Anticancer drugs: new insights revealed by a powerful and complementary multi-technique approach

Anthracyclines are widely used and highly effective chemotherapy drugs, clinically used to treat various types of cancer. Powerful characterisation techniques were combined by researchers from the University of Manchester, University of Warsaw and the Institut Laue-Langevin (ILL) in order to better understand the interactions between anthracycline drugs and the cell membrane at the molecular level. The results provide new insights into key processes that determine the uptake of drugs, information that could contribute to the future design of more effective therapeutic strategies.
 

Although cell-based experiments can indicate the efficacy of an anticancer drug, they provide little information about how exactly the drug works. Molecular level information on the interaction mechanisms between anthracycline drugs and cell membranes can be revealed by characterisation techniques such as neutron reflectometry, Brewster angle microscopy and the Langmuir technique. However, a simpler model than real cells is required for such studies. Lipids are the primary component of cell membranes, and lipid monolayers on the surface of water can serve as a simplified model system of real cell membranes. As cancerous cells are known to have more charged than neutral lipids, two pure lipid monolayers were studied: neutral DMPC and negatively charged DMPS representing, respectively, crude models for healthy and cancerous cell membranes. As the proportion of charged lipids in real cancerous cells is estimated to be 20-30%, experiments were also carried out on a mixed (7:3 ratio) DMPC:DMPS lipid monolayer.

The molecular level interaction was studied between these lipid monolayers and two anthracyclines: doxorubicin (DOx) and idarubicin (IDA). Both drugs are widely used but differ significantly in their lipophilicity – the ability of a molecule to mix with an oil rather than with water. “Idarubicin is more lipophilic than doxorubicin and thus better able to penetrate the cell membrane into the tumour by a process referred to as passive diffusion, one of the key methods of drug transport in cancer treatment,” explains Dr Dorota Matyszewska, researcher at the Biological and Chemical Research Centre at the University of Warsaw.

The drug-cell membrane interactions were studied at different lipid monolayer surface pressures, including at physiologically-relevant values that mimic the surface pressure of real cell membranes. A Langmuir trough was used where lipids were spread on a water surface. From these measurements, the researchers were able to infer that the interactions of both drugs were minimal with DMPC but far more pronounced with DMPS. “An increase in surface pressure at a given area on the trough is generally assumed to be caused by the presence of a small amount of the drug at the surface,” explains Dr Richard Campbell, a Senior Lecturer in the Division of Pharmacy and Optometry at the University of Manchester. “That even more of the drug has gone to surface and it has removed some of the lipid is a possibility that is not routinely considered by researchers because, for more than a century, the surface science community has conducted Langmuir trough experiments assuming that the quantity of lipids remains constant because they are so insoluble themselves.”

Results from the Langmuir trough that inferred stronger drug interactions with DMPS than DMPC were directly and quantitatively resolved using neutron reflectometry (NR). Dynamic compression-expansion cycles of the drug-lipid monolayer systems in a Langmuir trough were performed during neutron measurements on the FIGARO instrument at the ILL. Particularly valuable insight was provided by a novel implementation of NR called the low-Q_z method, developed by Campbell and co-workers in 2016 during the time he worked at the ILL as first responsible scientist for FIGARO – a world-leading instrument that is optimised for successfully carrying out the low-Q_z method due to its exceptionally high neutron flux at the smallest angles of reflection measured. “The standard implementation of neutron reflectometry for biological systems involves deuterating the materials at the interface or the solvent such that their amounts and locations can be distinguished, and this process usually takes a couple of hours,” explains Campbell. “The low-Q_z method is much faster and more straightforward, and although the locations of the components are not resolved, the amounts of lipid and drug can be accurately quantified with respect to the surface area on the minute time scale.”

The novel low-Q_z method provided definitive evidence that for all systems studied, lipid is expelled from the monolayer during surface area compression, inferred by the researchers to be in the form of drug-lipid aggregates. “Neutrons not only revealed that some of the lipids are expelled from the model cell membrane by the drug but also allowed their amounts to be quantified for different systems at different pressures for the first time,” explains Campbell.

The neutron data was also necessary to place into context optical images from Brewster angle microscopy (BAM) showing domains of aggregates observed for DOx but not for IDA. The combined findings, published in the Journal of Colloid and Interface Science in 2021, reveal that though the mechanisms of interaction of DOx and IDA with DMPS monolayers are similar at low surface pressure, significant differences exist at the physiologically-relevant surface pressure. “NR data revealed that the doxorubicin clusters are due to a squeeze-out of the drug from the lipid monolayer, a process which could hinder the penetration and thus uptake of the drug at physiological surface pressure,” explains Matyszewska. “The greater lipophilicity of idarubicin, however, enables the molecule to penetrate the oily chains of the model membrane, which gives us a clue about its passive diffusion and increased uptake.”

The combination of multiple powerful characterisation techniques enabled new insights to be revealed about how two different anthracycline drugs interact with pure and mixed lipid monolayers. The findings improve our understanding of key processes that determine the uptake of drugs, providing information that could contribute to the future design of more effective therapeutic strategies. The experience gained by the researchers, meanwhile, has been dedicated to further investigating model membrane interaction of statins, a commonly prescribed drug to lower cholesterol levels in blood, involving neutron reflectometry experiments at both the ILL and the ISIS Neutron and Muon Source (UK).