The most common malignant tumor of astrocytic origin is the highly aggressive glioblastoma multiforme. The median survival time measured from the time of the diagnosis is only approximately one year due to the late stage of diagnosis and the lack of efficacy of currently available therapies. However, significant progress in uncovering cancer biology during the past decades has prompted extensive research within novel classes of anticancer drugs, exploiting molecular differences between the tumor and the normal tissue for inducing specific cancer cell toxicity. Drug carriers can be directed to molecules aberrantly expressed by the cancer cells or the tumor vasculature for inducing direct cancer cell toxicity or regression of the tumoral blood supply, respectively. Liposomal systems comprise one main type of targeted drug carriers and have potential for transporting a wide variety of drugs at a high load per carrier. A recent development within liposomal cancer therapy is to combine several types of liposomes directed to different targets or to use multitargeted liposomes, in order to achieve a synergistic targeting effect. In glioblastoma multiforme, this strategy could be used for simultaneous targeting of cancer cells and tumor endothelial cells, in order to benefit from the separate advantages of the two targeting strategies.
The subject of this thesis has been to investigate liposomes as a possible drug carrier system for targeting cancer cells and tumor endothelial cells of glioblastoma multiforme. One of the primary aims was to develop and characterize an intracranial animal model of human glioblastoma multiforme. Cells of a human glioblastoma multiforme cell line, U-87 MG, were inoculated into the striatum of immunodeficient nude mice which lead to tumor development in two thirds of animals. Histological investigations of brain sections revealed extensive growth and local invasion of the tumor xenografts. As a measure of vascular permeability, the intratumoral accumulation of intraperitoneally injected peroxidase and endogenous mouse albumin was investigated. Both substances demonstrated a diffuse pattern of accumulation in the tumor area, compatible with increased leakiness of the tumor vessels. In summary, the intracranial U-87 MG tumor xenograft model was observed to recapitulate important features of human glioblastoma multiforme such as aggressive growth, local invasion, and probably also some degree of vascular leakiness.
Furthermore, a protocol for preparation of immunoliposomes was developed, based on the post insertion technique. Using this protocol cancer cell and vascular targeted liposomes was prepared. For targeting of U-87 MG cells liposomes was conjugated with the EGFR-antibody Erbitux® and for targeting of murine brain endothelial cells liposomes was conjugated with VCAM-1 antibody. Erbitux®-liposomes demonstrated significant cellular uptake by the U-87 MG cells in contrast to unconjugated liposomes. However, no uptake of anti-VCAM-1 liposomes by activated murine brain endothelial cells could be detected. One major reason for this was probably the fact that a much lower amount of antibody was conjugated to the anti-VCAM-1 liposomes compared to the Erbitux®-liposomes. Methods for assessing the tissue distribution of fluorescent liposomes in vivo were investigated, since the next is investigation of the liposome behavior in vivo. Fluorescence microscopy of tissue sections was hampered by a high background signal and fluorescent liposomes could only be detected in the liver and the spleen. Fluorescence spectroscopy of tissue homogenates yielded promising results, however due to time constraints data have only been obtained for some tissues. After further optimization and validation of these methods, it would be interesting to explore the in vivo characteristics of the Erbitux®-liposomes in the established U-87 MG tumor xenograft model, perhaps along with liposomes targeting the tumor endothelial cells via an alternative to VCAM-1.