Article pubs.acs.org/Langmuir
PEGylation of Phytantriol-Based Lyotropic Liquid Crystalline ParticlesThe Effect of Lipid Composition, PEG Chain Length, and Temperature on the Internal Nanostructure Christa Nilsson,† Jesper Østergaard,† Susan Weng Larsen,† 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 ‡ Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Helsinki 19392, Finland § School of Pharmacy, University of Eastern Finland, Kuopio 70211, Finland ABSTRACT: Poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3phosphoethanolamines (DSPE-mPEGs) are a family of amphiphilic lipopolymers attractive in formulating injectable long-circulating nanoparticulate drug formulations. In addition to long circulating liposomes, there is an interest in developing injectable long-circulating drug nanocarriers based on cubosomes and hexosomes by shielding and coating the dispersed particles enveloping well-defined internal nonlamellar liquid crystalline nanostructures with hydrophilic PEG segments. The present study attempts to shed light on the possible PEGylation of these lipidic nonlamellar liquid crystalline particles by using DSPE-mPEGs with three different block lengths of the hydrophilic PEG segment. The effects of lipid composition, PEG chain length, and temperature on the morphology and internal nanostructure of these self-assembled lipidic aqueous dispersions based on phytantriol (PHYT) were investigated by means of synchrotron small-angle X-ray scattering and Transmission Electron Cryo-Microscopy. The results suggest that the used lipopolymers are incorporated into the water−PHYT interfacial area and induce a significant effect on the internal nanostructures of the dispersed submicrometer-sized particles. The hydrophilic domains of the internal liquid crystalline nanostructures of these aqueous dispersions are f unctionalized, i.e., the hydrophilic nanochannels of the internal cubic Pn3m and Im3m phases are significantly enlarged in the presence of relatively small amounts of the used DSPE-mPEGs. It is evident that the partial replacement of PHYT by these PEGylated lipids could be an attractive approach for the surface modification of cubosomal and hexosomal particles. These PEGylated nanocarriers are particularly attractive in designing injectable cubosomal and hexosomal nanocarriers for loading drugs and/or imaging probes.
1. INTRODUCTION Certain amphiphilic lipids self-assemble upon contact with aqueous environments to form nonlamellar liquid crystalline phases.1−7 Among these liquid crystalline phases, the reverse bicontinuous cubic (V2) and (H2) hexagonal phases1,3,4,6−10 offer various advantages, including the formation of tunable nanostructures with biological relevance, and the capability to incorporate various drugs and imaging agents.4,5,8,11−17 However, these liquid crystalline phases are highly viscous and therefore not suitable as injectable nanocarriers. An interesting approach is the emulsification of these liquid crystalline phases in the presence of a stabilizing agent, most commonly the nonionic polymeric stabilizing agent Pluronic F127, to obtain dispersions consisting of submicrometer-sized particles where the highly ordered internal nanostructures are enveloped.4,8,18−22 The most investigated lipidic particles with internal nonlamellar liquid crystalline nanostructures are cubosomes (aqueous dispersions of the V2 phase) and hexosomes (aqueous dispersions of the H2 phase).4,8,13,17,18,23 © 2014 American Chemical Society
Many of the characteristics of the liquid nonlamellar crystalline phases are preserved, while the emulsification procedure now has rendered them suitable for injection. In recent years, there has been an increasing interest in improving the in vivo stability of cubosomes and hexosomes in biological fluids by means of surface modification of these lipidic nanocarriers with lipopolymers.8,16,17,24−26 By surface modification, the nanocarriers escape recognition and clearance by macrophages from the reticuloendothelial system (RES).27 In a recent study,17 a successful radiolabeling method for PHYT-based hexosomes was introduced. In vivo biodistribution studies of these radiolabeled hexosomes suggested the formation of a depot in the subcutaneous adipose tissue upon subcutaneous (s.c.) injection. A promising noninvasive visualization tool was introduced, which could allow the formation of Received: April 14, 2014 Revised: May 15, 2014 Published: May 15, 2014 6398
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Figure 1. Molecular structures of phytantriol (PHYT); 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy (poly(ethylene glycol))-350] (ammonium salt) (DSPE-mPEG350); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (poly(ethylene glycol))-750] (ammonium salt) (DSPE-mPEG750); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (poly(ethylene glycol))-2000] (ammonium salt) (DSPE-mPEG2000).
