Self-assembled Lyotropic Liquid Crystalline Phase Behavior of

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Self-assembled lyotropic liquid crystalline phase behaviour of monoolein-capric acid-phospholipid nanoparticulate systems Jiali Zhai, Nhiem Tran, Sampa Sarkar, Celesta Fong, Xavier Mulet, and Calum J. Drummond Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04045 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 20, 2017

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Self-assembled lyotropic liquid crystalline phase behaviour of monoolein-capric acid-phospholipid nanoparticulate systems Jiali Zhai,1a Nhiem Tran,1a* Sampa Sarkar,a Celesta Fong,a, b Xavier Mulet,b Calum J. Drummonda* a

School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3000 Australia b

1

CSIRO Manufacturing, Clayton, Victoria 3168 Australia

These authors contributed equally to the work.

Corresponding authors: Prof. Calum J. Drummond School of Science, RMIT University, 124 La Trobe Street, Melbourne 3000, Victoria, Australia Email: [email protected] Phone: +61 3 9925 4265

Dr. Nhiem Tran School of Science, RMIT University, 124 La Trobe Street, Melbourne 3000, Victoria, Australia Email: [email protected] Phone: +61 3 9925 2131

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Abstract We report here the lyotropic liquid crystalline phase behaviour of two lipid nanoparticulate systems

containing

mixtures

of

monoolein,

capric

acid

and

saturated

diacyl

phosphatidylcholines dispersed by the Pluronic F127 block copolymer. Synchrotron small angle X-ray scattering (SAXS) was used to screen the phase behaviour of a library of lipid nanoparticles in a high-throughput manner. It was found that adding capric acid and phosphatidylcholines had opposing effects on the spontaneous membrane curvature of the monoolein lipid layer and hence the internal mesophase of the final nanoparticles. By varying the relative concentration of the three lipid components, we were able to establish a library of nanoparticles with a wide range of mesophases including at least the inverse bicontinuous primitive and double diamond cubic phases, the inverse hexagonal phase, the fluid lamellar phase and possibly other phases. Furthermore, the in vitro cytotoxicity assay showed that the endogenous phospholipid-containing nanoparticleswere less toxic to cultured cell lines compared to monoolein-based counterparts, improving the potential of the non-lamellar lipid nanoparticles for biomedical applications.

Keywords: monoolein, fatty acid, phospholipid, self-assembly, nanoparticles, cubosomes, hexosomes,

SAXS,

high-throughput,

lyotropic


liquid

crystals

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1. Introduction Amphiphilic lipids in water display a rich polymorphism with one-, two- and threedimensional nanostructures such as the lamellar, cubic and hexagonal polymorphs occurring as a function of the interplay between the hydrophobic effect and geometric packing constraints.1, 2, 3 Over the past two decades, lipid nanoparticles with the inverse hexagonal and the inverse cubic nanoarchitectures, named hexosomes and cubosomes respectively, have attracted considerable interest in the development of next generation nanocarriers for drug delivery.4, 5, 6 A wide variety of compounds including hydrophobic and hydrophilic drugs,7, 8, 9, 10

nucleic acids,11,

12, 13

proteins,14,

15, 16, 17

and imaging agents18,

19, 20, 21

have been

incorporated into hexosomes and cubosomes. The amphiphilic nature and complex structure of the internal mesophases in hexosomes and cubosomes offers increased solubilisation and protection of the payloads. The large internal surface area (up to 400 m2g-1)22 and aqueous pore network of the cubic and the hexagonal phase enable enhanced payload and controlled release from these systems.23,

24, 25

Emerging in vitro and in vivo studies have reported

improved therapeutic efficacy of encapsulated drugs7, mechanisms of nanoparticle-cell interactions27,

8, 26

28, 29, 30, 31

and also provided insights into and

behaviours in biological

bodies.8, 32 From the physicochemical to the pharmacokinetics and biodistribution properties , it was identified that the internal lyotropic liquid crystalline nanostructure of lipid-based nanoparticles is a key parameter that may be exploited for manufacturing “fit-for-function” materials as nanoparticulate drug delivery systems. The design and fine control of nanoscale structure is still a major challenge. Demand for personalised medicine through integrating excipients for multi-platform capabilities in therapeutics and diagnostics is driving the desire for smarter nanomaterials with controllable multi-functionality. A much used strategy for engineering nanostructure is the compositional 3 ACS Paragon Plus Environment

