Spiros Kotopoulis

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

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.

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

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 30min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5min. SonoVue® was injected intravenously (0.5ml followed by 5ml saline every 3.5min) 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±6.0cycles, versus 13.8±5.6cycles, 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 8.9months to 17.6months (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.

Sonoporation at a low mechanical index

AbstractThe 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 therap...

Microfoam formation in a capillary

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.

Laser-nucleated acoustic cavitation in focused ultrasound

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 0.5×10(6) 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.

Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype

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 5×10⁵ 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.

Open-source, high-throughput ultrasound treatment chamber.

Abstract 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.

Ultrasound- and microbubble-enhanced chemotherapy for treating pancreatic cancer: A phase I clinical trial

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 ultrasound scanner was targeted at the largest slice of the tumour using modified non-linear contrast imaging 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 control...

Evaluation of the effects of clinical diagnostic ultrasound in combination with ultrasound contrast agents on cell stress: Single cell analysis of intracellular phospho-signaling pathways in blood cancer cells and normal blood leukocytes

Clinical diagnostic ultrasound has been known as one of the safest imaging modalities available, yet very little is known about the cellular response to such acoustic conditions. With the increased interest in therapeutic ultrasound it is becoming ever more important to understand the effects of ultrasound on cells.In our work here we investigate the effect of clinical diagnostic ultrasound on several cell signalling proteins (p38 p-Thr180/p-Tyr182, ERK 1/2 p-Thr202/p-Tyr204 and p53 ac-Lys382) on leukaemia cells (MOLM-13) and monocytes. Our results show that leukaemia cells and monocytes react differently to ultrasound and microbubbles. A relatively small increase in p38 signalling was seen in the leukemic cells, and only at higher intensities in combination with microbubbles. In contrast the monocytes showed an increase in p38 signalling at all acoustic intensities with microbubbles and at the high acoustic intensity without microbubbles. Furthermore, the leukemic cells showed an overall increase in ERK 1/2 signalling whereas the monocytes showed a decrease. These results indicate that the leukaemia cells are less sensitive to stress induced by ultrasound and microbubbles when compared to normal monocytes. In conclusion, our results show that clinical diagnostic ultrasound does have a measurable effect on intracellular signalling but may differ drastically between different cell types. This may affect the conditions necessary for therapeutic ultrasound.

Measured acoustic intensities for clinical diagnostic ultrasound transducers and correlation to the Thermal Index.

To investigate if the thermal index for bone (TIB) displayed on screen is an adequate predictor for the derated spatial‐peak temporal‐average (ISPTA.3) and spatial‐peak pulse‐average (ISPPA.3) acoustic intensities in a selection of clinical diagnostic ultrasound machines and transducers.