Journal of Colloid and Interface Science 605 (2022) 146–154

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Comparison of cubosomes and hexosomes for the delivery of phenytoin

to the brain
Younus Mohammad a,1, Richard N. Prentice a,1, Ben J. Boyd b, Shakila B. Rizwan a,⇑
a
School of Pharmacy, University of Otago, Dunedin, New Zealand
b
Drug Delivery, Disposition and Dynamics and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences,
Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o a b s t r a c t

Article history: The ability to formulate cubosomes and hexosomes with a single lipid by changing only the colloidal sta-
Received 13 April 2021 biliser presents a unique opportunity to directly compare the biological performance of these uniquely
Revised 8 July 2021 structured nanoparticles. This was explored here via the encapsulation and brain delivery of a model
Accepted 9 July 2021
anti-seizure drug, phenytoin, in selachyl alcohol cubosomes and hexosomes. Nanoparticles were pre-
Available online 18 July 202118 July 2021
pared with PluronicÒ F127 or Tween 80Ò as the stabiliser and characterised. The internal nanostructure
of nanoparticles shifted from hexosomes when using PluronicÒ F127 as the stabiliser to cubosomes when
Keywords:
using Tween 80Ò and was conserved following loading of phenytoin, with high encapsulation efficiencies
Phenytoin
Cubosomes
(>97%) in both particle type. Cytotoxicity towards brain endothelial cells using the hCMEC/D3 line was
Hexosomes comparable regardless of stabiliser type. Finally, in vivo brain delivery of phenytoin encapsulated in cubo-
hCMEC/D3 somes and hexosomes after intravenous administration to rats was studied over a period of 60 min,
Tween 80Ò showing cubosomes to be superior to hexosomes, both in terms of brain concentrations and brain to
PluronicÒ F127 plasma ratio. While the role of stabiliser and/or internal nanostructure remains to be conclusively deter-
Brain mined, this study is the first in vivo comparison of cubosomes and hexosomes for the delivery of a ther-
apeutic drug molecule across the BBB and into the brain.
Ó 2021 Published by Elsevier Inc.

1. Introduction

⇑ Corresponding author. Delivery of therapeutics to the brain is a formidable challenge

E-mail address: shakila.rizwan@otago.ac.nz (S.B. Rizwan). due to the strict regulation of molecular trafficking across the
1
Younus M. and R.N. Prentice contributed equally as first author of the manuscript. blood-brain barrier (BBB). Targeted drug-loaded nanoparticles