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mix of various lipids that have different molecular shapes to vary the effective critical packing parameter (CPP)3,

33

and hence the interfacial curvature. The CPP can provide a

rough indication to the self-assembly morphology as it defines amphiphiles by their molecular shapes, whereby CPP = v/aHL, where v is the hydrophobic tail volume, aH is the headgroup area, and L is the hydrophobic chain length. For example, if the amphiphiles consist of a hydrophilic headgroup and a hydrophobic tail with a similar cross-section area (CPP ≈ 1), they will form the lamellar phase (Type 0).1 If the amphiphile headgroup is smaller than the tail (CPP > 1), inverse phases (Type II) with negatively curved surfaces are formed.1 A theoretical sequence of mesophases arranged with increasing surface negative curvature is presented in Figure 1. Various mesophases including the lamellar, the inverse bicontinuous cubic phases with the Im3m (QIIP) and Pn3m (QIID) crystallographic space group, the inverse hexagonal phase (HII), and the inverse micellar phase (L2) may be obtained through compositional variation.34, 35, 36, 37, 38

Figure 1: Some commonly observed lyotropic liquid crystalline phases of amphiphilic lipid self-assemblies. In order of increasing negative interfacial curvature, these are the fluid lamellar phase (Lα), the inverse bicontinuous cubic phases with crystallographic space group Im3m (QIIP) and Pn3m (QIID), the inverse hexagonal phase (HII) and the inverse micellar phase (L2).


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For example, oils and fatty acids when added to amphiphilic lipids such as monoolein (MO) and phytantriol have been shown to drive the original inverse cubic phases toward more negatively curved structures.36, 37, 39 For example, capric acid (CA) is known to drive a cubic to hexagonal phase transition in MO nanoparticles.40 Coexisting mesophases in discrete lipid nanoparticles were also observed in the MO-CA system that contains bi-phasic QIIP-QIID or QIID-HII nanostructures within a single nanoparticle.41 In contrast, selected phospholipids and cholesterol are believed to swell the membranes and direct the formation of mesophases with less curved surfaces.42 For example, a highly swollen cubic structure with large tunable pore size was observed in a ternary mixture of MO, cholesterol and charged phospholipids.42 The synergistic effect of cholesterol that promotes membrane stiffness together with the electrostatic repulsion of the headgroups of the charged phospholipids, led to the formation of a swollen QIIP phase with a lattice parameter (LP) a = 470 Å, that has potential for solubilising large therapeutic proteins and vaccine molecules.42 Study of complex ternary lipid systems is therefore highly desirable due to the greater flexibility to fine tune the internal nanostructures so that they are “fit-for-function”. However, the number of ternary lipid systems in dispersed form is still limited. The aim of the study is to investigate the compositional space for cubosomes and hexosomes that are based on MO but enriched with two endogenous membrane lipid additives, CA and saturated diacyl phosphatidylcholines (Figure 2) using combinatorial high-throughput formulation and analysis. It was of interest to understand how incorporating two common endogenous lipids, having different molecular shapes, to MO would fine tune the lipid membrane curvature and change the cytotoxicity profile of the formed cubosomes and hexosomes that may facilitate biological studies. Specifically, we report the lyotropic liquid crystalline phase behaviour of two lipid nanoparticulate systems, namely MO-CA-DLPC and MO-CA-DSPC, dispersed by block copolymer Pluronic F127 in water. CA, a saturated fatty acid with a decyl (C10)


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hydrocarbon chain, was added to increase the negative surface curvature of the Type II “parent” MO cubic phase, as indicated in our previous study.36, 40 In contrast, saturated diacyl phosphatidylcholines, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), have a preference for less negatively curved surfaces and thus are predicted to drive towards the formation of a lamellar liquid crystalline phase. These ternary systems allowed us to access a wide range of the inverse cubic and hexagonal nanostructures in lipid nanoparticles while comprising up to 30 wt% of lamellar-forming endogenous phospholipids. Following the discovery, a prospective cytotoxicity assay on Chinese Hamster Ovary (CHO) cells demonstrated the potential of improved cell viability with these lipid systems. This finding provides incentives for further exploration of ternary lipid mixtures that will facilitate fine tuning of the lyotropic non-lamellar mesophases in the nanoparticulate form and their subsequent biomedical applications.