conjugation of PEG chains to these phospholipids, which are one of the major phospholipids of biological membranes, amphiphilic molecules with a modified hydrophilic moiety are synthesized. In liposomes, the amphiphilic nature of these PEPEGs allows the penetration of the hydrophobic moiety into the lipid bilayer, while the hydrophilic PEG chain remains in the aqueous environment.30,33,34 The main attention of the present work was to shed light on the possible formation of PEGylated lipidic nanocarriers consisting of internally well-defined self-assembled nonlamellar nanostructures. The effects of PEG chain length, lipid composition, and temperature on the PHYT-based nanocarriers were investigated by means of synchrotron small-angle X-ray scattering (SAXS). Further, investigations of the morphological characteristics were performed by Transmission Electron CryoMicroscopy (cryo-TEM). Figure 1 shows the molecular structure of the investigated lipids.
theranostic cubosomal and hexosomal nanocarriers. However, further investigations are required to develop efficient longcirculating PEGylated cubosomal and hexosomal formulations that can be used as injectable nanocarriers for delivering drugs after intravenous (IV) and subcutaneous (s.c.) administration. Lipopolymers are a family of lipids that have been covalently grafted with hydrophilic and flexible biocompatible polymers for increasing the hydrophilicity degree, and improving the performance in different pharmaceutical and biomedical applications.28−30 They have an amphiphilic core, consisting of the lipid, and a hydrophilic shell comprising the grafted polymer.28,31,32 There is growing interest in the use of lipopolymers in the development of new nanocarriers for targeting and delivery of diagnostic and therapeutic agents.16,17,25,30−33 An attractive strategy for the formation of lipopolymers is to use the biocompatible hydrophilic polymer poly(ethylene glycol) (PEG).30,34,35 PEG was introduced as a surface-modifying agent in the 1990s for improving the pharmacokinetics of IV administered liposomes.33−36 With the introduction of different PEG-chains as surface modifying agents, a reduced immunogenicity as well as an increased blood circulation time has been observed.8,30 PEGylation reduces adsorption of various opsonins, such as complement factors onto the surface of the nanoparticles resulting in an increased biological stability.37,38 PEG can also be used as a linker for the covalent attachment of different active targeting moieties.32,38,39 The most promising PEGylated lipid candidates for biomedical and pharmaceutical applications are PEG-phosphatidylethanolamines (PE-PEGs).30,32,34,40−42 By the covalent
2. MATERIALS AND METHODS 2.1. Materials. Phytantriol (PHYT) with a nominal purity of >96.4% (from product specifications by gas chromatography) was a gift from DSM Nutritional Products Ltd. (Basel, Switzerland). Impurities include a tiny amount of water (about 0.07%), sulfated ash, heavy metals, and a diastereomer of phytantriol (3,7,11,15tetramethyl-1,2,3,4-tetrahydroxyhexadecane), which is a compound that differs from PHYT in that it has a hydroxy group in position 4, resulting in five chiral C atoms, as opposed to 4 chiral C atoms in PHYT. The following phospholipids: 1,2-distearoyl-sn-glycero-3phosphoethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (poly(ethylene glycol))-750] (ammonium 6399
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Table 1. Structure Parameters As Derived from SAXS Investigations on Aqueous Dispersions Based on PHYT or Binary PHYT/ DSPE-mPEGx Mixtures at 25 °C sample no. 1
PHYT/DSPE-mPEGx (weight ratio) 100:0
mean a (Å)
space group Pn3m and H2
2
98:2a
Pn3m; H2 and traces of Im3m
3 4
98:2b 95:5b
Pn3m Pn3m
5
90:10b
Pn3m and Im3m
6 7
98:2c 95:5c
Pn3m Pn3m and Im3m
8 9
90:10c 98:2d
Im3m Pn3m and Im3m
10 11
95:5d 90:10d
Im3m Im3m
65.6 49.4 66.2 48.8 69.1 69.2 73.6 84.9 107.4 70.0 72.4 92.9 105.2 70.7 94.2 95.1 94.5 90.5
(Pn3m) (H2) (Pn3m) (H2) 85.7 (traces of Im3m) (Pn3m) (Pn3m) (Pn3m) (Im3m)
Bonnet ratio aIm3m/aPn3m
∼1.29
∼1.27
(Pn3m) (Im3m)
∼1.28
(Pn3m) (Im3m)
∼1.33
(Im3m) (Im3m)
a The binary lipid mixture is PHYT/DSPE. bThe binary lipid mixture is PHYT/DSPE-mPEG350. cThe binary lipid mixture is PHYT/DSPEmPEG750. dThe binary lipid mixture is PHYT/DSPE-mPEG2000.
