1. |
Dimcevski G.♦, Kotopoulis S.♦, Bjånes T.♦, Hoem D.♦, Schjøt J.♦, Gjertsen B.T.♦, Biermann M.♦, Molven A.♦, Sorbye H.♦, McCormack E.♦, Postema M., Gilja O.H.♦, A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer,
Journal of Controlled Release, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2016.10.007, Vol.243, pp.172-181, 2016Abstract: Background:
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.
Methods:
Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30 min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5 min. SonoVue® was injected intravenously (0.5 ml followed by 5 ml saline every 3.5 min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy.
Results:
The combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n = 10) tolerated an increased number of gemcitabine cycles compared with historical controls (n = 63 patients; average of 8.3 ± 6.0 cycles, versus 13.8 ± 5.6 cycles, p = 0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n = 10) was also increased from 8.9 months to 17.6 months (p = 0.011).
Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma. Keywords: Ultrasound, Microbubbles, Sonoporation, Pancreatic cancer, Image-guided therapy, Clinical trial Affiliations:
Dimcevski G. | - | Haukeland University Hospital (NO) | Kotopoulis S. | - | Haukeland University Hospital (NO) | Bjånes T. | - | Haukeland University Hospital (NO) | Hoem D. | - | Haukeland University Hospital (NO) | Schjøt J. | - | Haukeland University Hospital (NO) | Gjertsen B.T. | - | University of Bergen (NO) | Biermann M. | - | Haukeland University Hospital (NO) | Molven A. | - | Haukeland University Hospital (NO) | Sorbye H. | - | Haukeland University Hospital (NO) | McCormack E. | - | Haukeland University Hospital (NO) | Postema M. | - | IPPT PAN | Gilja O.H. | - | Haukeland University Hospital (NO) |
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2. |
Yddal T.♦, Gilja O.H.♦, Cochran S.♦, Postema M.♦, Kotopoulis S.♦, Glass-windowed ultrasound transducers,
Ultrasonics, ISSN: 0041-624X, DOI: 10.1016/j.ultras.2016.02.005, Vol.68, pp.108-119, 2016Abstract: 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. Keywords: Ultrasound transducer, De-fouling, Optical window, Acoustic field simulation Affiliations:
Yddal T. | - | Haukeland University Hospital (NO) | Gilja O.H. | - | Haukeland University Hospital (NO) | Cochran S. | - | University of Dundee (GB) | Postema M. | - | other affiliation | Kotopoulis S. | - | Haukeland University Hospital (NO) |
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3. |
Kotopoulis S.♦, Dimcevski G.♦, McCormack E.♦, Postema M.♦, Gjertsen B.T.♦, Gilja O.H.♦, Ultrasound and microbubble-enhanced chemotherapy for treating pancreatic cancer: a phase I clinical trial,
JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA, ISSN: 0001-4966, DOI: 10.1121/1.4950209, Vol.139, No.4, abstract, pp.2092, 2016Abstract: Experimental research of ultrasound to induce or improve delivery has snowballed in the past decade. In our work, we investigate the use of low-intensity ultrasound in combination with clinically approved microbubbles to enhance the therapeutic efficacy of chemotherapy. Ten voluntary patients with locally advanced or metastatic pancreatic adenocarcinoma were consecutively recruited. Following standard chemotherapy protocol (intravenous infusion of gemcitabine over 30 min), a clinical ultrasoundscanner was targeted at the largest slice of the tumour using modified non-linear contrastimaging settings (1.9 MHz center frequency, 0.27 MPa peak-negative pressure), and SonoVue® was injected intravenously. Ultrasound and microbubble treatment duration was 31.5 min. The combined therapy did not induce any additional toxicity or increase side effect frequency when compared to chemotherapy alone. Combination treated patients were able to tolerate an increased amount treatment cycles when compare historical controls (n = 63); average of 8.3±6.0 cycles, versus 13.8±5.6 cycles. The median survival also increased from 7.0 months to 17.6 months (p = 0.0044). In addition, five patients showed a primary tumor diameter decrease. Combined treatment of ultrasound,microbubbles, and gemcitabine does not increase side effects and may have the potential to increase the therapeutic efficacy of chemotherapy in patients with pancreatic adenocarcinoma. Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Dimcevski G. | - | Haukeland University Hospital (NO) | McCormack E. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation | Gjertsen B.T. | - | University of Bergen (NO) | Gilja O.H. | - | Haukeland University Hospital (NO) |
