Three-Dimensional Quantitative Co-Mapping of Pulmonary Morphology and Nanoparticle Distribution with Cellular Resolution in Nondissected Murine Lungs

Abstract

Deciphering biodistribution, biokinetics and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools.Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in non-dissected ex vivo murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 ± 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 ± 0.37) for instillation. After inhalation most of NPs were observed in proximal part of the acini as predicted by Computational Fluid Dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity-dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues facilitating both nanotoxicology studies and the development of nanomedicines. Keywords

Multimodal Precision Imaging of Pulmonary Nanoparticle Delivery in Mice: Dynamics of Application, Spatial Distribution, and Dosimetry

“…Lungs from nine independent slow‐instillation experiments were stratified for preferentially left and right as well as uniform (global) NP deposition in the lung. The ratios of the fluorescence intensities from the right and left lung, which are normalized to their respective right and left lung volume to account for the effect of volume‐dependent lung ventilation, reveal that for global delivery the ratio is equal to unity and a 3.7‐fold preferential targeting of the right (3.7) and left (3.7 = 1/0.27) half of the lung was accomplished, respectively (Figure e). Finally, the delivered dose was determined from quantitative fluorescence analysis using dose‐intensity conversion curves based on two reference lungs containing known amounts of polystyrene‐SkyBlue NPs (using regular intratracheal instillation) and three nontreated lungs for tissue‐autofluorescence correction .…”

“…While CT scan and LSFI offer 3D imaging capabilities, they do not allow for NP localization within individual cells. We recently demonstrated that ex vivo LSFM allows for quantitative co‐mapping of lung architecture and NP deposition with single‐cell resolution in nondissected but optically cleared lungs . Here we apply this method to study the pulmonary distribution of 100 µL of an NP suspension after intratracheal slow‐instillation.…”

“…It is noteworthy that from quantitative spectrofluorometry of the lung slices (LSFI) one obtains a deposited aerosol fraction of 15.6 ± 2.8 % (whole lung) (Figure j), which is significantly larger than the 9.3 ± 2.3% (trachea + lung lobes) measured in lung/trachea homogenates, probably due to the higher light attenuation in sliced lung tissue. Since we consider tissue homogenization as more accurate method of LSFI, we report 8.6 and 12.9 nL s −1 as pulmonary and tracheal aerosol deposition (delivery) rate, resulting in 5.6 and 8.3 µL NP liquid out of applied 150 µL volume deposited in the lung and trachea, respectively. Not surprisingly, in vivo PB‐PCXI showed no iodine‐related signal during aerosol application (Figure a) due to the low iodine delivery rate and the extremely uniform distribution of (aerosolized) iodine throughout the lung.…”

“…The acquired images include attenuation and phase information, with the attenuation effects rendering the bones and highly absorbing contrast agents visible, and the phase effects rendering the lungs and airways visible . Here, PB‐PCXI was applied to the in vivo real‐time monitoring of controlled NP‐liquid delivery to different regions of the lung, and subsequent ex vivo CT and fluorescence imaging (here epifluorescence imaging of the whole or dried‐sliced lung or tissue‐cleared light sheet fluorescence microscopy (LSFM)) further provided the 3D NP distribution profile throughout the entire lung in both a qualitative and a quantitative manner. The advantages and limitations of each of these techniques are discussed and we demonstrate the potential of this multimodal imaging platform for resolving mechanisms of pulmonary drug delivery and specific features of the NP distribution profile observed for three of the most widely used preclinical modes of pulmonary application, namely, intratracheal instillation, ventilator‐assisted aerosol inhalation, and nasal aspiration.…”

Severe dysregulation of TNF expression leads to multiple inflammatory diseases and embryonic death

“…The tissue was imaged using a Zeiss Z.1 lightsheet microscope equipped with a 5x/0.16 detection objective. Blood vessels were detected using hemoglobin autofluorescence excited with the 405nm laser line and detected using a 500-545nm GFP band pass filter (24). Multiview data sets were acquired at 120-degree angles and fused using the Multiview Reconstruction plugin in FIJI (25).…”