Simultaneous Evaluations of pH and Enzyme Activity with a CEST MRI Contrast Agent

Chathuri J. Kombala, Sanjaya D. Lokugama, Aikaterini Kotrotsou, Tianzhe Li, Alyssa C. Pollard, Mark D. Pagel

Research output: Contribution to journalArticlepeer-review

Abstract

The extracellular tumor microenvironment of many solid tumors has high acidosis and high protease activity. Simultaneously assessing both characteristics may improve diagnostic evaluations of aggressive tumors and the effects of anticancer treatments. Noninvasive imaging methods have previously been developed that measure extracellular pH or can detect enzyme activity using chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). Herein, we developed a single-hybrid CEST agent that can simultaneously measure pH and evaluate protease activity using a combination of dual-power acidoCEST MRI and catalyCEST MRI. Our agent showed CEST signals at 9.2 ppm from a salicylic acid moiety and at 5.0 ppm from an aryl amide. The CEST signal at 9.2 ppm could be measured after selective saturation was applied at 1 and 4 μT, and these measurements could be used with a ratiometric analysis to determine pH. The CEST signal at 5.0 ppm from the aryl amide disappeared after the agent was treated with cathepsin B, while the CEST signal at 9.2 ppm remained, indicating that the agent could detect protease activity through the amide bond cleavage. Michaelis–Menten kinetics studies with catalyCEST MRI demonstrated that the binding affinity (as shown with the Michaelis constant KM), the catalytic turnover rate (kcat), and catalytic efficiency (kcat/KM) were each higher for cathepsin B at lower pH. The kcat rates measured with catalyCEST MRI were lower than the comparable rates measured with liquid chromatography–mass spectrometry (LC–MS), which reflected a limitation of inherently noisy and relatively insensitive CEST MRI analyses. Although this level of precision limited catalyCEST MRI to semiquantitative evaluations, these semiquantitative assessments of high and low protease activity still had value by demonstrating that high acidosis and high protease activity can be used as synergistic, multiparametric biomarkers.

Original languageEnglish (US)
Pages (from-to)4535-4544
Number of pages10
JournalACS Sensors
Volume6
Issue number12
DOIs
StatePublished - Dec 24 2021

Keywords

  • CEST
  • Michaelis−Menten kinetics
  • enzyme activity
  • magnetic resonance imaging
  • pH

ASJC Scopus subject areas

  • Bioengineering
  • Instrumentation
  • Process Chemistry and Technology
  • Fluid Flow and Transfer Processes

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