Biomaterials xxx (2012) 1e11

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Metal-free and MRI visible theranostic lyotropic liquid crystal nitroxide-based

nanoparticles
Benjamin W. Muir a, **, Durga P. Acharya a, Danielle F. Kennedy a, Xavier Mulet a, b, Richard A. Evans a,
Suzanne M. Pereira a, Kim L. Wark a, Ben J. Boyd b, Tri-Hung Nguyen b, Tracey M. Hinton c,
Lynne J. Waddington a, Nigel Kirby d, David K. Wright e, Hong X. Wang e, Gary F. Egan e,
Bradford A. Moffat f, *
a

CSIRO Materials Science and Engineering, Bayview Avenue, Clayton 3168, Australia
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville 3052, Australia
c
CSIRO Livestock Industries, Australian Animal Health Laboratory, East Geelong 3219, Australia
d
Australian Synchrotron, Clayton 3168, Australia
e
Howard Florey Institute, The University of Melbourne, 3010, Australia
f
The University of Melbourne, Department of Radiology, Parkville 3050, Australia
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 November 2011
Accepted 6 December 2011
Available online xxx

The development of improved, low toxicity, clinically viable nanomaterials that provide MRI contrast
have tremendous potential to form the basis of translatable theranostic agents. Herein we describe
a class of MRI visible materials based on lyotropic liquid crystal nanoparticles loaded with a paramagnetic nitroxide lipid. These readily synthesized nanoparticles achieved enhanced proton-relaxivities
on the order of clinically used gadolinium complexes such as Omniscan without the use of heavy metal
coordination complexes. Their low toxicity, high water solubility and colloidal stability in buffer resulted
in them being well tolerated in vitro and in vivo. The nanoparticles were initially screened in vitro for
cytotoxicity and subsequently a dened concentration range was tested in rats to determine the
maximum tolerated dose. Pharmacokinetic proles of the candidate nanoparticles were established
in vivo on IV administration to rats. The lyotropic liquid crystal nanoparticles were proven to be effective
liver MRI contrast agents. We have demonstrated the effective in vivo performance of a T1 enhancing,
biocompatible, colloidally stable, amphiphilic MRI contrast agent that does not contain a metal.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Cubosome
Contrast agent
Lyotropic liquid crystal
Cytotoxicity
Maximum tolerated dose
High-throughput

1. Introduction
The development of various nanoparticle formulations as
potential MRI contrast agents is an area of ever growing research.
The benets of developing nanoparticle formulations include the
potential to target these agents to diseased tissues such as tumors.
Previously this has been achieved passively via the enhanced
permeability and retention effect (EPR) [1e3] or actively via
attachment of targeting ligands such as folate [4], peptides [5] or
antibodies [6]. In addition, the use of nanoparticles may enable
multimodal theranostic agents to be developed for both imaging
diseased tissue, and treating the disease by controlled release of
drugs [7,8]. Few nanoparticle formulations have been clinically
* Corresponding author. Fax: 61 393428369.
** Corresponding author. Fax: 61 395452515.
E-mail addresses: ben.muir@csiro.au (B.W. Muir), brad.moffat@rmh.org.au (B.
A. Moffat).

approved due to the immense physico-chemical, biological and

regulatory hurdles that confront the nanotechnologist when
devising successful nanoparticle and coating formulations [8].
The main aim of scientists developing new and improved MRI
theranostic materials is to produce nanoparticles which provide
strong signal contrast while maintaining low toxicity and ease of
intra-venous delivery (small volume bolus, stability in saline,
increased blood half life and low viscosity). Contrast agents are used
clinically when poor contrast of the diseased tissue is observed
during MRI scanning (as seen commonly when imaging brain and
liver lesions). Signal enhancement is achieved by contrast agents
that decrease the water spin-lattice relaxation time (T1) during the
acquisition of T1 weighted MR images, while signal suppression is
achieved by agents that decrease the water spin-spin relaxation time
(T2) during the acquisition of T2 weighted MR images. The contrast
agent relaxivity (r1 or r2) is a measure of the relative effectiveness of
a given contrast agent and has units of s
1mM
1. To date most
nanoparticle MRI contrast agent formulations (whether T1 or T2

0142-9612/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.12.018

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

enhancing) have been used as liver contrast agents due primarily to

uptake by the reticuloendothelial system (RES) after bolus delivery
[8,9]. Most clinically used T1 enhancing agents contain gadolinium
(Gd) [10,11] which is highly toxic as a free trivalent ion [12]. Although
the Gd metal in clinically used contrast agents are chelated [10] the
metal may still potentially leach. Certain agents may cause adverse
reactions in patients with renal failure resulting in a debilitating
disease called nephrogenic systemic brosis [13]. As a result,
phasing out the use of heavy metal based contrast agents and nding
suitable alternatives is of great interest to the eld. To date, nonheavy metal based materials investigated as T1 enhancing agents
have essentially been limited to 19F [14,15] and nitroxide enriched
compounds [16e18].
One of the greatest challenges to the materials scientist in the
preparation of suitable MRI contrast agent nanoparticles is the
production of highly stable colloidal dispersions that have negligible cytotoxic properties [8]. To this end we have developed the
use of lyotropic liquid crystal nanoparticles that contain a paramagnetic nitroxide lipid to provide T1 contrast rather than the
conventionally used gadolinium based compounds. This may allow
the eld to move away from the toxicity issues associated with
gadolinium based contrast agents. Previous reports have shown the
applicability of using gadolinium functionalized lipids incorporated
into lyotropic mesophase liquid crystal nanoparticles as potential
MRI agents in vitro [19,20]. The use of amphiphilic lipids such as
phytantriol and glyceryl monooleate (GMO) (structure in Fig. 1)
result in the formation of distinct lyotropic mesophases of varying
complexity and dimensionality. Glycerol monooleate contains an
ester group that is susceptible to hydrolysis and therefore biodegradation in vivo. The common phases seen when using lyotropic
liquid crystal materials include lamellar phase (L) (1-D) comprised
of stacked bilayer sheets, the hexagonal phase (H2) (2-D) which can
be conceptualized as innitely long hexagonally packed rods with
an aqueous interior and nally the cubic phase (Q2) (3-D) consisting of a bi-continuous network of hydrophilic and hydrophobic
domains containing two continuous water channels. The Q2 phase
represents a family of closely related structures, where the underlying crystal lattice can be described by the gyroid (G), diamond (D)
and primitive (P) minimal surfaces, which correspond to the Ia3d
(G), Pn3m (D) and Im3m (P) crystallographic space groups,

