Specular neutron reflectometry is a powerful technique to resolve interfacial compositions and structures in soft matter. Surprisingly however, even after several decades, a universal modeling approach for the treatment of data of surfactant and phospholipid monolayers at the air/water interface has not yet been established. To address this shortcoming, first a systematic evaluation of the suitability of different models is presented. The result is a comprehensive validation of an optimum model, which is evidently much needed in the field, and which we recommend as a starting point for future data treatment. While its limitations are openly discussed, consequences of failing to take into account various key aspects are critically examined and the systematic errors quantified. On the basis of this physical framework, we go on to show for the first time that neutron reflectometry can be used to quantify directly in situ at the air/water interface the extent of acyl chain compaction of phospholipid monolayers with respect to their phase. The achieved precision of this novel quantification is ∼10%. These advances together enhance significantly the potential for exploitation in future studies data from a broad range of systems including those involving synthetic polymers, proteins, DNA, nanoparticles and drugs.

We quantify directly here for the first time the extents of interactions of two different anthracycline drugs with pure and mixed lipid monolayers with respect to the surface pressure and elucidate differences in the resulting interaction mechanisms. The work concerns interactions of doxorubicin (DOx) and idarubicin (IDA) with monolayers of the zwitterionic DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and negatively charged DMPS (1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt)) as well as a 7:3 mixture of the two lipids. These drugs are used in current cancer treatments, while the lipid systems were chosen as phosphocholines are the major lipid component of healthy cell membranes, and phosphoserines are the major lipid component that is externalized into the outer leaflet of cancerous cell membranes. It is shown that DOx interacts with DMPS monolayers to a greater extent than with DMPC monolayers by lower limits of a factor of 5 at a surface pressure of 10 mN/m and a factor of 12 at 30 mN/m. With increasing surface pressure, the small amount of drug (~0.3 µmol/m²) bound to DMPC monolayers is excluded from the interface, yet its interaction with DMPS monolayers is enhanced until there is even more drug (~3.2 µmol/m²) than lipid (~2.6 µmol/m²) at the interface. Direct evidence is presented for all systems studied that upon surface area compression lipid is reproducibly expelled from the monolayer, which we infer to be in the form of drug-lipid aggregates, yet the nature of adsorption of material back to the monolayer upon expansion is system-dependent. At 30 mN/m, most relevant to human physiology, the interactions of DOx and IDA are starkly different. For DOx, there is a conformational change in the interfacial layer driven by aggregation, resulting in the formation of lateral domains that have extended layers of drug. For the more lipophilic IDA, there is penetration of the drug into the hydrophobic acyl chain region of the monolayer and no indication of lateral segregation. In addition to the Langmuir technique, these advances were made as a result of direct measurements of the interfacial composition, structure and morphology using two different implementations of neutron reflectometry and Brewster angle microscopy. The results provide new insight into key processes that determine the uptake of drugs such as limited drug penetration through cell membranes by passive diffusion as well as activation of drug removal mechanisms related to multidrug resistance.

The interactions of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA), with phospholipid monolayers used as simple models of cell membranes, were investigated. The results of Langmuir experiments together with Brewster angle microscopy showed that both drugs strongly affect cancer cell membranes composed of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS). Electrostatic interactions allow positively charged DNR and IDA to interact with negatively charged DMPS polar heads but increased lipophilicity of IDA allows it to penetrate the layer more effectively than DNR and prevents from its expulsion at higher surface pressures. The analysis of the thermodynamical functions of hysteresis proves the presence of the enthalpically favorable interactions within the monolayer during its compression in the presence of idarubicin, which may form aggregates with DMPS molecules. The influence of the drugs was significantly less pronounced for a healthy cell model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) due to the lack of strong electrostatic attractions. The interactions of drugs with pre-compressed phospholipid monolayers were also examined. The physical state of the monolayer and its packing determined only to some extent the penetration of anthracyclines. Since drug molecules first approach the polar region of the monolayer, the increase in surface pressure in time was more pronounced for negatively charged DMPS monolayers than for zwitterionic DMPC. Additionally, idarubicin was able to penetrate the precompressed DMPS monolayers more effectively than daunorubicin due to increased lipophilicity. This property of the drug was also responsible for IDA better penetration of hydrocarbon chains of supported DMPS monolayers compared to DNR, as shown by electrochemical studies.