Toward Cell Membrane Biomimetic Lipidic Cubic Phases: A High

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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Toward Cell Membrane Biomimetic Lipidic Cubic Phases: A HighThroughput Exploration of Lipid Compositional Space Sampa Sarkar,† Nhiem Tran,† Md Harunur Rashid,‡,§ Tu C. Le,‡ Irene Yarovsky,‡ Charlotte E. Conn,*,† and Calum J. Drummond*,† †

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School of Science, College of Science, Engineering and Health, RMIT University, GPO Box 2476, Melbourne, Victoria 3001, Australia ‡ School of Engineering, RMIT University, GPO Box 2476, Melbourne, Victoria 3001, Australia S Supporting Information *

ABSTRACT: The bicontinuous lipidic cubic phase (LCP), which is based on the fundamental structure of the lipid bilayer, is increasingly used in a range of applications including drug delivery, in meso crystallization of membrane proteins, biosensors, and biofuel cells. The majority of LCPs investigated to date have been formulated from a single lipid or a combination of two lipids in water. Such systems lack tunability, with only a narrow range of lattice parameters adopted. In addition, the lipid bilayer of these materials lacks the complexity of natural cell membranes, which are composed of hundreds of different lipids and which may be essential to retaining the functionality of proteins embedded within them. In this work, we investigate the phase behavior of quaternary lipid−water systems consisting of three different lipids (monoolein−cholesterol−phospholipid) and water using a combination of experimental and simulation techniques. This study provides a large library of lipidic materials with bilayer compositions, which more effectively mimic the native cell membrane and significantly increased tunability based on nanostructural parameters such as lattice parameter, aqueous channel size, and bilayer thickness. Importantly, the library contained several extremely swollen cubic phases with a maximum lattice parameter of up to 342.5 Å. Many of these cubic phases were successfully dispersed into highly swollen cubosomes. The swollen cubic phases described in this article contain only uncharged lipids and are therefore particularly useful for applications with a high salt concentration, including encapsulation of larger therapeutic proteins and peptides for in vivo delivery, or for the crystallization of large membrane proteins such as GPCRs.1 KEYWORDS: monoolein, cholesterol, phospholipids, self-assembly, lipidic cubic phase, nanoparticles, cubosome, SAXS, high throughput, lyotropic liquid crystals, molecular modeling

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INTRODUCTION

bicontinuous cubic phases retain the fundamental lipid bilayer structure. Design rules have been developed to assist in predicting the phase adopted by a specific amphiphile, or combination of amphiphiles.13,14 A key strategy used to rationalize the mesophase adopted is to consider the critical packing parameter (CPP), which is related to the intrinsic interfacial curvature of the system15 and can be defined as

The cell membrane environment, which is abundant in biological systems, provides an exceptional model to devise “smart nanostructures” based on the molecular self-assembly of biological macromolecules such as carbohydrates, lipids, nucleic acids, and proteins.2,3 Amphiphilic biomolecules such as lipids, in particular, are known to self-assemble into liquid crystalline nanostructures of distinct geometry in the presence of water due to the hydrophobic effect. The unique nanostructure of these materials can provide an ideal lipid bilayer matrix for numerous applications in nanoscience such as imaging,4 physiological studies,5,6 drug delivery systems,3,7 nanoreactors,8 biosensors,9 and synthetic cells.10,11 The most common of these nanostructures is the lamellar phase, which is analogous to the lipid bilayer structure of the cell membrane. More complex architectures include the inverse bicontinuous cubic phase, inverse hexagonal phase, and ordered micellar phases. A disordered fluid of micelles (fluid isotropic phase) can also be adopted.12 Of these, only the lamellar and © XXXX American Chemical Society

CPP =

V a 0l

(1)

where V is the hydrophobic tail volume, a0 is the area of the hydrophilic headgroup, and l is the effective length of the hydrocarbon chain. The specific CPP value can therefore rationalize the morphology of the self-assembled nanostructure Received: September 17, 2018 Accepted: December 7, 2018 Published: December 7, 2018 A

DOI: 10.1021/acsabm.8b00539 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

Figure 1. Graphic presentation of commonly formed lyotropic liquid crystalline phases of amphiphiles. Phases are presented in order of increasing negative mean curvature: Lα−QIIP−QIID−QIIG−HII−L2.

by a consideration of the overall “shape” of the amphiphile or amphiphiles. The theoretical order of mesophases organized with an increasing negative curvature of the interface is presented in Figure 1. The fluid lamellar phase has a zero mean curvature and is typically adopted by molecules having a CPP value close to 1. With an increasing value of the CPP, mesophases of an increasingly negative curvature are adopted, that is, mesophases where curvature is toward the aqueous region. These include, with an increasing negative curvature, the inverse bicontinuous cubic phases (QIIP, QIID, QIIG), the inverse hexagonal phase (HII), and micellar phases (L2).16,17 The bicontinuous cubic phases, in particular, have attracted significant research interest in a range of applications including in meso-crystallization,18,19 in drug delivery,2 and as biosensors.20 The three bicontinuous cubic phases (Figure 1) are the Q224 (Pn3m), Q229 (Im3m), and Q230 (Ia3d), which reveal morphologically as the diamond (QIID), primitive (QIIP), and gyroid (QIIG) cubic phases, respectively.21−23 These consist of a single lipid bilayer convoluted over a three-dimensional space and separating space into two interpenetrating water networks.23 Their unique material properties include an amphiphilic 3D nanostructure consisting of coexisting aqueous and oil-like regions, high internal surface area, and good thermodynamic stability. Their fundamental bilayer structure also naturally mimics the native cell membrane, making them ideal for the encapsulation of membrane-associated and integral membrane proteins.24 Due to its biomimetic nature, the lipidic cubic phase can provide an ideal environment for the encapsulation of hydrophilic and hydrophobic proteins,25−27 peptides28,29 and nucleic acids30 with a range of molecular weights. Lipidic cubic phase systems formulated to date have been mainly based on a single lipid, or ternary systems comprising two lipids and water. The fundamental bilayer structure of such lipidic cubic phases is not overly representative of the native cell membrane, which can contain up to one hundred different lipids.22,31,32 In addition, the relatively small number of lipids, which are known to adopt the bicontinuous cubic phase,