characteristic distance. The composition of the investigated samples, the lattice parameters derived from SAXS data analysis, and the identified liquid crystalline phases are presented in Table 1. 2.4. Transmission Electron Cryo-Microscopy (Cryo-TEM). For the cryo-TEM analysis, vitrified samples were prepared as follows: approximately three microliter aliquots of the investigated sample were applied with a pipet onto glow discharged Lacy 300 holey carbon film grids (Ted Pella Inc., California, U.S.A.) maintained at ambient temperature and 100% relative humidity in a fully automated controlled environment vitrification device (Vitrobot, FEI, Eindhoven, The Netherlands). After automatic blotting of the grid inside the vitrification device, the grids were vitrified by rapid plunging into liquid-nitrogen cooled ethane. A Gatan 626 cryoholder (Gatan Inc., Warrendale, U.S.A.) was used to observe the samples in an FEI Tecnai G2 transmission electron microscope (FEI, Eindhoven, The Netherlands) at 200 kV under low-dose conditions (99.0% was obtained from Avanti Polar Lipids, Inc. (Alabaster, U.S.A.). Pluronic F127 was a gift from BASF SE (Ludwigshafen, Germany). Phosphate buffer solution (PBS) with a concentration of 67 mM at pH 7.4 was used. All ingredients were used without further purification. 2.2. Preparation of PHYT- and PHYT/DSPE-mPEGx-Based Aqueous Dispersions. The lipidic samples were prepared by heating PHYT at 50 °C. After the addition of DSPE-mPEG350, DSPEmPEG750, or DSPE-mPEG2000 at appropriate concentrations to the melted PHYT, the sample was homogenized by vigorous vortexing and then the polymeric stabilizing agent F127 was added. The emulsification of the aqueous dispersions was done in excess phosphate buffered saline (PBS, pH 7.4, 67.0 mM) using the ultrasonic processor Sonics Vibracell VCX 130 (Sonics & Materials Inc., Newton, Connecticut, U.S.A.) for 4−6 min in pulse mode (7 s pulses interrupted by 3 s breaks) at 80% of its maximum power. The formed milky aqueous dispersions were prepared with fixed total F127 and lipid concentrations of 1 and 10 wt %, respectively. 2.3. Synchrotron Small Angle X-ray Scattering (SAXS) and Data Analysis. X-ray measurements were performed at the beamline I911-SAXS (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 single-photon-counting hybrid pixel detector (Pilatus 1M, Dectris Ltd., Baden, Switzerland) using collection time of 120 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 0.001 to 0.6 Å−1. Silver behenate (CH3(CH2)20COOAg with a d spacing value of 58.4 Å) was used as a standard to calibrate the angular scale of the measured intensity. The samples were measured in custom-made glass capillaries. The measurements were performed at different temperatures, and the temperature was controlled (±0.1 °C) by the aid of a Peltier element. The lattice parameters of the V2 phases of the symmetries Pn3m and Im3m, and the H2 phase were deduced from the reflections with strong intensity applying standard procedures. The scattering profile of the reverse micellar (L2) phase is characterized by a single broad peak. For these phases, d = 2π/q is called the
3. RESULTS AND DISCUSSION 3.1. PEGylation of PHYT-Based Nanostructured Aqueous Dispersions: Effects of Lipid Concentration and PEG Chain Length. The internal nanostructures of different aqueous dispersions of the binary PHYT/PEGylated DSPE mixtures were investigated by synchrotron SAXS at 25 °C. The main focus in the present work is on the PEGylation of cubosomes and hexosomes, and therefore, possible structural transitions to lamellar or normal micellar solutions at high PEGylated DSPEs content are not discussed. Figure 2 presents the obtained SAXS patterns taken from the prepared dispersions. In the absence of the lipopolymer, the SAXS pattern taken from the PHYT-based dispersion displays six peaks characteristic for the bicontinuous cubic phase of the diamond type (V2, the space group Pn3m) with a lattice parameter of 65.6 Å (Table 1, sample 1). This is in good agreement with the previous findings by Dong et al.43 They 6400