Figure 2: Chemical structures of monoolein (MO), capric acid (CA), and 1,2-dilauroyl-snglycero-3-phosphocholine (DLPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). *Note: effective headgroup size of CA is dependent of pH. The structure depicted here represents CA at low pH, such as in this study. At higher pH, deprotonation of fatty acid molecules leads to a larger headgroup. 43 6 ACS Paragon Plus Environment

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2. Materials and Methods 2.1 Materials Monoolein (MO, 1-isomer) was obtained from Nu-check-Prep, Inc (Elysian, MN, USA). with purity >99% as determined by gas liquid chromatography. Capric acid (CA), Pluronic F127, ethanol, and chloroform were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-sn-glycero3-phosphocholine (DSPC) were purchased from Avanti Lipids (Alabaster, AL, US). Milli-Q water (18.2 MΩ) was used for all aqueous preparations. All compounds were used without further purification. 2.2 High throughput preparation of the ternary lipid nanoparticulate systems MO and CA stock solutions were prepared in ethanol. DLPC and DSPC stock solutions were prepared in chloroform. Desired mixtures of lipids were added to each well of a 96-well deep well block (Greiner Bio-One, Kremsmünster, Austria). The organic solvents were then removed overnight using a centrifugal evaporator (GeneVac, Ipswich, UK). The amounts of phospholipids and/or CA added to the ternary lipid nanoparticulate systems are defined by the following ratios: α represents the ratio of the amount of phospholipid (either DLPC or DSPC) to the total amount of the ternary lipid mixture in weight; β represents the ratio of the amount of CA to the amount of MO lipid in weight. A 96-well plate setup for the composition of each of the sample of the material libraries is given in Supporting Information Table S1.


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Pluronic F127 in milli-Q water was added to the dried lipid mixtures at 10 wt% to the total amount of bulk lipids. The final total lipid concentration in the dispersed sample was fixed at 20 mg/ml. The samples were then sonicated by an automated probe sonicator on a Chemspeed platform (Chemspeed Technologies, Füllinsdorf, Switzerland) in a highthroughput manner as previously described.36 A 10 wt% concentration of Pluronic F127 has been a common approach to make nanoparticulate dispersions of a range of lipid mixtures in previous studies.40, 44 Representative particle sizes and polydispersities of the nanoparticles is also provided in the Supporting Information. After sonication, the plate was sealed and the resultant libraries were kept at room temperature for further examination. To validate the high-throughput screening procedure and show repeatability, the libraries of materials in this study were formulated independently at least twice using the Chemspeed automated platform and screened using the synchrotron SAXS setup. Furthermore, selected samples (Table 1) were manually prepared using a microprobe sonicator (Qsonica, Newtown, CT, USA) following a protocol of 30% amplitude, 5 second-on, 5 second-off, and 3 minute duration. 2.3 High-throughput small angle X-ray scattering (SAXS) characterisation Synchrotron SAXS experiment were performed at the SAXS/wide-angle X-ray scattering (WAXS) beamline at the Australian Synchrotron at 25 °C. 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 chosen as 1.6 m which provided 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 Decris-Pilatus 1-M detector using an in-house IDL-based ScatterBrain software.45 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). The exposure time for each sample was 1 s. 8 ACS Paragon Plus Environment

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Prepared samples were loaded in a 96-well, half-area UV-clear plate (Greiner Bio-One) and positioned in the high-throughput plate holder at the beamline with temperatures controlled via a recirculating water bath as previously described. SAXS data were analysed using the IDL-based AXcess software package to determine the identity and the LP of the internal lyotropic liquid crystalline mesophase. Nanoparticle mesophases were identified using the spacing ratios of peaks in the one dimensional plot (Supporting Information Figure S1).40 46 2.4 Cryogenic-Transmission electron microscopy (Cryo-TEM) Cryo-TEM was used to visualise the formulated nanoparticles. Copper grids (200mesh) coated with perforated carbon film (Lacey carbon film, ProSci Tech, 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, grids were blotted manually by filter paper for approximately 3 s. 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 120KV. 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 a FEI Eagle 4k x 4k CCD camera at magnifications ranging from 15000x to 50000x. 2.5 Cell viability assay Chinese Hamster Ovary (CHO: ATCC CCL-061) cells were grown in MEM α modification

media

supplemented

with

10%

fetal

bovine

serum

and

1%

penicillin/streptomycin, and adjusted to pH 7.2 (Thermo Fisher Scientific, Scoresby, Vic, Australia). Cells were grown at 37 °C with 5% CO2 and subcultured twice weekly. Prior to the experiment, selected nanoparticles (Table 1) were serially diluted with milli-Q water. CHO cells were seeded at 5000 cells per well in 96-well tissue culture plates together with 9 ACS Paragon Plus Environment

