Studies on the Interactions of 2-Hydroxyoleic Acid with Monolayers

Jun 19, 2019 - Drug delivery in cationic liposomes seems to be a promising therapeutic approach in cancer treatment. The rational design of the positi...
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Article Cite This: Langmuir 2019, 35, 9084−9092

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Studies on the Interactions of 2‑Hydroxyoleic Acid with Monolayers and Bilayers Containing Cationic Lipid: Searching for the Formulations for More Efficient Drug Delivery to Cancer Cells Karolina Olechowska,† Marzena Mach,† Katarzyna Ha̧c-Wydro,‡ and Paweł Wydro*,† †

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Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland ‡ Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland S Supporting Information *

ABSTRACT: Drug delivery in cationic liposomes seems to be a promising therapeutic approach in cancer treatment. The rational design of the positively charged lipid vesicles as anticancer drug carriers should be supported by a detailed analysis of the interactions of the carrier components with anticancer drugs. In the present work, 2-hydroxyoleic acid (2OHOA; Minerval), a membrane lipid therapy drug, was incorporated into positively charged mono- and bilayer membranes containing 1-palmitoyl2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC), the synthetic cationic lipid, and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The intermolecular interactions, fluidity, and miscibility of the studied monolayers were analyzed by utilizing Langmuir balance experiments. The morphology of two-dimensional films was inspected using a Brewster angle microscopy technique. The properties of the liposomes were investigated by dynamic light scattering (DLS) and zeta potential measurements, steady-state fluorescence anisotropy experiments, and the spectrofluorimetric titration of calcein-encapsulated vesicles with a lysis-inducing agent. According to the collected results, 2OHOA intercalation into films of pure phospholipids or a binary EPOPC/DOPC film is thermodynamically favorable. Surprisingly, no significant effect of the presence of unsaturated 2OHOA chains on the EPOPC/DOPC monolayer order was observed. The experiments carried out for 2OHOA-inserted cationic EPOPC/DOPC (1:4) liposomes indicate effective incorporation of the drug into the liposome bilayer and the formation of stable vesicles without affecting their properties markedly. On the basis of the obtained results, EPOPC/DOPC/ 2OHOA cationic liposomes with 15% 2OHOA content in the phospholipid bilayer seem to be the most suitable for potential biomedical applications.

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INTRODUCTION

osomes, micelles, nanoparticles, carbon nanotubes, and dendrimers are extensively studied as drug carriers, and some of them have been approved for clinical application.4−9 Among the various carriers, liposomes are probably the most widely studied. Different liposomal formulations are generally characterized by biocompatibility, the ability to encapsulate a hydrophilic drug into the aqueous space or to incorporate hydrophobic substances into the lipid bilayer, protection of the

Cancer remains one of the most common causes of death in the world.1 Despite the fact that a lot of effort has been put into the development of antitumor therapies, there are still many limitations to be overcome to carry out efficient and safe anticancer therapy. The effect of many antitumor drugs is often limited by low aqueous solubility, lack of stability, short blood circulation times, poor accumulation in tumor tissue, and nonselective distribution.2,3 To overcome these limitations, novel therapeutic approaches such as lipid therapy and the use of drug delivery systems have been proposed as reducing side effects to provide more successful cancer treatment. Lip© 2019 American Chemical Society

Received: May 5, 2019 Revised: June 13, 2019 Published: June 19, 2019 9084

DOI: 10.1021/acs.langmuir.9b01326 Langmuir 2019, 35, 9084−9092

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synthesis), and thus no antiproliferative or other cytotoxic effects have been reported for normal cells.24,30,34 In this study, in-depth analysis of the properties of lipid monolayers and bilayers, after the incorporation of Minerval into positively charged EPOPC-containing systems, was performed on the basis of the assumption that the 2OHOAinserted cationic liposomes may enhance the antitumor efficiency as compared to 2OHOA administered alone and become a potentially valuable therapeutic approach. Additionally, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was utilized in the present investigations as a neutral helper lipid that is widely used in liposomal formulations.11,35 As an important preliminary step for further biological experiments, we recognized the studies of the molecular organization, stability, and fluidity of binary and ternary monolayer systems composed of 2OHOA, EPOPC, and DOPC. Applied in this work, the Langmuir monolayer technique is extensively used to describe phenomena occurring in two-dimensional films, and the obtained results can be related to bilayer systems.36−39 Taking into account that the lipid composition and size of the liposomes are important factors in the efficacious delivery of the antitumor agents to the cancer cells,40 the physicochemical properties of EPOPC/DOPC/2OHOA vesicles with different drug content were investigated. Studies of the molecular organization, thermodynamic stability, fluidity, and permeability of such systems may be helpful in the rational design of the nanocarriers, optimization of the liposomes composition, and dose of the incorporated drug.