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4. |
Yddal T.♦, Cochran S.♦, Gilja O.H.♦, Postema M.♦, Kotopoulis S.♦, Open-source, high-throughput ultrasound treatment chamber,
Biomedical Engineering-Biomedizinische Technik, ISSN: 1862-278X, DOI: 10.1515/bmt-2014-0046, Vol.60, No.1, pp.77-87, 2015Abstract: Studying the effects of ultrasound on biological cells requires extensive knowledge of both the physical ultrasound and cellular biology. Translating knowledge between these fields can be complicated and time consuming. With the vast range of ultrasonic equipment available, nearly every research group uses different or unique devices. Hence, recreating the experimental conditions and results may be expensive or difficult. For this reason, we have developed devices to combat the common problems seen in state-of-the-art biomedical ultrasound research. In this paper, we present the design, fabrication, and characterisation of an open-source device that is easy to manufacture, allows for parallel sample sonication, and is highly reproducible, with complete acoustic calibration. This device is designed to act as a template for sample sonication experiments. We demonstrate the fabrication technique for devices designed to sonicate 24-well plates and OptiCell™ using three-dimensional (3D) printing and low-cost consumables. We increased the pressure output by electrical impedance matching of the transducers using transmission line transformers, resulting in an increase by a factor of 3.15. The devices cost approximately €220 in consumables, with a major portion attributed to the 3D printing, and can be fabricated in approximately 8 working hours. Our results show that, if our protocol is followed, the mean acoustic output between devices has a variance of <1%. We openly provide the 3D files and operation software allowing any laboratory to fabricate and use these devices at minimal cost and without substantial prior know-how. Keywords: Sonoporation, experimentation devices, rapid prototyping, ultrasound transducers Affiliations:
Yddal T. | - | Haukeland University Hospital (NO) | Cochran S. | - | University of Dundee (GB) | Gilja O.H. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation | Kotopoulis S. | - | Haukeland University Hospital (NO) |
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5. |
Kotopoulis S.♦, Johansen K.♦, Gilja O.H.♦, Poortinga A.T.♦, Postema M.♦, Acoustically Active Antibubbles,
ACTA PHYSICA POLONICA A, ISSN: 0587-4246, DOI: 10.12693/APhysPolA.127.99, Vol.127, No.1, pp.99-102, 2015Abstract: In this study, we analyse the behaviour of antibubbles when subjected to an ultrasonic pulse. Speci cally, we derive oscillating behaviour of acoustic antibubbles with a negligible outer shell, resulting in a Rayleigh Plesset equation of antibubble dynamics. Furthermore, we compare theoretical behaviour of antibubbles to behaviour of regular gas bubbles. We conclude that antibubbles and regular bubbles respond to an acoustic wave in a very similar manner if the antibubble's liquid core radius is less than half the antibubble radius. For larger cores, antibubbles demonstrate highly harmonic behaviour, which would make them suitable vehicles in ultrasonic imaging and ultrasound-guided drug delivery. Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Johansen K. | - | University of Bergen (NO) | Gilja O.H. | - | Haukeland University Hospital (NO) | Poortinga A.T. | - | Eindhoven University of Technology (NL) | Postema M. | - | other affiliation |
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6. |
Johansen K.♦, Kotopoulis S.♦, Postema M.♦, Ultrasonically driven antibubbles encapsulated by Newtonian fluids for active leakage detection,