respectively. The 3-dimensional structure affords a self-assembled

scaffold with a remarkably high surface area and extensive
porosity. These properties, coupled with the liquid crystalline
nature of the phase, result in a structure that was found to not be
susceptible to osmotic or mechanical rupture in contrast to the
properties of liposomes [21] or micelles. The thermodynamic
stability of the Q2 phase affords a structure that co-exists in equilibrium with excess water over a broad temperature range. The
dispersion of the bulk gel-like cubic phases can be achieved by
mechanical or ultrasonic treatment and results in the formation of
nanometer-sized particles that retain the internal cubic structure of
the parent bulk cubic phase. The incorporation of additives to such
materials may result in the formation of other phases such as the
inverse hexagonal phase (H2) due to an alteration in the spontaneous curvature of the lyotropic liquid crystal assemblies. Dispersions of hexagonal phase nanoparticles are commonly called
hexosomes and cubic phase nanoparticles are referred to as
cubosomes [22e26].
The aim of this study was to investigate the benet of incorporating a myristic nitroxide lipid (structure in Fig. 1) into lyotropic
liquid crystal nanoparticles. Nitroxides are stable, organic free
radicals with an unpaired (paramagnetic) electron and are therefore capable of shortening the MRI relaxation times [18]. Once
inside the body, an equilibrium exists between the paramagnetic
(contrast enhancing) nitroxide and the reduced non-paramagnetic
(non-contrast enhancing) hydroxylamine [27]. Previous studies
have shown that these compounds can be useful for imaging
intracellular redox metabolism by MRI [28,29], because the ratio of
the two states was dictated by the local oxygen and redox environment. In addition these compounds have been shown to have
potential for controlling hypertension and weight, preventing
damage from reperfusion injury, and treating neurodegenerative
diseases and ocular damage [29,30]. It was hypothesized that
encapsulating the nitroxide lipid inside the lyotropic mesophase
liquid crystal nanoparticles would extend the nitroxide radicals
half-life in vivo making it an effective MRI contrast agent with
an acceptable cytotoxicity prole. Furthermore, the presence of
conned water channels in cubic and hexagonal phase nanoparticles, and their greater surface area compared to liposomes may
result in enhanced relaxivities of the nitroxide lipid due to rotational correlation constant and proton exchange processes.
To investigate these hypotheses, nanoparticles were synthesized using two different bulk cubic phase forming lipids. In this
study the effect of nanoparticle structure on relaxivity, cytotoxicity,
maximum tolerated dose in rats and efcacy of the contrast agents
in vivo for nitroxide loaded cubosomes and hexosomes, was
investigated. The cubic and hexagonal phase nanoparticles have
a viscosity approximately equal to water, which makes it desirable
for bolus delivery of MRI contrast agents. Previous formulations of
these types of nanoparticles have shown them to have high
colloidal stability and low cytotoxicity through the appropriate
selection of the amphiphile used to form the lyotropic liquid crystal
phase [31].
2. Materials and methods
2.1. Self-assembly of the lyotropic liquid crystal nitroxide containing nanoparticles

Fig. 1. Chemical structure of phytantriol amphiphile (top structure), glyceryl monooleate amphiphile (middle structure, the main component of Myverol) and the
nitroxide lipid (bottom structure) used to make lyotropic liquid crystal, MRI contrast
agent nanoparticles.

Two bulk cubic phase forming lipids were used in this work, phytantriol (DSM
Nutritional Products, GmbH) and Myverol (Bronson & Jacobs, Sydney) which were
used as received. Myverol was used as a commercially available source of GMO
which was the main lipid component of this product. Samples for screening the T1
and T2 relaxivities were prepared in a high-throughput manner using a Chemspeed
Accelerator SLTII robotic synthesis platform equipped with a 4-needle head and
probe sonicator tools. As previously reported [32], stock solutions of the phytantriol,
Myverol and nitroxide lipid were prepared in a poly(ethylene) 96 well plate (2 mL
capacity per well) containing chloroform, 0.5 mg of bulk lipid and an appropriate

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

volume of nitroxide lipid. For each bulk lipid solution transfer into the wells an extra
100 mL and a 10 mL air-gap were used. To achieve higher accuracy in dispensing
nitroxide lipid solutions, no air gap was used for these low volume transfers. A
concentration gradient of the nitroxide lipid in each bulk lipid multi-well plate zone
was prepared between 0 and 14 wt% nitroxide lipid. The plates were removed from
the platform in order to remove the solvent using a Genevac centrifugal evaporator. The pluronic was then added at a concentration of 10 wt% to each well in 1 mL
of water and sonicated on the Chemspeed platform to produce milky dispersions of
nanoparticles.
Bulk samples for relaxivity testing, cytotoxicity testing and injection in animals
were prepared individually. For each sample, 1 g of bulk lipid (phytantriol or
Myverol) and the required amount of nitroxide lipid were heated to 60  C and mixed
until a homogeneous gel was observed. The required amount of pluronic (F127) was
then added to a nal concentration of 15 wt%. The mixture was then heated to 70  C
for 5 min followed by vortex mixing. This process was repeated 4 or 5 times. This hot
mixture was added dropwise to water (at w65  C) under shear generated by
a Polytron shear homogenizer using a 20 mm probe rotating at 20,000 rpm. The
dispersion was further homogenized at 65  C with three passes in an Avastin
pressure homogenizer to obtain the nal dispersed nanoparticle solutions. The
fraction of nonaqueous components in the solution of lipid nanoparticles was
determined by thermogravimetric analysis (Mettler). About 35 mg of nanoparticle
solution was placed in a 70 mL silica crucible and analysed in the temperature range
of 25e800  C. The average of three results was taken to determine the fraction of
nonaqueous components in the solution and the concentration of phytantriol or
Myverol in the bulk nanoparticle solution.
2.2. Nitroxide lipid synthesis
The nitroxide lipid was made by dissolving 4-hydroxy-TEMPO (2 g) in
dichloromethane (DCM, 40 mL) with 2.4 mL of triethylamine. Myristol chloride
(2.6 g) in DCM (5 mL) was added dropwise at room temperature. The reaction was
stirred at room temperature under argon for 4 h. The reaction was worked up by
washing with water, dilute hydrochloric acid and brine before drying with sodium
sulphate and evaporation to give 3.6 g of crude oil that partially crystallized. The
material was then chromatographed on silica (hexane:ether 70:30). The product
fraction was collected and evaporated down to give 2.5 g (45% yield) of red oil. Due
to the paramagnetic nature of the nitroxide the NMR spectra was poor in terms of
resolution, sensitivity and integration. 1H NMR (CDCl3, 400 MHz), d 1.59 (br. s), 1.97
(br. s., CH2 CH3), 2.34 (br. s. CH2) 2.99 (2H, CH2CO) ppm. 13C NMR (CDCl3) d 15.4,
23.70, 26.00, 30.09, 30.25, 30.31, 30.41, 30.54, 30.58, 30.64, 32.90, 38.57, 172.97
(C]O) ppm. Mass Spectroscopy experiments were carried out using a ThermoQuest
MAT95XP, employing Electron Impact at 70 eV and peruorokerosene was used as
a reference standard. Mass spectral analysis conrmed the nitroxide lipid had been
successfully synthesized with a measured m/z of 382.3318.
2.3. Cryo-transmission electron microscopy (Cryo-TEM)
A humidity-controlled vitrication system was used to prepare the nanoparticles for imaging in a thin layer of vitried ice using cryo-TEM. Humidity was
kept close to 80% for all experiments, and ambient temperature was 22  C. 200mesh copper grids coated with perforated carbon lm (Lacey carbon lm: ProSciTech, Qld, Australia) were used for all experiments. Aliquots of the sample (4 mL)
were pipetted onto each grid prior to plunging. After 30 s of adsorption time the grid
was blotted manually using Whatman 541 lter paper for approximately 2 s. Blotting time was optimized for each sample. The grid was then plunged into liquid
ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until
required. The samples were examined using a Gatan 626 cryoholder (Gatan,
Pleasanton, CA, USA) and Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. At all times low dose
procedures were followed, using an electron dose of 8e10 electrons/2 for
all imaging. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus) using magnications in the range
60,000e110,000.
2.4. Dynamic light scattering analysis
Particle size measurements were performed using a Zetasizer-Nano instrument
(Malvern, UK). Particle sizes were measured in Milli-Q water using samples
appropriately diluted. The analysis was performed at 25  C and for each sample, the
mean diameter and polydispersity index (PDI) of six measurements was calculated.
2.5. Synchrotron SAXS measurements
Small-angle X-ray scattering (SAXS) experiments were performed on the SAXS/
WAXS beamline at the Australian Synchrotron. Special glass capillaries (1.5 mm)
containing sample solutions were placed in a temperature controlled sample holder
maintained at 37  C by a recirculating water bath. Samples were exposed to the
12 keV X-ray beam of dimensions 2500 mm  130 mm and a typical ux of
5  1012 photons/s and diffraction patterns were recorded using a Pilatus 1M

detector (Dectris, Switzerland). A silver behenate standard was used to calibrate the
reciprocal space vector for analysis. Data reduction (calibration and integration) was
performed using AXcess, a custom-written SAXS analysis program written by Dr.
Andrew Heron from Imperial College, London [33]. The bicontinuous cubic phases
with Pn3m symmetry (diamond) and hexagonal phase observed in this study were
identied from the positions of diffracted peaks at O2, O3, O4, O6, O8, O9 and at 1, O3,
O4 respectively.
2.6. MRI relaxivity measurements
A high throughput MRI screening technique was used to evaluate the NMR
relaxation properties of the nanoparticle solutions at 3 Tesla using a method similar
to that previously reported [34]. For initial MRI relaxivity measurements, the plates
produced in the Chemspeed robotic platform were imaged at 23  C in a Siemens
(Germany) 3 Tesla TRIO MRI scanner using a body transmit radio frequency coil and
a 12 channel radio frequency receiver coil. For MRI relaxivity measurements on bulk
produced samples, 1 mL of each lyotropic liquid crystal nanoparticle formulation
was placed in a 96 well plate and serially diluted to measure T1, T2 and calculate
relaxivities.
For quantifying the spin-spin relaxation rates (R2 1/T2) a multiple spin echo
sequence was used to acquire 32 images at echo times (TE) ranging from 11.5 to
310.5 ms with a repetition time (TR) of 3 s. To quantify the spin-lattice relaxation
rates (R1 1/T1) a spin echo inversion recovery imaging sequence was used to
acquire images at nine different inversion times (TI) ranging from 20 ms to 5 s (TR/
TE 10,000/10 ms). All images were acquired with a 3 mm slice thickness, 100 mm
FOV, 192  154 matrix size and 2 averages. Zero lling was used to reconstruct all
images to a matrix size of 256  256. To calculate R2 the signal from regions of
interest (>40 voxels) centered within each well was averaged and plotted as
a function of TE. The R2 values were then calculated (as the decay constants) by
numerically tting (using a non-linear least squares algorithm, Matlab, MA, USA)
the data to a mono-exponential equation:
S S0 exp
TE  R2

(1)

where S0 is the signal when TE  1/R2.

