Liquid Crystalline Systems of Phytantriol and Glyceryl

Monooleate Containing a Hydrophilic Protein:

Characterisation, Swelling and Release Kinetics
S.B. RIZWAN,1 T. HANLEY,2 B.J. BOYD,3 T. RADES,1 S. HOOK1
1
New Zealand’s National School of Pharmacy, University of Otago, Dunedin, New Zealand
2
Bragg Institute, Australian Nuclear Science and Technology Organisation, Menai, NSW 2234, Australia
3
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,
Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia

Received 16 December 2008; accepted 22 January 2009

Published online 1 April 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21724

ABSTRACT: Swelling and phase behaviour of phytantriol and glyceryl monooleate

(GMO) matrices with varying water loadings were investigated. Release of a model
protein, FITC-Ova was subsequently examined. Polarised light microscopy and small
angle X-ray scattering analysis showed that the addition of FITC-Ova only altered the
liquid crystalline structure of phytantriol matrices at low water loadings, and that
postrelease study, the phase structure of matrices at both low and high loading reflected
that of the binary system. Addition of FITC-Ova to GMO matrices also altered the liquid
crystalline structure when compared to the respective binary system at low but not at
high loading. All samples analysed after the release study had transformed to the
reverse hexagonal phase (HII). Swelling studies revealed a faster and more extensive
swelling of GMO when compared to phytantriol. Release of FITC-Ova from phytantriol
matrices was faster and occurred to a greater extent most likely due to the conversion of
GMO matrices into the HII phase. No effect on release as a function of initial water
content was observed for either lipid. We have confirmed that phytantriol based liquid
crystalline matrices can sustain the release of a hydrophilic protein, suggesting its
suitability as a potential sustained antigen-delivery system. ß 2009 Wiley-Liss, Inc. and the
American Pharmacists Association J Pharm Sci 98:4191–4204, 2009
Keywords: controlled release; diffusion; kinetics; lipids; protein delivery

INTRODUCTION other conditions, these liquid crystal forming

Lyotropic liquid crystalline structures are often
formed by polar lipids in an aqueous environment.
These systems can display a rich array of
polymorphism in their lipidic self-assembly.
Depending on amphiphile structure, concentra-
tion of solvent or amphiphile, temperature or
Correspondence to: S. Hook (Telephone: þ64-3-479-7877;
Fax: þ64-3-479-7034;
E-mail: sarah.hook@stonebow.otago.ac.nz)
Journal of Pharmaceutical Sciences, Vol. 98, 4191–4204 (2009)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
lipids (mesogens) may self-assemble into thermo-
dynamically stable lyotropic phases such as the
lamellar (La), the reverse hexagonal (HII), and the
bicontinuous or discrete micellar cubic phases.1
Due to its unique microstructure the bicontin-
uous cubic phase (referred to hereafter as simply
‘cubic phase’ or Q) has received considerable
attention as a promising delivery system for
various actives ranging from low molecular weight
drugs to proteins, peptides, amino acids and
nucleic acids.2,3 The combination of hydrophilic
and hydrophobic domains in the cubic mesophase
provides an ideal environment to solubilise