means that the ability to tune these systems to a required nanostructure is limited. The majority of published research on complex lipid mixtures forming the lipidic cubic phase has focused on mixtures of two lipids and water. The effect of various lipids, including sterols and amphiphiles having single or multiple alkyl chains, on the structure of the bicontinuous cubic phase has been summarized in a recent review.13 To date, a small number of studies have investigated the structure of more complex mixtures consisting of three or more lipids in aqueous conditions.33,34 Recently Tran et al. have shown that the addition of capric acid and a saturated fatty acid to monoolein promotes phase transitions to more curved phases in the sequence from an inverse bicontinuous cubic phase to hexagonal phase and then emulsified microemulsion.15 In general, the majority of phase transitions observed can be rationalized by two main factors: (1) the shape or critical packing parameter of the lipid additives and (2) the headgroup charge.13,14 Amphiphiles with CPP < 1 were found to promote the formation of less curved phases such as the lamellar phase including highly swollen cubics, while amphiphiles with CPP ≫ 1 were found to induce more curved phases such as the inverse hexagonal and micellar phases. The largest cubic phase formed to date is the QIIP phase with a lattice parameter of 480 Å and was formed from a ternary mixture of MO, cholesterol, and the charged lipid DOPS.33 The aim of the current study was to produce a library of lipid materials that retain the cubic phase suitable for encapsulation of any bioactive molecules, but with lipid bilayer compositions that are more biologically relevant. We therefore investigated the phase behavior of quaternary systems consisting of three lipids and water. MO, which is the most commonly used cubic phase forming lipid, was used as a base lipid. MO was doped with cholesterol as a second component as cholesterol is the most common sterol in all eukaryotic cell membranes (although lacking in prokaryotic cells). The cholesterol hydroxyl group can form a hydrogen bond with the carbonyl oxygen of phospholipids and, as a result, can increase the rigidity and/or fluidity of the membrane B


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Figure 2. Phase adopted and associated lattice parameter at 25 °C of MO-based lipid systems following the addition of PC phospholipids having C12−C18 saturated and singly unsaturated (C18:1) chains (A−O). Data are presented at three cholesterol concentrations (0 mol %; 15 mol %; 30 mol %). The identity of different phases is as follows: lamellar (Lα); diamond bicontinuous cubic (QIID); primitive bicontinuous cubic (QIIP). “−” indicates that the specific composition was not run. “X” indicates that no Bragg peaks were observed for that sample. The accuracy of the reported lattice parameter values is ±0.5 Å for all phases. The concentration of lipids is calculated in mol %.

(depending on the overall composition).35 The third component was a phospholipid, again due to the high prevalence of these lipids in the cell membrane. Phospholipids having PC, PE, and PS headgroups with a range of physiologically relevant chain lengths from C12 to C18 were investigated. All phospholipids contained saturated chains except for the singly unsaturated (C18:1) oleoyl chain. The systematic variation in lipid composition allowed us to extract the effect of different physiologically relevant lipids on the lipid mesophase nanostructure and lattice parameter.

Previously, it has been difficult to generate the phase behavior of a large library of complex mixtures using traditional sample preparation and characterization methods. Recent advances in high-throughput (HT) methodologies, which allow for the formulation and structural characterization of thousands of samples per day,36−38 have allowed us to characterize a large library of lipidic materials with physiologically relevant bilayer compositions. The extra degrees of freedom introduced by the additional lipid components made it possible to exert a much finer control over the nanostructure C


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Figure 3. Phase adopted and associated lattice parameter at 25 °C of MO-based lipid systems following the addition of PE phospholipids having C12−C18 saturated and singly unsaturated (C18:1) chains (A−O). Data are presented at three cholesterol concentrations (0 mol %; 15 mol %; 30 mol %). The identity of different phases is as follows: lamellar (Lα); diamond bicontinuous cubic (QIID); primitive bicontinuous cubic (QIIP) and hexagonal (HII). “−”indicates that the specific composition was not run. The accuracy of the reported lattice parameter values is ±0.5 Å for all phases. The concentration of lipids is calculated in mol %.

(DLS) techniques. Molecular simulations have been previously shown to advance our understanding of membrane defects,39,40 ion-bilayer interactions,41−43 membrane tension,44,45 and alteration of the lipid bilayer structure.46 Molecular simulation studies employed herein enabled us to obtain a detailed understanding of the bilayer structural and dynamics properties including bilayer thickness, lipid diffusion, and clustering and provided additional molecular level information not available via the experimental studies.

of the resulting lipid nanomaterial, e.g., unit cell size, aqueous channel size, bilayer thickness. This may allow for the selection of a lipid nanomaterial with the precise characteristics required for a specific application. The internal structure of the lipidic materials was investigated using high-throughput synchrotron small-angle X-ray scattering (SAXS).38 Dispersed nanoparticles were assessed using a combination of SAXS, cryo-transmission electron microscopy (cryo-TEM), and dynamic light scattering D


