Ralf Seip

Ultrasound-triggered release of materials entrapped in microbubble-liposome constructs: a tool for targeted drug delivery.

We investigated the preparation of ultrasound-triggered drug delivery system, based on a pendant complex of microbubble coated with liposomes. Biotinylated decafluorobutane microbubbles were coated with biotinylated liposomes via a streptavidin linker. Liposomes were prepared incorporating calcein and thrombin. Based on initial concentration of calcein, over 1 um(3) payload volume per each microbubble-liposome particle was achieved, when 100 nm liposomes were used. Insonation of microbubble-liposome pendants in vitro resulted in the complete destruction of microbubbles and triggered release of a significant fraction of the entrapped material. Treatment with 1MHz ultrasound (5 pulses, 100 ms, 7 MPa peak negative acoustic pressure) resulted in the release of ~30% of entrapped calcein, as estimated by the fluorescence quenching assay. Thrombin release from liposomes complexed with microbubbles (11% of entrapped material) due to ultrasound treatment was estimated by a chromogenic substrate study. Prior to insonation, substrate hydrolysis was at background level. Ultrasound-triggered release of thrombin from the pendant complexes caused an acceleration of blood clotting.

Targeted Ultrasound-Mediated Delivery of Nanoparticles: On the Development of a New HIFU-Based Therapy and Imaging Device

Ultrasound-mediated delivery (USMD) is an active research topic, as researchers develop applications for therapeutic ultrasound in addition to thermal ablation. In USMD, ultrasound is used in conjunction with microbubbles and drugs, nanoparticles, siRNA, pDNA, stem cells, etc., to facilitate their cellular delivery and uptake using pressure and temperature-mediated mechanisms to bring about a desired therapeutic effect. To investigate the potential of targeted USMD of nanoparticles, pDNA, and stem cells for cardiovascular and other applications, a general-purpose preclinical research tool, therapy imaging probe system (TIPS) was designed. It consists of a wideband annular array, a small-animal acoustic coupler, a motorized positioning system, integrated control software for ultrasound image-guided treatment planning and execution, and triggering electronics that allow ECG and respiration-gated ultrasound exposures. TIPS was then used to enhance delivery of nanoparticles into the murine myocardium and heart vessel walls to demonstrate the feasibility of the technology, pave the way for additional basic research in cardiovascular USMD, and begin to explore the requirements that USMD devices will have to meet to be useful in a clinical setting.

Ultrasound therapy transducers with space-filling non-periodic arrays

Ultrasound transducers designed for therapeutic purposes such as tissue ablation, histotripsy, or drug delivery require large apertures for adequate spatial localization while providing sufficient power and steerability without the presence of secondary grating lobes. In addition, it is highly preferred to minimize the total number of channels and to maintain simplicity in electrical matching network design. To this end, we propose array designs that are both space-filling and non-periodic in the placement of the elements. Such array designs can be generated using the mathematical concept of non-periodic or aperiodic tiling (tessellation) and can lead to reduced grating lobes while maintaining full surface area coverage to deliver maximum power. For illustration, we designed two 2-D space-filling therapeutic arrays with 128 elements arranged on a spherical shell. One was based on the two-shape Penrose rhombus tiling, and the other was based on a single rectangular shape arranged non-periodically. The steerability performance of these arrays was studied using acoustic field simulations. For comparison, we also studied two other arrays, one with circular elements distributed randomly, and the other a periodic array with square elements. Results showed that the two space-filling non-periodic arrays were able to steer to treat a volume of 16 × 16 × 20 mm while ensuring that the grating lobes were under -10 dB compared with the main lobe. The rectangular non-periodic array was able to generate two and half times higher power than the random circles array. The rectangular array was then fabricated by patterning the array using laser scribing methods and its steerability performance was validated using hydrophone measurements. This work demonstrates that the concept of space-filling aperiodic/non-periodic tiling can be used to generate therapy arrays that are able to provide higher power for the same total transducer area compared with random arrays while maintaining acceptable grating lobe levels.

Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: A case for bolus injections

Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200 μg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimb skeletal muscles were exposed to ultrasound from a non-focused transducer (1 MHz, 1.25 MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated/control muscles of up to 101× were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery.

Enhanced gene expression of systemically administered plasmid DNA in the liver with therapeutic ultrasound and microbubbles

Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 μg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p <; 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into clinical practice.