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009 4191
4192 RIZWAN ET AL.
materials of varying polarity.2–8 The potential of
the cubic phase to provide a slow release matrix
for drugs of varying size and polarity, including
peptidic drugs has been reported.2–10 The release
of the incorporated active is thought to be
controlled partly by the unique microstructure,
where the active has to diffuse through the
structure to access the external solution. The
pore size and tortuosity of the water channels and
the stiffness and high viscosity of the cubic phase
contributes to the sustained release of the
entrapped active.
A number of different classes of polar lipids,
including phospholipids, alkyl glycerates11,12 and
glycolipids13 have been investigated for their
ability to form cubic phases. However the majority
of work has focused primarily on the unsaturated
monoglycerides, in particular glyceryl monooleate
(GMO) and mixtures of GMO with other lipids or
structural derivatives based on GMO.3,5,6,14 Phy-
tantriol is a lipid of more recent interest that also
forms cubic liquid crystalline phases, and whose
phase diagram was first described by Barauskas
and Landh in 2003.15 Phytantriol is commonly
used as an ingredient in the cosmetics industry for
improving moisture retention.15,16 Structurally it
is different to GMO (Fig. 1), however interestingly
it displays a very similar phase behaviour as a
function of water concentration and tempera-
ture.17,18 As fatty acid based materials such as
GMO are susceptible to esterase catalysed hydro-
lysis, the phytanyl backbone of phytantriol has
been proposed as a structurally stable alterna-
tive.11 Additionally most commercially produced
sources of GMO vary significantly in purity
making this material difficult to characterise
and control, whereas phytantriol is generally
available at 95% nominal purity.18 However
variations in the phase behaviour of phytantriol
sourced from different manufacturers has been
recently reported.19
Figure 1. Chemical structures of phytantriol
(3,7,11,15-tetramethyl-1,2,3-hexadecanetriol) (A) and
glyceryl monooleate (GMO) (B).
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
Several studies have investigated the swelling
behaviour of GMO-based liquid crystalline sys-
tems and the release of various actives from such
systems.6,11,14 However to the best of our knowl-
edge there have been no reports on the swelling
behaviour and release of protein bioactives from
phytantriol based mesophases. It is pertinent to
understand the physicochemical properties of
phytantriol such as swelling upon contact with
excess water and the mechanisms of release of
entrapped actives if it is to be used as the basis of
a sustained release formulation. Consequently,
the aim of this study was to investigate how the
swelling, initial water content, presence of actives
and the type of liquid crystalline phase formed
influence the release of entrapped active from
phytantriol based liquid crystalline systems.
GMO-based systems served as a comparison.
MATERIALS AND METHODS
Materials
Phytantriol (3,7,11,15-tetramethyl-1,2,3-hexade-
canetriol) was purchased from A & E Connock
(Hampshire, England) and glyceryl monooleate
(GMO) (RYLO MG 20 PHARMA), a distilled
monoglyceride (min. 95%) with a high GMO
content (90%) was a gift from Danisco (Brabrand,
Denmark). Both lipids were used as received.
RYLO MG 20 constitutes a similar composition
to other GMO-based products published in the
literature with its phase behaviour shown to be
representative of GMO.20,21 Ovalbumin (Ova)
(chicken egg derived, grade V, Sigma, St. Louis,
Missouri) was conjugated to fluorescein isothio-
cyanate (FITC) (Isomer I, Sigma) as described
previously.22 Triton1 X-100 (octylphenol-poly-
ethylene glycol ether) and phosphate buffered
saline (PBS) sachets (pH 7.4, 0.01 M) were
obtained from Sigma–Aldrich (Auckland, New
Zealand). All water was ion exchanged, distilled
and passed through a Milli-Q water purification
system (Millipore, Bedford, MA).
Characterisation of Liquid Crystalline Systems of
Phytantriol and GMO
Preparation of Liquid Crystalline Systems for
Phase Behaviour Studies
Binary mixtures were prepared by weighing
phytantriol or GMO (approximately 300 mg) into
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 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO
glass vials and adding water at appropriate
concentrations (0–35%, w/w). The samples were
then heated briefly in a water bath at 958C to
obtain a homogenous mixture (low-viscosity L2
phase), which was then vortexed. The heating and
vortex mixing was repeated for three cycles and
the samples were then centrifuged at 1500 g for
1 h23 and allowed to equilibrate for 1 week at room
temperature prior to further investigations.
Polarizing Light Microscopy
The preparations of phytantriol and GMO were
examined at ambient temperature by polarizing
light microscopy (PLM) using a Nikon Optiphot
PFX (Japan) fitted with crossed polarizing filter.
Images were captured with a digital camera
(Nikon CoolPix 990, Japan) and classified accord-
ing to the visualised textures of lyotropic meso-
phases described by Rosevear.24
Cryo-Field Emission Scanning Electron
Microscopy (Cryo-FESEM)
Samples were loaded into brass stubs and plunge
frozen in liquid nitrogen or propane. The samples
were transferred into the cryo-chamber (Gatan,
Alto 2500, UK) of the microscope (JEOL, JSM-
6700F, Japan), which was held at a temperature of
1408C. The samples were then sublimed at
908C for 2–5 min and then coated with platinum
for 2 min. Once coated the samples were viewed at
1408C, at an acceleration voltage of 3 kV and a
working distance of 6 mm.
Small Angle X-Ray Scattering (SAXS)
Measurements were performed on a Bruker
Nanostar SAXS camera with pin-hole collimation
for point focus geometry. The instrument source
was a copper rotating anode (0.3 mm filament)
operating at 45 kV and 110 mA, fitted with a cross
coupled Göbel mirror, resulting in Cu Ka radia-
tion wavelength of 1.54 Å. The SAXS camera was
fitted with a Hi-star 2D detector (effective pixel
size 100 mm). The sample to detector distance was
chosen as 650 mm which provided a q-range
of 0.008–0.32 Å1. Samples were loaded into
a custom made stainless steel gel holder with
Kapton windows and temperature was controlled
by a Peltier cooled sample stage within 0.18C.
The samples were equilibrated at 258C for 30 min
prior to collection of scattering pattern for 30 min.
Optics and sample chamber were under vacuum
to minimise air scatter. Scattering files were
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
4193
background subtracted and normalised to sample
transmission and then integrated using Bruker
AXS software v4.1.18. The diffraction patterns
obtained were converted to plots of intensity
versus q-value, which enabled the identification of
peak positions, and their correlation with Miller
indices, to identify the different liquid crystalline
structures and space groups for the dominant
internal nanostructure of the sample.25
Swelling Studies of Phytantriol and
GMO-Based Liquid Crystalline Systems
Bulk liquid crystalline phases for swelling studies
were prepared with varying initial water content.
Phytantriol and GMO were heated at 458C to
lower their viscosity, weighed into 5 mL vials
(approximately 300 mg) and allowed to cool to
378C. Over each sample, water (0–35% w/w of
lipid) was gently layered onto the surface of the
lipids and vials were sealed and stored at room
temperature for at least a week to equilibrate
prior to swelling study experiments.
The uptake of water from both phytantriol and
GMO matrices was determined gravimetrically. A
layer of water (4 mL, 378C) was gently placed over
the equilibrated samples, while ensuring that the
integrity of the surface was not compromised
(visual inspection). The samples were placed in
a water bath with a horizontal shaker (378C,
60 strokes/min). At fixed time intervals all the
excess water from the samples was removed,
the surface of the samples blotted with a fine
weave paper to remove any excess water and then
weighed. Once weighed, 4 mL of water was again
placed onto the surface of the samples and the
sample was placed back in the water bath. The
water uptake data was fitted to first-order kinetic
(according to Eq. 1) and second order kinetic
(Eq. 2) equations below and as described else-
where in the literature6,14,26,27
W1
ln ¼ kt (1)
W1  W
t 1 t
¼ 2
þ (2)
W kW1 W1
where W1 is the maximum water uptake, W is the
water uptake at time t, (W1  W) is the unrealised
water uptake, and k is the rate constant. For
Eq. (2), the initial rate of swelling is the reciprocal
of the y-intercept of the plot of t/W versus t and
maximum or equilibrium water uptake (W1) is
described by the reciprocal of the slope.
4194 RIZWAN ET AL.
Release of FITC-Ova from Phytantriol and
GMO-Based Liquid Crystalline Systems
Samples containing the model protein FITC-Ova
were prepared in a similar manner to the swelling
study samples. Phytantriol or GMO were heated
at 458C, to this a concentrated solution of FITC-
Ova (1 mg/10 mL) at 10% (w/w) with respect to the
lipid was added and the sample was mixed on
a magnetic stirrer (250–300 rpm) until it was
visually homogenous, forming a low viscosity
phase. The samples were then weighed into
5 mL vials as described above (300 mg) and water
was added to the samples (378C) such that the
final concentrations were 10%, 20%, 25%, 30% and
35% (w/w) with respect to the lipid. The vials were
sealed and stored at room temperature for a week
to allow samples to equilibrate, prior to release
study experiments.
Release studies were conducted under condi-
tions identical to the swelling study. Briefly, PBS
(4 mL and 378C) was gently layered on the surface
of samples and at fixed time intervals 100 mL
sample was removed, diluted with 5% (w/w)
Triton1 X-100 in PBS (pH 7.4) and analysed by
spectrofluorimetry (Shimadzu Rf-540, Japan) at
lexcitation ¼ 490 nm and lemission ¼ 520 nm. Triton1
X-100 was added to the PBS to lyse any lipid
fragments. The volume removed was replaced
with fresh PBS. Absorbance values obtained were
analysed against standard curves prepared on
each day of analysis from FITC-Ova solutions kept
under the release study conditions. All experi-
ments were conducted in triplicate unless speci-
fied otherwise. The phases of selected samples
were studied postrelease using SAXS to determine
any changes to the liquid crystalline structure.
Statistical Analysis
Standard curves were constructed and assessed
using regression analysis. A one-way analysis of
variance (ANOVA) followed by Tukey’s pairwise
comparisons was used to assess statistical sig-
nificances where required. All statistical analysis
was performed using Minitab1 Statistical Soft-
ware Release 12.1 (Minitab Inc., State College,
Pennsylvania).
RESULTS
Cryo-FESEM
The microstructure of a binary mixture of GMO
and water (30%, w/w) is shown in Figure 2, panels
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
A and B. Cryo-FESEM revealed a tortuous
internal structure with the lipidic regions sepa-
rated from the network of water channels, which
appear to open to the exterior of the structure.
The surface appeared rough with a nodule like
appearance, with ‘holes’ in the structure most
likely caused by the channels that are open to the
exterior of the structure. The structure of the
GMO cubic phase shown in the micrographs is
comparable to that of phytantriol, in Figure 2,
panels C and D reported previously.28
Phase Determination by Polarizing Light Microscopy
and Visual Appearance
The appearance and consistency of the phases
formed at ambient temperature by binary systems
of phytantriol and GMO at various water ratios,
with or without FITC-Ova, were investigated and
are summarised in Table 1.
Phytantriol and GMO binary systems showed
similar phase sequences with increasing water
concentration. At 5% (w/w) water both GMO and
phytantriol show a low viscosity isotropic phase
(most likely the L2 phase). Samples of phytantriol
with 10 and 15% (w/w) water were soft, opaque
gels with medium flow, however GMO samples at
15% (w/w) were stiff and did not flow. These
samples were optically anisotropic (PLM) with
the presence of oily streak textures and Maltese
crosses (Fig. 3A and B) indicating the presence of
the La phase. Further increase in water content
(20%, w/w) transformed the samples into vis-
cous, transparent and optically isotropic gels,
generally indicative of the formation of the cubic
phase.
Phytantriol matrices containing FITC-Ova
showed the same phase behaviour to their
respective binary systems at all water loadings
investigated. GMO matrices containing FITC-Ova
were generally comparable to their corresponding
binary systems except for samples at 20% and 25%
(w/w) water loadings, which were anisotropic
with the presence of fan like textures (Fig. 3C),
indicative of the formation of the HII phase rather
than the cubic phase due to the presence of the
FITC-Ova.
Phase Determination by Small Angle
X-Ray Scattering
Small angle X-ray scattering (SAXS) was used to
determine the internal structure of the liquid
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 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO 4195

