BIOMED Master's Thesis Defense
Development of an Apoptosis Inducing Ultrasound Contrast Agent|
Date: June 27, 2006
at 11:00 AM
Location: Bossone Research Enterprise Center, Room: 708
Lauren M. Leodore
Margaret A. Wheatley, Ph.D.
Contrast-enhanced echography has been shown to be a powerful technique in the imaging of vessels in developing tumors. Over the years ultrasound contrast agents have been developed that can be used for such imaging. Ultrasound contrast agents have now been modified to create a targeted contrast agent, which can ultimately be used in the early detection, diagnosis, and treatment of a variety of pathologies. This study focuses on the development of a targeted polymer microcapsule ultrasound contrast agent that can specifically target and induce an apoptotic cascade to cancer cells. Apoptosis is a cell mechanism for suicide that is regulated by a variety of cellular signaling pathways. This mechanism has been shown to be a primary route of cytotoxicity by many forms of oncologic therapy. TNF-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, is particularly interesting for its unique ability to induce cancer cell death while sparing most normal cells, which implies its use as a potent anti-cancer agent. Two TRAIL receptors, TRAIL-R1, also known as death receptor 4 (DR4) and TRAIL-R2 also known as death receptor 5 (DR5) induce death following TRAIL binding. Cross-linking of TRAIL with the two death receptors results in activation of caspase-8 at the level of the death-inducing signaling complex. Activated caspase-8 then initiates the apoptosis executing caspase cascade.
During the development of the contrast agent several various parameters were assessed regarding the contrast agent. First was the effect of polymer type on agent preparation, specifically the effect of use of polymer with the free acid (PLA-COOH) rather than a polymer with an ester end-cap on the carboxylic acid group (PLA). This was a necessary assessment because use of polymer with the free carboxylic acid was expected to present more of these groups for the conjugation reaction to work and produce a contrast agent that carried greater density of apoptosis inducing ligand. After producing agent with this type of polymer, it was necessary to evaluate its echogenicity in vitro. After an echogenic contrast agent was produced, size was evaluated to make sure the microcapsules fell within our desired size range of less than 8 m. Lastly, morphology was evaluated to make sure the microcapsules were smooth so we could modify the surface of the contrast agent and still be sure the ligand was accessible to the target receptor, not buried in a surface cleft.
It was found that in order to develop PLA-COOH microcapsules that fulfilled all of the necessary parameters, the volume of the organic phase in the W/O/W emulsion was increased and the molarity of the porogen used was decreased. TRAIL was attached to these microcapsules using carbodiimide chemistry. These modified microcapsules were then cultured with breast cancer cells in vitro for 16 hours. Apoptosis was measured using the LIVE/DEAD assay under fluorescent microscopy. The TRAIL-modified microcapsules did in fact induce apoptosis on breast cancer cells at various different concentrations of TRAIL. The TRAIL-modified microcapsules show promise as candidates to be used as a drug delivery vehicle.
The Bossone Research Enterprise Center is located at the corner of 32nd and Market Streets.