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

Abstract

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. Graphical abstract

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

“…aeruginosa biofilm-infected cystic fibrosis patient, a protocol was established to address especially three underrepresented conditions in such models: (1) growing a bacterial biofilm on top of tight monolayers of human bronchial epithelial cells, (2) air–liquid interface conditions to enable repeated deposition of anti-infective drugs as aerosols, and (3) monitor both bacterial growth and killing, as well as host cell viability and epithelial barrier function under such treatment for several days. In contrast to the lung in vivo , where biofilms may take a long time to develop, , growing bacterial biofilms on cultured epithelial cells is not trivial, as the latter will typically die within hours due to rapid bacterial overgrowth. , To overcome this problem, biofilms were pre-grown separately for 72 h and then transferred to monolayers of CFBE41o- cells, which had also been grown separately for 7 days under ALI conditions (Figure ).…”

“…The biofilm suspension is then incubated for 1 h on top of the cells to allow sedimentation. Experiments from a previous publication have shown that longer incubation times than 1 h lead to disruption of the barrier and premature subsequent cell death, pointing to the fact that keeping living bacteria and host cells in equilibrium for a long time is not trivial . After that, ALI conditions were restored by removing the remaining liquid from the apical compartment.…”

“…For sample analysis via confocal laser scanning microscopy (CLSM), an infection experiment was conducted as described . Instead of PAO1, PAO1-GFP was used.…”

“…Other groups have explored the formation of biofilms on epithelial cells after long-time exposure to planktonic Staphylococcus aureus (4 days) or Haemophilus influenza (7 days) . Our group recently presented a new system that could show 24 h pre-grown biofilm parts infecting CFBE41o- cells treated with ciprofloxacin nanoparticles . While air–liquid interface (ALI) conditions are quite common in pulmonary cell culture models, , this significant study showed the feasibility of cells being infected under ALI conditions and treated via aerosolized particles.…”

Drug delivery for fighting infectious diseases: a global perspective

“…Thiyagarajan et al enhance the pulmonary delivery potential of antimycobacterial nanopharmaceuticals by spray-drying the nanoparticles to form lactose-leucine microparticles with potentially more favorable aerodynamic properties [15]. Along the same delivery route, the challenge of delivering large antiinfectious biologics, antibodies, is presented by Mayor et al [16] while Juntke et al present a novel model of human bronchial epithelial cells cultivated at the air-liquid interface (ALI) and infected with a Pseudomonas aeruginosa biofilm that could have a large impact on the testing of such aerosolized anti-infective nanoparticle delivery systems [17]. The use of lipid delivery systems and their potential to ameliorate antimicrobial therapeutic effects are expounded in Clive Prestidge's "Nano-fats for bugs" review [18] and highlighted again by Ryan et al exploration of solid lipid nanoparticles for delivery of dual-acting antimicrobial peptides [19].…”