Visualising biodistribution and biokinetics of inhaled biologicals: application of light sheet imaging technology

Abstract

The therapeutic utility of systemic “biologicals” (i.e. recombinant proteins), in particular monoclonal antibodies, has rekindled interest in development of diverse inhaled biological platforms to treat lung diseases. However, very little is known about the specific patterns and mechanisms of their uptake, epithelial transfer, clearance and redistribution. Here we compare conventional PK analysis with whole-of-lung biodistribution by 3D light-sheet microscopy (LSM) and FACS profiling of macrophages using a human NGAL lipocalin protein as a test probe.

Methods: Unlabelled NGAL was microsprayed into male Balb/c mice lungs. At 1, 2, 4, 6, & 24h, blood, PBS lung washes and lung lobes were collected. Separately, fluoro tagged NGAL-647 was microsprayed into mouse lungs, harvested, optically cleared and imaged by LSM and lung macrophages analysed by FACS.

Results: Instilled NGAL displayed first order elimination kinetics. Appeared in the blood in a dose proportional manner (T_max=2h,& C_max=1859 ng/ml). LSM confirmed progressive redistribution from the airway lumen into the interstitium; most likely by paracellular transport. As previously demonstrated, instilled protein macrophage uptake was inferred from a punctate cell biodistribution pattern and redistribution at later time points. This was confirmed by FACS as the majority of NGAL+ cells were alveolar monocyte/macrophages cells.

Conclusion: Consistent with previous reports, NGAL delivered directly to the lung by microspray undergoes first order elimination, transit from lumen to submucosa in airways and lumen to interstitium and blood from the alveoli as well as macrophage uptake and clearance via the mucociliary escalator.