Development of Targeted Therapeutics for the Treatment of Glioblastoma

Glioblastoma is the most aggressive cancer of the brain. Despite recent advances in cancer biology and multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome of patients with high grade glioma remains fatal. The major drawback of current glioma chemotherapeutics is their inability to cross the blood brain barrier, lack of tumour specificity agents and their consequent side effects. Matrix metalloprotease (MMP) activity is central to cancer development, angiogenesis and invasion. They are highly active in the tumour environment and absent or inactive in normal tissues, therefore they represent viable targets for cancer drug discovery. A better understanding of the role of MMPs in human gliomas could potentially have diagnostic, prognostic and therapeutic implications. This study aims to assess the expression of specific MMPs in preclinical human glioma models and clinical glioma samples; evaluate in silico docking to rationalise substrate binding preferences of homologous MMPs; rationally design MMP-subtype-selective tumour activated prodrugs; and determine the feasibility of targeting MMP-selective anticancer prodrugs conjugated to graphene oxide as a local drug delivery approach for glioblastoma. This study found significant overexpression of MMP-10 in glioma relative to histologically normal brain tissues. Strong correlation was observed between MMP-10 protein and gene expression of glioma cell lines relative to low expression in a normal brain cell line. MMP-10 activity, as measured by fluorogenic substrate cleavage assay, also demonstrated a strong correlation between MMP-10 activity and gene expression levels. Following demonstration of selective overexpression of MMP-10 in glioma, a reiterative in silico proteolytic docking coupled in vitro biochemical assessment was utilised to rationalise functional similarity and differentiate substrate binding selectivity of homologous MMPs. The binding modes of MMP-substrates within the active site of closely related MMPs were able to accurately predict the cleavage subsites by specific MMPs, as confirmed by in vitro cleavage assay. The success of computational and experimental methodology provided a robust tool for identifying MMP-subtype differences and subsequent development of MMP-10 selective peptide prodrugs. MMP-subtype selective and MMP-10 selective prodrugs were designed by rational exploitation of MMP-docked complexes of substrates. Peptide residues were modified to achieve selectivity for MMP-2 and MMP-10 (over MMP-3 and MMP-9) demonstrating predicted cleavage at distinct subsites. This selectivity was further exploited to attain MMP-10 selectivity, over MMP-2, MMP-3 and MMP-9. The rationally designed peptide prodrugs were synthesised and were shown to be preferentially cleaved by MMPs at predicted subsites and demonstrated no activation by engineered-out MMPs, as predicted. Compared to MJ02 (MMP-2 and MMP-10 selective doxorubicin prodrug), MJ04 (MMP-10 selective doxorubicin prodrug) demonstrated selective metabolism by glioma cell lines to release chemotherapeutic agents. This therapeutic approach against glioma cell lines depended upon the involvement of MMPs, confirmed using pharmacological inhibition. MJ04 demonstrated negligible activity in the presence of an MMP-10 selective inhibitor, suggesting MMP-10 selective activation of the prodrug in glioma cells relative to normal glial cells. Following successful development of MMP-10 selective prodrugs, the feasibility of targeting glioma tumour with local delivery of chemotherapeutics from functionalised graphene-oxide tethered prodrug implants, was assessed as a therapeutic strategy to circumvent the blood brain barrier. Graphene oxide conjugated prodrug was synthesised which is shown to be preferentially cleaved in MMP expressing glioma cell lines relative to normal glial cells. This study demonstrates that MMP-10 is overexpressed in glioblastoma and can be used to metabolise anticancer prodrugs that can be activated selectively by local tumour environment

Development of Liposome Drug Delivery Systems for Anti-Glioma Therapy

This study aims to investigate the potential of ethanol-based proliposomes in generating paclitaxel-loaded liposome delivery in vitro, by employing various phospholipid compositions. Liposomes prepared using ethanol-based proliposome method successfully generated multilamellar vesicles. Three different lipid phases: SPC:Chol, HSPC:Chol or DPPC:Chol in 1:1 mole ratio were used in each liposomal formulation to compare their size, size distribution, zeta potential, pH and morphology. The size of the liposomes was then reduced into nanometre size range. DPPC-liposomes entrapped 70-85% of the available paclitaxel compared to only 46-75% and 26-67% entrapped by liposomes made from SPC and HSPC respectively, using a range of paclitaxel concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of paclitaxel to modify surface charge. In vitro studies revealed that paclitaxel alone was more toxic to U87-MG as well as SVG-P12 cell lines than liposome formulations. The cytotoxicity of liposomes was dependent on their entrapment efficiency and sustained drug release. Thus, DPPC-liposomes had a more cytotoxic effect on the cells than SPC and HSPC liposomes. However, Drug-free liposomes proved to be non-toxic to the cells, indicating that liposomes might enhance the efficacy of the entrapped drug. The properties of different liposome formulations were essential in understanding their drug delivery mechanism