Jean-Michel Escoffre

Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.

Ultrastructural modifications of cell membranes and organelles induced by sonoporation

Sonoporation increases transiently the native cell membrane permeability. However, the exact mechanism involved in the membrane permeabilization remains to be elucidated. While, no consensus is reached, the pore formation is usually hypothesized as having a central role in the membrane permeabilization induced by sonoporation. In this study, we investigate the sonoporation effect on the plasma membrane and organelles using electron microscopy. Adherent U-87 MG cells were insonated at 1 MHz, 1 W/cm2 acoustic intensity, 20% duty cycle for 10 or 60 s. BR14® microbubbles were added at a bubble/ cell ratio of 5. SYTOX® Green was used as a permeabilization marker. With these US conditions in combination with BR14®, flow cytometry results showed a permeabilization rate of 60%. The ultrastructural modifications of the cells were monitored by scanning and transmission electron microscopy (SEM/TEM) either immediately or 15 min post-sonoporation. Based on SEM images, the control cells (No US) showed a regular plasma membrane with microvilli, while the insonified cells (US+BR14®) exhibited circular and dark spots on their surfaces, suggesting pore-like structures. The number of these structures increased in the presence of BR14® and with the insonation time. The porelike structures size distribution is heterogeneous, and ranged from 10 nm to 160 nm. However, these structures might also correspond to caveolae or clathrin endocytic vesicles. To investigate the transient character of these structures, the cells were fixed 15 min after sonoporation. The results showed that the number of these pore-like structures decreased strongly indicating that the cells are still metabolically active. The maximal size of these pores is 100 nm and we suggest that small pores still require more time to reseal. Sonoporation effects on the organelles structure were investigated. Based on TEM images, insonified cells (US+BR14®) presented a stimulation of endocytosis pathways compared to control cells. In addition, the sonoporated cells showed less dense cytoplasm, suggesting a decrease of their viscosity, which would facilitate the intracellular traffic of delivered molecules. In conclusion, these microscopic observations reveal that the sonoporation effects are not confined to the membrane only, but also occur at the intracellular level.

Role of thermal and mechanical effects on drug release from thermosensitive nanocarriers

The combination of focused ultrasound (FUS) with thermosensitive liposomes (TSL) is a promising method for drug delivery since it allows a localized release upon moderate heating with ultrasound. Besides thermal effects, FUS also induces mechanical stresses on drug nanocarriers. We propose in this study to examine the influence of both effects (thermal and mechanical) on drug release. For this, an in-vitro setup allowing liposomal drug delivery using FUS was first evaluated. Calcein was used as a model drug. FUS experiments were performed in water at 37°C using a 1 MHz transducer focused at 48 mm, at 1 kHz PRF and 40% duty cycle. The driving pressure and the insonation duration were varied from 1 to 2 MPa and from 0 to 30 min, respectively. Thermal heating using a water-bath was also performed with temperatures from 37 to 49°C. For TSL, the release reaches a plateau above 42°C (45%) after 10 min heating while no release is observed for non-thermosensitive liposomes (NTSL). Using FUS, a rapid calcein release is observed for pressures from 1 to 1.5 MPa (from 0% to 49%) for TSL. Above 1.5 MPa, the release increases slightly (59% at 2 MPa). For NTSL, a weak calcein release is measured for acoustic pressures higher than 1.5 MPa. This release is attributed to the mechanical stress generated by FUS which is sufficient to destabilize the liposomal membrane. Mechanical stress alone can enhance the calcein release by up to 17% for pressures higher than 1.75 MPa.

Ultrasound-induced temperature elevation for in-vitro controlled release of temperature-sensitive liposomes

Drug loaded temperature-sensitive liposomes (TSLs) release their payload with mild hyperthermia near their phase transition temperature (Tmu2009=u200943–45u2009°C). Such a release may improve therapeutic efficacy and reduce toxic side effects in cancer treatment. In the present work, two different approaches are considered where focused ultrasound is used to induce the required temperature elevation for the release of doxorubicin from TSLs: (a) primary heating due to thermo-viscous absorption of ultrasound in absorptive media (oil, glycerol) and (b) secondary heating in non-absorptive media (blood, cell medium) due to heat transfer from the surroundings. Fine-wire thermocouple readings where in close agreement with theoretical predictions of temperature elevation with the Bioheat equation. Pulsing schemes to elevate and maintain the temperature at the desired value were designed with the Bioheat equation and validated with experiments. Fluorescence spectroscopy was used to assess the release of free doxorubicin that ...

Use of high intensity focused ultrasound for localized activation of thermosensitive liposomes for drug delivery.

Localized drug delivery holds great promise in improving drug efficacy in cancer treatment. Newly developed temperature‐sensitive liposomes (TSLs) loaded with doxorubicin have been shown to release their payload with mild hyperthermia near their phase transition temperature (Tm = 43–45 °C). In the present work, high intensity focused ultrasound is used to induce the required temperature elevation for the release of the drug from TSLs. A theoretical model based on Pennes’ bioheat equation was initially used to calculate the conditions for temperature elevation in fluids and tissue phantoms under conditions for drug activation. Acoustic pressures of 1–2 MPa at the focus with varying duty cycle (typically 50%) at 1 MHz frequency were calculated. Measurements of temperature rise were found in good agreement with our theoretical predictions. Fluorescence measurements were used to assess the release of free doxorubicin that exhibits higher fluorescence intensity than the liposomal formulation. In vitro experime...