carried agent from the external media, and the lipidcomposition-dependent modulation of the surface charge.10,11 Liposomes are considered to undergo passive accumulation in tumor tissue with extended vascular permeability with subsequent retention resulting from ineffective lymphatic drainage from the tumor. The phenomenon, called the enhanced permeability and retention (EPR) effect, increases to some extent the therapeutic effectiveness of the drug carried in the liposomes.12 Currently, the attention of some researchers has been focused on cationic liposomes, which seem to demonstrate quite specific targeting of tumor vasculature.13 Because of the positive surface charge, they interact with negatively charged phosphatidylserine headgroups, overexpressed on the surface of the tumor endothelium, and may facilitate cellular adhesion to cancer cells.14−16 Cationic liposomes have been successfully applied to codelivery formulations of chemotherapeutic drugs and nucleic acids for more effective cancer treatment as compared to individually administrated agents.17−19 A compound that, because of its amphiphilic structure and positive charge, may be considered to be a candidate for an effective anticancer drug carrier is investigated herein: 1palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC), a synthetic cationic phosphatidylcholine triester (O-ethyl-phosphatidylcholine, EPC). What is important in the context of potential biomedical applications is that EPOPC, like all groups of EPCs as derivatives of naturally occurring phosphatidylcholines, shows diminished cellular toxicity and is metabolized by cells.20−23 Because of potential applications in drug delivery, comprehensive knowledge of the interactions of EPOPC with membrane lipids or antitumor agents and the organization of such structures are necessary. 2-Hydroxyoleic acid (2OHOA; Minerval), which is employed in this work, is a membrane lipid therapy (MLT) drug. Because alterations in the lipid composition or structure have been found for membranes of pathological cells, MLT drugs, as an alternative to conventional chemotherapy agents, are intended to target the cell membrane and to treat conditions associated with membrane disorders. Modification of the lipid environment influences the localization and activity of membrane proteins and thereby modulates cellular functions, cell signaling pathways, and gene expression.24,25 2OHOA is a synthetic analogue of oleic acid, designated as an orphan drug for the treatment of glioma.26 Because of the amphiphilic structure of the molecule, Minerval is spontaneously incorporated into the plasma membrane with a subsequent increase in its fluidity and has a nonlamellar phase propensity.27−29 This direct interaction with lipid membranes is accompanied by the regulation of the membrane composition by the activation of sphingomyelin synthase (SMS), an enzyme that controls the phosphatidylethanolamine (PE) to sphingomyelin (SM) ratio in plasma membranes.29,30 It is known that cancer cells show disturbances in SM and PC levels that affect membrane lipid-associated signaling pathways supporting the pathological cells’ proliferation. 2OHOA treatment leads to a reduction in SM/PC disproportion to the level found in nontumor cells. It has been shown that this regulatory influence contributes to cell cycle arrest, differentiation, autophagy, and/or apoptosis of glioma, leukemia, lung cancer, and breast cancer cells.30−33 In contrast, 2OHOA does not induce significant changes in the membrane composition of nontumor cells that already contain high SM content and low PC content (with PC being a substrate in SM

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EXPERIMENTAL SECTION

Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-ethyl-phosphocholine (chloride salt) (EPOPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (purity >99%) were purchased from Avanti Polar Lipids. 2-Hydroxyoleic acid (2OHOA, Minerval) was provided by Sigma-Aldrich. Also, Sephadex G-50, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein (calcein), 1,6-dphenyl-1,3,5-hexatriene (DPH), N,N-dimethylformamide (DMF), Triton X-100, chloroform, and methanol (both HPLC grade, ≥99.9%) were supplied by SigmaAldrich. In all experiments, ultrapure water (Milli-Q, resistivity 18.2 MΩ·cm) was used. Langmuir Balance Experiments. The surface pressure−area isotherms were recorded with the application of a Langmuir trough (NIMA) of nominal area 300 cm2. Stock solutions of the investigated phospholipids and 2OHOA were obtained by dissolving the samples in a mixture of chloroform and methanol (9:1 v/v). The monolayers were prepared by spreading the solution onto the air/water interface with a Hamilton microsyringe (±2.0 μL). After solvent evaporation, the monolayer was compressed by the movable barrier at a constant rate of 20 cm2/min. The changes in surface pressure during monolayer compression were automatically recorded (with an accuracy of ±0.1 mN/m) by a surface pressure sensor equipped with a Wilhelmy plate made of chromatographic paper (ashless Whatman Chr1). The subphase temperature (20 °C) was controlled by a Julabo thermostat. The state of the monolayers was verified on the basis of compression modulus values calculated as follows41 i dπ y CS−1 = − Ajjj zzz k dA { p , T

(1)

where A is the mean area per molecule at a given surface pressure π. The miscibility and interactions between investigated lipids and 2OHOA were assessed on the basis of the excess mean area per molecule calculated according to eqs 2 and 3 (for binary monolayers) or eq 4 (for ternary films)42 Aexc = A − Aid 9085

(2) DOI: 10.1021/acs.langmuir.9b01326 Langmuir 2019, 35, 9084−9092

Langmuir Aid = A1X1 + A 2 X 2

(3)

Aid = A12 (X1 + X 2) + A3X3

(4)

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RESULTS Interactions in Binary Systems. The surface pressure/ area isotherms recorded for one-component monolayers and binary EPOPC/2OHOA, DOPC/2OHOA, and EPOPC/ DOPC mixed systems are shown in Figure 1. The experiments

where A is the mean area per lipid molecule in the mixed monolayer (determined directly from the isotherms), A1, A2, and A3 are the mean molecular areas of the respective lipids in their one-component films at a given surface pressure, A12 is the mean area per molecule in a binary EPOPC/DOPC monolayer, and X1, X2, and X3 are the mole fractions of the respective compounds in the mixtures. BAM Experiments. The morphological changes resulting from the compression of the investigated films were visualized with the application of a Brewster angle microscope (UltraBAM, Accurion GmbH) placed on an antivibration table. The microscope was equipped with laser-emitting light with a wavelength of 658 nm which was polarized linearly, in the plane of incidence, by a Glan Thomson polarizer. The spatial resolution of the microscope was 2 μm. Liposome Preparation. Multilamellar lipid vesicles were prepared by the hydration of the dry lipid film. The dry lipid film was formed by solvent evaporation (under a gentle stream of nitrogen) from mixed solutions with appropriate compositions of lipids and 2OHOA. Then the lipid film was hydrated with PBS (pH 7.4) and vortex mixed. The obtained suspension of multilamellar liposomes was subjected to five cycles of freezing and thawing (from liquid-nitrogen temperature to about 60 °C) and then extruded (13 times) through a polycarbonate filter with a 100 nm pore size using a manual extruder purchased from Avestin. The final concentration of lipids was 1.27 μmol/mL. Liposome Size and Zeta Potential Determination. A Malvern Zetasizer Nano ZS apparatus was used for dynamic light scattering (DLS) and zeta potential measurements. The sample was placed in a disposable folded capillary cell (DTS1070) and the measurements were repeated three times at 20 °C. The mean hydrodynamic diameter (dz) (z-average), polydispersity index (PDI) and zeta potential (ζ) of the liposome suspensions were calculated using the software supplied by Malvern. Steady-State Fluorescence Anisotropy. The fluorescence probe was incorporated into the liposomal bilayer by the following method. The solution of DPH in DMF was added to the liposome suspension to maintain the DPH final concentration of 0.14 μM. The mixtures were then incubated in the dark for 1 h. The fluorescence intensity was measured with a Hitachi F-7100 spectrofluorimeter equipped with fluorescence polarizers. Samples were excited at 350 nm, and emission intensities at 430 nm were registered. The measurements were repeated three times. The steady-state anisotropy (r) was calculated according to the equation r=

IVV − GIVH IVV + 2GIVH

Figure 1. Surface pressure−area isotherms obtained for the mixed monolayers of EPOPC/2OHOA (a), DOPC/2OHOA (b), and DOPC/EPOPC (c).

were performed at the air−water interface. In Table 1, the maximal compression modulus values calculated from the π/A curves for pure films and the studied mixed systems are summarized. Finally, in Figure 2 the excess area per molecule values calculated at three different surface pressures are

(5)

where IVV is the intensity of fluorescence parallel to the excitation plane, IVH is the intensity of fluorescence perpendicular to the excitation plane, and G is an instrumental correction factor determined as follows:43 G=

IVH IHH

Article

Table 1. Maximal Compression Modulus Values of Binary Mixed Films

(6)

Calcein-Release Measurements. Calcein-loaded liposomes were prepared similarly to the procedure described in section 2.3; however, the dry lipid film was hydrated with calcein solution (in PBS, pH 7.4) in a self-quenching concentration (60 mM). The molecules of calcein which were not trapped inside the liposomes were separated with the application of size-exclusion chromatography on a Sephadex G-50 column with PBS buffer as an eluent. The changes in the fluorescence intensity connected with calcein release from the liposomes during the titration of the suspensions with a 0.6% solution of Triton X-100 (membrane lysis agent) were recorded using a Hitachi F-7100 spectrofluorimeter. The fluorescence emission of calcein was measured at 520 nm, and the excitation wavelength was set at 490 nm.

