The role of ultrasound-driven microbubble dynamics in drug delivery : from microbubble fundamentals to clinical translation

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future

Ultrafast Microscopy Imaging of Acoustic Cluster Therapy Bubbles: Activation and Oscillation

Acoustic Cluster Therapy (ACT®) is a platform for improving drug delivery and has had promising pre-clinical results. A clinical trial is ongoing. ACT® is based on microclusters of microbubbles–microdroplets that, when sonicated, form a large ACT® bubble. The aim of this study was to obtain new knowledge on the dynamic formation and oscillations of ACT® bubbles by ultrafast optical imaging in a microchannel. The high-speed recordings revealed the microbubble–microdroplet fusion, and the gas in the microbubble acted as a vaporization seed for the microdroplet. Subsequently, the bubble grew by gas diffusion from the surrounding medium and became a large ACT® bubble with a diameter of 5–50 μm. A second ultrasound exposure at lower frequency caused the ACT® bubble to oscillate. The recorded oscillations were compared with simulations using the modified Rayleigh–Plesset equation. A term accounting for the physical boundary imposed by the microchannel wall was included. The recorded oscillation amplitudes were approximately 1–2 µm, hence similar to oscillations of smaller contrast agent microbubbles. These findings, together with our previously reported promising pre-clinical therapeutic results, suggest that these oscillations covering a large part of the vessel wall because of the large bubble volume can substantially improve therapeutic outcome.publishedVersio

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40-2.00 ml PS101/kg body weight (n = 8-15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated

Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine

Successful delivery of drugs and nanomedicine to tumors requires a functional vascular network, extravasation across the capillary wall, penetration through the extracellular matrix, and cellular uptake. Nanomedicine has many merits, but penetration deep into the tumor interstitium remains a challenge. Failure of cancer treatment can be caused by insufficient delivery of the therapeutic agents. After intra-venous administration, nanomedicines are often found in off-target organs and the tumor extracellular matrix close to the capillary wall. With circulating microbubbles, ultrasound exposure focused toward the tumor shows great promise in improving the delivery of therapeutic agents. In this review, we address the impact of focused ultrasound and microbubbles to overcome barriers for drug delivery such as perfusion, extravasation, and transport through the extracellular matrix. Furthermore, we discuss the induction of an immune response with ultrasound and delivery of immuno-therapeutics. The review dis-cusses mainly preclinical results and ends with a summary of ongoing clinical trials.publishedVersio

Possible expansion of blood vessels by means of the electrostrictive effect

In cases when it is desirable to transport medication through blood vessels, especially when dealing with brain cancer being confronted with the narrow arteries in the brain, the blood-brain barrier makes the medical treatment difficult. There is a need of expanding the diameters of the arteries in order to facilitate the transport of medicaments. Recent research has pointed to various ways to improve this situation; in particular, the use ultrasound acting on microbubbles in the blood stream has turned out to be a promising option. Here, a different possibility of enlarging the diameters of arteries is discussed, namely to exploit the electrostrictive pressure produced by internal strong, ultrashort and repetitive laser pulses. Each pulse will at first give rise to inward directed optical forces, and once the pulse terminates there will be a hydrodynamical bouncing flow in the outward radial direction giving an outward impulse to the vessel wall. In the absence of friction a symmetric oscillation picture emerges. Clearly, a supply of repetitive pulses will be needed (at parametric resonance) to make the effect appreciable. The effect has to our knowledge not been discussed before. We give an approximate optical and hydrodynamical theory of it. The calculations indicate promising results for the wall pressure, although experimental work is desirable to show whether the idea can be useful in practice. Our calculation is made from a general physical perspective, not necessarily linked to medical applications.Comment: 19 pages, latex 2e, no figures. To appear in Symmetr

Acoustic Cluster Therapy (ACT) enhances the therapeutic efficacy of paclitaxel and Abraxane® for treatment of human prostate adenocarcinoma in mice.