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nanoparticles. The highest and lowest nanoparticle concentrations tested were 200 µg/mL and 6.25 µg/mL respectively. Non-treated cells were used as controls. After adding the nanoparticles at the desired concentration, the cells were incubated for an additional 48 hours in the standard cell culture environment Alamar Blue assay (Thermo Fisher Scientific, Scoresby, Vic, Australia) is a colorimetric assay for evaluating cell viability. In order to reduce interference of nanoparticles with the assay, the cells were rinsed with PBS to remove excess nanoparticles. After the final rinse, 200 µL of fresh cell media was added to each well together with 20 µL Alamar Blue solution. The cells were incubated at 37 °C for another 3 hours then analysed using a spectrophotometer at 562 nm absorbance. The absorbance was compared to the absorbance at a reference wavelength of 630 nm. Optical densities were normalised to the non-particle control and plotted against lipid concentration. 2.6 Statistical analysis The cell viability experiments were performed in triplicates and were repeated three times. Statistical analysis was performed using two-tail Student’s t-test with p < 0.05 considered statistically significant. The concentration of lipid nanoparticles required to reduce the cell viability by half (IC50) was calculated using the ED50 Plus v1.0 online software.


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3. Results and Discussion 3.1 Effect of adding capric acid (CA) The MO-CA system demonstrated preference for mesophases with negative interfacial curvature (Type II), namely QIIP, QIID and HII phases depending on the ratio of CA to MO, (β) (Figure 3). Consistent with previous report,40 MO cubosomes stabilised by Pluronic F127 exhibited a QIIP phase (α = 0, β = 0) with a LP of 138 Å that tolerated the addition of 5 wt% CA (α = 0, β = 0.05) without perturbation of the nanostructure (Figure 3A&B). A preference for increasing negative interfacial curvature was observed with higher CA concentrations. Specifically, a phase progression from QIIP to QIID and HII occurred at 11 wt% CA (α = 0, β = 0.11) and at 25 wt% CA (α = 0, β = 0.25) respectively (Figure 3C&D). Cyro-TEM images (Figure 4) confirmed the presence of the ordered pattern of the cubic phase in the pure MO nanoparticles (α = 0, β = 0) and the characteristic fingerprint pattern of the hexagonal phase in the MO-CA nanoparticles (α = 0, β = 0.25). The LP decreased gradually as a function of CA concentration: QIIP (138 Å) > QIIP (119 Å) > QIID (80 Å) > HII (49 Å) (Supporting Information Table S2). This phase progression can be rationalised by consideration of the CPP. Amphiphiles such as MO have CPP > 1 as it has a relatively small headgroup area compared to its hydrophobic chain volume, to promote the formation of inverse Type II structures.47 The addition of CA to MO-based system effectively increases the CPP as demonstrated by the QIIP→QIID→HII phase transition. It should be noted that previous studies have shown that fatty acids such as CA and oleic acid (OA) are pH sensitive.40, 43 The ionisation behaviour of long chain fatty acids, such as CA, residing within amphiphile self-assemble objects is markedly different from that observed for short chain analogues in aqueous solution.48, 49, 50 The apparent pKa is significantly higher than the pKa in water due to interfacial 11 ACS Paragon Plus Environment

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microenvironment and surface charge effects. Under these circumstances at a solution pH in the range of 4.8 to 5.0 (Supporting Information Table S3), CA in the will exist in the protonated form.48 Therefore, it is likely that the phase transition reported in this study may be largely due to the concentration effect of CA instead of ionization of the headgroup, consistent with previous studies.40, 51

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Figure 3: The synchrotron SAXS diffraction pattern of the ternary MO-CA-DLPC nanoparticulate system dispersed by Pluronic F127. α represents the ratio of the amount of phospholipids (DLPC) to the total amount of the ternary lipid mixtures in weight; β represents the ratio of the amount of CA to the amount of MO lipid in weight.

