Abstract
Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.
Highlights
For studying complex cell populations, fluorescence-based multi-parameter cytometric analysis techniques are routinely used for monitoring the expression of multiple phenotypic markers at a single cell level
3.1 Imaging and quantification of diattenuation, optical density, and Cy5 fluorescence of crystal-like drug inclusions (CLDIs) using multi-parameter imaging system To demonstrate the capabilities of this multi-parameter imaging system, the drug clofazimine was chosen as a model compound
After analyzing isolated CLDIs from mice treated with CFZ, we decided to probe the utility of this instrument in studying live cells isolated from animals fed the drug for a period of eight weeks
Summary
For studying complex cell populations, fluorescence-based multi-parameter cytometric analysis techniques are routinely used for monitoring the expression of multiple phenotypic markers at a single cell level. For this purpose, fluorescence-based flow and image cytometry instruments can be used to quantify the expression of different molecular markers down to the level of individual cells, using fluorescently tagged antibodies that recognize specific cell surface receptors, intracellular proteins or other cellular targets of interest. Polarization microscopy can be used to obtain information about how the molecules within these subdomains are oriented, providing insight into the underlying molecular order of the material [9,10,11]
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