Thesis
SERS & efficacy assessment of anti-cancer drug-nanoparticle conjugates
- Creator
- Rights statement
- Awarding institution
- University of Edinburgh.
- University of Strathclyde
- Date of award
- 2019
- Thesis identifier
- T15292
- Person Identifier (Local)
- 201458148
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Non-small cell lung cancer (NSCLC) is the result of tumour development from genetically-mutated epithelial cells lining the surface of the lung. Clinical response to treatments is generally gauged by comparing tumour sizes pre- and posttreatment on computerised tomography. If no response is observed, it is difficult to understand why the patient is not responding to treatement. Measuring the success of the drug at the intracellular level, for example: drug entry, accumulation and binding to its active site, can be difficult to observe directly when the drug is not labelled. Being able to determine the intracellular accumulation (concentration), biological effect and fate (metabolism or degradation) of the drug could inform preclinical development of future generations of drugs. The purpose of this study was to functionalise a nanoparticle (NP) with an anticancer drug to be intracellularly imaged without the use of a dye. The aim was to track the uptake, distribution and release kinetics of the small molecule drug from the NP conjugate within the cell at clinically relevant levels. Here, the inherent chemical signature of the small molecule tyrosine kinase inhibitor (SM-TKI) erlotinib (EL) that targets a mutant epidermal growth factor receptor (EGFR), was selected as a proof of concept for optical imaging in cancerous cells. The alkyne bond within EL was detected with the vibrational spectroscopy technique, Raman microspectroscopy. Raman scattered signals can be enhanced when the analyte is associated with the surface of a NP (surface enhanced Raman spectroscopy (SERS)). Two metals were compared for signal detection, namely gold and silver NPs. Once EL conjugate imaging was achieved, the anti-proliferative efficacy and cytotoxicity of the NP conjugated erlotinib to the free drug was assessed. EL-NP conjugates were characterised in solution and mapped in fixed cells by SERS at 300 nM EL. This was a high resolution time-lapsed intracellular localisation study. The alkyne signal from silver conjugates were found to occupy more of the cell volume over time 32.6% versus gold 0.2% of the cell volume at 24 hours. The dynamic release of EL from the silver NP was observed from 4 hours by tracking a Raman shift of the alkyne vibration from bound to free-EL. The intracellular uptake was confirmed via correlative dark-field (DF) microscopy, single particle inductively coupled plasma- mass spectrometry (spICP-MS) and TEM. Conjugates were present at 1-10,000 particles per cell and a final concentration of 39 nM EL was delivered. The fate of gold NPs were illustrated by TEM to be different when compared to the silver, as they tend to accumulate more in membrane bound organelles than the silver NPs. This is likely to have consequences for the efficacy of drug responses. A SERS & Efficacy Assessment of Anti-Cancer Drug-Nanoparticle Conjugates v microfluidics device was also employed to demonstrate the potential for 3D SERS imaging at physiological conditions to monitor conjugated EL in real-time. This is the first example of a dye-free SERS approach for drug detection within cells using 3D mapping and a microfluidic device for live cell imaging. Cell viability was assessed during a fluorescent image time-course study to evaluate the cytotoxicity and efficacy of the nano-delivery method employed versus parent (free) drug formulation. It was found that the silver NP conjugate was almost as effective as the free drug. In contrast, the gold NP conjugate was completely ineffective on EL-sensitive cells. It was shown that that NP toxicity on cells must be determined empirically per NP and per cell type, as the EL-insensitive cell line was far more susceptible to silver NP toxicity than the EL-sensitive cell line. Examining the inhibition of EGFR phosphorylation elucidated reduced expression levels in EL-silver NP conjugate treated cells, the gold counterpart had no effect on inhibiting activation of EGFR. This was demonstrated by immunofluorescent imaging for localisation and Western immunoblots for quantitation (of phosphorylated/total EGFR (p/tEGFR) protein). Functional proteomics in the form of reverse phase protein array (RPPA) analysis was carried out to assess the downstream effects of the conjugates compared with unconjugated (free) EL on drug-sensitive cells. Hierarchical cluster analysis (HCA) and interactive network maps of RPPA for downstream signal transduction phosphorylation studies confirmed the earlier efficacy results from cell viability studies. EGFR and associated signalling events were similarly disrupted in free-EL and silver conjugate but were unaffected in the gold conjugate samples. Interestingly, RPPA highlighted another key difference in that free-EL induced more of a DNA damage response than in the conjugate-treated EL-sensitive cells. In conclusion, it was determined that nanoparticles could be useful as the next generation of dye-label-free theranostics for multiple purposes, for example: as diagnostic, treatment and prognostic markers. To enable this, the NPs must be rendered safe enough for use in humans, specifically targeted to the cancerous cells and loaded with a drug moiety that can be released near the active site. Although these studies have revealed potential utility in drug imaging, there are clearly a number of key hurdles to be overcome if NP-drug conjugates are to be used in a clinical setting for cancer treatment. However, their potential to aid in the tracking the intracellular fate of the drug could help in nanoformulation drug discovery pipelines.
- Advisor / supervisor
- Graham, Duncan, (Professor of Chemistry)
- Frame, Margaret
- Faulds, Karen
- Wark, Alastair
- Resource Type
- Note
- This thesis was previously held under moratorium from 8 November 2019 to 8 November 2021.
- DOI
- Date Created
- 2019
- Former identifier
- 9912733493302996
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