Development of carbon nanotubes as delivery systems for biomedical applications.

摘要

In the last few years the field of nanobiotechnology has been revolutionised with the emergence of a variety of novel engineered nanomaterials such as quantum dots, fullerenes and carbon nanotubes (CNT). CNT are single- or multi-walled cylindrical graphene structures of diameters ranging from a few to hundreds of nanometres, while their length can be up to a few micrometers. The development of CNT for biomedical applications is in the nascent stages, however is thought to lead to novel types of constructs to be used in diagnostic, therapeutic and regenerative medicine. One such approach is the development of CNT as delivery systems that was thoroughly studied in this thesis using in vitro and in vivo models. The ability of water-dispersible 1,3-dipolar cycloaddition-functionalised CNT (f- CNT) to enter cells and traffick intracellularly and the evaluation of critical in vivo parameters, namely the toxicological and pharmacological profiles of f-CNT constitute the focus of this work. The interaction between cells and f-CNT was investigated by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). f-CNT exhibited a capacity to be uptaken by mammalian cells and intracellularly traffick through different cellular barriers. Energy-independent cellular uptake mechanisms are explained based on the cylindrical shape and high aspect ratio of f-CNT that can allow their penetration through the plasma membrane, similar to a 'nanosyringe'. From in vivo tissue distribution studies, body clearance and toxicity profiles of f- CNT were evaluated after intravenous administration of therapeutically-relevant doses in mice and rats. Radiolabelled f-CNT were dynamically tracked by small-animal single photon emission computed tomography (SPECT) imaging. f-CNT circulated in systemic blood circulation, accumulated in the renal cortex, rapidly trespassed the glomerular filtration system and were excreted in urine in the absence of any tissue accumulation or damage resulting in healthy animals. Gross observation and histological examination of all organs was carried out and the extent of nanotube accumulation and damage in different tissues was monitored. TEM analysis of kidney tissue sections indicated that CNT were capable of positioning themselves vertically to the axis of the glomerular filter. Finally, the potential of f-CNT as new vectors for delivery of plasmid DNA (pDNA) to cells and organs was investigated. The complexation of f-CNT and pDNA was monitored by fluorescence spectrophotometry and gel electrophoresis, and the gene expression was evaluated in cancer cells and syngeneic tumour models. The f-CNT exhibited the capacity to condense effectively and deliver pDNA to cells and tissues. Although no improvements were achieved compared to commercially available transfection reagents, the f-CNT were compatible with the biological milieu and showed minimal toxicity in vitro and in vivo. Overall, the studies in this thesis contribute to a greater understanding of the interaction of f-CNT with cells (in vitro) and tissues (in vivo). The capacity of f-CNT to be uptaken by cells and get cleared from blood circulation without undesirable side effects was shown, illustrating the potential of these novel nanomaterials to be further developed in order to construct novel platforms with therapeutic, diagnosis or imaging applications in nanomedicine.