Lipid–PEG Conjugates Sterically Stabilize and Reduce the Toxicity of

Sep 11, 2015 - CSIRO Biosecurity Flagship, Australian Animal Health Laboratory, ... CSIRO Manufacturing Flagship, 343 Royal Parade, Parkville, VIC 305...
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Lipid−PEG Conjugates Sterically Stabilize and Reduce the Toxicity of Phytantriol-Based Lyotropic Liquid Crystalline Nanoparticles Jiali Zhai,*,† Tracey M. Hinton,‡ Lynne J. Waddington,§ Celesta Fong,†,∥ Nhiem Tran,† Xavier Mulet,† Calum J. Drummond,∥ and Benjamin W. Muir*,† †

CSIRO Manufacturing Flagship, Private Bag 10, Clayton, VIC 3169, Australia CSIRO Biosecurity Flagship, Australian Animal Health Laboratory, 5 Portarlington Road, East Geelong, VIC 3219, Australia § CSIRO Manufacturing Flagship, 343 Royal Parade, Parkville, VIC 3052, Australia ∥ School of Applied Sciences, College of Science, Engineering and Health, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia ‡

ABSTRACT: Lyotropic liquid crystalline nanoparticle dispersions are of interest as delivery vectors for biomedicine. Aqueous dispersions of liposomes, cubosomes, and hexosomes are commonly stabilized by nonionic amphiphilic block copolymers to prevent flocculation and phase separation. Pluronic stabilizers such as F127 are commonly used; however, there is increasing interest in using chemically reactive stabilizers for enhanced functionalization and specificity in therapeutic delivery applications. This study has explored the ability of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with poly(ethylene glycol) (DSPE-PEGMW) (2000 Da ≤ MW ≤ 5000 Da) to engineer and stabilize phytantriolbased lyotropic liquid crystalline dispersions. The poly(ethylene glycol) (PEG) moiety provides a tunable handle to the headgroup hydrophilicity/hydrophobicity to allow access to a range of nanoarchitectures in these systems. Specifically, it was observed that increasing PEG molecular weight promotes greater interfacial curvature of the dispersions, with liposomes (Lα) present at lower PEG molecular weight (MW 2000 Da), and a propensity for cubosomes (QIIP or QIID phase) at MW 3400 Da or 5000 Da. In comparison to Pluronic F127-stabilized cubosomes, those made using DSPE-PEG3400 or DSPE-PEG5000 had enlarged internal water channels. The toxicity of these cubosomes was assessed in vitro using A549 and CHO cell lines, with cubosomes prepared using DSPE-PEG5000 having reduced cytotoxicity relative to their Pluronic F127-stabilized analogues.



INTRODUCTION

phase (Lα) to the inverse bicontinuous cubic phase (QII) and the inverse hexagonal phase (HII).10,11,16 These liquid crystalline phases are thermodynamically stable and can be mechanically dispersed to form nanoparticles, termed “liposomes”, “cubosomes”, and “hexosomes”, respectively.17−19 The structure of the lipid lyotropic liquid crystalline phases has been described in detail elsewhere; in addition, several reviews have summarized the bioactives which have been incorporated into these matrices for drug delivery to date.2,20,21 Chong et al. recently reviewed and categorized the different types of steric stabilizers that have been reported in the literature for preparing cubosomes and hexosomes.22 Pluronic F127, an amphiphilic block copolymer, is the most commonly used stabilizer and comprises poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO−PEO) blocks (Figure 1), wherein the PPO hydrophobic block

The therapeutic and diagnostic applications of lipid lyotropic liquid crystalline phases and their corresponding nanoparticles as drug delivery systems have attracted growing interest.1−3 Studies have shown that these nanoparticles retain the porous structure of the “parent” liquid crystalline phase for improved drug loading and release and provide a nonviscous formulation for easy administration.4−8 In particular, the inherent high surface area and extensive mesoporosity of the inverse bicontinouse cubic (QII) and the inverse hexagonal (HII) phases provide advantages such as enhanced payload for the delivery of both hydrophilic and hydrophobic drugs, protection against enzymatic degradation, and controlled release.4−9 Lipids such as glyceryl monooleate (GMO) and phytantriol self-assemble in excess water to form lyotropic liquid crystalline architectures that are characterized by long-range periodicity, while simultaneously demonstrating short-range disorder at atomic distances.10−14 The phase behavior of these lipids is well characterized,15 and under conditions of increasing temperature and/or water content, there is a conversion of the lamellar © 2015 American Chemical Society

Received: July 30, 2015 Revised: September 9, 2015 Published: September 11, 2015 10871

DOI: 10.1021/acs.langmuir.5b02797 Langmuir 2015, 31, 10871−10880

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conventional liposomes.37 With the broad availability and flexibility in terms of hydrophilicity and functionality (maleimide, thiol, amine, biotin) of the PEGylated phospholipids, there are many possibilities for the use of these materials in this fashion; therefore, it is imperative to understand how DSPE-PEG’s interact and stabilize nanoparticles in a systematic manner. The current work aimed to improve understanding of the formation and stability of phytantriol-based lyotropic liquid crystalline dispersions, stabilized by DSPE-PEG’s without the need for Pluronic F127 with a focus on the influence of the PEG chain length and concentration. The molecular structures of phytantriol and the stabilizers used in this study are given in Figure 1. The internal liquid crystalline nanostructures were examined by synchrotron small-angle X-ray scattering (SAXS), and cryogenic-transmission electron microscopy (cryo-TEM) was used to study their morphology. Finally, preliminary assessments of their stability and cytotoxicity were conducted. Specifically, the stability of the internal mesophases and the size distribution of the nanoparticles were examined in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and the cytotoxicity toward two common cell lines was tested. Analogous studies have been conducted upon Pluronic F127-stabilized nanoparticles for comparison.

