3.1. The Structure of OLA-OA Binary Monolayers
The surface pressure (π, mN/m) versus area per molecule (A, Å
2/molecule) dependence was determined for pure OLA and OA monolayers and mixed (OLA:OA 5:1, 2:1, 1:1, 1:2 and 1:5) systems and are depicted in
Figure 1A. The isotherm-specific parameters (such as A
lift-off, A
lim, or π
coll, see
Figure S2) are summarized in
Table S1 in the
Supplementary Materials. Conducted isotherm experiments confirm that both pure and mixed systems create insoluble monolayers at the air/water interface, and every individual system shows features that strongly depend on the film composition. The shape of the isotherms of OLA and OA reflects the extremely different molecular structures of these chemical substances. It can be seen that the A
lift-off value for the OA monolayer is equal to 34 Å
2/molecule and 75 Å
2/molecule for the film of OLA. Upon the monolayer compression, the surface pressure increases as the mean area per molecule decreases, until reaching the π
coll value of 31 mN/m for OA, which corresponds to the monolayer collapse. The case of the oleanolic acid monolayer is more complex. Due to the specific molecule structure, oleanolic acid creates a rigid film at the interface, and the monolayer exhibits stability up to 10 mN/m. The presence of the hydrophilic hydroxyl and carboxyl groups on the opposite sites of the molecule, regardless of the arrangement of molecules at the air/water interface, makes the assembly energetically unfavorable. Thus, above 10 mN/m, the OLA π–A isotherms are not repeatable, due to instabilities of the molecule’s orientation and structure [
5,
6,
7,
27]. The collapse of the OLA monolayer starts at π
coll value of 41 mN/m.
The shape and position of the π–A isotherms of mixed systems are clearly dependent on the monolayer composition. The curves of mixed systems are located between the pure OA and OLA isotherms. Increasing the content of OA shifts the mixed monolayer isotherms towards a lower molecular area corresponding to values reminiscent of fatty acid isotherm. The systems where OLA is the dominant constituent over OA, especially OLA:OA 5:1, are qualitatively similar to the isotherms of pure OLA monolayer. On the other hand, the systems with excess concentrations of OA (OLA:OA 1:2 and OLA:OA 1:5) and equimolar systems do not exceed the π
coll value of pure OA isotherm. However, the observed fluctuations of OLA:OA 2:1, 1:1 and 1:2 curves reveal signs of instability (double collapse regions, disrupted isotherm shape). In fact, two collapse regions can be distinguished in these systems, which are 30 mN/m and 35 mN/m, 25 mN/m and 29 mN/m, 27 mN/m and 29 mN/m, respectively. This phenomenon is associated with a lack of miscibility among mixed monolayer components [
28,
29,
30]. In the case of a monolayer of miscible components, the value of the collapse surface pressure is in the range of π
coll for pure monolayers. Otherwise, when the monolayer components do not mix with each other or the miscibility is limited, two collapse regions may occur within the course of the π–A isotherm, which indicates the phase separation of the mixed monolayer. The miscibility aspects of the binary systems will be discussed in detail further in the paper.
The molecules conformation at the interface and the interactions between OLA and OA molecules are reflected in the compression modulus graph [
Figure 1B]. The Cs
−1 is strongly related to the isotherm course and molecule’s orientation. The more rapid the surface pressure increase, the higher the compression modulus is; thus, a higher monolayer compression is possible.
Due to the presence of an unsaturated bond within the hydrocarbon chain, the OA monolayer does not exceed the value of Cs
−1 = 50 mN/m; thus, referring to the study of Rideal and Scott [
31], it remains in a liquid-expanded (LE) state. The unsaturation enhances the monolayer fluidity and prevents the molecules from being tightly compressed.
The slope of the OLA isotherm changes up to a value of π = 10 mN/m and becomes steeper, which influences the Cs−1 values. In the G and LE phases, orientation B of the OLA molecule is more favorable (tilted above the air/water interface). Based on the molecular dimension, the area occupied by a single molecule is estimated as ca. 72 Å2, which corresponds to the value of OLA Alift-off. Above a surface pressure value of 10 mN/m, the monolayer undergoes an LE–LC phase transition, and orientation A starts to predominate within the monolayer. For nearly vertical OLA orientations, with the main axis towards the air/water interface, the molecular area value equals ca. 40 Å2, which, in turn, corresponds to the value of mean area per molecule at the collapse of the monolayer, where the molecules are packed the tightest. Taking this into account, as well as the value of Alim of OLA monolayer, it can be concluded that in liquid states of the monolayer both of the OLA molecule orientations coexist. According to the phase classification, the OLA monolayer at the maximal compression is in the LC phase.
