DS0413 - Technologies pour la santé

Cavitation Regulation In-vivo - Application to the Blood Brain Barrier Opening by Ultrasound – CARIBBBOU

CARIBBBOU

Cavitation regulation in-vivo: application to the blood brain barrier opening by ultrasound

Objectives and scientific barriers

It is now commonly admitted that the combination of focused ultrasound and microbubbles could temporarily deforms endothelial cells and change the integration of tight junctions, leading to the temporarily disruption of the blood brain barrier (BBB) and thereby increases the efficiency of drug delivery into the central nervous system. This technology, suited for transcranial delivery of water-soluble drugs, relies on the interaction of microbubbles with focused ultrasound that may occasionally be accompanied by side effects, such as brain tissue damage induced by ultrasound over-excitation or microbubble overdosing. These drawbacks are induced by the complexity of the cavitation phenomenon, i.e. the nucleation and oscillation (stable cavitation) before possible collapses (inertial cavitation) of bioengineered or free gas bubbles. Recent studies revealed that cell modification and molecular internalization could also occur when working only in the stable cavitation regime, preferable as possibly resulting in lower tissue damages. We hypothesize that ultrasound could rapidly appear as an important brain tumor treatment modality, particularly if we demonstrate the added value of: (i) controlling the various nonlinear aspects of cavitation phenomenon, in real-time during exposure, both in-vitro and in-vivo, (ii) a better understanding of the mechanisms underlying molecular uptake stimulation and ultrasound-based cell modification into brain tissue and (iii) the opportunity of a targeted and controlled local delivery of therapeutic agents into the brain.

To fully discriminate the origin of ultrasound-induced cell molecular uptake and modification, it is necessary to control the contributions of the different mechanical phenomena induced by ultrasound: acoustic wave itself, soft cavitation or inertial cavitation. The main challenge is the possibility of controlling soft bubble oscillation, a phenomenon that can stochastically co-exist with bubble collapses. Then, a regulation loop based on a soft cavitation indicator with minimization of bubble collapse during sonication should be designed and implanted in real-time on both in-vitro and in-vivo devices. Even if inertial cavitation (bubble collapse) is commonly considered as the main mechanism underlying cell membrane permeabilization through the creation of transient pores, the use of the controlled-cavitation device would allow discriminating the cell response modulation to each mechanical stress (acoustic only, stable or inertial cavitation), and thus differentiating the induced bioeffects regarding the targeted application (sonotransfection, BBB opening…). In order to have a better understanding of the mechanisms underlying BBB opening and gene therapy by drug delivery, the ultrasound-induced cell activity (proliferation, apoptosis) or cell molecular uptake stimulation by acoustic cavitation will be investigated on cell monolayers having the advantage of mimicking the blood brain barrier permeability. Finally, the cavitation-controlled device will be implemented on in-vivo devices dedicated to brain gene delivery. The main technical barrier concerns the possibility of detecting the real-time feedback signal for cavitation control in-vivo, and to confirm that the soft cavitation indicator is discriminating in patterns for BBB-intact and BBB-opening conditions.

Theoretical and experimental investigations have been performed and have shown the origin of the subharmonic component of a interacting bubble cloud possible oscillating on their surface modes. This subharmonic emission was then used as a target indicator in a real-time feedback control strategy to regulate subharmonic emission during pulsed sonication. This feedback loop acts with a fast (250µs) rate allowing the control of bubble oscillations in time. The efficiency and high sensitivity of this technique was demonstrated experimentally through the correlation of this indicator with the BBB opening by ultrasound. A murine model of Parkinson's disease has consequently been developed to continue experimental trials to prove the efficiency of a controlled ultrasound sequence in the treatment of this disease.

At this stage it is expected a better control of the trials reproducibility, as well as a better efficiency, according to the real-time in-vivo control of ultrasound cavitation.

