Characterization of Oil-Free and Oil-Loaded Liquid-Crystalline

Article pubs.acs.org/Langmuir

Characterization of Oil-Free and Oil-Loaded Liquid-Crystalline Particles Stabilized by Negatively Charged Stabilizer Citrem Christa Nilsson,† Katarina Edwards,‡,§ Jonny Eriksson,‡ Susan Weng Larsen,† Jesper Østergaard,† Claus Larsen,† Arto Urtti,∥ and Anan Yaghmur*,† †

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡ Department of Chemistry, BMC, Uppsala University, Husargatan 3, SE-751 23 Uppsala, Sweden § FRIAS, School of Soft Matter Research, University of Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany ∥ Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, FI-00790 Helsinki, Finland S Supporting Information *

ABSTRACT: The present study was designed to evaluate the effect of the negatively charged food-grade emulsifier citrem on the internal nanostructures of oil-free and oil-loaded aqueous dispersions of phytantriol (PHYT) and glyceryl monooleate (GMO). To our knowledge, this is the first report in the literature on the utilization of this charged stabilizing agent in the formation of aqueous dispersions consisting of well-ordered interiors (either inverted-type hexagonal (H2) phases or inverted-type microemulsion systems). Synchrotron smallangle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) were used to characterize the dispersed and the corresponding nondispersed phases of inverted-type nonlamellar liquid-crystalline phases and microemulsions. The results suggest a transition between different internal nanostructures of the aqueous dispersions after the addition of the stabilizer. In addition to the main function of citrem as a stabilizer that adheres to the surface of the dispersed particles, it has a significant impact on the internal nanostructures, which is governed by the following factors: (1) its penetration between the hydrophobic tails of the lipid molecules and (2) its degree of incorporation into the lipid−water interfacial area. In the presence of citrem, the formation of aqueous dispersions with functionalized hydrophilic domains by the enlargement of the hydrophilic nanochannels of the internal H2 phase in hexosomes and the hydrophilic core of the L2 phase in emulsified microemulsions (EMEs) could be particularly attractive for solubilizing and controlling the release of positively charged drugs.

1. INTRODUCTION Lyotropic liquid-crystalline (LLC) systems have very appealing properties that can be exploited in different pharmaceutical, agricultural, food, and cosmetics applications.1−6 They are a unique family of self-assembled phases that originate upon the exposure of amphiphilic lipids, such as monoglycerides, glycolipids, and phospholipids, to water.3,7−9 The hydrationinduced self-assembly process of these surfactant-like lipids includes the organization of hierarchically ordered nanostructures to form a rich polymorphism of lamellar and nonlamellar liquid-crystalline phases and inverted-type micellar solutions.7−16 The phase transitions in these systems are affected by different factors, including the molecular shape of the investigated lipid, the lipid composition, the electrostatic interactions, and the applied experimental conditions.11,15,17−21 In the lamellar structure, the lipid molecules are arranged in a 1D periodic lattice consisting of planar bilayers.11 The invertedtype nonlamellar phases include the bicontinuous cubic (Q2) and the hexagonal (H 2) phases.3,7,8,10,15 Q 2 is a 3D © 2012 American Chemical Society

bicontinuous phase composed of curved bilayers and has infinite periodic minimal surfaces (IPMS) whereas H2 is a 2D phase consisting of hydrophilic nanochannels embedded in a continuous hydrophobic medium. These lyotropic nonlamellar self-assembled nanostructures have attracted much interest in pharmaceutical research because they have a high potential to control the release of solubilized drugs and other bioactive materials.1,3,9,12,13,22−24 The great interest in utilizing these nanostructures in different biotechnological applications is also motivated by their close resemblance to structures found in biologically relevant systems and the biodegradability and biocompatibility of the lipids used in their formation.25,26 For different pharmaceutical applications, one major drawback of the lipidic inverted-type cubic and hexagonal liquidReceived: May 24, 2012 Revised: July 2, 2012 Published: July 25, 2012 11755

dx.doi.org/10.1021/la3021244 | Langmuir 2012, 28, 11755−11766

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Figure 1. Molecular structures of (A) phytantriol (PHYT), (B) monoolein (GMO), and (C) citrem.

to cover the outer surfaces of submicrometer particles consisting of Q2 or H2 phases during the emulsification procedure. Herein, it is essential to maintain the stability and integrity of these liquid-crystalline particles for their storage and application. In this regard, there is great interest in evaluating the use of stabilizing agents capable of keeping the dispersions stable over time without significantly disrupting or destroying their inner nanostructures. Various stabilizers have been employed for the stabilization of these liquid-crystalline particles,3,38−46 and among them, Pluronic F127 has emerged as an efficient hydrophilic stabilizer for a variety of dispersed lipids.3,21,27,38,47 It is capable of forming stable colloidal dispersions with well-defined nonlamellar interiors and is therefore the most commonly utilized stabilizing agent.3,40,48−52 A characteristic feature of this stabilizer is its effect on the internal nanostructures of aqueous dispersions based on monoolein (MO). It was reported that F127 affects the inner nanostructure of cubosomes by inducing a phase transition from the fully hydrated bicontinuous cubic phase with space group Pn3m (the diamond type, QD) to a bicontinuous cubic phase with space group Im3m (the primitive type, Qp).6,27 However, it acts as an efficient stabilizer for both monolinolein14,53,54 (MLO)- and PHYT51,55-based systems because it mainly covers the outer surface of the dispersed particles without significantly affecting the cubic Pn3m

