Manipulating the Ordered Nanostructure of Self ... - ACS Publications

Article Cite This: Langmuir 2018, 34, 2764−2773

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Manipulating the Ordered Nanostructure of Self-Assembled Monoolein and Phytantriol Nanoparticles with Unsaturated Fatty Acids Nhiem Tran,*,†,‡,⊥ Xavier Mulet,*,‡ Adrian M. Hawley,§ Celesta Fong,†,‡ Jiali Zhai,† Tu C. Le,∥ Julian Ratcliffe,‡ and Calum J. Drummond*,† †

School of Science and ∥School of Engineering, RMIT University, Melbourne, Victoria 3001, Australia ‡ CSIRO Manufacturing, Clayton, Victoria 3149, Australia § Australian Synchrotron, ANSTO, Clayton, Victoria 3149, Australia S Supporting Information *

ABSTRACT: Mesophase structures of self-assembled lyotropic liquid crystalline nanoparticles are important factors that directly influence their ability to encapsulate and release drugs and their biological activities. However, it is difficult to predict and precisely control the mesophase behavior of these materials, especially in complex systems with several components. In this study, we report the controlled manipulation of mesophase structures of monoolein (MO) and phytantriol (PHYT) nanoparticles by adding unsaturated fatty acids (FAs). By using high throughput formulation and small-angle X-ray scattering characterization methods, the effects of FAs chain length, cis−trans isomerism, double bond location, and level of chain unsaturation on self-assembled systems are determined. Additionally, the influence of temperature on the phase behavior of these nanoparticles is analyzed. We found that in general, the addition of unsaturated FAs to MO and PHYT induces the formation of mesophases with higher Gaussian surface curvatures. As a result, a rich variety of lipid polymorphs are found to correspond with the increasing amounts of FAs. These phases include inverse bicontinuous cubic, inverse hexagonal, and discrete micellar cubic phases and microemulsion. However, there are substantial differences between the phase behavior of nanoparticles with trans FA, cis FAs with one double bond, and cis FAs with multiple double bonds. Therefore, the material library produced in this study will assist the selection and development of nanoparticle-based drug delivery systems with desired mesophase.

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INTRODUCTION Self-assembled lyotropic liquid crystalline nanoparticles have a great potential as drug delivery systems because of their customizable internal nanostructures and functionalizable surfaces. Their mesoporous internal structures have allowed for the encapsulation of a wide range of hydrophobic and hydrophilic compounds, including chemotherapeutic drugs, antimicrobial agents, proteins, peptides, and nucleic acids.1−3 Previous studies have demonstrated that the nanostructures directly influence the release rate of drugs and biomolecules.4−11 For example, the release rate of hydrophilic compounds was found to be much faster in the cubic phase than in the hexagonal phase, micellar cubic phase, and microemulsion.12 Additionally, it has been shown that the internal nanostructures also affect cellular response such as cell uptake of nanoparticles, hemolysis, and cytotoxicity.13−17 More importantly, the in vivo behavior of nanoparticles such as biodistribution appears to be regulated by their nanostructures.18 Consequently, the phase behavior of the self-assembled lipid nanoparticles and the ability to tune it has been the subject © 2018 American Chemical Society

of intense research interest to achieve drug delivery systems with desirable physical and biomedical properties. Among the studied lipids, monoolein (MO) and phytantriol (PHYT) have received high-level attention because of their biocompatibility and propensity to form nonlamellar phases in water.19−22 Depending on the environmental conditions and presence of additives, these lipids adopt various polymorphs including fluid lamellar phase (Lα), inverse bicontinuous cubic phase of diamond (QDII ) and primitive (QPII) symmetry, inverse hexagonal (H2) phase, micellar cubic phase (I2), and microemulsions (L2).23,24 One simple factor that provides a strong indication of the mesophase structure is the critical packing parameter (cpp).25 For an amphiphile, the cpp is defined as cpp =

V al

Received: October 10, 2017 Revised: January 29, 2018 Published: January 30, 2018 2764

DOI: 10.1021/acs.langmuir.7b03541 Langmuir 2018, 34, 2764−2773

Article

Langmuir Table 1. Twenty Unsaturated FAs That Were Added to MO and PHYT Nanoparticles to Study Their Phase Behavior sample number

