Design, synthesis and development of biologically inspired polymeric nanomedicines for the treatment of advanced atherosclerosis

Cardiovascular disease (CVD) is a leading cause of death world-wide. The major cause of CVD is atherosclerosis, which is an inflammation driven chronic disease of the arteries, whereby cholesterol accumulates in the arterial wall leading to the build-up of atherosclerotic plaques. Accumulation of cholesterol in early lesions in the form of oxidized low-density lipoproteins (oxLDL) leads to the formation of macrophage foam cells that ingest free cholesterol, eventually resulting in the presence of intra- and extracellular cholesterol crystal in advanced atherosclerotic plaques.

Over time, growth of the necrotic core leads to plaque destabilization and vessel narrowing, which in turn increases the risk of rupture and thrombosis leading to heart attacks and strokes. Currently atherosclerosis treatment mainly involves the use of small-molecule drugs such as statins that lower systemic levels of LDL, and more recently antibody treatments against the proprotein convertase subtilisin/kexin type 9 (PCSK9) enzyme have been developed to achieve the same effect but without major improvements.

Cardiovascular disease is still the leading cause of death worldwide, and new targeted anti-inflammatory therapies that resolve inflammation via endogenous counter-regulatory processes have the potential to be beneficial and complementary to LDL-lowering therapies. This thesis presents the development and investigation of three anti-atherosclerotic nanodrug delivery platforms to address this need.

In the first instance, the development of novel bioinspired polyethylene glycol (PEG) crosslinked nanogel nanobiocatalysts formed from a single simultaneous cross-linking and co-polymerization step in water without the requirement for organic solvent, high temperature or sheer stress is presented. The nanogel synthesis also incorporates in situ non-covalent electrostatically driven template polymerization around an innate anti-inflammatory and anti-oxidizing paraoxonase-1 (PON-1) enzyme payload - the release of which is triggered due to matrix metalloproteinase (MMP) responsive elements instilled in the PEG crosslinker monomer.

Results obtained demonstrate the potential of triggered release of PON-1 enzyme and its efficacy against the production of ox-LDL and therefore a reduction in macrophage foam cell and reactive oxygen species formation in vitro. This study demonstrates the potential of stimuli-responsive nanotherapy strategies incorporating potent anti-oxLDL acting innate enzymes.

Secondly, the potential of MMP responsive anti-inflammatory nanogels incorporating a potent anti-inflammatory peptide (Ac2-26) to reduce the detrimental inflammatory effects associated with stroke is presented. Using a mouse model of cerebral ischaemia reperfusion-injury, we can found that administration of MMP responsive Ac2-26 nanogels had a protective effect and could traverse the BBB as assessed by cranial intravital fluorescence microscopy and favourably effect leukocyte biology.

Finally, reactive oxygen (ROS)-scavenging antioxidative nanoparticles that can serve as an effective therapy for atherosclerosis were developed. The antioxidant ROS-eliminating modality was synthesized via RAFT polymerization. The copolymer was synthesized using different feed ratios of 2,2,6,6-Tetramethyl-4-piperidyl methacrylate and glycidyl methacrylate monomers.

The antioxidant and anti-ROS nitroxyl radical polymer was prepared via oxidation. The copolymer was further conjugated with a 6-aminofluorescein via a ring opening reaction. All synthesized copolymers were blended to create nanoparticles in a single self-assembly step in aqueous conditions. The nanoparticles were shown to be effectively taken up by macrophages and biocompatible even at high dose levels.

Finally, the antioxidant nanoparticles effectively inhibited foam cell formation in macrophages by decreasing internalization of oxLDL. Polymeric nanotherapies developed as part of this thesis have the potential to target key atherosclerotic biological mechanisms, not as yet addressed by conventional therapies and results obtained demonstrate the potent power of biological based and stimuli-responsive nanomedicines for the treatment of atherosclerosis and associated complications such as stroke.