A similar approach was used to calculate R1 except the data was plotted as
a function of TI and tted to the following equation:
S S0 1
2exp
TI  R1

(2)

The respective relaxation rates, R1 and R2, were plotted as a function of nitroxide
lipid concentration and linear least squares analysis (Matlab, MA, USA) was used to
quantify the relaxivities: r1 and r2 respectively.
2.7. Cytotoxicity testing of the lyotropic liquid crystal nitroxide containing
nanoparticles
CHO-GFP and HEK293 (ATCC No. CRL1573) cells were seeded at 1 104 cells in
96-well tissue culture plates in triplicate and grown overnight at 37  C with 5% CO2.
Toxicity was measured using the Alamar Blue reagent (Invitrogen USA) according to
manufacturers instructions. In short, 20 mL of Alamar Blue was added to each well
containing 200 mL of media and incubated for 4 h at 37  C with 5% CO2. The assay
was read on an EL808 Absorbance microplate reader (BIOTEK, USA) at 540 nm and
620 nm. Cell viability was determined by subtracting the optical density measurement at 620 nm from that at 540 nm. Results are presented as a percentage of
untreated cells.
2.8. Pharmacokinetics of phytantriol and Myverol-based lyotropic liquid crystalline
nanoparticles
These experiments were approved by the Monash Institute of Pharmaceutical
Science Animal Ethics Committee. To assess the circulatory behavior of the lyotropic
liquid crystalline nanoparticles, a radiolabelled tracer molecule (3H-dioleyl phosphatidylcholine) was incorporated into phytantriol and Myverol based liquid crystalline nanoparticles as was commonly employed to study circulation of liposomes.
Two levels of colloidal stabilizer were assessed to determine whether the total
pluronic concentration inuenced particle removal from systemic circulation due to
inhibition of protein binding. The dispersions consisted of 5% w/v lipid, containing
3
H-PL equivalent to 0.5 mCi per dose (<0.1% w/v). The combined lipid and radiolabel
was added drop wise to an isotonic saline solution containing 8 or 15 wt% pluronic
F127. The mixture was ultrasonicated (Misonix XL 2000, Misonix, NY) non-stop for
1 min followed by a pulsing mode, (1 second pulse interrupted by 1 s pauses for
20 min) to disperse the lipid, resulting in a uniform milky mixture with no visible
signs of aggregates.
Rats were cannulated in the carotid artery for sampling, and jugular vein for
administration of nanoparticles using polyethylene tubing (0.96  0.58 mm, Paton
Scientic, Victor Harbour, Australia). On the following day they were administered
the 0.5 mCi dose over 1 min. Plasma samples (100 mL) were retrieved at times as

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indicated in results and mixed with scintillation cocktail (2 mL Starscint) and

counted on a Packard Tri-Carb 2000CA liquid scintillation analyzer (Meriden, CT).
2.9. Maximum tolerated dose testing of lyotropic liquid crystal nitroxide containing
nanoparticles
Maximum tolerated dose (MTD) testing was performed by MIR (Michigan, USA)
and approved by their UCUCA. According to body weight, 10 mL/kg of nanoparticle
solutions were administered to female sher 344 rats (130e145 g Charles River
Laboratories, Indiana, USA). The animals were fed irradiated Rodent Diet 5053
(LabDiet) and water ad libitum. Animals were housed in static cages with BedOCobs bedding inside Biobubble clean rooms that provide HEPA ltered air into
the bubble environment at 100 complete air changes per hour. All treatments and
body weight determinations were carried out in the bubble environment at
a temperature of 21  C and a humidity range of 30e70%. Cubosome and hexosome
solutions were administered via tail vein injection into animals at a maximum
concentration of 365 mg/kg and monitored for abnormal effects. If a death occurred
due to nanoparticle toxicity, the nanoparticle solutions were diluted by 25% (v/v)
and then to a 10% (v/v) dilution of stock and dosing was repeated in nave animals
until tolerated doses were reached for each compound.
2.10. In vivo imaging
Approval for this was obtained from the Florey Neuroscience Institutes animal
ethics committee. An adult male SpragueeDawley rat weighing 423 g was anesthetized with 5% isourane in a 1:1 mixture of medical grade air and oxygen and
prepared for surgery. Anesthesia was maintained throughout the procedure and
subsequent imaging with 1e2.5% isourane delivered through a nosecone placed
over the animals snout. The femoral vein was exposed, cannulated and the incision
closed with silk sutures while the animal was immediately prepared for MRI.
Anesthetized animals were laid supinely in an animal holder with respiration
continuously monitored throughout the experiment using a pressure sensitive
probe positioned over the rats diaphragm. The cradle was inserted into a transmit/
receive coil xed inside a BGA12S gradient set for imaging with a 4.7 Tesla Bruker
Biospec 47/30 scanner. The scanning protocol consisted of a three-plane localizer
sequence followed by multi-slice axial, coronal and sagittal scout images to ascertain
the orientation and position of the kidneys and liver. Two oblique slices were
selected; the rst orientated to image the midline of both kidneys while the second
dissected the liver. T1-weighted images were acquired prior to, and after, injection of
contrast enhancing hexosomes using a rapid acquisition, relaxation enhanced
(RARE) sequence with the following imaging parameters: repetition time (TR)
500 ms, RARE factor 4, effective echo time (TEeff) 15.63 ms, in-plane resolution 137  137 mm, number of slices 2, slice thickness 2 mm, averages
(NEX) 4 and a total scan time of 4 min and 16 s.

dynamic light scattering (DLS, intensity vs. size) of all nanoparticle

formulations used in this work were found to be consistently around
200  30 nm in dimension and have polydispersity indices of
between 0.13 and 0.18 (data not shown). The nanoparticle sizes and
polydispersity indices did not vary signicantly between the two
bulk lipids (phytantriol and Myverol) or incorporated nitroxide
concentration and no trend in size or polydispersity index was
observed.
Cryo-TEM was further used to investigate the dispersed lyotropic liquid crystalline nanoparticles. The cryo-TEM images (Fig. 2)
conrm the presence of discrete colloidal nanoparticles spanning
a size range of 60 nm and 300 nm which was typical for these
systems and was consistent with the DLS traces observed. The
phytantriol sample containing 2% nitroxide lipid shows nanostructured particles displaying a periodic internal structure typical
of cubic phase materials. As the percentage loading of nitroxide
lipid was increased, distinctly different structures known as onion
rings and multi-faceted nanoparticles were visible, typical of
hexagonal phase materials. This indicated that a cubic to hexagonal
phase change occurred upon increased loading of nitroxide lipid.
To investigate this phase change in more detail and gain further
insight into the nanostructural changes occurring in these nanoparticles from increasing the nitroxide lipid incorporation, SAXS
was performed on the different formulations. This technique

2.11. Intravenous administration of contrast agent

After acquiring a baseline scan, 600 mL of a 40 mg/mL solution of Myverol
hexosomes containing 14.5 wt% nitroxide lipid in 0.1 M NaCl was intravenously
(10 mL/s) injected followed by 0.3 mL of saline to ush the cannula line. This was
followed by a post injection scan, acquired immediately after administration of the
contrast agent.