As can be seen from
Figure 1B, the compressibility of mixed systems is strongly dependent on their composition. The mixed systems with excess OLA concentrations reach Cs
−1max values higher than the pure OLA monolayer. Furthermore, the values of Cs
−1max indicate that the mixed compositions of OLA:OA 5:1, 2:1 and 1:1 reach the LC thermodynamic state, while OLA:OA 1:5 and 1:2 are on the line between LE and LC states. It can be noticed that even a small amount of OLA in the OA monolayer causes a condensing effect and enhances the compressibility of mixed monolayers.
Analysis of the Brewster Angle Microscopy images provides evidence for the phase separation within the compressed monolayers. BAM was performed for both pure and mixed monolayers of OLA and OA during the π–A isotherm compression/expansion cycles. In the images, the very dark regions correspond to the water subphase, while the monolayers are visible as gray areas of various brightness depending on differences in orientation-induced monolayer thickness. The brighter the region, the thicker the monolayer is.
Figure 2 depicts obvious differences in the monolayer structures of pure OLA and OA. Within the OLA monolayer, there are angular, tile-like domains of various size and brightness. This fact may correspond with the coexistence of the two orientations of the OLA molecule in the monolayer. There is a difference in the OLA film thickness depending on which polar moiety of the molecule is anchored in the water subphase. Therefore, the monolayer domains of diverse brightness can be observed. Although upon the monolayer compression, domains are fused (at ca. 29.73 mN/m), the surface inhomogeneity (brighter regions and wrinkles) can be observed during the whole process.
On the other hand, at the air/water interface, OA forms characteristic domains of rounded shape (so-called “foam-like” structures). These microdomains are clearly seen for a relatively short time during the monolayer compression because a slight increase in surface pressure due to the compression leads to the fusion of the microdomains. The OA monolayer is homogenous even at surface pressures close to πcoll (ca. 27.43 mN/m). The binary monolayers exhibit intermediate features between the morphology of pure substances. The mixed systems with the excess of OA correspond to the pure OA monolayer and do not show indications of collapse or phase separation when compressed. The system of OLA:OA 5:1 reveals an analogous morphology as pure OLA, but the edges of the tiles are smoother. But, more importantly, in contrast to OLA after compression, the OLA:OA 5:1 monolayer morphology is uniform, devoid of domains or aggregates. However, higher OA content in the OLA monolayer (OLA:OA 2:1 and 1:1) leads to lace-like structures at the interface, followed by the bright, elongated aggregates demonstrating phase separation.
3.2. Interactions among Monolayers–Thermodynamic Analysis of the Miscibility
The miscibility of a binary Langmuir monolayer is determined by the interactions between its components. Analogous to mixtures in bulk systems, film components can be immiscible, partially miscible, or completely miscible. If the monolayer components are fully immiscible or their mixture is ideal, the dependency of the mean area per molecule on the mixed system composition is linear [
Figure 3A]. However, mixed monolayers usually exhibit a non-ideal behavior due to the interactions between their components. Positive deviation evidences the presence of repulsive interactions, while negative deviations indicate attractive interactions between the components. Positive deviation values can also indicate phase separation within the binary monolayer [
28].
The thermodynamic analysis of the mixed systems as a graph of the excess free energy of mixing ΔG
exc vs. the molar fraction of OLA (X
OLA) is shown in
Figure 3B. A monolayer composed of perfectly miscible substances exhibits a ΔG
exc value of zero. The deviations plotted in
Figure 3B indicate the presence of various interaction types between the components depending on the composition. The negative values of ΔG
exc signify stronger attraction compared with repulsion between the binary monolayer components in comparison to single-component monolayers. Moreover, the more negative the value, the more pronounced the stability of the mixed monolayer. On the other hand, the positive value of ΔG
exc indicates weaker attraction (stronger repulsion) between the molecules in the binary monolayers compared with pure substances.
Strong evidence of the phase separation in the OLA:OA 1:2 system was found. The positive values of ΔGexc reached by the mixed system OLA:OA 1:2 in the whole range of surface pressure values indicate the presence of strong repulsive interactions between the components. The same behavior can be noticed in the case of the systems OLA:OA 1:5, 1:1, and 2:1 at π value above 10 mN/m. However, within a relatively loosely packed monolayer (at 5 and 10 mN/m), those systems are miscible. On the other hand, the binary system OLA:OA 5:1 exhibits the negative values of ΔGexc in the whole range of surface pressures tested. It is most likely that in this system, attractive interactions between the molecules occur, which may cause relatively good miscibility of the monolayer components. What is more, sufficiently low values of ΔGexc support the stability of the monolayer in time. This observation may find similarities in the analysis of the relaxation measurements.