crystalline phases is their high viscosity, which makes them unsuitable as injectable drug carriers. In addition, the injection of these phases could induce irritation upon direct contact with the biological environment. Therefore, the emulsification of these fully hydrated liquid-crystalline phases to form submicrometer-sized particles with well-defined internal nanostructures in excess buffer3,6,27−29 and the in situ formation of these liquid-crystalline phases at the administration site12,30,31 are two promising approaches for pharmaceutical applications. These approaches may provide simple and inexpensive methods for solubilizing drugs and controlling their release without inducing severe toxic side effects. In recent years, increasing interest in the formation of lipidic nanostructured aqueous dispersions such as cubosomes (aqueous dispersions of Q2 phases) and hexosomes (aqueous dispersions of H2 phase) as drug nanocarriers is due to their unique properties.3,10,27 These low-viscosity nanoparticulate formulations can be tailored to solubilize different therapeutic agents.3,29,32−37 Surprisingly, only a modest number of studies in the literature have dealt with the pharmaceutical applications of such drug-loaded nanoparticulate dispersions. Cubosomes and hexosomes are formed upon the emulsification of surfactant-like lipids with a nonlamellar propensity in the presence of excess water.3,27,38 There is a need to apply high-energy input and to use an efficient amphiphilic stabilizer 11756


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nanostructure. In a recent study,56 a wide range of triblock PEO-PPO-PEO copolymers (Pluronics) were investigated and evaluated for their ability to stabilize cubosomes and hexosomes based on MO and PHYT. It was found that F108 is superior to F127 as a stabilizer for MO-based aqueous dispersions. Furthermore, this polymeric stabilizer was able to preserve the internal diamond-type cubic phase and to keep the dispersion stable when used at low concentrations. Recent studies also suggested the use of colloidal clay platelets (Laponite), which are capable of stabilizing cubosomal dispersion at a neutral pH without causing disruption, thus the internal Pn3m cubic phase can be easily retained.57,58 One drawback with F127 was detected in a recent study by Murgia et al.59 It was reported that MO-based particles stabilized by F127 display toxicity toward three different cell lines. It has also been shown that Pluronics could activate the immune system in humans, though there is a need to investigate the cause behind such activation further.60 In the quest for a new nontoxic, sufficient stabilizing agent, different systems have been investigated.10,34,61,62 These compounds include lipids covalently attached to poly(ethylene glycol) (PEG),34,61 such as PEG (660)-glycerol monooleate (GMO)61 and PEG2000-grafted 1,2-distearoyl-sn-glycero-3phosphatidylethanolamine (DSPE-PEG2000).34 Previous studies also demonstrate the use of proteins as stabilizers for these dispersions.10,62 For instance, the highly amphiphilic, flexible protein β-casein provides steric stabilization to cubosomes without affecting the internal nanostructure.10,62 The aim of this work is to assess the ability of citrem to provide stabilization for different oil-free and oil-loaded lipidic nanostructured aqueous dispersions and to assess the possibility of modulating the internal nanostructures. Citrem is an anionic citric acid ester of monoglycerides that has been widely used as a small-molecule emulsifying agent in different food products including margarines, sausages, and confectionary.63−66 It is listed as an acceptable food additive by the FDA. 67 Furthermore, citrem has been proven to have an antioxidant effect in recent studies.65,66,68 In the present work, SAXS experiments and cryo-TEM observations were used to characterize the oil-free and oil-loaded PHYT- and GMObased aqueous dispersions and to determine the effect of the citrem content on the internal nanostructures. The molecular structures of investigated lipids PHYT, GMO, and citrem are illustrated in Figure 1.