https://doi.org/10.1016/j.jcis.2021.07.070
0021-9797/Ó 2021 Published by Elsevier Inc.
Y. Mohammad, R.N. Prentice, B.J. Boyd et al.
have been suggested as a promising strategy to circumvent this
[1,2]. The approach is largely based on the idea of a biocompatible
particle coated with a surface ligand that either interacts with
endothelial cells at the BBB to facilitate entry into the brain or
adsorbs plasma proteins which make the particle appear to be car-
rying endogenous cargo, thereby mediating entry. A prominent
example of the latter is the surfactant Tween 80Ò, which has been
shown to adsorb apolipoprotein E and increase brain delivery of
therapeutics such as dalargin, doxorubicin and loperamide [3–5].
Amphiphilic lipids when in contact with aqueous environments
can self-assemble to produce a variety of thermodynamically
stable lyotropic liquid crystalline systems. Of these systems, non-
lamellar phases, such as reverse bicontinuous cubic (V2) and
reverse hexagonal (H2) phases, have received considerable interest
in drug delivery research due to their compartmentalised, ordered
internal structure and for their ability to incorporate and sustain
the release of a wide range of molecules [6–9]. Dispersing the V2
phase and the H2 phase in the presence of a colloidal stabiliser
results in the formation of liquid crystalline nanoparticles (LCNs)
termed cubosomes and hexosomes, respectively [10,11]. Phy-
tantriol (Fig. 1) or glyceryl monooleate (GMO) are two lipids that
are typically used as building blocks in the production of LCNs
[12,13]. More recently, endogenous lipids such as selachyl alcohol
have also shown promise in this regard [14,15].
Selachyl alcohol (Fig. 1) belongs to the family of alkylglycerols,
which are natural ether lipids that exhibit several biological activ-
ities with distinct mechanisms. Of particular interest, co-
administration of alkylglycerols with chemotherapeutic drugs has
been shown to dramatically increase their transport across the
BBB in both healthy and tumour-afflicted brains, an effect which
has been attributed to transient opening of tight junctions [16–
18] and has been well-established with short-chain alkylglycerols.
Most other biological effects of alkylglycerols have been estab-
lished using long-chain molecules, however, neither the effect on
mediating drug delivery across the BBB, nor their potential as
self-assembling particulate brain delivery vehicles have yet been
investigated for long-chain alkylglycerols.
Selachyl alcohol is a long-chain alkylglycerol that forms the H2
phase when in excess water, which can be dispersed into hexo-
somes with the commonly used stabiliser PluronicÒ F127 [14,19].
Recently we have shown that when selachyl alcohol dispersions
are stabilised with Tween 80Ò the nanoparticles possess an inter-
nal V2 phase structure [15]. Whilst a lot has been published in
the area of formulation and physicochemical characterisation of
LCNs, studies of the biological performance of cubosomes and hex-
osomes as drug delivery systems in vivo is lacking. Moreover, to the
best of our knowledge, there are no reports in the literature that
directly compare the biological performance of these two LCNs.
The need to alter the lipid composition of the particles to induce
a V2 to H2 phase change makes this a challenging task [20]. There-
fore, the ability to formulate cubosomes and hexosomes as with
selachyl alcohol by changing only the stabiliser presents a unique
opportunity to directly compare the in vivo performance of these
differently structured nanoparticles. Consequently, the overall
aim of the current study was to compare the performance of sela-
chyl alcohol cubosomes and hexosomes, by comparing their encap-
sulation and brain delivery of a model anti-seizure drug, phenytoin
(Fig. 1).
Phenytoin is used intravenously [21,22] as a first-line treatment
for status epilepticus [23], a life-threatening, prolonged state of
seizure associated with high morbidity and mortality [24,25].
Given its poor water solubility (weak acid, pKa 8.33 and aqueous
solubility of 7.53
105 M) [26], the commercial parenteral form
of phenytoin is dissolved in 40% propylene glycol and 10% ethanol
and adjusted to a pH of 12 to maintain solubility [27]. Conse-
quently, intravenous administration often results in complications
147
Journal of Colloid and Interface Science 605 (2022) 146–154
like severe pain [28,29], tissue necrosis [30,31] and ‘‘purple glove”
syndrome (oedema, discoloration, and pain distal to the site of
intravenous administration) [32,33]. Such complications could
potentially be avoided by encapsulation of phenytoin in aqueous
dispersible LCNs and delivery in a particulate formulation may also
facilitate targeting of drug to the brain, thereby reducing off-target
side effects.
Whilst dispersions of selachyl alcohol stabilised with PluronicÒ
F127 or Tween 80Ò result in the formation of cubosomes and hex-
osomes [14,19], respectively, the addition of guest molecules are
known to disrupt the structure of liquid crystalline systems. There-
fore, we first sought to encapsulate phenytoin in selachyl alcohol
and then investigate its effect on particle nanostructure. The
well-characterised and commonly studied synthetic lipid phy-
tantriol was used for comparison in these studies (Fig. 1). Cubo-
somes and hexosomes of selachyl alcohol and encapsulating
comparable amounts of phenytoin, where only the surface sta-
biliser differed were successfully prepared. Following on from the
formulation studies, selachyl alcohol cubosome and hexosome for-
mulations with respect to in vitro cellular toxicity in a human cere-
bral microvascular endothelial cell line (hCMEC/D3) and in vivo
brain delivery of phenytoin in rats were then compared in order
to gain insight into the effect of internal structure on drug delivery
across the BBB.
2. Materials and methods
Selachyl alcohol was obtained from Haihang Industry Co., Ltd,
(China). Phytantriol was purchased from A & E Connock (Hamp-
shire, England). PluronicÒ F127 was obtained from BASF (Lud-
wigshafen, Germany). TweenÒ 80, and phenytoin were purchased
from Sigma-Aldrich (Australia). Propylene glycol (99.0%), chloro-
form, methanol (HPLC grade) and acetonitrile (HPLC grade) were
sourced from Merck (Germany). All water used in this study was
ion exchanged distilled and passed through a Milli-Q water purifi-
cation system (Millipore, Bedford, MA, USA). All the chemicals
were used as sourced, without any further purification.
2.1. Preparation of dispersions
The liquid precursor technique [34] was used to prepare the
dispersions. Briefly, selachyl alcohol (250 mg), stabiliser (PluronicÒ
F127 or Tween 80Ò) (37.5 mg) and a co-solvent, propylene glycol
(625 mg) with or without phenytoin (2.5 mg) were weighed into
glass vials. The mixture was dissolved in excess chloroform, which
was then removed by evaporation under vacuum at 45 °C leaving
behind a lipid mixture. The lipid mixture was then dispersed in
excess water (5 mL) by vortex mixing with glass beads typically
for 15 min. All formulations were stored at room temperature.
2.2. Particle size analysis
Dynamic light scattering (DLS) was used to measure the size
(expressed as Z-average) and polydispersity index (PDI) of disper-
sions using a Malvern ZetaSizer NanoÒ (ATA Scientific, Australia)
instrument. To adjust the signal level a 5 lL aliquot of the disper-
sions was diluted to 1 mL with water. Three sets of measurements,
each comprising 10 runs of 10 s were performed for each sample at
25 °C and a scattering angle of 173°.
2.3. Small angle X-ray scattering (SAXS)
Phase structure of dispersions was investigated using the SAXS/
WAXS beamline at the Australian Synchrotron using a previously
described method [35]. A 96-well plate pre-loaded with samples
Y. Mohammad, R.N. Prentice, B.J. Boyd et al.
Phytantriol
Selachyl alcohol
Phenytoin
Fig. 1. Chemical structures of the lipids (selachyl alcohol and phytantriol), drug (phenytoin) and colloidal stabilisers (Tween 80Ò and PluronicÒ F127) used in this study.
(200 lL) and sealed with an adhesive film was mounted vertically
in the beam path. The samples were exposed to the X-rays (13 keV)
for 1 s at 25 °C and a Pilatus 1 M detector (total area:
169
180 mm; pixel size: 172 lm
170 lm) placed at a distance
of 1532 mm from the samples was used to generate two dimen-
sional SAXS patterns. Scatterbrain software was used to integrate
two-dimensional diffraction patterns into one-dimensional scat-
tering functions and were plotted as intensity (I) versus the magni-
tude of the scattering vector (q) plots. The relative positions of the
Bragg peaks, which correspond to their Miller indices were used to
define the phase space groups and the lattice parameters were cal-
culated using the appropriate expressions [36].
2.4. Cryogenic transmission electron microscopy (cryo-TEM)
Our previously reported cryo-TEM method was used to image
the morphology of the nanoparticles [37]. A 5 lL aliquot of the dis-
persion was applied to a glow discharged 400 mesh R2/2 Quantifoil
grid (Quantifoil GmbH, Germany). After allowing 10 s adsorption
time, excess sample was blotted with filter paper (Whatman grade
1) for 3 s to obtain a thin liquid film. The grid was then vitrified by
rapidly plunging into liquid ethane maintained at 180 °C (Reich-
ert KF80 cryo-fixation device) and stored in liquid nitrogen until
analysis. Samples were viewed using a JEOL 2200FS TEM with a
Gatan side-entry cryo-stage equipped with a TVIPS F416 CMOS
camera at 20 000
magnification. IMOD software (University of
Colorado, Boulder, CO, USA) was used for image analysis.
2.5. Quantification of phenytoin encapsulated in nanoparticles
Our previously described gel-permeation chromatography
method [37] was used to determine the encapsulation of pheny-
toin in the dispersions. Briefly, dispersions were diluted in water
(0.5–2 mL) and passed through PD-10 columns filled with Sepha-
dex G-25 (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA).
The column was eluted with 24 mL of water and eluents were col-
lected in 3 mL aliquots. Subsequently, methanol (20 mL) was used
to elute the column and 5 mL fractions were collected. The elution
profile of unencapsulated (free) phenytoin was obtained by passing
the phenytoin solution (2.5 mg of phenytoin, 625 mg of propylene
glycol were dissolved in chloroform, chloroform was evaporated
and mixture was suspended in 5 mL water) through the column.
The eluents were dissolved in 2:1 acetonitrile: methanol (Merck,
Germany) (to solubilise nanoparticle components and phenytoin)
and were filtered through 0.2 lm nylon filters prior to HPLC anal-
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Journal of Colloid and Interface Science 605 (2022) 146–154
Tween 80®
(w+x+y+z) = 20
Pluronic® F127
ysis for quantification of phenytoin using a method modified after
[38,39]. An Agilent HPLC (Agilent Technologies 1200 series)
equipped with UV detector was used. Samples were subjected to
a separation on a Phenomenex Gemini C18 column (150 mm
4.
6 mm, 5 lm) column maintained at 25 °C. A mobile phase contain-
ing 60% v/v methanol in water in isocratic mode with a flow rate of
1 mL/min was used. A 25 lL of sample was injected into system
from the auto sampler which was maintained at 25 °C. Validation
data for the HPLC assay used to quantify the phenytoin is provided
in Supplementary Information 2. The standard concentration range
of phenytoin was 0.5 to 50 lg/mL (Supplementary Fig. 2). The col-
umn eluents were monitored at a wavelength of 225 nm. Pheny-
toin eluted at 4.5 min (Supplementary Fig. 3).
2.6. In vitro cytotoxicity
Human cerebral microvascular endothelial cells (hCMEC/D3)
(Cedarlane, Canada) between passage 23 to 34 were grown in
flasks pre-coated with collagen type 1 (FalconTM) and in EBM-2
medium (Lonza, Basel, Switzerland) supplemented with 5% fetal
bovine serum (Life Technologies, NZ), 1% penicillin-streptomycin
(Life Technologies, NZ), 1.4 lM hydrocortisone (Sigma-Aldrich),
5 lg/mL ascorbic acid (Sigma-Aldrich), 0.001% chemically defined
lipid concentrate (Life Technologies, NZ), 10 mM HEPES (Life Tech-
nologies, NZ) and 1 ng/mL human basic fibroblast growth factor
(Sigma-Aldrich). Cells were grown at a density of 5
104 cells
per well at 37 °C with 5% CO2 and saturated humidity. Cell culture
medium was replaced every 2 to 3 days and studies were con-
ducted when the cells reached 70% confluency, typically 3 to 4 days
after seeding.
A propidium iodide (PI) assay was used to determine cell viabil-
ity. Cells were washed three times with Dulbecco’s phosphate buf-
fered saline (Invitrogen Corporation) and incubated with
formulations diluted in media at various concentrations of lipid
(12.5, 25, 37.5 and 50 lg/mL without phenytoin; 10, 15, 20, 25
and 30 lg/mL with phenytoin) for 2 h at 37 °C. The cells were then
washed with flow assisted cell sorting (FACS) buffer (1% bovine
serum albumin and 0.1% sodium azide in phosphate buffered sal-
ine) to remove any remaining formulation. TrypLE (Gibco, Den-
mark) was added to each well and left for 10 min at 37 °C to
allow the cells to detach. The cells were re-suspended with FACS
buffer and centrifuged for 10 min at 160 g. Supernatants were dis-
carded and the cells were re-suspended in 100 lL of FACS buffer
containing PI. Cells were then analysed using flow cytometry. PI
fluorescence was quantified using a FACS CantoTM II flow cytome-
Y. Mohammad, R.N. Prentice, B.J. Boyd et al. Journal of Colloid and Interface Science 605 (2022) 146–154