Figure 1. Molecular structures of phytantriol, 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol)] (ammonium salt) (DSPE-PEGMW), and Pluronic F127. The number of the PEG units in the DSPE-PEG’s is represented in n.

partitions into the lipid bilayer, and the long hydrophilic PEO block is extended from the lipid layer into the aqueous medium to form a sterically repulsive corona around the nanoparticle surface (commonly known as PEGylation).23,24 In addition to steric stabilization, PEGylation has an additional “stealth” effect in vivo by interfering with plasma protein adsorption and macrophage-mediated foreign body responses, thus improving the pharmacokinetics and biocompatibility of nanoparticlebased drug delivery systems.25−28 The “low-fouling” nature of PEO (“PEG”) is attributed to a number of effects including an enhanced steric entropic repulsive interaction induced by density-dependent polymorphism of the hydrophilic extended chain, i.e., the “brush-mushroom model”.29,30 Lipids that are conjugated with PEG have emerged as a promising class for steric stabilization applications.31−33 For example, Chong et al. have synthesized a class of PEGylatedphytanyl copolymers with a large range of PEG size (200−14 000 Da) and found the optimal molecular weight range to be 3000−6000 Da for preparing stable lyotropic liquid crystalline nanoparticles.34 Nilsson et al. have examined 1,2-distearoyl-snglycero-3-phosphoethanolamine conjugated to PEG (DSPEPEGMW where MW = 350, 750, or 2000 Da). They observed that Pluronic F127 was also required to obtain kinetically stabilized phytantriol-based dispersions, possibly due to the insufficient hydrophilic length from the investigated DSPEPEG.35 With the burgeoning of multiplatform therapies and theranostics there is an increasing need for alternative stabilizers that are chemically reactive to enhance the targeting specificity of nanoparticles for use in biomedical applications.1 A few studies have used reactive lipid-PEG (containing a reactive group on the PEG chain end) such as maleimide(triethylene glycol)ether lipid,32 N-[methoxy(poly(ethylene glycol)] biotin (DSPE-PEG-biotin),33 and oleate-PEG-maleimide36 to make surface-functionalized lipid lyotropic liquid crystalline nanoparticles. Pluronic F127 remained a necessary ingredient for stabilizing the dispersions in these works. Previously, it was shown that DSPE-PEG3400-maleimide was not only effective in stabilizing phytantriol-based liquid crystalline nanoparticles but that the maleimide functionality was key to providing a chemical handle for conjugation to targeting antibodies, with efficiencies that were superior to



MATERIALS AND METHODS

Materials. Phytantriol (98%) was a gift from DSM Nutritional Products. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(poly(ethylene glycol)] (ammonium salt) with PEG molecular weight of 2000 Da (DSPE-PEG2000), 3400 Da (DSPEPEG3400), and 5000 Da (DSPE-PEG5000) was purchased from Nanocs Inc. Pluronic F127 and phosphate buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich. Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies. Milli-Q H2O (18.2 MΩ) was used for all aqueous preparations. All compounds were used without further purification. Preparation of Lipid Lyotropic Liquid Crystalline Nanoparticles. Desired amounts of stabilizers in molar percentage to phytantriol using 10 mg of phytantriol were solubilized in chloroform, and the mixtures were placed under vacuum overnight for complete solvent removal (Table 1). PBS was then added to the mixture of phytantriol and stabilizers (at 50 °C) to achieve a final phytantriol concentration of 60 mM. The phytantriol concentration was kept constant in all samples to investigate the effect of PEG chain length and stabilizer concentration on the lyotropic liquid crystalline phases. Samples were then sonicated by a probe sonicator (Heat systemsUltrasonics, Inc., model W-220F) at a frequency of 20 kHz and an output power of 40 W, with a 5 s on, 5 s off mode for a total of 2 min. Incubation with DMEM plus FBS. Phytantriol-based disperions stabilized by either DSPE-PEG or Pluronic F127 were incubated with 50% or 80% DMEM supplemented with 10% FBS (DMEM/10% FBS) at 25 °C for 24 h before dynamic light scattering (DLS), synchrotron SAXS, and cryo-TEM measurements. Dispersions in DMEM/10% FBS were studied at 25 and 37 °C by DLS and synchrotron SAXS. DMEM/10% FBS was chosen for the purpose of this study as it is a common medium formulation for many cell lines. Particle Size Measurement. Particle size distribution and polydispersity were measured using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Samples were diluted 20 times either with PBS buffer or DMEM supplemented with 10% FBS and transferred to disposable low-volume plastic cuvettes. Samples were analyzed at 25 or 37 °C with refractive indices of phytantriol (nD = 1.47) and either PBS buffer (n = 1.333) or DMEM supplemented with 10% FBS (nD = 1.345) as the bulk diluent to calculate the hydrodynamic particle sizes. A viscosity of water η = 7.0 × 10−4 Nsm2 and DMEM medium η = 8.4 × 10−4 Nsm2 were used for analysis.38 Temperature was controlled 10872