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found to be 49.4 Å (Table 1, sample 1). The coexistence of the cubic Pn3m phase with the H2 phase has previously been reported and discussed in ref 17. Here it was stated that the coexistence of the cubic Pn3m phase with the H2 phase is most likely attributed to the presence of small amounts of impurities in the used lipid. In this context, it was reported that the phase behavior of the lyotropic liquid crystalline phases based on PHYT, which is an amphiphilic compound with a propensity to form nonlamellar phases at ambient temperatures,7 is sensitive to the presence of impurities such as 3,7,11,15-tetramethyl1,2,3,4-tetrahydroxyhexadecane. The presence of small amounts of impurities could thus have a significant effect on the phase transition temperatures.44 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) is a saturated phospholipid that forms lamellar and nonlamellar phases in the presence of water.45,46 The fully hydrated DSPE has been found to form one of two different phases at low temperature, either a metastable lamellar gel (Lβ) or a crystalline subgel (Lc) phase. Upon increasing temperature, the fully hydrated DSPE undergoes structural transitions in the following sequence: a metastable Lβ phase → a mixture of the fluid lamellar (Lα) phase and the Lc phase → Lα → H2 phase.45,46 In the presence of a small amount of DSPE (sample 2 in Table 1), the SAXS pattern taken from the aqueous PHYT/DSPE dispersion still shows six peaks that are compatible with the cubic Pn3m phase (spacing ratios of √2:√3:√4:√6:√8:√9) as well as three peaks that are compatible with the H2 phase (Figure 2A). In addition, a weak peak indicating the formation of a new phase is observed at qvalue of approximately 103.7 Å−1. This detected peak most likely indicates the formation of traces of an additional bicontinuous cubic phase of the primitive type (V2, space group Im3m). Indexing of this newly formed phase is based on SAXS experiments performed on few PHYT/DSPE-based aqueous dispersions formed at different PHYT/DSPE weight ratios (data not shown). The characteristic peaks of the cubic Pn3m phase are very slightly shifted to lower q values with a subsequent increase in the structure parameter, from 65.6 to 66.2 Å. The minimal surfaces of the cubic Pn3m and the cubic Im3m curved bilayers in excess water should have the same averaged Gaussian curvature under equilibrium conditions, i.e., the average radii of curvature should remain the same throughout the phase transition.47−49 Hence, the Bonnet (aIm3m/aPn3m) ratio between the lattice parameters of the coexisting cubic phases was calculated. It was approximately 1.29 (Table 1), which is close to the theoretical value of 1.279.47 The calculated Bonnet values in the present work are furthermore consistent with those reported in the literature for different lipid/water systems.48,50,51 It was reported that different long circulating liposomes containing DSPE-mPEGs at various lipid composition and PEG chain lengths are stable without using a stabilizing agent. In this study, stabile dispersions were however not obtained by merely replacing a part of PHYT with different DSPE-mPEGs but required the introduction of the stabilizing agent Pluronic F127. In Figure 2A, the effect of the partial replacement of PHYT with a small amount of DSPE-mPEG on the internal nanostructure of the aqueous dispersions (binary PHYT/ DSPE-mPEGx mixture prepared at a fixed weight ratio of 98/2) is shown. The lattice parameter of the cubic Pn3m phase increases from 65.6 to 69.1 Å in the presence of DSPEmPEG350 (Table 1, sample 3). The hydrophilic nanochannels of the V2 phase are thus f unctionalized, i.e., the hydrophilic
Figure 2. Effects of the lipopolymer concentration and its PEG chain length on the internal nanostructure of binary PHYT/DSPE-mPEGx aqueous dispersions. The SAXS experiments were performed at 25 °C on nanostructured aqueous dispersions with the following binary lipid composition: PHYT/DSPE-mPEGx at weight ratios of 98:2 (A); 95:5 (B); and 90:10 (C).
reported a formation of cubosomes with internal cubic Pn3m phase having a lattice parameter of 68.6 Å. As seen in both Figure 2A and in Table 1, this internal bicontinuous cubic Pn3m phase coexists with an inverse type hexagonal (H2) phase. The assignment of the H2 phase is based on the appearance of the three characteristic reflections at the spacing ratios of √1:√3:√4. The lattice parameter for this phase is 6401
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Figure 3. Cryo-TEM images of the aqueous dispersions based on the binary PHYT/DSPE-mPEG750 (A−C) and PHYT/DSPE-mPEG2000 (D−F) mixtures after vitrification at ambient temperature. The total lipid content is 10 wt % for all investigated samples. The micrographs display nanoparticles based on the binary PHYT/DSPE-mPEG750 and PHYT/DSPE-mPEG2000 mixtures formed at the following weight ratios: 98:2 (panels A and D); 95:5 (panels B and E); and 90:10 (panels C and F), respectively. Scale bar: 100 nm (A−C) and 200 nm (D−F).
