Blood-brain barrier disruption with focused ultrasound enhances delivery of dopamine transporter tracer (PE2I) into the brain

International audiencePE2I is one of the most selective ligands for dopamine transporter. However it is associated with blood-brain barrier (BBB) permeability limitations. The aim of this study was to investigate the use of ultrasound and microbubbles to increase its delivery through the BBB and by determining the optimal experimental conditions that achieve a transient and safe BBB disruption. First, we stablished the ultrasound conditions that achieved a transient BBB disruption in rats using a non-permeant marker, Evans blue. Hence SonoVue® (450μL/kg) and Evans blue (100mg/kg) were intravenously administered. BBB leakage was obtained using ultrasound insonation through the rat skull at 1.6MPa, PRF 1Hz, duty cycle 1%, burst 10ms during 120sec. BBB disruption was observed in all treated animals (N=4) by histological analysis. The same experimental conditions were applied to enhance brain uptake of PE2I. Biological samples were analyzed using a scintillation counter apparatus. The results showed 50% and 20% increase of 125I-PE2I uptake in the striatum and cerebral cortex, respectively, in the treated rats (N=5) versus control (N=4). Similar enhancements were observed using SonoVue® at half concentration. This innovative method provides a great potential for intracerebral delivery of molecular ligands that could be used for the therapy of brain diseases

Visualization of membrane loss during the shrinkage of giant vesicles under electropulsation

We study the effect of permeabilizing electric fields applied to two different types of giant unilamellar vesicles, the first formed from EggPC lipids and the second formed from DOPC lipids. Experiments on vesicles of both lipid types show a decrease in vesicle radius which is interpreted as being due to lipid loss during the permeabilization process. We show that the decrease in size can be qualitatively explained as a loss of lipid area which is proportional to the area of the vesicle which is permeabilized. Three possible mechanisms responsible for lipid loss were directly observed: pore formation, vesicle formation and tubule formation.Comment: Final published versio

NAP1L1-MLLT10 is a rare recurrent translocation that is associated with HOXA activation and poor treatment response in T-cell acute lymphoblastic leukaemia

International audienceno abstrac

Doxorubicin-liposomes loaded microbubbles for ultrasound-triggered doxorubicin delivery

International audienceDoxorubicin (Dox) is a potent chemotherapeutic whose severe side effects limit its clinical efficacy. Microbubble-assisted ultrasound has become a promising strategy for non-invasive local drug delivery to increase the drug concentration locally and to reduce systemic side effects. The aim of this study is to evaluate the effectiveness of administration of Dox-liposomes loaded on MB combined with ultrasound in human glioblastoma cells. Experiments were carried out with free Dox or Dox-loaded MBs on a cell suspension of U-87MG cells. Ultrasound waves were transmitted at 1MHz frequency with a pulse repetition period of 100µs, 40 cycles per pulse and for 30s. Cell viability was evaluated by Trypan blue assay 24h and 48h later. Using Dox alone, the cell viability was 63±3% and 26±2% at 24h and 48h later, respectively. The combination of ultrasound at 600 kPa and Dox-loaded MBs induced a 2.5-fold decrease of cell viability compared to the incubation of Dox-loaded MBs alone at 24h and 48h after treatment, respectively. At 24h, this combination was 3 times more efficient than the doxorubicin treatment alone. The conclusions drawn from this study show the potential of this strategy for a controlled, efficient, and safe drug delivery. Project funded by the EU Project SONODRUGS (NMP4-LA-2008-213706)

Development and characterization of an enhanced nonviral expression vector for electroporation cancer treatment

Nonviral plasmid DNA gene therapy represents a promising approach for the treatment of many diseases including cancer. Intracellular delivery of DNA can be achieved with the application of electroporation, which facilitates the initial transport of exogenous DNA across the cell membrane into the cytoplasm. However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches. To overcome these expression difficulties, we developed a proof-of-concept vector enhanced expression vector (EEV), which incorporates elements from viral systems including nuclear localization sequences and a viral replicase from the Semliki Forest virus. The replicase allows for cytoplasmic mRNA expression and bypasses the need for nuclear localization to generate high levels of gene expression. We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types. Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model. Using a novel endoscopic electroporation system, EndoVe, we demonstrate and compare, for the first time, both standard cytomegalovirus (CMV) promoter-driven plasmid and EEV gene expression in intraluminal porcine tissues. Our EEV plasmid displays reliable and superior expression capability, and due to its inherent induced oncolytic activity in transfected cells, it may enhance the efficacy and safety of several cancer immunogene therapy approaches

Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood–brain and blood–spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments

Calcium electroporation and electrochemotherapy for cancer treatment:Importance of cell membrane composition investigated by lipidomics, calorimetry and in vitro efficacy

Abstract Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy

A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

We report a new and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (EggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of the latter with cholesterol and dioleoylphosphatidylethanolamine (DOPE). Our data confirm previous results for EggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of DOPE is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. Presumably, interlipid hydrogen bonding lies in the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner