Karin Hensel

Magnetic Microbubbles: Magnetically Targeted and Ultrasound‐Triggered Vectors for Gene Delivery in Vitro

Based on the concept of magnetofection, we prepared lipid shell microbubbles loaded with highly positively charged iron oxide magnetic nanoparticles through electrostatic and matrix affinity interactions. These magnetic microbubbles showed strong ultrasound contrast. When the magnetic microbubbles were mixed with plasmid DNA encoding a reporter gene, gene delivery to HeLa cells was achieved only when ultrasound was applied. Gene transfer efficiency strongly depended on the application of a gradient magnetic field. Treatment of HeLa cells with the microbubbles and ultrasound resulted in strong concentration‐dependent cytotoxic effects, whereas ultrasound alone, lipid microbubbles alone, magnetic nanoparticles or magnetic microbubbles alone did not significantly affect cell viability. These magnetic microbubbles could be used as magnetically targeted diagnostic agents for real‐time ultrasound imaging or for cancer therapy, therapy of vascular thrombosis and gene therapy.

Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma.

Sonodynamic therapy, in combination with ultrasound contrast agents, proved to enhance the uptake of chemotherapeutics in malignant cells. HT1080 fibrosarcoma cells were treated in vitro with a combination of ultrasound SonoVue™-microbubbles and taurolidine (TRD) plus tumor necrosis factor related apoptosis inducing ligand (TRAIL). Apoptosis was measured by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and fluorescence activated cell sorting (FACS) analysis. Gene expression was analysed by RNA-microarray. The apoptotic effects of TRD and TRAIL on human fibrosarcoma are enhanced by sonodynamic therapy and additional application of contrast agents, such as SonoVue™ by 25%. A broad change in the expression of genes related to apoptotic pathways is observed when ultrasound and microbubbles act synchronously in combination with the chemotherapeutics (e.g. BIRC3, NFKBIA and TNFAIP3). Some of these genes have already been proven to play a role in programmed cell death in human fibrosarcoma (HSPA1A/HSPA1B, APAF1, PAWR, SOCS2) or were associated with sonication induced apoptosis (CD44). Further studies are needed to explore the options of sonodynamic therapy on soft tissue sarcoma and its molecular mechanisms.

Evaluation of Ferucarbotran (Resovist®) as a photoacoustic contrast agent / Evaluation von Ferucarbotran (Resovist®) als photoakustisches Kontrastmittel

Abstract Photoacoustic imaging combines the resolution of ultrasound imaging with the contrast of optical imaging, while maintaining a penetration depth up to a few centimeters. Inorganic gold nanorods can be employed as photoacoustic contrast agents. However, the toxicological properties of such nanoparticles are still under investigation. At the same time, there is an increasing need for clinically established photoacoustic contrast agents. In this paper, therefore, we investigate the photoacoustic properties of Ferucarbotran, which is a clinically established nanoscale contrast agent for magnetic resonance imaging. Gelatin phantoms containing cubes with different gelatin-Ferucarbotran mixture concentrations were prepared and irradiated by a Nd:YAG laser (1064 nm). First, the photoacoustic signals were acquired by a single element ultrasound transducer (7.5 MHz) and evaluated quantitatively. In a second setup, photoacoustic imaging of Ferucarbotran with a modified clinical scanner was demonstrated. The experiments showed that in order to achieve a 6 dB gain of received photoacoustic signal energy, compared to the sensitivity threshold of the used system, a Ferucarbotran concentration of 1.9 μmol Fe/ml is needed. The photoacoustic imaging was successful and showed a contrast-to-background ratio of 15.7 dB for a concentration of 11.63 μmol Fe/ml. However, for imaging in tissue the signal-to-noise ratio has to be increased. Zusammenfassung Die Photoakustik kombiniert die Auflösung der Ultraschallbildgebung mit dem Kontrast der optischen Bildgebung, wobei eine Eindringtiefe von bis zu einigen Zentimetern erhalten bleibt. Insbesondere können anorganische Gold-Nano-Rods auf Grund ihrer besonders starken optischen Absorption vorteilhaft als photoakustische Kontrastmittel eingesetzt werden. Die toxikologischen Eigenschaften dieser Partikel sind jedoch noch nicht eingehend geklärt. Gleichzeitig entsteht aber ein zunehmender Bedarf an klinisch zugelassenen photoakustischen Kontrastmitteln. Daher sollen ihm Rahmen dieser Arbeit die photoakustischen Eigenschaften von Ferucarbotran, einem klinisch eingesetzten Nanopartikel-Kontrastmittel für die Magnetresonanztomographie, evaluiert werden. Es wurden Gelatinephantome hergestellt, die einen Kubus mit verschiedenen Ferucarbotrankonzentrationen erhielten und mittels eines Nd:YAG-Lasers (1064 nm) bestrahlt wurden. Zunächst wurden photoakustische Signale durch einen Einzelelementultraschallwandler (7,5 MHz) aufgenommen und quantitativ ausgewertet. Anschließend wurde die photoakustische Bildgebung von Ferucarbotran mit einem modifizierten klinischen Ultraschallgerät demonstriert. Die quantitativen Experimente zeigen, dass, um einen Gewinn der Signalenergie aus dem Ferucarbotranblock von 6 dB gegenüber der Sensitivitätsgrenze des eingesetzten Systems zu erhalten, eine Konzentration von 1,9 μmol Fe/ml notwendig ist. Weiterhin wurden die Phantome erfolgreich photoakustisch abgebildet und ein Kontrast der Zielregion zum Hintergrund von 15,7 dB für eine Konzentration von 11,63 μmol Fe/ml erreicht. Für die Bildgebung im Gewebe muss das Signal-Rausch-Verhältnis des Systems jedoch noch weiter verbessert werden.

Coencapsulation of lipid microbubbles within polymer microcapsules for contrast applications

In this work, the authors examine the acoustic response generated by the coencapsulation of phospholipid shelled microbubbles within the aqueous core of polymer microcapsules and its feasibility as an ultrasound contrast agent. The addition of the polymer shell provides the added benefit of approximately doubling the inertial cavitation threshold of the microbubbles contained within. The feasibility of the utilisation of the coencapsulated contrast agent as a drug delivery vehicle is also discussed. It is concluded that the coencapsulated contrast agent provides contrast similar to that of unencapsulated microbubbles, both in acoustic response and image intensity of contrast to tissue.

Evaluation of subharmonic emission from encapsulated microbubbles as an indicator for sonoporation of cell monolayers

Sonoporation is the ultrasound induced transient opening of cell membranes. Large scale oscillation of nearby microbubbles (MBs) is considered to be the primary effect in sonoporation whereas MB destruction correlates with lower sonoporation efficiency and increased cell damage. The scale of MB oscillation in a cloud can be determined by monitoring subharmonic emission. In this study, SW480 cells are grown in Opticell containers, which are filled with a growth medium containing SonoVue MBs and propidium iodide (PI). Each container is placed in water in the focus of a single element transducer emitting 10 cycles sine-bursts at 3.3 MHz. The peak negative pressure is varied from 75 to 750 kPa. Scattered signals are recorded by a broadband transducer to monitor subharmonic-to-fundamental ratio (SFR) during therapy. A fluorescence microscope is focused on the therapy region to monitor PI fluorescence during and after acoustic excitation. PI cannot overcome intact cell membranes and changes its fluorescence properties when bound to intracellular fluid. A cell is considered sonoporated, if it reveals a sharp rise of fluorescence intensity and a subsequent decrease. A cell is considered permanently stained, if fluorescence intensity is concentrated at the cell nucleus and rises to a plateau. For maximum peak negative excitation pressure, maximum sonoporation (4.9%) and maximum permanent poration (2.8%) is observed. Correcting the number of sonoporated cells by the number of permanently stained cells, best sonoporation efficiency is achieved for the amplitude of 520 kPa. The same holds for the SFR, which significantly rises to a maximum of −26,6 dB at 520 kPa, too. The relationship of sonoporation efficiency and SFR reveals that subharmonic emission from ultrasound contrast agent is an indicator for sonoporation efficiency of cell monolayers.

Monitoring of Insonicated Microbubble Behavior and their Effect on Sonoporation Supported Chemotherapy of Fibrosarcoma Cells

Fibrosarcoma shows low response rates to cytotoxic agents. A method to improve chemotherapeutic effects could be microbubble (MB) enhanced sonoporation, which acts through the transient opening of cell membranes due to ultrasound. In this study the behavior of insonicated MB clouds is acoustically monitored and their effect on sonoporation supported chemotherapy of fibrosarcoma cells is analyzed.

Microcapsules: Reverse Sonoporation and Long-lasting, Safe Contrast

We present a novel vehicle designed to serve the dual roles of enhanced ultrasound contrast and ultrasound-triggered drug delivery. The vehicle is comprised of a microcapsule that is filled with water in whose aqueous core a population of freely floating, phospholipid-coated microbubbles is suspended. At ultrasound intensities below the inertial cavitation threshold of the microbubbles, the microbubbles provide enhanced ultrasound contrast. The measured contrast is comparable in strength with SonoVue®. Encapsulation of microbubbles within microcapsules putatively eliminates – or at least significantly slows – dissolution of gas in the bulk aqueous medium, thereby avoiding disappearance of microbubbles that would otherwise occur due to pressure-induced gas diffusion across the surfactant monolayer coating the microbubble-water interface. Results suggest that our vehicle might provide longer lasting contrast in a clinical setting. We demonstrate that encapsulation of the microbubbles within microcapsules causes at least a doubling of the ultrasound intensity necessary to induce inertial cavitation. Moreover, no cell death was observed when cells were insonified in the presence of microbubble-containing microcapsules, whereas appreciable cell death occurs with unencapsulated microbubbles. These results point toward a potential safety benefit during ultrasound contrast imaging by using encapsulated microbubbles. Studies are underway to investigate the feasibility of ultrasound-triggered release of drug from the microcapsules, owing to inertial- or stable-cavitation, or both. Whereas leakage from polymeric microcapsule shells, such as poly(lactic acid), seemingly requires shell rupture and is exceedingly difficult to achieve, leakage across a lipid bilayer microcapsule shells appears feasible. Leakage across a bilayer shell has the additional benefit that the leakage mechanism can be tuned via phase behavior (liquid-ordered versus liquid-disordered) and cavitation mechanism (stable versus inertial).