3. Results and discussion

3.1. Self-assembly of the lyotropic liquid crystal nitroxide
containing nanoparticles
The paramagnetic nitroxide lipid (Fig. 1) capable of providing MRI
contrast used in this work was synthesized using a fatty reactive acyl
chloride with 4-hydroxy TEMPO as described in the experimental
section. The compound was easily chromatographically puried and
then incorporated into two lyotropic liquid crystal forming bulk lipid
materials, namely phytantriol and Myverol. Cubosome and hexosome dispersions were easily generated from these bulk gels via the
addition of F127 in water acting as a steric stabilizer followed by
sonication of the solution. Phytantriol-pluronic-nitroxide lipid and
myverol-pluronic-nitroxide lipid dispersions were prepared at
various concentrations of nitroxide lipid from 0 to 14 wt % in a highthroughput manner using a Chemspeed Accelerator SLTII robot. An
automated formulation method allowed for the rapid production
and screening of nanoparticles to determine the effects of small
changes in additive composition upon the lyotropic liquid crystal
nanoparticle properties. The average particle size measured by

Fig. 2. Cryo-TEM images of phytantriol cubosomes containing 2 wt% nitroxide lipid

(A) with inset showing periodic internal structure inside a single cubosome and
hexosomes containing 14.5 wt% nitroxide lipid (B) with inset showing characteristic
onion ring structures (scale bar is 200 nm).

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

allowed a detailed investigation of the possible internal lamellar,

cubic and hexagonal phase symmetries of the dispersed nanoparticles produced from phytantriol and Myverol (Fig. 3). The
lattice parameters of cubic (Q2) and hexagonal (H2) phases
detected (Fig. 3) were calculated from the scattering curves at
different loadings of the nitroxide lipid (data not shown). The SAXS
patterns of nanoparticles made from phytantriol (lattice parameter
67.3 ) and Myverol (lattice parameter 86.3 ) without any addition of nitroxide lipid indicated a cubic lattice symmetry with
a Pn3m space group (double diamond) typical of these bulk lipids.
As the concentration of nitroxide lipid increased, a Q2 to H2 transition as indicated by a two phase region commencing at a nitroxide lipid concentration of 4 wt% in phytantriol and 2 wt% in
Myverol was observed (Fig. 3). The lattice parameter of both the Q2
and H2 phases decreased with increasing concentration of nitroxide lipid. The cubic and hexagonal lattice parameter of the

phytantriol nanoparticles was around 1e2 nm smaller than that of

the Myverol nanoparticles. The nitroxide lipid appears to drive an
increase in curvature in both bulk lipids which results in the inverse
cubic (Q2) to inverse hexagonal (H2) phase change observed. This
decrease in lattice parameter with increasing additive loading
(increasing curvature) has been observed for other systems and
was typical of aliphatic additives [32,35]. These results indicate that
the nitroxide lipid was more readily incorporated into phytantriol
than Myverol. The robustness of phytantriol to retain its original
cubic phase in the presence of higher levels of additives when
compared to Myverol has been observed by us previously [32]. In
summary, the DLS, Cryo-TEM and SAXS data demonstrate that
addition of small amounts of the nitroxide lipid result in signicant
nanostructural changes (specically a Q2 to H2 phase transition
and decrease in lattice parameter) within the lyotropic liquid
crystal nanoparticle population.

70

Q2 Phase

68
two phase region

53

H2 phase

66

51

64

49

62

47

45

60
0

Lattice parameter of Q2 phase (A)

Lattice parameter of H2 phase (A)

55

6
4
8
10
Wt % of nitroxide lipid in Phytantriol

12

14

88

66
Q2 Phase

86

64

H2 phase

84

62

two phase
region

82

60

80

58

Lattice parameter of H2 phase (A)

Lattice parameter of Q2 phase (A)

56

78
0

10

12

14

Wt % of nitroxide lipid in Myverol

Fig. 3. Synchrotron source SAXS lattice parameter () analysis and liquid crystal phase data of phytantriol (A) and Myverol (B) nanoparticles with increasing wt% nitroxide lipid
concentration. SAXS lattice parameter data of selected bulk gel compositions are shown as a comparison.

Please cite this article in press as: Muir BW, et al., Metal-free and MRI visible theranostic lyotropic liquid crystal nitroxide-based nanoparticles,
Biomaterials (2012), doi:10.1016/j.biomaterials.2011.12.018

B.W. Muir et al. / Biomaterials xxx (2012) 1e11

3.2. Magnetic resonance relaxion rates and relaxivities

The nitroxide lipid in this work was paramagnetic because it
contains a stable unpaired electron. Previous work has shown that
liposomes with a nitroxide lipid incorporated into their structure
may induce a slight enhancement in MRI relaxation rates [17].
However, to date there are limited reports on the observed relaxivities of nitroxide nanoparticles. The use of these liposomal-based
materials has been limited in the past [36,37] due to the fact the
nitroxyl group was rapidly reduced in vivo producing diamagnetic
N-hydroxy compounds that will not provide extended and effective
contrast in vivo [17]. Therefore it was hypothesized that lyotropic
mesophase nanoparticles containing nitroxides should be capable
of also enhancing the MRI relaxation rates whilst also protecting the
nitroxide groups from oxidation in vivo for an extended period
[38e40]. The use of a fatty nitroxide lipid inside a cubic or hexagonal
phase nanoparticles, as reported here, was expected to show
a signicant increase in relaxivity compared to the free nitroxyl
group. It is postulated that this may be due to either or both rotational correlation constant effects from the reduced tumbling of the