EPOPC/2OHOA

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−1

DOPC/2OHOA −1

DOPC/EPOPC

X2OHOA

Cs [mN/m]

X2OHOA

Cs [mN/m]

XEPOPC

Cs−1 [mN/m]

0 0.1 0.333 0.5 0.667 0.9 1

96 103 108 116 106 73 59

0 0.1 0.333 0.5 0.667 0.9 1

138 135 115 111 90 71 59

0 0.1 0.2 0.333 0.5 0.667 0.9 1

138 125 123 117 113 108 97 96

DOI: 10.1021/acs.langmuir.9b01326 Langmuir 2019, 35, 9084−9092

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This attraction causes the reduction of intermolecular distance and the same strengthening of van der Waals interactions between the hydrophobic parts of the molecules, provoking some increase in acyl chain ordering. Further growth of 2OHOA content (more than 50 mol %) causes a drop in Cs−1 values, indicating a decrease in the hydrophobic chain order. With regard to DOPC/2OHOA monolayers (Figure 1b), similar to the system discussed above, the addition of the drug leads to the systematic shift of the curves to the isotherm obtained for a one-component 2OHOA film. However, in contrast to EPOPC/2OHOA mixed monolayers, even a small content of 2OHOA causes a drop in the compression modulus values, revealing the disordering effect of this compound on DOPC molecules. Despite the fact that DOPC molecules also possess an unsaturated acid residue, they are characterized by the highest values of Cs−1 among the compounds analyzed in this work, and hence it is not surprising that the addition of 2OHOA with a high tendency to loosely pack results in a gradual decrease in the analyzed parameter. To get a basis for the analysis of the intermolecular interactions in three-component films, the isotherms for EPOPC/DOPC have also been recorded (Figure 1c). For this system, with increasing cationic lipid content, the shift of the curves toward higher surface area and an inconsiderable decrease in the collapse surface pressure values can be observed. The Cs−1 values (Table 1) show that all of the examined systems are in a liquid state; however, the ordering of phospholipid acyl chains decreases in an EPOPCconcentration-dependent way. To analyze the miscibility and intermolecular interactions between components in the foregoing mixed systems, the mean area per molecule values at various surface pressures were compared with the values resulting from the additivity rule. For EPOPC/2OHOA mixed systems (Figure 2a), negative values of AExc are observed for all of the investigated surface pressures and over the whole range of monolayer composition. This fact indicates the miscibility of the monolayer components and suggests that interactions between 2OHOA and EPOPC are more favorable than the interactions between molecules in the respective pure films. This is a consequence of the attractive electrostatic interactions between the oppositely charged hydrophilic groups of the molecules. The lowest values of this parameter are observed at X2OHOA = 0.5, where the most attractive interactions between their components occur, making the film the most condensed. Also, the analysis of ΔGExc proves the highest thermodynamic stability of the system with equimolar contents of 2OHOA and EPOPC (Figure S1a, Supporting Information). As can be seen in Figure 2b, negative deviations from ideality observed for binary DOPC/2OHOA films occur over the whole range of surface pressure and system composition. This suggests that 2OHOA and DOPC molecules are miscible and the mixed monolayers are more condensed than the respective ideal mixture. The minimal value of AExc and therefore the strongest intermolecular interactions are obtained for 50 mol % 2OHOA. Figure 2c shows that for all EPOPC/DOPC mixtures the excess area of mixing values differs from zero. For surface pressures of 15 and 30 mN/m, in the molar ratios of cationic lipid from 0.1 to 0.5 (and in the whole range of monolayer composition for π = 5 mN/m), negative deviations from ideal mixing are visible. These deviations from ideality indicate the mixing of monolayer components and the presence of

Figure 2. Excess area per molecule vs monolayer composition plots for the mixed systems of EPOPC/2OHOA (a), DOPC/2OHOA (b), and DOPC/EPOPC (c). The solid lines connecting the points are only guides for the eye.

presented. The dashed lines in Figure 2 correspond to ideal mixing or the immiscibility of the film components. Analyzing the π/A curve obtained for the 2OHOA monolayer, it can be seen that the lift-off area is ca. 44 Å2/ molecule and the collapse surface pressure is about 43 mN/m. As found earlier, 2OHOA molecules form at the air/water interface of the film in the liquid expanded state.44 BAM pictures taken at different stages of the monolayer compression evidenced total homogeneity of the 2OHOA film up to the collapse point (data not presented), which is consistent with previously published results.44 Also, results collected for a onecomponent EPOPC film is in agreement with our previous findings: the surface pressure starts to rise at A ≈ 150 Å2/ molecule, and the film collapses at πcoll ≈ 45 mN/m.45 EPOPC molecules form the film in a liquid state showing a uniform texture during the compression. As can be observed in Figure 1a, with the increase in 2OHOA concentration, the isotherms registered for the mixed EPOPC/2OHOA monolayers systematically shift toward smaller areas and their shape becomes similar to the curve of a pure Minerval film. By analyzing the data collected in Table 1, one can see that the Cs−1 values for two-component systems increase slightly up to 50 mol % 2OHOA in the mixtures. This suggests a small increase in the monolayer order as compared to that of a pure EPOPC film. These findings may be a consequence of electrostatic interactions between positively charged EPOPC and negatively charged 2OHOA molecules. 9087

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Langmuir interactions between the investigated lipids that are different from those in the one-component films. Negative AExc values demonstrate the reduction of the area per molecule and more attractive (or less repulsive) interactions between the components of the mixed monolayer as compared to those in the respective pure films. On the other hand, the positive values of the analyzed parameter obtained for mixtures containing more than 50% EPOPC suggest the appearance of less beneficial or repulsive interactions between system components and the expansion of the mixed film. The greatest positive deviation from ideal behavior occurs at 90 mol % cationic lipid content, while the most negative values of AExc, and thus the most condensed monolayer in comparison with the ideal state, are obtained for XEPOPC = 0.333. However, for π = 30 mN/m, that is, for the region of surface pressure which is of biological significance,46 the values of AExc of up to 50% EPOPC are comparable. For the discussion of two-component systems in this section, BAM images have been recorded. However, the analyzed monolayers are characterized by complete homogeneity; therefore, the microscopy photographs have not been shown in the present work. Influence of 2OHOA on the EPOPC/DOPC Monolayer System. For further research involving three-component monolayer and bilayer systems, the mixture of EPOPC/ DOPC with a 1/4 molar ratio has been selected because the system with such a composition shows thermodynamic stability and allows us to obtain liposomes with high, positive surface charge (discussed below). As can be observed in Figure 3a, π/A isotherms for EPOPC/DOPC/2OHOA monolayers are localized between those for the above-mentioned binary mixed film and one-component 2OHOA film. To analyze the effect of 2OHOA on the molecular order of a two-component membrane, Cs−1 vs the surface pressure plots have been depicted in Figure 3b. The values of Cs−1 indicate that EPOPC/DOPC forms a monolayer of a liquidlike state. Such a film is also characterized by complete homogeneity within a wide range of surface pressures (Figure 4). A small amount of 2OHOA in the mixture does not change the compression modulus values, indicating no influence on monolayer order. The 50 mol % content of the drug decreases Cs−1 only insignificantly (from 126 to 118 mN/m). For mole fractions higher than 0.5, a sudden increase in the acyl chain disordering is observed. Figure 3c exhibits AExc/X2OHOA plots calculated from EPOPC/DOPC/2OHOA isotherms. The plots for ideal mixing are introduced as a dashed line. Negative deviations from ideal behavior suggest more favorable interactions between the components of the mixed monolayer than in binary EPOPC/DOPC and one-component 2OHOA films. Moreover, similar to the case of two-component EPOPC/ 2OHOA and DOPC/2OHOA systems, the strongest intermolecular interactions are noticeable for the film containing 50% 2OHOA. Taking into account the course and shape of π/ A isotherms, favorable interactions between films components, and the high thermodynamic stability of the system (Figure S1d), it is not surprising that recorded BAM images are homogeneous, and no phase separation or domain formation is observed (Figure 4). Properties of the EPOPC/DOPC/2OHOA Bilayer System. Table 2 presents the results obtained by dynamic light scattering and zeta potential measurements for EPOPC/ DOPC/2OHOA liposomes. According to the presented data,

Figure 3. Surface pressure−area curves (a), compression modulus vs the surface pressure plots (b), and excess area per molecule values (c) for the EPOPC/DOPC/2OHOA system. The solid lines connecting the points (in c) are only a guide for the eye.

the values of the mean hydrodynamic diameter of the liposomes remain practically unchanged. Regardless of the composition of the vesicles, dz is about 100 nm. The values of the polydispersity index range between 0.061 and 0.077, revealing the homogeneity of the liposome sizes and no aggregation process. As can be predicted, the zeta potential of the liposomes tends to decrease in a 2OHOA contentdependent manner, from 52.8 mV for 2OHOA-free liposomes to −16.5 mV for a ternary system with the highest acid concentration. Values of the zeta potential above +30 mV, obtained for the majority of the investigated systems, indicate the stability of the studied liposomes due to repulsive forces between particles preventing their aggregation.47 The stability of the prepared vesicles was also confirmed by monitoring the values of the mean hydrodynamic diameter thereof during storage of the suspensions in 4 °C for 14 days (Table S1). No significant changes in the size of the liposomes were observed except for the system with the highest 2OHOA content exhibiting the growth of dz after 2 weeks. The fluidity of the liposome bilayer was assessed on the basis of the steady-state fluorescence anisotropy experiments. The measurements were performed using DPH as a fluorescent probe, with molecules incorporating into the lipid bilayer structure that are sensitive to the order and packing of the surrounding acyl chains.48 Changes in the membrane rigidity result in the limitation or enhancement of the rotational movements of the dye with the subsequent alteration of 9088

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Figure 4. BAM images recorded for EPOPC/DOPC and EPOPC/DOPC/2OHOA systems containing 33.3% 2OHOA.

addition of 2OHOA. Because 2OHOA has a cis double bond, bending the hydrocarbon residue, and it is well known that the presence of unsaturated fatty acid molecules has a fluidizing effect on lipid membranes,49−51 it could be expected that the incorporation of its molecules into the EPOPC monolayer should induce acyl chain disordering and film expansion. On the other hand, the 2OHOA molecule possesses a carboxylic group undergoing dissociation and becoming negatively charged in the aqueous solution. Therefore, the increase in the monolayer condensation and thermodynamic stability (Figure 2) is a consequence of the appearance of favorable electrostatic interactions between positively charged EPOPC and negatively charged 2OHOA molecules. This effect is the most pronounced for the equimolar mixture of the compounds in which compensation of the opposite charges may occur. These beneficial interactions bring molecules closer to each other, inducing an enhancement in the van der Waals attractions between the hydrophobic parts of the molecules and provoking a slight increase in the acyl chain ordering. This effect is reflected in a small increase in the compression modulus values. The foregoing findings indicate that in the EPOPC/2OHOA system, in which X2OHOA < 0.9, attractive electrostatic interactions between polar headgroups prevail over the steric effect associated with the molecular structure of 2OHOA. The results obtained for EPOPC/2OHOA mixtures can be compared with those published previously for POPC/ 2OHOA films.44 Although EPOPC and POPC possess the same hydrophobic parts, the effect of 2-hydroxyoleic acid on the EPOPC monolayer is different from that on the POPC film. This is particularly visible in the changes of the compressional modulus values with the variation in the monolayer composition. In the case of the POPC/2OHOA system, Cs−1 values gradually decrease with the increase in 2OHOA, which is consistent with the fluidizing effect of 2OHOA observed for the majority of lipid films. On the other hand, the addition of 2OHOA to the EPOPC monolayer causes an increase in the Cs−1 values, which achieve their maximum for the equimolar mixture. These findings confirm that the observed differences result from the electrostatic attractions between positively charged EPOPC molecules and negatively charged 2-hydroxyoleic acid. Slightly different outcomes are gained after mixing 2OHOA with DOPC. Namely, the gradual decrease in Cs−1 values with increasing 2OHOA content is observed. On the other hand, negative values of AExc and ΔGExc (Figure 2 and Figure S1b) are

