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Article

Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging

1
Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
2
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
*
Author to whom correspondence should be addressed.
Pharmaceutics 2020, 12(2), 132; https://doi.org/10.3390/pharmaceutics12020132
Received: 1 January 2020 / Revised: 20 January 2020 / Accepted: 2 February 2020 / Published: 5 February 2020
(This article belongs to the Special Issue Molecular Imaging in Targeted Drug Delivery)
Many tools for studying the pharmacokinetics of biologics lack single-cell resolution to quantify the heterogeneous tissue distribution and subsequent therapeutic degradation in vivo. This protocol describes a dual-labeling technique using two near-infrared dyes with widely differing residualization rates to efficiently quantify in vivo therapeutic protein distribution and degradation rates at the single cell level (number of proteins/cell) via ex vivo flow cytometry and histology. Examples are shown for four biologics with varying rates of receptor internalization and degradation and a secondary dye pair for use in systems with lower receptor expression. Organ biodistribution, tissue-level confocal microscopy, and cellular-level flow cytometry were used to image the multi-scale distribution of these agents in tumor xenograft mouse models. The single-cell measurements reveal highly heterogeneous delivery, and degradation results show the delay between peak tumor uptake and maximum protein degradation. This approach has broad applicability in tracking the tissue and cellular distribution of protein therapeutics for drug development and dose determination. View Full-Text
Keywords: antibody–drug conjugates; pharmacokinetic imaging; single cell measurements; residualization antibody–drug conjugates; pharmacokinetic imaging; single cell measurements; residualization
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MDPI and ACS Style

Nessler, I.; Cilliers, C.; Thurber, G.M. Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging. Pharmaceutics 2020, 12, 132. https://doi.org/10.3390/pharmaceutics12020132

AMA Style

Nessler I, Cilliers C, Thurber GM. Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging. Pharmaceutics. 2020; 12(2):132. https://doi.org/10.3390/pharmaceutics12020132

Chicago/Turabian Style

Nessler, Ian, Cornelius Cilliers, and Greg M. Thurber. 2020. "Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging" Pharmaceutics 12, no. 2: 132. https://doi.org/10.3390/pharmaceutics12020132

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