Longitudinal relaxation rate, R1 (sec -1)

nitroxide group inside the nanoparticle and water connement/

exchange effects in the bi-continuous water channels throughout
the cubosomes. Previously [17], maximal in vivo contrast enhancement of the nanoparticles was achieved when these tumbling rates
were similar to the MRI Larmor precessional frequency.
The effect of the nitroxide lipid concentration on the R1 and R2
relaxation rates in nanoparticle dispersions made with phytantriol
and Myverol is shown in Fig. 4A and B respectively. In the phytantriol system, a rapid rise of R1 was observed from 0 to 1 wt%
nitroxide lipid followed by a linear increase of R1 with increasing
nitroxide lipid (Fig. 4A). The phytantriol system also demonstrated
a possible plateau of R1 between 4 and 8 wt% nitroxide lipid
loading which corresponds with the two phase region (Q2 and H2)
as detected via SAXS. Above 8 wt% there was a further increase in
relaxivity with increasing nitroxide lipid content. In the Myverol
system no signicant increase in R1 was observed until a concentration of 4 wt% was reached. The rate of increase in R1 with
nitroxide concentration in the Myverol system was less than that of
the phytantriol system. The R2 relaxation rates observed in both
nanoparticle systems followed a similar trend as for the R1s as

Phytantriol
2.5

Myverol

2
1.5
1
0.5
0
0

10

12

14

10

12

14

4.5

Transverse relaxation rate, R2 (sec-1)

Wt% nitroxide lipid

Phytantriol
Myverol

3.5
3
2.5
2
1.5
1
0.5
0
0

6
8
Wt% nitroxide lipid

Fig. 4. Longitudinal (A) and Transverse (B) relaxation rates of phytantriol and Myverol nanoparticles with increasing wt% nitroxide lipid concentration.

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

Table 1
Relaxivity values of selected phytantriol and Myverol self assembled lyotropic liquid crystal nanoparticles containing varying levels of a nitroxide lipid. The nanoparticle
formulation liquid crystal phases as determined via SAXS are also reported.
Bulk lipid

Nitroxide lipid concentration (wt%)

r1 (mM
1s
1)

r2 (mM
1 s
1)

r1/r2

Liquid crystal phase

Phytantriol
Phytantriol
Myverol
Myverol

4.0
14.5
2
14.5

0.59
0.27
0.14
0.08

1.35
0.43
0.32
0.17

0.43
0.43
0.44
0.49

Cubic (Q2)
Hexagonal (H2)
Cubic (Q2)
Hexagonal (H2)

a function of the nitroxide lipid concentration (Fig. 4B). To further

investigate the two bulk lipid systems, the r1 and r2 relaxivities
(mM
1 s
1) of two select nitroxide containing samples (in the cubic
and hexagonal phase region respectively) of both bulk lipid systems
were calculated (Table 1).
The relaxivity was calculated by measuring the gradient of the
linear best t of the R1 and R2 versus nitroxide lipid concentration
(mM) plots. The relaxivity of the cubic phase nanoparticles for
both bulk lipids was approximately double that of the hexagonal
phase nanoparticles in each system. The highest r1 relaxivity
(0.59 mM
1 s
1) was measured in the phytantriol system at a nitroxide lipid concentration of 4 wt%. As a comparison the relaxivities of
small molecule nitroxyl contrast agents used in functional MRI
imaging were on the order of 0.1 mM
1 s
1 (4.7 T) with half lives of
a few minutes [41]. In a study by Gussoni et al. poly(amidoamine)
based polymers conjugated with amino-TEMPO recorded relaxivities

similar to those reported here on the order of 0.4 mM
1 s
1 in vitro
but no data on efcacy in vivo has been reported of such systems [42].
It was quite striking that the cubic phase nanoparticles containing less nitroxide lipid (4 wt%) were signicantly more effective
at enhancing the relaxation rates compared to the hexagonal phase
nanoparticles containing greater concentrations of nitroxide lipid
(14.5 wt%). This indicates a strong effect of lyotropic mesophase
upon nanoparticle relaxivities. As the nanoparticles have very
similar size distributions and polydispersities, rotational correlation constant effects should not be a signicant factor in the
enhanced relaxivity observed in the cubic phase nanoparticles.
Therefore the nanostructure of the cubic phase nanoparticles must
be playing an important role in enhancing the MRI relaxation rates.
This observation was consistent with the plateau observed in the
phytantriol nanoparticles R1 at nitroxide loadings between 4 and
8 wt% where there was a cubic to hexagonal phase nanoparticle

A
CHO-GFP cells

Cell Viability (%)

no Nitroxide lipid (Q2)

3% Nitroxide lipid (Q2)
6% Nitroxide lipid (Q2 & H2)
9% Nitroxide lipid (H2)
14% Nitroxide lipid (H2)

120

HEK cells

100
Cell Viability (%)

100
90
80
70
60
50
40
30
20
10
0

no Nitroxide lipid (Q2)

80

3% Nitroxide lipid (Q2)

6% Nitroxide lipid (Q2 & H2)

60

9% Nitroxide lipid (H2)

40

14% Nitroxide lipid (H2)

20

80

60

40

20

10

Phytantriol Lipid Concentration (ug/ml)

80

60

40

20

10

0
Phytantriol Lipid Concentration (ug/ml)

2.1% Nitroxide lipid (Q2)

14.6% Nitroxide lipid (H2)

Myverol Lipid Concentration (ug/ml)

HEK cells
no Nitroxide lipid (Q2)
2.1% Nitroxide lipid (Q2)
14.6% Nitroxide lipid (H2)

0
50
75
10
0
12
5
15
0
17
5
20
0
22
5
25
0
27
5
30
0
32
5
35
0
40
0

no Nitroxide lipid (Q2)

100
90
80
70
60
50
40
30
20
10
0

Cell Viability (%)

CHO-GFP cells

0
50
75
10
0
12
5
15
0
17
5
20
0
22
5
25
0
27
5
30
0
32
5
35
0
40
0

Cell Viability (%)

100
90
80
70
60
50
40
30
20
10
0

Myverol Lipid Concentration (ug/ml)