Figure 4: Representative cyro-TEM of multilamellar vesicles (A, Sample α = 0.4, β = 0.25), cubosomes (B, Sample α = 0, β = 0), and hexosomes (C, Sample α = 0, β = 0.25).


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3.2 Effect of adding DLPC and DSPC Building upon our study on the binary MO-CA system,36 we have extended the compositional space by the further addition of saturated diacyl chain-containing phosphatidylcholines (DLPC and DSPC), that are the building blocks of cell membranes. Membrane-forming phosphatidylcholines generally have similar cross-sectional areas for the headgroup and the two acyl tails (CPP = 0.8 to 1.0)52 and thus their molecular geometry is more or less cylindrical. These phosphatidylcholines are known to arrange in lamellar layers,52,

53, 54

and in this study their addition to the binary lipid MO-CA systems was

anticipated to decrease negative interfacial curvature, opposing the effect of CA. Whilst keeping the MO-CA ratio fixed in a particular binary system, we replaced the MO-CA binary mixture with DLPC or DSPC in a 10 wt% stepwise manner up to 100 wt%, at which point the system contains only phospholipids. In doing so we were able to obtain a gradual nonlamellar to lamellar phase transition with finely-tuned LPs in these ternary lipid mixture systems, namely MO-CA-DLPC and MO-CA-DSPC. Furthermore, we were able to demonstrate improved cellular tolerance in nanoparticles containing more endogenous phospholipids than the pure MO or the binary MO-CA systems to increase the compositional window for studying cellular interactions with cubosomes and hexosomes. 3.2.1 MO-CA-DLPC nanoparticulate system Figure 3 presents the phase behaviour of the MO-CA-DLPC nanoparticulate system dispersed by Pluronic F127. The LP of all the observed non-lamellar phases is given in Supporting Information Table S2. The results revealed the opposing effect of DLPC and CA in changing the interfacial curvature. While CA increased the negative interfacial curvature as discussed above, DLPC decreased the curvature and hence supressed the non-lamellar phase. In the absence of CA, replacing 10 wt% of MO with DLPC (α = 0.1, β = 0) was sufficient to disrupt the MO QIIP phase and the system displayed no long-range periodic


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structure or ordering (Figure 3A). Increasing DLPC composition in the lipid mixture led to the appearance of a broad diffraction peak due to the increased population of liposomes with the fluid lamellar phase, consistent with previous studies.55, 56 As the CA contribution increased relative to MO, increased DLPC was also required to promote the transition to non-lamellar nanostructures. At 5 wt% CA to MO (Figure 3B), coexistence of QIIP (LP = 119 Å) and QIID (LP = 90 Å) nanostructure was observed. The incorporation of 10 wt% DLPC led to the formation of QIID (LP = 90 Å) with an increase in the LP to 146 Å. At 15 wt% DLPC (α = 0.15, β = 0.05) the presence of the cubic phase was completely suppressed. At 11 wt% CA to MO (Figure 3C), the initial QIID (LP = 80 Å) underwent a gradual transition to the QIIP with increasing DLPC concentration, up to 20 wt% (α = 0.2, β = 0.11), before a complete loss of cubic phase at 25 wt% DLPC (α = 0.25, β = 0.11). At 25 wt% CA to MO, the nanoparticles exhibited HII phase behaviour (α = 0, β = 0.25) (Figure 3D). Interestingly, the HII phase was retained up to 20 wt% DLPC incorporation, although at this point, a less curved QIID phase also appeared. The LP of the HII phase increased from 49 Å in the binary MO-CA mixture to a maximum of 56 Å in the presence of 20 wt% DLPC. As anticipated, the incorporation of DLPC led to a gradual decrease in the membrane curvature. Specifically, the internal mesophase showed a phase transition from QIID at α = 0.2 to QIIP at α = 0.3 then to a lamellar phase. The associated LP increased in the order HII (49 – 56 Å) < QIID (93 Å) < QIIP (135 Å). In several samples of the MO-CA-DLPC system, co-existence of multiple phases are observed. For example, both QIIP and QIID were present in the sample of (α = 0.1, β = 0.11). For the system (α = 0.2, β = 0.25), two phases (QIID and HII) were also present. These may imply anisotropy within some of the nanoparticles as previously observed for the MO-CA system41 though this was not explored further.