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RESULTS AND DISCUSSION In the absence of additives, MO adopted a QIID phase with a lattice parameter of ∼104 Å, at 60% (w/w) water (excess water conditions). The results presented below illustrate the structural effects exerted on the MO cubic phase, as determined using SAXS, following the addition of up to two lipid additives, cholesterol, and a phospholipid. Phospholipids having PC, PE, and PS headgroups with a range of physiologically relevant chain lengths from C12 to C18 were investigated. All phospholipids contained saturated chains [lauryl (C12); myristyl (C14); palmityl (C16); stearyl (C18)] except for the singly unsaturated (C18:1) oleoyl chain. The phase adopted and associated lattice parameter of each of these additives in excess water was determined using SAXS and is presented in Table S1. Representative 2D diffraction images and 1D spectra of intensity vs q are provided in Figures S1−S5 for selected bulk phase and cubosome materials . MO−Cholesterol Systems. In excess water, ∼60% (w/w), cholesterol forms a crystalline lamellar (Lc) phase with a lattice parameter of 34 Å (Table S1). The effect of cholesterol on the phase behavior of MO in excess water is provided in Figure S6. With an increasing cholesterol concentration, a transition from the original QIID phase to a QIIP phase was observed, in agreement with a previous study.47 The lattice parameter of both the QIID and QIIP phases consistently increase with an increasing cholesterol concentration, with a maximum lattice parameter of 198 Å observed for the QIIP phase at 50 mol % cholesterol. MO−Cholesterol−PC Systems. The phase behavior of MO following the addition of saturated and unsaturated PC headgroup lipids containing 12−18 carbons per chain was determined using synchrotron SAXS and is presented in Figure 2 at 0, 15, and 30 mol % cholesterol. All of the PC lipids used have a CPP value ∼1 and generally adopt the lamellar phase in excess water as presented in Table S1. In the absence of cholesterol (Figure 2A−E), an initial increase in lattice parameter of the QIID phase was observed with an increasing concentration of PC phospholipid, followed by a subsequent transition to a Lα phase. For short-medium saturated chains (C12−C16), the system had transitioned to the Lα phase by 20 mol % PC phospholipid. For longer saturated (C18) and unsaturated (C18:1) chains, the Lα phase was initially observed at a higher PC concentration (30 or 40 mol %). For all saturated chain PC lipids, the Lα phase remained stable up to 50 mol % of PC molecules (Figure 2), which was the highest concentration studied, consistent with previous research.47 For DSPC, an additional QIIP phase was observed, coexisting with the Lα phase, at 30 and 40 mol % DSPC. Data are also provided in the presence of cholesterol at 15 mol % (Figure 2F−J) and 30 mol % (Figure 2K−O). For the shorter and medium saturated chain PC lipids, a transition to a Lα phase is still generally observed at 20 mol %, despite the addition of 15 or 30 mol % cholesterol. For DSPC (C18) and DOPC (C18:1), addition of cholesterol appears to prevent the formation of the lamellar phase in most cases; coexisting QIIP and QIID phases are observed at intermediate phospholipid concentrations. The lattice parameter of the observed Lα phase is similar to that of the relevant pure lipids dispersed in water (e.g., DLPC Lα = 61.6 Å (pure DLPC = 55.4 Å); DMPC Lα = 63.5 Å (pure DMPC = 64.5 Å); DPPC Lα = 62.1 Å (pure DPPC = 62.01 Å); DSPC Lα = 63.6 Å (pure DSPC = 64.52

Å); DOPC Lα = 61.3 Å (pure DOPC = 59.62 Å)), suggesting that at higher concentrations significant phase separation of the lipids occurs.47 We note that the combined effect of cholesterol and PC phospholipid causes the original QIID phase to swell to much higher lattice parameters than for either cholesterol or phospholipid alone. The effect appears to be dependent on the cholesterol concentration; at a DMPC concentration of 10 mol %, the QIID phase adopted a lattice parameter of 263.5 Å at 15 mol % cholesterol, but a much higher lattice parameter of 342.5 Å at 30 mol % cholesterol. Similarly, at 30 mol % cholesterol and 30 mol % DOPC, a highly swollen QIID phase was observed (223.87 Å), representing a ∼50% increase in lattice parameter compared to the same system in the absence of cholesterol. MO−Cholesterol−Phosphatidylethanolamine (PE) Systems. The phase behavior of MO following the addition of lipids having a zwitterionic PE headgroup is presented in Figure 3 at 0, 15, and 30 mol % cholesterol. All four of the saturated chain lipids (DLPE; DMPE; DPPE; DSPE) form a lamellar phase in excess water, Table S1, with CPP values ∼1. In contrast, DOPE has a CPP value greater than 1 and adopts the highly curved inverse hexagonal phase in excess water, Table S1. In the absence of cholesterol, the addition of all saturated PE headgroup lipids containing 12−18 carbons per chain to hydrated MO resulted in an eventual transition to a fluid lamellar (Lα) phase, Figure 3A−D. In all cases, the observed fluid lamellar phase (Lα) coexisted with the original QIID phase. The coexisting QIID and Lα phases were retained up to 50 mol % PE lipids (the highest concentration studied). For all four saturated chain PE lipids, the lattice parameters of the coexisting lamellar phase are essentially identical to that of the pure lipids (e.g., DLPE Lα = 42.5 Å (pure DLPE = 43.9 Å); DMPE Lα = 50.2 Å (pure DMPE= 50.3 Å); DPPE Lα = 55.2 Å (pure DPPE = 55.0 Å); DSPE Lα = 60.9 Å (pure DSPE = 60.1 Å)) (Table S1), again indicating that significant phase separation of these lipids had occurred. The coexisting Lα phase was initially observed at a higher concentration for the shorter chain lipids (50 and 40 mol % for DLPE and DMPE, respectively). For medium (DPPE-C16) and longer chain (DSPE-C18) saturated lipids, the QIID phase coexisted with the Lα phase from 10 mol % additive, indicating a limited uptake of these lipids into the MO bilayer. Beyond the solubility limit of the hydrated MO, the lattice parameter of the Q IID phase remained constant, consistent with phase separation of the lipids. For the singly unsaturated C18 chain (DOPE), the QIID phase was retained up to 30 mol % of the DOPE additive. From 40 mol % of DOPE concentration, the system transitioned to a sharp and well-defined HII phase, which was retained up to 50 mol %, in good agreement with previous work.47 The phase behavior of the quaternary system consisting of MO−cholesterol−PE lipid under excess water conditions is presented in Figure 3F−O. Following the addition of 15 mol % or 30 mol % cholesterol, a transition to an Lα phase coexisting with the QIID phase was observed at 30 mol % for the shorter chain PE lipids (DLPE and DMPE). The main difference observed was that the combined effect of cholesterol and PE phospholipid induced the phase separation at lower phospholipid concentrations. Again, the lattice parameter of the Lα phase in the quaternary system was similar to that of the E


