Interactions of Lipidic Cubic Phase Nanoparticles with Lipid

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Interactions of Lipidic Cubic Phase Nanoparticles with Lipid Membranes Elzḃ ieta Jabłonowska,† Ewa Nazaruk,† Dorota Matyszewska,‡ Chiara Speziale,§ Raffaele Mezzenga,§ Ehud M. Landau,∥ and Renata Bilewicz*,† †

Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Ż wirki i Wigury 101, 02-089 Warsaw, Poland § Department of Health Sciences & Technology, ETH Zurich, 8092 Zurich, Switzerland ∥ Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland ‡

S Supporting Information *

ABSTRACT: The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air−solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air−water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur.

1. INTRODUCTION Phospholipid monolayers and bilayers have been used as model systems for biological membranes in studies of the effect of drug and drug carriers on the properties of the biomembranes.1,2 Self-assembled nanostructures such as liposomes and especially liquid crystalline nanoparticles have been studied extensively as therapeutic and diagnostic agents and delivery systems due to their biocompatibility, ease of drug encapsulation, and targeting.1−4 There have been several approaches in recent years to use cubosomes, which are dispersed lipidic cubic phase nanoparticles stabilized by a nonionic block copolymer such as Pluronic F-127, as efficient drug delivery systems.4−6 Their advantage over liposomes lies in the liquid crystalline internal structure, which is a bicontinuous phase composed of lipid bilayers and aqueous channels, thereby providing a large internal surface area able to efficiently bind large amounts of drugs. The properties and structures of lipidic cubic phase nanoparticles have been recently extensively studied.7−10 © XXXX American Chemical Society

First reports on the spreading of liposomes at the air−water interface were from the Pattus group11 and the mechanism and kinetics of transformation of the liposomes were described by Schindler et al. and Ivanova et al.12,13 The interactions of liposomes with Langmuir−Blodgett monolayers of 1,2dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA) compressed to 5 mN/m were studied by Leonard-Latour et al.3 They observed progressive incorporation of 1,2-dimyristoilo-sn-glycero-3-phosphocholine (DMPC) from the liposomal suspension into the lipid monolayer at the air−water interface. The DMPA/DMPC mixed monolayer could be transferred without formation of aggregates or mesophases onto solid substrate. Nylander et al. have shown that liquid crystal nanoparticles (SPC/GDO/P80) adsorb strongly on cationic surfaces covered by DOPC bilayers14,15 while on Received: May 6, 2016 Revised: July 16, 2016

A

DOI: 10.1021/acs.langmuir.6b01746 Langmuir XXXX, XXX, XXX−XXX

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the subphase containing cubosomes. We also reveal the mechanistic steps during the cubosome−lipid layer interactions and image them with Brewster angle microscopy.

hydrophobic ones the attractive interaction between lipids and the surface completely disrupts the cubosomes structure and a mixed monolayer is formed at the air−water interface.16 At a hydrophilic or charged surface much thicker films are formed retaining to some extent the original cubosome structure. Adsorption behavior depends also on the internal structure and lipid composition of the nanoparticles. Adsorption of phytantriol (PT) and monoolein (GMO)-based cubosomes and hexosomes on various substrates was studied. Hexosomes showed a higher rate of adsorption but lower mass upon saturation of adsorption. Moreover PT-based cubosomes showed stronger adhesion than GMO-based cubosomes.17 Null ellipsometry and neutron reflectivity were used to study the interaction of cubosomes with model lipid bilayer, revealing that cubosomes first adsorb to the DOPC bilayer and next the exchange of lipids takes place. After sufficient time of such exchange the cubosomal lipids were desorbed from the interface.14 It was further reported that exchange of material takes place regardless of the initial bilayer coverage and evolution toward lamellar phase occurs.18 Study on PT-based cubosomes and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine− phytantriol (DPPS-PT) vesicles interaction with model lipid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidylcholine (POPC) showed that DPPS-PT vesicles exhibit stronger interaction and preferential attachment and fusion to lipid membrane than the cubosomes.19 Shen et al. suggested that interactions of multicomponent dispersions of lyotropic liquid-crystalline particles with cell membranes involve a multistep process involving attachment and fusion as well as lipid mixing. These interactions were related to cellular uptake and hence cytotoxicity of the particles. PT-based cubosomes accumulate at the POPC bilayer in the intact form, which was confirmed by the appearance of a Bragg peak in the neutron reflectivity data. Moreover, confocal microscopy showed that DPPS-containing formulations had increased membrane affinity in the case of HeLa cell membranes. Monte Carlo simulations also point to the adsorption of amphiphilic nanoparticles on the lipid bilayer resulting in the destabilization of the bilayer.20 Handa and co-workers studied the interactions of GMObased cubosomes with plasma components in vitro.21 They found that GMO cubosomes disintegrated in the presence of plasma components such as albumin.22 Upon incubation with low-density lipoprotein the disintegration could be prevented. In vitro cytotoxicity studies showed that PT-based cubosomes possess greater toxicity than GMO-based, which was related to the disruption of the membrane integrity and oxidative stress. In the present investigation we present the effect of cubosomes on a lipid Langmuir monolayer treated as one leaflet of a model membrane. This approach relies on the formation of a highly ordered monolayer of precise thickness at the air−water interface. The Langmuir method enables the study of monolayers of varying packing densities corresponding to different surface pressures. It should be also underlined that the properties of the monolayer are not affected by any solid substrate, which is the case when a lipid layer is self-assembled on a solid support. Most importantly, the fluidity and organization of the system are not affected by the support. This resembles the conditions of a biomembrane, and the approach can be treated as a complementary method in the studies of drug carrier−membrane interactions. We follow the changes of surface pressure−area per molecule isotherms during formation of the monolayer while it is in contact with

Scheme 1. Structures of Lipids Used for the LangmuirBlodgett Monolayer and Cubosome Formationa

a (1) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (2) (1(cis-9-octadecenoyl)-rac-glycerol (Monoolein, GMO), (3) ([poly(ethylene oxide)] 100 -[poly(propyleneoxide)] 65 -[poly(ethylene oxide]100) (PEO100-PPO65−PEO100, Pluronic® F-127).

2. MATERIALS AND METHODS 2.1. Materials. Monoolein (1-oleoyl-rac-glycerol) (GMO), Pluronic F-127, and chloroform of analytical grade were from SigmaAldrich. Lipids: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2dihexadecanoyl-3-trimethylammonium-propane (chloride salt) (DPTAP), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DPEPC) were from Avanti Polar Lipids. Inc. Water was distilled and passed through Hydrolab HLP and then Milli-Q purification system (MQ-water). 2.2. Methods. 2.2.1. Preparation and Characterization of Cubosomes. Cubosomes were prepared with the use of top-down technique.23 Initially, bulk cubic phase was prepared by mixing melted monoolein with water in an appropriate ratio according to the phase diagram.24 The ratio GMO/water was 60/40 wt %. Bulk cubic phases were left at room temperature for a few days to equilibrate. To obtain cubosomes, the cubic phase was weighed, and then added to the appropriate amount of Pluronic F-127. The Pluronic F-127 solution in water was added to a final GMO concentration of ca. 3% (w/v). The cubic phase was fragmented by high-shear dispersing emulsifier (IKA T10 homogenizer) followed by sonication. The final composition of cubosomes was 96.3/3.0/0.7 wt % of water/GMO/Pluronic F-127. Vials were covered with aluminum foil to protect samples from light. Cubosome suspensions were diluted 5-fold (v/v) with MQ-water and vortexed for 15 min prior to the Langmuir−Blodgett experiment. The average size, polydispersity index, and zeta potential (ζ) of the cubosomes were determined at 25 °C by dynamic light scattering (DLS Zetasizer Nano ZS Malvern. UK) with the use of disposable cuvettes (DTS1070). Cubosome suspensions were diluted (v/v) with MQ-water and then vortexed for ca. 30 min before measurements. The refractive indexes used for lipid and water were 1.48 and 1.33, respectively. Phase identity and structural parameters of the lipidic samples were determined by SAXS measurements performed on a Bruker AXS Micro instrument with a microfocused X-ray source, operating at voltage and filament current of 50 kV and 1000 μA, respectively. The Cu Kα radiation (λCu Kα = 1.5418 Å) was collimated by a 2D Kratkycollimator, and the data were collected by a 2D Pilatus 100 K detector. The scattering vector q = (4π/λ) sin θ, with 2θ being the scattering angle, was calibrated using silver behenate. Data were collected and B