FA

MW

common name

structurea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

trans-9-hexadecenoic acid trans-9-octadecenoic acid trans-11-octadecenoic acid trans-11-eicosenoic acid trans-13-docosenoic acid trans-9,12-octadecadienoic acid cis-9-hexadecenoic acid cis-9-trans-11-octadecadienoic acid cis-11-octadecenoic acid cis-9-octadecenoic acid cis-11-eicosenoic acid cis-13-docosenoic acid cis-15-tetracosenoic acid cis-9,12-octadecadienoic acid cis-9,12,15-octadecatrienoic acid cis-6,9,12-octadecatrienoic acid cis-11,14,17-eicosatrienoic acid cis-5,8,11,14-eicosatetraenoic acid cis-5,8,11,14,17-eicosapentaenoic acid cis-4,7,10,13,16,19-docosahexaenoic acid

254 282 282 311 339 280 254 280 282 282 311 339 367 280 278 278 307 305 302 329

palmitelaidic acid elaidic acid trans vaccenic acid trans gondoic acid brassidic acid linoelaidic acid palmitoleic acid rumenic acid vaccenic acid oleic acid gondoic acid erucic acid nervonic acid linoleic acid α-linolenic acid γ-linolenic acid ETA arachidonic acid EPA DHA

trans-16:1, n − 7 trans-18:1, n − 9 trans-18:1, n − 7 trans-20:1, n − 9 trans-22:1, n − 9 trans-18:2, n − 6 cis-16:1, n − 7 cis,trans-18:2, n − 7 cis-18:1, n − 7 cis-18:1, n − 9 cis-20:1, n − 9 cis-22:1, n − 9 cis-24:1, n − 9 cis-18:2, n − 6 cis-18:3, n − 3 cis-18:3, n − 6 cis-20:3, n − 3 cis-20:4, n − 6 cis-20:5, n − 3 cis-22:6, n − 3

Note: FA structure is described as “cis/trans-a:b, n − c”, where a is the number of carbons in the FA molecule, b is the number of double bonds, and c is the location of the first unsaturated carbon. a

in which V is the effective volume of the hydrophobic part of the amphiphile, a is the effective cross-sectional area of the head group, and l is the effective length of the hydrocarbon chain. As a result, inverse phases with negative Gaussian curvatures are usually formed when cpp > 1, whereas the lamellar phase with a flat bilayer structure has a cpp = 1.25,26 The addition of foreign molecules to an amphiphile system may change the effective cpp of the system and directs the transition into different mesophases.27 Additives such as fatty acids (FAs), oils, cholesterol, vitamin E acetate, and phospholipids have been added to MO and PHYT in both bulk and dispersion forms to modify the nanostructures.28−33 For instance, the addition of an FA to MO nanoparticles at low pH appeared to increases the effective cpp of the system, resulting in mesophases with higher negative curvatures.34−36 It was evident from the phase sequence of MO nanoparticles in the presence of increasing amount of FAs from QPII to QDII , to H2, and to L2. In contrary, doping phospholipids such as DLPC and DSPC with large headgroups to MO nanoparticles reduced effective cpp, which was apparent from a phase sequence of H2 → QDII → QPII → Lα.37,38 Previously, we have used high throughput screening techniques to examine the influence of 12 saturated FAs on the mesophases of MO, PHYT, and monopalmitolein nanoparticles. 39 The data showed that increasing the FA concentration in nanoparticles resulted in phase sequences that preferred mesophases with higher negative curvatures, suggesting elevated effective cpps. It appeared that the FA chain length had a similar effect on nanoparticle mesophases with longer chain FAs encouraging the formation of more negatively curved interfaces. To further investigate the influence of FAs on lipid nanoparticles, herein, we report the phase behavior of MO and PHYT nanoparticles doped with 20 different unsaturated FAs (Table 1). These FAs, which are typically generally regarded as safe, differed in chain length, cis−trans isomerism, double-bond location, and level of chain unsaturation. The

material library created here will be valuable for the design of advanced lipid systems for drug delivery.