Figure 2. Cryo-FESEM micrographs of the bulk cubic phase formed by addition of

30% w/w water to GMO (A and B). Panels C and D show bulk cubic phase of phytantriol
formed by addition of excess water, for comparison from reference 28. All samples were
equilibrated for a minimum of 1 week prior to analysis. The bar represents 1 mm.

Table 1. Summary of Physical and Optical Properties of the Liquid Crystalline Phases of Phytantriol (PHYT) and
GMO/Water Systems With or Without FITC-Ova (1 mg:100 mg lipid, Indicated by Italics and Shaded) Determined
Visually and by Polarizing Light Microscopy

Physical Optical Phase

Water Content
(%, w/w) PHYT GMO PHYT GMO PHYT GMO
0 C, FF C, FF þ S Iso Iso þ crystals L2 L2 þ crystals
5 C, FF C, FF Iso Iso L2 L2
10 O, MF O, MF Aniso Aniso La La
10 O, MF O, MF Aniso Aniso La La
15 O, MF C, NF Aniso Aniso La La
20 C, NF C, NF Iso Iso Q Q
20 C, NF C, NF Iso Aniso Q H
25 C, NF C, NF Iso Iso Q Q
25 C, NF C, NF Iso Aniso Q H
30 C, NF C, NF Iso Iso Q Q
30 C, NF C, NF Iso Iso Q Q
35 C, NF C, NF Iso Iso Q Q
35 C, NF C, NF Iso Iso Q Q
Physical properties: C, clear; O, opaque; S, solid; FF, free flow; MF, medium flow and NF, no flow.
Optical properties: Iso, isotropic; Aniso, anisotropic.
Phase identification: L2, reversed micellar; La, lamellar; H, reverse hexagonal; Q, cubic.

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4196 RIZWAN ET AL.

for both lipids at 10% (w/w) initial water loading

Figure 3. Representative polarizing light micro-
graphs (magnification 100) showing maltese crosses
(panel A) and a combination of maltese crosses and oily
streaks (panel B) observed in binary mixtures of phy-
tantriol and GMO with initial water loadings of 10%
and 15% (w/w) and in pseudo-ternary systems of phy-
tantriol and GMO containing 10% (w/w) water and
FITC-Ova. A representative micrograph of GMO
matrices containing 20% and 25% (w/w) water and
FITC-Ova showing fan like textures is shown in
panel C.
is indicative of the La phase, although the
underlying diffuse hump may indicate some co-
existing L2 phase. The lattice parameters for
the La phase formed by phytantriol and GMO at
10% (w/w) water were 30 and 38.1 Å, respectively
(Tab. 2). Samples of both lipids at 35% (w/w) water
exhibited multiple peaks with relative positions at
ratios of H2:H3:H4:H6:H8:H9 indicative of a
diamond type cubic phase (Pn3m space group)25
with lattice parameters of 67.2 and 86.8 Å,
respectively (Tab. 2).
The phase behaviour of representative samples
of phytantriol (Fig. 5A) and GMO (Fig. 5B) at
10 and 35% w/w initial water content, containing
the model protein FITC-Ova, were also analysed
using SAXS before (‘pre’) and after (‘post’) the
release study. Comparing the ‘pre’ release sam-
ples with the binary samples enables an initial
assessment of the nature of any interaction of
FITC-Ova with liquid crystalline structure. Addi-
tion of FITC-Ova to phytantriol samples had no
effect on the liquid crystalline structure (‘pre-
release’) at 35% (w/w) water. However at 10% (w/
w) water, the La phase structure observed in the
absence of FITC-Ova (Fig. 4) was replaced by
the L2 phase in the presence of FITC-Ova
(Fig. 5A), with a reduction in the lattice parameter

crystalline systems. Figure 4 shows plots of
intensity versus the scattering vector, q obtained
for representative binary samples of phytantriol
and GMO with water, containing either 10% or
35% (w/w) initial water loading. Diffraction peaks
Table 2. Lattice Parameters of the Binary and
Pseudoternary Mixtures (Containing FITC-Ova) of
Phytantriol and GMO Analysed Pre- and Post-release
Study
Phase Structure

