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Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

University of Bergen

Exact Therapeutics AS

In research and industrial processes, it is increasingly common practice to combine multiple measurement modalities. Nevertheless, experimental tools that allow the co-linear combination of optical and ultrasonic transmission have rarely been reported. The aim of this study was to develop and characterise a water-matched ultrasound transducer architecture using standard components, with a central optical window larger than 10 mm in diameter allowing for optical transmission. The window can be used to place illumination or imaging apparatus such as light guides, miniature cameras, or microscope objectives, simplifying experimental setups. Four design variations of a basic architecture were fabricated and characterised with the objective to assess whether the variations influence the acoustic output. The basic architecture consisted of a piezoelectric ring and a glass disc, with an aluminium casing. The designs differed in piezoelectric element dimensions: inner diameter, ID=10 mm, outer diameter, OD=25 mm, thickness, TH=4 mm or ID=20 mm, OD=40 mm, TH=5 mm; glass disc dimensions OD=20-50 mm, TH=2-4 mm; and details of assembly. The transducers' frequency responses were characterised using electrical impedance spectroscopy and pulse-echo measurements, the acoustic propagation pattern using acoustic pressure field scans, the acoustic power output using radiation force balance measurements, and the acoustic pressure using a needle hydrophone. Depending on the design and piezoelectric element dimensions, the resonance frequency was in the range 350-630 kHz, the -6 dB bandwidth was in the range 87-97%, acoustic output power exceeded 1 W, and acoustic pressure exceeded 1 MPa peak-to-peak. 3D stress simulations were performed to predict the isostatic pressure required to induce material failure and 4D acoustic simulations. The pressure simulations indicated that specific design variations could sustain isostatic pressures up to 4.8 MPa.The acoustic simulations were able to predict the behaviour of the fabricated devices. A total of 480 simulations, varying material dimensions (piezoelectric ring ID, glass disc diameter, glass thickness) and drive frequency indicated that the emitted acoustic profile varies nonlinearly with these parameters.

Glass-windowed ultrasound transducers.

The primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival.

A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with survival averaging only 3months if untreated following diagnosis. A major limitation in effectively treating PDAC using conventional and targeted chemotherapeutic agents, is inadequate drug delivery to the target location, predominantly due to a poorly vascularised, desmoplastic tumour microenvironment. Ultrasound in combination with ultrasound contrast agents, i.e., microbubbles, that flow through the vasculature and capillaries can be used to disrupt such mechanical barriers, potentially allowing for a greater therapeutic efficacy. This phenomenon is commonly referred to as sonoporation. In an attempt to improve the efficacy of sonoporation, novel microbubble formulations are being developed to address the limitation of commercially produced clinical diagnostic ultrasound contrast agents. In our work here we evaluate the ability of a novel formulation; namely Acoustic Cluster Therapy (ACT®) to improve the therapeutic efficacy of the chemotherapeutic agent paclitaxel, longitudinally in a xenograft model of PDAC. Results indicated that ACT® bubbles alone demonstrated no observable toxic effects, whilst ACT® in combination with paclitaxel can transiently reduce tumour volumes significantly, three days posttreatment (p=0.0347-0.0458). Quantitative 3D ultrasound validated the calliper measurements. Power Doppler ultrasound imaging indicated that ACT® in combination with paclitaxel was able to transiently sustain peak vasculature percentages as observed in the initial stages of tumour development. Nevertheless, there was no significant difference in tumour vasculature percentage at the end of treatment. The high vascular percentage correlated to the transient decrease and overall inhibition of the tumour volumes. In conclusion, ACT® improves the therapeutic efficacy of paclitaxel in a PDAC xenograft model allowing for transient tumour volume reduction and sustained tumour vasculature percentage.

Sonoporation with Acoustic Cluster Therapy (ACT®) induces transient tumour volume reduction in a subcutaneous xenograft model of pancreatic ductal adenocarcinoma.

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (-0.03), CREB (+0.0), STAT3 (-0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.

Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death worldwide. Poor drug delivery to tumours is thought to limit chemotherapeutic treatment efficacy. Sonoporation combines ultrasound (US) and microbubbles to increase the permeability of cell membranes. We assessed gemcitabine uptake combined with sonoporation in vitro in three PDAC cell lines (BxPC-3, MIA PaCa-2 and PANC-1). Cells were cultured in hypoxic bioreactors, while gemcitabine incubation ± sonoporation was conducted in cells with operational or inhibited nucleoside membrane transporters. Intracellular active metabolite (dFdCTP), extracellular gemcitabine, and inactive metabolite (dFdU) concentrations were measured with liquid chromatography tandem mass spectrometry. Sonoporation with increasing US intensities resulted in decreasing extracellular gemcitabine concentrations in all three cell lines with inhibited membrane transporters. In cells with inhibited membrane transporters, without sonoporation, dFdCTP concentrations were reduced down to 10% of baseline. Sonoporation partially restored gemcitabine uptake in these cells, as indicated by a moderate increase in dFdCTP concentrations (up to 37% of baseline) in MIA PaCa-2 and PANC-1. In BxPC-3, gemcitabine was effectively inactivated to dFdU, which might represent a protective mechanism against dFdCTP accumulation in these cells. Intracellular dFdCTP concentrations did not change significantly following sonoporation in any of the cell lines with operational membrane transporters, indicating that the gemcitabine activation pathway may have been saturated with the drug. Sonoporation allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention.