3.3. Mixed Monolayer Stability Investigated by Relaxations
The stability of the monolayers over time was assessed based on the relaxation measurements at constant surface pressures of 5 and 10 mN/m, due to the stable assembly of OLA at the interface in this π range. The dependence of the relative molecular area A/A
0 as a function of time for both values of π is presented in
Figure 4. The rates of monolayer disruption of pure OLA and OA differ significantly for both levels of tested surface pressures. For the OLA monolayer, the relative surface area decreased by only 10% and 12% in 150 minutes for 5 and 10 mN/m, respectively. The relaxation curve of OLA at both values of surface pressure decreases in the initial stage of the measurement, but it stabilizes after ca. 30 min. Due to the unsaturated bond in the hydrocarbon chain, the stability of the OA monolayer at the interface is markedly limited—in 150 min, A/A
0 dropped by 67% at 5 mN/m, and at 10 mN/m, the monolayer existed at the interface only for 60 min. It has been found that the stability of the binary system depends on the monolayer composition. For both surface pressures investigated, the tendency in the disruption kinetics is quite consistent. The system of OLA:OA 5:1 exhibits a relaxation curve at an even higher A/A
0 level than the pure OLA monolayer, contrary to OLA:OA 1:5, where the addition of a small amount of triterpenoid to the oleic acid monolayer accelerates the disruption of the monolayer. Bearing in mind the miscibility of the mixed systems analysis, the enhanced stability of OLA:OA 5:1 is likely caused by the presence of attractive interactions between the monolayer components, while the repulsive interactions within OLA:OA 1:5 causes the stability decrease. For the systems of OLA:OA 2:1, the relaxation curves correspond to the pure OLA monolayer graph. The disruption of this system is relatively low in comparison to the other mixed systems, and even the stabilization of A/A
0 occurs. The course of the equimolar system’s relaxation also stabilizes, but at lower values of A/A
0 and after a relatively long time. The system of OLA:OA 2:1 exhibits analogous features to the relaxation curve of oleic acid, but at a higher relative surface area. What is more, at π = 10 mN/m this system achieved a constant A/A
0 value.
3.4. Dilatational Rheological Properties of the Mixed Systems
The rheological properties of the therapeutic system play a significant role in the selection of the appropriate route of administration of medicinal substances to the human body [
32]. Thus, the viscoelastic properties become an important consideration of the physicochemical studies of the OLA:OA binary system. The dilatation rheology measurements were performed using the barrier oscillation method to follow the relaxation processes. The elastic dilatational modulus E’ and dilatational viscous modulus E’’ of each pure and mixed system are plotted over time t [
Figure 5, top panels]. The rheological data are supported with the relaxation plots to track the changes in viscoelastic and thermodynamic properties simultaneously [
Figure 5, lower panels]. Oscillations were repeated cyclically every 10 minutes. Relaxation data were recorded during the oscillation itself, as well as during the waiting time, when π was kept constant at 5 mN/m [
Figure S1 Supplementary Materials]. We observed the asymmetry of the oscillation peaks towards the desired value [
Figure S3 Supplementary Materials], which is in line with the feature attributed to the dilatational rheology. In the dilatation strain, the compression step induces a quantitatively different outcome than the expansion step leading to a different response in surface pressure values. According to the fact that the force of the oscillating barriers applied to the monolayer causes a monolayer degradation (at a rate depending on the composition), the relaxation curves without additional stress were added for a broader view. Moreover, we also present the values of the surface pressure vs. time during the oscillation/relaxation experiment, because it was noted that barrier movement causes enormous variations in surface pressure for systems with substantial OLA content.
From
Figure 5, it can be seen that the values of elastic modulus (E’) are higher than viscous modulus (E’’), which means that both OLA and OA monolayers exhibit more elastic than viscous properties. However, despite the similar dependence for pure substances, the values of the viscoelastic moduli and alterations over time differ significantly. E’ and E’’ rate changes are associated with the physicochemical properties of the film. Kinetics of the A/A
0 decrease (as an effect of the oscillation) is characteristic of specific film compositions. It is evident that the relaxation behavior is highly sensitive to film composition.