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MATERIALS AND METHODS

Materials. MO and 20 unsaturated FAs listed in Table 1 were obtained from Nu-Chek-Prep, Inc (Elysian, MN, USA). PHYT is from TCI (Tokyo, Japan). The purity of MO and FAs is greater than 99% except arachidonic acid, whose purity is over 90%. The purity of PHYT is at least 95%. Pluronic F127 and ethanol were also purchased from Sigma-Aldrich. FAs Selection and Categorization. Among 20 selected unsaturated FAs, 6 of them contain only trans isomers, 13 contain only cis isomers, and 1 contain both cis and trans isomers. Their chemical structures are provided in Figure 1. Nine FAs have more than one double bond with docosahexaenoic acid (DHA) having the highest number of double bonds (six double bonds). It is generally expected that the behavior of trans FAs more closely resembles saturated chain FAs compared to cis FAs.40,41 It is therefore sensible to assume that the cpps of trans FAs are smaller than those of cis FAs. Among trans FAs with similar degree of unsaturation, it is expected that the longer the hydrocarbon chain, the higher the cpp. This assumption is derived from our previous study suggesting that the longer chain saturated FAs triggered phase transition in MO nanoparticles at lower FA concentration than that of shorter chain saturated FAs.39 Among cis FAs, it is also reasonable to assume that FAs with more double bonds will have higher cpp. Considering these assumptions, the FAs are categorized as in Table 1. FAs 1 to 6 contain trans isomers. FAs 7−13 contain cis isomers with single double bond and increasing molecular weight. An exception is 8 with a cis and a trans isomer making it likely to behave similar to 9 and 10 (vaccenic acid and oleic acid). FAs 14−20 contain more than one all-cis double bond with increasing molecular weight and a degree of unsaturation. By arranging the FAs in this order, it is easier to compare the influence of these FAs on MO and PHYT nanoparticle mesophases. Preparation of Nanoparticles. A materials library consisting of 442 unique nanoparticle samples was formulated by using high throughput methods. Briefly, MO, PHYT, and FAs were dissolved in absolute ethanol separately. Lipids and FAs solutions were combined in a well of a 2 mL 96-well master block (Greiner Bio-One, Interpath Inc., VIC Australia). The MO and PHYT weights were fixed at 10 mg per well, whereas the FA weights varied. The ratios rMO and rPHYT are 2765


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1.128 Å (11.0 keV) with a typical flux of approximately 1013 photons/ s. The sample to detector distance (camera length) was chosen as 0.9 m which covers a q-range of 0.01−0.6 Å−1 (scattering vector q = 4π sin(θ)/λ where θ is the scattering angle and λ is the wavelength). Twodimensional X-ray diffraction images were recorded on a DectrisPilatus 1-M detector using in-house IDL-based ScatterBrain software. The scattering images were integrated into one-dimensional plots of intensity versus q for phase identification. A silver behenate standard (d = 58.38 Å) was used for calibration of the camera length. The exposure time for each sample was 1 s. Prepared nanoparticles (100 μL) were loaded into a clear 96-well half-area polystyrene plate (Greiner Bio-One, Interpath Inc., VIC, Australia). The plate was placed in a holder perpendicularly to the Xray beam. The sample holder was moved by motors that were controlled by ScatterBrain software for automatic SAXS screening. The sample temperature was controlled by a circulating water bath. SAXS Data Analysis. Phase identification of self-assembled lipid nanoparticles was based on the relative distance of the Bragg peaks in the scattering profile, which corresponds to the diffraction planes defined by their (hkl) Miller indices. For this, the one-dimensional SAXS profiles were analyzed by an in-house developed RapidPhaseIdent software and an IDL-based AXcess software package.42 These programs identify lyotropic liquid crystalline phases and calculate the lattice parameter (LP) of each lyotropic liquid crystalline phase. LP a was calculated using the equation a = d(h2 + k2 + l2)1/2 for cubic phase or a = d(h2 + k2 + hk)1/2 for hexagonal phase where d is the spacing between the diffraction planes, defined by Bragg’s law d = 2π/q. Cryogenic Transmission Electron Microscopy. Cryogenic transmission electron microscopy (cryo-TEM) was used to visualize the formulated nanoparticles. Copper grids (200 mesh) coated with perforated carbon film (lacey carbon film, ProSciTech, Australia) were glow-discharged in nitrogen to render them hydrophilic and placed in a laboratory-built humidity-controlled vitrification system. Aliquots of samples were applied onto the grids and after 30 s adsorption time, the grids were blotted manually by filter paper for approximately 3 s. The grids were then plunged into liquid ethane cooled by liquid nitrogen. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. At all times, low-dose procedures were followed using an electron dose of 8−10 electrons Å−2 for all imaging. Images were recorded using an FEI Eagle 4k × 4k charge-coupled device camera at magnifications ranging from 15 000× to 50 000×. The cryo-TEM images were analyzed using ImageJ software (NIH).