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Cryo-TEM Imaging. Cryo-TEM was used to study the morphology of the samples either in PBS buffer or in DMEM supplemented with 10% FBS. Copper grids (200 mesh) 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. Images were recorded using a Tecnai 12 TEM operating at 120 kV, equipped with an FEI Eagle 4k × 4k CCD (FEI, Eindhoeven, The Netherlands). At all times low dose procedures were followed limiting the electron dose to no more than 10 electrons/Å2. Cell Lines. Adenocarcinomic human alveolar basal epithelial cells (A549; ATCC No.CCL-185) were grown in DMEM supplemented with 10% FBS, 10 mM HEPES, 2 mM glutamine, 0.01% penicillin, and 0.01% streptomycin at 37 °C with 5% CO2 and subcultured twice weekly. Chinese hamster ovary (CHO) cells (kindly received from K. Wark; CSIRO MSE Australia) were grown in MEMα modification supplemented with 10% FBS, 10 mM HEPES, 0.01% penicillin, and 0.01% streptomycin at 37 °C with 5% CO2 and subcultured twice weekly. Cytotoxicity Assay. A549 and CHO cells were seeded at 1 × 104 cells per well in 96-well tissue culture plates (Greiner) and grown overnight at 37 °C with 5% CO2. Nanoparticles were serially diluted to the required concentration (between 0 and 100 μg/mL) and added to three wells in the 96-well culture plates for each sample and incubated for 72 h at 37 °C in 200 μL of standard media. Toxicity was measured using the Alamar Blue reagent (Invitrogen USA) according to manufacturer’s instructions and described previously.41 Results are presented as a percentage of untreated cells, and the data are representative of three separate experiments in triplicate.

Table 1. Phase Parameters and Particle Sizes of PhytantriolBased Dispersions Derived from SAXS and DLS Data at 25 °C stabilizer

mol %a

DSPE-PEG2000

0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0

DSPE-PEG3400

DSPE-PEG5000

Pluronic F127

Phytantriol bulk phase in excess H2O

phaseb

Lα Lα Lα QIIP QIIP Lα+QIIP Lα+QIIP Lα+QIIP Lα+QIIP QIID QIID Lα + QIID Lα + QIID Lα + QIID Lα + QIID QIID QIID Lα + QIID Lα + QIID Lα + QIID Lα QIID

ac (Å)

98.5 98.5 98.7 99.0 99.1 99.9 70.6 71.3 71.8 72.5 72.2 72.4 69.4 68.4 68.1 68.4 67.8

size (nm)

340 346 376 270 273 232 165 156 140 339 269 279 278 266 244 198 146 126 116 114 116

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 8 13 9 12 15 13 8 5 8 5 11 13 7 10 3 3 3 1 1 1

PDId

0.20 0.16 0.31 0.17 0.21 0.27 0.27 0.26 0.24 0.14 0.13 0.11 0.14 0.20 0.24 0.09 0.08 0.10 0.11 0.12 0.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.05 0.07 0.04 0.02 0.02 0.05 0.01 0.00 0.07 0.02 0.04 0.04 0.03 0.04 0.02 0.01 0.01 0.01 0.01 0.01



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RESULTS DSPE-PEG-Stabilized Phytantriol-Based Lyotropic Liquid Crystalline Nanoparticles. This study examined the ability of three PEGylated phospholipids, specifically DSPE conjugated with 2000, 3400, and 5000 Da PEG, corresponding to 45, 70, and 113 PEG units, respectively, to stabilize phytantriol dispersions in PBS buffer. The commonly used block copolymer, Pluronic F127 (100 PEG units), was also studied side by side for comparison. The four stabilizers were added in a concentration range of 0.5−3 mol % to the bulk lipid phytantriol, which was fixed at a final concentration of 60 mM in all formulations. Keeping the concentration of phytantriol constant allowed us to study the effect of varying the PEG chain length and concentration on the stability and nanostructure of the phytantriol nanoparticle dispersions. The ability of the DSPE-PEG to stabilize and disperse phytantriol nanoparticles increased as a function of the PEG chain length and thus the hydrophilic fraction of the PEGylated phospholipids. Specifically, DSPE-PEG2000 was only able to satisfactorily disperse phytantriol when greater than 2 mol % was added, whereas both DSPE-PEG3400 and DSPE-PEG5000 yielded opalescent, stable dispersions at all concentrations investigated (0.5−3 mol %). The particle size distribution and structure of the dispersions were characterized using DLS and synchrotron SAXS. Figure 2 shows the 2D scattering patterns and integrated intensities as a function of q for each of the dispersions. Table 1 lists the relevant self-assembled liquid crystalline mesophase, lattice parameter (a), particle diameter, and polydispersity (PDI) of the nanoparticle formulations. For DSPE-PEG2000-stabilized dispersions containing ≥2 mol % stabilizers, the SAXS patterns displayed the characteristic first-, second-, and the third-order peaks at 0.04, 0.08, and 0.12 Å−1 of the lamellar phase (Lα)