1. Experimental evidence of nonlinear interactions between spherical and nonspherical oscillations of microbubbles. M Guedra, C Inserra, C Mauger, B Gilles. Physical Review E, 94: 053115, 2016.
2. A derivation of the stable cavitation threshold accounting for bubble-bubble interactions. M. Guedra, C. Cornu, C. Inserra. Ultrasonics Sonochemistry, 38: 168-173, 2017.
3. Real-time monitoring and control of cavitation activity for enhancing ultrasound transfection and bubble-cell interactions. C. Inserra, JC. Bera, P. Muleki-Seya, WS Chen. 16th International Symposium on Therapeutic Ultrasound, Tel Aviv, Israel, March 14-16, 2016.
4. Real-time control of the stable cavitation activity in free- and vessel-confined bubble cloud. C Cornu, M Guedra, JC Bera, WS Chen HL Liu, C Inserra. 17th International Symposium on Therapeutic Ultrasound, Nanjing, China, May 31- June 2, 2017
5. An acoustic emission-feedback planar ultrasound system for localized blood-brain barrier opening and monitoring. YX Lin, YC Lin, C Inserra, WS Chen, HL Liu. 17th International Symposium on Therapeutic Ultrasound, Nanjing, China, May 31- June 2, 2017
6. Development of an a-synuclein (SNCA)-based mouse model for Parkinson’s disease by ultrasound-guide CNS Delivery. CY Lin, YC Lin, HL Liu. 17th International Symposium on Therapeutic Ultrasound, Nanjing, China, May 31- June 2, 2017
7. Real-time monitoring and control of stable cavitation activity in pulsed sonication. C. Cornu, M Guédra, JC Béra, C Inserra. 173rd Meeting of the Acoustical Society of America and the 8th Forum Acusticum, Boston, USA, June 25-29, 2017

Scientific background: It has been recognized that the blood brain barrier (BBB) presents a major obstacle to the entry of therapeutic molecules into the central nervous system (CNS). Recently, it has been discovered that, using ultrasound exposure with the presence of microbubbles to locally enhance acoustic cavitation in CNS capillaries, the CNS-blood permeability can be significantly enhanced due to the temporal opening of the blood brain barrier, thus providing a promising strategy to increase delivery of therapeutic agents into brain tumors. In consequence, understanding the mechanisms underlying molecular uptake stimulation and ultrasound-based cell modification in brain tissue is crucial, with the key requirement of a better control of the diverse aspects of the cavitation phenomenon. Given the complexity of acoustic cavitation phenomenon and drug delivery issue into brain, it is important to (i) monitor and control in-vitro and in-vivo cavitation activity, (ii) study the ultrasound-induced uptake stimulation effect on BBB-mimicking cell monolayers and (iii) evaluate the in-vivo performance of ultrasound-based BBB penetration.
Description of the project: We plan to design and implement a cavitation-controlled device to facilitate ultrasound-based cell therapy, with a particular focus on the BBB opening by ultrasound. Firstly, we propose to conceive and implement an acoustic cavitation control system based on the detection of the bubble cloud, the differentiation between oscillating and collapsing bubbles and the quantification of bubble activity. This requires to study the physical aspect of cavitation phenomenon in both stable (oscillating) or inertial (collapsing) cavitation regimes. Once the appropriate indicator of each cavitation regime identified, its real-time monitoring and feedback control will be implemented on diverse technological devices for in-vitro or in-vivo applications. Secondly, the cavitation regime enhancing either brain gene therapy or BBB permeabilization would be clarified by using two specific cell line monolayers that have been found to be useful predictors of blood brain barrier permeability. The cell growth, proliferation and the molecular uptake changes induced by a given cavitation state will be followed through video-microscopy in classical two-dimensional (2D) cell cultures on classical culture substrata. The possible intracellular pathways for cavitation-induced molecular uptake will be explored by studying three major endocytosis pathways and the ultrasound-induced modulation of gene expression within cells. Finally, by employing the cavitation-controlled device, BBB will be temporarily compromised to allow successful delivery of DNA into the brain parenchyma and subsequent gene expression in brain. To reach subsequent amount of gene expression, the possibility of using smartly designed polymers will be explored to control the efficiency and expression duration of transfected DNA. The efficiency of such molecule delivery into the brain by real-time controlled focused ultrasound will be evaluated in-vivo using neurodegenerative disease animal model.
Expected results: Using the proposed approach, we believe that the ultrasound-induced bioeffects could be significantly increased, as the chaotic behavior of cavitation process would be controlled. We should be able to highlight relevant biophysical and acoustical parameters quantifying ultrasound-induced cell modification and molecular uptake stimulation, depending on the external mechanical stress which cells were submitted to. Concerning the applications, brain tumors are characterized by marked resistance to radiation and chemotherapy, so that novel therapeutic approaches should be developed. By controlling cavitation process and the induced bioeffects, this project and the in-vivo technological designs are expected to enlarge the potential of therapeutic ultrasound in the field, with real societal impact.

Project coordination

Claude Inserra (Application des Ultrasons à la Therapie (LabTAU))

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CGU Chang Gung University
ILM - CNRS Institut Lumière Matière
NTUH National Taiwan University Hospital
LabTAU Inserm UMR_S 1032 Application des Ultrasons à la Therapie (LabTAU)

Help of the ANR 395,322 euros
Beginning and duration of the scientific project: December 2015 - 42 Months

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