Figure 1. Chemical structures of the selected unsaturated FAs listed in Table 1. These structures were generated by Chem3D software. defined as the molar ratios of FA to MO and PHYT in the mixture, respectively. The specific compositions of the studied MO and PHYT nanoparticles are provided in the Supporting Information Tables S1 and S2, respectively. Ethanol was then evaporated overnight using a centrifugal evaporator (Genevac, NSW, Australia). Pluronic F127 (500 μL) in Milli-Q water was added to the dried mixture at 10% to MO or PHYT weight. Nanoparticles were produced by sonicating the mixtures for 3 min at the instrument amplitude of 30 using a high throughput 24-tip sonicator (Q700, QSonica, CT, USA). After sonication, the plate was sealed and the nanoparticles were kept at room temperature for further characterization. The presence of homogeneous opaque dispersions without sedimentation confirmed the effectiveness of the F127 steric stabilizer. Small-Angle X-Ray Scattering. Small-angle X-ray scattering (SAXS) experiments were performed at the SAXS/wide-angle X-ray scattering (WAXS) beamline at the Australian Synchrotron (Clayton, VIC, Australia). The instrument used an X-ray of wavelength of λ =

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RESULTS AND DISCUSSIONS In this study, unsaturated FAs were mixed with either MO or PHYT to study their influence on the mesophase structure lipid nanoparticles. The list of 20 unsaturated FAs is given in Table 1. All solutions contain nanoparticles that appeared to be milky white without visible precipitation. This method of preparation has been shown to be successful in creating lipid nanoparticles with a hydrodynamic diameter around 200 nm.16,39 Repre-

Figure 2. Representative cryo-TEM images of cubosomes (A), hexosomes (B), and microemulsions (C) of MO−linoelaidic acid nanoparticles at different acid concentrations. 2766


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SAXS scattering profiles of MO−linoelaidic acid nanoparticles are provided in Figure S2 of the Supporting Information. The FA content in the mixture is represented by rMO, which is defined as the molar ratio of FA to MO. As expected, the addition of unsaturated FAs to MO nanoparticles greatly altered their mesophase structure. It should be noted that the original phase of MO nanoparticles stabilized with Pluronic F127 is the primitive cubic phase QPII. An overall trend observed here is the formation of nanostructures with higher negative curvatures with increasing amount of FAs in the nanoparticles. This result indicates that the addition of unsaturated FAs increases the effective cpp of the system (Figure 4). Specifically, for nanoparticles with increasing amounts of trans FAs, there is an appearance of the QDII phase, replacing the QPII phase of MO. This is followed by a transition into an inverse hexagonal phase H2. For FAs 1 and 4 (palmitoelaidic and trans brassidic acid), a lamellar crystalline phase Lc is found at the highest concentration of FAs. The Lc phase is evident by the spotty lamellar X-ray scattering pattern.39 The phase behavior observed here for trans FA−MO nanoparticles is similar to that of saturated FA−MO systems.39 However, because of the presence of the double bond, there is a permanent kink in the hydrocarbon chain, which enlarges and alters the geometry of the effective tail volume. Consequently, the trans FAs here were incorporated into MO bilayers in a much easier way and at higher amounts compared to saturated FAs of similar chain length. For example, the Lc phase was found in all MO nanoparticles added with palmitic acid (16:0) at an rMO higher than 0.2.39 However, in the case of palmitelaidic acid (trans-16:1), the Lc phase is only found at rMO = 1.4. For MO nanoparticles doped with FA 6, a trans FA with two double bonds, at the highest tested FA concentration, the microemulsion (L2 phase) with a single broad scattering peak is observed. The forming of L2 instead of Lc at this concentration is likely because of the presence of two double bonds, which enlarges the effective tail volume and lowers the melting point of 6 and makes it easier to be incorporated into the MO bilayer. A molecular modeling of the chemical structure of 6 in vacuum, which shows the bent hydrocarbon chain because of the two double bonds, is provided in Figure S3. The appearance of QDII phase at lower FA concentration in 4, 5, and 6 compared to 1, 2, and 3 suggests that 4, 5, and 6 have higher cpp and thus affect the original QPII phase of MO more strongly than 1, 2, and 3. The phase behavior of MO nanoparticles added with 7 (cis16:1) and 8 (cis,trans-18:2) is similar to that of 6 and shortchain saturated FAs (C8−C12). A phase transition sequence

sentative hydrodynamic diameters and polydispersity index (PdI) of MO−linoelaidic acid nanoparticles at various acid concentrations were measured and provided in Figure S1. The mean diameters for these nanoparticles were around 220 nm and gradually increased with the FA content to around 300 nm in the microemulsion sample with highest linoelaidic acid concentration. Representative cryo-TEM images of cubosomes, hexosomes, and microemulsion of MO−linoelaidic acid nanoparticles at different acid concentrations are presented in Figure 2. The cubosomes show a typical cubic symmetry when analyzed by fast Fourier transformation (Figure 2A), whereas the hexosome exhibits sharp edges and a hexagonal symmetry (Figure 2B). Some other hexosomes are also found with “finger-print” patterns, which are commonly observed in these nanoparticles. The microemulsion show no ordered internal structure (Figure 2C). Phase Behavior of MO−FA Nanoparticles. The phase diagram for FA−MO nanoparticles at 25 °C is obtained directly from SAXS data and is presented in Figure 3. Representative

Figure 3. Partial phase diagram for MO nanoparticles with increasing amounts of unsaturated FAs at 25 °C. The list of 20 unsaturated FAs is provided in Table 1. The FA content in the mixture is represented by rMO, which is defined as the molar ratio of FA to MO.