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Figure 4. Phase adopted and associated lattice parameter at 25 °C of MO-based lipid systems following the addition of PS phospholipids having C12, C14, C18 saturated and C18 singly unsaturated (C18:1) chains (A−L). Data are presented at three cholesterol concentrations (0 mol %; 15 mol %; 30 mol %). The identity of different phases is as follows: lamellar (Lα); diamond bicontinuous cubic (QIID); primitive bicontinuous cubic (QIIP). The accuracy of the reported lattice parameter values is ±0.5 Å for all phases. The concentration of lipids is calculated in mol %.

unsaturated chain PS lipids at room temperature (Figure 4). The saturated lipids included DLPS, DPPS, and DSPS. The addition of the unsaturated lipid DOPS was also investigated. The phase adopted by these lipids in excess water is given in Table S1. In the absence of cholesterol, the addition of PS had a significant effect on the phase behavior of hydrated MO, Figure 4A−D. The addition of saturated and unsaturated PS headgroup lipid-containing 12, 16, and 18 carbons per chain to hydrated MO resulted in an immediate phase transition from the QIID phase to the QIIP phase at the lowest additive concentration studied (1 mol %). The lattice parameter of the QIIP phase increased with an increasing additive concentration, from approximately ∼144 Å to a maximum value of ∼170 Å (at 5 mol % DPPS). The QIIP phase was typically retained up to 5 mol % additive followed by a phase transition to a Lα phase, in good agreement with a previous study.47 The phase behavior of the quaternary system consisting of MO−cholesterol−PS lipid under excess water conditions was investigated for the same PS phospholipids, Figure 4E−L. The

lamellar phase formed by pure DLPE and DSME in water, suggesting phase separation had occurred. For medium and long chain saturated PE lipids (DPPE and DSPE), the Lα phase appeared at a slightly higher phospholipid concentration (20 mol %) in the presence of cholesterol compared to that in the absence of cholesterol (10 mol %). While the effect is small, this may suggest that cholesterol could increase the capacity of MO to load these phospholipids. At both cholesterol concentrations, the lattice parameter of the QIID phase initially increases with an increasing phospholipid concentration before decreasing again. This decrease is either coincident with or occurs just before the appearance of the Lα phase and may be linked to the solubility limit of the phospholipid in the MO−cholesterol bilayer. A coexisting QIIP phase was also observed for DLPE, DMPE, and DPPE, but only at 15 mol % cholesterol. MO−Cholesterol−Phosphatidylserine (PS) Systems. The effect of a phospholipid having a negatively charged PS headgroup on the QIID phase adopted by MO in excess water was investigated using SAXS for both saturated and F


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ACS Applied Bio Materials PS phospholipid was studied at a lower range of concentrations (up to 10 mol %) as the effect of charged lipids on phase behavior can be significant, even at very low concentrations. In the presence of cholesterol, an immediate phase transition to a QIIP phase was again observed; note that at 15% cholesterol QIID and QIIP phases coexist at 1 mol % PS additive. However, for saturated chain PS lipids, a subsequent transition to a Lα phase is not observed in the presence of cholesterol. As for uncharged phospholipids (PC and PE), the increase in lattice parameter of the QIIP phase is more pronounced in the presence of cholesterol, although this effect is not dramatic. For shorter chain PS lipids (DPPS and DLPS), the QIIP phase is retained up to 10 mol %, the highest concentration studied. For the longer chain lipid, DSPS at 10 mol %, no Bragg diffraction peaks are observed in the presence of cholesterol, indicating the loss of long-range order within the sample. For the unsaturated lipid DOPS (C18:1), a similar phase sequence (QIID−QIIP−Lα) was observed with or without cholesterol. The main difference was that the lattice parameter of the QIIP phase is higher in the presence of cholesterol. Dispersion of Bulk Lipid Mixtures into Cubosomes. Many of the current and projected uses for lipid nanoparticles require dispersion of the material into submicron or nanoparticles. Dispersed nanoparticles of the lamellar, cubic, and hexagonal phases are termed liposomes, cubosomes, and hexosomes, respectively. We therefore investigated the structure of selected lipid mixtures upon dispersion in water (Table 1). Some samples were also dispersed in PBS at pH 7.4 for physiological relevance (Table 1). Representative 2D diffraction images and 1D spectra of intensity vs q are provided in the Supporting Information for selected cubosome dispersions (Figures S3, S4, and S5). Lipid mixtures were selected from ternary and quaternary bulk lipid systems with a range of lattice parameters (∼45−198 Å). The cubic structure of the cubosomes was confirmed by SAXS analysis (Table 1). Pure MO formed cubosomes of QIIP symmetry with a typical lattice parameter of 142 Å, in agreement with the literature.48 The phase change from QIID (bulk MO) to QIIP (MO cubosomes) is due to addition of the triblock PEO−PPO-PEO copolymer Pluronic F127, which is used to impart steric stabilization and prevent flocculation of cubosomes in solution. Addition of such stabilizers is known to impact the phase behavior of the lipid, due to partitioning of the hydrophobic domain of the polymer into the lipid bilayer. The effect is related to the hydrophilic−lipophilic balance (HLB) of the polymer; polymers with a HLB < 23 (including F127 used herein) are known to induce a phase transition to a QIIP phase for monoolein.49 Cubosomes formed from mixtures of two lipids in water were generally well-structured and diffracted well. At least three well-resolved Bragg peaks were typically observed with relative positions in a ratio of √2 √4 √6, characteristic of the QIIP phase (Table 1 and Figures S3− 5). The cubosomes investigated adopted a range of lattice parameters from 142 Å (MO−cholesterol 90:10 mol %) to 217 Å (MO−DSPC 70:30 mol %) (Table 1). A direct comparison between cubosome and bulk data is not possible due to the phase transition from QIID to QIIP upon dispersion (due to the presence of the steric stabilizer F127 in the cubosome formulation). However, in general, the lattice parameter of the cubosomes followed the same trend as that of the bulk phases. We note that many of the cubosomes formed from mixtures of three lipids and water did not diffract well (e.g., MO−