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Langmuir azimuthally averaged using the Saxsgui software to yield onedimensional intensity versus scattering vector q, with a q range from 0.004 to 0.5 Å−1. Dispersion samples were filled into 2 mm diameter quartz capillaries which were sealed with epoxy glue (UHU). Measurements were performed at 22 °C; scattered intensity was collected over 5 h. The lattice parameter (a) was calculated using the following equation: a=

2π 2 h + k2 + l 2 q

(1)

where q is the scattering vector, h, k, and l are Miller indices of the Bragg peak. Cryo-SEM measurements were carried out using an Auriga Cross-beam microscope (Carl Zeiss microscopy, GmbH. For cryo-SEM measurement the sample was frozen in liquid nitrogen and then transferred into the cryochamber of the microscope which was held at −140 °C. Before measurement, the sample was sublimed at −90 °C for ca. 3 min. 2.2.2. Langmuir Technique. Surface pressure versus molecular area isotherms were recorded using the KSV LB trough 5000 equipped with hydrophilic barriers. The experiment was controlled with software version KSV 5000. A Wilhelmy balance was used as a surface pressure sensor (filter paper changed after each experiment). Surface pressure was recorded as a function of molecular area. The accuracy of measurements of area per molecule was 1 Å2 and that of surface pressure was 1 mN/m. After the solution was spread, it was left for 15 min for solvent evaporation. Compression was accomplished with a barrier speed of 7.5 cm2/min (10 mm/min). Temperature was kept at 22 ± 1 °C unless otherwise stated. 2.2.3. Brewster Angle Microscopy (BAM). Brewster angle microscopy experiments were performed using a Nanofilm Ep3 instrument (AccurionGmbH, Göttingen, Germany) equipped with an UltraBam objective (10× magnification), 50 mW solid-state laser emitting p-polarized light at a wavelength of 658 nm and a CCD camera. The lateral resolution of the UltraBam objective is 2 μm. Images were captured during the compression of the monolayers at the air−water interface and each picture represents the area of 320 μm × 410 μm.

Figure 1. SAXS pattern for GMO/Pluronic F-127 cubosomes. Inset: Cryo-SEM micrograph of a GMO/Pluronic F-127 cubosome.

indicates a tortuous structure resembling the proposed mathematical models of cubosomes. 3.2. Behavior of Cubosomes at the Air−Water Interface. Surface pressure recorded as a function of area per surfactant molecule allows a determination of the monolayer state and area occupied by one molecule in the most organized form of the monolayer. When injected into the subphase of a Langmuir trough, cubosomes approach the air− water interface and spread forming a monolayer whose isotherm and compression modulus are almost identical to those obtained for a mixture of monoolein and the Pluronic F127 polymer spread directly at the interface from the chloroform solution (Figure 2A,B). An identical composition of the components to those constituting the cubosomes was used. The similarity of the surface pressure area per molecule isotherm indicates that cubosomes tend to spread at the air− water interface to form a monolayer. The reference isotherms with GMO (Supporting Information, Figure 1S) or Pluronic F127 (Figure 2S) only and stability of monolayer at different surface pressures are shown in Supporting Information. Based on the changes of isotherms recorded for different ratios of components, a noninteracting two-component monolayer is formed. Mixed monolayer formation is indicated by the reversibility of the compression−decompression isotherms. Moreover, BAM micrographs show a homogeneous surface without aggregates. The compression modulus, Cs−1, calculated based on the following equation,28

3. RESULTS AND DISCUSSION 3.1. Cubosomes Properties. Cubic phase nanoparticles were prepared by fragmentation of bulk cubic phase obtained at a GMO/water ratio of 60/40 wt %. The bulk cubic-Pn3m phase at room temperature exhibited a lattice parameter of 9.7 nm.25 To obtain a suspension of cubic nanoparticles the bulk cubic phase was fragmented in the presence of 0.7 wt % of Pluronic F-127. SAXS measurements were performed to identify the type and structural parameters of the cubosome formulation. Figure 1 displays the SAXS pattern for dispersed cubosome particles. Three distinct Bragg peaks with relative peak positions of √2, √4, and √6 that can be indexed as hkl 110, 200, and 211 were observed. This pattern corresponds to the cubic-Im3m structure with a lattice parameter of 14.0 nm. This is in accord with the finding that the GMO-based cubic phase, when dispersed and stabilized with Pluronic F-127, may undergo a phase transition from the diamond Pn3m type to the more swollen primitive cubic phase of Im3m symmetry,26 although this depends on the size of the cubosomes. The properties of the cubosome formulation are presented in Table 1. The particle size of the cubosomes determined with DLS was ca. 180 nm, with a PDI value close to 0.2. The zeta potential obtained for the cubosome was ca. −19 mV although GMO and Pluronic F-127 are not charged. Negative zeta potential of the cubosome may be connected with contamination of GMO with oleic acid or adsorption of hydroxyl ions on the lipid/water interface.27 The cryo-SEM graph (Figure 1)

Cs−1 = −A

dπ dA

(2)

is plotted versus surface pressure and shown in Figure 2 as the inset. Two local minima can be recognized in the plot for the cubosomes. The one at 13 mN/m appears at slightly higher surface pressure than for the pure Pluronic F-127, which is at 11 mN/m. The second minimum of compression modulus seen at ca. 38 mN/m corresponds to the removal of Pluronic F127 from the monolayer. The maximal value of Cs−1 obtained for the cubosome is similar to that of monoolein and indicates a liquid expanded layer. 3.3. Interactions of Cubosomes with DPPC Monolayers at the Air−Water Interface. The behavior of cubosomes approaching the interface covered by lipid-forming C


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Langmuir Table 1. Properties of the GMO/Pluronic F-127 Cubosome Formulation sample

symmetry

unit cell [nm]

size [nm]

PDI

zeta potential [mV]

GMO/Pluronic F-127 cubosome

Im3m

14.0

183.7 ± 15

0.20 ± 0.05

−19.0 ± 2.6

Figure 2. (A) Comparison of isotherms: (a, black trace) GMO/ Pluronic F-127 cubosome formulation injected on the subphase; the final ratio of GMO/Pluronic F-127 in subphase was 0.996/0.004 w/w %; (b, red trace) monolayer of the mixture of GMO/Pluronic F-127 (0.996/0.004 w/w %) in chloroform; (c, green trace) GMO monolayer formed on pure water subphase; (inset) compression modulus vs surface pressure for the investigated monolayers. (B) (d, orange trace) Isotherm of Pluronic F-127; (inset) compression modulus vs surface pressure.

Figure 3. (A) Surface pressure vs area per molecule isotherms of DPPC on subphases containing increasing concentration of GMO/ Pluronic F-127 cubosomes: (a) 0; (b) 0.10; (c) 0.15; (d) 0.25; (e) 0.35; (f) 0.45 mgGMO dm−3; (inset) compression modulus vs surface pressure for the studied monolayers. (B) Surface pressure vs area per molecule isotherms of DPPC on subphases containing increasing concentration of mixture GMO/Pluronic F-127 (0.996:0.004) dissolved in chloroform (a) 1:0, (b) 0.9:0.1, (c) 0.8:0.2, (d) 0.7:0.3; (inset) compression modulus vs surface pressure for the studied monolayers.

