Efficient bioactive delivery of aerosolized drugs to pulmonary cells at the air-liquid interface: Validation of the ALICE-CLOUD technology

Introduction: A major bottleneck for testing drugs for inhalation therapy is the lack of realistic and simple to operate in vitro systems. Here we present an easy-to-use in vitro system (ALICE-CLOUD) for efficient delivery of aerosolized aqueous drugs to lung epithelial cells cultured under realistic air-liquid interface (ALI) conditions.

Methods: The ALICE-CLOUD utilizes the principles of cloud motion for fast and efficient droplet-to-cell delivery. The drug delivery performance of the ALICE-CLOUD was evaluated with a photometric assay using fluorescein as surrogate drug. The ALICE-CLOUD was functionally validated with the FDA-approved proteasome inhibitor Bortezomib (Velcade®), which has not been used for inhalation therapy yet. The anti-inflammatory efficacy of Bortezomib was assessed using human epithelial lung cells (A549) containing a stably transfected luciferase reporter gene under the control of the IL-8 promoter. A549 cells were stimulated with TNF- α to induce a 7-fold activation of the IL-8 promoter after 24h and therapeutic efficacy of Bortezomib was assessed based on the reduction of IL-8 promoter activation.

Results: The ALICE-CLOUD delivers 83.4 ± 7.8% (SD) of the invested drug within 3.5 min to a standard multi-well plate (here: 6-well plate), which corresponds to a cell-delivered drug fraction of 17%. Dose application is highly repeatable (9%; SD) and well-to-well variability is less than 5% (SD). Bortezomib reduced proteasome activity by more than 90% without impairing cell viability (WST-1 and LDH assays). Both aerosolized and non-aerosolized Bortezomib had a pronounced anti-inflammatory effect on A549 cells.

Discussion: Cell-based therapeutic efficacy of aerosolized Bortezomib at ALI conditions was similar for aerosolized/ALI and non-aerosolized/submerged conditions. This indicates that (1) the IL-8 response of A549 cells does not strongly depend on cell culture conditions, (2) Bortezomib is a promising candidate for anti-inflammatory inhalation therapy and (3) the ALICE-CLOUD is a reliable tool for inhalable drug screening, which combines ease-of-handling with rapid, efficient and dosimetrically accurate drug-to-cell delivery. This may pave the way for high-throughput screening of inhalable drugs under physiologically more relevant and hence potentially more predictive conditions than the currently used submerged cell culture systems.