the obtained isotropic solutions with preheated PBS was achieved by holding the binary mixtures of PHYT/water and GMO/water and the ternary GMO/citrem/water systems at 50−65 °C for 5−10 min and then homogenizing them by vigorous vortex mixing. The binary PHYT/vitamin E (vit E) and GMO/MCT mixtures were prepared at different solubilized oil (vit E or MCT) concentrations in the range of 10−50 wt %. The effect of loading citrem on the nanostructures of the PHYT/vit E- and the GMO/MCT-based fully hydrated systems was also investigated at low concentrations of citrem. The oil-free and oilloaded fully hydrated samples were formed at a fixed total PBS concentration of 50 wt % and incubated at 37 °C for 10−14 days before carrying out the SAXS measurements. 2.3. Formation of PHYT- and GMO-Based Aqueous Dispersions. As discussed above, investigated lipid PHYT or GMO and citrem were held at 40−50 °C and homogenized by vigorous vortex mixing to obtain an isotropic solution. This solution was then emulsified in excess PBS by means of ultrasonication. The emulsification was done by using a Sonics Vibracell VCX 130 (Sonics & Materials Inc., Newton, CT, USA) ultrasonic processor for 10−15 min in pulse mode (10 s pulses interrupted by 2 s breaks) at 80% of the maximum power. The formed milky dispersions were prepared with a total citrem concentration in the range of 1.5−4.0 wt % and a total lipid concentration in the range of 2.0−5.0 wt %. 2.4. Small-Angle X-ray Scattering (SAXS) Measurements and Data Analysis. X-ray measurements were performed at beamline I911-4 (MAX II storage ring, MAX-lab synchrotron facility, Lund University, Sweden) at an operating electron energy of 1.5 GeV. The scattering patterns were recorded with a 2D image plate detector (165 mm MarCCD, MarResearch, Norderstedt, Germany) using collection times of 30−240 s. The camera was kept under vacuum during data collection to minimize the background scattering. The detector covered the q range (q = 4π sin θ/λ, where λ is the wavelength and 2θ is the scattering angle) of interest from about 0.1 to 0.65 Å−1. Silver behenate (CH3-(CH2)20-COOAg with a d spacing value of 5.84 nm) was used as a standard to calibrate the angular scale of the measured intensity. The samples were measured in custom-made glass capillaries and sample holders for the aqueous dispersions and the fully hydrated samples, respectively. The measurements were performed at 25 or 37 °C (±0.1 °C) by the aid of a Peltier element. This beamline is described thoroughly in ref 69. The lattice parameters of L2, Q2 of symmetry Pn3m, and the H2 phases were deduced from the strongintensity reflections by applying standard procedures. 2.5. Cryogenic Transmission Electron Microscopy (CryoTEM). Cryogenic transmission electron microscopy (cryo-TEM) investigations were performed using a Zeiss Libra 120 (Carl Zeiss NTS, Oberkochen, Germany). The microscope was operated at an accelerating voltage of 80 kV in zero-loss bright-field mode. Digital images were recorded under low-dose conditions with a slow-scan CCD camera (TRS GmbH, Moorenweis, Germany) and iTEM software (Olympus Soft Imaging Solutions GmbH, Mü nster, Germany). An underfocus of 1 to 2 μm was used to enhance the image contrast. The cryo-TEM specimen preparations were performed in a custom-built climate chamber at 25 °C and approximately 98− 100% relative humidity. A small drop of sample was deposited on a copper grid covered with a carbon-reinforced holey polymer film. Excess liquid was thereafter removed by means of blotting with filter paper, leaving a thin film of the solution on the grid. Immediately after blotting, the sample was vitrified in liquid ethane and held just above its freezing point of −182 °C. Samples were kept below −165 °C and protected against atmospheric conditions during both transfer to the TEM and examination, where representative images for the samples were acquired. A more comprehensive description of the technique can be found in ref 70. 2.6. Size and Zeta Potential. The average particle size distribution of a GMO-based aqueous dispersion was determined by dynamic light scattering using the photon correlation spectroscopy (PCS) technique. The surface charge of the particles was estimated by analyzing the zeta potential (laser Doppler electrophoresis). Four measurements were performed, which prior to the measurement was diluted 100-fold in PBS. The sample was measured immediately after

2. MATERIALS AND METHODS 2.1. Materials. The commercial distilled Myverol 18-99K (GMO) was a gift from Kerry Bio-Sciences (Almere, The Netherlands). This distilled food-grade emulsifier consists of 93% monoglycerides including 60.0% monoolein and about 21.0% monolinolein. Medium chain triglycerides (MCT) consisting of approximately 60% caprylic acid and 40% capric acid were kindly donated by Brøste (Lyngby, Denmark). Phytantriol (PHYT, purity ≥85%) was purchased from Fluka Chemie GmbH (Buchs, Switzerland). α-Tocopherol (vitamin E, purity ≥96%) was purchased from Sigma-Aldrich Denmark A/S (Brøndby, Denmark). Grinsted citrem LR10, which is the citric acid ester of monoglycerides made from sunflower oil, was received as a gift from Danisco A/S (Copenhagen, Denmark). Phosphate buffer solution (PBS) with a concentration of 0.067 M at pH 7.4 was used. All ingredients were used without further purification. 2.2. Preparation of Fully Hydrated PHYT- and GMO-Based Self-Assembled Systems. The oil-free samples were prepared by holding investigated lipids PHYT and GMO and citrem at 40−50 °C. Fully hydrated GMO-based samples were prepared at different total citrem concentrations in the range of 0−5.0 wt %. The hydration of 11757


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preparation (day 0) and after 7, 14, and 21 days of incubation at room temperature. Both types of measurements were performed at room temperature using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) equipped with a 633 nm laser and 173° detection optics. Malvern DTS v. 6.34 software (Malvern instruments, Worcestershire, U.K.) was used for data acquisition and analysis. For the viscosity and refractive index, the values of pure water were used.

coexistence of the cubic Pn3m phase with traces of the bicontinuous cubic phase with symmetry Ia3d (gyriod type, Q230). The assignment of the cubic Ia3d phase is based on the appearance of the first two characteristic reflections, (211) and (220).16 The lattice parameter of the Ia3d phase was found to be 100.5 Å as seen in Table 1, which is in good agreement with previous findings.16 The Bonnet ratio, which is identified as the ratio of the mean lattice parameter of the cubic Ia3d (aIa3d) to that of the cubic Pn3m (aPn3m) phase, was found to be 1.558, which is close to the theoretical value of 1.57671 for the two coexisting cubic phases. This is in good agreement with the fact that the minimal surfaces of both the cubic Ia3d and the cubic Pn3m curved bilayers should have the same averaged Gaussian curvature under equilibrium conditions.16,47,71,72 It should be noted that the fully hydrated sample was prepared 2 weeks prior to the SAXS experiments; therefore, it is interesting to detect that a complete transition from a cubic phase with the space group of Ia3d to a cubic phase with the space group of the Pn3m nanostructure was not fully achieved at 25 °C. The cubic Ia3d phase vanished upon increasing the temperature to 37 °C (unpublished data). In the presence of citrem, the SAXS patterns taken from the PHYT aqueous dispersions show peaks that are compatible with the first three reflections of an inverted-type hexagonal (H2) phase (Figure 2). The emulsification of the fully hydrated PHYT system is thus accompanied by a phase transition from a biphasic sample consisting of the cubic Pn3m phase coexisting with traces of the cubic Ia3d phase to H2 phase with an a value of 55.4 Å (formation of hexosomes) at a total citrem concentration of 2.0 wt % (Table 1). The formation of milky hexosomal dispersions is related to the significant effect of citrem on the internal nanostructures of the kinetically stabilized aqueous dispersions of PHYT. The obtained results suggest, as discussed below in detail, that a considerable amount of citrem penetrates the internal nanostructure and another part of these molecules is present at the outer surface of the dispersed particles with the hydrophobic tails anchored in the inner hydrophobic core of the dispersed particles. With increasing citrem content, the three characteristic peaks of the H2 phase (Figure 2) are still observed, but they are slightly shifted to lower q values with a subsequent slight increase in the corresponding structure parameters (Table 1). For instance, the mean lattice parameter, a, of the H2 phase increases from 55.4 to 55.8 Å as the stabilizer concentration is increased from 2.0 to 3.0 wt %. It is clear that there is no indication of the formation of cubosomes in the presence of citrem. In 1976, Israelachvili et al.73 introduced the critical packing parameter (CPP) or the wedge shape factor that describes how the surfactant molecular shape is useful in predicting which type of self-assembled system is formed upon exposure of the surfactant to water or oil. CPP is described as follows v CPP = s a 0l (1)