Table 2. Values of the Mean Hydrodynamic Diameter (dz), Polydispersity Index (PDI), and Zeta Potentials (ζ) of the EPOPC/DOPC/2OHOA Liposomes and the Fluorescence Anisotropy of DPH X2OHOA 0 0.05 0.1 0.15 0.33

dz[nm] 101 100 102 102 101

± ± ± ± ±

1 1 1 2 2

PDI 0.074 0.065 0.067 0.061 0.077

± ± ± ± ±

0.012 0.016 0.014 0.018 0.010

ζ[mV] 52.8 49.4 43.6 34.9 −16.5

± ± ± ± ±

3.0 3.2 3.1 2.5 2.3

DPH anisotropy 0.070 0.069 0.067 0.065 0.062

± ± ± ± ±

0.003 0.002 0.003 0.004 0.004

fluorescence anisotropy. The results of these experiments are shown in Table 2. As can be seen, the values of the fluorescence anisotropy practically do not change with the increase in the Minerval mole fraction. These findings reveal that 2OHOA (to 33% in the system) does not affect the liposome bilayer fluidity. The changes in fluorescence intensity, resulting from calcein leakage from the liposome interior during the titration of the suspension with Triton X-100, are presented in Figure 4. The addition of the surfactant to the initial dispersion leads to a gradual increase in the fluorescence intensity. At a molar ratio of Triton X-100 to lipids of about 0.1, the maximal value of the fluorescence intensity is reached as a result of the total disintegration of the vesicles and the dilution of the initially self-quenched dye into the external medium. With the increase in the 2OHOA content in the system, the curves are slightly shifted to higher values of the Triton X-100/lipids molar ratio. Nevertheless, this effect can be considered to be negligible, and one can conclude that 2OHOA does not influence the permeability of the studied lipid bilayers.

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DISCUSSION In the context of the potential application of 2OHOAcontaining cationic liposomes as anticancer drug carriers, the purpose of the present study was to examine the effect of 2OHOA on the molecular organization and properties of monolayer and bilayer systems composed of EPOPC and DOPC. Langmuir monolayer experiments carried out for EPOPC/ 2OHOA mixed films showed that the incorporation of the drug, at a dose not exceeding 50 mol %, causes cationic lipid monolayer condensation and ordering (Figure 1, Table 1). This effect is followed by the growth of the fluidity with further 9089

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Langmuir observed over the whole range of the monolayer composition, which suggests that the interactions between components of this system are dominated by the beneficial effect of the separation of the negatively charged 2OHOA headgroups by phospholipid molecules. Because of the fact that in the analyzed system no expansion of the film is observed, the decline of the compression modulus values seems to be caused by the disorganization of the acyl chains of the DOPC, related to the structure of the 2-hydroxyoleic acid molecules discussed above, namely, the kink in the hydrocarbon chain. A very similar effect was previously observed for the POPC/2OHOA system,44 which indicates that the presence of an additional cis unsaturated bond in the DOPC, in comparison to the POPC molecule, practically does not affect its interactions with 2OHOA. With regard to DOPC/EPOPC monolayers (Figure 2), with XEPOPC ≤ 0.5, the beneficial effect associated with the separation of like-charged EPOPC ions by DOPC appears. Strong electrostatic repulsions occurring in the singlecomponent EPOPC monolayer are weakened by DOPC molecules, which is favorable from the thermodynamic point of view and is manifested by the negative values of the AExc and ΔGExc parameters. A large concentration of cationic lipid molecules (XEPOPC = 0.9), in turn, causes the expansion of the film as a result of the insufficient number of DOPC molecules that could shield the positive charge of the EPOPC hydrophilic groups. After getting the first insight into the interactions of 2OHOA with EPOPC or DOPC molecules, the properties of ternary mixed films have been examined. On the basis of the obtained results, it can be concluded that the influence of Minerval on the ordering and packing of EPOPC/DOPC 1:4 monolayer is conditioned by the intermolecular interactions in two-component systems. It has been evidenced that a 2OHOA concentration lower than 50% does not affect the Cs−1 values. Therefore, the effect connected with the attractive interactions between oppositely charged headgroups and the increase in membrane order (observed for EPOPC/2OHOA system) is balanced by the disordering effect associated with the construction of the 2OHOA hydrophobic chain (obtained for the DOPC/2OHOA system). What is important from the point of view of possible applications of EPOPC/DOPC/ 2OHOA systems is that the results of ΔGExc calculations show that the addition of 2OHOA to the EPOPC/DOPC system is thermodynamically favorable (Figure S1d). Finally, the properties of EPOPC/DOPC/2OHOA bilayers in various compositions have been verified. The gradual decrease in the values of the zeta potential demonstrates that 2OHOA was effectively incorporated into the bilayer, notwithstanding, according to the DLS results, the liposome size not being affected (Table 2). It is worth emphasizing that liposomes with a diameter of about 100 nm are considered to have the optimal size for the most effective drug delivery to the tumor tissue.40 Because the values of the DPH anisotropy fluorescence remain unchanged, the presence of 2OHOA molecules appears not to influence the rotational movements of the fluorescent dye. Thus, it can be concluded that 2OHOA has no impact on the EPOPC/DOPC liposome bilayer fluidity. These findings agree with the results obtained for Cs−1 calculations carried out for the analogous monolayer system. According to the results obtained by utilizing spectrofluorimetric titration with Triton X-100 (Figure 5), 2OHOA seems not to enhance the calcein permeation through the liposomal bilayer despite the fact that unsaturated compounds tend to