nanochannels of the internal cubic Pn3m phase are significantly enlarged in the presence of the used DSPE-mPEG. This enlargement of the hydrophilic nanochannels can enhance the solubilization of hydrophilic guest materials, such as drugs or imaging agents in the internal nanostructures of the lipidic aqueous dispersions. When increasing the concentration of the DSPE-mPEG350 in the binary PHYT/DSPE-mPEG350 mixture from 2 to 5 wt %, there is no significant change in the lattice parameter for the already existing cubic Pn3m phase (Figure 2 and Table 1). However, an additional cubic Pn3m phase with a lattice parameter of 73.6 Å (Table 1, sample 4) appears with increasing DSPE-mPEG350 concentration. Thus, the SAXS data indicate the formation of an aqueous dispersion enveloping two coexisting cubic Pn3m phases at a PHYT/ DSPE-mPEG350 weight ratio of 95/5. The enlargement of the hydrophilic nanochannels in both nonlamellar phases suggests that the lipopolymer is incorporated into the water−PHYT neutral interfacial area. The steric repulsion between the hydrophilic PEG segments of the lipopolymer causes an increase in the headgroup area and promotes the solubilization of a higher amount of water in the hydrophilic domains of the internal cubic Pn3m phase. Upon further increase of DSPEmPEG350 concentration in the aqueous dispersion from 5 to 10 wt % lipid, a structural transition is induced causing the destabilization of one of the cubic Pn3m phases and the evolvement of a new cubic Im3m phase with a lattice parameter of 107.4 Å due to the significant effect of DSPE-mPEG350 on the internal nanostructure. The incorporation of DSPEmPEG350 into the water−PHYT interfacial area increases the amount of solubilized water in the hydrophilic nanochannels of the liquid crystalline phase (Table 1, sample 5). The calculated
Bonnet ratio is approximately 1.27, which agrees well with the theoretical value.47 The phase transition from the cubic Pn3m phase into the cubic Im3m phase can be attributed to the fact that there is a limit to the ability of the cubic Pn3m phase to swell water while still retaining the internal nanostructural arrangement. The SAXS pattern for the binary PHYT/DSPE-mPEG750 sample at a weight ratio of 98:2 shows peaks that are compatible with the first six reflections of the cubic Pn3m phase with a lattice parameter of 70.0 Å (Table 1, sample 6). Thus, increasing the PEG chain length (the replacement of DSPEmPEG350 by DSPE-mPEG750) enhances a slight increase in the lattice parameter of the internal nanostructure of the dispersion from 69.1 to 70.0 Å. The SAXS pattern for the binary PHYT/ DSPE-mPEG2000 aqueous dispersion at a 98:2 weight ratio, however, shows a coexistence of two cubic phases: cubic Pn3m and Im3m phases with lattice parameters of 70.7 and 94.2 Å, respectively (Table 1, sample 9). Increasing DSPE-mPEG750 concentration to 5 wt % in the binary lipid mixture (Figure 2B) leads to an increase in the lattice parameter of the cubic Pn3m phase to 72.4 Å (Table 1, sample 7). In addition, a newly formed phase displaying the first three characteristic peaks of a cubic Im3m phase with a lattice parameter of 92.9 Å is present in the sample. The SAXS pattern for the dispersion based on the binary PHYT/DSPEmPEG2000 at a 95:5 weight ratio displays peaks characteristic for a cubic Im3m phase with lattice parameter of 95.1 Å (Table 1, sample 10). It is also evident that the inclusion of DSPEmPEG2000 consisting of 45 monomeric EO units results in a significant increase in amount of solubilized water in the hydrophilic nanochannels and leads to the formation of a neat cubic Im3m phase. 6402
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monoglycerides.58,59 The coexistence of particles of highly ordered inner H2 phase with vesicles was also observed by cryoTEM in aqueous dispersions consisting of dioleoylphosphatidylethanolamine (DOPE) and a small amount of DSPEmPEG750.42 At the lowest content of the lipopolymer (PHYT/ DSPE-mPEG750 98/2, Figure 3A), there is no significant visible morphological change of the dispersed particles as compared to the PHYT-based aqueous dispersion (results not shown). In Figure 3B,C, the DSPE-mPEG750 concentration is further increased to 5 and 10 wt %, respectively. The effect of the lipopolymer on the morphology is not detectable in the micrographs. But the cryo-TEM observations indicate the formation of more vesicles due to the tendency of DSPEmPEG750 to form bilayers at moderate concentrations.40 Furthermore, the aqueous dispersions based on the binary PHYT/DSPE-mPEG2000 dispersions (samples 9−11, Table 1) were characterized. As for the binary PHYT/DSPE-mPEG750 samples, the micrographs for the PHYT/DSPE-PEG2000 (Figure 3D−F) comprise submicrometer-sized particles in the range of a few hundred nanometers, which