title: Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging.
abstract: Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm(-1) at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Nirav
authors.initials: N
authors.lastname: Barapatre
authors.affiliation: Institute of Biological and Medical Imaging, Helmholtz Zentrum München and Technische Universität München, 85764 Neuherberg, Germany.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Symvoulidis
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Winfried
authors.initials: W
authors.lastname: Möller
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Prade
authors.affiliation: Institute of Biological and Medical Imaging, Helmholtz Zentrum München and Technische Universität München, 85764 Neuherberg, Germany; Fraunhofer Project Group for Automation in Medicine and Biotechnology, 68167 Mannheim, Germany.
authors.forename: Nikolaos C
authors.initials: NC
authors.lastname: Deliolanis
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig-Maximilians-University, 81377 Munich, Germany; Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hertel
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig-Maximilians-University, 81377 Munich, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Winter
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Ali Ö
authors.initials: AÖ
authors.lastname: Yildirim
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Stoeger
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany; University Hospital, Ludwig-Maximilians-University, Marchioninistraße 15, 81377 Munich, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Eickelberg
authors.affiliation: Institute of Biological and Medical Imaging, Helmholtz Zentrum München and Technische Universität München, 85764 Neuherberg, Germany.
authors.forename: Vasilis
authors.initials: V
authors.lastname: Ntziachristos
authors.affiliation: Comprehensive Pneumology Center, Member of the German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany. Electronic address: otmar.schmid@helmholtz-muenchen.de.
authors.forename: Otmar
authors.initials: O
authors.lastname: Schmid
mesh_terms: D000280:Administration, Inhalation; D000818:Animals; D016616:Cryoultramicrotomy; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008168:Lung / Q000737:chemistry*; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000008:administration & dosage; D017452:Receptors, IgG / Q000008:administration & dosage / Q000032:analysis; D013388:Succinimides / Q000008:administration & dosage / Q000032:analysis*
chemical_list: C502599:Alexa Fluor 750; C499173:FCGR2B protein, human; D017452:Receptors, IgG; D013388:Succinimides
keywords: Cryoslicing Imaging; Drug delivery; Fluorescence; Mouse model; Tissue attenuation coefficient