Label-free spectral imaging to study drug distribution and metabolism in single living cells

Qamar A. Alshammari,Rajasekharreddy Pala,Surya M. Nauli,Nir Katzir

doi:10.1038/s41598-021-81817-0

Abstract

During drug development, evaluation of drug and its metabolite is an essential process to understand drug activity, stability, toxicity and distribution. Liquid chromatography (LC) coupled with mass spectrometry (MS) has become the standard analytical tool for screening and identifying drug metabolites. Unlike LC/MS approach requiring liquifying the biological samples, we showed that spectral imaging (or spectral microscopy) could provide high-resolution images of doxorubicin (dox) and its metabolite doxorubicinol (dox’ol) in single living cells. Using this new method, we performed measurements without destroying the biological samples. We calculated the rate constant of dox translocating from extracellular moiety into the cell and the metabolism rate of dox to dox’ol in living cells. The translocation rate of dox into a single cell for spectral microscopy and LC/MS approaches was similar (~ 1.5 pM min−1 cell−1). When compared to spectral microscopy, the metabolism rate of dox was underestimated for about every 500 cells using LC/MS. The microscopy approach further showed that dox and dox’ol translocated to the nucleus at different rates of 0.8 and 0.3 pM min−1, respectively. LC/MS is not a practical approach to determine drug translocation from cytosol to nucleus. Using various methods, we confirmed that when combined with a high-resolution imaging, spectral characteristics of a molecule could be used as a powerful approach to analyze drug metabolism. We propose that spectral microscopy is a new method to study drug localization, translocation, transformation and identification with a resolution at a single cell level, while LC/MS is more appropriate for drug screening at an organ or tissue level.

Highlights

We propose that spectral microscopy is a new method to study drug localization, translocation, transformation and identification with a resolution at a single cell level, while Liquid chromatography (LC)/mass spectrometry (MS) is more appropriate for drug screening at an organ or tissue level
Because evaluation of drug and its metabolite is an essential process to understand drug activity, stability, toxicity and distribution during drug development[23], we compared the use of our novel technique to liquid chromatography and mass spectrometry (LC/MS), which is the standard analytical tool for screening and identifying drug m
Without labeling drugs and destroying the samples, spectral imaging is a novel method that allows us to analyze the dynamics of drug distribution and metabolism in single living cells (Supp Fig. S7)

Summary

Introduction

The spectral microscopy indicated that unlike dox and dox’ol, rhodamine was mainly localized in cytoplasm. We further validated the spectral microscopy system using rhodamine (Fig. 5a). While other chemicals with well-characterized spectra could be used, the main reason rhodamine was selected to validate our system was that dox and rhodamine possessed similar spectra. The spectra (or wavelengths) of dox and rhodamine were distinct from each other. Dox has a peak spectrum at 592 nm, while rhodamine has a peak at 580 nm. The significant of our studies using spectral information (rather than fluorescent) was to eventually distinguish the identity of two closely resemble molecules; ie. The significant of our studies using spectral information (rather than fluorescent) was to eventually distinguish the identity of two closely resemble molecules; ie. dox vs. dox’ol

Methods

Results

Discussion

Conclusion