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It was found that regardless of the MO-CA ratio, when more than 35 wt% of the total lipid mixture was replaced with DLPC, the internal phase structure exhibited mainly the fluid lamellar liquid crystalline phase characterised by a broad peak over a large q range (0.12 to 0.22). A random sample (α = 0.4, β = 0.25) representing the characteristic lamellar SAXS profile was examined under the cryo-TEM, which confirmed the presence of the multilamellar vesicles (Figure 4A). It is also worth noting the possible occurrence of the less ordered sponge L3 phase in the MO-CA-DLPC nanoparticulate system. Although cryo-TEM experiment did not directly discover any sponge phase in the current study, several studies have previously shown a phase transition from the ordered MO cubic phase to the less ordered sponge phase when dioleoylphosphatidyalglycerol (DOPG) or diglycerol monooleate (DGMO) was added to the final lipid mixture.57,

58

The sponge phase remains the

bicontinuous nature of the lipid layer but does not display the long-range crystalline order and therefore may occur before the final transition to the fluid lamellar phase when DLPC was gradually added to the MO-CA binary system. In order to search for the sponge phase if of interest, it is recommended to conduct careful SAXS fitting and cryo-TEM experiments in the compositional space of the low DLPC-containing samples where the cubic or the hexagonal patterns start to disappear. 3.2.2 MO-CA-DSPC nanoparticulate system Figure 5 presents the phase behaviour of the MO-CA-DSPC nanoparticulate system dispersed by Pluronic F127 in water. The LP of all the observed non-lamellar phases is given in Supporting Information Table S2. Similar to the DLPC system, the addition of DSPC to MO-CA mixture promoted flatter interfacial curvatures with the lamellar phase dominant at high DSPC content. However there are some interesting features of the MO-CA-DSPC system relative to MO-CA-DLPC. Firstly, DSPC was more effective in disrupting the nonlamellar phase than DLPC at similar concentrations. For example, Figure 5B shows that it


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required 10 wt% (equivalent to 5 mol%) of DSPC to supress the QIIP phase of the starting MO-CA binary system (α = 0.1, β = 0.05), 5wt% less than DLPC which supresses the QIIP phase at 15 wt% (equivalent to 10 mol%). When DSPC was incorporated into the QIID phase of the MO-CA binary system (α = 0, β = 0.11), the QIID phase was disrupted significantly and, unlike the DLPC-based system, the transition to the QIIP phase was weak (Figures 3C and 5C) with only very small Bragg peaks corresponding to a QIIP phase observed at 15 wt% (equivalent to 8.8 mol%) DSPC (α = 15, β = 0.11). In the MO system enriched with 25 wt% CA (α = 0, β = 0.25), the HII phase was preserved up to higher concentrations of DSPC (up to 30 wt% or 24 mol%) compared to DLPC (up to 20 wt% or 18 mol%). (Figures 3D and 5D). -

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Figure 5: The synchrotron SAXS diffraction pattern of the ternary MO-CA-DSPC nanoparticulate system dispersed by Pluronic F127. α represents the ratio of the amount of phospholipids (DSPC) to the total amount of the ternary lipid mixtures in weight; β represents the ratio of the amount of CA to the amount of MO lipid in weight.

The difference in the lyotropic liquid crystalline behaviour of the two nanoparticulate systems, namely MO-CA-DLPC and MO-CA-DSPC, may be explained by the different lengths of the double acyl chains (C18 for DSPC versus C12 for DLPC). Kumar studied the CPP values of phosphatidylcholines as a function of the saturated acyl chain length but only


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showed a marginal increase from 0.75 for DLPC to 0.80 for DSPC.52 However, the longer tails of DSPC can “extend” and “fill” the void space existing in between the hexagonally arranged cylinders in the HII phase. It is estimated that a hexagonally packed symmetry generates around 9% void space.59 The extension into the void space by the long tails of DSPC is believed to relieve the higher degree of packing frustration in the HII phase relative to the cubic phase.33 Therefore, in contrast to the MO-CA-DLPC system, which displayed a gradual phase transition in the sequence of HII→QIID→QIIP→Lα (Figure 3D), retention of the HII phase in the MO-CA-DSPC system was observed (Figure 5D). A similar trend was observed in a previous study where DSPC showed higher propensity for packing into the HII phase than DLPC when fatty acids were added to the lipids under elevated temperatures above 50 °C.60 Another interesting observation of the MO-CA-DSPC system is in the range of 50-80 wt% DSPC, where the broad lobe in the scattering pattern at high α ratio, this was overlaid with fine structure corresponding to a mixed population of lipid vesicles. The small undulation of broad peaks corresponding to the Lα phase is consistent with previous reports from SAXS experiments on some phospholipid based unilamellar or multilamellar liposomes.61,