Lattice Parameter (Å)

Composition L2 La Pn3m HII

Phytantriol
10% w/w binary mixture 30
35% w/w binary mixture 67.2
10% FITC-Ova ‘pre-release’ 27.8
35% FITC-Ova ‘pre-release’ 67.2
10% FITC-Ova ‘post-release’ 67.9
35% FITC-Ova ‘post-release’ 67.0
GMO
10% w/w binary mixture 38.1
35% w/w binary mixture 86.8
Figure 4. Intensity versus q plots obtained from 10% FITC-Ova ‘pre-release’ 31.2a 50.2a
SAXS measurements of selected binary mixtures of 35% FITC-Ova ‘pre-release’ 86.8
phytantriol (PHYT) and GMO matrices containing 10% FITC-Ova ‘post-release’ 55.4
initial water loadings of 10% and 35% (w/w). Samples 35% FITC-Ova ‘post-release’ 57.3
were equilibrated at room temperature for a minimum
a
of 1 week prior to analysis. Denotes detection of co-existing phases.

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 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO 4197

both lipids at low water content in a similar

manner, but at high water content no significant
interaction was apparent.
Postrelease, the phytantriol formulation with
10% (w/w) initial water content had converted to
the Pn3m cubic phase by the end of the release
study (Fig. 5A), as expected under excess water
conditions in which the structure is free to absorb
water from the release medium to reach its full
swelling capacity at 37% (w/w) water (determined
in swelling data in Tab. 3). Both GMO samples
(initially containing 35% or 10% w/w water),
showed a transition from the cubic Pn3m to
the HII mesophase (Fig. 5B) under the release
study conditions and in the presence of FITC-Ova.

Figure 5. Intensity versus q plots obtained from
SAXS measurements of phytantriol (A) and GMO (B)
matrices at initial water loadings of 10% and 35% (w/w),
containing 1 mg FITC-Ova per 100 mg of lipid analysed
pre- (solid lines) and postrelease (dashed lines) study.
from 30 to 27.8 Å. Addition of FITC-Ova also
altered the phase structure for GMO at 10% (w/w)
water, shifting the peak for the La phase from
q  0.17 (Fig. 4), to q  0.12 (Fig. 5B), represent-
ing a change in lattice parameter from 38.1 to
50.2 Å (Tab. 2), and the L2 diffuse peak at q  0.20
was more pronounced (Fig. 5B). Taken together
these results indicate that FITC-Ova did interact
with the liquid crystalline structures formed by
Swelling Study
The swelling rate and resulting mesophase
formed by swelling amphiphiles can impact the
kinetics of release of incorporated actives.14
Therefore the swelling behaviour of GMO and
phytantriol over time was examined. Figure 6, left
hand panels show plots of increase in weight of
phytantriol (Fig. 6A) or GMO (Fig. 6B)/water
matrices with differing initial water contents
over the duration of the swelling study, whilst
Figure 6, right hand panels show the data within
the first 6 h. Both lipids quickly absorbed water
until they approached their respective equili-
brium water content. As expected, samples with
lower initial water content (0% and 15%, w/w)
hydrated rapidly whilst those with higher initial
water loading hydrated more slowly. Maximum
swelling was achieved within 24 h for GMO
matrices and within approximately 48 h for
phytantriol matrices.
Fits to Eq. (1) [ln(W1/(W1  W)) vs. t] for
swelling by a first order mechanism for either

Table 3. Swelling Kinetic Parameters of Binary Mixtures of Phytantriol and GMO at 378C Calculated from
Swelling Data Using a Second Order Rate Equation (Eq. 2)

Sample Swellingrate (g/g h) Swellingmax (g/g) Correlation Coefficient

Phytantriol
0% (w/w) 0.07 0.37 0.998
15% (w/w) 0.02 0.21 0.995
25% (w/w) 0.01 0.11 0.987
GMO
0% (w/w) 0.12 0.39 0.995
20% (w/w) 0.07 0.17 0.995
25% (w/w) 0.05 0.12 0.991
30% (w/w) 0.04 0.07 0.997

