Synthesis, surface modifications and biomedical applications of colloidal goldnanoparticles, towards controlled regulation of angiogenesis in conjunctionwith photo-thermal therapy

摘要

The unique optical properties of colloidal gold nanoparticles: high extinction coefficient and adjustable plasmon band, as well as their excellent biocompatibility (low toxicity and a natural affinity towards thiols and amines) make them very attractive materials for biomedical research. In this project, two types of gold nanoparticles (NPs): spherical and anisotropic were utilised. Spherical NPs predominantly absorb in the visible range of the electromagnetic spectrum, while the plasmon band of anisotropic NPs, e.g. rod-like is shifted into the longer wavelengths. Such variety in the optical signatures of NPs enables their usage in a number of imaging and therapy techniques. Biological activity of NPs is determined by the chemical composition of the organic corona, surrounding the gold core. Appropriate surface capping provides NPs with substantial stability against aggregation (and loss of their properties) in physiological media. Hence, crude colloids were stabilised with a variety of organic molecules, strongly interacting with the gold surface. A selection of functional peptides was incorporated into the organic corona of NPs, in order to achieve desired biological activity of colloids. Functionalised NPs interacted with a certain type of human cells in a selective manner. Such selectivity was obtained by precise recognition between peptides attached to NPs and cell receptors. In this project, human umbilical vein endothelial cells (HUVECs) were studied. These cells build the interior layer of blood vessels in the entire circulatory system and participate in many important physiological processes, as well as pathological developments, e.g. blood vessels formation (angiogenesis). In angiogenesis, several cell receptors are involved. Two of them are: vascular endothelial growth factor receptor type 1 (VEGFR-1) and neuropilin receptor type 1 (NRP-1). Both, VEGFR-1 and NRP-1 were targeted with peptide functionalised NPs. Selectivity of the binding was investigated. Following the binding event, physiological changes in cell metabolism were triggered. The nature and efficiency of these changes were studied, by measuring the expression levels of several angiogenesis related genes, as well as the ability of cells to form capillaries using in vitro angiogenesis assays. HUVECs targeted with anisotropic NPs were illuminated with near-infrared (NIR) laser light. Light at this frequency penetrates human tissue and is absorbed by associated with cells anisotropic NPs. NPs convert the absorbed light into heat, which causes an increase in the local temperature. Increased temperatures result in cell damage and/or stimulate cellular response. Physiological changes in HUVECs upon a moderate heat stimuli were assessed by measuring the expression levels of two genes (ELAM-1 and ELAM-1), which are responsive to the temperature changes. Elevated heat resulted in thermal damage of HUVECs, the degree of which was studied by cell viability assays.In this project, a dual approach towards controlled regulation of HUVECs physiology was demonstrated. Not only regulation of cell metabolism, but the whole angiogenesis process was achieved with peptide functionalised NPs. Targeted with anisotropic NPs cells were illuminated with the NIR laser, which induced the heat shock response in the cell or at the extreme led to death. This work possesses a clear potential and relevance for therapy of various disorders related to insufficient or excessive angiogenesis of endothelial cells