Table 1. Phase Adopted and Associated Lattice Parameter of Lipid Mixtures Dispersed into Sub-Micron Particles in Either Water or PBS lipid material MO-10 chol MO-30 chol MO-50 chol MO-10 DLPC MO-10 DMPC MO-10 DSPC MO-20 DSPC MO-10 DPPE MO-10 DSPE MO-5 DPPS MO-10 DOPS MO-30 DSPC MO-15 chol-10 DPPS MO-30 chol-1 DPPS MO-30 chol-5 DPPS MO-15 chol-10 DLPE MO-15 chol-20 DLPE MO-15 chol-30 DLPC MO-15 chol-10 DPPC MO-30 chol-10 DPPC MO-15 chol-5 DMPC MO-30 chol-10 DMPS MO-15 chol-5 DLPS MO-15 chol-10 DLPS MO-15 chol-30 DSPC MO-30 chol-30 DSPC MO-30 chol-10 DOPC MO-30 chol-20 DOPC MO-30 chol-30 DOPC MO-30 chol-10 DOPS

phase in H2O QIIP QIIP QIIP QIIP QIIP QIIP QIIP QIIP QIIP QIIP QIIP

d spacing (Å) in H2O

phase in PBS

d spacing (Å) in PBS

142.4 146.3 197.5 153.7 154.4 162.0 163.0 156.8 163.1 142.8 no diffraction 217.1 no diffraction

QIIP QIIP QIIP QIIP

144.3 166.9 155.3 155.3 not run 17.3 199.9 157.1 163.8 not run no diffraction no diffraction no diffraction

QIIP QIIP QIIP QIIP

Lα

35.6

not run

Lα

35.0

not run

QIIP

141.4

micelles no diffraction QIIP, micelles

QIIP

QIIP

153.4

QIIP

184.6

sponge

168.2

not run

no diffraction

no diffraction QIIP

142.2

158.8

no diffraction

no diffraction

QIIP

153.5

not run

QIIP, micelles sponge

170−175

not run sponge

no diffraction

no diffraction

QIIP, micelles micelles

175

no diffraction

QIIP, micelles

175.2

micelles no diffraction

no diffraction

QIIP

150.7

cholesterol−DPPS 75:15:10 mol %; MO−cholesterol−DPPC 60:30:10 mol %), although cubosomes were observed for some samples (e.g., MO−cholesterol−DLPE 75:15:10 mol %; MO− cholesterol−DMPC 75:15:10 mol %). The lattice parameter adopted by the cubosomes was also significantly lower than that of the corresponding bulk phase in some cases. We suggest that the dispersion process may have prevented unstable large cubic phases from successfully dispersing into cubosomes. For some systems, e.g., MO−cholesterol−DPPC 75:15:10 mol %, Bragg peaks corresponding to a cubic phase are superimposed on a broad peak. The correlation length of the broad peak is approximately 47.1 Å, characteristic of coexisting micelles, and confirming the instability of some systems upon G


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In general, a good agreement between the phase adopted in water and in PBS was observed for uncharged lipids as expected. Diffraction was sometimes observed for only one condition, in either water or PBS, and differences of up to 10 Å in lattice parameter were observed. For uncharged lipids, this is unlikely to reject any fundamental change in phase behavior in PBS. Rather, highly swollen cubosomes are likely to be unstable and may not form reproducibly. Representative cryo-TEM images of selected cubosomes are presented in Figure 5. The fast Fourier transforms (FFT) acquired from the optical diffractogram of the cryo-TEM images (shown as an inset in Figure 5) confirmed the existence of internal cubic symmetry.48 In Figure 5A, the characteristic well-ordered cubosomes formed by pure MO are observed. The characteristic motifs and reflections of a QIIP phase are confirmed in the FFT analysis. For this QIIP phase, the distance between the d200 planes is visible with a spacing of 7.02 nm, corresponding to a calculated lattice parameter of 14.02 nm. This value is in good agreement with the corresponding lattice parameter measured by SAXS analysis (14.3 nm). In Figure 5B, MO doped with 10 mol % cholesterol, many wellstructured cubosomes are observed, with some evidence of smaller sponge-like particles, in agreement with the reduced mean particle diameter as determined by DLS. The average size of cubosomes consisting of MO doped with 50 mol % cholesterol (Figure 5C) particles is much larger than that of the original MO QIIP phase (Figure 5A). The calculated lattice parameter value by FFT is 19.27 nm, with some evidence of smaller sponge-like particles in Figure 5C. In addition, we note

dispersion. The existence of micelles was also observed for MO−cholesterol−DLPS 75:15:10 mol %, along with some Bragg peaks which do not index as any known mesophase and may represent a more disordered architecture based on the cubic phase. For MO−cholesterol−DSPC 65:15:30 mol %, a broad hump is visible to a lower angle, potentially consistent with the formation of a sponge phase. The existence of coexisting micelles and the sponge phase was confirmed by cryo-TEM and is described below. The average cubosome size as determined by dynamic light scattering (DLS) is presented in Table 2. MO cubosomes have Table 2. Mean Hydrodynamic Diameter (MDD) and Polydispersity Index (PDI) of Selected Lipid Nanoparticles Measured by Dynamic Light Scattering cubosomes

MMD (nm)

PDI

MO MO−cholesterol 90:10 mol % MO−cholesterol 50:50 mol% MO−cholesterol−DLPC 75:15:10 mol % MO−cholesterol−DLPE 75:15:10 mol % MO−cholesterol−DLPS 75:15:10 mol %

208.8 174.0 206.8 1576 161.8 168.1

0.149 0.096 0.205 0.096 0.105 0.129

a mean diameter of 208 nm, in good agreement with previous studies.50 Cubosomes formed from lipid mixtures were generally smaller than those formed from a single lipid having mean diameters in the range 150−210 nm.

