Nebulizable azithromycin into anti-biofilm superstable nanovesicles

ALTUBE MARÍA JULIA; MARTINEZ MELINA MARÍA BELÉN; MAFFIA PAULO ; MORILLA MARÍA JOSE; ROMERO EDER LILIA

Basilea

Conferencia; 11Th European and global for clinical Nanomedicine; 2018

CLINAM

Introduction: Azithromycin is a macrolide antibiotic of broad-spectrum of action and immunomodulatory activity that is used for the treatment of chronic pulmonary diseases like cystic fibrosis (CF). CF is a life-limiting autosomal recessive genetic disorder. The disease is caused by mutation of a gene that encodes a chloride-conducting transmembrane channel which regulates anion transport. One of the consequences of this dysfunction is the impediment of the mucociliary clearance in the airways. This allows the development of the pulmonary disease characterized by mucus retention, bacterial infections and airway inflammation. Pulmonary disease is the major cause of mortality and therefore the major focus of clinical attention and research (Elborn, 2016). The first lung infections of CF patients are produced by colonies of Staphylococcus aureus and during the development of the infection; there is a co-infection with Pseudomonas aeruginosa. The infection with P. aeruginosa becomes persistent by adaptive mechanisms that elude the immune system defense and resist antibiotic therapy, like bacterial biofilm formation (Ciofu et al., 2015). Treatment with oral AZ is recommended for patients with CF for up to six months, since one of the main drawbacks to the prolonged use of oral AZ is its tolerability, being the most serious side effect in cardiac toxicity (Altenburg, 2011). The inhaled administration of AZ could be an alternative to minimize the systemic adverse effects and maximize the accumulation of the drug in the target site of the disease. However, due to the chemical characteristics of AZ, it is difficult to achieve an adequate suspension for nebulization (Hickey et al., 2006).The pharmacokinetics and pharmacodynamics of inhaled antibiotics could be modified using delivery systems to improve tolerability, increase the retention time in the lungs, as well as minimize the premature mucociliary clearance of drugs (Zhou et al., 2015). Currently, there are two formulations of liposomal antibiotics, which are in advanced clinical phases for use in CF: Lipoquin (liposomal ciprofloxacin) and Arikace (liposomal amikacin). In addition, one of the major barriers against efficient CF therapy is the fact that the CF patients have thickened and viscous mucus that adheres to the airway surface. Antibiotic delivery with inhaled nanovesicles capable of penetrating the thick mucus layer, could improve interaction of antibiotics with bacterial cells. For this, nanovesicles must be able to avoid adhesion to mucin fibers and being small enough to avoid significant steric inhibition produced by the mucin dense fiber mesh (Nafee et al. 2014). Archaeosomes (ARC) are nanovesicles made of total polar archaeolipidis (TPA). Archaeolipids are novel biomaterials extracted from a non-conventional source (neither animal and vegetal nor bacterial), the hyperhalophilic archaebacterial Halorubrum tebenquichense. The presence of these archaeolipids, particularly that of the major component 2,3-di-O-phytanyl-sn-glycero-1-phospho-(30-sn-glycerol-10-methylphosphate), renders the nanovesicles resistant to physical, chemical, and enzymatic attacks, including to the shear stress of nebulization (Altube et al., 2016). Previous studies have shown that ARC and related structures are more extensively captured by macrophages than conventional liposomes (Perez et al., 2014). Furthermore, no in vitro deleterious effects on pulmonary surfactant activity have been detected for ARC (Altube et al., 2017).Based on the above-mentioned advantages that nanovesicles could provide to lung delivery, in this work we propose, for the first time, the incorporation of AZ on nebulized nanovesicles aiming to achieve a nebulizable formulation, to provide long term-action and reduced systemic adverse effects, which could result from the oral administration. To that aim, AZ was loaded in highly stable nanovesicles -ARC- and its performance was compared with that of ordinary hydrogenated phosphatidylcholine liposomes -L-. The formulations were tested for stability