AbuHammad, Ala
(2024)
Investigation of metabolic effects of polymer nanomedicines using advanced metabolomics methods.
PhD thesis, University of Nottingham.
Abstract
Breast cancer is the leading cause of death among women and the most frequently diagnosed female cancer worldwide, owing to its complex and heterogenous nature. Despite the intensified research that has been devoted to identifying the best treatment modalities for breast cancer, the mortality rate is still high due to frequent chemoresistance resulting in relapses and tumour metastasis. It is well known that chemoresistance, which is the failure of tumour cells to respond to chemotherapy, is the main challenge for any successful cancer treatment, and is not yet fully understood, since it originates from impairment of several biochemical pathways. In order to improve chemotherapies for breast cancer, intensive research showed promising results for nanomedicine applications. In particular, there is a focus on polymer nanoparticles for improving the therapeutic efficacy of the conventional chemotherapeutic agents for breast cancer. There is also an increasing attention towards the utilisation of natural products such as thymoquinone (THY), in combination-therapies with other chemotherapeutics agents, and is considered as a promising approach for enhancing the therapeutic efficacy of the conventional chemotherapies, by evading chemoresistance and reducing related severe side effects. At the same time, there is an urgent need to identify molecular targets involved in breast cancer carcinogenesis and drug resistance, and to develop selective drug delivery systems with optimal physicochemical characteristics that can simultaneously circumvent drug resistance and confer molecular targeting. However, despite the progress achieved to date in these areas, there is still a compelling requirement for deeper and more comprehensive understanding of the mechanisms involved in chemoresistance, and the interactions between polymer nanoparticles (PNPs) and cells at the molecular level, since the conventional cytotoxicity biochemical can measure cell metabolic activity as a proxy of cell-viability. Therefore, the information that can be derived from single end-point assays will fall short of providing a comprehensive depiction of complete biological responses or emphasising the impacted biochemical pathways following any exposure to treatments/materials. To address these challenges, new advanced tools such as the emerging metabolomics approaches are being recently employed to study nanomedicine related biological responses at the molecular level.
In this project, docetaxel (DTX) based nanoparticles were formulated using Methoxy poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-b-PCL) polymer for examination against triple negative breast cancer (TNBC) cell-lines, to assess the enhancement effect of encapsulating our model drug DTX within a nanocarrier, as well as co-encapsulating DTX with THY. Data showed enhanced cytotoxic activity of the encapsulated combination (NP-COMB) over both the free and encapsulated DTX (NP-DTX) in all cell-lines.
In the next stage, the conventional cytotoxicity assay results were taken further so that the same DTX based treatments including NP-COMB were tested again against TNBC cell-lines for LC-MS based global metabolic profiling, to explore the metabolic pathways perturbed upon receiving treatments. DTX-based treatments significantly altered metabolites, in the three cell-lines, crucial and central metabolic pathways, such as Alanine, aspartate, and glutamate metabolism pathway, which is closely related to energy metabolism and metabolic rewiring observed in cancer cells and is directly linked to the mechanism of action of DTX. Moreover, the BRCA1 mutant MDA-MB-436 cell-line exhibited least alterations being the most resistant to DTX, which is in line with literature. Additionally, which could be attributed mainly to the differences in uptake and trafficking methods for both treatments, which is in accord with previous literature. Interestingly, NPCOMB had distinct metabolic signatures, compared to DTX and NP-DTX, in the treated cells, such as the upregulation of metabolites involved in maintaining redox balance (e.g. glutathione metabolism), pro-apoptotic AMPK activation, and amino acid metabolism (i.e. alanine, aspartate, and glutamate metabolism pathway). We also reported several unknown mass ions that were distinctively and drastically upregulated due to NP-COMB exposure in TNBC cell-lines. These unique metabolic signatures suggest the NP-COMB superior antitumour activity and potential synergism, and could be also attributed to the differences in the uptake and trafficking mechanisms of the different treatments.
Finally, in-situ 3D OrbiSIMS single cell-based metabolomics approach was employed to further investigate the metabolic effect of DTX based nanomedicine against a TNBC cell-line model. With this new MS-based technique, and unlike the previously mentioned LC-MS method, metabolomics experiments can be conducted using only a single cell with greater sensitivity and resolution, much simpler sample preparation, and perseverance to cell integrity prior to analysis.
Herein, the 3D OrbiSIMS technique was successfully employed to unravel a number of biomarkers that are specific to treating MDA-MB-231 cells with NP-DTX. The results demonstrated the enhanced cytotoxic effect of NP-DTX over the free drug in MDA-MB-231. NP-DTX uniquely and significantly altered the levels of key bioactive lipids, ROS production and redox balance, induced apoptosis as well as ATP consumption, causing unique metabolic signature. It is worth noting that the ionisation mechanisms in both techniques are different since 3D OrbiSIMS and LCMS techniques operate hard and soft ionisation modes, respectively. Therefore, there is great potential in employing both techniques to obtain different spectral information, thus different metabolic information. We believe that employing metabolomics techniques will accelerate drug development by building up cumulative knowledge contributing to more accurate designs of future nanomedicine which can advance current treatment options to eradicate breast tumour effectively.
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