Fig. 5. Results of Alamar blue cytotoxicity assay testing (expressed as a percentage of viable cells relative to a positive control) of nitroxide lipid (A: phytantriol & B: Myverol)
nanoparticle formulations on Green Fluorescent Protein expressing Chinese Hamster Ovary cells (CHO-GFP) and Human Embryonic Kidney cells (HEK293). The gure legends
display the wt% of nitroxide lipid in the nanoparticle formulations and the nanoparticle liquid crystal phase determined via SAXS analysis is shown in brackets (Q2 Cubic,
H2 Hexagonal). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

transition with signicantly lower relaxivities. In the two phase

region with increasing wt% nitroxide lipid more of the nanoparticles being generated consist of the lower relaxivity hexagonal
phase material. Therefore, as the proportion of higher relaxivity
cubosomes decreases despite the increasing nitroxide loading, the
average R1 in the system plateaus. Finally, as the total concentration
of nitroxides in the system increases signicantly past 8 wt%
nitroxide lipid, the relaxivity of the nanoparticles begins to
increase. The higher relaxivity of the cubic phase nanoparticles was
likely due in part to the greater surface area of the bi-continuous
water channels in these structures compared to the hexagonal
phase nanoparticles.
The lattice parameter of the phytantriol cubosomes used in this
study was around 1e1.5 nm smaller than the Myverol cubosomes
and the relaxivity of phytantriol cubosomes was signicantly
greater. This may be due to a higher rate of water exchange in the
inner and/or second coordination sphere of the nitroxide lipid
headgroup in the smaller water channels of the phytantriol cubic
phase compared to the Myverol cubic phase. The diameter of the
water channels in the bulk cubic phase nanoparticles in phytantriol
can be theoretically calculated to be approximately 2.3 nm
compared to 4.3 nm in the Myverol system, based on the method of
Briggs et al. [43]. In more recent work Ritman et al. conrmed this
calculation experimentally using atomic force microscopy to
measure the internal water channels in excess water of phytantriol
and monoolein [44]. To summarise, the increased connement of
water inside the bi-continuous water channels of the bulk cubic
phase of phytantriol may result in an increased relaxivity due to
faster exchange of the inner sphere water molecules with bulk
water, compared to the hexagonal phase water that was located in
columns. In addition, the smaller bi-continuous water channels in
the cubic phase of phytantriol compared to Myverol may create an
environment that enhances lifetimes (decreased mobility) of inner
sphere water protons. Interestingly, this effect of increasing relaxivity in conned systems has recently been reported for gadolinium
based MRI contrast agents [45].

3.3. Cytotoxicity and maximum tolerated dose study

The toxicity of lyotropic mesophase nanoparticles is unclear
despite the large number of in vitro and in vivo studies published,
since only a limited number of studies have reported experimental data on the toxicity of such nanoparticles [46e49]. Prior to
testing of lead compounds in animals, cytotoxicity testing was
performed in vitro using Chinese hamster ovary green uorescent
protein expressing (CHO-GFP) cells and human embryonic kidney
(HEK293) cells. The results of an Alamar blue cytotoxicity assay on
phytantriol and Myverol nanoparticles with increasing concentrations of nitroxide lipid are shown in Fig. 5A. Both cell lines
demonstrated that the phytantriol nanoparticles were toxic at
a concentration of 20 mg/mL and were highly toxic above 40 mg/mL.
The cytotoxicity of phytantriol based nanoparticles was observed to
increase with nitroxide lipid concentration. Shen et al. [46] reported
a similar cytotoxicity of phytantriol containing cubosomes in L929
broblast cells at concentrations greater than 40 mg/mL using an
MTT assay. Lyotropic mesophase lipid nanoparticles may interact
with the lipid bilayer of cells and result in membrane fusion and lipid
exchange that can disrupt membrane integrity and cause cell lysis.
Whilst in vitro testing is considered the gold standard in the
initial phase of toxicity testing, it is also limited in its ability to
address complex, living systems. Hence it was deemed necessary to
perform in vivo testing based on the preliminary data to determine
the MTD. The static nature of in vitro tests and the added
complexity of the system once these nanoparticles were delivered
in vivo, not to mention their mode of delivery (oral, buccal, dermal,
bolus), make it very difcult to ascertain from in vitro cell based
assays the maximum nanoparticle concentration that can safely be
delivered in vivo. A signicant difculty in assessing nanoparticle
toxicity is the absence of a standard assay or cell line. As such we
performed MTD testing in rats on phytantriol based nanoparticles
and found that the MTD in rats of phytantriol cubosomes was
350 mg/kg while for phytantriol cubosomes containing 4 wt%
nitroxide lipid the MTD decreased to 80 mg/kg. Phytantriol

100000

DPM/mL

Phytantriol + 8% PF127
Myverol + 8% PF127
Phytantriol + 15% PF127
Myverol + 15% PF127

10000

100

200

300

400

500

Time (min)
Fig. 6. Plasma concentration (disintegrations per minute (DPM)) versus time pharmacokinetic proles for phytantriol (lled symbols) and Myverol (open symbols) nanoparticles after intravenous administration in rats. Circles denote dispersions prepared using 8 wt% F127 and triangles denote 15 wt% F127. Particles contained 0.5 mCi
3
H-dioleylphosphatidylcholine as a radioactive tracer.

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B.W. Muir et al. / Biomaterials xxx (2012) 1e11

