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

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.

Enhancing scanning electrochemical microscopy's potential to probe dynamic co-culture systems via hyperspectral assisted-imaging

“…Hyperspectral imaging widens the scope of research with the use of tunable filters, which allow users to increase spectral discrimination between wavelengths in comparison to the use of standard gratings. Spectral imaging has been used to understand cellular dynamics with respect to pharmacological responses, 26 single-cell viability, 27,28 and carcinogenesis. [28][29][30][31][32] While most methods depend on labeling the analyte of interest, others have used spectral imaging to characterize analytes with known spectral properties, such as the experimental drug doxorubicin which has known fluorescence properties.…”

“…[28][29][30][31][32] While most methods depend on labeling the analyte of interest, others have used spectral imaging to characterize analytes with known spectral properties, such as the experimental drug doxorubicin which has known fluorescence properties. 26 Hyperspectral imaging is momentous to the field of biological imaging as it allows for the differentiation of biochemical complexes that are known to display unique spectral signatures. 33 Hyperspectral imaging systems are characterized by their ability to collect hundreds of spectral bands; the temporal and spatial resolution of a hyperspectral imaging system are based on the limitations of the system's optical and mechanical components.…”

The use of advanced spectral imaging to reveal nanoparticle identity in biological samples

“…We recently introduced a spectral imaging system in which broader spectral characteristics are first identified, characterized and stored in a library. 20 Unlike other previous studies, however, our current studies did not identify a molecule based merely on the spectral intensity, relative intensity, or spectral points. Our new strategies using a higher spectral resolution and artificial intelligence for point recognition improved our capability to differentiate two very similar molecules.…”

“…Using spectral imaging, we were able to visualize fluorescent molecules fairly easily based mainly on their predominant fluorescence spectra (data not shown). 20 Note that the spectral identities might be different between non-labeled and fluorescent-labeled NPs. A fluorescence microscope might be more practical to detect the fluorescent-labeled NPs.…”

“…A broader spectrum is required to extract more precise characteristics of a single molecule. 20 Specifically, this approach provides a more specific fingerprinting for each NP.…”