LECTURE NOTES IN ENGINEERING AND COMPUTER SCIENCE, ISSN: 2078-0958, Vol.2216, pp.750-754, 2015Abstract: An antibubble consists of a liquid droplet, surrounded by a gas, often with an encapsulating shell. Antibubbles of microscopic sizes suspended in fluids are acoustically active in the ultrasonic range. In this study, a Rayleigh-Plesset-like model is derived for micron-sized antibubbles encapsulated by Newtonian fluids. The theoretical behaviour of an encapsulated antibubble is compared to that of an antibubble without an encapsulating shell, a free gas bubble, and an encapsulated gas bubble. Antibubbles, with droplet core sizes in the range of 60– 90% of the equilibrium antibubble inner radius were studied. Acoustic pressures of 100kPa and 300kPa were studied. The antibubble resonance frequency, the phase difference of the radial oscillations with respect to the incident acoustic pulse, and the presence of higher harmonics are strongly dependent of the core droplet size. The contribution to the radial dynamics from a zero-thickness shell is negligible for the bubble size studied, at high acoustic amplitudes, antibubbles oscillate highly nonlinearly independent of core droplet size. This may allow for active leakage detection using harmonic imaging methods. Keywords: Active leakage detection, Antibubble, Bubble resonance, Microbubble, Nonlinear dynamics Affiliations:
Johansen K. | - | University of Bergen (NO) | Kotopoulis S. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation |
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7. |
Kotopoulis S.♦, Delalande A.♦, Popa M.♦, Mamaeva V.♦, Dimcevski G.♦, Gilja O.H.♦, Postema M.♦, Gjertsen B.T.♦, McCormack E.♦, Sonoporation-enhanced chemotherapy significantly reduces primary tumour burden in an orthotopic pancreatic cancer xenograft,
Molecular Imaging and Biology, ISSN: 1536-1632, DOI: 10.1007/s11307-013-0672-5, Vol.16, pp.53-62, 2014Abstract: Purpose
Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50 % are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer.
Experimental Design
An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound.
Results
Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10 % demonstrated in comparison to the control groups.
Conclusion
Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation. Keywords: Sonoporation, Pancreatic cancer, Ultrasound, Chemotherapy, 3D ultrasound, Bioluminescence Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Delalande A. | - | CNRS (FR) | Popa M. | - | KinN Therapeutics (NO) | Mamaeva V. | - | University of Bergen (NO) | Dimcevski G. | - | Haukeland University Hospital (NO) | Gilja O.H. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation | Gjertsen B.T. | - | University of Bergen (NO) | McCormack E. | - | Haukeland University Hospital (NO) |
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8. |
Kotopoulis S.♦, Dimcevski G.♦, Gilja O.H.♦, Hoem D.♦, Postema M.♦, Treatment of human pancreatic cancer using combined ultrasound, microbubbles, and gemcitabine: A clinical case study,
Medical Physics, ISSN: 0094-2405, DOI: 10.1118/1.4808149, Vol.40, No.7, pp.072902-1-9, 2013Abstract: Purpose:
The purpose of this study was to investigate the ability and efficacy of inducing sonoporation in a clinical setting, using commercially available technology, to increase the patients’ quality of life and extend the low Eastern Cooperative Oncology Group performance grade; as a result increasing the overall survival in patients with pancreatic adenocarcinoma.
Methods:
Patients were treated using a customized configuration of a commercial clinical ultrasound scanner over a time period of 31.5 min following standard chemotherapy treatment with gemcitabine. SonoVue® ultrasound contrast agent was injected intravascularly during the treatment with the aim to induce sonoporation.
Results:
Using the authors’ custom acoustic settings, the authors’ patients were able to undergo an increased number of treatment cycles; from an average of 9 cycles, to an average of 16 cycles when comparing to a historical control group of 80 patients. In two out of five patients treated, the maximum tumor diameter was temporally decreased to 80 ± 5% and permanently to 70 ± 5% of their original size, while the other patients showed reduced growth. The authors also explain and characterize the settings and acoustic output obtained from a commercial clinical scanner used for combined ultrasound microbubble and chemotherapy treatment.
Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available clinical ultrasound scanners to increase the number of treatment cycles, prolonging the quality of life in patients with pancreatic adenocarcinoma compared to chemotherapy alone. Keywords: Ultrasound, Microbubbles, Sonoporation, Chemotherapy Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Dimcevski G. | - | Haukeland University Hospital (NO) | Gilja O.H. | - | Haukeland University Hospital (NO) | Hoem D. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation |
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9. |
Delalande A.♦, Kotopoulis S.♦, Postema M.♦, Midoux P.♦, Pichon C.♦, Sonoporation: Mechanistic insights and ongoing challenges for gene transfer,
Gene, ISSN: 0378-1119, DOI: 10.1016/j.gene.2013.03.095, Vol.525, pp.191-199, 2013Abstract: Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed. Keywords: Ultrasound, Microbubbles, Physical gene delivery method, Gene therapy Affiliations:
Delalande A. | - | CNRS (FR) | Kotopoulis S. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation | Midoux P. | - | CNRS (FR) | Pichon C. | - | CNRS (FR) |
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10. |
Kotopoulis S.♦, Eder S.D.♦, Greve M.M.♦, Holst B.♦, Postema M.♦, Lab-on-a-chip device for fabrication of therapeutic microbubbles on demand,
Biomedical Engineering-Biomedizinische Technik, ISSN: 1862-278X, DOI: 10.1515/bmt-2013-4037, Vol.58, No.S1, Supplement, pp.#4037-1-2, 2013Abstract: Electron beam lithography (EBL) was used to fabricate microchannels to produce microbubbles with highly homogeneous size distributions. Using the EBL technique, microchannels can be prototyped at a fast and cost effective rate allowing for evaluation of various microbubble shell materials. Keywords: Microbubbles, EBL, Targeted drug delivery Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Eder S.D. | - | University of Bergen (NO) | Greve M.M. | - | University of Bergen (NO) | Holst B. | - | University of Bergen (NO) | Postema M. | - | other affiliation |
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11. |
Kotopoulis S.♦, Wang H.♦, Cochran S.♦, Postema M.♦, High-frequency transducer for MR-guided FUS,
Biomedical Engineering-Biomedizinische Technik, ISSN: 1862-278X, DOI: 10.1515/bmt-2012-4135, Vol.57, pp.S1, 2012Abstract: Introduction
High-intensity focused ultrasound is finding increasing therapeutic use. However, the frequencies at which it operates are typically limited to below 5 MHz, preventing research into therapy with ultrahigh spatial precision. A reason for this is that the design and fabrication of high-frequency biomedical ultrasound transducers to produce high intensities is an engineering challenge, especially for operating frequencies above 30 MHz, primarily because of the acoustic impedance mismatch and the high attenuation of water of 6dB/cm at 50 MHz leading to a low penetration depth. Commonly used materials such as PZT do not have the ability to produce a high enough intensity, due to de-poling or cracking. A potential application of high-intensity high-frequency ultrasound is non-invasive microsurgery.
Methods
To overcome these problems, we used Y-36o Lithium Niobate (LiNbO3). This crystal has a high Curie temperature and is much more difficult to de-pole at high-power inputs. In addition, Y-36o LiNbO3 has a resonant frequency of 3.3 MHz mm-1, thus allowing for much thicker elements at higher frequencies compared to PZT. A bowl transducer was manufactured using a total of 7 0.5-mm thick elements (4 hexagonal and 5 pentagonal) with a maximum width of 25 mm. The bowl had a curvature radius of 50 mm. The transducer was microballoon-backed in order to simplify the manufacturing process. The pentagonal elements were linked and driven by a 50-dB amplifier, whereas the hexagonal elements were linked and driven by a 55-dB amplifier. To test the available working frequency; single element transducers were manufactured with element thickness ranging from 500 μm to 200 μm, having working frequencies between 6.6 MHz and 20 MHz.
