Dynamic fluorescence microscopy of cellular uptake of intercalating model drugs by ultrasound-activated microbubbles

The 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. SYTOX 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. Ultrasound-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 x 10(-3) to 2.2 x 10(-3) s(-1) for 1 and 20 mu M, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. These 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

Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.publishedVersio

Developing a Standard Set of Patient-Centred Outcomes for inflammatory Bowel Disease-an international, cross-disciplinary consensus

Background and Aims: Success in delivering value-based healthcare involves measuring outcomes that matter most to patients. Our aim was to develop a minimum Standard Set of patient-centred outcome measures for inflammatory bowel disease [IBD], for use in different healthcare settings. Methods: An international working group [n = 25] representing patients, patient associations, gastroenterologists, surgeons, specialist nurses, IBD registries and patient-reported outcome measure [PROM] methodologists participated in a series of teleconferences incorporating a modified Delphi process. Systematic review of existing literature, registry data, patient focus groups and open review periods were used to reach consensus on a minimum set of standard outcome measures and risk adjustment variables. Similar methodology has been used in 21 other disease areas [www.ichom.org]. Results: A minimum Standard Set of outcomes was developed for patients [aged =16] with IBD. Outcome domains included survival and disease control [survival, disease activity/remission, colorectal cancer, anaem

MICROBUBBLE-ASSISTED ULTRASOUND-INDUCED TRANSIENT PHOSPHATIDYLSERINE TRANSLOCATION

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Correction for photobleaching in dynamic fluorescence microscopy : application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally. Photobleaching rates up to 5.0 10(-3) s(-1) were measured when applying power densities up to 0.2 W.cm(-2). By applying the three-compartment model, the model drug uptake rate of 6.0 10(-3) s(-1) was measured independent of the applied laser power. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization

CORRECTION OF PHOTOBLEACHING FOR THE ASSESSMENT OF PHARMACOKINETIC PARAMETERS USING DYNAMIC FLUORESCENCE MICROSCOPY

Local drug delivery in oncology aims at depositing high doses of anticancer agents while limiting their toxic side effects. Biological barriers, such as cell plasma membranes, hinder their delivery and requires strategies to address this challenge. Previously [1], we demonstrated the feasibility to monitor in real-time, with dynamic fluorescence microscopy, the in-tracellular delivery of a hydrophilic model drug mediated by ultrasound (US), and to quantify the pharmacokinetic parameters derived from a two-compartment model. We evaluate here the impact of the photobleaching (PB) effect experimentally , and compute the PB-corrected uptake kinetics

Correction for Photobleaching in Dynamic Fluorescence Microscopy: Application in the Assessment of Pharmacokinetic Parameters in Ultrasound-Mediated Drug Delivery

Purpose. We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates.Procedures. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally.Results. Photobleaching rates up to 5.0 10-3 s-1 were measured when applying power densities up to 0.2 W.cm-2. By applying the three-compartment model, the model drug uptake rate of 6.0 10-3 s-1 was measured independent of the applied laser power.Conclusions. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization