Live-cell FTIR spectroscopy as a novel bioanalytical tool for anti-diabetic drugs research

Abstract

The incidence and mortality rate of diabetes is increasing globally against the variety of currently available anti-diabetic drugs in clinical practice. Unfortunately, current pre-clinical approaches in drug discovery of novel anti-diabetic compounds have not met the increasing demand associated with a high cost and time-consuming research. Therefore, a cost-effective, reliable, and high throughput method that provides critically essential mechanistic information on the drug-cell interaction is urgently needed in the pre-clinical selection of new anti-diabetes drug candidates. This thesis aims to develop a new screening approach based on live-cell FTIR spectroscopy. And the purpose is that this low-cost technique provides information regarding the biomarker alteration between cell and anti-diabetic drugs comparable to the conventional method.

We developed the measurement approach in the first result chapter (chapter 3). We illustrated that multi-reflection ATR FTIR spectroscopy could acquire high-quality spectra of live cells in their aqueous culture medium containing different glucose concentrations for the first time. The results were compared with the reference chemical spectra and confirmed with results from the literature. Hepatoma cancer cell line, namely HepG2, was investigated with a medium of high and low glucose concentration. The difference spectra showed significant spectral changes in the IR absorbance bands between low and high glucose treatment. Glycogen and ADP: ATP spectra are the cellular changes from high glucose treated cells that can be evidence of metabolism alteration according to increasing high cellular glucose uptake. In contrast, low glucose treated cells have shown the rising of phosphate, and nucleic acid, which refers to normal cell growth.

In chapter 4, we applied this approach to study the cellular changes of insulin sensitivity and insulin-resistance HepG2 cells when exposed to a high glucose environment. The experiment was categorized into four sections: normal glucose with/without insulin and high glucose with/without insulin-containing culture medium. The spectral changes between insulin and normal glucose-treated cells have shown significant differences with a broad feature of carbohydrate and phosphate regions in the loadings plot. This refers to the sensitivity of HepG2 cells in normal glucose conditions after insulin addition. However, the high glucose treated insulin has shown different spectral changes compared to the normal glucose treatment indicating that the response from insulin resistance HepG2 to insulin can be distinguished by the live-cell FTIR method. The insulin resistance HepG2 model has been set up and tested against two insulin sensitizers, metformin, and resveratrol. The significantly increasing glycogen absorbance peaks after adding insulin sensitizers indicate an increasing hepatic glucose uptake compared to the control. Moreover, the FTIR results are also comparable with the conventional glycogen assay.

In chapter 5, we applied the developed method to study a new anti-diabetic drug with a different mechanism of action. PF—04991532, a novel glucokinase activator compound, has been tested to study the mechanism of the glucose-lowering effect in diabetic HepG2 cells. However, the spectral change between high glucose and PF-04995132 with high glucose treated cell were insignificant for all treatment time. This is because the HepG2 cell line lacks gene expression regarding this compound's mechanism of action. This insignificant result confirms that the live-vell approach is a specific method that can show negative results when the drug is ineffective because the mechanism of action is not activated.

In conclusion, the work presented in this thesis has successfully shown that live-cell FTIR spectroscopy combined with PCA can discriminate biochemical alteration in cells under treatment of different glucose levels, insulin, and drugs. Therefore, this technique can be an alternative low-cost screening tool for studying the anti-diabetic drug in vitro. Furthermore, further study of this label-free technique can provide information regarding other diabetes cells, e.g., pancreas or kidney, combined with automated measurement and machine learning to establish the tool for diabetes drug screening.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	King's College London
Supervisor	Andrew Chan (Supervisor) & Cristina Legido Quigley (Supervisor)