Results
The multi-element focused transducer generated a modulated sound field with an enveloped wavelength of 550 kHz at a frequency of 6.6 MHz with a maximum peak-to-peak pressure of 24.3 MPa; equivalent to mechanical index of 4.7. The modulation could be varied by changing the phase of either the pentagonal or hexagonal linked elements. The microballoon-backed transducers had a 5% reduced acoustic output compared to the air-backed transducer. Single- element transducers produced a maximum peak-to-peak pressure of 14 MPa at 6.3 MHz in the acoustic focus at 12 mm. These transducers were capable of producing over 6 MPa and 4 MPa at the 3rd and 5th harmonics, respectively, corresponding to frequencies of 21 MHz and 35 MHz.
Conclusion
We have established that manufacturing a high frequency, high intensity, multi-element, focused ultrasound transducer using LiNbO3 is feasible. We have also shown it is possible to use the resonant frequency and up to the 5th harmonic to achieve higher working frequencies. Keywords: High-frequency ultrasound, Ultrasound transducer, MR-guided Focussed Ultrasound Surgery Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Wang H. | - | other affiliation | Cochran S. | - | University of Dundee (GB) | Postema M. | - | other affiliation |
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12. |
Kotopoulis S.♦, Wang H.♦, Cochran S.♦, Postema M.♦, Lithium Niobate Transducers for MRI-Guided Ultrasonic Microsurgery,
IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL, ISSN: 0885-3010, DOI: 10.1109/TUFFC.2011.1984, Vol.58, No.8, pp.1570-1576, 2011Abstract: Focused ultrasound surgery (FUS) is usually based on frequencies below 5 MHz—typically around 1 MHz. Although this allows good penetration into tissue, it limits the minimum lesion dimensions that can be achieved. In this study, we investigate devices to allow FUS at much higher frequencies, in principle, reducing the minimum lesion dimensions. Furthermore, FUS can produce deep-sub-millimeter demarcation between viable and necrosed tissue; high-frequency devices may allow this to be exploited in super cial applications which may include dermatology, ophthalmology, treatment of the vascular system, and treatment of early dysplasia in epithelial tissue. In this paper, we explain the methodology we have used to build high-frequency high-intensity transducers using Y-36°-cut lithium niobate. This material was chosen because its low losses give it the potential to allow very-high- frequency operation at harmonics of the fundamental operating frequency. A range of single-element transducers with center frequencies between 6.6 and 20.0 MHz were built and the transducers’ e ciency and acoustic power output were measured. A focused 6.6-MHz transducer was built with multiple elements operating together and tested using an ultrasound phantom and MRI scans. It was shown to increase phantom temperature by 32°C in a localized area of 2.5 × 3.4 mm in the plane of the MRI scan. Ex vivo tests on poultry tissue were also performed and shown to create lesions of similar dimensions. This study, therefore, demonstrates that it is feasible to produce high-frequency transducers capable of high-resolution FUS using lithium niobate. Keywords: Lithium Niobite, Ultrasound Transducer, MRI-Guided ultrasound, Microsurgery Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Wang H. | - | other affiliation | Cochran S. | - | University of Dundee (GB) | Postema M. | - | other affiliation |
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13. |
Delalande A.♦, Bouakaz A.♦, Renault G.♦, Tabareau F.♦, Kotopoulis S.♦, Midoux P.♦, Arbeille B.♦, Uzbekov R.♦, Chakravarti S.♦, Postema M.♦, Pichon C.♦, Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype,
Journal of Controlled Release, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2011.08.020, Vol.156, pp.223-230, 2011Abstract: The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5 × 10^5 BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods. Keywords: Gene delivery, Sonoporation, Tendon Affiliations:
