic against Staphylococcus epidermidis [24]. This strategy was based on two points. First, combination therapy of vancomycin and rifampicin helps avoid the emergence of rifampin-resistant strains. Indeed, numerous studies have already reported the antibiofilm activities of rifampin in combinations with other antibiotics [25]; [26]; [27]. Second, rifampicin fails alone to eradicate bacterial biofilm [28]. Nevertheless, the developed liposomal combination was ineffective to eradicate S. epidermidis‘s biofilm. The authors attributed this result to the lack of liposomal adsorption or low penetration into the bacterial biofilm [24]. A more adjusted formulation with enhanced penetration behavior into the biofilm may lead to the initially expected effect.
Protection against enzymatic degradation and inactivation by polyanionic compounds
Nanoparticulate delivery systems provide a physical barrier shielding the entrapped antibiotic from aggregation and inactivation with polyanionic compounds, such as bacterial endotoxins e.g. LPS and LTA. Additionally, encapsulation may protect antibiotics against enzymatic degradation by β-lactamases, macrolide esterases and other bacterial enzymes [31]. Two decades ago, Lagacé et al. demonstrated that liposomal encapsulation of ticarcillin or tobramycin reverse the resistance of P. aeruginosa strains towards these both antibiotics [32]. Growth inhibition of ticarcillin- and tobramycin- resistant strains was achieved using ticarcillin and tobramycin liposomal formulations at 2 % and 20 % of their respective MIC. Liposomal formulations were as effective against the β-lactamase -producing strains as βlactamase -non producing ones. Recently, Alipour et al. demonstrated the versatility of liposomal encapsulation in protecting tobramycin or polymyxin B from inhibition by LPS, LTA, neutrophil-derived DNA, actin filaments (F-actin) and glycoproteins e.g. mucin, common components in the CF-patients ‘sputa [33]. Being polycationic, tobramycin and polymyxin B can bind to these polyanionic compounds and thereby have their bioactivity reduced. The authors postulated that “liposomes are able to reduce the antibiotic contact with polyanionic factors in the sputum and to enhance bacteria-antibiotic interactions” [33]. In vitro stability studies revealed that liposomal formulations were stable after an 18 h-incubation at 37°C with i) a supernatant of biofilm-forming P. aeruginosa, ii) a combination of DNA, F-actin, LPS and LTA or iii) an intact or an autoclaved patient’s sputum. No significant differences with respect to control (before incubation) were observed. Furthermore, the antibacterial potency of liposomal antibiotics were checked after both short (3 h) and prolonged (18 h) exposure to a combination of DNA/F-actin or LPS/LTA at different concentrations. It was found that for both free and liposomal drugs the antibioactivity was reduced in a concentration-dependent manner. However, much higher concentrations (100 to 1000 mg/L) and (500 to 100 mg/L) of LPS/LTA and DNA/F-actin, respectively, were needed to inhibit liposomal forms in comparison to free drugs. The authors explained this finding by the increased viscoelasticity induced by the high concentrations of polyanionic elements that may hinder the interaction of liposomes with bacteria. Indeed, the early leakage of antibiotics from liposomes cannot be used as a plausible cause of the inactivation of liposomal antibiotic because in vitro stability studies showed that liposomal vesicles were not disrupted [33]. To further confirm the superiority of liposomal forms, the authors studied the bactericidal activity of liposomal formulations versus free forms against P. aeruginosa found in CFpatients’ sputa. The antibacterial activities of liposomal formulations were 4-fold higher when compared to the free drugs, despite the presence of different bacterial strains in the patient’s sputum. It is of note that liposomal tobramycin reduced growth at a high concentration (128 mg/L), whereas liposomal polymyxin B did it at a markedly lower concentration (8 mg/L). The dissimilar activities of tobramycin and polymyxin B was attributed to their different sites of action. The different behaviors of liposomal formulations as a function of the encapsulated drug will be discussed thoroughly in the following sections of this review. The same research group conducted a meticulously detailed study confirming the inhibiting effect of the polyanionic compounds in CF-patients ‘sputa, i.e. neutrophil-derived DNA, mucoid P. aeruginosa-produced alginates and mucins, on the antibacterial activities of free and liposomal aminoglycosides [34]. It was found that bactericidal concentrations of aminoglycosides were increased by 8- to 256-folds against biofilm-forming strain, while the treatment with alginate lyase (AlgL) improved the eradication of this latter. The activity of the tested aminoglycosides, i.e. tobramycin, gentamicin and amikacin, was significantly increased by the concomitant use of recombinant human DNase or AlgL. However, liposomal antibiotic formulations did not display an additional effectiveness with respect to the free drugs, unless used in combination with AlgL.