Ultrasound- and Microbubble-Assisted Gemcitabine Delivery to Pancreatic Cancer Cells.

The use of ultrasound (US) and microbubbles (MB), usually referred to as sonoporation, has great potential to increase the efficacy of chemotherapy. However, the molecular mechanisms that mediate sonoporation response are not well-known, and recent research suggests that cell stress induced by US + MBs may contribute to the treatment benefit. Furthermore, there is a growing understanding that the effects of US + MBs are beyond only the cancer cells and involves the tumour vasculature and microenvironment. We treated pancreatic cancer cells (MIA PaCa-2) and stromal cells, fibroblasts (BJ) and human umbilical vein endothelial cells (HUVECs), with US ± MB, and investigated the extent of uptake of cell impermeable dye (calcein, by flow cytometry), viability (cell count, Annexin/PI and WST-1 assays) and activation of a number of key proteins in important intracellular signalling pathways immediately and 2 h after sonoporation (phospho flow cytometry). Different cell types responded differently to US ± MBs in all these aspects. In general, sonoporation induces immediate, transient activation of MAP-kinases (p38, ERK1/2), and an increase in phosphorylation of ribosomal protein S6 together with dephosphorylation of 4E-BP1. The sonoporation stress-response resembles cellular responses to electroporation and pore-forming toxins in membrane repair and restoring cellular homeostasis, and may be exploited therapeutically. The stromal cells were more sensitive to sonoporation than tumoural cells, and further efforts in optimising sonoporation-enhanced therapy should be targeted at the microenvironment.

Low-Intensity Sonoporation-Induced Intracellular Signalling of Pancreatic Cancer Cells, Fibroblasts and Endothelial Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the modern world, in part due to poor delivery of chemotherapeutics. Sonoporation can be used to enhance the efficacy of standard of care therapies for PDAC. Using xenograft models of PDAC we investigate sonoporation using four ifferent ultrasound contrast agents (UCAs) and two ultrasound regimens to identify the ideal parameters to increase therapeutic efficacy. MIA-PaCa2 xenografts in over 175 immunodeficient mice were treated with gemcitabine and paclitaxel and subjected to low or high power ultrasound (60 and 200 mW/cm respectively) in conjunction with one of four different UCAs. The UCAs investigated were Definity®, SonoVue®, Optison™ or Sonazoid™. Tumor volumes, vascularity, hemoglobin, and oxygenation were measured and compared to controls. High power treatment in conjunction with Sonazoid sonoporation led to significantly smaller tumors when started early (tumors ~50mm;  = .0105), while no UCAs significantly increased efficacy in the low power cohort. This trend was also found in larger tumors (~250mm) where all four UCA agents significantly increased therapeutic efficacy in the high power group ( < .01), while only Definity and SonoVue increased efficacy in the low power cohort ( < .03). Overall, the higher power ultrasound treatment modality was more consistently effective at decreasing tumor volume and increasing vascularity characteristics. In conclusion, Sonazoid was the most consistently effective UCA at decreasing tumor volume and increasing vascularity. Thus, we are pursuing a larger phase II clinical trial to validate the increased efficacy of sonoporation in conjunction with chemotherapy in PDAC patients.

Selecting the optimal parameters for sonoporation of pancreatic cancer in a pre-clinical model.

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue resulted in a significant decrease in weight vs. DOX alone ( = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone ( = 0.0004), whilst SonoVue ( = 0.0001) and Sonazoid™ ( < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an "ultrasound alone" group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys.

The use of ultrasound and microbubbles to enhance therapeutic efficacy (sonoporation) has shown great promise in cancer therapy from in vitro to ongoing clinical studies. The fastest bench-to-bedside translation involves the use of ultrasound contrast agents (microbubbles) and clinical diagnostic scanners. Despite substantial research in this field, it is currently not known which of these microbubbles result in the greatest enhancement of therapy within the applied conditions. Three microbubble formulations-SonoVue, Sonazoid™, and Optison™-were physiochemically and acoustically characterized. The microbubble response to the ultrasound pulses used in vivo was simulated via a Rayleigh-Plesset type equation. The three formulations were compared in vitro for permeabilization efficacy in three different pancreatic cancer cell lines, and in vivo, using an orthotopic pancreatic cancer (PDAC) murine model. The mice were treated using one of the three formulations exposed to ultrasound from a GE Logiq E9 and C1-5 ultrasound transducer. Characterisation of the microbubbles showed a rapid degradation in concentration, shape, and/or size for both SonoVue and Optison™ within 30 min of reconstitution/opening. Sonazoid™ showed no degradation after 1 h. Attenuation measurements indicated that SonoVue was the softest bubble followed by Sonazoid™ then Optison™. Sonazoid™ emitted nonlinear ultrasound at the lowest MIs followed by Optison™, then SonoVue. Simulations indicated that SonoVue would be the most effective bubble using the evaluated ultrasound conditions. This was verified in the pre-clinical PDAC model demonstrated by improved survival and largest tumor growth inhibition. In vitro results indicated that the best microbubble formulation depends on the ultrasound parameters and concentration used, with SonoVue being best at lower intensities and Sonazoid™ at higher intensities.

Spiros Kotopoulis

Department of Clinical Medicine

University of Bergen

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

Spiros Kotopoulis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Clinical Medicine

University of Bergen

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

Department of Clinical Medicine