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4198 RIZWAN ET AL.
Figure 6. Water uptake as a function of time for
selected binary samples of phytantriol (A) and GMO
(B) with initial water loading of 0% (^), 15% (*), 20%
(&), 25% (~) and 30 (*) (w/w) (right hand panels) over
the swelling study period. Inserts in the left hand panels
show water uptake by either lipid over the first 6 h.
lipid were poor (Fig. 7A and B), with typical
correlation coefficient values of around 0.80.
However, the swelling of both lipids to form a
cubic phase could be described well using second
order kinetics, as a linear relationship was
obtained when swelling data were plotted as t/W
versus t, according to Eq. (2) (Fig. 7C and D).
Important kinetic parameters calculated from
plots of t/W vs. t are summarised in Table 3.
Correlation coefficients from plots of t/W versus
t were 0.99 at all initial water contents
investigated. The maximum swelling capacities
for phytantriol and GMO obtained experimentally
were 0.36 and 0.37 g/g, respectively. Maxi-
mum swelling capacity calculated using Eq. (2)
(Swellingmax, Tab. 3) for phytantriol and GMO
were 0.37 and 0.39 g/g, respectively. Generally the
initial rate of swelling for both lipids increased as
the water loadings decreased.
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In Vitro Release Study
In vitro release of FITC-Ova from phytantriol and
GMO matrices containing different initial water
contents (therefore different starting liquid crys-
talline phase) were investigated (Fig. 8). In pilot
experiments, the matrices were prepared by
mixing powdered FITC-Ova into molten lipids;
however this resulted in heterogeneous mixtures
of lipid and FITC-Ova evident from the presence
of large FITC-Ova aggregates (data not shown).
Therefore FITC-Ova was incorporated as a solu-
tion in the low viscosity L2 phase to which water
was added at required concentrations to obtain
the various liquid crystalline phases with differ-
ing initial water contents.
Release of FITC-Ova was faster and the extent
of release was greater from phytantriol than from
GMO matrices, with approximately 30% and 10%
released respectively over 3 weeks ( p < 0.05).
Release from both lipid systems was independent
of initial water content ( p < 0.05) and thus the
initial liquid crystalline phase formed. Release
from both liquid crystalline systems plateaued
after 3 weeks.
Matrices were examined visually and under the
PLM to investigate any changes to the liquid
crystalline structure under release study condi-
tions. There was no evidence of erosion of the
matrices upon visual examination. PLM investi-
gation of postrelease matrices at 10% and 35% (w/
w) water content (initial water) revealed presence
of birefringence for GMO matrices whilst phytan-
triol matrices at 10% and 35% were isotropic
postrelease (data not shown), which is consistent
with the SAXS findings in Table 2 and Figure 5.
When plotted against the square root of time,
the release was linear for at least the first 48 h for
both phytantriol and GMO-based systems (Fig. 9).
The linear fit (r2  0.99) demonstrates that the
release kinetics can be explained by Higuchi’s
equation29 thus release of hydrophilic FITC-Ova
from both phytantriol and GMO matrices was
under diffusion control at the early time points.
Extrapolation of the curves in Figure 9 down to 0%
release reveals that for phytantriol there was a 6 h
delay in release, which was not evident in the case
of GMO.
The diffusion coefficient of FITC-Ova from
phytantriol and GMO matrices at 35% (w/w) were
calculated according to5,29,30 and were 0.0088 
108 and 0.0011  108 cm2 s1, respectively.
Diffusion of FITC-Ova was therefore greater from
phytantriol compared to GMO-based matrices.
DOI 10.1002/jps
 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO 4199

Figure 7. Swelling kinetics of selected binary samples of phytantriol and GMO with
initial water loading of 0% (^), 15% (*), 20% (&), 25% (~) and 30% (*) (w/w). Data
fitted to the first order equation (Eq. 1) are shown in panel A for phytantriol and in panel
B for GMO. Data fitted to the second order equation (Eq. 2) are shown in panels C
(phytantriol) and D (GMO).