Down-regulation of bacteria’ oxidative-stress resistance genes
Bacterial adaptation to oxidative and nitrosative stress could be considered as a resistance mechanism to host defenses [44]. Indeed, innate immune cells generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as superoxide and peroxynitrite, respectively, in order to kill phagocyted bacteria [45]. Consistently, pathogenic bacteria resist to host-mediated oxidative stress by up-regulating the expression of their antioxidant enzymes [46]. Importantly, it was claimed that many antibiotics exert their bactericidal effects via the production of hydroxyl radicals, regardless of their molecular targets [47]. Recently, it was found that metal NP, namely zinc oxide-NP (ZnO-NP), exert by themselves bactericidal effects on GPB and GNB [48]. A synergistic killing effect on acid fast bacteria (i.e. Mycobacterium bovis-BCG) was also observed for ZnO-NP when used in combination with rifampicin [48]. Moreover, ZnO-NP effectively killed MRSA clinical strains [48]. Several mechanisms were found to be involved in ZnO-NP antibacterial activities. Most importantly, ZnO-NP were found to down-regulate the transcription of oxidative stress resistance genes in S. aureus. Strictly speaking, the treatment with 300 µg/mL of ZnO-NP decreased the transcription of peroxide stress regulon katA and perR genes by 10- and 3.1- folds, respectively, when compared to untreated bacteria [48]. These results highlight the importance of ZnO-NP in fighting drug-resistant bacteria. It is of note that ZnO-NP induced oxidative stress response on macrophages, as ROS and NO production was markedly increased, thus reinforcing their bacterial killing capacity [48]. Generally speaking, metal NP, such gold or silver NP, are known to induce oxidative stress in host cells, which is considered as one of mechanisms involved in their toxic effects [49]; [50]. However, to the best of our knowledge, Pati et al. were the first to demonstrate the opposite effect on bacterial cells [48].
Passive fusion “stalk mechanism”
Taking into account the barriers and the interaction driving forces, NP were designed in order to give rise to different types of interaction with bacteria or their biofilms. Passive fusion is the commonly described interaction mechanism with the bacterial cell-wall for NP in general but also particularly for fusogenic liposomes. The formulation of fusogenic liposomes is based on the combined use of lipids forming hexagonal II (HII) phase and lipids forming lamellar phase [55]. The lipids forming HII phase, such as phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidic acid (PA), have a small polar head showing a cone shaped-molecule. When used in formulation, the negative curvature stress leads to the spontaneous formation of inverted micelles (HII phase) [56]. In contrast, lipids with similar packing ratios for the polar head and the hydrophobic queue, such as phosphatidylcholine (PC), phosphatidylglycerol (PG) or phosphatidylinositol (PI), have a cylindric shape and produce a small or no curvature stress. They form lamellar phases [57]. The principal fusion mechanism as identified by Markin et al. was called the stalk mechanism [58]. The approaching membranes form an hourglass-shaped structure, called stalk, generating a local stress and spontaneous curvature in these membranes followed by bilayer reorganization. The stalk formation is promoted by an HII forming lipids since they stabilize the hemi-fusion intermediate structures. This hypothesized mechanism was subsequently confirmed thanks to TEM. The intermediate structure consisting in lamellar/HII transition phase was observed in a mixture of PC/PE after dehydration [59]. It is of note that there is a category of lipids called lysolipids, characterized by a bully polar head, molecularly shaped as inverted cone and so form hexagonal I (HI) phase. They produce a positive curvature stress in the membranes and inhibit the stalk formation of approaching membranes and thereby their fusion [60]. Accordingly, the design of fusogenic liposomes requires a careful qualitative and quantitative choice of phospholipids as it directly influences the stability of lamellar/HII transition phase [55].
Fusion triggered by specific “ligand-receptor” recognition
In an attempt to target a specific type of bacteria, researchers focused on the outer membrane composition of the considered bacterium. This is with the aim to find a membrane component that specifically interacts with a ligand grafted on NP surface. Accordingly, Bardonnet et al. took benefit of the fact that some strains of Helicobacter pylori has an outer membrane protein (BabA2 adhesin) that binds with the fucosylated Lewis b (Leb) histo-blood group antigen expressed by human gastric epithelial cells [62]. Based on this phenomenon, Bardonnet et al. elaborated liposomes containing a synthesized glycolipid (Fuc-E4-Chol) composed of cholesterol as an anchor part, four ethylene glycol residues as a linker, and fucose as an exposed part at the surface of liposomes [63]. These liposomes were used as delivery systems for ampicillin and metronidazole and were found to be effective against both the spiral and the coccoid bacterial forms. In this study, the authors attributed the interactions H. pylori-liposomes to four events [63]. The first one is the incorporation cholesterol in the formulation of liposomes which enhances their interaction with H. pylori, because of the specific affinity of this latter to this steroid. The second phenomenon is the electrostatic interaction as H. pylori is negatively-charged. The authors found that liposomes exhibiting a lower negative zeta-potential (between -2.9 and -4.3 mV) were more efficient than those with a higher one (between -12.2 and -20 mV). The third phenomenon is the specific interaction of fucosylated liposomes-BabA2 adhesin, since better results were obtained with liposomes grafted with the synthesized glycolipid Fuc-E4- Chol. Finally, the fourth important point was the age of the bacterial culture or, in other words, the bacterium phenotype. During aging, the morphology of H. pylori evolves from the spiral to the coccoid resistant form. Fucosylated liposomes were found to interact with both phenotypes, whereas analogous ones without Fuc-E4-Chol were only able to interact with H. pylori spiral form. Consistently, the authors supposed that both phenotypes express BabA2 adhesin on the outer membrane [63].
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Table des matières
LISTE DES ABREVIATIONS
INTRODUCTION GENERALE
CHAPITRE 1 INTERACTIONS NANOPARTICULE-BACTERIE: DE NOUVEAUX HORIZONS POUR COMBATTRE LA RESISTANCE AUX ANTIBIOTIQUES
“INSIGHTS IN NANOPARTICLE- BACTERIUM INTERACTIONS: NEW FRONTIERS TO BYPASS BACTERIAL RESISTANCE TO ANTIBIOTICS”
ABSTRACT
1. INTRODUCTION
2. HOW CAN NANOPARTICLES HELP TO BYPASS BACTERIAL DRUG RESISTANCE?
2.1. Alteration of bacteria’s efflux pump activity
2.2. Antibiofilm activity
2.3. Enhanced penetration through biofilms
2.4. Protection against enzymatic degradation and inactivation by polyanionic compounds
2.5. Intracellular bacterial killing
2.6. Specific targeting and sustained-release
2.7. Down-regulation of bacteria’ oxidative-stress resistance genes
3. MECHANISMS OF NANOPARTICLE- BACTERIUM INTERACTIONS
3.1. Internalization mechanism of liposomes
3.2. Internalization mechanisms of polymeric and inorganic nanoparticles
4. FACTORS AFFECTING NANOPARTICLE- BACTERIUM INTERACTION
4.1. Nanoparticle-related factors
4.2. Antibiotic-related factors
4.3. Bacterium-related factors
REFERENCES
CHAPITRE 2 DEVELOPPEMENT DES LIPOSOMES STABILISES STERIQUEMENT COMME VECTEURS DU SNITROSOGLUTATHION POUR CIBLER LES MACROPHAGES
“ELABORATION OF STERICALLY STABILIZED LIPOSOMES FOR S NITROSOGLUTATHIONE TARGETING TO MACROPHAGE”
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Materials
2.2. Encapsulation screening
2.3. Preparation of liposomes
2.4. Characterization of liposomes
2.5. In vitro release
2.6. Cytotoxicity studies
2.7. Cell uptake studies
2.8. Liposomal GSNO antibacterial activity assessments
3. RESULTS AND DISCUSSION
3.1. Screening the liposome manufacturing process
3.2. Physico-chemical characterization of liposomes
3.3. In vitro release kinetics
3.4. Cytotoxicity studies
3.5. Cell uptake studies
3.6. Liposomal GSNO antibacterial activity studies
4. SUMMARY
REFERENCES
CHAPITRE 3 MICROENCAPSULATION DE LA RIFAMPICINE EN UTILISANT LE PALMITATE DE SACCHAROSE COMME TENSIOACTIF ALTERNATIF A L’ALCOOL POLYVINYLIQUE POUR CIBLER LES MACROPHAGES ALVEOLAIRES
“FORMULATION AND IN VITRO CHARACTERIZATION OF INHALABLE POLYVINYL ALCOHOLFREE RIFAMPICIN-LOADED PLGA MICROSPHERES PREPARED WITH SUCROSE PALMITATE AS STABILIZER: EFFICIENCY FOR EX VIVO ALVEOLAR MACROPHAGE TARGETING”
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Materials
2.2. Microsphere preparation
2.3. Microsphere characterization
2.4. Morphology analysis
2.5. In vitro RIF release studies
2.6. Aerodynamic evaluations
2.7. Alveolar macrophage cells
2.8. RIF uptake by alveolar macrophage
2.9. Toxicity assay
3. RESULTS AND DISCUSSION
3.1. Preparation and characterization of RIF-loaded microspheres
3.2. Morphology analysis
3.3. In vitro RIF release studies
3.4. Aerodynamic behavior assessment of RIF-loaded microspheres
3.5. Rifampicin uptake by alveolar macrophage
3.6. Cell toxicity
4. CONCLUSION
REFERENCES
CONCLUSION GENERALE
RESUME
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