a

Mol % refers to the molar percentage of stabilizers added to 60 mM phytantriol. bPhytantriol self-assembled liquid crystalline mesophases: QIIP (the primitive cubic phase); QIID (the double-diamond cubic phase). ca refers to the lattice parameter of nonlamellar bicontinueous cubic phase. dPDI refers to the polydispersity index. inside the measuring chamber of the Malvern Zetasizer. An equilibration time of 15 min was allowed before the measurements. Hydrodynamic diameter and size distribution (polydispersity indexPDI) of the nanoparticles were averaged from triplicate measurements of two individually prepared samples, and the results were reported as a mean ± range, n = 2. Synchrotron SAXS Measurement. Synchrotron SAXS measurements were performed at the small-angle/wide-angle X-ray scattering (SAXS/WAXS) beamline at the Australian Synchrotron. The X-ray had a beam with a wavelength of λ = 1.128 Å (11.0 keV) with a typical flux of approximately 1013 photons/s. The sample-to-detector distance was chosen as 1.5 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). 2D X-ray diffraction patterns were recorded on a Dectris-Pilatus 1-M detector of 10 modules. A silver behenate standard (d-spacing = 58.38 Å) was used for calibration. The exposure time for each sample was 1 s. Samples were loaded in a 96-well, half-area UV clear plate (Greiner Bio-One) and positioned in a custom-designed plate holder with temperatures controlled via a recirculating water bath as previously described.39 To study the phase of cubosomes in DMEM at 37 °C, samples were heated from 25 to 37 °C in situ, and an equilibration time of 15 min was allowed before the measurements. Samples were vortex mixed before SAXS assessment. Dilution of the cubosomes with a higher amount of DMEM made the samples too diluted for accurate SAXS measurements. SAXS data were analyzed using the IDL-based AXcess software package to identify the phase and to determine the lattice parameter of the internal phytantriol lyotropic liquid crystalline mesophase.40 10873

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Figure 2. SAXS patterns of phytantriol-based dispersions stabilized by (A) DSPE-PEG2000, (B) DSPE-PEG3400, (C) DSPE-PEG5000, and (D) Pluronic F127 at various molar concentrations (0.5−3%). The experiments were performed at 25 °C.

lattice parameters of the QIIP phase in DSPE-PEG3400-stabilized cubosomes. Although not as prominent as the DSPE-PEG3400stabilized nanoparticles, there was an increase in the diffraction signal at the lower angle with increasing DSPE-PEG5000 incorporation into the formulation. The high peak intensity at the position of √2 could potential mask the increase in the signal at the lower angle indicative of lamellar phase with increasing stabilizer concentration. There was also a gradual decrease in the particle size as a function of the DSPE-PEG concentration, suggesting liposomes are likely to be present though undetectable by SAXS. Similar to the dispersions of DSPE-PEG5000-stabilized nanoparticles, the Pluronic F127-stabilized phytantriol dispersions also demonstrated a QIID phase, with lattice parameters of between 67.8 and 69.4 Å, which is in good agreement with previous studies.12,43 For F127 concentrations >2 mol %, a shoulder appeared at q ∼ 0.05 Å−1 that was increasingly dominant at 2.5−3 mol %, which is attributed to the coexistence of liposomes. Mortensen and Talrnon have identified a correlation peak for F127 micelles at similar scattering lengths for F127 concentrations more than 5% at 25 °C.44 While micelles formed from F127 are known and have been previously observed,44,45 cryo-TEM used in the current work supports the presence of liposomes. In general, the particle size of the Pluronic F127-stabilized dispersions decreased with increasing F127 concentration, though were generally much smaller ( 1 as it has a relatively small headgroup area compared to its hydrophobic 10876

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Stability of PEGylated Phytantriol Cubosomes in Cell Culture Medium. It is interesting to note that despite their larger particle size and polydispersity, DSPE-PEG3400 or DSPEPEG5000-stabilized phytantriol cubosomes have a similar colloidal stability to their Pluronic F127 analogues, as can be seen by both the DLS and SAXS study. However, the density increase from the solvating environment of DMEM/10% FBS gradually led to creaming of the cubosomes when incubated for periods longer than 24 h. To avoid creaming and possible perturbation of therapeutic cargo, it is suggested that such phytantriol cubosomes should be freshly diluted with the cell medium just before adding them to cell lines for in vitro assays. The protein corona effect, which is the attachment of proteins from complex biological fluids to the nanoparticle surface to form a corona layer, is a concern for drug delivery as this may influence physicochemical and therapeutic features of the nanoparticle system.53 PEGylation has been reported to prevent proteins from binding to the particle surface and therefore reduce the protein corona effect.54 Although this study observed no significant impact on the particle size and polydispersity of nanoparticles incubated in medium within 24 h, it cannot eliminate the possibility of proteins from the medium attaching to the nanoparticles. However, the retention of the cubic phase in the medium indicates no disruption in the structure of the internal phase and the loaded therapeutic cargos when testing phytantriol cubosomes as drug delivery systems in vitro. Reduced Toxicity of PEGylated Phytantriol Cubosomes in Vitro. When developing a drug delivery system, it is vital to consider the inherent toxicity of the delivery vehicle to cells as well as the ability of it to interact either actively or passively with cells. Currently nanoparticle toxicity, particularly in lipid based systems, is not well understood. In an in vitro environment nanoparticle toxicity can be affected by the size, internal nanostructure, and molecular composition of the particle and thus can vary greatly from the toxicity exhibited by the same compound in bulk phase form. As an emerging drug delivery system, the in vitro toxicity of lipid lyotropic liquid crystalline nanoparticles is of rising interest to investigators,41,55 mainly because these nanostructured mesophase architectures are present in biomembranes and have been observed in many cell types and organelles.14,56 The relative ease of the lamellar to cubic phase transition in biomembranes is postulated to promote membrane fusion and assist in transbilayer transport.57,58 In addition, studies on model cell membranes and hemolysis assays have shown that cubosomes can disrupt membranes and are hemolytic.41,57 In particular, Hinton et al. reported that phytantriol cubosomes stabilized by Pluronic F127 became highly toxic to A549 and CHO cells (lower than 20% viability) at 50 μg/mL, whereas cells retained over 80% viability when treated with cubosomes prepared from monoolein.41 Another study explored the cytotoxicity of monoolein-based lyotropic liquid crystalline nanoparticles containing different mesophases and reported that 2D inverse hexagonal phase containing nanoparticles were less toxic than cubic phase containing analogues.59 These studies suggest that lipid lyotropic liquid crystalline mesophases and the effectiveness of the stabilizers are two important factors that may determine the in vitro cytotoxicity profiles. The current study showed that phytantriol cubosomes stabilized by Pluronic F127 and DSPE-PEG3400 became highly toxic to A549 and CHO cells (lower than 20% viability) at

