The liver has a wide range of capabilities and is the key organ for drug metabolism, detoxification and elimination. Molecules are absorbed from the GI tract into the bloodstream and transported to the liver through the portal vein circulation system. Most of the metabolic functions within the liver are processed by hepatocytes. The project aim was to test two drugs (5-Fluorouracil (5-FU) and diclofenac) with different metabolic pathways, interpret their toxic effects in hepatocytes and relate these to mechanical and morphological alterations in the cells. The findings of this project thereby allow development of a cell mechanics model for toxicity profiling and drug efficacy at a subcellular level. Hepatocellular carcinoma (HCC) is the second most common cancer in the world and the most frequent type of liver cancer. Therefore, a great amount of effort has been aimed at the discovery of anticancer compounds to treat it. The majority of conventional chemotherapeutic drugs work on the principle of halting DNA synthesis, and 5-FU follows this principle. This drug is also commonly used for treatment for most gastrointestinal tract cancers. When 5-FU is administered, the toxic adverse effects need to be considered as there is a possibility of severe side effects. To characterise toxic changes in hepatocytes, an Atomic Force Microscope (AFM) was used, which can produce high-resolution images by probing the surface of the cell, to provide information on the cell’s mechanical properties (such as Young’s modulus). However, with the complexity of this technique it has proved to be challenging to measure relative Young’s modulus values that minimise artefacts which affected the image quality of the cells. Using 5-FU as a model drug, apoptosis was detected by relating surface morphology and mechanical measurements. The surface morphology of HepG2 cells was examined with AFM, and the images produced showed cells exhibiting networking lines of a fibrous nature on the cell surface and protrusions from the cell membrane after the application of 5-FU. This is thought to be related to apoptotic behaviour occurring within the cell but this is not conclusive and further investigations need to be conducted. Mitochondria are the main source of adenosine triphosphate (ATP), which is vital for energy, and therefore control all active processes within the cell. Mitochondrial injury often occurs due to drug toxicity, causing altered metabolic function within the cells. Diclofenac is a widely prescribed NSAID, which may cause serious hepatotoxicity, and is thought to be a mitochondrial toxicant. When altering the physiological conditions from glucose containing medium to galactose containing medium, it was shown that the growth and metabolic function of HepG2 cells decreased. Diclofenac caused a depletion of ATP within the cells. When imaging the cell with AFM, after treatment with diclofenac, there were alterations at surface of HepG2 cells. When the cantilever was separated from the cell surface, the retraction curves showed intermolecular interactions occurring, after treatment with diclofenac which were not observed if the cells were untreated. Microscopic evidence suggested apoptosis may have occurred and it is proposed that the changes in the cell surface reflect this. Liposomes consisting of lipid bilayers can encapsulate a wide range of drugs, and their behaviour can be controlled by modifying their surface properties. This research also studied the production of liposomes in order to understand their interaction with cells. Liposomal delivery systems are used to improve the bioavailability of drugs and can reduce toxic effects. The liposomes were shown to engage with the cell surface by use of AFM but did not influence the cells viability, suggesting that they had potential as a non-toxic delivery system. This thesis has produced initial data to suggest that changes in cell mechanical properties can be used to detect changes in cell behaviour, such as apoptosis, but the method is still fraught with complexity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:720324 |
| Date | January 2017 |
| Creators | MacNeill, Olivia Lee |
| Publisher | University of Strathclyde |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=28414 |
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