ter (BD Biosciences, California, USA) and data acquired using Cell- using unpaired two-tailed t-tests. All the data was analysed in
QuestPro software (BD Biosciences). Data analysis was conducted GraphPad PrismÒ with P < 0.05 considered statistically significant.
using FlowJoÒ 7.6 software (Tree Star, Oregon, USA). Cell viability
was defined as the percentage of PI-negative cells within the single
3. Results
cell population.

3.1. Physicochemical properties of phenytoin containing dispersions

2.7. Intravenous administration of LCNs to animals

Phenytoin is a poorly water-soluble crystalline drug which can

Plasma and brain concentrations of phenytoin and its major
be seen as anisotropic, birefringent crystals under the cross
metabolite, 4-HPPH (5-(4-hydroxyphenyl)-5-phenylhydantoin),
polarised light microscope (CPLM), where crystals as small as
were measured in rats up to 60 min after intravenous administra-
0.2 lm can be observed [41]. CPLM was used to estimate the max-
tion of selachyl alcohol LCNs. The study was approved by the
imum loading of phenytoin in selachyl alcohol and phytantriol by
University of Otago Animal Ethics Committee. Male Wistar rats
the detection of residual drug crystals (method and results are
(average weight ~280 g) were sourced from the Hercus Taieri
summarised in Supplementary Information 1, Supplementary
Resource Unit. LCNs were administered as an intravenous bolus
Table 1 and Supplementary Fig. 1). Based on CPLM studies, concen-
to rats at a dose of 1 mL/kg (equivalent to 0.5 mg/kg phenytoin)
trations of 1% w/w (relative to lipid) of phenytoin was selected and
via a lateral tail vein using a 0.5 mL Lo-dose U-100 insulin syringe
selachyl alcohol and phytantriol dispersions containing phenytoin
with 29 G
12.7 mm needle (BD Biosciences).
were formulated. Table 1 summarises the resulting particle size
distribution and encapsulation efficiency of phenytoin in the dif-
2.8. Tissue collection
ferent dispersions. Encapsulation of phenytoin into nanoparticles
prepared with either selachyl alcohol or phytantriol did not affect
Animals were euthanased by guillotine decapitation after one of
the mean particle size and PDI (P > 0.5) when compared to their
three time points (15, 30 or 60 min) following the injection (n = 4
blank counterparts.
to 5 animals per group for each formulation). Trunk blood was col-
The effect of phenytoin on the nanostructure of the LCNs was
lected in a 6 mL tube coated with sodium heparin (BD Biosciences)
investigated by SAXS (Table 1 and Fig. 2) and cryo-TEM (Fig. 3).
at the time of euthanasia and centrifuged at 2000 g for 10 min at
Phytantriol dispersions stabilised with PluronicÒ F127, and con-
ambient temperature (Heraeus Multifuge X3FR, Thermo Scientific),
taining no phenytoin displayed four Bragg peaks with relative posi-
after which the plasma supernatant was extracted. Brains were p p p p
tions at ratios of 2: 3: 4: 6 indicating the presence of V2 phase
also dissected and rinsed in PBS. Both plasma and brain samples
particles with Pn3m space group. The lattice parameter of 70.5 ± 0.
were frozen at 80 °C until required for LC-MS analysis.
12 Å (Table 1) calculated from the relative peak positions was in
agreement with our previous findings [15]. Phytantriol cubosomes
2.9. Quantification of phenytoin and 4-HPPH in plasma and brain
stabilised with Tween 80Ò displayed three Bragg peaks with rela-
tissue by LC-MS p p p
tive positions at ratios of 2: 4: 6 indicating the presence V2
phase particles but with Im3m space group. The lattice parameter
Quantification of phenytoin and its major metabolite 4-HPPH in
of 120.8 ± 0. 6 Å for these particles is also consistent with our pre-
plasma and brain tissue was performed in accordance with a vali-
vious findings [37].
dated LC-MS method modified after Tanaka et al. [40]. Briefly, ali-
Incorporation of phenytoin in either selachyl alcohol or phy-
quots of 100 mL plasma or brain (homogenised in 2 mL/g of water)
tantriol LCNs did not cause dramatic changes in the phase struc-
were spiked with d10-phenytoin (internal standard). Phenytoin and
ture with Bragg peaks appearing at similar relative positions to
4-HPPH were extracted by adding 200 mL of chilled acetonitrile and
their blank counterparts. However, a decrease in the lattice param-
800 mL of tert-butyl methyl ether (TBME) (Merck, Germany), cen-
eter (Table 1) of selachyl alcohol LCNs prepared in the presence of
trifuged (17,200 g for 20 min at 4 °C) and then the supernatant
both stabilisers was observed with the addition of phenytoin. In
transferred to a new tube which was then evaporated to dryness
contrast, incorporation of phenytoin in phytantriol LCNs showed
in a centrifugal evaporator and reconstituted in methanol.
a mixed effect in the presence of the two stabilisers used. PluronicÒ
Samples were analysed using an Agilent 1290 HPLC system con-
F127 stabilised dispersions showed an increase in the lattice
nected to an AbSciex QTRAP 5500 mass spectrometer with Turbo
parameter whilst a decrease in the lattice parameter was observed
Spray ion source in positive mode. The ion pairs monitored were
for dispersions stabilised with Tween 80Ò (Table 1), when com-
253/182 (phenytoin), 263/192 (d10-phenytoin) and 269/198 (4-
pared to their blank counterparts.
HPPH). Mobile phase A consisted of 0.1% formic acid in water.
Selachyl alcohol dispersions containing phenytoin were
Mobile phase B was 0.1% formic acid in 2:1 acetonitrile:methanol.
selected for ultrastructural analysis by cryo-TEM. Representative
Analysis was performed at a flow rate of 0.25 mL/min by injecting
electron micrographs are shown in Fig. 3. In Panel A onion type
5 mL of sample into a Kinetex EVO 5 mm 100 Å C18 (150
2.1 mm)
vesicles with curved striations typical of hexosomes were observed
column maintained at 40 °C. The gradient was started at 80% A, 20%
for dispersions prepared in the presence of PluronicÒ F127 [15]. In
B, where it was held for 30 s before shifting to 5% A, 95% B over
contrast, dispersions stabilised with Tween 80Ò (Panel B) showed
7 min to elute the analytes. The auto-sampler was maintained at
the characteristic internal cubic structure of cubosomes when
a temperature of 20 °C during analysis. Data was collected in Ana-
observed under TEM.
lystÒ software and analyte/internal standard ratio was used to con-
struct calibration curves and analyse the data in Microsoft Excel.
3.2. In vitro cell viability studies using human brain endothelial cells
2.10. Statistical analysis
Selachyl alcohol LCNs not only showed significantly higher
Encapsulation efficiencies and DLS data were compared statisti- encapsulation of phenytoin compared to phytantriol LCNs, but
cally between selachyl alcohol and phytantriol nanoparticles using the nanostructure of the particles (i.e. cubosome and hexosome)
two-way ANOVA combined with Sidak’s multiple comparison test. was retained upon the addition of the model drug. As a result, sela-
Tissue concentration and cell culture data was compared statisti- chyl alcohol cubosomes and hexosomes were selected for further
cally between formulations at each concentration or time point biological testing in vitro and in vivo. The effect of cubosomes
149
Y. Mohammad, R.N. Prentice, B.J. Boyd et al. Journal of Colloid and Interface Science 605 (2022) 146–154