Monitoring and modeling of microbubble behavior during ultrasound mediated transfection of cell monolayers

The large scale oscillation of insonified microbubbles (MBs) is considered to be the primary effect for sonoporation and thus enhances cell transfection in gene therapy. MB destruction on the other hand is suspected to lead to lower transfection rates. For future in vivo therapy, online acoustic monitoring could be used to identify these effects and to determine optimal pulse sequence parameters adaptively. As a first step, we monitor MB cloud behavior optically and acoustically during ultrasound mediated transfection of cell monolayers. Opticellreg containers are used to grow monolayers of 293T cells. The containers are filled with a medium containing GFP expressing plasmid DNA and SonoVuereg MBs. Each container is placed in water in the focus of a single element transducer emitting 5 cycles sine-bursts at 1.1 MHz repeated 150 times at 3 Hz. The peak negative pressure varies from 0.29 to 1.53 MPa. A second transducer (1 MHz center frequency) detects transmitted signals on the opposite side. The transducers horizontally scan the entire cell monolayer in a rectangular grid with a spacing of 6 mm. Transmitted and backscattered therapy signals are recorded. For optical MB monitoring, a microscope coupled to a high speed camera is used. The transfection rate is determined by flow cytometry after incubating the cells for 48 hours. The acoustical transmission results reveal MB destruction, which is confirmed by optical MB monitoring. Furthermore, an exponential model of MB destruction in suspensions can be fitted to the monolayer situation. A correlation of the point in time of the maximum of the backscattered signal with the point in time of maximum bubble expansion can be identified. Transfection efficiency, bubble extension and the maximum of the backscattered signals at MB resonance frequency rise with increasing peak negative pressure. In this study, the correlation of sonoporation efficiency and MB extension at cell layers is demonstrated by online monitoring. MB cloud dynamics are acoustically monitored and identified during sonoporation therapy for different excitation peak negative pressures. This is a first step towards adaptively optimizing transfection efficiency in sonoporation therapy by online acoustic monitoring.

Registration of Intraoperative 3D Ultrasound with MR Data for the Navigated Computer Based Surgery

Computer based navigated surgery assists the spatial orientation of the surgeon. Our system registers preoperative data like CT or MR with intraoperative ultrasound data to get the coordinate transformation between the preoperative and the intraoperative data. With a surface volume registration we avoid a difficult surface segmentation in the ultrasound data. To prevent radial exposure and to get more details in the soft tissue the use of MR data for the operation planning is common. Extracting the bone surface in MR data is more difficult than in CT data because MR data has no normalized gray values. To register the ultrasound with the MR data at the knee we detected distinctive anatomic regions in the ultrasound data. We selected an adequate MR sequence in which we could segment the bone surface at the specific region. We evaluate the registration with 1000 random starting positions. 99.2% of the 1000 trails reached the optimum with an error less than 1 mm.

1E-2 Experimental Characterization of Ferucarbotran (Resovist ®) as a Photoacoustic Nanoparticle Contrast Agent

Photoacoustics (PA) combines the high contrast of optical imaging modalities with the high resolution of clinical ultrasound. Photoacoustic contrast agents improve the maximum imaging depth of photoacoustic imaging and may render it possible to use this modality for molecular imaging. Organic dyes, like indocyanine green, or inorganic nanoparticles are used as photoacoustic contrast agents. However, the toxicological properties of many nanoparticles, like gold, have still not been fully investigated. Therefore, in this paper the photoacoustic properties of Ferucarbotran are investigated. Ferucarbotran is a clinically established contrast agent for magnetic resonance imaging. It consists of superparamagnetic iron oxide nanoparticle cores coated with carboxydextran. In order to evaluate the photoacoustic application of Ferucarbotran, gelatin phantoms with different Ferucarbotran concentrations were fabricated. These phantoms, which were mounted on a three-dimensional positioning system, were irradiated by a Nd:YAG laser (1064 nm) and the photoacoustic signals were acquired by a single element ultrasound transducer (7.5 MHz). The experiments showed that in order to achieve a 6 dB gain of photoacoustic received signal energy compared to signals from gelatin a Ferucarbotran concentration of 1.9 mumol Fe/ml is needed. This concentration is equivalent to the quadruple in vivo concentration of Ferucarbotran in the human liver