· · · Repository hosted by TU Delft Library

GSD1a, the most prevalent type among the glycogen storage disease families, is caused by an inherited glycogen-6-phosphatase gene defectresulting in an impaired glycogen to glucose conversion pathway. Strict dietary management continues to be the only treatment for GSD1apatients. Recently, the advent of targeted ultrasound mediated delivery (USMD) of pDNA to the liver of GSD1a patients in conjunction with microbubbles may provide an alternative treatment option. As thesuccess of USMD of agents is largely dependent on the accessibilityof the targeted tissue by the ultrasound beam, there is a need to quantitatively determine this parameter. For this reason, this study focused on determining the acoustically accessible liver volume in GSD1a patients using transducer models of various apertures and focallengths using suitable CT and MRI datasets, following a geometry-driven approach. Results show that GSD1a patients generally have a larger acoustic accessible liver volume compared to a normal patient group for a given transducer geometry, and that transducers with a smaller aperture and longer focal length would be better suited for these applications. This data is a necessary initial design criterion for ultrasound mediated delivery systems for liver applications in general, and USMD pDNA delivery systems to the liver for GSD1a patientsin particular.

Glycogen storage disease type Ia (GSDIa) is caused by an inherited single-gene defect resulting in an impaired glycogen to glucose conversion pathway. Targeted ultrasound mediated delivery (USMD) of plasmid DNA to liver in conjunction with microbubbles may provide a potential treatment for GSDIa patients. As the success of USMD treatmentsis largely dependent on the accessibility of the targeted tissue bythe focused ultrasound beam, this study presents a quantitative approach to determine the acoustically accessible liver volume in GSDIapatients. Models of focused ultrasound beam profiles for transducers of varying aperture and focal lengths were applied to abdomen models reconstructed from suitable CT and MRI images. Transducer manipulations (simulating USMD treatment procedures) were implemented via transducer translations and 2D rotations with the intent of targetingand exposing the entire liver to ultrasound. Results indicate thatacoustically accessible liver volumes can be as large as 60% of theentire liver volume for GSDIa patients and on average 3 times largercompared to a normal group due to GSDIa patients’ increased liver size. Detailed descriptions of the evaluation algorithm, transducer-and abdomen models will be presented, together with implications forUSMD treatments of GSDIa patients and transducer designs for USMD applications.

Background, Motivation and Objective: Localized drug delivery couldimprove the therapeutic efficacy for treatment of pathological lesions and reduce toxic exposure to healthy organs and tissues. The objective is to develop image-guided, ultrasound-activated tumor chemotherapy with intravenously administrated doxorubicin-liposome-microbubble complexes. Statement of Contribution/Methods: Decafluorobutanemicrobubbles were prepared by sonication and stabilized with phosphatidylcholine/PEG stearate/biotin-PEG-PE shell. They were decoratedwith biotinylated liposomes via a streptavidin link. Liposomes in the complex were loaded with doxorubicin using remote loading ammoniumcitrate protocol (average drug load 0.6 pg per particle). Subcutaneous murine (C57BL/6) hindleg tumor model (MC38 colon adenocarcinomacells, provided by J. Schlom, NIH) was used (6 animals per experimental group). After average tumor size reached 5mm, anesthetized micewere subjected to ultrasound treatment (day 1). First, 3 mg/kg doxorubicin entrapped in microbubble-liposome complex was injected intravenously under ultrasound imaging control with imaging system (iE33; Philips). Immediately following injection, entire tumor was insonated in a spiral pattern intermittently (15s on and 10s off, for microbubble replenishment) for 6 minutes with 1.2MHz ultrasound (2MPa, 10000 cycle, 10 Hz PRF, TIPS system with 1x1x6 mm3 focal zone). Ultrasound treatment combined with 1 mg/kg doxorubicin-liposome-microbubble complex was repeated on day 7. In a control group, animals received doxorubicin entrapped in the carrier without TIPS insonation. Saline-injected animals served as another control. Tumor size and bodymass were monitored. Results: Ultrasound imaging allowed direct observation of the circulating drug carrier. Combination of TIPS ultrasound treatment with doxorubicin-liposome-microbubble complexes resulted in tumor growth suppression, in comparison with both control groups, and showed statistically significant tumor growth inhibition (p<0.05) for days 5-9 of the 10-day study, with a maximal tumor size reduction of 31.6% on day 9. Control animals showed unimpeded tumor growth. Relative body mass change during the study was not statistically significant for animals in all groups. Discussion and Conclusions: Ultrasound activation of the drug-liposome-microbubble complex in the tumor vasculature results in the retardation of tumor growth.The novel liposome-microbubble complex may enable not only localizeddrug release inside the tumor but also enhanced drug delivery across biological barriers, achieving chemotherapeutic effect. This is astep towards next-generation targeted image-guided therapy.

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 liver. 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 Sonovue microbubbles, while the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and subsequently sacrificed after 2 days when liver samples were extracted. RT-PCR assays using Taqman probes were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed toevaluate protein expression. Ultrasound exposed animals showed a four-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a two-fold increased protein expression in ultrasound exposedanimals after two days (p<0.05). A second pilot study was performedwith G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks prior to tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. Theseresults demonstrate that ultrasound mediated delivery can increasegene expression of systemically injected naked pDNA in liver and also provide insight into the development of realistic approaches thatcan be translated into clinical practice.