Figure 4. Schematic representation of the effect of increasing concentration of FA on molecular packing, surface curvature, and phase behavior of MO and PHYT. 2767


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Figure 5. Lattice parameter (LP) of MO nanoparticulate dispersion added with increasing amount of unsaturated FAs. LPs of several mesophases including primitive cubic (QPII of Im3̅m space group, blue square), diamond cubic (QDII of Pn3̅m space group, green diamond), hexagonal (H2, ), micellar cubic (I2 of Fd3m ̅ space group, ), microemulsion (L2, red circle), and lamellar crystal (Lc, ) was determined by SAXS. A nanoparticle solution ( ) with mixed phases of L2, sponge phase (L3), and fluid lamellar phase (Lα) was also detected. Because the microemulsion phase (L2) lacks the long-range order, the correlation distances were calculated instead of LP.

from QPII → QDII → H2 → L2 is observed with the increasing FA concentration. It is counter-intuitive that 6, a trans FA with two double bonds, can induce phase transition from QPII to QDII at lower concentration than 7, a cis FA. This result indicates that the kink in the molecular structure of trans FA 6 may have a significant effect on increasing its effective tail volume and cpp compared to its saturated FA counterpart or cis FA 7. Further molecular modeling to calculate the exact values of the cpp of these FAs may elucidate this observation. FAs 9−13 each possesses one cis double bond. The increasing addition of these FAs to MO nanoparticles also induces a phase transition from QPII to QDII and to H2. However, at the highest FA concentration tested, a micellar cubic phase (I2) of the space group Fd3̅m is found. Previous studies have shown the existence of an I2 phase in MO−oleic acid bulk and dispersion systems.28,35,43 It is likely that because of the structural similarity of these FAs with oleic acid and MO, they were able to form an I2 phase. The rarity of the I2 phase reported in the literature makes this composition worth highlighting. Furthermore, in MO nanoparticles with 12 (erucic acid, cis-22:1) at an rMO of 0.13, the coexistence of QPII, QDII , and H2 is detected using SAXS. Recently, observations of stable Janus lipid nanoparticles with coexisting QDII /QPII phases or QDII /

H2 phases were reported.36 This raised a question regarding the existence of a nanoparticle with three coexisting phases. Consequently, we have surveyed these nanoparticles using cryo-TEM, however, no such nanoparticles were found. 14−20 are cis FAs with more than one double bond. Among these FAs, only the phase behavior of MO nanoparticles added with 14 (linoleic acid, cis-18:2) is similar to that of FAs with one double bond. As the concentration of 14 increased, the nanoparticles went through a phase sequence from QPII → QDII → H2 → L2. For cis FAs 15−20, the phase transition from QPII to QDII occurred at low FA concentration, and the window in which two phases coexist is wider than that for other FAs (1− 14) with lower degree of unsaturation. For example, in MO nanoparticles added with 18 (arachidonic acid, cis-20:4), the QDII /QPII coexistence was observed up to rMO = 0.22, whereas this value for FAs 1−14 was less than 0.144. Curiously, the nanoparticles added with 15−20 did not transform into a hexagonal phase as the FA concentration increased. Instead, the SAXS profiles of these nanoparticles showed a single strong peak at concentrations higher than rMO = 0.271 on top of a broad peak (Figure S4a). The broad peak is typical of an L2 phase, which has no long-range order. However, phase identification from the single strong peak is difficult because 2768