Table 1
Physicochemical properties (Z-average, PDI, encapsulation efficiency (EE%) of phenytoin, lattice parameters and phase structure obtained from SAXS measurements) of selachyl
alcohol and phytantriol dispersions with (+) and without () phenytoin. Dispersions contained 15% w/w stabiliser (relative to lipid). Data presented are the mean ± SD of three
independent experiments. #P < 0.0001.

Formulation Composition Z-average (nm) PDI EE (%) Lattice parameter (Å) Phase structure
Lipid Stabiliser Phenytoin
Selachyl alcohol PluronicÒ F127  174 ± 3 0.14 ± 0.01 58.0 ± 0.1 H2
þ 162 ± 7 0.13 ± 0.02 97.5 ± 2.4# 56.6 ± 0.1
Tween 80Ò  155 ± 6 0.12 ± 0.02 131.6 ± 0.6 V2 (Im3m)
þ 144 ± 4 0.12 ± 0.03 96.7 ± 2.4# 128.3 ± 0.8
Phytantriol PluronicÒ F127  152 ± 5 0.15 ± 0.02 70.5 ± 0.1 V2 (Pn3m)
þ 150 ± 10 0.20 ± 0.03 68.3 ± 3.1 74.1 ± 0.6
Ò
Tween 80  131 ± 5 0.18 ± 0.01 120.8 ± 0.6 V2 (Im3m)
þ 143 ± 14 0.15 ± 0.05 72.6 ± 2.7 116.7 ± 0.5

Phytantriol 10–30 lg/mL was then investigated (Fig. 4). The results show that
2 incorporation of phenytoin at the tested concentrations into cubo-
3 somes or hexosomes did not alter its cytotoxicity towards hCMEC/
4
6
D3 cells.
3
2
4 + Phenytoin
6 3.3. In vivo brain and plasma profiles of phenytoin administered in
Pluronic® F127 selachyl alcohol cubosomes and hexosomes
2

4
6
Brain and plasma concentrations of phenytoin after administra-
tion in cubosomes and hexosomes are shown in Fig. 5. Plasma con-
Arbitrary intensity units

