Author: Ali Habib
Habib, Ali, 2025 Development of Ferroptosis-Inducing Liposomes to Target Multiple Myeloma, Flinders University, College of Medicine and Public Health
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Multiple myeloma (MM) is an incurable malignancy, characterised by the uncontrolled proliferation of clonal plasma cells, usually in the bone marrow. MM accounts for 10% of all haematological malignancies diagnosed worldwide and has a significant impact on both patients and healthcare systems. The poor outcome of patients with MM highlights the urgent need for novel treatment strategies that overcome resistance to more conventional apoptosis-based regimens.
Ferroptosis is a recently characterised form of non-apoptotic programmed cell death. Phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) play a crucial role as ferroptosis substrates as they are readily oxidised to form toxic lipid reactive oxygen species (ROS). Despite evidence suggesting ferroptosis may represent a novel approach for the treatment of cancer, targeting tumour cells while minimising off-target effects, is a significant challenge. One way in which this has been achieved is through the development of monoclonal antibodies raised against proteins expressed by the tumour cells, which by themselves or by directing therapeutics to the tumour cells, result in greater tumour cell death whilst minimising toxicities. Regimens incorporating monoclonal antibodies have proven highly effective for a range of cancers, including MM.
In this study, ferroptosis was induced by treating cells with RSL3, which inhibits glutathione peroxidase 4 (GPX4), a key enzyme in the primary lipid antioxidant system within cells. Ferroptosis was confirmed as the mechanism of cell death by using the synthetic antioxidant, liproxstain-1. Using a range of scientific techniques, we identified a strong correlation between the PL profile of MM and diffuse large B cell lymphoma (DLBCL) cells and their sensitivity to ferroptosis. Our findings demonstrate that DLBCL cells, which contain high proportions of PLs containing PUFAs are sensitive to ferroptosis-mediated cell death, while MM cells, which generally contain high proportions of PLs containing monounsaturated fatty acids (MUFAs), are relatively resistant.
Altering the lipidome of MM cells through exogenous supplementation with PL-PUFAs identified by our lipidome profiling was sufficient to induce ferroptosis-mediated cell death and sensitise the cells to RSL3. Liposomes, predominantly comprised of the same PL-PUFAs, were subsequently manufactured and loaded with RSL3. Uptake, cytotoxicity and lipid ROS studies showed these novel liposomes were readily taken up and triggered ferroptosis-mediated cell death of MM cells. Furthermore, liposomes containing RSL3 were significantly more effective at inducing ferroptosis than empty liposomes or free RSL3; IC50 values for RSL3 were on average between 7.1 and 14.5-fold lower in the liposomal formulation compared to free RSL3, representing a reduction from a micromolar to a nanomolar effective dose range.
In order to specifically target MM cells, the liposomes were modified to enable conjugation to the FDA-approved anti-CD38 monoclonal antibody, daratumumab. These functionalised liposomes were readily taken up by MM cells but interestingly and moreover, importantly, the presence of daratumumab reduced uptake and cytotoxic effects of the liposomes against the FH9 healthy B cell line, suggesting a potential reduction in the effects of the liposomes against healthy, non-CD38 expressing cells.
The findings of this study significantly increase our understanding of the mechanisms associated with the resistance of MM cells to ferroptosis. The data presented are proof of the concept that antibody-conjugated liposomes may represent an effective and targeted means of delivering key substrates and drugs to trigger ferroptosis in cancers that are insensitive to this form of programmed cell death, such as MM.
Keywords: Multiple Myeloma, Ferroptosis, Lipids, Nanotechnology, Liposomes.
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Professor Craig Wallington-Gates