Figure 5. Cell viability of phytantriol cubosomes stabilized by different stabilizers in vitro. Cubosomes at indicated lipid concentrations were added to (A) A549 cells or (B) CHO-GFP cells. The data are presented at % untreated cells representative of three separate experiments in triplicate ± SD.

footprint.47,48 The incorporation to the phytantriol layer of amphiphilic molecules such as DSPE-PEG2000 or DSPEPEG3400, which have lower CPPs (approximately 0.05) due to their large hydrophilic PEG headgroup,49,50 promotes the formation of mesophases with reduced negative interfacial curvature. The fact that DSPE-PEG3400 and DSPE-PEG2000 can be incorporated into the internal matrix of the phytantriol nanoparticles suggests fewer PEG units are available at the surface to impart steric stabilization. This manifests as an increase in the particle size and polydispersity of the dispersions compared to DSPE-PEG5000 or Pluronic F127-stabilized dispersions. It has been proposed that the nanostructure of the mesophase impacts performance.51,52 In the current study both the bicontinuous cubic QIID and QIIP phases are formed by varying the PEG chain length of the PEGylated phospholipids and possess different structural characteristics that may be used to advantage depending on the requirement. For example, the QIIP phase of the DSPE-PEG3400 system contains larger water channels (radius around 15.1 Å) that are better suited for the encapsulation of large biotherapeutics such as antibodies and proteins than the QIID phase. On the other hand, the QIID phase with its small water channels (radius around 13.9−14.5 Å) may be advantageous in systems that require retardation of therapeutics to provide a more controlled release profile. Moreover, DSPE-PEG molecules with chemically reactive functional groups can offer a platform to formulation antibody-conjugated liquid crystalline nanoparticles for targeted drug delivery.37 10877

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concentrations as low as 12.5 μg/mL. However, there was a significant reduction in the toxicity of phytantriol cubosomes stabilized by DSPE-PEG5000. It is postulated that increasing the PEG chain length in this formulation provides an increased density of PEG chains on the nanoparticle surface to impart an improved steric−entropic barrier to confer better efficacy against nanoparticle−cell membrane interactions and hence cytotoxicity. It is interesting to note that as Pluronic F127 provides two PEG chains, each containing 100 PEGunits, per molecule one may have expected that this would provide effective surface coverage of the nanoparticles to reduce toxicity. Perhaps somewhat counterintuitively, Pluronic F127stabilized cubosomes are more toxic in the Alamar Blue assays than those stabilized by DSPE-PEG5000. This could be due to the increased number of liposomes as opposed to cubosomes in the dispersion, reducing the effective surface coverage of the Pluronic F127 chains per particle than the DSPE-PEG5000 analogues. Moreover, the cryo-TEM study has shown that the surfaces of the Pluronic F127-stabilized nanoparticles are not enveloped by lamellar layers. Hence, the effective surface coverage of the Pluronic F127 or PEG chains of the stabilizer plays an important role in the in vitro toxicity of the nanoparticles. The current study has added more information to the current knowledge of the in vitro toxicity of lipid lyotropic liquid crystalline nanoparticles. However, it should also be noted that the link (if any) between the in vitro toxicity and in vivo toxicity of the lipid lyotropic liquid crystalline nanoparticles is still not clear.



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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (J.Z.). *E-mail [email protected] (B.W.M.). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was undertaken, in part, on the SAXS/WAXS beamline (Proposal AS143/SAXS/8509) at the Australian Synchrotron, Victoria, Australia. Jiali Zhai is supported by a CSIRO OCE Postdoctoral Fellowship. Nhiem Tran is supported by a SIEF John Stocker Postdoctoral Fellowship.



