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Visualization of a Bruton's Tyrosine Kinase Inhibitor Using Fluorescence and Raman Microscopy.

Andrew S Merchant1, William J Tipping1, Duncan Graham1

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Raman imaging offers a more accurate way to track drugs in cells than traditional fluorescence microscopy. This technique reveals drug retention and metabolic changes, providing better insights into drug effectiveness and side effects.

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Area of Science:

  • Cellular and Molecular Imaging
  • Pharmacokinetics and Pharmacodynamics
  • Chemical Biology

Background:

  • Accurate cellular imaging is vital for understanding drug behavior, requiring unmodified drugs to avoid altering their properties.
  • Traditional fluorescence microscopy uses bulky tags that can cause off-target effects and alter drug pharmacokinetics.
  • Minimally perturbing imaging techniques are needed to study cellular systems and native drug kinetics.

Purpose of the Study:

  • To assess the localization of ibrutinib, a Bruton's tyrosine kinase inhibitor, using both fluorescent and bio-orthogonal Raman labeled analogues.
  • To compare the advantages of Raman scattering techniques against fluorescence microscopy for drug imaging and assessing drug-induced effects.
  • To investigate drug-induced phenotypic shifts in lipid metabolism using hyperspectral stimulated Raman scattering (SRS).

Main Methods:

  • Utilized fluorescence and Raman microscopies to determine the spatial localization of fluorescently and Raman-labeled ibrutinib analogues.
  • Employed stimulated Raman scattering (SRS) microscopy for high-resolution, high-speed cellular imaging.
  • Applied spectral phasor analysis of hyperspectral SRS data to evaluate drug effects on lipid metabolism.

Main Results:

  • Inhibitor retention was found to be proportional to Bruton's tyrosine kinase (BTK) expression.
  • Fluorescent analogue showed significant nonspecific binding, independent of kinase expression.
  • BTK inhibition led to increased lipid content and altered lipid type, proportional to kinase expression, as revealed by SRS.

Conclusions:

  • Raman scattering techniques, particularly SRS, offer superior advantages over fluorescence microscopy as companion imaging tools for drug localization.
  • Raman imaging accurately reflects drug retention based on target expression and minimizes concerns about nonspecific binding.
  • This study demonstrates Raman scattering's capability to assess phenotypic shifts in lipid metabolism induced by anticancer drugs like ibrutinib.