Delalande A. | - | CNRS (FR) | Bouakaz A. | - | Université François Rabelais (FR) | Renault G. | - | CNRS (FR) | Tabareau F. | - | CHR, Service d'anatomie et cytologie pathologiques (FR) | Kotopoulis S. | - | Haukeland University Hospital (NO) | Midoux P. | - | CNRS (FR) | Arbeille B. | - | Université François Rabelais (FR) | Uzbekov R. | - | Université François Rabelais (FR) | Chakravarti S. | - | Johns Hopkins School of Medicine (US) | Postema M. | - | other affiliation | Pichon C. | - | CNRS (FR) |
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14. |
Gerold B.♦, Kotopoulis S.♦, McDougall C.♦, McGloin D.♦, Postema M.♦, Prentice P.♦, Laser-nucleated acoustic cavitation in focused ultrasound,
REVIEW OF SCIENTIFIC INSTRUMENTS, ISSN: 0034-6748, DOI: 10.1063/1.3579499, Vol.82, pp.044902-1-9, 2011Abstract: Acoustic cavitation can occur in therapeutic applications of high-amplitude focused ultrasound. Studying acoustic cavitation has been challenging, because the onset of nucleation is unpredictable. We hypothesized that acoustic cavitation can be forced to occur at a specific location using a laser to nucleate a microcavity in a pre-established ultrasound field. In this paper we describe a scientific instrument that is dedicated to this outcome, combining a focused ultrasound transducer with a pulsed laser. We present high-speed photographic observations of laser-induced cavitation and laser- nucleated acoustic cavitation, at frame rates of 0.5×106 frames per second, from laser pulses of energy above and below the optical breakdown threshold, respectively. Acoustic recordings demonstrated inertial cavitation can be controllably introduced to the ultrasound focus. This technique will contribute to the understanding of cavitation evolution in focused ultrasound including for potential therapeutic applications. Keywords: Laser-nucleated acoustic cavitation, focussed ultrasound Affiliations:
Gerold B. | - | University of Dundee (GB) | Kotopoulis S. | - | Haukeland University Hospital (NO) | McDougall C. | - | other affiliation | McGloin D. | - | other affiliation | Postema M. | - | other affiliation | Prentice P. | - | University of Dundee (GB) |
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15. |
Kotopoulis S.♦, Postema M.♦, Therapeutic ultrasound and sonoporation,
Biomedical Engineering-Biomedizinische Technik, ISSN: 1862-278X, DOI: 10.1515/bmt.2011.525, Vol.56, No.S1, Supplement, pp.#525-1-2, 2011Abstract: Introduction
Focussed ultrasound surgery can heat tissue to a temperature that causes protein denaturation and coagulative necrosis. For high-resolution focussed ultrasound microsurgery, high working frequencies are necessary. The manufacture of such equipment is technically challenging. Low acoustic amplitudes have been associated with increased cellular drug and gene uptake.
We studied tissue, microbubbles and cells, using commercial and self-built transducers operating at various frequencies, using very low or very high acoustic amplitudes.
Methods
We manufactured a high-frequency, high-intensity focussed ultrasound transducer, using lithium niobate as the active element.
To study acoustic cavitation, we designed and built a scientific instrument combining a pulsed laser and a high-intensity focussed ultrasound transducer, capable of nucleating at precise locations. The cavitation dynamics were recorded using high-speed cameras. At high acoustic intensities, interacting cavitation clouds were formed.
Results
Clusters formed at a quarter wavelength apart owing to radiation forces. We observed cluster coalescence and translation towards the capillary wall.
Microbubbles under sonication have been observed to create transient pores in adjacent cell membranes, also known as sonoporation. We observed lipid-shelled microbubbles near cancer cells under quasi-continuous low-amplitude sonication. Typically within a second of sonication, microbubbles were seen to enter the cells and dissolve. This new explanation of sonoporation was verified using high-speed photography and confocal fluorescence microscopy.
