The effects of heparin functionalised cerium oxide nanoparticles on angiogenesis

Abstract

Angiogenesis is a vital step of cancer growth and metastasis and has become a promising target for cancer therapy. Due to the abnormal vascularisation and poor blood supply, tumours are usually hypoxic and lack nutrients, which triggers tumour aggressiveness and hampers efficient drug delivery. The success of traditional anti-angiogenic strategies, which focuses on decreasing the vascular supply to tumours, is limited by insufficient drug delivery or tumour resistance. Thus, vascular promotion therapy based on promoting angiogenesis is emerging as a complementary cancer therapy. Heparin is a linear negatively charged polysaccharide that, in addition to its anticoagulant action, can promote angiogenesis by potentiating angiogenic growth factors. Thus, heparin is a promising bioactive for cancer drug delivery applications. Cerium oxide nanoparticles (CNPs), a cytocompatible redox biomaterial, has gained attention as it actively scavenges reactive oxygen species (ROS) in biological systems which are related to cancer aggressiveness and tumour hypoxia. In this thesis, CNPs were functionalised with heparin with different chain lengths and density, aiming to investigate their mechanisms of interaction with the vasculature as blood vessel-targeting cancer therapeutics. CNPs were synthesised via precipitation and then functionalised with different amounts of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) via an organosilane linker. CNPs and heparin-CNPs were approximately 12 nm in crystal size, exhibited face-centred cubic phase structure in morphology and as determined by high-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD), respectively. The successful functionalisation of CNPs with heparin was qualitatively verified by attenuated total reflectance-Fourier transform infra-red spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Additionally, thermogravimetric analysis (TGA) demonstrated the conjugation of heparin onto the surface of CNPs with approximately 1.5 μmol UFH, and either 1.8 μmol or 5.7 μmol LMWH conjugated per gram of CNPs. In addition, CNPs and heparin-CNPs showed a dose-dependent cytotoxicity in human umbilical vein endothelial cells (HUVECs), indicating that concentrations of heparin-CNPs above 5 μg/mL can act as anti-angiogenic agents. Heparin-CNPs at a dose of 1.5 μg/mL had limited redox activity which correlated with their limited effect on vascular endothelial growth factor receptor 2 (VEGFR2) expression, a key receptor in angiogenesis regulated via redox. However, the heparin-CNPs signalled fibroblast growth factor 2 (FGF-2) in vitro and in vivo, a key growth factor in angiogenesis, indicating their potential to support angiogenesis. In vivo analyses of the heparin-CNPs indicated that they dose-dependently promoted angiogenesis in the CAM assay at doses up to 1 mg/mL. Moreover, compared to CNPs, heparin-CNPs reduced intracellular ROS level in melanoma cells (MM200), reducing the hypoxic level of tumour cells, which indicated their potential as an anti-cancer agent. A human tumour model in the CAM was developed in this thesis to investigate the effects of nanoparticles on tumour angiogenesis and their interactions with the tumour vasculature. It was found that heparin-CNPs, compared to CNPs, dose-dependently promoted angiogenesis in the human tumour model in the CAM and exhibited a higher level of penetration into the tumour mass via light sheet microscopy and histological analysis. Overall, heparin-CNPs dose-dependently promoted angiogenesis by modulating key processes in angiogenesis and address tumour aggressiveness, showing their potential application in cancer therapeutics.