REFERENCES

(1) Mulet, X.; Boyd, B. J.; Drummond, C. J. Advances in drug delivery and medical imaging using colloidal lyotropic liquid crystalline dispersions. J. Colloid Interface Sci. 2013, 393, 1−20. (2) Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid Drug Delivery. Acc. Chem. Res. 2011, 44, 147−156. (3) Namiki, Y.; Fuchigami, T.; Tada, N.; Kawamura, R.; Matsunuma, S.; Kitamoto, Y.; Nakagawa, M. Nanomedicine for cancer: lipid-based nanostructures for drug delivery and monitoring. Acc. Chem. Res. 2011, 44, 1080−1093. (4) Angelova, A.; Angelov, B.; Drechsler, M.; Garamus, V. M.; Lesieur, S. Protein entrapment in PEGylated lipid nanoparticles. Int. J. Pharm. 2013, 454, 625−632. (5) Dong, Y.-D.; Larson, I.; Barnes, T. J.; Prestidge, C. A.; Boyd, B. J. Adsorption of Nonlamellar Nanostructured Liquid-Crystalline Particles to Biorelevant Surfaces for Improved Delivery of Bioactive Compounds. ACS Appl. Mater. Interfaces 2011, 3, 1771−1780. (6) Jain, V.; Swarnakar, N. K.; Mishra, P. R.; Verma, A.; Kaul, A.; Mishra, A. K.; Jain, N. K. Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy. Biomaterials 2012, 33, 7206−7220. (7) Murgia, S.; Bonacchi, S.; Falchi, A. M.; Lampis, S.; Lippolis, V.; Meli, V.; Monduzzi, M.; Prodi, L.; Schmidt, J.; Talmon, Y.; Caltagirone, C. Drug-Loaded Fluorescent Cubosomes: Versatile Nanoparticles for Potential Theranostic Applications. Langmuir 2013, 29, 6673−6679. (8) Swarnakar, N. K.; Thanki, K.; Jain, S. Bicontinuous Cubic Liquid Crystalline Nanoparticles for Oral Delivery of Doxorubicin: Implications on Bioavailability, Therapeutic Efficacy, and Cardiotoxicity. Pharm. Res. 2014, 31, 1219−1238. (9) Bye, N.; Hutt, O. E.; Hinton, T. M.; Acharya, D. P.; Waddington, L. J.; Moffat, B. A.; Wright, D. K.; Wang, H. X.; Mulet, X.; Muir, B. W. Nitroxide-Loaded Hexosomes Provide MRI Contrast in Vivo. Langmuir 2014, 30, 8898−8906. (10) Barauskas, J.; Landh, T. Phase behavior of the phytantriol/water system. Langmuir 2003, 19, 9562−9565. (11) Clogston, J.; Rathman, J.; Tomasko, D.; Walker, H.; Caffrey, M. Phase behavior of a monoacylglycerol: (Myverol 18−99K)/water system. Chem. Phys. Lipids 2000, 107, 191−220. (12) Dong, Y.-D.; Larson, I.; Hanley, T.; Boyd, B. J. Bulk and Dispersed Aqueous Phase Behavior of Phytantriol: Effect of Vitamin E Acetate and F127 Polymer on Liquid Crystal Nanostructure. Langmuir 2006, 22, 9512−9518. (13) Seddon, J. M. Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochim. Biophys. Acta, Rev. Biomembr. 1990, 1031, 1−69. (14) Larsson, K. Cubic lipid-water phases: structures and biomembrane aspects. J. Phys. Chem. 1989, 93, 7304−7314. (15) Kaasgaard, T.; Drummond, C. J. Ordered 2-D and 3-D nanostructured amphiphile self-assembly materials stable in excess solvent. Phys. Chem. Chem. Phys. 2006, 8, 4957−4975.

CONCLUSION

This study has shown that phytantriol cubosomes may be stabilized by PEGylated phospholipids such as DSPE-PEG, wherein the hydrophobic phospholipid component, DSPE (a C18 lipid), resides within the phytantriol (a C16 lipid) bilayer, and the hydrophilic PEG block is able to reside in either the outer external aqueous solvating medium or the internal water channels depending on its size. The DSPE-PEG with PEG chains of 2000, 3400, and 5000 Da was able to effectively stabilize phytantriol dispersions with nanoarchitectures ranging from Lα to QIIP and QIID, respectively, which were a function of PEG chain length. Although increasing the length and concentration of PEG enhances the steric stabilization, this study showed that a high concentration of the incorporated stabilizers could lead to the formation and coexistence of liposomes that may compromise drug encapsulation and release profiles. It is therefore essential to optimize the concentration of the stabilizer to find a balance between sufficient stabilization and homogeneity of the lyotropic liquid crystalline mesophases. The QIIP and the QIID phases of the dispersions were stable at 37 °C and have been assessed in vitro against two cell lines for cytotoxicity. The DSPE-PEG5000-stabilized cubosomes were less toxic than the DSPE-PEG3400 or Pluronic F127-stabilized dispersions which is attributed to the increased surface coverage of PEG chains on the particle surface, implicating the role of PEGylation in future in vivo investigations in these systems. In summary, the PEG chain length has an important effect on the resulting nanostructure within the nanoparticles, including the internal water channel size. The stability of the internal mesophase and particle size as well as the in vitro toxicity of the nanoparticles were also impacted. 10878

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Length, and Temperature on the Internal Nanostructure. Langmuir 2014, 30, 6398−6407. (36) Wu, H.; Li, J.; Zhang, Q.; Yan, X.; Guo, L.; Gao, X.; Qiu, M.; Jiang, X.; Lai, R.; Chen, H. A novel small Odorranalectin-bearing cubosomes: Preparation, brain delivery and pharmacodynamic study on amyloid-β25−35-treated rats following intranasal administration. Eur. J. Pharm. Biopharm. 2012, 80, 368−378. (37) Zhai, J.; Scoble, J. A.; Li, N.; Lovrecz, G.; Waddington, L. J.; Tran, N.; Muir, B. W.; Coia, G.; Kirby, N.; Drummond, C. J.; Mulet, X. Epidermal growth factor receptor-targeted lipid nanoparticles retain self-assembled nanostructures and provide high specificity. Nanoscale 2015, 7, 2905−2913. (38) Liu, S.-L.; Karmenyan, A.; Wei, M.-T.; Huang, C.-C.; Lin, C.-H.; Chiou, A. Optical forced oscillation for the study of lectin-glycoprotein interaction at the cellular membrane of a Chinese hamster ovary cell. Opt. Express 2007, 15, 2713−2723. (39) Mulet, X.; Conn, C. E.; Fong, C.; Kennedy, D. F.; Moghaddam, M. J.; Drummond, C. J. High-throughput development of amphiphile self-assembly materials: fast-tracking synthesis, characterization, formulation, application, and understanding. Acc. Chem. Res. 2013, 46, 1497−1505. (40) Seddon, J. M.; Squires, A. M.; Conn, C. E.; Ces, O.; Heron, A. J.; Mulet, X.; Shearman, G. C.; Templer, R. H. Pressure-jump X-ray studies of liquid crystal transitions in lipids. Philos. Trans. R. Soc., A 2006, 364, 2635−2655. (41) Hinton, T. M.; Grusche, F.; Acharya, D.; Shukla, R.; Bansal, V.; Waddington, L. J.; Monaghan, P.; Muir, B. W. Bicontinuous cubic phase nanoparticle lipid chemistry affects toxicity in cultured cells. Toxicol. Res. 2014, 3, 11−22. (42) Chang, D. P.; Jankunec, M.; Barauskas, J.; Tiberg, F.; Nylander, T. Adsorption of Lipid Liquid Crystalline Nanoparticles: Effects of Particle Composition, Internal Structure, and Phase Behavior. Langmuir 2012, 28, 10688−10696. (43) Chong, J. Y. T.; Mulet, X.; Waddington, L. J.; Boyd, B. J.; Drummond, C. J. High-Throughput Discovery of Novel Steric Stabilizers for Cubic Lyotropic Liquid Crystal Nanoparticle Dispersions. Langmuir 2012, 28, 9223−9232. (44) Mortensen, K.; Talmon, Y. Cryo-TEM and SANS Microstructural Study of Pluronic Polymer Solutions. Macromolecules 1995, 28, 8829−8834. (45) Lam, Y.-M.; Grigorieff, N.; Goldbeck-Wood, G. Direct visualisation of micelles of Pluronic block copolymers in aqueous solution by cryo-TEM. Phys. Chem. Chem. Phys. 1999, 1, 3331−3334. (46) Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J. Incorporation of the dopamine D2L receptor and bacteriorhodopsin within bicontinuous cubic lipid phases. 1. Relevance to in meso crystallization of integral membrane proteins in monoolein systems. Soft Matter 2010, 6, 4828− 4837. (47) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of selfassembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525−1568. (48) Fong, C.; Le, T.; Drummond, C. J. Lyotropic liquid crystal engineering-ordered nanostructured small molecule amphiphile selfassembly materials by design. Chem. Soc. Rev. 2012, 41, 1297−1322. (49) Ashok, B.; Arleth, L.; Hjelm, R. P.; Rubinstein, I.; Onyuksel, H. In vitro characterization of PEGylated phospholipid micelles for improved drug solubilization: effects of PEG chain length and PC incorporation. J. Pharm. Sci. 2004, 93, 2476−2487. (50) Hak, S.; Helgesen, E.; Hektoen, H. H.; Huuse, E. M.; Jarzyna, P. A.; Mulder, W. J. M.; Haraldseth, O.; Davies, C. d. L. The Effect of Nanoparticle Polyethylene Glycol Surface Density on Ligand-directed Tumor Targeting Studied in vivo by Dual Modality Imaging. ACS Nano 2012, 6, 5648−5658. (51) Lee, K. W. Y.; Nguyen, T.-H.; Hanley, T.; Boyd, B. J. Nanostructure of liquid crystalline matrix determines in vitro sustained release and in vivo oral absorption kinetics for hydrophilic model drugs. Int. J. Pharm. 2009, 365, 190−199.