Figure 5. Cryo-TEM images and fast Fourier transformation of MO-based lipid nanoparticles. (A) Pure MO, (B) MO−cholesterol 90:10 mol %, (C) MO−cholesterol 50:50 mol %, (D) MO−cholesterol−DMPC 75:15:10 mol %, (E) MO−cholesterol−DLPE 75:15:10 mol %, (F) MO− cholesterol−DLPS 75:15:5 mol %. H


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thickness was most significant for MO−cholesterol−DOPS, compared to MO−cholesterol−DOPC and MO−cholesterol. A significant reduction in lateral diffusion of MO is also observed in the MO−cholesterol−DOPS system, Table 3. The reduction in the diffusion coefficient for MO in the mixed lipid system can be linked to the increase of headgroup area of MO in these systems (Table 3). We observed that this large increase in MO headgroup area in the MO−cholesterol− DOPS system (Table 3) is due to hydrogen bond formation (a weak electrostatic interaction) between the hydroxyl group of O4 and O3 atom of MO and the O13B oxygen atom of DOPS, (Figure 6A), which causes “bending” or “lifting” of the MO headgroup out of the plane of the bilayer. The corresponding reduction in CPP and compression of the chain area drives the increase in bilayer thickness. However, the frequently formed hydrogen bonds are not long-lived as observed over the course of the simulation. Therefore, to provide further evidence for the formation of a hydrogen bond between MO and DOPS, we plotted the RDF (radial distribution function) for an O3 atom of MO and O13B atom of DOPS. Indeed, the RDF shown in Figure 6B exhibits a sharp peak at 2.52 Å, corresponding to the H-bond. The DOPS molecule can also interact with cholesterol via the phosphate oxygen and cholesterol headgroup oxygen, a common interaction in the lipid−cholesterol system (Figure 6A). However, these interactions may be reduced or prevented by intermolecular hydrogen bonding, which has been reported between the amino group (NH3) and phosphate group (PO4−) in DOPS.56 The interaction between MO and DOPS can therefore actually strengthen the interaction between DOPS and cholesterol, by reducing fluctuations in the NH3 group, which can weaken the DOPS−cholesterol interaction. The interactions of MO and cholesterol with DOPS both contribute to the increase in hydrocarbon thickness. In contrast, in the MO−cholesterol−DOPC system, MO must compete with cholesterol for the hydrogen bonding interaction with the phosphate oxygen (Figure 6C) due to the absence of another hydrogen bond acceptor in the headgroup. Our simulations show that the fluctuations of methyl (CH3) groups in DOPC prevent cholesterol from forming hydrogen bonds with the phosphate oxygen. Less frequent hydrogen bond formation is also observed between MO and DOPC. The electrostatic nature of the interaction between MO and DOPS is confirmed by the addition of the Na+ ion to the system, which screens this interaction. A corresponding increase in bilayer thickness is not observed in the presence of the Na+ ion.55

the more varied morphology of the particles, compared to characteristic square faceting observed for pure MO cubosomes. The FFT analysis shows a spacing d200 of 9.63 nm, in agreement with SAXS analysis. For MO cubosomes doped with cholesterol and either PC, PE, or PS phospholipids (Figure 5D−F, respectively), many cubosomes displaying a characteristic cubic symmetry are observed. For MO− cholesterol−DMPC 75:15:10 (Figure 5D) and MO−cholesterol−DLPE 75:15:10 mol % (Figure 5E), numerous wellstructured cubosomes were observed with some evidence of smaller, sponge-like particles. In Figure 5F (MO−cholesterol− DLPS 80:15:5 mol %), cubosomes display square facetting with some evidence of sponge-like particles. In general, there was a good agreement between the size distributions observed in the cryo-TEM images (Figure 5) and those determined from DLS measurements (Table 2). Molecular Simulation of Complex Lipid Bilayers with Monoolein, PC, PE, PS, and Cholesterol. The modeling of selected complex lipid mixtures provided complementary information on the influence of components on the bilayer thickness and diffusion coefficient of MO in these systems. Such information is essential to progress the exploitation of these systems, particularly in terms of biomedical applications where bioactive molecules are encapsulated.51 Research has shown that the bilayer thickness has a significant influence on the conformation and activity of encapsulated membrane proteins and peptides, while the diffusion coefficient for MO is linked to the rate of protein or peptide diffusion within the bilayer. Both factors have been shown to influence the rate and mechanism of protein crystallization within a lipidic cubic phase.52−54 Modeling was carried out on three systems, which retain the fundamental lipid bilayer structure: MO−cholesterol 60:40 mol %; MO−cholesterol−DOPC 60:30:10 mol %; and MO− cholesterol−DOPS 60:30:10 mol %. The corresponding PE headgroup system (MO−cholesterol−DOPE) was not suitable for modeling as it forms a hexagonal phase, which is not bilayer-based. The bilayer hydrocarbon thickness for MO was estimated as 26.2 Å (in excess water) by subtracting the headgroup size from the known bilayer thickness, as described in more detail in Figure S7 and the associated text. Note that the bilayer hydrocarbon thickness has been determined rather than the full bilayer thickness due to the absence of a common heavy atom in the lipid headgroups, which would allow us to calculate the bilayer thickness. In all cases, the bilayer hydrocarbon thickness increased for mixed lipid phases relative to MO (Table 3).55 The increase in bilayer hydrocarbon

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DISCUSSION In general, phase transitions in lipid mesophases can be rationalized by consideration of (1) the shape or critical packing parameter (CPP) of the lipids additives and (2) the headgroup charge.13 The observed phase behavior described herein for both two- and three-lipid systems is generally consistent with known physicochemical studies on the effect of specific molecular architectures, and charge, on the curvature of the bilayer.14,13 Type 0 lipids, including cholesterol, all PC lipids, and the saturated PE lipids investigated here, are approximately cylindrical in shape and have CPP values close to 1. Such additives are known to drive a reduction in interfacial curvature and resulting phase transitions in the sequence QIID−QIIP−Lα. This phase sequence was generally observed following addition of all type 0 lipids, although the