upon storage, nebulization and mucins contact. In addition, biological aspects were evaluated like safety of epithelial and macrophage cells as well as maintained anti-bacterial activity against S. aureus and P. aureuginosa.Results: nanovesicles with AZ were prepared by the film hydration method, conventional liposomes were made of HSPC:colesterol:AZ 7.5:2.5:5:4 w:w (L-AZ) and archaeosomes were made of TPA:AZ 10:4 (ARC-AZ). Our results showed that only ARC-AZ were stable upon 6 months of storage at 4ºC, while L-AZ rendering variable drug load and undergoing aggregation. Evaluation of the formulation stability during the nebulization process is a key factor to achieve an efficient delivery of an inhalable formulation system. (Lehofer et al., 2014). Although the vibration mesh nebulizer is a dispositive recommended for nebulization of liposome formulations, it produces mechanical stress due to shears forces during the nebulization process (Cipolla, Gonda, & Chan, 2013). Although very rigid HSPC-cholesterol liposomes can support this process well, the incorporation of AZ drastically changed this behavior, causing the formulation to be retained in the vibration mesh nebulizer and the reminder that managed to be nebulized (~ 50 % LT) had a size in the order of micrometers. On the other hand, the formulation with ARQ-AZ had excellent nebulization efficiency without significant losses of mass and with colloidal stability (Figure 1).The incubation of empty nanovesicles or with AZ with mucins during 4 hours at a mucin:nanovesicle 1:1 (w:w) ratio, showed no significant change on the nanovesicles size. ARC were not mucoadhesive: the size distribution of the nanoparticles/mucin mixtures is in the same size range of nanoparticles alone. Instead, we found that ARC were more mucus penetrating than L upon 9 h, ARC diffusing significantly deeper (80± 4%) than L (58 ± 2%) through a mucin layer.Cell viability was evaluated on two cell types representative of the alveolar structure. One was the human lung adenocarcinoma cell line A549, which has characteristics of type II alveolar cells and the other was the human macrophage cell line, THP-1. In our work, no decrease in cell viability was recorded by the MTT method by both ARC-AZ and L-AZ after 24 hours incubation up to a concentration of 45 μg/mL of AZ and 225 μg/mL of LT. After 48 hours of incubation, cell viability slightly decreased (~ 15%) with these concentrations. On the other hand, for THP-1 cells, free AZ induced a significantly decreased of viability (~ 40%) with 5 μg/mL of drug. The antibacterial activity of free AZ and incorporated on nanovesicles was evaluated on S. aureus ATCC25923 and P. aeruginosa PAO1, planktonic and forming biofilms. For S. aureus, a MIC of 4 μg/mL was determined for free AZ or in nanovesicles and for P. aeruginosa a MIC of 32 μg/mL for free AZ and of 8 μg/mL for ARQ-AZ and 16 μg/mL for L-AZ. In addition, it was evaluated the ability of AZ in nanovesicles to inhibit S. aureus biofilm formation. At 1/2 of the MIC, both free drug and on the nanovesicles inhibited ~50 % of planktonic bacteria growth, but only when it was incorporated into nanovesicles the amount of biofilm generated decreased by 40%. Finally, when AZ is incorporated in nanovesicles remained its anti-biofilm activity, decreasing 70 % of viable biofilm bacteria.Conclusion: Despite ARC-AZ formulation showed comparable anti-bacterial activities with L-AZ and free AZ, only ARC-AZ remained structurally stable upon nebulization process and storage as well as dsiplaying deeper mucopenetration. Therefore, the advantages of ARC-AZ formulation over free AZ on ordinary AZ liposomal formulations constitute a promising tool for inhalatory treatment of diseases characterized by bacterial biofilm formation.ReferencesAltenburg et al. (2011). Respiration, 81, 75-87.Altube et al. (2016). Nanomedicine, 11, 2103-2117.Altube et al. (2017). Journal of Materials Chemistry B, 5, 8083-8095.Ciofu et al. (2015). Advanced drug delivery reviews, 85, 7-23.Elborn, J. S. (2016). Lancet, 388, 2519-2531. Hickey et al. (2006). Journal of aerosol medicine, 19, 54-60.Nafee et al. (2014). Journal of Controlled Release, 192, 131-140.Perez et al. (2014). International journal of nanomedicine, 9, 3335.Zhou et al. (2015). Advanced drug delivery reviews, 85, 83-99.