2 centrations (Panel A) of phenytoin was significantly higher when

4
6 administered in cubosome formulations as compared to hexo-
1
Selachyl alcohol
1
2
4
6
2 ratio below 1.0 at 15 min, but at 30 and 60 min, the average ratio
4
6
0.05 0.10 0.15
Fig. 2. SAXS diffractograms of selachyl alcohol and phytantriol dispersions
stabilised with PluronicÒ F127 or Tween 80Ò (15% w/w to lipid) without phenytoin
(black curve) and dispersions containing 1% w/w phenytoin (blue curves). Relative
peak positions for each formulation are shown. Note that the scattering profiles are
displaced on the vertical axis for clarity of presentation.
and hexosomes on cell viability as a function of the concentration
of selachyl alcohol and the internal structure of the LCNs, was
investigated using a PI staining flow cytometry assay (Fig. 4). Cell
viability of greater than 80% of untreated control was considered
non-cytotoxic as previously described [42,43]. Selachyl alcohol
cubosomes and hexosomes were found to be non-cytotoxic to
hCMEC/D3 up to a concentration of 37.5 lg/mL of selachyl alcohol.
No differences (P > 0.05) in cytotoxicity between cubosomes and
hexosomes was observed.
As the hCMEC/D3 cells could tolerate up to 37.5 lg/mL of sela-
chyl alcohol, the cytotoxicity of selachyl alcohol cubosomes and
hexosomes containing phenytoin within a concentration range of
+ Phenytoin somes at 15 min (184.1 ng/mL vs 102.9 ng/mL, p = 0.0239), but
not at 30 and 60 min, where plasma concentrations decreased with
Tween 80®
both formulations. Brain concentrations (Panel B) appeared mark-
edly higher after administration of the cubosome formulations at
15 min (159.0 ng/g vs 58.5 ng/g) and 30 min (151.9 ng/g vs
74.1 ng/g), but the difference was only statistically significant at
3
4 + Phenytoin 30 min (p = 0.0308), and not 15 min (p = 0.0705). At 60 min, the
difference in plasma concentration of phenytoin for the cubosome
treated group as compared to the hexosome group (64.6 ng/g vs
3
Pluronic® F127 44.7 ng/g) was no longer significant.
4
Brain to plasma ratio for each individual animal and the average
was calculated and is shown in Panel C. Both formulations had a
+ Phenytoin
for cubosomes was 1.23 and 1.20, respectively, which exceeded 1.0
and indicated accumulation of phenytoin in the brain. The ratio for
hexosomes also continued to rise after 15 min but remained below
Tween 80®
1.0 (0.82 at 30 min and 0.91 at 60 min) and was significantly lower
than the cubosomes (p = 0.0115 and 0.0097 for 30 and 60 min,
0.20 0.25 0.30 0.35 0.40 respectively).
q(Å-1)
To gain further insight into the delivery of phenytoin encapsu-
lated in cubosomes and hexosomes, the concentration of its major
metabolite 4-HPPH was simultaneously measured in the plasma
and brain tissue. Fig. 5D shows that 4-HPPH was present in plasma
at 15 min (44.1 ng/mL (cubosomes), 27.6 ng/mL (hexosomes)),
appeared to rise slightly at 30 min (60.7 ng/mL (cubosomes),
47.6 ng/mL (hexosomes)) and fall again at 60 min (41.8 ng/mL (cu-
bosomes), 44.7 ng/mL (hexosomes)), but did not vary significantly
between the formulations at any time point (P > 0.05). No 4-HPPH
was detected in the brain tissue within the quantifiable range of
the assay.
4. Discussion
The aim of this study was to explore the encapsulation and
brain delivery of phenytoin in LCNs composed of the endogenous
alkylglycerol lipid, selachyl alcohol. Qualitatively, CPLM studies
indicated greater solubility of phenytoin in selachyl alcohol than
in phytantriol. This is consistent with previous studies using the
150
Y. Mohammad, R.N. Prentice, B.J. Boyd et al.
Fig. 3. Representative cryo-TEM micrographs of selachyl alcohol dispersions containing phenytoin (1% w/w) and stabilised with 15% w/w PluronicÒ F127 (A) or Tween 80Ò
(B). Panel A shows onion-type vesicles with curved striations (hexosomes) whereas in Panel B nanoparticles with distinct cubic symmetry (cubosomes) are evident. Scale
bar = 200 nm.
110
100
Cell viability (%)
90
80
70
60
50
10
0 to an increase in the lattice parameter by ~4 Å. In contrast, in the
0 10 20 30 40 50
Selachyl alcohol ( g/mL)
Fig. 4. Viability (%) of hCMEC/D3 cells after exposure to selachyl alcohol cubosomes
(squares) and hexosomes (triangles) containing: no phenytoin (black curves) or 1%
w/w phenytoin (black curves). Data presented are the mean ± SD of three
independent experiments.
poorly water-soluble drug, cinnarizine [9]. Additionally, encapsula-
tion efficiency of phenytoin was significantly greater in selachyl
alcohol LCNs compared to phytantriol LCNs, collectively suggesting
higher solubility in selachyl alcohol than phytantriol. The
hydrophobic tail group chain architecture in selachyl alcohol and
in phytantriol are likely to be responsible for the differences in sol-
ubilisation of phenytoin. The more rigid phytanyl chain in phy-
tantriol may have permitted less accommodation of phenytoin in
its structure (cubic), compared to the oleate chain (with a kink)
in selachyl alcohol, resulting in lower solubilisation of phenytoin
(and thus lower encapsulation).
The decrease in the lattice parameter observed in selachyl alco-
hol LCNs containing phenytoin compared to their non-drug con-
taining counterparts, suggests that encapsulation of hydrophobic
phenytoin in the lipid bilayer leads to an increase in mean negative
curvature. Encapsulation of other hydrophobic drugs such as pacli-
taxel and docetaxel in GMO and phytantriol based LCNs, respec-
tively, have also resulted in an increase in the negative curvature
leading to a complete phase transformation from a V2 to an H2
phase [44,45]. Compared to the effects seen with paclitaxel and
151
