Biological studies of matrix metalloproteinase sensitive drug delivery systems

Abstract

Cancer, which is a group of diseases characterized by cells with elevated replication rate and compromised DNA damage response, is often treated with cytotoxic drugs, chemotherapeutics, inducing DNA damage that results in cell death. The use of chemotherapeutics in the clinic, however, is limited due to severe side effects as a result of drug distribution to healthy tissues. To enhance ecacy of treatment and improve life quality of patients, tumor specific drug delivery strategies, such as liposome encapsulated drugs, which accumulate in tumor tissue, has gained increased attention. Several strategies have been developed to target tumor tissue, however, liposomal systems developed so far rely on passive drug diffusion or unspecific association between liposomes and cells after accumulation in tumor tissue, resulting in low drug efficiency. Therefore, development of rationally designed systems for delivery of drugs to specific tissues or cells utilizing biological knowledge of cancer tissue is getting increased attention. In this thesis a novel matrix metalloproteinase-2 (MMP-2) sensitive poly-ethylene glycol (PEG) coated liposomal drug delivery system for treatment of cancer was developed. The system exploits the increased MMP-2 activity present in tumor tissue as a site-specific trigger of liposomal activation and controlled drug release after accumulation due to the enhanced permeability and retention effect. Enzymatic activity of MMP-2 results in shedding of a novel PEG coating, consisting of a negatively charged lipopeptide-PEG conjugates containing a MMP-2 cleavable peptide, which leads to cationic liposomes with enhanced ability to interact with negatively charged cell membranes. Activation of the liposomal formulation developed here resulted in enhanced association of liposomes with cancer cells in vitro and in addition, the liposomes were not associated with phagocytosing cells in human blood without prior enzymatic activation. Encapsulation of oxaliplatin resulted in significantly increased toxicity compared with both the free drug and oxaliplatin encapsulated in PEG-coated neutral liposomes in vitro. High MMP-2 expression and activity was furthermore observed in human clinical samples of different types of cancer as well as in patient-derived colon cancer xenografts in mice, whereas only low concentrations of pro-enzymes were observed in healthy mouse tissues. These data support the use of MMP- 2 as a trigger for liposomal activation in tumor tissue. Thus, this new strategy provides a promising system for specific delivery of encapsulated drugs and controlled release in tumor tissues, resulting in enhanced drug bioavailability and decreased systemic side effects. In addition, we investigated the interaction between liposomes and cell populations in the blood, resulting in a novel liposomal system for specific targeting to CD14+ monocytes. Monocytes play an important role in in ammatory diseases, which are commonly treated with steroids, through their secretion of proin ammatory cytokines, such as TNF-alpha and IL-1beta. However, the use of these drugs has limitations due to side effects as a result of distribution to healthy tissues, which can be circumvented by the use of specific drug delivery systems targeting the in ammatory cells. We found that liposome formulations with a positive charge between 7.5 - 10 % were optimal for targeting specific to monocytes and it was observed that this association rapidly occurred in freshly drawn human blood. It was furthermore revealed that the use of newly drawn blood was essential for mimicking in vivo conditions closest possible, as the monocytes ability to phagocyte liposomes relatively fast declined after the blood was drawn. In addition, we demonstrated that these formulations were not toxic to cells in vitro. The formulations described in this study enable specific targeting to monocytes potentially resulting in enhanced in ammatory effect and decreased side effects of anti-in ammatory drugs.