envelope highly ordered inner nanostructures at the compositions of 98:2 (Figure 3D) and 95:5 (Figure 3E). However, the particles do not display the same highly ordered internal nanostructure with a further increase in DSPE-PEG2000 content (Figure 3F). It is evident from the cryo-TEM observations that the partial replacement of PHYT by a relatively large amount of DSPEmPEG2000 (binary PHYT/DSPE-mPEG20000 mixture at a weight ratio of 90:10) induces a significant effect on the dispersed lipidic particles (Figure 3E). Clearly, there is considerable increase in the formation of vesicles that adhere to the outer surface of the dispersed particles. The internal nanostructure is surrounded by a less dense shell comprising vesicular-like structures, a behavior that also has been observed for different nanoparticulate systems prepared in the presence of normal micelle- or lamellar-prone lipids.56 In a recent report,56 citrem-based nanoparticles formed in the presence of MO-PEG460 or MO-PEG860 were investigated. The cryo-TEM observations of the dispersions revealed submicrometer-sized nanoparticles surrounded by a less dense shell comprising vesicular-like structures. It was stated that the shell was enriched with PEGylated MO, and the dense inner core was most likely enriched with citrem. In the present study, the behavior is similar, with the dense inner core most likely being enriched with PHYT and the shell being enriched in DSPEmPEG2000. Even though the SAXS results for this sample display the coexistence of two cubic Im3m phases (Table 1, sample 11), the development of the ordered inner nanostructure might be dependent on the size of the dispersed particles as well as the time that has passed from production of the sample until it is investigated by cryo-TEM, as reported by Johnsson and Edwards.42 In SAXS experiments, it is not surprising that the vesicles are not well detected due to the weak signal, compared to that of the nonlamellar liquid crystalline particles. It is most likely that the arising weak signal is due to the possible formation of vesicles containing small amounts of lipids. 3.3. Influence of Temperature. To obtain a general understanding of the effect of temperature on these binary PHYT/DSPE-mPEGx dispersions, the effect of increasing temperature was investigated on four different PHYT-based aqueous dispersions. The samples were investigated in the temperature range of 25−60 °C. A few representative SAXS patterns for the investigated samples are displayed in Figure 4.
In Figure 2C the SAXS patterns for dispersions prepared at a binary PHYT/DSPE-mPEGx composition of 90:10 are displayed. Upon the increase of the amount of monomeric units from 7 (DSPE-mPEG350) to 16 (DSPE-mPEG750), the SAXS pattern displays peaks characteristic for a cubic Im3m phase with corresponding lattice parameter of 105.2 Å (Table 1, sample 8). In addition, further increase in the amount of EO units from 16 to 45 (DSPE-mPEG2000), gives rise to the coexistence of two cubic Im3m phases with lattice parameters of 94.5 and 90.5 Å, respectively. The critical packing parameter (CPP) or the wedge shape factor is useful in predicting the formed self-assembled nanostructures in water.52 The CPP value relates molecular parameters (headgroup area, chain length, and hydrophobic tail volume) and intensive variables (temperature, ionic strength, level of hydration, and so forth) to surfactant molecular shape.1,52−54 It is described as follows:52
CPP = vs/a0l
(1)
where vs is the volume of the hydrophobic chain, a0 is the effective area of the headgroup, and l is the length of the hydrophobic chain.52 The effect of PEGylation on the CPPvalue of surfactant-like lipids has recently been reported.22,24,25,55,56 As described in a recent report by Hedegaard et al.,56 the covalent conjugation of PEG with Mn ≈ 460 and Mn ≈ 860 to MO was used to obtain more hydrophilic surfactants in order to be able to tune the selfassembled nanostructure from inverted (type II) to normal (type I). The mixing of lipids favoring the formation of nonlamellar phases with lipids favoring the formation of planar bilayers has proven to be a very effective strategy for controlling the self-assembled nanostructure.48,49,51,55 It is possible to convert DSPE to a more hydrophilic surfactant by grafting a hydrophilic segment such as PEG.57 For instance, the increased hydrophilicity inflicts the formation of normal spherical micelles (L1 phase) upon mixing DSPEmPEG2000 in water.37,57 The contribution of grafting hydrophilic PEG to the molecular structure of the amphiphilic lipid DSPE is thus dominating. It was stated in a previous report by Kenworthy et al.40 that DSPE-mPEG350 is more cylindrical than DSPE-mPEG2000 and therefore its binary mixture with distearoylphosphatidylcholine (DSPC) tends to form bilayers in water; whereas DSPEmPEG750 has an intermediate shape, forming bilayers with DSPC at moderate concentrations and mixed micelles at higher concentrations. 