Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

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 mu M of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 mu M) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.18.5.056010

Confocal Raman Microscopy to image targeted chemotherapy

International audienceThe Side effects of chemotherapy in cancer treatments are unavoidable due to the lack of anticancer drug’s specificity and it has a painful impact on patient quality of life. Over the past 30 years, increasing efforts is done to optimize chemotherapy dosing to reduce drug toxicity while increasing its efficacy. A new study proves stem cells can act as a drug reservoir and they will release anticancer drug in its original form in nearby area of cancer cells. Stem cells, due to their capacity to uptake drug, can control the drug toxicity. Dental Pulp Stem Cells DPSCs are able to uptake Paclitaxel PTX and could release it in the culture medium gradually. The conditioned culture medium (culture medium plus released PTX from DPSC) is transferred to the breast cancer cells MCF7. Visualizing the drug uptake intracellular could provide us mechanism of action of the drug. Confocal Raman microscopy as a noninvasive label free method is being used to trace drugs intracellular [1,2]. Applying Confocal Raman Microscopy, anticancer drug uptake by MCF7 is imaged. Surprisingly MCF7 - without any direct contact with PTX- showed drug uptake. It proves the stem cells carry and deliver anticancer drug without its modification. It could be a revolution in chemotherapy to avoid the side effects and increase the drug efficacy.[1] H. Salehi et al. Label-free detection of anticancer drug paclitaxel in living cells by Confocal Raman Microscopy. Applied physics letter. 2013, 102, 113701.[2] H. Salehi et al. Confocal Raman data analysis to comparison of apoptotic and non-apoptotic MCF-7 cells caused by anticancer drug paclitaxel. Journal of Biomedical Optics. 2013,18(5), 056010

Dental pulp stem cells used to deliver the anticancer drug paclitaxel

International audienceAbstractBackground: Understanding 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.Methods: Drug 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.Results: In 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.Conclusions: Applications 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.Keywords: Raman spectroscopy, Dental pulp stem cells, Cancer, Paclitaxel, Apoptosi

Confocal Raman Microscope for the study of anti-cancer drug delivery by dental pulp stem cells

International audienceUsing stem cells as anticancer drug carriers aims for targeting tumors and reducing the side effects of chemotherapy. Coupling the multipotential capacities of stem cells with the nanomedical properties of the drugs provides an attractive platform for cancer therapy. Raman microscopy with its high spatial resolution and sensitivity is used as a unique tool to trace drugs and cellular components without labeling. Drug uptake, transfer, and release by stem cells, in addition to the effect on tumor cells were studied by Confocal Raman Microscope.Moreover, to further understand the intra-cellular effect of paclitaxel drug exposure and stem cell treatment upon the stem and cancer cells, we have traced different enzymes and biomarkers intra-cellularly. By software-aided Raman analysis, we were able to detect and measure the enzymes: cytochrome c in the mitochondria and Matrix Metalloproteinase MMP-1, as cellular markers pre and post-treatment. This was performed by calculating the Pearson correlation coefficient for the selected reference spectrum to the spectral correlation matrix. We investigated the cellular viability by means of confocal Raman microscope, also verified by apoptosis and cytotoxic assays.Our results prove the promising use of dental pulp stem cells as cellular vehicles, and significant phenotypic change in cellular marker expression after treatment with paclitaxel. Biochemical imaging of intracellular drug uptake using Confocal Raman would help to have a better understanding of drugs mechanism of action and cellular behavior, including cellular interaction mechanisms

Precise nanodiamonds localization in cellular environment

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Detection and localization of luminescent nanodiamonds in cellular environment using Raman imaging

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Nanodiamonds internalization in MCF7 cells monitored by cell membrane stiffness changes and their luminescent signal

International audienceLuminescent nanodiamonds (ND) are attractive tools for nanoscale biologic cellular imaging allowing both photoluminescence (PL) and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright NDs with high content of nitrogen-vacancy centres [2] that are anticipated to serve as a cell probes. In this work we present novel method of NDs detection in cellular environment. We demonstrate simultaneous visualization of NDs and of the non- labeled nucleus of living cells based on Raman and PL detection as a new tool for the localization of internalized nanoparticles.To this end, NDs of size ranging from ultra-small particles ~ 5 nm to 60 nm were used, prepared from Ib synthetic diamond. Cells used for this experiment were from mammalian breast cancer (MCF7). We report on accomplishing to successfully internalize ND particles in MCF7 cells. We show that ND internalization can be monitored by Raman imaging method using K-mean cluster analysis. Whilst standard Raman imaging methods of NDs make use of the sp3 diamond Raman signal, which limits their use to 100 nm size particles or bigger [3], here we employ Raman imaging in a novel way to detect small near-IR cellular probe. Changes of cells stiffness were detected by force measurement in atomic force microscopy after incubating cells with NDs, suggesting cell membrane hardening upon ND uptake.[1] L. Moore, M. Nesladek et al., Nanoscale (2014)[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013) [3] C.-Y. Cheng et al., Applied Physics Letter (2007

Morphological and nanomechanical characterization of dental pulp stem cells in neuron co-culture and neurogenic differentiation

International audiencePeripheral nerve injuries are serious traumas resulting in loss of motor and sensory functions of nerves. The post-traumatic neuropathies are often chronic and mostly resistant to current treatments. Our objective is to develop new strategies to regenerate nerve after trauma by biomimetic approaches and go towards in vitro neurogenesis. For this we use neural crest derived human multipotent dental pulp stem cells (hDPSC) in co-culture with sensory neurons from mice dorsal root ganglia (DRG), and an in vitro neurogenic differentiation of hDPSC [3].In order to explore nanomechanical and morphological characteristics of human DPSC and mouse DRG neurons atomic force microscopy was employed. We have shown that the mechanical properties of the soma and sub cellular growth cone regions correlate well with the amount, organization and dynamics of the underlying cytoskeletal structure [1,2]. Here we present preliminary data about hDPSC in co-culture, with mDRG neurons and during an in vitro neurogenic differentiation gathered by nanomechanical mapping.[1] O. Benzina, V. Szabo, O. Lucas, M-b. Saab, T. Cloitre, F. Scamps, C. Gergely and M. Martin (2013) Changes induced by peripheral nerve injury in the morphology and nanomechanics of sensory neurons, J. Biomedical Optics 18(10)[2] M. Martin, O.Benzina, V. Szabó, A. G. Végh, O. Lucas, T. Cloitre, F. Scamps and C. Gergely (2013) Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury, Plos One , 8 (2)[3] M. Király, B. Porcsalmy, A. Pataki, K. Kádár, M. Jelitai, B. Molnár, P. Hermann, I. Gera, WD. Grimm, B. Ganss, A. Zsembery, G. Varga, (2009) Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons, Neurochem. Int. 55 (323-332

Confocal Raman spectroscopy to monitor intracellular penetration of TiO2 nanoparticles

Confocal Raman microscopy, a noninvasive, label-free, and high-spatial resolution imaging technique, in combination with K-mean cluster analysis and a correlation coefficient map, was employed to trace titanium dioxide (TiO2) nanoparticles in living MCF-7 and TERT cells. The penetration of TiO2 nanoparticles into cells revealed a gradual time-dependent diffusion of nanoparticles over the entire cell. Cell apoptosis was monitored by tracing cytochrome c diffusion into the cytoplasm. A comparison with the mitochondrial clustering indicated that cytochrome c was inside the mitochondria for TiO2 concentration of 2 mu gml(-1). This result demonstrates that the presence of TiO2 particles within a cell does not induce apoptosis. We demonstrated that confocal Raman microscopy allow to follow penetration of TiO2 particles in cell and to monitor the apoptotic status of the penetrated cells. Copyright (c) 2014 John Wiley & Sons, Ltd