Table 3. Lattice Parameter, Simulated Bilayer Thickness, Headgroup Area, and Lateral Diffusion Coefficient of MO for Selected Lipid Systems

lipid system MO− cholesterol MO− cholesterol− DOPC MO− cholesterol− DOPS

lattice parameter (Å)

bilayer thickness (Å)

MO headgroup area (Å2)

D in 10−8 cm2/s

169.0 (QIIp)

36.6 ± 1.1

44.0 ± 0.1

19.5

223.9 (QIID)

35.2 ± 0.7

57.1 ± 0.6

18.1

45.8 (Lα)

41.5 ± 0.7

63.1 ± 2.5

15.4

I


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Figure 6. (A) Simulation snapshot showing the hydrogen bond interaction of MO and cholesterol with DOPS lipid molecule. The surrounding bilayer and water molecules are not shown for clarity. (B) RDF for O13B and O3 atoms as labeled in part A. (C) Simulation snapshot illustrating typical MO and cholesterol orientation around a DOPC lipid molecule in the MO−cholesterol−DOPS system.

intermediate QIIP phase was not observed for all samples. Following addition of cholesterol, which also drives the formation of lower curvature phases, a similar phase sequence was typically observed as expected, with the QIID phase transitioning to either a Lα or a QIIP phase (Figure 2).35 The addition of cholesterol did have a small effect on the phospholipid concentration, at which specific phase transitions occurred. The increase in bilayer thickness observed in the presence of cholesterol is consistent with previous research.57 The addition of some phospholipids, particularly saturated PE lipids, to MO resulted in a significant phase separation; the Lα phase is believed to be highly enriched in the pure phospholipid due to similarities between the lattice parameter of the Lα phase and the lamellar phase formed by the pure phospholipid. A previous study on phospholipid−cholesterol mixtures also showed a significant phase separation of cholesterol.58 The addition of type 0 lipids is also associated with the formation of highly swollen cubic phases (with reduced interfacial curvature) and this effect was consistently observed for many samples. In particular, the combined effect of cholesterol and a type 0 phospholipid resulted in highly swollen QIID and QIIP phases. The maximum lattice parameter observed for a QIID phase was 342.5 Å (for the system MO− cholesterol−DMPC 60:30:10 mol %). This lattice parameter is of similar magnitude to recent highly swollen cubic phases formed by MO in the presence of charged lipids (300 Å at 5 mol % of DOPS and 350 Å at 5 mol % DOPG).33 However, we note that swollen cubic phases formed in the presence of a charged lipid are anticipated to be highly unstable in the presence of salt, rendering them unsuitable for many of the applications of swollen cubic phases including in vivo applications such as drug delivery and the crystallization of larger integral membrane proteins in high salt concentrations. Previous research has shown that the presence of salt can impact phase behavior, even for zwitterionic lipids.18,59 The effect is even greater for charged lipids as the salt effectively screens the electrostatic repulsion between charged lipids as demonstrated recently by van’t Hag et al. for the addition of charged peptides to the cubic phase.60 A recent study by Brasnett et al. systematically investigated the structural behavior of cubic phases based on MO in the presence of mono- and divalent salts.61 The addition of salt to cubic phases containing the anionic lipid DOPS was found to dramatically reduce the swelling effect, with the effect dependent on both the valency and concentration of the cation.61

Type II lipids, such as the singly unsaturated DOPE, which has a CPP value >1, promoted an increase in interfacial curvature, consistent with the decrease in lattice parameter of the QIID phase and subsequent formation of the HII phase observed for this system in the presence and absence of cholesterol. Cholesterol appears to facilitate the transition to the HII phase at lower DOPE concentrations (10 mol %). This is slightly counterintuitive as cholesterol itself is a “type 0” lipid and should drive the reverse transition. However, in this case, the small molecule cholesterol may act to relieve the high packing frustration typically associated with the HII phase, thereby facilitating its formation. For PS lipids, the effect of the charged headgroup is expected to dominate phase behavior, with CPP playing a more minor role. The addition of all PS headgroup lipids resulted in a phase transition from QIID−QIIP−Lα with an increasing PS concentration, consistent with a decrease in interfacial curvature. Compared to the quaternary systems containing uncharged phospholipids (PC and PE), the addition of cholesterol had a minor effect on the overall phase behavior. This is consistent with the dominant effect of charged lipids on phase behavior.62,13 The lattice parameter of the Lα phase is again similar to the corresponding value measured for pure lipids in water, indicative of phase separation of the lipids. We note that, while swelling of the QIIP phase is observed in the presence of charged lipids, the lattice parameter of the swollen QIIP phase is significantly less than those recently reported for MO doped with DOPS and DOPG.33,47 Previous studies reported that the cubic phase formed by MO could swell to a lattice parameter of 300 to 400 Å due to electrostatic interactions when doped with charged lipids.33 This may reflect variability in solubility of the charged lipids into the bilayers. We also note that the highly swollen cubic phases previously reported were mainly observed above 35 °C. In general, phase behavior in dispersions closely reflected phase behavior in the corresponding bulk phase. However, highly swollen cubic phases appeared to be relatively unstable and did not always disperse well. Modeling results indicated that headgroup interactions drive changes in phase behavior via modifications to the CPP. Specifically, a strong electrostatic and hydrogen bond interaction between MO and DOPS, which causes a bending or lifting of the MO headgroup out of the plane of the bilayer, reduces the CPP, increasing the bilayer thickness and simultaneously reducing the self-diffusion rate for MO in the MO−cholesterol−DOPS system. As diffusion of membrane J