DISCUSSION systems can be viewed clearly in three dimensions

Phase Behaviour and Structure
Several techniques are described in the literature
to characterise the nanostructure of the various
liquid crystalline phases.31 Cryo-FESEM, PLM
and SAXS were used in this work to characterise
the phase behaviour of phytantriol and GMO in
binary and pseudo ternary mixtures containing
the model protein FITC-Ova.
We have previously shown the advantages of
using cryo-FESEM as a technique for describing
the three dimensional structure of both the bulk
and dispersed liquid crystalline phases.28,32
Figure 2 shows the cryo-FESEM micrographs of
the cubic liquid crystalline phases of phytantriol
and GMO. The features of the liquid crystalline
and the benefits of cryo-FESEM as a technique
in describing the structures becomes apparent.
Descriptions of the internal microstructure of
cubic liquid crystals to date are based largely on
minimal surfaces, where a single continuous lipid
bilayer is contorted such that it divides space into
two congruent and nonintersecting water chan-
nels. Previous cryo-FESEM micrographs have
supported this model demonstrating two distinct
regions, one most likely the water channels, that
appear to open to the exterior of the surface,
whilst the other appears to be the lipid regions
of the cubic phase. The description in the
literature of a highly tortuous structure, made
evident by cryo-FESEM, describe a highly twisted
internal structure. This has major implications for
the release of entrapped actives.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
4200 RIZWAN ET AL.
Figure 8. Release of FITC-Ova from phytantriol
(open symbols) and GMO (closed symbols) matrices
with initial water loadings of 10% (&), 20% (&), 25%
(~), 30% (*) and 35 () (w/w) at 378C in PBS, pH 7.4
( p > 0.05, p < 0.05 (ANOVA), n ¼ 3).
The effect of FITC-Ova on the liquid crystalline
behaviour of phytantriol and GMO was addition-
ally investigated. Selected samples analysed by
PLM show that in general the phase behaviour
after addition of FITC-Ova in phytantriol
matrices at all water loadings was comparable
to that of the binary mixtures. However surpris-
ingly for GMO samples containing FITC-Ova at
20% and 25% (w/w) there was a conversion from
isotropic (in binary mixtures) to fan textures
(in the presence of FITC-Ova), which was not
Figure 9. Release of FITC-Ova from phytantriol
(open symbols, dotted line) and GMO (closed symbols,
solid line) matrices with initial water loading of 10%
(&), 20% (&), 25% (~), 30% (*) and 35 () (w/w) as a
function of the square route of time for the first 48 h of
the release study (378C in PBS at pH 7.4). The correla-
tion coefficient for both curves were at least 0.99. The
linear fit is indicative of diffusion controlled release for
at least the first 48 h.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
observed for samples at higher water levels. The
fan textures indicate the presence of the HII phase
and may be due to incomplete phase transition or
inhomogeneity of the samples analysed. Problems
in metastability of cubic phases, particularly of
monoacylglycerides-water systems have been
reported in literature.33 Problems reported
included difficulties in the preparation of homo-
genous samples when the water content of the
systems was less than that required for complete
hydration. This can be minimised by repeated
cycles of heating and vortex mixing followed by
centrifugation.23 Inhomogeneity may be preva-
lent for formulations of GMO at 20% and 25% (w/
w) as full hydration occurs at around 36% (w/w).
Additionally samples containing FITC-Ova were
prepared without repeated cycles of heating
and centrifugation, due to the labile nature of
ovalbumin, increasing the likelihood of small
inhomogeneous areas even though samples were
equilibrated for at least a week prior to analysis.
However, the strong correlation of the release
data with square root of time indicates that
release is from a homogeneous matrix.
SAXS data on selected binary and pseudo
ternary mixtures confirmed the results obtained
from PLM. Binary mixtures of phytantriol and
GMO (Fig. 4) at 10% and 35% (w/w) water content
correspond to the La and the diamond cubic phase
as described elsewhere in the literature.15,25
Addition of FITC-Ova to samples of phytantriol
and GMO at 10% and 35% (w/w) water content
examined prior to the release study show phase
behaviour comparable to the binary mixtures,
indicating the presence of FITC-Ova did not alter
the liquid crystalline structures of either lipid at
35% (w/w) water, however significant differences
were observed at the lower 10% water content,
indicating a greater sensitivity of the structure to
interaction with FITC-Ova when in a relatively
dehydrated state. Interestingly, GMO samples
containing FITC-Ova postrelease had converted
from the isotropic cubic to the birefringent
HII phase, whilst no changes were observed for
phytantriol matrices containing FITC-Ova. These
results are in agreement with the obtained lattice
parameters shown in Table 1.
Several authors have found that addition of
drugs or solvents to GMO/water system have lead
to modifications of the liquid crystalline struc-
ture,34–37 whilst others have shown no effect.38,39
Caboi et al. have investigated the effects of adding
various hydrophilic and lipophilic compounds to
the GMO/water system. They showed that most of
DOI 10.1002/jps
 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO
the additives examined retained a stable La or
cubic liquid crystalline structure for a few months
but then transformed to the HII phase.20 In a
subsequent publication they demonstrated by
13
C NMR that the mechanism of this conversion
was due to the presence of glycerol and oleic acid
from hydrolysis of the GMO.40 The mechanism of
conversion of pseudoternary mixtures of GMO
into the HII phase under the release study
conditions can be explained similarly. Free oleic
acid in the system can increase the apparent
hydrophobic volume of the lipid leading to an
increase in the packing parameter ( P) and favour
a transition from the cubic to the HII phase. The
location of the additive(s) within the liquid
crystalline mesophase is determined by factors
such as polarity and molecular structure. The
active may preferentially locate within the aqu-
eous water channels, the hydrocarbon chain
region or the interfacial head group region,
affecting P differently and having a variable
impact on the liquid crystalline structure.6,20 In
this study it is likely that both the instability of
the GMO and the presence of the active contribute
to the transition, which does not occur with
phytantriol.
There are at least two possible types of
interaction of FITC-Ova with system components
that might lead to changes in liquid crystal
structure; one for which the hydrophobic portions
of the protein interact with the lipids, and one
where the protein is acting as a competitor to
solvation of the lipid headgroups, reducing the
water activity. Based on the fact that only the low
water content samples showed an influence of
FITC-Ova on phase structure, and that FITC-Ova
is a soluble globular protein rather than a
transmembrane protein, the latter is most likely
the mechanism at play.
Matrix Swelling and Release of Bioactives
Swelling for most polymer based matrices has
been shown to follow first order kinetics,27
however swelling kinetics for both phytantriol
and GMO could be described more accurately in
this study using second order kinetics. This
finding is consistent with the literature, where
several authors have shown that monoglycerides
like GMO swell according to second order
kinetics6,14 and the present work demonstrates
that the same holds true for phytantriol. Max-
imum swelling capacity of phytantriol obtained
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
4201
experimentally was in close agreement to the
