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Quantitative Determination of Intracellular Bond Cleavage

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Quantitative Analysis of Cellular Drug Transport, Disposition, and Delivery

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

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Abstract

Cleavable crosslinkers that respond to specific intracellular cues (pH, reducing environments, enzymes, etc.) are a critical component of drug delivery systems, which drives the subcellular localization and processing of therapeutic cargo. With numerous stimuli-responsive drug delivery systems in development, a quantitative measurement of their intracellular processing is of paramount importance. In this chapter, we discuss methods for determining the intracellular rate of bond cleavage and highlight a recent framework developed in our group for quantifying the rate of intracellular bond degradation in the endocytic pathway. This quantitative method involves the use of fluorescent FRET probes built on an oligothioetheramide (oligoTEA) trifunctional linker scaffold, that when attached to antibodies can report compartment specific cleavage events. This method involves the synthesis and site-specific bioconjugation of a reduction-sensitive FRET-based crosslinker to a variety of targeting ligands. We demonstrate this concept with trastuzumab, a humanized monoclonal antibody against the HER2 receptor. Furthermore, this chapter details a kinetic model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model, developed in conjunction with live-cell experiments, can be used to extract the rate constant for intracellular bond degradation. We present an example of a trastuzumab FRET-probe conjugate bearing a reduction-sensitive disulfide bond. The framework outlined in this chapter is applicable to the quantification of drug delivery systems that employ alternative endocytosis pathways, bond types, and cell types.

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Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, CA, USA

    Joshua A. Walker

  2. Berkeley Lights Inc.,, Emeryville, CA, USA

    Michelle R. Sorkin

  3. Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    Christopher A. Alabi

Authors
  1. Joshua A. Walker
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  2. Michelle R. Sorkin
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  3. Christopher A. Alabi
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Corresponding author

Correspondence to Christopher A. Alabi .

Editor information

Editors and Affiliations

  1. College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    Gus R. Rosania

  2. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA

    Greg M. Thurber

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Walker, J.A., Sorkin, M.R., Alabi, C.A. (2021). Quantitative Determination of Intracellular Bond Cleavage. In: Rosania, G.R., Thurber, G.M. (eds) Quantitative Analysis of Cellular Drug Transport, Disposition, and Delivery. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1250-7_14

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  • DOI: https://doi.org/10.1007/978-1-0716-1250-7_14

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  • Print ISBN: 978-1-0716-1249-1

  • Online ISBN: 978-1-0716-1250-7

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