Transferring Microclusters of P. aeruginosa Biofilms to the Air-Liquid Interface of Bronchial Epithelial Cells for Repeated Deposition of Aerosolized Tobramycin.

As an alternative to technically demanding and ethically debatable animal models, the use of organotypic and disease-relevant human cell culture models may improve the throughput, speed, and success rate for the translation of novel anti-infectives into the clinic. Besides bacterial killing, host cell viability and barrier function appear as relevant but seldomly measured readouts. Moreover, bacterial virulence factors and signaling molecules are typically not addressed in current cell culture models. Here, we describe a reproducible protocol for cultivating barrier-forming human bronchial epithelial cell monolayers on Transwell inserts and infecting them with microclusters of pre-grown mature Pseudomonas aeruginosa PAO1 biofilms under the air-liquid interface conditions. Bacterial growth and quorum sensing molecules were determined upon tobramycin treatment. The host cell response was simultaneously assessed through cell viability, epithelial barrier function, and cytokine release. By repeated deposition of aerosolized tobramycin after 1, 24, and 48 h, bacterial growth was controlled (reduction from 10 to 4 log10 CFU/mL), which leads to epithelial cell survival for up to 72 h. E-cadherin's cell-cell adhesion protein expression was preserved with the consecutive treatment, and quorum sensing molecules were reduced. However, the bacteria could not be eradicated and epithelial barrier function was impaired, similar to the currently observed situation in the clinic in lack of more efficient anti-infective therapies. Such a human-based in vitro approach has the potential for the preclinical development of novel anti-infectives and nanoscale delivery systems for oral inhalation.

Testing of aerosolized ciprofloxacin nanocarriers on cystic fibrosis airway cells infected with P. aeruginosa biofilms.

The major pathogen found in the lungs of adult cystic fibrosis (CF) patients is Pseudomonas aeruginosa, which builds antibiotic-resistant biofilms. Pulmonary delivery of antibiotics by inhalation has already been proved advantageous in the clinic, but the development of novel anti-infective aerosol medicines is complex and could benefit from adequate in vitro test systems. This work describes the first in vitro model of human bronchial epithelial cells cultivated at the air-liquid interface (ALI) and infected with P. aeruginosa biofilm and its application to demonstrate the safety and efficacy of aerosolized anti-infective nanocarriers. Such a model may facilitate the translation of novel therapeutic modalities into the clinic, reducing animal experiments and the associated problems of species differences. A preformed biofilm of P. aeruginosa PAO1 was transferred to filter-grown monolayers of the human CF cell line (CFBE41o-) at ALI and additionally supplemented with human tracheobronchial mucus. This experimental protocol provides an appropriate time window to deposit aerosolized ciprofloxacin-loaded nanocarriers at the ALI. When applied 1 h post-infection, the nanocarriers eradicated all planktonic bacteria and reduced the biofilm fraction of the pathogen by log 6, while CFBE41o- viability and barrier properties were maintained. The here described complex in vitro model approach may open new avenues for preclinical safety and efficacy testing of aerosol medicines against P. aeruginosa lung infection.

A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells in vitro.

The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air-liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air-liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 µl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.

A pulmonary mucus surrogate for investigating antibiotic permeation and activity against Pseudomonas aeruginosa biofilms.

BACKGROUND: Pulmonary infections associated with Pseudomonas aeruginosa can be life-threatening for patients suffering from chronic lung diseases such as cystic fibrosis. In this scenario, the formation of biofilms embedded in a mucus layer can limit the permeation and the activity of anti-infectives. OBJECTIVES: Native human pulmonary mucus can be isolated from endotracheal tubes, but this source is limited for large-scale testing. This study, therefore, aimed to evaluate a modified artificial sputum medium (ASMmod) with mucus-like viscoelastic properties as a surrogate for testing anti-infectives against P. aeruginosa biofilms. METHODS: Bacterial growth in conventional broth cultures was compared with that in ASMmod, and PAO1-GFP biofilms were imaged by confocal microscopy. Transport kinetics of three antibiotics, tobramycin, colistin, and ciprofloxacin, through native mucus and ASMmod were studied, and their activity against PAO1 biofilms grown in different media was assessed by determination of metabolic activity and cfu. RESULTS: PAO1(-GFP) cultured in human pulmonary mucus or ASMmod showed similarities in bacterial growth and biofilm morphology. A limited permeation of antibiotics through ASMmod was observed, indicating its strong barrier properties, which are comparable to those of native human mucus. Reduced susceptibility of PAO1 biofilms was observed in ASMmod compared with LB medium for tobramycin and colistin, but less for ciprofloxacin. CONCLUSIONS: These findings underline the importance of mucus as a biological barrier to antibiotics. ASMmod appears to be a valuable surrogate for studying mucus permeation of anti-infectives and their efficacy against PAO1 biofilms.

Disease Models: Lung Models for Testing Drugs Against Inflammation and Infection.

Lung diseases have increasingly attracted interest in the past years. The all-known fear of failing treatments against severe pulmonary infections and plans of the pharmaceutical industry to limit research on anti-infectives to a minimum due to cost reasons makes infections of the lung nowadays a "hot topic." Inhalable antibiotics show promising efficacy while limiting adverse systemic effects to a minimum. Moreover, in times of increased life expectancy in developed countries, the treatment of chronic maladies implicating inflammatory diseases, like bronchial asthma or chronic obstructive pulmonary disease, becomes more and more exigent and still lacks proper treatment.In this chapter, we address in vitro models as well as necessary in vivo models to help develop new drugs for the treatment of various severe pulmonary diseases with a strong focus on infectious diseases. By first presenting the essential hands-on techniques for the setup of in vitro models, we intend to combine these with already successful and interesting model approaches to serve as some guideline for the development of future models. The overall goal is to maximize time and cost-efficacy and to minimize attrition as well as animal trials when developing novel anti-infective therapeutics.

Spray-dried multidrug particles for pulmonary co-delivery of antibiotics with N-acetylcysteine and curcumin-loaded PLGA-nanoparticles.

Nowadays, the resistance of bacterial biofilms towards the available antibiotics is a severe problem. Therefore, many efforts were devoted to develop new formulations using nanotechnology. We have developed an inhalable microparticle formulation using spray-drying combining multiple drugs: an antibiotic (tobramycin, ciprofloxacin or azithromycin), N-acetylcysteine (NAC), and curcumin (Cur). The use of PLGA nanoparticles (NP) also allowed incorporating curcumin to facilitate spray drying and modify the release of some compounds. The aerosolizable microparticles formulations were characterized in terms of size, morphology, and aerodynamic properties. Biocompatibility when tested on macrophage-like cells was acceptable after 20 h exposure for concentrations up to at least 32 µg/mL. Antibacterial activity of free drugs versus drugs in the multiple drug formulations was evaluated on P. aeruginosa in the same range. When co-delivered the efficacy of tobramycin was enhanced compared to the free drug for the 1 µg/mL concentration. The combinations of azithromycin and ciprofloxacin with NAC and Cur did not show an improved antibacterial activity. Bacteria-triggered cytokine release was not inhibited by free antibiotics, except for TNF-α. In contrast, the application of NAC and the addition of curcumin-loaded PLGA NPs showed a higher potential to inhibit TNF-α, IL-8, and IL-1ß release. Overall, the approach described here allows simultaneous delivery of antibacterial, mucolytic, and anti-inflammatory compounds in a single inhalable formulation and may therefore pave the way for a more efficient therapy of pulmonary infections.

PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus.

Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration.

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L5 content (10-20%, w/w), and (ii) the L5:siRNA ratio (10,1-30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

Triple co-culture of human alveolar epithelium, endothelium and macrophages for studying the interaction of nanocarriers with the air-blood barrier.