Journal of Colloid and Interface Science 605 (2022) 146–154
docetaxel, a less dramatic effect on phase structure resulting only
in a decrease in the lattice parameter was observed in the current
study with phenytoin (which is smaller in size). With phytantriol,
encapsulation of phenytoin in the presence of the two stabilisers
had different effects on the lattice parameter suggesting that sta-
bilisers also play a role in accommodating guest molecules in the
lipid bilayer. Tween 80Ò is believed to be more internalised into
the lipid bilayer compared to PluronicÒ F127 which is believed to
be mostly surface associated [15,37,46,47]. Incorporation of pheny-
toin into phytantriol dispersions stabilised with PluronicÒ F127,
where it is assumed that the stabilizer is surface-associated
resulted in greater swelling of the lipid bilayer, thereby leading
presence of the more internalised Tween 80Ò, encapsulation of
phenytoin in the phytantriol lipid bilayer resulted in an increase
in the negative curvature, with a corresponding ~4 Å reduction in
the lattice parameter.
Cellular toxicity of selachyl alcohol cubosomes and hexosomes
towards brain endothelial cells has not been previously reported.
LCNs with different internal structures have been reported to show
differences in their interaction with lipid membranes of cells by
membrane fusion and lipid exchange, resulting in membrane dis-
ruption and cell death [43,48,49]. Despite this, comparable cell via-
bility following exposure to selachyl alcohol hexosomes and
cubosomes in this study suggested a similar mode of interaction
with cells. This was an interesting finding considering that a recent
review of LCNs by Tan et al. [50] concluded that nanostructures
with higher negative curvature (i.e. the H2 phase with respect to
our study) demonstrated better tolerability across different cell
lines. It was postulated that this might be due to a difference in vis-
coelastic behaviour, with hexosomes being less rigid than cubo-
somes leading to a lower degree of lipid mixing with cell
membranes. However, the authors did acknowledge that incorpo-
ration of additional dopant lipids, changes in the stabiliser or mod-
ification of the surface charge in the reviewed studies meant that
the effect of internal structure on cellular interactions is by no
means clear-cut. For instance, Tran et al. [51] studied GMO-based
LCNs and reported that less curved V2 phase nanoparticles were
more toxic than H2 phase nanoparticles towards fibroblasts. This
was tested by adding increasing amounts of the endogenous lipid
capric acid to induce the structural transformation from V2 to H2
phase, however the capric acid control solution showed the highest
Y. Mohammad, R.N. Prentice, B.J. Boyd et al.
400
A B
PhenytoinPlasma (ng/mL)
300 * 300
200
100
0 0
15 30 60
Time (min)
2.0
C D
* *
Brain:Plasma ratio
1.5
1.0
0.5
0.0
15 30 60
Time (min)
Fig. 5. Plasma (A) and brain (B) concentrations of phenytoin, brain to plasma ratio of phenytoin (C) and plasma concentrations of 4-HPPH (D) in rats 15, 30 and 60 min after
intravenous administration of selachyl alcohol cubosomes (circles) or hexosomes (triangles) encapsulating phenytoin (1% w/w relative to lipid). *P < 0.05, n = 4 to 5 rats per
group.
cellular viability, so the effect of internal structure could not easily
be dissociated from the potentially protective effect of the increas-
ing amount of capric acid in the system. In contrast, in this study
we were able to alter the phase from V2 to H2 by only modifying
the stabiliser. While previous studies have suggested that stabiliser
modification can alter the cell toxicity profile depending on the
balance of surface coverage and interaction of the stabiliser with
the bilayer [50], we did not find a difference. Whatever the case,
our findings offered a point of confidence for progressing towards
in vivo comparisons of brain uptake of phenytoin from these differ-
ently structured nanoparticles without the confounding effect of
differences in cytotoxicity of the formulation components.
The final element of this study was to investigate in vivo brain
delivery of phenytoin using the two differently structured selachyl
alcohol LCNs, cubosomes and hexosomes. Average concentrations
of phenytoin in the brain and plasma were consistently higher
when administered in cubosomes compared with hexosomes.
The differences were statistically significant at 15 min in plasma
and 30 min in the brain, which can be interpreted as a greater
retention of cubosomes in the plasma relative to hexosomes, fol-
lowed by a higher relative accumulation in the brain. This finding
is further supported by the average brain to plasma ratio of pheny-
toin which exceeded 1.0 (indicating accumulation in the brain rel-
ative to plasma) at 30 min after cubosome administration, but not
hexosomes, a difference that was maintained up to 60 min. These
results therefore support the hypothesis that selachyl alcohol
cubosomes enhance drug delivery to the brain compared with hex-
osomes prepared using the same lipid.
As touched on above, comparing different types of LCNs, most
commonly cubosomes and hexosomes as in this study, is a chal-
lenging task given that the lipid composition usually needs to be
modified in order to alter bilayer curvature enough to induce a
phase change [50]. The data presented here is unique in that it
compares cubosomes and hexosomes composed entirely of one
lipid, selachyl alcohol, and encapsulating the same amount of drug
(phenytoin) with 97–98% efficiency, where only the surface sta-
152
Journal of Colloid and Interface Science 605 (2022) 146–154
400
PhenytoinBrain (ng/g)