62, 63

Sharp peaks corresponding to the crystalline lamellar phase (Lc) were

observed in two MO-CA-DSPC mixtures (α = 0.6/β = 0.11 and α = 0.8/β = 0.25 in Figure 5). This occurrence of the Lc phase is attributed to the high crystalline-to-gel transition temperature (Tm = 55 °C) of DSPC.53 Therefore, at the temperature (25 °C) of the SAXS experiment, locally concentrated crystalline DSPC bilayers from non-homogeneous mixing may have crystalline packing of the acyl chains, resulting in the Lc phase scattering. In contrast, DLPC has a Tm of −1 °C,53 so that these form Lα bilayers as indicated by the broad diffraction peak (Figure 3).


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A previous study incorporated charged and/or unsaturated phospholipids such as dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylserine (DOPS) to MO and also reported a preference for the flat lamellar structure.42 Interestingly these systems adopted a QIIP phase with LP up to 365 Å, attributed to swelling effect of the electrostatic repulsion between the charged lipid bilayers.42 Logically, zwitterionic bilayers of DLPC and DSPC investigated in this study have limited electrostatic repulsion and hence did not show this swelling effect when incorporated into the MO-CA mixture, resulting in tuneable LPs across various compositions (Supporting Information Table S2). 3.3 Cytotoxicity of the nanoparticles and implications The applicability of cubosomes and hexosomes as drug delivery system depend largely on their toxicity. The in vitro cytotoxicity and haemolysis assays have been a standard preliminary test to provide quick safety information for nanoparticle-based materials. Recent studies have shown the relative cytotoxic and haemolytic effect of the MO-based cubosomes and hexosomes..28, 31, 40, 64, 65, 66, 67 Multiple factors may come into play in determining their cytotoxicity profiles. Tran et al (2015) investigated the MO-CA nanoparticle toxicity to L929 fibroblast cells and proposed that the mesophase nanostructure may influence the cytotoxicity effect.40 CA-enriched MO-based hexosomes and emulsions were found to be less toxic than cubic phase counterparts. Other studies found that in vitro cytotoxicity properties of cubosomes may be moderated by better surface coverage using stabilisers other than the tri-block copolymer Pluronic F127.31, 65 Both lipid-PEG conjugates31 and custom-made brush polymers65 can stabilise cubosomes of less toxicity than Pluronic F127 counterparts. Another study reported an increased cytotoxicity to L-929 fibroblasts when phytantriol cubosomes were incorporated with dipalmitoyl phosphatidylserine (DPPS), despite DPPS being a naturally occurring lipid in mammalian cell membranes.64 The authors postulated that the counterintuitive effect was due to the mixed population of phytantriol cubosomes and DPPS


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vesicles, promoting membrane fusogenic potential and the cubic-lamellar phase transition.64 On the other hand, cubosomes made of soy phosphatidylcholines (SPC) and food-grade emulsifier Citrem were found to be less haemolytic and to have less activation of the complement effector functions than the MO-based cubosomes, suggesting the role of naturally occurring phospholipids in the particles’ toxicity profile.68, 69 However, most studies realised that there might be several factors that can affect cytotoxicity of nanoparticles, including phase, size, charge, and protein adsorption on nanoparticle surfaces. Table 1: Composition, phase structure and lattice parameters (LP) of selected samples MOCA, MO-CA-DLPC and MO-CA-DSPC nanoparticulate systems. MO-CA Sample MO-CA#1

α 0

β 0

Phase

LP (Å)

P

139

D

80

QII

MO-CA#2

0

0.11

QII

MO-CA#3

0

0.25

HII

α

β

DXPC#4

0.2

0.11

QIIP

147

Lα

-

DXPC#5

0.2

0.25

HII/QIID

56/87

HII

56

135

HII

60

DXPC#6

0.3

0.25

49

MO-CA-DLPC Phase LP (Å)