model membranes was studied by recording surface pressure vs area per molecule isotherms for DPPC (Figure 3). The DPPC isotherms are shifted toward larger molecular areas, that is, undergo expansion as a function of increased concentration of cubosomes in the subphase (Figure 3A). Moreover, upon increasing the concentration of cubosomes the shape of the isotherms is transformed toward that recorded for a monoolein/Pluronic F127 sample dissolved in chloroform (Figure 3B). It is noteworthy that the characteristic phase transition of the DPPC monolayer at ca. 7 mN/m disappears upon increased cubosome concentration and the compression modulus decreases, reflecting a more fluidic character of the monolayer. For increasing cubosome concentration characteristics, new minima reflecting a sharp decrease of the Cs−1 value appear (inset in Figure 3A). The one at 10.5 mN/m corresponds to the one seen for the Pluronic F-127 polymer,

and is connected with structural changes in the layer.29 The decrease in the Cs−1 value observed at 18 mN/m can be attributed to the affected phase transition of DPPC in the presence of cubosomes. This indicates incorporation of GMO into the layer since in a mixed GMO−DPPC layer such a minimum also appears at higher values of surface pressure than that observed for the DPPC phase transition itself (Supporting Information, Figure S3). At 35 mN/m the Pluronic F-127 layer collapses, and hence in the mixed layer recorded in the presence of cubosomes in solution it is squeezed out from the layer. At 48 mN/m the change in the slope of the layer corresponds to the point of GMO monolayer collapse. This behavior indicates partitioning of the cubosomes’ lipids into the DPPC monolayer at the air−water interface. The decrease in the final collapse pressure with increasing concentration of D


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Table 2. Characteristics of the DPPC Monolayer in the Presence of GMO/Pluronic F-127 Cubosomes in the Subphase concn of cubosomes (mgGMO dm−3)

A/Å2 at 35 mN/m ± 1

Acoll (Å2 ± 1)

Πcoll (mN/m ± 1)

Cs−1max (mN/m ± 5)

DPPC/pure water GMO/pure water 0.10 0.15 0.25 0.35 0.45

46.1 44.8 53.9 56.4 59.9 65.1 69.6

39.5 28.8 43.7 46.0 41.8 43.5 41.0

58.3 47.2 58.5 57.3 56.5 56.5 56.5

260 98 190 166 130 125 115

uniform monolayer at a surface pressure of approximately 20 mN/m. At even higher surface pressures (ca. 30 mN/m) the presence of very bright areas corresponding to the condensed phase are clearly visible. The obtained BAM pictures are typical for the DPPC monolayers forming the LC phase.30 In contrast, GMO monolayers yielded significantly different images (Figure 4 middle column). Even at very low surface pressures the air− water interface is covered by a very thin layer. Upon increasing the surface pressure to 4 mN/m some small round-shaped brighter regions, corresponding to a thicker lipid layer in a liquid state, can be observed. Further compression of the monolayer leads to growth of these regions and to the formation of a monolayer of liquid character. However, some condensation resulting in bright spots can be observed at approximately 30 mN/m, which corresponds to the surface pressure at which the compression modulus attains the maximum value for GMO monolayers. The observed differences in the morphology of DPPC and GMO monolayers are caused by different surface properties of the two lipids: DPPC forms liquid-condensed monolayer which is much better organized and thicker than a GMO monolayer, which is in the liquid-expanded state. The surface behavior of DPPC monolayers spread on a subphase that contains cubosomes is markedly different from that of DPPC layers formed on pure water (Figure 4). At low surface pressures the small round-shaped domains of the liquidexpanded phase are clearly visible. Compression of the monolayer leads to the formation of bigger domains, which partially form relatively uniform liquid regions at lower surface pressures. This type of morphology may be attributed to partitioning of GMO from the cubosomes in the subphase into the air−water interface, thereby enhancing its liquid character. However, despite the presence of the cubosomes in the subphase and GMO at the air−water interface the process of the DPPC domain formation corresponding to the liquidcondensed phase is not entirely inhibited. The domains typical for DPPC can be still observed at a surface pressure of 4 mN/m but the size and the number of the domains is smaller compared to the images obtained for DPPC monolayers formed on the pure water subphase. The fact that DPPC domains remain despite the presence of monoolein in the monolayer may be explained by the limited miscibility of GMO and DPPC. Additionally, the images obtained at the highest surface pressures (approximately 32 mN/m) are similar to those of GMO since the number of bright spots due to condensation of the monolayer is very limited, akin to the case of liquid GMO monolayers. Therefore, BAM pictures confirm the fluidizing effect of cubosomes on DPPC monolayers observed in Langmuir studies. 3.4. Interactions of Cubosomes with Precompressed DPPC Monolayers. To simulate a biologically more relevant state in which drug carriers interact with cell membranes over a

cubosomes observed for this multicomponent monolayer indicates interactions between the components of the layer affecting its parameters (Table 2). BAM images were taken at selected points during the compression in order to visualize changes in the DPPC monolayer morphology that result from cubosome incorporation from the subphase (Figure 4). DPPC monolayers were

Figure 4. BAM images obtained for DPPC monolayers on pure water subphase (left column); GMO monolayers on pure water subphase (middle column); DPPC monolayers on a water subphase containing 0.15 mgGMO dm−3 cubosomes (right column).

formed either on pure water subphase or subphase containing 0.15 mgGMO dm−3 cubosomes. Additionally the BAM pictures were also recorded for a pure GMO monolayer formed on water. Pictures obtained for DPPC monolayers formed on pure water show the coexistence of the gaseous and liquid-expanded phase at low values of surface pressure (Figure 4 left column). Upon increasing the surface pressure the formation of the characteristic, flower-shaped domains is observed and continues in the plateau region at approximately 4 mN/m, which corresponds to the transition from liquid expanded to liquid condensed phase. The domains grow in size and finally form a E


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Figure 5. Time dependence of the surface pressure: (A) air−solution interface over subphases containing: (a) 0.25; (b) 0.35; (c) 0.45; (d) 1.00; (e) 2.05; (f) 3.00 mgGMO dm−3 cubosomes; (B) DPPC monolayer compressed to 5 mN/m over subphases containing: (a) 0.0; (b) 0.10; (c) 0.20; (d) 0.25; (e) 0.30; (f) 0.40; (g) 0.45 mgGMO dm−3 cubosomes. (C) DPPC monolayer compressed to 35 mN/m over subphases containing (a) 0.0; (b) 0.01; (c) 0.025; (d) 0.05; (e) 0.15; (f) 0.25; (g) 0.35 mgGMO dm−3 cubosomes. (D) plots of steady state surface pressure vs log of cubosome concentration (a) air−solution interface; (b) DPPC monolayer compressed to 5 mN/m; (c) DPPC monolayer compressed to 35 mN/m. (A−C: cubosome concentrations used were varied to demonstrate the different saturation values for each interface).

Figure 6. Time-dependent BAM images of DPPC monolayers compressed to 5 mN/m on water subphase (top row), and on water subphase containing 0.35 mgGMO dm−3 cubosomes (bottom row).

investigated. The monolayers were first compressed to surface pressures of 5 and 35 mN/m, which correspond to different

longer period of time, interactions of cubosomes with DPPC monolayers precompressed to selected surface pressures were F


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Langmuir organization of the lipidic membrane and the changes in surface pressure as a function of time were recorded (Figure 5). As a control, the time dependent increase in surface pressure due to the presence of various concentrations of cubosomes in the subphase without the lipid spread at the air−water interface was also recorded (Figure 5A). The cubosomes themselves are surface active, as can be observed by their partitioning from the subphase to the air− water interface when it is not covered by a phospholipid monolayer. This results in a significant increase in surface pressure with time, an effect that is also concentration dependent (Figure 5A). When the monolayer at the air− solution interface is compressed to a certain state over cubosome solutions in the subphase, the surface pressure changes with time due to the interactions of cubosomes with DPPC monolayers (Figure 5B,C). Surface pressure changes occur for both investigated initial surface pressures and were shown to depend on the concentration of the cubosomes in the subphase. The relative increase in surface pressure is much more pronounced for DPPC monolayers precompressed to 5 mN/m, when the monolayer is in the liquid expanded phase, than when it is compressed to the liquid condensed phase (35 mN/m). At low surface pressure the monolayers are less organized and thus incorporation of cubosomes from the subphase results in higher energy gain than in case of highly packed monolayers at 35 mN/m. The changes in the DPPC monolayer morphology as a function of time were visualized by means of Brewster angle microscopy (Figure 6). Lipid monolayers compressed to 5 mN/m on a pure water subphase form typical domains corresponding to the phase transition from a liquid-expanded to liquid-condensed state (Figure 6 top row).31,32 The shape of these domains changes with time after the compression has been stopped, reflecting the observed slight decrease in the surface pressure (Figure 5B). When cubosomes are present in the subphase, the DPPC domains are much smaller, and a thin layer of liquid material filling the space between the domains is evident (Figure 6 bottom row). This layer may be attributed to the incorporated cubosomes, or more specifically to GMO molecules, which contribute to the monolayer at the air−water interface. These fill the available interfacial space and form a type of matrix for DPPC domains. This thin liquid lipid layer is not completely uniform, as can be observed by the dark spots that correspond to the pure subphase. In this case the DPPC domains also change their shape with time, and simultaneously additional lipidic material from the subphase adsorbs to the air−water interface, and relatively thick, brighter local aggregates with a high refractive index can also be observed after 15 min (Figure 6 bottom row). This may explain the observed increase in surface pressure (Figure 5B). When the surface pressure of the monolayer is 35 mN/m (Figure 5C), the cubosomes are initially incorporated into the monolayers and the surface pressure increases. After ca. 6 h it starts to decrease, indicating partial removal of the components of the monolayer. As shown in Figure 5D the higher is the initial surface pressure the lower is the saturation concentration Csat defined as the value of a concentration above which the surface pressure does not change (or decrease). It reflects the maximum concentration at which the incorporation of cubosomes into the lipidic monolayer occurs. Csat values for the two selected initial surface pressures as well as for the adsorption of the cubosomes from the subphase to the air− water interface are shown in Table 3.

Table 3. Saturation Concentration of GMO Incorporated into the Monolayer

a

Π0a (mN/m)

Csat (mgGMO dm−3)

no lipid 5 35

0.52 0.42 0.32

Initial surface pressure to which DPPC monolayer was compressed.

The penetration of GMO molecules from the cubosomes into the lipid layer depends on the availability of free adsorption sites in the layer, that is, on the molecular packing of the monolayer. The increase of surface pressure can be described by eqs 3 and 4:33,34 Γ=

⎡ δ(π − π0) ⎤ 1 ⎢ ⎥ 2.303RT ⎣ δ log C ⎦

T

(3)

1 (4) NΓ where π0 is the initial surface pressure to which the DPPC layer was compressed, RT is thermal energy, N is Avogadro number, and C is the concentration of GMO in the cubosome formulation in molGMO dm−3. The value of area per molecule is 0.43 nm2 and approaches that of a GMO molecule at critical surface pressure because then the Pluronic F127 polymer is removed from the monolayer. Only a small percentage of cubosome material is inserted into the layer while the rest stays in the form of cubosome at the interface and at longer time scale exchanges material with that of the monolayer at the air− water interface (which results in the decrease of surface pressure). 3.5. Interactions of Cubosomes with Precompressed Lipid Layers of Different Charge. Cubosomes show a small negative value of zeta potential (Table 1) which implies also negative surface charge; therefore their interaction with lipidic monolayers should depend on the charge of the lipid (structures of lipids used are presented in Scheme 1S). Figure 7 shows the plot of surface pressure changes with time for A=

Figure 7. Time dependence of the surface pressures of various monolayers compressed to 35 mN/m over cubosome-containing subphases: (a) DPPA, (b) DOPS, (c) DPPC, (d) DPEPC, (e) DPTAP precompressed to 35 mN/m following addition of cubosomes under the monolayer. Dashed lines: same plots but in the absence of cubosomes in the subphase. G


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aggregates of still intact cubosomes. Following concentration dependent delay time, tdelay, the adsorption of GMO from disintegrated cubosomes starts to predominate and a linear increase of surface pressure is observed. The more packed and organized the layer is, the lower is the saturation value, Csat, which is the concentration of cubosomes that can be still spread at the lipid covered water−air interface. Above concentration certain critical surface pressure (48 mN/m), the cubosomes remain close to the lipid interface in the intact form, and at long time-scale, the exchange of material between lipid layer and cubosome is observed leading to a slow decrease of surface pressure. The results indicate that for more porous lipid layers, for example, corresponding to cancer cell environment, the action of the cubosomes disintegration will be more intensive and delivery of the drug can be more efficient. These results are significant, as they enhance our understanding of the molecular interactions between model membranes and cubosomes as alternative, biocompatible drug delivery systems.

differently charged monolayers. At longer times some lipids show a decrease of surface pressure due to surface oxidation or interactions with the environment. At short times corresponding to recording of the isotherm reproducible isotherms are obtained indicating that nothing is changed during the compression and decompression of the monolayer. The monolayers were compressed to an initial surface pressure of 35 mN/m and left in contact with the cubosome containing subphase. The interaction with the cubosome results first in an increase of surface pressure, reaching a maximum, followed by a decrease at longer times. The time at which the maximal surface pressure is reached depends on the charge of the lipid’s headgroup: it is shortest for negatively charged head groups. (Table 4) When the monolayer is positively charged the Table 4. Dependence of Time of Attaining Maximal Surface Pressure on the Lipid Charge lipid (state of monolayer at 35 mN/m)

charge at pH 5

time at which maximum occurs (h)

DPPA (solid) DOPS (liquid) DPPC (liquid condensed) DPEPC (liquid expanded) DPTAP (liquid condensed)

−1 −0.25 0 +1 +1

2.5 4.4 5.5 6.0 8.0

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01746. Figures showing surface pressure vs area per molecule isotherms of GMO and Pluronic F127 on pure water subphase, area per molecule as a function of time recorded at different surface pressures; surface pressure− area per molecule isotherms for DPPC, GMO, and mixture of DPPC:GMO 2:8 on aqueous subphase; surface pressure−area per molecule isotherms for the lipids studied at pure aqueous subphase; compression modulus vs surface pressure for the lipid monolayers (PDF)

electrostatic attraction of the cubosome increases the time of contact of the cubosome with the layer. This dependence of the time of attaining a maximal surface pressure on the charge of the lipid’s headgroup suggests that at high surface pressure exchange of a lipid component between the monolayer and the cubosome in its intact form may occur. It should be noted that this behavior depends also on the state of the monolayer at the air−water interface and its stability over time. Isotherms of each of the lipids used (Supporting Information, Figure 4S) allow a comparison of the states of the monolayer at the initially compressed surface pressure of 35 mN/m, while the dashed lines in Figure 7 indicate the stabilities of the layers over time on pure aqueous subphase.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +48 22 55 26 357.

4. CONCLUSIONS Cubosomes are shown to disintegrate in contact with a DPPC monolayer treated as one leaflet of a biological membrane. The penetration of GMO molecules from the disintegrated cubosomes into the lipid layer depends on the availability of free adsorption sites in the layer, and hence on the molecular packing of the monolayer. Our monolayer study shows that whenever the membrane is porous the free place will be occupied by the lipid and polymer from the spread cubosome. It is proven here by the increase of surface pressure upon contact of cubosome solution with the layer and mixed monolayer formation. On the other hand, as also shown, the increase of surface pressure is smaller when we start the experiment with a more organized DPPC layer (hence, when the initial pressure of the DPPC monolayer is higher). When the DPPC monolayer is highly organized the cubosome cannot spread anymore and remain under the monolayerwe recognize it by the lighter color of the domains in BAM images and lack of changes of the surface pressure with time (monolayer organization is not affected). The light color of the BAM image in certain places indicates the layer becomes much thicker due to the presence of cubosomes but it is not a continuous second layer overlaying the first one, rather

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Sinergia project no. CRSII2_154451 financed by the Swiss National Science Foundation. EN acknowledges support from the National Science Centre of Poland (2013/09/D/ST5/03876). The BAM part of this study was carried out at the Biological and Chemical Research Centre University of Warsaw established within the project cofinanced by European Union from the European Regional Development Fund under the Operational Programme Innovative Economy 2007−2013.

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Interactions of Lipidic Cubic Phase Nanoparticles with Lipid

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