Our custom-built transducer was capable of creating 2.5×3.4 (mm)2 lesions without affecting surrounding tissue.
Such disruptive effects of ultrasound also have applications outside medicine. Since cyanobacteria contain gas vesicles, we hypothesised that these can be disrupted with the aid of ultrasound. During 1-hour sonication in the clinical diagnostic range, we forced blue-green algae to sink, thus promoting natural decay.
Conclusion
If drug and genes can be successfully coupled to acoustically active vehicles, sonoporation might revolutionise noninvasive therapy as we know it. Keywords: Therapeutic ultrasound, Sonoporation Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation |
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16. |
Delalande A.♦, Kotopoulis S.♦, Rovers T.♦, Pichon C.♦, Postema M.♦, Sonoporation at a low mechanical index,
Bubble Science, Engineering and Technology, ISSN: 1758-8960, DOI: 10.1179/1758897911Y.0000000001, Vol.3, No.1, pp.3-11, 2011Abstract: The purpose of this study was to investigate the physical mechanisms of sonoporation, in order to understand and improve ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation and resealing of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. The authors studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6?6 MHz ultrasound with a mechanical index of 0?2 and observed with a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell while entering. The authors have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic agent is added to the shell of the bubble or inside the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. In addition, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging. Keywords: Sonoporation, Low mechanical index, Microbubbles, Ultrasound contrast agent, HeLa cells, Cell penetration Affiliations:
Delalande A. | - | CNRS (FR) | Kotopoulis S. | - | Haukeland University Hospital (NO) | Rovers T. | - | other affiliation | Pichon C. | - | CNRS (FR) | Postema M. | - | other affiliation |
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17. |
Kotopoulis S.♦, Postema M.♦, Microfoam formation in a capillary,
Ultrasonics, ISSN: 0041-624X, DOI: 10.1016/j.ultras.2009.09.028, Vol.50, pp.260-268, 2010Abstract: The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source.
In this paper, we analyse how ultrasound contrast agent clusters are formed in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1– 10 MHz. Furthermore, we show high-speed camera footage of microbubble clustering phenomena.
We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces.
We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. It also just takes seconds to drive the clusters towards the capillary wall.
Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation. Keywords: Capillary blocking, Embolisation, Microfoam, Radiation forces, Ultrasound contrast agent Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Postema M. | - | other affiliation |
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18. |
Kotopoulis S.♦, Schommartz A.♦, Postema M.♦, Sonic cracking of blue-green algae,
APPLIED ACOUSTICS, ISSN: 0003-682X, DOI: 10.1016/j.apacoust.2009.02.003, Vol.70, No.10, pp.1306-1312, 2009Abstract: Algae are aquatic organisms classified separately from plants. They are known to cause many hazards to humans and the environment. Algae strands contain nitrogen-producing cells that help them float (heterocysts). It is hypothesized that if the membranes of these cells are disrupted by means of ultrasound, the gas may be released analogous to sonic cracking, causing the strands to sink. This is a desirable ecological effect, because of the resulting suppressed release of toxins into the water.
We subjected small quantities of blue-green algae of the Anabaena sphaerica species to ultrasound of frequencies and pressures in the clinical diagnostic range, and observed the changes in brightness of these solutions over time. Blue-green algae were forced to sink at any ultrasonic frequency we studied, supporting our hypothesis that heterocysts release nitrogen under ultrasound insonification in the clinical diagnostic range.
Although the acoustic fields we used to eradicate blue-green algae are perfectly safe in terms of mechanical index, the acoustic pressures surpass the NURC Rules and Procedures by over 35 dB. Therefore, caution should be taken when using these techniques in a surrounding where aquatic or semi-aquatic animals are present. Keywords: Ultrasonic algae eradication, Blue-green algae, Sonic cracking Affiliations:
Kotopoulis S. | - | Haukeland University Hospital (NO) | Schommartz A. | - | other affiliation | Postema M. | - | other affiliation |
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