AbstractThis thesis reports the investigation of spontaneous Raman Spectroscopy (RS) for its applicability in early drug discovery. A key focus has been to develop an understanding of the applicability of RS for the quantification and localisation of compound concentration inside mammalian cells. Further investigation into the use of Surface Enhanced Raman Spectroscopy (SERS) for research on Visceral Leishmaniasis (VL) and Leishmania donovani as well as investigating applicability for cancer research are decisive parts of this work.
The key work described in this thesis is the investigation of whole cell concentration of compounds inside THP-1 and Madin Darby Canine Kidney (MDCK) cells. For true quantification the Cell Silent Region (CSR) is used to measure without interference from cellular background signal. The model compound is erlotinib, an anti-cancer drug with an alkyne group expressing a peak in the CSR. The developed RS system is calibrated using the current gold standard technique Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). However, because of the single cell nature of the RS information on inter cell variability can be extracted. The RS measurements suggest that there is a large variation of concentration within single cell populations. The RS measurements can therefore give insight in single cell behaviour within a large cell population. Findings shows that washing cycles, before fixation, alter the intra-cellular concentrations significantly. This is hypothesised to be caused by the sudden change in concentration on the outside of the cell that applies an osmotic pressure, leading to loss of substance from inside the cell wall. Localisation of erlotinib is shown within THP-1 cells and points towards an accumulation inside the cell nucleus.
Later, internalised Au nano-particles in the range of 30 nm to 80 nm have been investigated for their enhancement effects and localisation inside THP-1 cells. Au nano-particles are found to be internalised easily by differentiated THP-1 cells and accumulate in lysosomes. This allows for a high local enhancement of the spontaneous Raman signal. However, no advantage for the detection of lysosomally trapped compounds (chloroquine, chlorpromazine) was achieved. The detection of substances without a signal in the CSR was achieved without enhancement. Nonetheless, compounds with intrinsic peaks in the CSR could benefit from this enhancement.
Lastly the RS system is explored for alternative uses in early drug discovery. This includes the detection of toxicity as well as the discrimination of cell types. Toxicity has been detected using optically trapped THP-1 cells and doxorubicin. Utilising Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) on these measured spectra, allowed for a clear discrimination of toxically influenced from healthy cells. Differences mainly show up in DNA content caused by the mode of action of doxorubicin and caused by the trapping, which generates most of the signal within the nucleus of the cell. Discriminating cancerogenic (DU145) from healthy prostate cells (PNT2) has been achieved by probing fixed cells and evaluating the acquired Raman spectra with a PCA/LDA combination. The accuracy of separation of these cells when tested with a 10-fold cross-validation technique, is above 98 %, allowing a good discrimination.
|Date of Award||2018|
|Supervisor||Kevin Read (Supervisor) & David McGloin (Supervisor)|
- Raman Spectroscopy
- Drug DIscovery
- Quantification of substances intra-cellular
- Single cell quantification
- Surface Enhanced Raman Spectroscopy
- Cancer research
- Cell Silent Region