In the case of pure OLA monolayers, rapid decreases in A/A
0 leads to an increase in the elastic modulus whereas the viscous modulus remains almost unchanged at 100 mN/m [
Figure 5A top panel]. The maximal value of E’ was 500 mN/m after 150 min. Only in the last stage of the measurement does the viscous modulus grow. The difference between the course of the relaxation curves when exposed to the oscillations and in the absence of stress is relatively insignificant [
Figure 5A lower panel]. It is worthwhile to note that although the relaxation experiments were conducted with a specification that π remain constant close to 5 mN/m, this was found to be difficult for the OLA system, even though the same control software could successfully maintain constant surface pressure values for other materials (for example, fatty acids). Instead, for the OLA system, the surface pressure varied between 3 and 13 mN/m. However, it does not affect the monolayer stability, as can be seen in [
Figure 5A lower panel]. Such high deviations are probably related to the bolaamphiphilic nature of OLA and the very stiff structure of the film [
5,
33]. As revealed using BAM, OLA monolayers in the form of tiles interacting with each other at the interface are susceptible to compression because of the very rigid structure. It is doubtful that excessively high values of the surface pressure achieved during oscillations and relaxations result from the feedback control loop utilized by the software. This is evidenced by the lack of such a phenomenon for other monolayers, such as, for example, oleic acid presented here.
Interestingly, even a minor addition of OA to the monolayer of OLA (OLA:OA 5:1) alters the trend of changes of viscoelastic moduli—over time, the E’ modulus decreases when E’’ stays at the steady level of ca. 100 mN/m [
Figure 5B top panel]. The maximum value of the elastic modulus for this system was about 400 mN/m. The addition of even a small amount of the OA makes the monolayer more ductile. The monolayer of OLA:OA 5:1 is resistant even to strong stress such as barrier oscillations, so as a consequence, the relaxation during oscillation and without additional barrier movements follows the same course [
Figure 5B lower panel]. Surface pressure amplitudes during the first few oscillations are as high as 11 mN/m, but in the initial stages of the relaxation experiment, they are at 5 ± 1 mN/m, and after 90 min, the monolayer becomes even more stable at 5 mN/m. Thus, we conclude that the addition of OA enhances the stability of the OLA monolayer.
Increasing the amount of OA in the mixture to OLA:OA 2:1 follows similar trends of the viscoelastic properties over time, but after 30 min the moduli stabilize [
Figure 5C top panel]. Surprisingly for the equimolar system, we noted the rapid increase in E’ according to decreasing A/A
0 [
Figure 5D]. E’’ values are close to 0 mN/m until ca. 60 min. When the oscillation/relaxation curve stabilizes, so does the viscoelastic modulus. For this monolayer composition, E’ reaches the highest values of all the investigated systems (650 mN/m), but the π variations are the largest as well. A slight decrease in the A/A
0 value can be observed [
Figure 5D lower panel]. Almost ideal consistency among oscillation/relaxation and relaxation curves for described binary systems at 5 mN/m may be related to the attractive interactions between the molecules among the monolayer and relatively good miscibility of the components.
The behavior of the pure oleic acid monolayer is quite different—E’ and E’’ moduli are very small [
Figure 5G top panel], and the area of monolayer coverage decreases very rapidly during the relaxation process [
Figure 5G lower panel]. The maximal values of E’ and E’’ of the OA monolayer captured at the initial stage of the process equal 25 mN/m and 3 mN/m, respectively. For the film compositions in which OA predominates, E’ moduli reach values of 250 mN/m and 100 mN/m for OLA:OA 1:2 and 1:5, respectively, while E’’ reaches 35 mN/m and 15 mN/m [
Figure 5E and F top panels]. In the initial stage of the experiment, the viscoelastic moduli are almost constant and then increase reaching the maximum. For OLA:OA 1:2 and 1:5 systems, surface pressure deviations are significantly limited and shifted after 60 and 150 min of the relaxation process [
Figure 5E and F lower panels]. What is more, the oscillation cycles strongly affect the shape of the relaxation curves. We note large differences between basic relaxation and oscillation/relaxation curves, where A/A
0 declines rapidly during the first 30 min of the measurement. This effect is even more pronounced for the pure OA monolayer—the barrier oscillations destabilized the monolayer and led to its large contraction after four oscillation cycles [
Figure 5G lower panel]. The susceptibility to the stress caused by the barrier movement is due to the
cis double bond in the hydrocarbon chain of the OA molecule.