Abstract
Raman spectroscopy has been used to observe uptake, metabolism and response of single-cells to drugs. Photodynamic therapy is based on the use of light, a photosensitiser and oxygen to destroy tumour tissue. Here, we used single-cell Raman spectroscopy to study the uptake and intracellular degradation of a novel photosensitiser with a diphenylacetylene structure, DC473, in live single-cells from colorectal adenocarcinoma cell lines SW480, HT29 and SW620. DC473 was seen to predominantly accumulate in lipid droplets, showing higher accumulation in HT29 and SW620 cells than in SW480 cells, with a broader DC473 peak shifted to higher wavenumbers. DC473 activation and effects were tracked on live single-cells for 5 minutes. Upon exposure to UV light, the DC473 signal intensity dropped, with remaining DC473 shifting towards higher wavenumbers and widening, with a lifetime of approximately 50 seconds. Morphologically, SW480 and SW620 cells showed changes upon photodynamic therapy, whereas HT29 cells showed no changes. Morphological changes correlated with higher remaining DC473 signal after UV exposure. Our research suggests that DC473 forms aggregates within the cells that disaggregate following activation, showing the potential of Raman spectroscopy for the study of time-dependent single-cell pharmacodynamics.
Highlights
Drug efficacy or resistance in vitro is often tracked using single-protein assays
Our experiments indicate that DC473 can be readily taken up by different Colorectal cancer (CRC) cell lines
Raman spectroscopy results indicated that accumulation mainly occurs in lipid droplets and the cytosol, and was significantly higher for more advanced adenocarcinoma HT29 and SW620 cells compared to SW480 cells
Summary
Drug efficacy or resistance in vitro is often tracked using single-protein assays. This approach may miss the bigger picture and lead to responses that are not consistent with in vivo results.[1] Raman spectroscopy provides the molecular signature of a sample using the inelastic scattering of monochromatic light, for the detection of chemical functional groups.[2,3] It is a label-free, non-destructive technique, with spatial resolution in the micrometre scale, allowing for singlecell measurements on live cells.[4,5,6,7,8] The target compound can be detected within a cell in a semi-quantitative manner and. Biorthogonal Raman tagging uses molecules that exhibit vibrational bands in the Raman silent region of cells (1800–2800 cm−1) such as alkynes, enabling more accurate detection of compounds in single cells.[16,17,18,21,22,23,24,25,26] Alternatively, non-linear RS, such as Coherent anti-Stokes RS (CARS)[27] or Stimulated RS (SRS)[28] can be used though they require a more complicated setup with multiple laser beams making their translation to medical and biological applications challenging.[29]
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