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that l is a constant in excess water condition, the decrease of LP because of increasing FA content in nanoparticles leads to smaller water channel size in cubic phase. This observation has several implications to the applicability of these lipid nanoparticles. Indeed, the relative size of a compound to the water channel will determine whether it can reside in the water channel or not. For example, Pluronic F127 induced phase changes in MO from QDII to QPII but not in PHYT. This was shown to be because of the larger water channel size of MO compared to that of PHYT, which led to the distribution of F127 inside MO water channels, reducing interfacial curvature and inducing phase transition.51 This size-dependent encapsulation is also applied for small molecular drugs, proteins, peptides, DNA, and siRNA. The relative size of the encapsulated compounds to the water channel size also has a significant influence on the release of the compounds.4 For soluble compounds whose release rate is controlled by diffusion, the smaller the compound, the faster the release rate. Similarly, it is expected that retardation in diffusion occurs more readily in matrices with smaller water channel size. The coexistence of two cubic phases QPII and QDII is an intriguing aspect that needs to be emphasized. Theoretically, the P-type and D-type minimal surfaces are related through the so-called Bonnet transformation,52,53 which indicates that minimal surfaces can be isometrically bent without stretching into one another while preserving the Gaussian curvature at all points. According to the minimal surface theory, the LP ratio of Bonnet related P-type and D-type surfaces should be 1.28.25 In MO nanoparticles with coexisting QPII and QDII , we found that the LP of each phase varied with the FA content, however, the LP ratio remained relatively unchanged. The average LP ratios of QPII/QDII (γP−D) for samples where coexisting cubic phases were detected were calculated and presented in Table 2. The LP ratio γP−D for MO nanoparticles spreads from 1.29 to 1.35, slightly larger than the theoretical value of 1.28. Our finding here supports several previous studies in which γP−D is found to be around 1.33.33,39,54−57 The results presented here are

of no visible secondary peaks. Upon further investigation using cryo-TEM, it is apparent that the solution consists of spongephase nanoparticles (L3) and multilamellar vesicles (Lα) in addition to microemulsions (L2) (Figure S4b). When the FA concentration was further increased, L2 phase was found in MO nanoparticles added with 15−18. The sponge-phase nanoparticles (L3) are usually regarded as swollen cubic phase, which represent the isotropic liquid phase between Lα and the cubic phase.44,45 However, there have been reports showing L3 sponge phase as an intermediate of the transition between H2 and L2 phases.46 The “missing” H2 phase here could be explained when considering the packing frustration. The concept of packing frustration is based on the idea of the optimized tiling (packing) of elements of uniform geometry, for example, cylinders into hexagonally close-packed (hcp) symmetries.24 Such an arrangement results in “void” space that has to be filled by amphiphile alkyl chains that must extend beyond their preferred conformation. Previous studies have shown that by adding oil into the “void” volume, relief of the packing frustration of the chains could be achieved.47,48 For cis FAs with a high level of unsaturation (15−20), the kinks in their structure may have resulted in (1) bent molecules with shorter effective chain length and higher cpp and (2) a hydrocarbon chain that is unable to fully stretch to fill the “void” space, hence, no H2 phase is formed. Similar to the addition of oil, temperature is known to have a strong effect on the formation of mesophases in these types of systems. When temperature rises, the fluidity of the hydrocarbon chain increases, leading to a larger effective tail volume and a higher cpp. Consequently, increasing the temperature is often associated with the formation of phases with higher negative surface curvature.49 The partial phase diagrams of MO−FA nanoparticles at 37 and 60 °C are provided in Figures S5 and S6, respectively. It is evident that the phase diagram is shifted through a phase sequence from QPII → QDII → H2 → L2. At this 60 °C, the presence of Lc phase is no longer detected. This is likely because 60 °C is higher than the melting points of all studied FAs. Similarly, the I2 phase is replaced by L2 in MO nanoparticles added with FAs 9−13. LP of MO−FA Nanoparticles. The LP for each mesophase was calculated from the SAXS data. LP is plotted against rMO and presented in Figure 5. It has been shown before that MO nanoparticles stabilized with Pluronic F127 in water exhibit a QPII phase with LP around 145 Å.39 As the concentration of FA increases, the LP of the nanoparticle gradually decreases until a second phase appears. This is a general trend with all FAs studied here, with the exception of a few FAs with a high degree of unsaturation such as 16 and 19. In any case, the LP of QPII, QDII , and H2 range from around 120−145, 80−110, and 50−65 Å, respectively. These values cluster around the expected LPs for cubic and hexagonal phases of MO.27 Previously, several theoretical models were developed to determine the water channel size from the LP data. For example, water channel radius of a cubic structure can be calculated based on minimal surface theory, which gives: 1/2

( ) σ

rw = − 2πχ

Table 2. Average LP Ratios for Coexisting QPII/QDII (γP−D)

a − l , where χ is Euler−Poincaré characteristic

and σ is the ratio of the minimal surface in a unit cell to the value (unit cell volume)2/3; a and l are LPs of the cubic phase and the monolayer thickness, respectively.20,50 Using this approach, the water channel radii of QPII and QDII phase are rw = (0.306)a − l and rw = (0.391)a − l, respectively. If we assume 2769