QII

P

MO-CA-DSPC Phase LP (Å)

Note: α represents the ratio of the amount of phospholipids (either DLPC or DSPC) to the total amount of the ternary lipid mixtures in weight; β represents the ratio of the amount of CA to the amount of MO lipid in weight. Refer to Table S2 for a complete analysis of all investigated nanoparticulate systems displaying non-lamellar mesophases. Samples DxPC#4#6 refer to sample names of either DLPC#4-#6 or DSPC#4-#6. Hydrodynamic diameters of these nanoparticles in water and cell culture media were measured by dynamic light scattering method. The data are presented in Supporting Information Table S4 and S5.

In order to study whether the incorporation of the phosphatidylcholines into cubosomes and hexosomes could change their in vitro cytotoxicity, we selected representative particles in the current material library, in particular samples that contained the highest amounts of


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DLPC or DSPC but retained either the cubic or the hexagonal phases to compare with the MO-based counterparts (Table 1). All formulations listed in Table 1 showed toxicities towards the CHO cells at concentrations above 100 µg/mL (Figure 6). At 12.5 µg/mL or lower, all formulations did not show significant toxicity with cell viabilities above 80%. Within the investigated range, the toxicity effect can then be quantified by the nanoparticle concentration needed to reduce half of the cell viability, i.e. the IC50 value. The binary MO-CA based nanoparticles exhibited marginally different toxicity with starting mesophases in the order of QIIP (MO-CA#1) > QIID (MO-CA#2) > HII (MO-CA#3), the corresponding IC50 values being 40 µg/mL, 43 µg/mL, and 46 µg/mL respectively (Figure 6C). Consistently, a previous study reported similar IC50 values of ca.40 µg/mL for cubosomes containing similar MO-CA binary mixture as sample MO-CA#1 and sample MO-CA#2.40 However, that study found hexosomes (MO-CA#3) exhibiting lower in vitro toxicity having an IC50 of ca. 64 µg/mL40 as compared to 46 µg/mL in the current study. The discrepancy may be attributed to different cell lines (fibroblast L929 versus CHO cells) and/or different toxicity assays (MTS assay versus Alamar Blue assay). When the binary MO-CA binary mixture was partially replaced by DLPC or DSPC, there was a significant effect of compositional variation on the in vitro toxicity. For example, in the MO-CA-DLPC nanoparticulate systems, at 50 µg/mL, cell viability reached 57% for 20 wt% DLPC (DLPC#4) and up to 70% for a 30 wt% DLPC (DLPC#4), a 24%-37% increase on the original MO-CA based cubosomes (Figure 6A). The IC50 values increased to 48 µg/mL and 55 µg/mL with 20 wt% and 30 wt% DLPC respectively (Figure 6C). In the MO-CA-DSPC system, it is worth noting that the nanoparticles (DSPC#5 and DSPC#6) had significantly lower toxicity than DSPC-absent counterpart (MO-CA#3) with cell viabilities reaching 70% at 50 µg/mL treatment and IC50 reaching 62 µg/mL.


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In summary, the cell viability assay on CHO cells suggests that by replacing monoolein with phospholipids nanoparticles of similar nanostructures can be obtained but with lower in vitro toxicity. However, whether the mechanism of such improvement is mesophase related remains unclear because the phase likely changes once the nanoparticles are added to cell media. To fully understand how mesophases influence the interaction with cells it is best to study the phase of nanoparticles once they are in contact with cells. However, this is a difficult task due to technological challenges with SAXS at low nanoparticle concentration, at which the background scattering noise of proteins in cell media may be higher than the scattering from the nanoparticle themselves. Additional in vitro studies including cellular uptake pathway of nanoparticles, haemolytic activity, and intracellular lipid profiles will help elucidate the mechanism of the cell-nanoparticle interactions.


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Figure 6: CHO cell viability of (A) MO-CA-DLPC and (B) MO-CA-DSPC nanoparticles measured by Alamar Blue assays. (C) IC50 values. Table 1 lists the composition for each nanoparticle and nomenclature. Data = mean ± SEM. (N=3). * p

Self-assembled Lyotropic Liquid Crystalline Phase Behavior of

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