(16) Dong, Y.-D.; Dong, A. W.; Larson, I.; Rappolt, M.; Amenitsch, H.; Hanley, T.; Boyd, B. J. Impurities in commercial phytantriol significantly alter its lyotropic liquid-crystalline phase behavior. Langmuir 2008, 24, 6998−7003. (17) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Cubic phase nanoparticles (cubosome†): principles for controlling size, structure, and stability. Langmuir 2005, 21, 2569−2577. (18) Hartnett, T. E.; Ladewig, K.; O’Connor, A. J.; Hartley, P. G.; McLean, K. M.; Size. and Phase Control of Cubic Lyotropic Liquid Crystal Nanoparticles. J. Phys. Chem. B 2014, 118, 7430−7439. (19) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Novel Process for Producing Cubic Liquid Crystalline Nanoparticles (Cubosomes). Langmuir 2001, 17, 5748−5756. (20) Spicer, P. T. In Dekker Encyclopedia of Nanoscience and Nanotechnology; Schwarz, A., Contescu, C. I., Eds.; CRC Press: New York, 2004. (21) Yaghmur, A.; Glatter, O. Characterization and potential applications of nanostructured aqueous dispersions. Adv. Colloid Interface Sci. 2009, 147−148, 333−342. (22) Chong, J. Y. T.; Mulet, X.; Boyd, B. J.; Drummond, C. J. In Advances in Planar Lipid Bilayers and Liposomes; Aleš Iglič, C. V. K., Michael, R., Eds.; Academic Press: New York, 2015; Vol. 21, pp 131− 187. (23) Abraham, T.; Hato, M.; Hirai, M. Polymer-dispersed bicontinuous cubic glycolipid nanoparticles. Biotechnol. Prog. 2005, 21, 255−262. (24) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Submicron Particles of Reversed Lipid Phases in Water Stabilized by a Nonionic Amphiphilic Polymer. Langmuir 1997, 13, 6964−6971. (25) Haran, G.; Cohen, R.; Bar, L. K.; Barenholz, Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim. Biophys. Acta, Biomembr. 1993, 1151, 201−215. (26) Owens, D. E., III; Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307, 93−102. (27) Rauscher, A.; Frindel, M.; Maurel, C.; Maillasson, M.; Le Saëc, P.; Rajerison, H.; Gestin, J. F.; Barbet, J.; Faivre-Chauvet, A.; MouginDegraef, M. Influence of pegylation and hapten location at the surface of radiolabelled liposomes on tumour immunotargeting using bispecific antibody. Nucl. Med. Biol. 2014, 41, e66−e74. (28) Vllasaliu, D.; Fowler, R.; Stolnik, S. PEGylated nanomedicines: recent progress and remaining concerns. Expert Opin. Drug Delivery 2014, 11, 139−154. (29) Owens Iii, D. E.; Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307, 93−102. (30) Jokerst, J. V.; Lobovkina, T.; Zare, R. N.; Gambhir, S. S. Nanoparticle PEGylation for imaging and therapy. Nanomedicine 2011, 6, 715−728. (31) Almgren, M.; Rangelov, S. Polymorph Dispersed Particles from the Bicontinuous Cubic Phase of Glycerol Monooleate Stabilized by PEG − Copolymers with Lipid − Mimetic Hydrophobic Anchors. J. Dispersion Sci. Technol. 2006, 27, 599−609. (32) Angelova, A.; Ollivon, M.; Campitelli, A.; Bourgaux, C. Lipid Cubic Phases as Stable Nanochannel Network Structures for Protein Biochip Development: X-ray Diffraction Study. Langmuir 2003, 19, 6928−6935. (33) Fraser, S. J.; Dawson, R. M.; Waddington, L. J.; Muir, B. W.; Mulet, X.; Hartley, P. G.; Separovic, F.; Polyzos, A. Development of Cubosomes as a Cell-Free Biosensing Platform. Aust. J. Chem. 2011, 64, 46−53. (34) Chong, J. Y. T.; Mulet, X.; Keddie, D. J.; Waddington, L.; Mudie, S. T.; Boyd, B. J.; Drummond, C. J. Novel Steric Stabilizers for Lyotropic Liquid Crystalline Nanoparticles: PEGylated-Phytanyl Copolymers. Langmuir 2015, 31, 2615−2629. (35) Nilsson, C.; Østergaard, J.; Larsen, S. W.; Larsen, C.; Urtti, A.; Yaghmur, A. PEGylation of Phytantriol-Based Lyotropic Liquid Crystalline ParticlesThe Effect of Lipid Composition, PEG Chain 10879

DOI: 10.1021/acs.langmuir.5b02797 Langmuir 2015, 31, 10871−10880

Article

Langmuir (52) Zabara, A.; Mezzenga, R. Modulating the crystal size and morphology of in meso-crystallized lysozyme by precisely controlling the water channel size of the hosting mesophase. Soft Matter 2013, 9, 1010−1014. (53) Lynch, I.; Dawson, K. A. Protein-nanoparticle interactions. Nano Today 2008, 3, 40−47. (54) Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J. Am. Chem. Soc. 2012, 134, 2139−2147. (55) Murgia, S.; Falchi, A. M.; Mano, M.; Lampis, S.; Angius, R.; Carnerup, A. M.; Schmidt, J.; Diaz, G.; Giacca, M.; Talmon, Y.; Monduzzi, M. Nanoparticles from Lipid-Based Liquid Crystals: Emulsifier Influence on Morphology and Cytotoxicity. J. Phys. Chem. B 2010, 114, 3518−3525. (56) Almsherqi, Z. A.; Kohlwein, S. D.; Deng, Y. Cubic membranes: a legend beyond the Flatland* of cell membrane organization. J. Cell Biol. 2006, 173, 839−844. (57) Barauskas, J.; Cervin, C.; Jankunec, M.; Špandyreva, M.; Ribokaitė, K.; Tiberg, F.; Johnsson, M. Interactions of lipid-based liquid crystalline nanoparticles with model and cell membranes. Int. J. Pharm. 2010, 391, 284−291. (58) Tilley, A.; Dong, Y. D.; Amenitsch, H.; Rappolt, M.; Boyd, B. J. Transfer of lipid and phase reorganisation in self-assembled liquid crystal nanostructured particles based on phytantriol. Phys. Chem. Chem. Phys. 2011, 13, 3026−3032. (59) Tran, N.; Mulet, X.; Hawley, A. M.; Hinton, T. M.; Mudie, S. T.; Muir, B. W.; Giakoumatos, E. C.; Waddington, L. J.; Kirby, N. M.; Drummond, C. J. Nanostructure and cytotoxicity of self-assembled monoolein-capric acid lyotropic liquid crystalline nanoparticles. RSC Adv. 2015, 5, 26785−26795.

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