FA

name

structure

γP−D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

palmitelaidic acid elaidic acid trans vaccenic acid trans gondoic acid brassidic acid linoelaidic acid palmitoleic acid rumenic acid vaccenic acid oleic acid gondoic acid erucic acid nervonic acid linoleic acid α-linolenic acid γ-linolenic acid ETA arachidonic acid EPA DHA

trans-16:1, n − 7 trans-18:1, n − 9 trans-18:1, n − 7 trans-20:1, n − 9 trans-22:1, n − 9 trans-18:2, n − 6 cis-16:1, n − 7 cis,trans-18:2, n − 7 cis-18:1, n − 7 cis-18:1, n − 9 cis-20:1, n − 9 cis-22:1, n − 9 cis-24:1, n − 9 cis-18:2, n − 6 cis-18:3, n − 3 cis-18:3, n − 6 cis-20:3, n − 3 cis-20:4, n − 6 cis-20:5, n − 3 cis-22:6, n − 3

1.35 1.31 1.34 1.32 ± 0.03 1.32 ± 0.01 1.30 1.34 1.34 1.30 1.33 1.33 1.30 1.30 1.33 1.33 1.33 1.32 1.32 1.29

± ± ± ± ± ± ± ± ± ± ±

0.03 0.03 0.06 0.01 0.01 0.02 0.01 0.02 0.02 0.01 0.06


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phase (Lc) is observed in PHYT nanoparticles with 2, 3, and 5 trans FAs. This phase behavior again resembles that of PHYT nanoparticles with saturated FAs with chain length longer than C12.39 Compared to the trans FAs, the cis FAs readily induce a phase transition in PHYT nanoparticles from H2 to the fluid isotropic phase L2. This reaffirms the notion that effective cpp of cis FAs is generally larger than that of trans FAs. An increase in temperature is also expected to have an influence on PHYT nanoparticle phase behavior. The partial phase diagrams of PHYT nanoparticles with unsaturated FAs at 37 and 60 °C are provided in Figures S7 and S8, respectively. As expected, the higher temperature drives the system toward mesophases with higher negative interfacial curvatures. This is clear from the replacement of QDII phases by H2 phases and associated higher effective cpp. Additionally, Lc phase is no longer detected in nanoparticles with 2, 3, and 5 trans FAs, most likely because 60 °C is higher than the melting point of all studied FAs. The LPs of PHYT nanoparticles with unsaturated FAs at 25 °C are presented in Figure 7. The PHYT nanoparticles without FAs showed a QDII phase with an LP of around 66 Å, similar to what has been reported.32,59 This QDII LP is smaller than that of MO QDII nanoparticles observed above, which ranged from 82 to 111 Å depending on the FA concentration and FA molecular structure. When the FA content increases, the LP of PHYT nanoparticles decreases to around 63 Å, at which a phase transition from QDII to H2 occurred. At the transition point, the LP of the H2 phase is around 48−49 Å. These LP values for QDII and H2 phases are very close to those reported in PHYTsaturated FAs nanoparticle systems at the same temperature.39 These results suggest that the LP of PHYT nanoparticles at the phase transition point is independent of the FAs molecular structure or the degree of unsaturation. As the concentration of FA further increases, the LP of H2 phase also decreases and eventually the nanoparticles lose their long-range order and transition into L2 phase. For all PHYT nanoparticles, at the transition point from H2 to L2 phase, the LP of H2 phase is around 43 Å.

consistent with the existence of periodic minimal surfaces within the formulated nanoparticles. Five occurrence of a micellar cubic phase I2 (space group Fd3̅m) was recorded in MO nanoparticles added with FAs 9− 15. The LP of these I2 phases appeared to be dependent on the chain length. As the chain length increased from C18 to C24, the LP of the I2 phase also gradually increased from 142 Å in system with FA 10 (oleic acid) to a maximum of 166 Å in system with FA 13 (nervonic acid). Phase Behavior of PHYT−FA Nanoparticles. Unlike MO, PHYT’s isoprenoid-type chain contains no double bond. However, because of the branching in the hydrocarbon backbone, PHYT molecule can be described as “bananashaped” with a strong tendency to form nonlamellar mesophase structures.22,58 Bulk PHYT in excess water showed an inverse bicontinuous cubic symmetry of diamond type (QDII ). This QDII structure is preserved when PHYT is dispersed and stabilized with Pluronic F127 up to 10% weight of PHYT.32 This behavior is different from that of MO, which changes from Pn3̅m (QDII ) symmetry in bulk to Im3̅m (QPII) symmetry in dispersion in the presence of 10 w/w % F127.51 The partial phase diagram of PHYT nanoparticles added with unsaturated FAs at 25 °C is presented in Figure 6. Overall, no obvious