Enhanced gene transfection of plasmid DNA in the liver with ultrasound and microbubbles

Ultrasound mediated delivery (USMD) in the presence of microbubbles is a potentially safe and effective method for gene therapy, offering many desired characteristics such as low toxicity, potential for repeated treatment, as well as organ specificity. In this study we tested the capability of USMD to improve gene transfection to mice livers for treating a rare genetic disorder, Glycogen Storage Disease Type 1a. An image guided therapeutic ultrasound system (TIPS, Philips) was used to provide therapeutic ultrasound to mice liver under IACUC approved protocols. Anesthetized healthy mice were placed supine on a heated pad and coupled to the TIPS transducer. Therapeutic plasmid DNA (FLAG-pG6Pase) was injected and microbubbles infused via the tail vein. Treated animals (n=4) received therapeutic ultrasound pulses (1 MHz, 2.5 MPa) over the entire liver. Control animals (n=5) received pDNA but not ultrasound treatment. Post treatment the animals were left to recover and subsequently sacrificed after 5 days. Tissues from the left, middle, caudal, right anterior, and right posterior liver lobes were harvested and stored. Quantitative PCR assays were then performed on the samples to quantify gene transfection. Ultrasound treated animals showed significantly higher levels of G6Pase transfection compared to control animals (p<0.05) in all five lobes of the liver. On average, the treated animals showed 5.4 times more pDNA accumulation in the liver compared to controls. Immunohistochemistry staining for FLAG tag showed increased transgene expression especially around the blood vessels in treated animals. No evidence of toxicity was found up to 5 days post treatment.

Acoustic Accessibility Investigation for Ultrasound Mediated Treatment of Glycogen Storage Disease Type Ia Patients

Glycogen storage disease type Ia (GSDIa) is caused by an inherited defect in the glucose-6-phosphatase gene. The recent advent of targeted ultrasound-mediated delivery (USMD) of plasmid DNA (pDNA) to the liver in conjunction with microbubbles may provide an alternative treatment option. This study focuses on determining the acoustically accessible liver volume in GSDIa patients using transducer models of various geometries with an image-based geometry-driven approach. Results show that transducers with longer focal lengths and smaller apertures (up to an f/number of 2) are able to access larger liver volumes in GSDIa patients while still being capable of delivering the required ultrasound dose in situ (2.5 MPa peak negative pressure at the focus). With sufficiently large acoustic windows and the ability to use glucose to easily assess efficacy, GSD appears to be a good model for testing USMD as proof of principle as a potential therapy for liver applications in general.

Therapeutic Ultrasound Research And Development From An Industrial And Commercial Perspective

The objective of this paper is to share the challenges and opportunities as viewed from an industrial and commercial perspective that one encounters when performing therapeutic ultrasound research, development, manufacturing, and sales activities. Research in therapeutic ultrasound has become an active field in the last decade, spurred by technological advances in the areas of transducer materials, control electronics, treatment monitoring techniques, an ever increasing number of clinical applications, and private and governmental funding opportunities. The development of devices and methods utilizing therapeutic ultrasound to cure or manage disease is being pursued by startup companies and large established companies alike, driven by the promise of profiting at many levels from this new and disruptive technology. Widespread penetration within the clinical community remains elusive, with current approaches focusing on very specific applications and niche markets. Challenges include difficulties in securin...

Ultrasound and Microbubble Mediated Doxil Delivery in a Murine Breast Cancer Model: Therapeutic Efficacy Dependence on Tumor Growth Rate

The effect of tumor growth rate and treatment repeats are examined as parameters in pressure-mediated ultrasound treatments with microbubbles and Doxil in a murine breast cancer model. For this purpose, mice with a tumor doubling time of 8 and 13 days respectively received either a single or two ultrasound treatments (at 1 MHz/1 MPa) in conjunction with Definity microbubbles (1:1 dilution) and Doxil (3 mg/Kg dose) after the tumor size reached 150 mm3. The tumor model was generated using MDA-MB-231-luc cells implanted into the lower mammary fat pad of SCID beige mice. At 15 days post-treatment, tumor size was reduced by 3±18%, 8±14%, and 20±10% as compared to control for the Doxil only, ultrasound + microbubbles + Doxil (single treatment), and ultrasound + microbubbles + Doxil (2 treatments) groups, respectively, in the mice with the slower growing tumors. The mice with the faster growing tumor yielded tumor size reductions of 46±27%, 71±10%, and 61±26%, respectively, for the same groups. We hypothesize that treatment efficacy is dependent on the dynamics of the tumor itself, even within the same cell line.