Evaluation of the acoustic properties of clots during sonothrombolysis

Sonothrombolysis with microbubbles (STL) is being explored as a promising therapeutic alternative in ischemic stroke. The objective of this study is to better understand the impact of STL +/-rt-PA on the acoustic properties of blood clot in vitro.

Microscopic observations of sonoporation mechanisms

BackgroundSonoporation promises a local gene/drug delivery with a high therapeutic efficacy and low toxicity level. However, the mechanisms orchestrating the molecules uptake are still unclear. Here, we investigate the effects of sonoporation on the plasma membrane of U-87 MG cells, either immediately or at different times post-sonoporation, using electron microscopy, and also the implication of cytoskeleton during the sonoporation process.MethodsIn our set-up, the U-87 MG cells were seeded on 18u2005mm diameter cover slips, placed in 24-well plates. The acoustic exposure conditions consisted of ultrasound pulses at 1u2005MHz, 1W/cm2 with duty cycle of 20% for 60 seconds. BR14® microbubbles were added to the cell medium before sonoporation at a microbubble/cell ratio of 5. These acoustic parameters were obtained as a result of a prior optimization experiments. Membrane permeabilization after sonoporation was assessed using SYTOX® Green dye (1u2005µM), as a model drug which does not cross the membrane of normal cells....

Ultrastructural sonoporation bio-effects: Comparative study on two human cancer cell lines

Sonoporation increases transiently the cell membrane permeability, enabling the therapeutic compounds internalization into the cells. Several investigations reported heterogeneities in the permeabilization and transfection efficacy depending on the ultrasound (US) settings and cell type. Here, we compare the sonoporation effects on two human cell lines, glioblastoma and breast cancer using scanning electron microscopy (SEM). Adherent U-87 MG and MDA-MB-231 cells were insonated at 1 W/cm2, during 60 s at 10% or 20% duty cycle, in the presence of BR14® microbubbles, added at a microbubble-cell ratio of 5. SYTOX® Green, a non-permeant fluorescent dye was used at 1μM, to quantify the membrane permeabilization using flow cytometry. The ultrastructural changes of the cell membrane morphology were monitored by SEM. Flow cytometry results show that the percentage of permeabilized U-87 MG cells reaches 60%, while this value doesnt exceed 40% for MDA-MB-231 cells. These results indicate that the percentage of permeabilized cells depends on the cell type. SEM observations were carried out to elucidate the differences in permeabilization rate between the two cell lines. The SEM analysis reveals that control cells show regular plasma membrane morphology. Their insonation in the presence of BR14® induce the formation of dark holes on their membrane surfaces (named here pore-like structures). However, the quantitative analysis of the SEM micrographs highlights noticeable differences in morphological changes post-sonoporation between the two cell lines. Thus, the mean number of pore-like structures is more abundant on U-87 MG cell membrane than on MDA-MB-231 cell membrane (645 vs. 290). In addition, the mean size of pore-like structures depends on the cell line. Indeed, the mean size on MDA-MB-231 cells was 40 ± 1.2 nm (30-60 nm) while this value reached 80 ± 0.9 nm (10 to 160 nm) for U-87 MG cells. In conclusion, the study confirms that the pore-like structures observed post sonoporation are directly associated to the cell permeabilization rate. Moreover, the observed differences in the permeabilization levels between both cell lines could be attributed to the differences in the number and size of pore-like structures that were seen on the cell membrane. This difference may be due to the fibroblastic nature of the U-87 MG cells in comparison to MDA-MB-231 cells.

Irinotecan delivery by microbubble-assisted ultrasound - A pilot preclinical study

Irinotecan is conventionally used for the treatment of colorectal cancer. However, its administration is associated with severe side effects. Targeted drug delivery using ultrasound (US) combined with microbubbles offers new opportunities to increase the therapeutic effectiveness of antitumor treatment and to reduce toxic exposure to healthy tissues. The objective of this study is to investigate the safety and efficacy of in-vivo delivery of irinotecan by microbubble-assisted US in human glioblastoma model (U-87 MG). In order to validate the potential of this new method in-vivo, subcutaneous tumors were implanted in the flank of nude mouse and treated when they reached a volume of 100 mm3. In the first study, the measured volumes with caliper and anatomic ultrasound imaging were compared for the monitoring and the quantification of tumor growth during 27 days. Ultrasound imaging measurements were positively correlated to caliper measurements. The tumor treatment consisted of an i.v. injection of irinoteca...

Delivery of dopamine transporter tracer (PE2I) through blood brain barrier with ultrasound and microbubbles

The blood-brain barrier plays a major role in controlling the delivery of therapeutic and imaging agents to the brain. 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 established the ultrasound conditions that achieved a transient BBB disruption in rats using a non-permeant marker, Evans blue. Hence SonoVue® (450 μL/kg) and Evans blue (100 mg/kg) were intravenously administered. BBB leakage was obtained using ultrasound insonation through the rat skull at 1.6 MPa, PRF 1 Hz, duty cycle 12%, burst 10 ms during 120 sec. 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.The blood-brain barrier plays a major role in controlling the delivery of therapeutic and imaging agents to the brain. 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 established the ultrasound conditions that achieved a transient BBB disruption in rats using a non-permeant marker, Evans blue. Hence SonoVue® (450 μL/kg) and Evans blue (100 mg/kg) were intravenously administered. BBB leakage was obtained using ultrasound insonation through the rat skull at 1.6 MPa, PRF 1 Hz, duty cycle 12%, burst 10 ms during 120 sec. 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 striat...