3.2. Cryo-TEM Investigations of Binary PHYT/DSPEmPEG Aqueous Dispersions. Morphological investigations were carried out for a few selected aqueous dispersions of the binary PHYT/DSPE-mPEGx mixture where x corresponds to PEG molecular weights of either 750 or 2000 Da, and the results are displayed in Figure 3. In Figure 3A−C, the micrographs for the binary PHYT/ DSPE-mPEG750 samples at three different lipid/lipopolymer weight ratios (samples 6−8, Table 1) are given. All micrographs show the formation of particles enveloping highly ordered internal nanostructures (defined as cubosomes by the SAXS investigations) with a size in the range of a few hundreds of nanometers. In addition, the micrographs show the coexistence of particles enveloping dense and highly ordered inner structures with empty vesicles. This coexistence has previously been observed in other cryo-TEM studies where F127 was used as a stabilizing agent for aqueous dispersions based on 6403
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Figure 5 shows the influence of increasing temperature on the lattice parameters of the internal nanostructures enveloped in different aqueous dispersions. As seen from Figure 5, the partial replacement of PHYT by DSPE-mPEGx leads to the formation of cubosomes with an internal cubic phase of the Pn3m or Im3m symmetry at different temperatures. This is of great interest when it comes to the development of thermally stable cubosomal nano-
particles. Generally, the lattice parameter of the internal nonlamellar liquid crystalline nanostructure in PHYT/DSPEmPEGx dispersions decreases with increasing temperature, as discussed below in detail. In the absence of DSPE-mPEGx, the PHYT-based dispersion consists of dispersed particles enveloping an internal cubic Pn3m phase with lattice parameter of 65.6 Å that coexists with traces of H2 phase having a lattice parameter of 49.4 Å at 25 °C (sample 1, Table 1). The appearance of traces of H2 phase is attributed most likely to the presence of small amounts of impurities in the used lipid, as described above in section 3.1. The lattice parameters of both phases decrease as the sample is heated from 25 to 50 °C. A transition to an internal neat inverse type micellar (L2) phase with a characteristic distance of 37.6 Å is detected at 55 °C. Heating this sample induces an increase in the CPP value and therefore the formation of selfassembled nanostructures with a more negative spontaneous curvature is favored, leading to a structural transition in the following sequence: cubic Pn3m + H2 → L2 (as seen in Figure 5). The results are in a good agreement with previous studies.7,22,43 For instance, Barauskas et al.7 investigated the effect of temperature on the fully hydrated PHYT system and reported a transition to the L2 phase at about 55−60 °C; whereas Fraser et al.22 investigated the ternary PHYT/F127/ water system at different temperatures and reported a transition from cubic Pn3m to L2 phase at a temperature of 60 °C. For the binary PHYT/DSPE-mPEG350 at a weight ratio of 95/5 (sample 4, Table 1), the two cubic Pn3m phases are retained with a subsequent decrease in lattice parameter upon increasing temperatures. Two new coexisting phases are formed at 50 °C, as seen in Figure 4A. The new coexisting phases are the cubic Im3m phase and the H2 phase, respectively. As seen from Figure 5, the lattice parameters for the newly formed phases slightly decrease with increasing temperature, with all four phases still present at 65 °C. For the binary PHYT/DSPEmPEG350 at a weight ratio of 90:10 (sample 5, Table 1) the two cubic phases of the Pn3m and Im3m symmetries are retained with a subsequent decrease in lattice parameter upon increasing temperatures. A new coexisting H2 phase is formed at 55 °C, as seen in Figure 4B. When investigating the effect of temperature on the binary PHYT/DSPE-mPEG750 dispersion at a composition of 90:10 (sample 8, Table 1 and Figure 4C), a single cubic Im3m phase is present at 25−50 °C. At 55 °C, an additional H2 phase is detected. It is clear that increasing the temperature from 25 to 55 °C induces a phase transition in the following sequence: cubic Im3m → cubic Im3m + H2. The significant influence of temperature on the internal nanostructures of dispersions based on the binary PHYT/ DSPE-mPEGx mixtures is attributed to a dehydration of the hydrophilic head groups of the PHYT and DSPE-mPEGs (a decrease in a0 value). This, in turn, leads to an increase in the CPP value, and therefore the lipids tend to self-assemble into nanostructures with a more negative spontaneous curvature (the formation of discontinuous H2 and L2 phases).