hexosomes containing more nitroxide lipid were not tested. All

subsequent tests on animals were performed below this concentration (80 mg/kg). The increased level of toxicity in the cubosomes
after addition of the nitroxide lipid correlated qualitatively with the
Alamar blue cytotoxicity assay data. The cytotoxicity of the Myverol
nanoparticles in the Alamar blue assay was signicantly less than
that of the phytantriol nanoparticles. Cytotoxicity effects were not
observed until concentrations above 75 mg/mL were reached
(Fig. 5B). Nanoparticles made from Myverol were also found to be
better tolerated by the CHO-GFP cells compared to the HEK293
cells. In addition, no toxic effects were observed in MTD testing of
nitroxide loaded Myverol nanoparticles up to the maximum
concentration tested (365 mg/kg).
3.4. Pharmacokinetic behavior of lyotropic liquid crystalline
nanoparticles
To investigate the behavior after IV injection of nanoparticles
made from Myverol and phytantriol a study of their pharmacokinetic behavior (blood half life) was performed in rats. The blood
plasma proles in Fig. 6 demonstrate the relative difference in
pharmacokinetic behavior of Myverol and phytantriol nanoparticles with two different levels of pluronic F127 stabiliser (8 and
15 wt%). The phytantriol nanoparticles were rapidly removed from
circulation on administration, whereas the Myverol nanoparticles
demonstrated a signicantly longer circulation prole with a half
life of approximately 90 min. At 2 h post injection there was
double the concentration of Myverol nanoparticles in circulation
relative to phytantriol nanoparticles. At longer times the concentration of nanoparticles in circulation converge, indicating that
some residual nanoparticles were still in circulation, albeit at a low
level (<1% of total dose). This behavior may complicate the utility
of phytantriol nanoparticles as a carrier for the nitroxide imaging
agent, and indicates that Myverol-based nanoparticles were likely
a better candidate for in vivo imaging. The rapid removal of
nanoparticles from circulation was principally a consequence of
binding of opsonins and subsequent removal by the reticuloendothelial. Steric stabilization of nanoparticles using PEG is often
used to extend the circulation time through inhibited initial
binding. The results indicate that the Myverol nanoparticles better
avoid recognition, indicating a more effective steric barrier of the
pluronic F127 on these nanoparticles to inhibit opsonin binding
and rapid removal from the bloodstream. This was consistent with
past observations that Myverol nanoparticle dispersions demonstrate improved colloidal stability compared to phytantriol
dispersions. This has been proposed to be linked to stronger
interactions of the pluronic stabilizer with the Myverol nanoparticles [50]. The half life of the nanoparticles in plasma was
however still well short of that of PEGylated liposomes measured
using identical methodologies indicating the relatively poor ability
of the pluronic to provide a stealth coating for liquid crystalline
nanoparticles [51,52]. Increasing the pluronic concentration did
not improve the circulation time for the phytantriol nanoparticles
indicating that they were likely saturated with pluronic. The
nature and strength of the interaction between the pluronic and
the nanoparticles governs the likelihood of recognition and uptake
by the RES in vivo. The Myverol nanoparticles were well tolerated
at the concentration administered, indicating an overall role for
Myverol-based nanoparticles as the preferred carrier for nitroxide
MR imaging contrast agents.
3.5. In vivo MRI study
Due to the fact the Myverol nanoparticles were found to be
signicantly less toxic than phytantriol nanoparticles and have

signicantly greater blood half lives, all in vivo magnetic imaging

experiments were performed using Myverol based nanoparticles.
The choice of this lipid was also justied by the fact that both the
bulk lipid and nitroxide lipid can readily undergo lipolysis in vivo
due to their ester bonds whereas phytantriol cannot be easily
biodegraded. The nitroxide lipid has the potential to function as an
MRI contrast agent by shortening T1 relaxation times that results in
brightening of T1 weighted MR images. It was envisioned that after
bolus delivery of Myverol nitroxide lipid nanoparticles, the RES
would transport these nanoparticles to organs such as the liver and
kidneys. As nanoparticulate contrast agents such as clinically used
superparamagnetic iron oxide nanoparticles (SPIOs) tend to be
found in the liver after injection due to the reasons described above,
they have been previously used in the clinic and in the laboratory
for diagnosis of small tumors, lesions, metastasis, brosis and
cirrhosis. Anticipating a similar distribution of the Myverol nanoparticles, T1 weighted MR images of the liver and kidneys were
taken both before and after IV injection of the Myverol nanoparticles (Fig. 7). The MRI signal increase in the liver was normalized to the surrounding mean muscle tissue signal and false colour
images were reported. No contrast was observed in the kidneys
post injection over the time course of this study but enhanced T1
weighted contrast in the liver was observed shortly after injection
of the nitroxide loaded nanoparticles.
These results demonstrate that Myverol nanoparticles can be
used as in vivo T1 enhancing MRI contrast agents, without the use of

Fig. 7. False color T1-weighted MR images of a rat liver prior to, and after, intravenous
injection of Myverol hexosomes containing 14.5 wt % nitroxide lipid (left and right
images, respectively). Signal intensity is normalized to the mean muscle tissue signal.
Color bar shows signal intensity in arbitrary units. No contrast enhancement was
observed in the kidneys (data not shown).

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10

B.W. Muir et al. / Biomaterials xxx (2012) 1e11

heavy metal containing compounds such as gadolinium chelates. In

addition, the in vitro and in vivo showed they had low cytotoxicity
and were readily biodegradable.
4. Conclusions
In summary, T1 enhancing nitroxide containing lyotropic liquid
crystal nanoparticles have been produced that provide effective
in vivo MRI contrast enhancement without containing potentially
hazardous heavy metals such as gadolinium. Phytantriol based
nanoparticles were found to have poor pharmacokinetic behavior
(low blood half life) when pluronic F127 was used as stabilizer
when compared to Myverol nanoparticles. In addition, phytantriol
based nanoparticles were found to be signicantly more toxic than
Myverol nanoparticles in vitro and in vivo. Unlike nitroxide loaded
liposomes that have been previously reported, lyotropic mesophase
nanoparticles loaded with nitroxide lipids were extremely effective
at providing MRI contrast in vivo. Interestingly, incorporation of
a nitroxide lipid into the liquid crystal nanoparticle structure
allowed for a greater enhancement of relaxivity when a Pn3m space
group cubic phase was present compared to the hexagonal phase
that appeared at higher nitroxide loadings. The Myverol based
nanoparticles were well tolerated in cells and live animals, and the
physical properties of the nanoparticles can be tuned by careful
control over additive composition.
Acknowledgements
The authors would like to acknowledge the work of Charles
River Discovery and Imaging Services for performing MTD testing
and in particular Vinod Kaimal, Patrick McConville and Lisa Repke.
The authors would also like to thank Charlotte Conn for useful
discussions and calculations on the size of the continuous water
channels in the cubic phase nanoparticles and Adrian Hawley for
help with SAXS analysis. This research was undertaken on the
SAXS/WAXS beamline at the Australian Synchrotron, Victoria,
Australia. This work was supported by Australian NHMRC Fellowships to GFE (#1003993) and BAM (#454790).
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Biomaterials (2012), doi:10.1016/j.biomaterials.2011.12.018