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

Cellular imaging is important in understanding drug pharmacokinetics and dynamics. As such, it is crucial that the drug is unmodified when performing these studies, to neither inhibit nor change its action and properties. Historically, fluorescence microscopy has been used for drug imaging due to its high sensitivity and ease of use, but bulky fluorescent tags have the potential to cause off-target effects and result in a change in the pharmacokinetic properties. The use and development of small optical tags are therefore attractive, as cellular systems can be probed with minimal perturbation to the cellular environment and the native kinetics of a drug. Bio-orthogonal Raman imaging makes use of molecular vibrations that are seldom observed in nature to determine spatial localization. Spontaneous Raman scattering can be used to achieve minimally labeled drug localization but is relatively slow and has a low spatial resolution when compared to fluorescence microscopy. Faster image acquisition and higher spatial resolution can be achieved by using stimulated Raman scattering (SRS), a powerful technique that is often used for native cellular imaging. The use of either intrinsically bio-orthogonal drugs or those with a small tag added allows Raman scattering to be used as a companion imaging diagnostic tool. This work assesses the localization of a covalently binding inhibitor of Bruton's tyrosine kinase, ibrutinib, using fluorescently labeled and bio-orthogonally Raman labeled analogues as companion diagnostic tools. Localization of these analogues was determined using fluorescence and Raman microscopies, and inhibitor retention was proportional to the expression of the kinase. Significant retention of the fluorescent analogue was observed independent of kinase expression, indicating significant nonspecific binding. Drug-induced effects were also explored using spectral phasor analysis of hyperspectral SRS data to assess lipid metabolism, where BTK inhibition was shown to cause an increase in the lipid content and change in the lipid type, which was proportional to kinase expression. This work showcases the advantages of Raman scattering techniques over fluorescence as companion imaging diagnostic tool and as a method of assessing phenotypic lipid shifts upon treatment with an anticancer drug.