Predictive in vitro models are valuable alternatives to animal experiments for evaluating the transport of molecules and (nano)particles across biological barriers. In this work, an improved triple co-culture of air-blood barrier was set-up, being exclusively constituted by human cell lines that allowed to perform experiments at air-liquid interface. Epithelial NCI-H441 cells and endothelial HPMEC-ST1.6R cells were seeded at the apical and basolateral sides of a Transwell® membrane, respectively. Differentiated THP-1 cells were also added on the top of the epithelial layer to mimetize alveolar macrophages. Translocation and permeability studies were also performed. It was observed that around 14-18% of 50-nm Fluorospheres®, but less than 1% of 1.0 µm-Fluorospheres® could pass through the triple co-culture as well as the epithelial monoculture and bi-cultures, leading to the conclusion that both in vitro models represented a significant biological barrier and could differentiate the translocation of different sized systems. The permeability of isoniazid was similar between the epithelial monoculture and bi-cultures when compared with the triple co-culture. However, when in vitro models were challenged with lipopolysaccharide, the release of interleukin-8 increased in the bi-cultures and triple co-culture, whereas the NCI-H441 monoculture did not show any proinflammatory response. Overall, this new in vitro model is a potential tool to assess the translocation of nanoparticles across the air-blood barrier both in healthy state and proinflammatory state. STATEMENT OF SIGNIFICANCE: The use of in vitro models for drug screening as an alternative to animal experiments is increasing over the last years, in particular, models to assess the permeation through biological membranes. Cell culture models are mainly constituted by one type of cells forming a confluent monolayer, but due to its oversimplicity they are being replaced by three-dimensional (3D) in vitro models, that present a higher complexity and reflect more the in vivo-like conditions. Being the pulmonary route one of the most studied approaches for drug administration, several in vitro models of alveolar epithelium have been used to assess the drug permeability and translocation and toxicity of nanocarriers. Nevertheless, there is still a lack of 3D in vitro models that mimic the morphology and the physiological behavior of the alveolar-capillary membrane. In this study, a 3D in vitro model of the air-blood barrier constituted by three different relevant cell lines was established and morphologically characterized. Different permeability/translocation studies were performed to achieve differences/similarities comparatively to each monoculture (epithelium, endothelium, and macrophages) and bi-cultures (epithelial cells either cultured with endothelial cells or macrophages). The release of pro-inflammatory cytokines (namely interleukin-8) after incubation of lipopolysaccharide, a pro-inflammatory inductor, was also evaluated in this work.

Polysaccharide Submicrocarrier for Improved Pulmonary Delivery of Poorly Soluble Anti-infective Ciprofloxacin: Preparation, Characterization, and Influence of Size on Cellular Uptake.

The majority of the currently used and developed anti-infectives are poorly water-soluble molecules. The poor solubility might lead to limited bioavailability and pharmacological action of the drug. Novel pharmaceutical materials have thus been designed to solve those problems and improve drug delivery. In this study, we propose a facile method to produce submicrocarriers (sMCs) by electrostatic gelation of anionic ß-cyclodextrin (aß-CD) and chitosan. The average hydrodynamic size ranged from 400 to 900 nm by carefully adjusting polymer concentrations and N/C ratio. The distinct host-guest reaction of cyclodextrin derivative is considered as a good approach to enhance solubility, and prevent drug recrystallization, and thus was used to develop sMC to improve the controlled release profile of a poorly soluble and clinically relevant anti-infective ciprofloxacin. The optimal molar ratio of ciprofloxacin to aß-CD was found to be 1:1, which helped maximize encapsulation efficiency (∼90%) and loading capacity (∼9%) of ciprofloxacin loaded sMCs. Furthermore, to recommend the future application of the developed sMCs, the dependence of cell uptake on sMCs size (500, 700, and 900 nm) was investigated in vitro on dTHP-1 by both flow cytometry and confocal microscopy. The results demonstrate that, regardless of their size, an only comparatively small fraction of the sMCs were taken up by the macrophage-like cells, while most of the carriers were merely adsorbed to the cell surface after 2 h incubation. After continuing the incubation to reach 24 h, the majority of the sMCs were found intracellularly. However, the sMCs had been designed to release sufficient amount of drug within 24 h, and the subsequent phagocytosis of the carrier may be considered as an efficient pathway for its safe degradation and elimination. In summary, the developed sMC is a suitable system with promising perspectives recommended for pulmonary extracellular infection therapeutics.

Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.

The air-blood barrier is mainly composed of alveolar epithelial cells and macrophages. Whereas the epithelium acts as a diffusional barrier, macrophages represent an immunological barrier, in particular for larger molecules and nanoparticles. This paper describes a new co-culture of human cell lines representing both cell types. Acquiring, culturing and maintaining primary alveolar epithelial cells presents significant logistical and technical difficulties. The recently established human alveolar epithelial lentivirus immortalized cell line, hAELVi, when grown on permeable filters, form monolayers with high functional and morphological resemblance to alveolar type I cells. To model alveolar macrophages, the human cell line THP-1 was seeded on pre-formed hAELVi monolayers. The co-culture was characterized regarding cellular morphology, viability and barrier function. Macrophages were homogenously distributed on the epithelium and could be kept in co-culture for up to 7 days. Transmission electron microscopy showed loose contact between THP-1 and hAELVi cells. When grown at air liquid interface, both cells were covered with extracellular matrix-like structure, which was absent in THP-1 mono culture. In co-culture with macrophages, hAELVi cells displayed similar, sometimes even higher, trans-epithelial electrical resistance than in mono-cultures. When exposed to silver and starch NPs, hAELVi mono-cultures were more tolerant to the particles than THP-1 mono-cultures. The viability in the co-culture was similar to that of hAELVi monocultures. Transport studies with sodium fluorescein in presence/absence of EDTA proved that the co culture acts as functional diffusion barrier. These data demonstrate that hAELVi-/THP-1 co-cultures represent a promising model for safety and permeability studies of inhaled chemicals, drugs and nanoparticles.

Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells.

BACKGROUND: Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. RESULTS: CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. CONCLUSION: M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.

Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis.

In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes.

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials.

Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO2, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1ß, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO2 or Au NPs. But once inflamed with IL-1ß, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation.

Inhalable Clarithromycin Microparticles for Treatment of Respiratory Infections.

PURPOSE: The aim of this work was to develop clarithromycin microparticles (CLARI-MP) and evaluate their aerodynamic behavior, safety in bronchial cells and anti-bacterial efficacy. METHODS: Microparticles containing clarithromycin were prepared as dry powder carrier for inhalation, using leucine and chitosan. CLARI-MP were deposited on Calu-3 grown at air-interface condition, using the pharmaceutical aerosol deposition device on cell cultures (PADDOCC). Deposition efficacy, transport across the cells and cytotoxicity were determined. Anti-antibacterial effect was evaluated against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. RESULTS: Microparticles were of spherical shape, smooth surface and size of about 765 nm. Aerosolization performance showed a fine particle fraction (FPF) of 73.3%, and a mass median aerodynamic diameter (MMAD) of 1.8 µm. Deposition on Calu-3 cells using the PADDOCC showed that 8.7 µg/cm(2) of deposited powder were transported to the basolateral compartment after 24 h. The safety of this formulation is supported by the integrity of the cellular epithelial barrier and absence of toxicity, and the antimicrobial activity demonstrated for Gram positive and Gram negative bacteria. CONCLUSIONS: The appropriate aerodynamic properties and the excellent deposition on Calu-3 cells indicate that clarithromycin microparticles are suitable for administration via pulmonary route and are efficient to inhibit bacteria proliferation.