calculated value of 0.37 g/g using Eq. (2). However
values obtained experimentally (0.36 g/g) for
GMO were slightly lower then the calculated
value (0.39 g/g) and those reported in the
literature.6,14,26 The maximum swelling capaci-
ties obtained experimentally and calculated at the
other water loadings for both lipids were overall
identical. The initial rate of swelling was also
calculated and it was found that the initial rate
of swelling increased as the water loadings
decreased. Maximum swelling capacity for phy-
tantriol, where a transition from the cubic (Pn3m)
to cubic þ excess water (Pn3m þ excess water)
occurs was reported by Barauskas and Landh15
to occur at around 29% (w/w) water. Interestingly
our results show that a higher water content
is necessary to access the Pn3m þ excess water
boundary thereby resulting in a maximum swel-
ling capacity of around 36% (w/w). This is
consistent with a recent finding reported by Dong
et al., where 34% (w/w) was required to access the
Pn3m þ excess water boundary. However differ-
ences could be due to the different sources of
phytantriol utilised in the studies.15,19
The release of actives as a function of initial
water content from GMO/water systems has been
described elsewhere in the literature.6,7,14,41 The
results of the various studies are conflicting,
where some investigators found that the greater
the initial water content, the greater the degree of
hydration of the matrices, and therefore the faster
the release of the entrapped active. Others have
found that there was no difference in the release
rates as a function of the initial water content of
the liquid crystalline phases due to the rapid
formation of the cubic phase.6,14,42 Chang and
Bodmeier14 found increased release of chlorphe-
niramine maleate with increasing initial water
content, with similar results observed for release
of salicylic acid6 and the peptide D-Ala2,D-Leu5]
enkephalin (DADLE)42 by other groups. These
findings were attributed to an increase in hydro-
philic channels available for the release of the
active as the water content increased. Therefore
more swollen matrices would provide more rapid
diffusion and faster release than less swollen
matrices with reduced channel diameter.
Statistical analysis using one-way ANOVA on
release of model hydrophilic protein FITC-Ova
from both lipids showed no difference ( p > 0.05) in
release as a function of initial water content. This
is perhaps not surprising in consideration of the
different time scales for swelling and release in
4202 RIZWAN ET AL.
the current study. Burrows et al.7 investigated the
release of several actives varying in polarity from
the GMO/water system and showed no significant
differences in release as a function of initial water
content. In the presence of excess solvent, such
as upon addition of release media, the matrices
absorb the solvent rapidly (supported by the
swelling studies) to form the cubic phase. The
incorporated active will therefore be released
primarily from the water channels of the cubic
phase, resulting in similar release profiles across
all initial water contents.7,43 For the systems
investigated in this study, the rate of release
of FITC-Ova was independent of initial water
content which allows for increased flexibility
when formulating, particularly when using them
as starting materials for dispersion into cubo-
somes.44
Release of FITC-Ova from phytantriol matrices
was significantly greater as compared to GMO.
Postrelease samples examined by PLM and SAXS
showed no changes in the liquid crystalline
structure of selected phytantriol based samples,
however a conversion of the GMO matrices from
the cubic to the HII phase was observed. Boyd
et al.11 have investigated the release of drugs
with varying polarity from liquid crystalline
systems based on lipids with glycerate head-
groups. Release of both hydrophobic and hydro-
philic was significantly greater from the cubic
phase when compared to the HII phase. The
observed differences were attributed to differ-
ences in the aqueous domains of the cubic phase
and the HII phase.
The release of FITC-Ova, a hydrophilic protein
would occur primarily through the aqueous
domains therefore access of the water channels
to the external aqueous environment is a crucial
factor in controlling release. Release of actives
from the cubic phase would occur from the two
nonintersecting water channels, which are shown
by cryo-FESEM to open to the exterior. However
the HII phase, thought to be composed of closed
infinitely long rod-like micelles,45 would trap
hydrophilic actives such as FITC-Ova efficiently
within their aqueous channels. The only mechan-
ism for release out of the matrix is postulated to
occur either by diffusion through the hydrophobic
regions to the exterior aqueous environment or
through random formation/destruction events of
the reversed micelles.11
Results observed in this study are similar to
those reported by Clogston and Caffrey5 who
described the release of amino acids, peptides and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
proteins from GMO-based systems and showed
that only 12% of entrapped ovalbumin released
over a period of 3 weeks. They hypothesised that
this was due to the size of the molecules and no
evidence was provided about the liquid crystalline
structure type from which release of ovalbumin
occurred. The diffusion coefficient calculated for
FITC-Ova in the GMO-based matrices in the
current study was 0.0011  108 cm2 s1, which
is in excellent agreement with the value of
0.0015  108 cm2 s1 for ovalbumin determined
by Clogston and Caffrey.5
Plots of release versus square root of time
particularly within the first 48 h, show diffusion
as the dominant mechanism of release, consistent
with other reports of release from liquid crystal-
line systems.6,7,11,14 Interestingly there was a lag
time before release occurred from phytantriol
matrices. This may be explained by the lower rate
of swelling (Tab. 3) for phytantriol matrices
(0.07 g/g h) compared to GMO. A plateau in the
release of FITC-Ova from GMO and phytantriol
based matrices was observed after 3 weeks. This
could not be due to saturation of the release media
as the study was conducted under sink conditions.
Incomplete release was reported for chlorphenir-
amine maleate from GMO-based matrices and
was attributed to interactions between GMO and
the drug.14 While possible interactions between
FITC-Ova and GMO/phytantriol matrices were
not specifically investigated in this study, it is
more likely that the plateau is due to very slow,
rather than incomplete release which is difficult
to quantify in a practical time frame. In vitro
‘incomplete’ release, in this case may not be reflec-
tive of what will happen in vivo where biodegra-
dation of the matrix would most likely occur.
CONCLUSION
The swelling kinetics and release of hydrophilic
ovalbumin incorporated into liquid crystalline
systems of both phytantriol and GMO was
investigated. Swelling of GMO-based liquid crys-
talline systems was generally faster compared to
phytantriol based matrices and in all cases the
rate of swelling was higher with lower initial
water content. Release of FITC-Ova from both
systems was sustained but was faster and
occurred to a greater extent from phytantriol
matrices than from GMO matrices due to the
conversion of GMO matrices from the cubic to
the HII phase. The initial water content of the
DOI 10.1002/jps
 LIQUID CRYSTALLINE SYSTEMS OF PHYTANTRIOL AND GMO
matrices had no effect on release of ovalbumin
from both matrices. Overall liquid crystalline
systems based on phytantriol, such as the cubic
phase, are an attractive alternative to GMO-based
systems for sustained release of hydrophilic
proteins and may serve as useful templates for
dispersion into cubosomes.
ACKNOWLEDGMENTS
The authors would like to thank the University