Figure 4. Temperature dependence of the lattice parameter of the internal liquid crystalline nanostructure for a selection of binary PHYT/DSPE-mPEGx samples. The temperature effect was investigated in the range of 25−60 °C. (A) The binary PHYT/DSPEmPEG350 sample at a weight ratio of 95:5; (B) the binary PHYT/ DSPE-mPEG350 sample at a weight ratio of 90:10; and (C) the binary PHYT/DSPE-mPEG750 sample with a weight ratio of 90:10.
4. CONCLUSIONS We report on the formation and structural characterization of lipopolymer-containing cubosomes based on PHYT. The used lipopolymers are DSPE-mPEGs with three different block lengths of the hydrophilic PEG segment. The effects of both the partial replacement of PHYT with DSPE-mPEG and 6404
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Figure 5. Temperature dependence of the lattice parameter, a (Å), of the internal liquid crystalline nanostructure of aqueous dispersions based on PHYT or selected binary PHYT/DSPE-mPEGx mixtures. The investigated temperature was in the range of 25−60 °C. For the PHYT-based sample, the temperature range was 25−55 °C. (A) Variation in the lattice parameters as a function of temperature for the PHYT-based sample (black), the binary PHYT/DSPE-mPEG350 sample with a weight ratio of 95:5 (pink), and the binary PHYT/DSPE-mPEG350 sample with a weight ratio of 90:10 (turquoise); and (B) lattice parameters for the binary PHYT/DSPE-mPEG750 sample with a fixed weight ratio of 90:10. The data were extracted from the SAXS experiments displayed in Figure 4. Both full and open triangles represent the internal cubic Pn3m phases; whereas full circles represent the cubic Im3m phase, and the full squares represent the H2 phase.
temperature on the internal nanostructures of the aqueous dispersions were investigated. It is evident from the results obtained by SAXS measurements and cryo-TEM investigations that the introduction of a lipopolymer into the PHYT-based aqueous dispersion f unctionalizes the internal cubic Pn3m and Im3m phases. This effect is ascribed to the considerable extent of incorporation of the investigated lipopolymer into the water−PHYT interfacial area. Its hydrophilic segments extend into the hydrophilic nanochannels of the internal liquid crystalline nanostructures. The effect of f unctionalization is dependent on the PEG chain length as well as the lipid composition. Clearly, longer PEG chains as well as higher lipopolymer content result in a more pronounced effect on the enlargement of the hydrophilic nanochannels of the internal liquid crystalline nanostructures of these dispersions. The incorporation of DSPE-mPEG into the
internal liquid crystalline nanostructure leads to a significant increase in the solubilized water content and enhances the stabilization of cubosomes at different temperatures. Finally, the partial replacement of PHYT by the investigated DSPE-mPEGs and the formation of PEGylated cubosomes could be attractive for the formation of injectable longcirculating drug nanocarriers and the solubilization of various drugs.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +45 35 33 65 41; fax: +45 35 33 60 30; e-mail: anan.
[email protected]. Notes
The authors declare no competing financial interest. 6405
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ACKNOWLEDGMENTS We thank Ramon Liebrechts and Michael Larsen at the Core Facility for Integrated Microscopy (CFIM, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark) for their valuable technical support with the cryo-TEM investigations. We further thank Sylvio Haas and Tomás S. Plivelic (MAX-lab, Lund University, Lund, Sweden) for their valuable technical support during the synchrotron SAXS beamtime. The research leading to these SAXS results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) CALIPSO under Grant Agreement No. 312284. C.N. would like to thank Esben Falk (Lambert Christensen ApS, Esbjerg, Denmark) for help with obtaining phytantriol. A.Y. thanks the Danish Natural Sciences Research Council (DanScatt) for the financial support of the SAXS experiments.
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