Figure 5. Changes in fluorescence intensity of calcein during the titration of the EPOPC/DOPC/2OHOA liposomal dispersions with Triton X-100 at pH 7.4. The solid lines connecting the points are only a guide for the eye.

disorganize the membrane, as manifested by the increase in its permeability. A surprising lack of 2OHOA impact on the EPOPC/DOPC properties may be associated with the effect detected for the monolayer system, namely, the favorable intermolecular interactions in the EPOPC/DOPC/2OHOA system which counteract steric effects. On the basis of the investigations presented in this work, the EPOPC/DOPC/2OHOA system containing 17% EDOPC and 15% 2OHOA seems to be of optimal composition for further investigation. This is the highest examined concentration of Minerval herein for which liposomes still have a relatively high positive surface charge ensuring stability. Taking into account the proven efficacy of 2OHOA in the treatment of certain types of cancer and the advantages of liposomes with respect to the positive surface charge as drug delivery systems, 2OHOA-inserted cationic liposomes can be considered to be promising candidates for anticancer drug carriers. It would be of interest to investigate whether antitumor agents additionally incorporated into 2OHOA-containing cationic liposomes show a synergistic effect against cancer. In this context, an additional advantage may be the fact that 2OHOA does not influence the properties of the EPOPC/DOPC system significantly and therefore should not determine the liposome loading capacity. However, this assumption needs to be verified.

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CONCLUSIONS In present study, results concerning the influence of 2hydroxyoleic acid, a membrane lipid therapy drug with anticancer activity, on monolayer and bilayer systems composed of EPOPC and DOPC have been shown. Langmuir balance measurements showed that 2OHOA intercalated into films of pure phospholipids or a binary EPOPC/DOPC film forms at the air/water interface thermodynamically stable monolayers and their components are entirely miscible. For the EPOPC/2OHOA system, composed of up to 50% 2OHOA, slight increases in condensation and acyl chain ordering can be noticed. The origin of this phenomenon can be related to the presence of electrostatic attractions between oppositely charged polar headgroups. In the case of DOPC/ 2OHOA monolayers, disorder in the acyl chains is provoked by the kinked structure of the 2OHOA hydrocarbon chain. In a ternary EPOPC/DOPC/2OHOA film, these effects are equilibrated. An analysis of the miscibility and intermolecular interactions in Langmuir films allowed us to select the system composition for further experiments in bilayer systems. The experiments carried out for EPOPC/DOPC (1:4) liposomes with 2OHOA indicate that the drug coforms stable vesicles 9090

DOI: 10.1021/acs.langmuir.9b01326 Langmuir 2019, 35, 9084−9092

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Langmuir

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without affecting their properties significantly. The fact that even a 5 mol % 2OHOA concentration causes the zeta potential values to decrease proves the high efficiency of drug incorporation into the liposome bilayer. Fluorescence anisotropy measurements do not show alterations in the fluidity of the studied systems, and spectrofluorimetric titration proved that the bilayer permeability practically does not change. These findings are in good agreement with the results obtained for monolayer systems. On the basis of the obtained results, EPOPC/DOPC/ 2OHOA cationic liposomes with 15% 2OHOA content in the phospholipid bilayer have been proposed to be the most promising for further studies and potential biomedical applications. According to the current knowledge, Minerval delivery in cationic liposomes may enhance its accumulation in tumor tissue and hence increase the therapeutic efficacy of the drug. The treatment utilizing an anticancer agent encapsulated in the examined liposomes and involving the simultaneous delivery of two anticancer drugs is also worth evaluating.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.9b01326. ΔGExc vs monolayer composition plots and changes in the mean hydrodynamic diameter of EPOPC/DOPC/ 2OHOA liposomes with different 2OHOA content, (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +48 (12)686 25 19. ORCID

Katarzyna Ha̧c-Wydro: 0000-0003-3470-0403 Paweł Wydro: 0000-0002-9145-1362 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financed by the National Science Centre, Poland (grant no. 2016/21/B/ST5/00266). REFERENCES

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