3. RESULTS AND DISCUSSION 3.1. PHYT-Based Aqueous Dispersions. Different studies, as discussed in the Introduction, showed that the emulsification of inverted-type nonlamellar phases in the presence of stabilizers most often involves a significant effect on the internal nanostructures.3,27,38,47 Therefore, it is of utmost importance in the present work to study the role of the selected stabilizer and its effects on the internal nanostructure of the dispersed phases. The internal nanostructures of the emulsified PHYT systems prepared at different citrem concentrations were investigated at 25 °C and compared with that of the corresponding fully hydrated nondispersed liquid-crystalline phase (Figure 2). It is

Figure 2. Effect of citrem content on the internal nanostructures of PHYT-based aqueous dispersions. The SAXS experiments were performed at 25 °C. The intensities have been shifted by a constant arbitrary factor for better visibility. The two observed peaks corresponding to the cubic Ia3d phase in the biphasic nondispersed fully hydrated sample are marked by asterisks.

clear that the SAXS pattern taken from the fully hydrated sample shows peaks characteristic for a cubic structure of the CD type with space group Pn3m with a lattice parameter of 64.5 Å. This is consistent with previous published studies. For instance, Barauskas and Landh16 reported on the formation of a fully hydrated cubic Pn3m phase at 25 °C with a lattice parameter of 66 Å. In another report, the phase behavior of PHYT from two different companies was investigated, and the lattice parameter of the fully hydrated cubic Pn3m phase was found to be 68 Å at 25 °C.55 It was reported that the slight change in the lattice parameter is due to the presence of impurities in PHYT that affect the self-assembled nanostructures. It is worth noting that our SAXS data also suggest the

where vs is the hydrophobic chain volume, a0 is the headgroup area, and l is the hydrophobic chain length.73 The CPP can also be described in terms of spontaneous curvature of the lipid interfacial film.11 This parameter is affected by different factors including the molecular shape of the surfactant, the investigated temperature, the hydration level, and the electrostatic interactions.10,14,15,73−77 In the present report, the obtained results suggest a dual effect of citrem on the value of CPP. The 11758


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Table 1. Structure Parameters as Derived from SAXS Investigations Carried Out on PHYT-Based Systems

a

sample no.

PHYT/vit E (weight ratio)

stabilizer content (wt %)

PHYT + vit E (wt %)

buffer content (wt %)

temperature (° C)

1

100/0

0

50

50a

25

2 3 4 5 6 7 8

100/0 100/0 100/0 100/0 90/10 90/10 90/10 90/10

0 2.0 2.4 3.0 0 1.5 2.6 3.25

50 4 3.6 3 50 5 3.9 3.25

50a 94b 94b 94b 50a 93.5b 93.5b 93.5b

37 25 25 25 25 25 25 25

space group

lattice parameter, a (Å)

Pn3m and traces of Ia3d Pn3m H2 H2 H2 H2 H2 H2 H2

64.5 (Pn3m) 100.5 (Ia3d) 62.8 55.4 55.8 55.8 46.0 51.8 55.2 56.4

Fully hydrated sample containing 50 wt % PBS. bEffect of citrem on the dispersed PHYT or PHYT/vit E-based systems.

also suggests that a greater fraction of citrem acts as a hydrophobic guest material and is preferably localized in the interstitial hydrophobic regions of the internal nanostructure; therefore, it promotes the formation of the H2 phase. A schematic description of the stabilization of these nanostructured aqueous dispersions in the presence of citrem is illustrated in Figure 3.

most pronounced effect is related to the high affinity of citrem to the hydrophobic tails of PHYT. This amphiphilic component acts as a guest hydrophobic agent; therefore, it induces a significant increase in the CPP value because of its penetration between the hydrophobic tails of PHYT, which leads to a significant increase in the effective hydrophobic chain volume (vs). This means that citrem molecules favor the formation of self-assembled nanostructures with a more negative spontaneous curvature; therefore, a phase transition following the sequence (cubic Pn3m + traces of cubic Ia3d) → H2 is observed. These results are consistent with previous investigations reported on the effect of solubilizing oils such as MCT, oleic acid, and tetradecane on fully hydrated monoglycerides.48,53,55,78,79 The solubilization of oil increases the CPP value because of an increase in the vs value with a simultaneous decrease in the solubilized water content of the self-assembled nanostructures (a reduction in the value of a0). It was reported that packing limitations are considered to be the main energy barrier to the formation of discontinuous phases74,79 such as H2. The results suggest that citrem behaves like an oil and therefore releases the packing frustration within the hexagonal lattice and stabilizes the newly formed H2 phase by filling out the interstitial hydrophobic regions.74,79 Figure 2, however, shows that the effect of citrem on the structure is different than that reported for oils because its penetration into the inner particle nanostructure leads to the formation of an H2 phase with relatively large hydrophilic nanochannels embedded in the hydrophobic continuous medium of the H2 phase. An enlargement of the hydrophilic nanochannels in this nonlamellar phase, as indicated by the observed shift of its characteristic SAXS peaks to low q values, is most likely attributed to the partial incorporation of citrem molecules into the water−PHYT neutral interfacial area. The repulsive forces between the penetrated negatively charged hydrophilic headgroups of citrem molecules in the interfacial area increase the headgroup area (a0)3,28 and most likely promote the solubilization of a higher amount of water in the hydrophilic domains of the internal H2 nanostructure. The penetration of citrem into the water−lipid interfacial film has a significant effect on the internal structure of PHYTbased aqueous dispersions. However, it is also clear that this effect is not dominant because flattening the bilayers of the cubic Pn3m/Ia3d phases to form planar bilayers (Lα phase) instead of the H2 phase is not achieved. It seems that the fraction of the citrem molecules that penetrate the water− PHYT interfacial area is not sufficient to induce the structural transition from cubic Pn3m/Ia3d phases to planar bilayers. This

Figure 3. Schematic description of the stabilization mechanism of citrem in PHYT-based aqueous dispersions.

In addition to the SAXS characterization of the internally nanostructured aqueous dispersions, a cryo-TEM investigation was carried out for a selected sample. Figure 4a presents the cryo-TEM image of an aqueous dispersion of PHYT containing 3.0 wt % citrem (sample 4, Table 1). The SAXS pattern of this hexosomal dispersion is shown in Figure 2. The obtained SAXS data clearly indicate the formation of hexosomes whereas the cryo-TEM image indicates the formation of submicrometersized particles enveloping an ordered internal nanostructure, but it was difficult because of the quality of the image to obtain further information on the emulsified nanostructure. It is also noteworthy that some of the observed dispersed particles are stabilized by a surface phase (arrows, Figure 4a) similar to that observed in the nanoparticulate aqueous dispersions of sponges. The formation of such nanostructured dispersions has previously been reported by Barauskas et al.80 Nonionic emulsifier polysorbate 80 (Tween 80) was used as a stabilizer for aqueous dispersions based on binary mixtures of diglycerol monooleate and glycerol dioleate. It was stated that these spongelike structures could also aid in the stabilization of the dispersed particles.80 Clearly, further cryo-TEM studies are needed to characterize these PHYT-based aqueous dispersions at different citrem contents in order to shed light on the influence of the formation of this surface phase on the 11759


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Figure 4. Cryo-TEM images of PHYT- and GMO/MCT-based aqueous dispersions after vitrification from 25 °C. (A) A PHYT-based aqueous dispersion consisting of PHYT containing 3.0 wt % citrem. (B) A GMO/MCT-based aqueous dispersion containing 1.5 wt % citrem and a binary GMO/MCT mixture with a weight ratio of 90/10. Scale bar = 100 nm. In panel A, some of the observed dispersed particles are stabilized by a surface phase (arrows). Figure 5. Effect of citrem content on the internal nanostructures of vitamin E (vit E)-loaded aqueous dispersions of PHYT. The SAXS experiments were performed at 25 °C. The intensities have been shifted by a constant arbitrary factor for better visibility.

stabilization of the submicrometer-sized particles. Figure 4a shows also that hexosomes are formed in the absence of vesicles, which is in good agreement with previous studies that showed hexosomes coexist with fewer vesicles than do cubosomes.14,81 It should also be noted that the present image does not allow us to extract further information about the internal nanostructure. In this regard, the application of a heat-treatment method as suggested by Johnsson et al.82 could be useful for improving the quality of the images (the observation of clear internal self-assembled nanostructures). This method was described82 as a valuable tool for obtaining good-quality cryo-TEM images by inducing the formation of fully developed, well-defined hexosomal particles. 3.2. Vit E-Loaded PHYT-Based Aqueous Dispersions. Figure 5 shows the SAXS pattern of emulsified PHYT/vit E systems prepared at different citrem concentrations compared to that of the corresponding fully hydrated nondispersed liquidcrystalline phase at 25 °C. The oil content is 10 wt % of the total lipid/oil concentration for the dispersed aqueous samples as well as the nondispersed fully hydrated sample. The SAXS pattern for the fully hydrated sample shows peaks that are compatible with the first three reflections of the H2 phase, with a lattice parameter of 46.0 Å (sample 5, Table 1). These results are consistent with investigations reported on the effect of solubilizing oils on fully hydrated monoglycerides, as previously described in Section 3.1 in depth. For instance, the effect of loading oil on the nanostructures of PHYT-based bulk nondispersed samples and the corresponding dispersed aqueous phases has been previously studied.55 Upon loading of vitamin E acetate, the internal nanostructure is transformed from the cubic Pn3m phase (cubosomes) to the H2 phase (hexosomes). In the presence of citrem, the SAXS patterns taken from the aqueous dispersions of binary PHYT/vit E still show peaks that are compatible with the H2 phase as the total citrem concentration is varied in the range of 1.5−3.25 wt % (Figure 5). However, these characteristic peaks of the H2 phase are

shifted to lower q values with a subsequent increase in the corresponding structure parameters (Table 1). For instance, the mean lattice parameter, a, of the H2 phase increases from 51.8 to 56.4 Å as the stabilizer concentration is increased from 1.5 to 3.25 wt %. The results suggest that a considerable amount of citrem penetrates the internal nanostructure, and another fraction of these molecules is present at the outer surface of the dispersed particles. This is also consistent with previous reported findings with respect to the use of citrem for the stabilization of oil-in-water (O/W) emulsions.63,67,68 It was suggested that these emulsions are stabilized in the following way: at the interface, the fatty acid groups of citrem are oriented into the oil phase whereas the negatively charged organic acid group extends into the aqueous phase, stabilizing the emulsion through electrostatic repulsion.66 The fact that the hydrophilic nanochannels of the H2 phase are enlarged with increasing citrem content makes the use of this charged emulsifier interesting for potential pharmaceutical and food applications as a means of functionalization. The functionalization of self-assembled nanostructures has previously been seen upon loading negatively charged surfactantlike lipids and peptides into neutral membranes of monoglycerides.1,18,47,77 3.3. GMO-Based Aqueous Dispersions. In Table 2, the lattice parameters for the internal nanostructures of the emulsified GMO-based systems prepared at different citrem concentrations are presented. The samples were investigated at 25 and 37 °C. For aqueous dispersions prepared with a citrem concentration in the range of 2.0−3.4 wt % at 25 °C, the SAXS patterns show three peaks that are compatible with the H2 phase (Figure S1 in the Supporting Information), but they are shifted to higher q values with a subsequent decrease in the corresponding structure parameters (Table 2). For instance, the 11760


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Table 2. Structure Parameters as Derived from SAXS Investigations Carried Out on GMO-Based Systems sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

GMO/MCT (weight ratio)

stabilizer content (wt %)

GMO + MCT (wt %)

buffer content (wt %)

temperature (° C)

space group

lattice parameter, a (Å)

100/0 100/0 100/0 100/0 100/0 100/0 100/0 100/0 100/0 90/10 90/10 90/10 90/10 90/10 90/10 90/10 85/15 85/15 85/15 85/15 75/25 75/25 60/40 60/40 50/50 50/50 100/0 100/0 100/0 90/10 90/10

0 2.0 2.0 2.4 3.0 3.0 3.4 3.4 4.0 0 0 1.5 1.5 2.6 2.6 3.25 0 1.5 2.6 3.25 0 1.5 0 1.5 0 1.5 1.0 2.0 5.0 2.0 5.0

50 4 4 3.6 3 3 2.6 2.6 2 50 50 5 5 3.9 3.9 3.25 50 5 3.9 3.25 50 5 50 5 50 5 49 48 45 48 45

50a 94b 94b 94b 94b 94b 94b 94b 94b 50a 50a 93.5b 93.5b 93.5b 93.5b 93.5b 50a 93.5b 93b 92b 50a 93.5b 50a 93.5b 50a 93.5b 50c 50c 50c 50c 50c

37 25 37 25 25 37 25 37 25 25 37 25 37 25 37 25 25 25 25 25 25 25 25 25 25 25 37 37 37 37 37

Pn3m H2 H2 H2 H2 H2 H2 H2 L2 H2 H2 H2 H2 H2 H2 L2 H2 H2 H2 L2 L2 L2 L2 L2 L2 L2 Pn3m Pn3m Pn3m H2 H2

84.0 65.6 65.2 64.8 64.1 62.6 64.0 n.i

d (Å)

59.6 60.0 55.0 62.6 61.1 63.9 63.6 57.5 55.5 61.4 61.9 57.5 49.3 nc 48.2 nc 47.1 nc 82.8 81.6 80.9 57.5 57.6

a

Fully hydrated samples containing 50 wt % PBS 7.4. bEffect of citrem on dispersed GMO or binary GMO/MCT systems. cEffect of citrem on fully hydrated GMO or binary GMO/MCT systems. nc: the values are not calculated.

mean lattice parameter, a, of the internal H2 phase decreases from 65.6 to 64.1 Å as the stabilizer concentration increases from 2.0 to 3.4 wt %. At a higher concentration of citrem (4.0 wt %), the obtained SAXS data indicate the formation of a dispersion with an internal biphasic nanostructure in which traces of the H2 phase coexist with an inverted-type water-in-oil (W/O) microemulsion that is characterized by a single broad peak in the observed SAXS pattern (data not shown). For the GMO-based systems, the experimental findings demonstrate that citrem has a significantly different effect on the internal nanostructures to what is seen above when PHYT is dispersed in excess water. The formation of aqueous dispersions with nanostructures that are shrinking upon loading with citrem is believed to be related to the structural molecular resemblance of citrem with GMO as seen in Figure 1. Citrem is an oil-soluble emulsifier composed of citric acid esters of monoglycerides. Previous studies by Berton et al.63,66 have shown the ability of citrem to stabilize oil-in-water (O/W) emulsions efficiently. It was found that citrem was almost lacking in the aqueous phase, which can be explained by its low water solubility.63 In these O/W emulsions, it was suggested that the charge of the emulsifying agent has a considerable effect on the oxidative stability because it governs attractive or repulsive forces between the interfaces.66

In addition to citrem’s function as a stabilizer, the obtained results suggest that a considerable amount of this charged stabilizer penetrates the hydrophobic regions of the internal nanostructure and behaves as a guest hydrophobic agent because of its high affinity for GMO. Citrem is an anionic stabilizer, and GMO is a nonionic lipid; therefore, the repulsive forces among charged citrem molecules at the neutral lipid− water interface are expected to affect the internal nanostructures of the dispersions, as described above for the PHYT-based dispersions. However, in the present study there is no indication of the incorporation of citrem molecules into the neutral GMO−water interfacial film. This is believed to be related to the fact that citrem has a higher affinity for GMO than for PHYT. In Table 2, the lattice parameters of the GMO-based fully hydrated nondispersed citrem-loaded samples at 37 °C are presented. The citrem content in these samples is varied between 1.0 and 5.0 wt %, and the total lipid concentration is varied between 45.0 and 49.0 wt %. The phase transition from the cubic Pn3m phase to the H2 phase that is seen for the internal nanostructures of the GMO-based dispersed samples upon loading with citrem is not evident for these fully hydrated samples. This is due to the fact that the fully hydrated samples are investigated at a lower citrem content than that used in the preparation of the aqueous dispersions. The dispersions have a 11761


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citrem-to-GMO ratio that varies between 0.5 to 2.0 whereas the ratio for the fully hydrated samples ranges from 0.02 to 0.11. It is still evident that loading citrem at relatively low concentrations affects the nanostructures under full hydration conditions. From the lattice parameters presented in Table 2, it is evident that loading the samples with citrem causes a decrease in the lattice parameter (shrinking the self-assembled nanostructure). The lattice parameter of the cubic Pn3m phase at 37 °C decreases from 84.0 (0 wt % citrem, sample 1 in Table 2) to 80.9 Å (5.0 wt % citrem, sample 23 in Table 2). The data for the dispersed samples show the ability of citrem to induce a phase transition in the sequence of cubic Pn3m → H2 (i.e., hexosomes are formed as the citrem content is varied in the range of 2.0−3.0 wt % (Table 2)). Briefly, the presence of citrem at relatively high concentrations in the aqueous dispersions causes an increase in the CPP and promotes the formation of nanostructures with a more negative spontaneous curvature. The particle size and zeta potential of a GMO-based dispersed sample containing 2.0 wt % citrem (sample 2, Table 2) were measured at different time intervals within 3 weeks of incubation. The mean value for the particle size is about 227 nm whereas the mean zeta potential is strongly negative with a value of −30 (±1.5) mV. The investigated dispersion remains stable during at least 3 weeks of storage at room temperature without any significant alteration in the mean values of the particle size and zeta potential. It is noteworthy that previous studies also demonstrated strongly negative zeta potential values for food-grade oil-in-water (O/ W) emulsions stabilized by citrem.83,84 3.4. MCT-Loaded GMO-Based Aqueous Dispersions. In Figure 6a, the SAXS patterns of the GMO/MCT-based dispersions are shown. These experiments were performed at 25 °C as the solubilized oil content was kept constant at 10.0 wt % (a binary GMO/MCT mixture with a lipid-to-oil weight ratio of 90/10) and the citrem content was varied from 1.5 to 3.25 wt %. In the absence of citrem, the lattice parameter of the fully hydrated, nondispersed H2 sample is 60.0 Å (sample 7 in Table 2). Upon loading of the GMO-based dispersed particles with MCT, the SAXS patterns indicate the formation of hexosomes with an internal H2 phase. The oil-loaded GMObased dispersions display the same behavior as seen for the oilloaded PHYT-based aqueous dispersions. Slight increases in the corresponding lattice parameter to 62.6 and 63.9 Å (samples 8 and 9 in Table 2) are obtained as the citrem concentration is increased from 1.5 to 2.6 wt % citrem, respectively. In addition to the effect of loading citrem on the internal nanostructure, it is also possible that a larger fraction of citrem adheres to the outer surface of the dispersed particles and induces a decrease in their size as more molecules of citrem are needed for the stabilization of relatively smaller particles. It is surprising to see that citrem has a very different effect than that found in the oil-free GMO-based aqueous dispersions (Table 2). This difference in behavior between the MCT-free and MCT-loaded aqueous dispersions can be attributed to the localization of MCT in the hydrophobic regions of the internal nanostructure. It seems that MCT forces some of the citrem molecules to migrate from the hydrophobic environment in the internal nanostructure to the interfacial lipid−water area with an overall effect similar to that observed in the internal nanostructures of the oil-loaded and oil-free aqueous dispersions of PHYT. At a higher citrem content (3.25 wt %, sample 10 in Table 2), it is clear that the internal H2 phase

Figure 6. Comparison of the SAXS scattering patterns of GMO/ MCT-based aqueous dispersions and the corresponding fully hydrated bulk samples at two different temperatures: (A) 25 and (B) 37 °C. The dispersed and nondispersed phases were prepared with a binary GMO/MCT mixture at a weight ratio of 90/10. The intensities have been shifted by a constant arbitrary factor for better visibility.

starts to vanish and an internal W/O microemulsion with a d value of 57.5 Å is evolved. The oil-induced hexosomes−EME transition is consistent with previous studies on the effect of solubilizing oil on the internal nanostructures of aqueous dispersions of monoglycerides.48,53,79 It was reported that a transition from cubosomes via hexosomes to micellar cubosomes and EMEs is enhanced by augmenting the solubilized oil content.48,53,79 Figure 4b shows a cryo-TEM image of an MCT-loaded aqueous dispersion containing 1.5 wt % citrem (sample 8 in Table 2). This MCT-loaded GMO-based dispersion consists of 11762


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4. CONCLUSIONS The present study described the structural characteristics of a series of oil-free and oil-loaded lipid-based nanoparticulate formulations that were stabilized by the food-grade anionic small-molecule emulsifier citrem. The obtained experimental findings demonstrate the enormous influence of citrem on the internal nanostructures of the oil-free and oil-loaded aqueous dispersions based on commercial lipids PHYT and GMO. Citrem has been shown to provide stabilization for the milky dispersions by adhering to the surfaces of the dispersed particles. This anionic stabilizer has also shown a potential for the functionalization of the internal H2 phase in hexosomes and the inverted microemulsion in EMEs. It is particularly interesting to find that the pattern of interaction of citrem with the inner particle nanostructures of the investigated aqueous dispersions of PHYT and oil-loaded GMO is significantly different from that found with the oil-free internal nanostructures based on GMO. The important role of citrem in functionalizing the internal nanostructures of both PHYT and oil-loaded GMO is most likely attributed to a greater degree of penetration of citrem molecules into the water−lipid interfacial area. However, this stabilizer is a chemical derivative of monoglycerides and therefore has a remarkably high degree of affinity for GMO. This explains the preferential interaction of citrem with the hydrophobic tails of GMO with an overall effect on the spontaneous curvature of the internal nanostructure similar to that observed upon the solubilization of oils and poorly water-soluble drugs.48,53,78 Future research activities include investigating the effect of citrem on aqueous dispersions based on model lipids favoring the formation of lamellar phase in order to determine the possibility of preparing cubosomes and also understanding the effect of varying pH on the stabilization of aqueous dispersions of monoglycerides and their internal nanostructures.

particles with a highly ordered internal nanostructure with a size in the range of a few hundred nanometers. Unlike the PHYT-based aqueous dispersion, the MCT-loaded GMObased aqueous dispersion displays a great tendency to adhere to the polymer film. This behavior has previously been seen in other monoglyceride-based aqueous dispersions70 such as monoelaidin (ME)-based cubosomes.21 Figure 6b shows the SAXS patterns of the GMO/MCTbased aqueous dispersions and the corresponding fully hydrated citrem-loaded GMO/MCT-based samples at 37 °C. Two fully hydrated samples containing 2.0 and 5.0 wt % citrem were characterized. The SAXS patterns for both the fully hydrated nondispersed and dispersed samples indicate the formation of an H2 phase. It is evident in this figure that citrem enlarges the hydrophilic nanochannels of the internal nanostructure. The lattice parameter of the internal H2 phase increases from 61.1 to 63.6 Å with an increasing citrem content of 1.5 to 2.6 wt % (samples 8 and 9 in Table 2). The effect of altering the oil content on the MCT-loaded aqueous dispersions prepared with a constant citrem concentration of 1.5 wt % and an oil content in the range of 10−40 wt % was also investigated (Figure S2 in the Supporting Information). With increasing oil content, the obtained results demonstrate a decrease in the lattice parameters of both the dispersed and the nondispersed phases (Table 2). In the presence of citrem, the lattice parameter of the internal H2 phase at 25 °C is subsequently increased from 60.0 to 62.6 Å and from 55.5 to 61.4 Å as the weight ratio of GMO/MCT is decreased from 90/10 to 85/15 (samples 7, 8, 11, and 12 in Table 2). At a higher MCT content (GMO/MCT weight ratio ≥75/25), a structural transition in the internal nanostructure from the H2 phase (hexosomes) to the W/O microemulsion phase (EMEs) is detected (Table 2). The d value of the nondispersed, fully hydrated W/O microemulsions decreases from 49.3 to 48.2 Å with increasing solubilized oil content from 25 to 40 wt %. In the aqueous dispersions, the addition of citrem induces an increase in the hydrophilic core of the internal inverted-type microemulsions. This leads to a significant broadening of the observed single peak of the internal W/O microemulsion (Figure S2 in the Supporting Information), which in turn makes it difficult to calculate an accurate d spacing of the internal microemulsions of these aqueous dispersions. The citrem-induced functionalization of the internal W/O microemulsion is consistent with the effect of citrem in hexosomes as discussed above. As seen in Figures 2, 5, and 6, the SAXS patterns for aqueous dispersions stabilized by citrem indicate a broadening of the second and third peaks of the H2 phase. These results suggest that the inclusion of citrem induces disorder in the internal selfassembled nanostructure of the aqueous dispersions, and this disorder is increased with increasing citrem content. The lower intensity and the broadening of the peaks compared to that of the fully hydrated samples might be attributed to a lower degree of order near the particle surface caused by the formation of a spongelike nanostructure surface phase (as seen in Figure 4a). The increased disorder in the internal nanostructure could also be related to the possible formation of small aggregates of the lipid with citrem, which coexist with the dispersed hexosomal particles. However, the obtained results do not show any indication of the formation of such aggregates. Further studies are needed to understand fully the effect of citrem on the dispersed and nondispersed phases of PHYT and GMO.

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ASSOCIATED CONTENT

* Supporting Information S

SAXS scattering patterns of the GMO-based aqueous dispersions and the corresponding fully hydrated bulk samples prepared with different citrem contents at two different temperatures. Effect of varying the GMO/MCT weight ratio on the internal nanostructures of GMO/MCT-based aqueous dispersions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45 35 33 65 41. Fax: +45 35336030. E-mail: aya@farma. ku.dk. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Tomás S. Plivelic and Ana Labrador (MAX-lab, Lund, Sweden) for their valuable technical support during the synchrotron SAXS experiments. We acknowledge the Danish Agency for Science, Technology and Innovation for the Zetasizer Nano ZS. K.E. gratefully acknowledges financial support from the Swedish Research Council and the Swedish Cancer Society. We also acknowledge the reviewers' comments. 11763


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