Bridging gaps between in vitro and in vivo data in pulmonary aerosol delivery with focus on pharmacokinetics

Pulmonary aerosolized delivery of drugs incorporated into nanocarriers (nanodrugs) by inhalation is a promising route for the prolonged treatment of lung diseases such as pulmonary hypertension or lung rejection after transplantation. Due to the lack of validated in vitro testing systems, the PK and efficacy of nanodrugs during the preclinical phase of drug testing has to be measured in animal models. This work investigates the particokinetics of two nanodrugs, Ptx-NP and L-CsA, which incorporate the active drug Paclitaxel (Ptx) and Cyclosporine A (CsA) into polymeric or liposomal nanocarriers, respectively. The goal of the study was to evaluate the potential of aerosolized drug delivery combined with physiological in vitro cell culture models of the lung to predict the clinical outcome with a focus on PK. The VITROCELL® Cloud 6 system was used to deliver the nanodrugs in aerosolized form to in vitro models of the healthy and diseased human alveolar air-blood barrier cultured at air-liquid interface (ALI) conditions, and to analyze the transbarrier transport of the incorporated drugs. ALI conditions showed a direct cell-nanodrug interaction, which allowed clarifying cellular uptake mechanisms (caveolae-mediated endocytosis and passive diffusion) and mirroring real cellular transport rates. In a direct comparison between the healthy and diseased models, no significant differences in the transbarrier transport rates were found, which highlight the prolonged tissue-association of drugs incorporated in nanocarriers. The obtained particokinetics were further combined with physiological-based PK (PBPK) modeling to predict the PK profile of CsA and Ptx (e.g. maximum drug concentration cmax in the blood; time until cmax is reached (tmax)) after inhalation. This confirmed that cmax levels after inhalation were achieved fast (< 0.25 h). Moreover, the modeling revealed that cmax levels after inhalation are typically low, which highlights the advantage of targeting lung diseases by inhalation therapy as this avoids high drug levels in the blood that could lead to systemic toxicities. Besides the PK, the efficacy of Ptx-NP was investigated. The analysis of Ptx doses in different compartments of the in vitro model demonstrated a cell-association of 30% of the initial dose 24 h after the aerosolized delivery of Ptx-NP. Accordingly, the potential of prolonged drug interaction with the diseased tissue in vitro could be highlighted. Moreover, a dose of 0.7 μg Ptx/cm² increased FoxO1 transcription – a hallmark of pulmonary hypertension - by a factor of 3 as compared to untreated control. Consequently, the aerosolized drug delivery to ALI cell culture models of the alveolar tissue barrier combined with PBPK modeling can support the development of drug formulations with a beneficial PK profile in the clinical settings. Moreover, these types of in vitro models are well suited to study cellular uptake and transport mechanisms

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ 12) and titanium dioxide nanoparticles (TiO 2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ 12 and TiO 2 NM-105 particles showed good linearity (R 2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ 12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO 2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO 2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials

A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m$^{2}$) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic In VItro Cell-stretch (CIVIC) “breathing” lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 μm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o$^{-}$) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation CV): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. a 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials