Tracking of Cell Nuclei for Assessment of In Vitro Uptake Kinetics in Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Derieppe, Marc; Kuijf, Hugo J.; Moonen, Chrit; Bos, Clemens

doi:10.1007/s11307-014-0726-3

Cited by 7 publications

(11 citation statements)

References 30 publications

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“…As depicted in Figure 1C, the permeabilization level to propidium iodide is 2 ± 0.01% in absence of ultrasound, indicating likely that these cells internalizing propidium iodide are presumably dead 36 . In agreement with previous investigations 38,39 , the permeabilization level to propidium iodide significantly increased using ultrasound and microbubbles (p < 0.05). As illustrated in Figure 1C, BG8214 microbubbles were more efficient than BG8610 microbubbles to induce membrane permeabilization to small and non-permeant molecule (78 ± 1% vs 53 ± 1%; p < 0.01) and both of them did not affect the cell viability (Figure 1A).…”

Section: Effects Of Bg8610 and Bg8214 On Cell Permeabilizationsupporting

confidence: 93%

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model

et al. 2019

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The combination of microbubbles and ultrasound is an emerging method for non-invasive and targeted enhancement of anticancer drugs uptake. This method showed to increase local drug extravasation in tumor tissue while reducing the systemic adverse effects in various tumor models. The present study aims into evaluating the therapeutic efficacy of this approach both in-vitro and in-vivo for Nab-paclitaxel delivery in a pancreatic tumor model. Ultrasound and microbubbles of different types in combination with Nab-paclitaxel showed a significant decrease in cell viability of human pancreatic cancer cells in comparison with Nab-paclitaxel treatment alone in in-vitro scenario. In-vivo, the results demonstrated that ultrasound and microbubbles in combination with Nab-paclitaxel induced a significant decrease in tumor growth in subcutaneous pancreatic adenocarcinoma xenograft model in nude mice in comparison to tumors treated with Nab-paclitaxel alone. The post-mortem anatomopathological analyses of tumor tissues partially confirmed these results. In conclusion, this study demonstrates that microbubble-assisted ultrasound is a promising method to improve the therapeutic efficacy of Nab-paclitaxel in a pancreatic cancer model.

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Section: Effects Of Bg8610 and Bg8214 On Cell Permeabilizationsupporting

confidence: 93%

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model

et al. 2019

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“…SYTOX Green uptake following USMB treatment was monitored in a population of cells using a fibered confocal fluorescence microscope (FCFM) (Cellvizio®, Mauna Kea Technologies, Paris, France), as previously described [ 22 ]. The FCFM microprobe has a field of view of 593 × 593 μm, and a 488 nm laser was used to excite SYTOX Green at 0.5 mW laser power.…”

Section: Methodsmentioning

confidence: 99%

“…To obtain fluorescence kinetics insensitive to motion, the individual cells in the FCFM data were segmented and tracked over the 30 min acquisition using MATLAB (MathWorks, USA), to determine the photobleaching rate constant k pb and the fluorescence rate constant k f , similar to Derieppe et al [ 22 ]. For a detailed description of this method, the reader is referred to the “ Electronic Supplementary Material ” of this manuscript.…”

Section: Methodsmentioning

confidence: 99%

Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles

et al. 2017

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PurposeThe combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs.ProceduresSYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line.ResultsUltrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10−3 to 2.2 × 10−3 s−1 for 1 and 20 μM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells.ConclusionsThese data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-016-1042-x) contains supplementary material, which is available to authorized users.

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“…Released-DOX penetration kinetics could be assessed upon imaging of cell-uptake kinetics of released DOX in the tumor interstitium and DOX diffusion coefficient, both in the same dataset. Here, 241 cell nuclei could be detected using the image processing pipeline developed previously [23], thus allowing a statistical analysis. Uptake time constants of 3 minutes were found after injection of 4 mg/kg DOX encapsulated in TSLs.…”

Section: Discussionmentioning

confidence: 99%

“…Cell uptake kinetics was assessed using the dedicated parametric pipeline described in Derieppe et al [23]. Briefly, this automated pipeline includes cell detection, which was facilitated by a nonlocal means algorithm [24], a cell tracking with the iterative-closest-point algorithm [25], and a fitting of the resulting uptake profiles by means of a two-compartment model where the fluorescence signal intensity ( I ) is as follows [26]:It=A1−e−kt−T,where A is the maximum DOX fluorescence signal, T the time of signal onset, and k the uptake rate.…”

Section: Kinetic Analysis Of Released-dox Penetrationmentioning

confidence: 99%

Assessment of Intratumoral Doxorubicin Penetration after Mild Hyperthermia-Mediated Release from Thermosensitive Liposomes

Derieppe

Escoffre

Houtum

et al. 2019

Contrast Media & Molecular Imaging

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In solid tumors, rapid local intravascular release of anticancer agents, e.g., doxorubicin (DOX), from thermosensitive liposomes (TSLs) can be an option to overcome poor extravasation of drug nanocarriers. The driving force of DOX penetration is the drug concentration gradient between the vascular compartment and the tumor interstitium. In this feasibility study, we used fibered confocal fluorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumor after its intravascular release from TSLs, Thermodox®. Cell uptake kinetics of the released DOX was quantified, along with an in-depth assessment of released-DOX penetration using an evolution model. A subcutaneous rat R1 rhabdomyosarcoma xenograft was used. The rodent was positioned in a setup including a water bath, and FCFM identification of functional vessels in the tumor tissue was applied based on AngioSense. The tumor-bearing leg was immersed in the 43°C water for preheating, and TSLs were injected intravenously. Real-time monitoring of intratumoral (i.t.) DOX penetration could be performed, and it showed the progressing DOX wave front via its native fluorescence, labeling successively all cell nuclei. Cell uptake rates (1/k) of 3 minutes were found (n=241 cells), and a released-DOX penetration in the range of 2500 µm2·s−1 was found in the tumor extravascular space. This study also showed that not all vessels, identified as functional based on AngioSense, gave rise to local DOX penetration.

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Tracking of Cell Nuclei for Assessment of In Vitro Uptake Kinetics in Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Cited by 7 publications

References 30 publications

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model

Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles

Assessment of Intratumoral Doxorubicin Penetration after Mild Hyperthermia-Mediated Release from Thermosensitive Liposomes

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