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CONCLUSIONS In summary, we have formulated and studied the phase behavior of self-assembled internally ordered lipid nanoparticles consisting of either MO or PHYT and unsaturated FAs. The study exemplified the benefit of high throughput methodologies, which allow us to both formulate and then characterize a large number of samples. The results illustrate that temperature, FA concentration, and FA molecular structure all have direct influence on the mesophase and LP of the nanoparticles. In the presence of increasing amounts of FAs, the effective cpps of both MO/FA and PHYT/FA systems increase, which lead to a phase transition process toward mesophases with higher Gaussian negative surface curvatures. In the studied FA concentration range, MO nanoparticles transformed from QPII → QDII → H2 → L2. Micellar cubic phase I2 is found in several MO nanoparticle formulations in between H2 and L2 phase. For PHYT, the phase sequence was QDII → H2 → L2. The phase diagrams and the calculated LPs suggest gradual transitions with the coexistence of mixed phases as the FA concentration increases. Notably, mixed SAXS signals of QPII/QDII /H2 were detected in MO nanoparticles with FA 13 (erucic acid). However, no nanoparticles with three coexisting phases were found in cryo-TEM. Additionally, the LP ratios of the two cubic phases (γP−D) were calculated to be between 1.29

Figure 6. Partial phase diagram PHYT nanoparticles with increasing amount of unsaturated FAs at 25 °C. The list of 20 unsaturated FAs is provided in Table 1. The FA content in the mixture is represented by rPHYT, which is defined as the molar ratio of FA to PHYT.

phase separation was detected. It is clear that within the studied compositional space, the phase behavior of PHYT is much less complex with fewer lyotropic liquid crystalline polymorphic states compared to that of MO nanoparticles. At low FA concentration, all nanoparticles exhibit a QDII phase, the same as the PHYT nanoparticles without FA. Similar to the case of MO nanoparticles, the addition of unsaturated FAs increases the Gaussian curvature of PHYT membrane surfaces. This is evident from the phase sequence from QDII to H2 and eventually L2 with increasing FA content. Notably, the phase diagram of PHYT nanoparticles is dominated by the H2 phase, which is present in all samples, spanning a large concentration window from rPHYT around 0.32 to 0.64. The H2 phase window is much larger in nanoparticles with trans FAs (1−6). Lamellar crystal 2770


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Figure 7. Lattice parameter (LP) of PHYT nanoparticulate dispersions with increasing amount of unsaturated FAs. LPs of several mesophases including diamond cubic (QDII of Pn3̅m space group, green diamond), hexagonal (H2, ), microemulsion (L2, red circle), and lamellar crystal (Lc, ) are determined by SAXS. Because the microemulsion (L2) lacks long-range order, the correlation distances were calculated instead of LP.

and 1.35. These γP−D values are consistent with the transformation between QPII and QDII , following the theoretical Bonnet transition. This study also emphasizes the complex phase behavior of self-assembled lipid systems with multiple components. As the requirement for multiple functionalities in drug delivery systems increases, a move beyond the binary compositional spaces is inevitable.27 In such a scenario, the material library, the characterization methods, and insights described herein may be valuable.

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with detailed compositions of MO−FA and PHYT−FA nanoparticles (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +61 3 9925 2131 (N.T.). *E-mail: [email protected] (X.M.). *E-mail: [email protected] (C.J.D.). ORCID

ASSOCIATED CONTENT

Nhiem Tran: 0000-0002-0209-2434 Calum J. Drummond: 0000-0001-7340-8611

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b03541. Representative hydrodynamic diameter, PdI, and SAXS patterns of MO−FA nanoparticles. SAXS profile and cryo-TEM images of MO nanoparticles with FA 18 (Arachidonic acid, cis-20:4) showing the presence of nanoparticles with sponge phase (L3) and multilamellar phase (Lα). Partial phase diagrams of MO−FA and PHYT−FA at temperatures from 37 and 60 °C. Tables

Present Address ⊥

School of Science, RMIT University GPO Box 2476, Melbourne, Vic 3000. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.T. is currently an RMIT University Vice-Chancellor’s Research Fellow and was supported by the Science and Industry Endowment Fund (SIEF) postdoctoral fellowship. 2771


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This research includes work undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, ANSTO, Victoria, Australia.

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