of Otago and the Australian Institute of Nuclear
Science and Engineering for funding the SAXS
studies in this manuscript under grant AIN-
GRA07016. Additionally the School of Pharmacy,
University of Otago is acknowledged for providing
financial assistance to S.R.
REFERENCES
1. Collings PJ. 2002. Liquid crystals nature’s delicate
phase of matter. 2nd edition. New Jersey: Princeton
University Press, Princeton.
2. Drummond CK, Fong C. 2000. Surfactant self-
assembly objects as novel drug delivery vehicles.
Curr Opin Colloid Interface Sci 4:449–456.
3. Clogston J, Craciun G, Hart DJ, Caffrey M. 2005.
Controlling release from the lipidic cubic phase by
selective alkylation. J Control Release 102:441–461.
4. Shah J, Sadhale Y, Chilukuri D. 2001. Cubic phase
gels as drug delivery systems. Adv Drug Deliv Rev
47:229–250.
5. Clogston J, Caffrey M. 2005. Controlling release
from the lipidic cubic phase. Amino acids, peptides,
proteins and nucleic acids. J Control Release 107:
97–111.
6. Lara MG, Bentley MV, Collet JH. 2005. In vitro
drug release mechanism and drug loading studies of
cubic phase gels. Int J Pharm 293:241–250.
7. Burrows R, Collett JH, Attwood D. 1994. The
release of drugs from monoglyceride-water liquid
crystalline phases. Int J Pharm 111:283–293.
8. Sadhale Y, Shah J. 1999. Stabilization of insulin
against agitation-induced aggregation by the GMO
cubic phase gel. Int J Pharm 191:51–64.
9. Sadhale Y, Shah J. 1999. Biological activity of
insulin in GMO gels and the effect of agitation.
Int J Pharm 191:65–74.
10. Shah MH, Paradkar A. 2005. Cubic liquid crystal-
line glyceryl monooleate matrices for oral delivery
of enzyme. Int J Pharm 294:161–171.
11. Boyd B, Whittaker D, Khoo S, Davey G. 2006.
Lyotropic liquid crystalline phases formed from
glycerate surfactants as sustained release drug
delivery systems. Int J Pharm 309:216–226.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
4203
12. Fong C, Wells D, I K, Booth J, Hartley PJ. 2007.
Synthesis and mesophases of glycerate surfactants.
J Phys Chem B 111:1384–1392.
13. Hato M, Minamikawa H. 1996. The effects of oligo
saccharide stereochemistry on the physical proper-
ties of aqueous synthetic lipids. Langmuir 12:1658–
1665.
14. Chang C, Bodmeier R. 1997. Swelling of and drug
release from monoglyceride-based drug delivery
systems. J Pharm Sci 86:747–752.
15. Barauskas J, Landh T. 2003. Phase behaviour of
the phytantriol/water system. Langmuir 19:9562–
9565.
16. Ribier A, Biatry B. 1998. Cosmetic or dermatologi-
cal composition in the form of an aqueous and stable
dispersion of cubic gel particles based on phytane-
triol and containing a surface-active agent which
has a fatty chain, as dispersing and stabilizing
agent. L’Oreal, Paris, United States.
17. Barauskas J, Johnsson M, Tiberg F. 2005. Self-
assembled lipid superstructures: Beyond vesicles
and liposomes. Nano Lett 5:1615–1619.
18. Dong Y, Larson I, Hanley T, Boyd BJ. 2006. Bulk
and dispersed aqueous phase behavior of phytan-
triol: Effect of vitamin E acetate and F127 polymer
on liquid crystal structure. Langmuir 22:9512–
9518.
19. Dong Y, Dong A, Larson I, Rappolt M, Amenitsch H,
Hanley T, Boyd BJ. 2008. Impurities in commercial
phytantriol significantly alter its lyotropic liquid-
crystalline phase behavior. Langmuir 24:6998–
7003.
20. Caboi F, Amico GS, Pitzalis P, Monduzzi M, Nylan-
der T, Larsson K. 2001. Addition of hydrophilic and
lipophilic compounds of biological relevance to the
monoolein/water system. I. Phase behavior. Chem
Phys Lipids 109:47–62.
21. Helledi LS, Schubert L. 2001. Release kinetics of
Acyclovir from a suspension of Acyclovir incorpo-
rated in a cubic phase delivery system. Drug Dev
Indus Pharm 27:1073–1081.
22. Könnings S, Copland M, Davies N, Rades T. 2002.
A method for the incorporation of ovalbumin into
immune stimulating complexes prepared by the
hydration method. Int J Pharm 241:385–389.
23. de Campo L, Yaghmur A, Sagalowicz L, Leser ME,
Watzke H, Glatter O. 2004. Reversible phase tran-
sitions in emulsified nanostructured lipid systems.
Langmuir 20:5254–5261.
24. Rosevear FB. 1954. The microscopy of the liquid
crystalline neat and middle phases of soaps and
synthetic detergents. J Am Oil Chem Soc 31:628–
639.
25. Hyde S. 2001. Chapter 16 Identification of lyotropic
liquid crystal mesophases. In: Holmberg K, editor.
Handbook of applied surface and colloid chemistry.
West Sussex, England: John Wiley & Sons Ltd.
pp. 299–331.
4204 RIZWAN ET AL.
26. Lee J, Choi SU, Yoon MK, Choi YW. 2003. Kinetic
characterization of swelling of liquid crystalline
phases of glyceryl monooleate. Arch Pharmacal
Res 26:880–885.
27. Schott H. 1992. Kinetics of swelling of polymers and
their gels. J Pharm Sci 81:467–470.
28. Rizwan SB, Dong Y, Boyd BJ, Rades T, Hook S.
2007. Characterisation of bicontinuous cubic liquid
crystalline systems of phytantriol and water using
cryo field emission scanning electron microscopy
(cryo FESEM). Micron 38:478–485.
29. Higuchi WI. 1967. Diffusional models useful in
biopharmaceutics. J Pharm Sci 56:315–324.
30. Lee KWY, Nguyen T, Hanley T, Boyd BJ. 2008.
Nanostructure of liquid crystalline matrix deter-
mines in vitro sustained release and in vivo oral
absorption kinetics for hydrophilic model drug. Int
J Pharm 365:190–199.
31. Muller-Goymann C. 2004. Physicochemical charac-
terization of colloidal drug delivery systems such as
reverse micelles, vesicles, liquid crystals and nano-
particles for topical application. Eur J Pharm Bio-
pharm 58:343–356.
32. Boyd BJ, Rizwan SB, Dong Y, Hook S, Rades T.
2007. Self-assembled geometric liquid-crystalline
nanoparticles imaged in three dimensions: Hexo-
somes are not necessarily flat hexagonal prisms.
Langmuir 23:12461–12464.
33. Qui H, Caffery M. 2000. The phase diagram of the
monoolein/water system: Metastability and equili-
brium aspects. Biomaterials 21:223–234.
34. Engström S, Engström L. 1992. Phase behaviour of
the lidocaine-monoolein-water system. Int J Pharm
79:113–122.
35. Lynch ML, Ofori-Boateng A, Hippe A, Kochvar K,
Spicer P. 2003. Enhanced loading of water-soluble
actives into bicontinuous cubic phase liquid crystals
using cationic surfactants. J Colloid Interface Sci
260:404–413.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 11, NOVEMBER 2009
36. Wang Z, Zheng L, Inouue T. 2005. Effect of sucrose
on the structure of a cubic phase formed from
monoolein/water mixture. J Colloid Interface Sci
288:638–641.
37. Engström S, Lindhal L, Wallin R, Engblom J. 1992.
A study of polar lipid drug carrier systems under-
going a thermosensitive lamellar-to-cubic phase
transition. Int J Pharm 86:137–145.
38. Nylander T, Mattisson C, Razumas V, Meizis Y,
Hakansson B. 1996. A study of entrapped enzyme
stability and substrate diffusion in a monoglycer-
ide-based cubic liquid crystalline phase. Colloids
Surf B 114:311–320.
39. Zheng L, Zhang J, Shui L, Chen F, Um J, Chung H.
2003. Component effects on the phase behavior of
monoglyceride-water mixtures studied by FT-IR
and X-ray diffraction. J Disp Sci Tech 24:773–
778.
40. Murgia S, Caboi F, Monduzzi M. 2001. Addition of
hydrophilic and lipophilic compounds of biological
relevance to the monoolein/water system II— 13C
NMR relaxation study. Chem Phys Lipids 110:
11–17.
41. Chang C, Bodmeier R. 1997. Effect of dissolution
media and additives on the drug release from cubic
phase delivery systems. J Control Release 46:215–
222.
42. Lee J, Kellaway IW. 2000. In vitro peptide release
from liquid crystalline buccal delivery systems. Int
J Pharm 195:29–33.
43. Engström S. 1990. Cubic phases as drug delivery
systems. Polymer Preprints 31:157–158.
44. Larsson K. 2000. Aqueous dispersions of cubic lipid-
water phases. Curr Opin Colloid Interface Sci 5:
64–69.
45. Shearman GC, Templer RH, Seddon JM. 2006.
Inverse lyotropic phases of lipids and membrane
curvature. J Phys: Condensed Matter 18:S1105–
S1124.
DOI 10.1002/jps