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sonicator in a high-throughput platform. The glass vials were sealed, and the resultant libraries were kept at room temperature for equilibration and further examination. Small-Angle X-ray Scattering (SAXS). Synchrotron SAXS experiments were performed at the small- and wide-angle X-ray scattering (SAXS/WAXS) beamline at the Australian Synchrotron. The X-ray had a beam with a wavelength of 1.033 Å (12.0 keV) with a typical flux of approximately 1013 photons/s. The sample to detector distance was 1 m, providing a q range of 0.01−0.5 Å−1 (scattering vector q = 4π sin (θ)/λ, where θ is the scattering angle and λ is the wavelength). Two-dimensional X-ray diffraction images were recorded on a Decree-Pilatus 1-M detector. The scattering images were integrated using an in-house IDL-based ScatterBrain software.63 The exposure time for each sample was 1 s. Prepared bulk samples were loaded in a 48-gel-well plate, where holes in the sample plate are covered with Kapton tape to enclose the samples. Nanoparticle samples were loaded in a standard polystyrene 96-well plate and positioned in the high-throughput plate holder at the beamline with temperatures controlled via a recirculating water bath as previously described.36 SAXS data were analyzed using the IDL-based AXcess software package to determine the identity and the lattice parameter (LP) of the lyotropic liquid crystalline mesophase.36 A silver behenate standard (LP = 58.38 Å) was used for calibration. Bilayer Model Setup and Simulation Protocol. The bilayer systems for MO−cholesterol 60:30 mol %, MO−cholesterol−DOPC 60:30:10 mol % and MO−cholesterol−DOPS 60:30:10 mol % were created using CHARMM GUI.64 All-atom molecular dynamics simulations were carried out with NAMD software (version 2.11)65 using CHARMM3666 force field and TIP3P water67 parameters. 3D periodic boundary conditions were applied to a unit cell of 9.3 × 9.7 × 8.1 nm3 dimensions. The simulations were performed in NPT ensemble with the temperature and pressure maintained at 293 K and 1 atm, respectively, via Langevin coupling with a damping coefficient of 5 ps−1. To compute the long-range electrostatic interactions with periodic boundary conditions, we employed the particle−mesh Ewald algorithm.68 Lennard−Jones interaction potential was switched off within 10−12 Å using a force-switching function. A nonbonded pair list cutoff of 16 Å was used, and a 2 fs time step was maintained throughout the simulations. The simulations were run for a total of 200 ns, and the results were analyzed for the last 100 ns following the systems’ equilibration. The lateral diffusion constant was calculated as

proteins and peptides has been shown to correlate closely with the lipid diffusion coefficient, this could have a knock-on effect on the use of these systems for applications such as in meso crystallization.53,54

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CONCLUSIONS A large library of self-assembled lipidic materials has been formulated and characterized using high-throughput protocols. These materials are based on the bicontinuous cubic phase, suitable for a range of applications including the encapsulation of bioactive molecules, but with more complex lipid compositions that more closely represent the native cell membrane. Materials with a wide range of lattice parameters are described suitable for end applications including in meso crystallization and drug delivery. Additional information on the bilayer thickness and lipid lateral diffusion coefficients was determined from molecular simulations. These parameters were found to depend on the strength of electrostatic and hydrogen bonding interactions between the lipid headgroups. A highlight of the study is the formulation of extremely swollen bicontinuous cubic phases, up to ∼342.5 Å. Some of these swollen lipidic mesophases have been successfully dispersed into swollen cubosomes. The swelling mechanism appears to be driven by the increase in curvature of the MO bilayer following the addition of one or more type 0 lipids, consistent with the lyotropic liquid crystal engineering design rules elucidated in a recent review.13 While swollen cubic phases have been previously observed, these typically contain charged phospholipids (DOPS and DOPG), which will be highly unstable under the high salt conditions associated with any physiological use.33 The highlighted swollen cubic phases formulated here contain only zwitterionic phospholipids, e.g., PC and/or cholesterol. They are therefore stable under physiologically relevant salt concentrations and may be of particular use for the encapsulation of larger therapeutic proteins and peptides, or for the crystallization of large membrane proteins such as GPCRs.

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EXPERIMENTAL SECTION

Materials. Monoolein (MO) was obtained from Nu-check-Prep, Inc. (Minnesota, USA) with purity >99% as determined by gas−liquid chromatography. Ethanol, Pluronic F127, and chloroform were purchased from Sigma-Aldrich (NSW, Australia). All phospholipids (DLPC, DMPC, DPPC, DSPC, DOPC, DLPE, DMPE, DPPE, DSPE, DOPE, DLPS, DPPS, DSPS, and DOPS) were purchased from Avanti Polar Lipids (Alabama, USA). Milli-Q H2O (18.2 MΩ) was used for all aqueous preparations. All compounds were used without further purification. High-Throughput Formulation. A stock solution of MO was prepared by dissolving pure MO in ethanol. All other phospholipids and cholesterol were dissolved in pure ethanol or ethanol−chloroform mixtures. Lipid solutions were mixed in an appropriate ratio within a glass vial. The organic solvents were then removed overnight using a centrifugal evaporator (GeneVac). The amounts of each additive added to the binary/ternary lipid systems are defined in mol % as exemplified below for MO: ij moles of MO yz zz × 100 mol % MO = jjj j total moles of lipid zz k {

D=

1 lim t →∞ < |x(t0) − x(t0 − t )|2 > 4

(3)

where x(t) is the position of the center of mass of a single lipid.41,69 Cryo-Transmission Electron Microscopy (Cryo-TEM). Sample preparation for cryo-TEM was done by using an FEI VITROBOT, where 2 μL of the nanoparticle sample was added to a C-flat Holey Carbon grid and kept for 30 s to dry, then blotted for 2 s, and straightaway dipped into liquid ethane; 65% humidity and 22 °C temperature were set on this device. The processed grids were kept in liquid nitrogen until put into a 626 model cryo holder and then imaged using an FEI Tecnai 12 transmission electron microscope operating at 120 kV. The samples were imaged at −190 °C, at a defocus level of 1.5−2 μm for all samples. The magnification used a range from 35k to 100k, and the electron dose was maintained

Toward Cell Membrane Biomimetic Lipidic Cubic Phases: A High

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