Acoustic cluster therapy (ACT) is a novel approach for ultrasound mediated, targeted drug delivery. In the current study, we have investigated ACT in combination with paclitaxel and Abraxane® for treatment of a subcutaneous human prostate adenocarcinoma (PC3) in mice. In combination with paclitaxel (12mg/kg given i.p.), ACT induced a strong increase in therapeutic efficacy; 120days after study start, 42% of the animals were in stable, complete remission vs. 0% for the paclitaxel only group and the median survival was increased by 86%. In combination with Abraxane® (12mg paclitaxel/kg given i.v.), ACT induced a strong increase in the therapeutic efficacy; 60days after study start 100% of the animals were in stable, remission vs. 0% for the Abraxane® only group, 120days after study start 67% of the animals were in stable, complete remission vs. 0% for the Abraxane® only group. For the ACT+Abraxane group 100% of the animals were alive after 120days vs. 0% for the Abraxane® only group. Proof of concept for Acoustic Cluster Therapy has been demonstrated; ACT markedly increases the therapeutic efficacy of both paclitaxel and Abraxane® for treatment of human prostate adenocarcinoma in mice

Molecular mechanisms of sonoporation in cancer therapy : Optimization of sonoporation parameters and investigations of intracellular signalling

Background: Sonoporation, which is treatment with ultrasound (US) and microbubbles (MB), has shown great potential for enhancing the therapeutic efficacy of chemotherapy in cancer therapy. However, there is still very little consensus regarding the mechanism or optimal experimental and therapeutic parameters. The original assumption was that pore formation in the cell membrane was responsible for the increased uptake of drugs, but it is currently understood that the mechanisms are far more complex. The field combines US physics, MB formulation and physics, (cell) biology, pharmacology, pharmacokinetics and the biodistribution of both drugs and MBs. Hence, there is an almost endless range of experimental parameters and potential bioeffects. The current literature includes a plethora of experimental setups and parameters, which complicates the clinical translation of sonoporation. Aims and methodology: In this thesis, the effects of low-intensity US and MB parameters were investigated in vitro using custom-made ultrasound chambers and correlating commonly used measures as uptakes of impermeable dye (i.e. flow cytometry) and viability to detect intracellular signalling responses to sonoporation in different cell types. Intracellular signalling responses to sonoporation are largely unknown, and their influence on key proteins in important signalling pathways have been elucidated using phosphoflow cytometry. To gain the understanding and translatability of US + MB parameters, three commercially available MB formulations were characterized, and important parameters, such as dose and formulation, were investigated in vitro and the in vivo enhancement of chemotherapy in a mural model of pancreatic ductal adenocarcinoma (PDAC). Results and conclusions: Effective sonoporation was achieved using commercial microbubbles and low-intensity US in the diagnostic range, both in vitro and in vivo. In the low-intensity US regimen, effective sonoporation required MBs, and the efficacy increased as US intensity and MB concentrations increased. The choice of optimal MBs depended on the US parameters used, and must be carefully chosen based on the therapeutic context. The findings in vivo were correlated to those in the in vitro experiments and to simulations on MB behaviour. Sonoporation induced the immediate, transient activation of intracellular signalling (MAPK-kinases; p38, ERK1/2, CREB, STAT3, Akt) as well as changes in the phosphorylation status of the proteins involved in protein translation (i.e. ribosomal protein S6, 4E-BP1 and eIF2α). The intracellular signalling response resembles cellular recovery after pore formation by electroporation and pore-forming toxins. Based on this observation, we hypothesize that sonoporation induces a cellular stress response that is related to the membrane repair and restoration of cellular homeostasis, and it may be exploited therapeutically. Varying responses in different cell types better represent the variability within a tumour, and they indicate that the effects on the tumour microenvironment may be important for sonoporation efficacy. In the present work, cellular stress was induced using low-intensity US below the intensity limit approved for diagnostic imaging, and healthy blood peripheral cells were minimally affected

SonoVue® vs. Sonazoid™ vs. Optison™: Which Bubble Is Best for Low-Intensity Sonoporation of Pancreatic Ductal Adenocarcinoma?

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.publishedVersio