BackgroundUnderstanding stem cell behavior as a delivery tool in cancer therapy is essential for evaluating their future clinical potential. Previous in-vivo studies proved the use of mesenchymal stem cells (MSCs) for local delivery of the commonest anticancer drug, paclitaxel (PTX). Dental pulp is a relatively abundant noninvasive source of MSCs. We assess dental pulp stem cells (DPSCs), for the first time, as anticancer drug carriers. Confocal Raman microscopy is a unique tool to trace drug and cell viability without labeling.MethodsDrug uptake and cell apoptosis are identified through confocal Raman microscope. We traced translocation of cytochrome c enzyme from the mitochondria, as a biomarker for apoptosis, after testing both cancer and stem cells. The viability of stem cells was checked by means of confocal Raman microscope and by cytotoxicity assays.ResultsIn this study, we prove that DPSCs can be loaded in vitro with the anticancerous drug without affecting their viability, which is later released in the culture medium of breast cancer cells (MCF-7 cells) in a time-dependent fashion. The induced cytotoxic damage in MCF-7 cells was observed consequently after PTX release by DPSCs. Additionally, quantitative Raman images of intracellular drug uptake in DPSCs and MCF-7 cells were obtained. Cytotoxic assays prove the DPSCs to be more resistant to PTX as compared to bone marrow-derived MSCs, provided similar conditions.ConclusionsApplications of dental stem cells for targeted treatment of cancer could be a revolution to reduce morbidity due to chemotherapy, and to increase the efficacy of systemic cancer treatment.
Abstract. Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.
In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.
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