*
200
100
15 30 60
Time (min)
400
4-HPPHPlasma (ng/mL)
300
200
100
0
15 30 60
Time (min)
biliser differs. The question remaining, however, is whether the
internal structure or stabiliser, or both, are responsible for the
apparently increased circulation time in plasma and drug delivery
to the brain. Previous literature on the proposed apolipoprotein-
mediated brain uptake mechanism associated with Tween 80Ò
[5,52], which formed the basis of the hypothesis, lends weight to
the stabiliser being the responsible factor. The internal nanostruc-
tures of cubosomes and hexosomes may also impart different drug
release profiles which could have played a role, but no direct com-
parison of these with a hydrophobic drug was found in the litera-
ture which leans heavily towards investigating the release of
hydrophilic compounds only [53–55]. Furthermore, there are
potential interactions of the LCNs with blood plasma components
leading to changes in internal structure, as has been previously
reported [56,57], which must also be considered when correlating
internal structure with drug delivery and investigations into this
are currently ongoing in our lab.
The major metabolite of phenytoin, 4-HPPH, was also quantified
in plasma and brain in order to give some extra insight into the
delivery of phenytoin in LCNs. There are a few interesting points
that can be taken from the results. Firstly, for phenytoin to be
effective, it must be released from the LCNs which the presence
of the metabolite in the plasma indicates happened for both for-
mulations. Given the absence of 4-HPPH in the brain, which is con-
sistent with previous studies [58,59], this metabolism must have
occurred due to free drug in the plasma. Whether the release of
the participating free phenytoin occurred in the plasma before
the particles reached the brain, or occurred in the brain, followed
by an equilibration-related movement out into plasma where the
metabolism occurred is not clear just by tracking the concentra-
tions of 4-HPPH. When phenytoin concentration is considered in
conjunction with this, however, it can be seen that the proportion
of 4-HPPH relative to phenytoin in the plasma and brain is signif-
icantly lower in the cubosome group at 15 and 30 min, respec-
tively, given that no significant difference was found between the
4-HPPH concentrations after each formulation, but was with
Y. Mohammad, R.N. Prentice, B.J. Boyd et al.
phenytoin concentrations at these time points. This suggests a
preferential distribution of phenytoin to the brain while encapsu-
lated in the cubosomes and a subsequently lower proportion of
drug being metabolised to 4-HPPH, in turn supporting the conclu-
sions drawn earlier from the phenytoin concentration data. Collec-
tively, our data suggest that selachyl alcohol cubosomes can
deliver drug cargo to the brain. However, precisely how the drug
cargo is transported across the BBB will need to be elucidated in
future studies. Whilst no toxic effects were observed via beha-
vioural observations in animals after intravenous injection of sela-
chyl alcohol LCNs, a detailed toxicity study (including haemolytic
studies) is warranted in the future as LCNs formulated with other
liquid crystal forming lipids such GMO/PluronicÒ F127 have shown
evidence of cytotoxicity [54]. Finally, the therapeutic efficacy of the
new cubosomes formulations which repurpose old drugs such as
phenytoin in the case of this study, should also be compared
against marketed formulations.
5. Conclusions
Even though cubosomes and hexosomes are two uniquely
structured lipid nanoparticles with immense potential to deliver
therapeutics, they have not been compared for their ability to deli-
ver drugs in vivo [60]. Studies on the cytotoxicity and cellular
uptake of cubosomes and hexosomes in different cell lines have
been reported, however, often differing conclusions are reported
[43,48–51]. A direct comparison of cubosomes and hexosomes is
a challenging task as the lipid composition often needs to be
altered to induce a phase change which likely contributed to the
differences observed in the in vitro studies. Herein is the first
reported study of the comparison of cubosomes and hexosomes
composed entirely of a single lipid, selachyl alcohol, for the deliv-
ery of a therapeutic molecule in vivo. Of interest for our research
group is using LCNs to deliver therapeutics across the BBB as there
remains a pressing need to find better ways to deliver molecules at
therapeutic concentrations to the brain to treat the many types of
destructive neurological disorders currently affecting human
health. Consequently, cubosomes and hexosomes were compared
for their ability to deliver the model drug phenytoin to the brain.
We demonstrate for the first time that cubosomes are superior to
hexosomes for delivery of a therapeutic molecule in the context
of drug delivery to the brain. Our findings demonstrate the tremen-
dous drug delivery potential of cubosomes. However, there
remains much to discover about the link, not only between the
internal nanostructure but also the surface architecture, and the
performance of these complex colloids in vivo.
CRediT authorship contribution statement
Younus Mohammad: Conceptualization, Methodology, Data
curation, Formal analysis, Validation, Visualization, Writing – orig-
inal draft, Writing - review & editing. Richard N. Prentice: Concep-
tualization, Methodology, Data curation, Formal analysis,
Validation, Visualization, Writing – original draft, Writing - review
& editing. Ben J. Boyd: Methodology, Writing - review & editing,
Funding acquisition, Supervision, Writing - review & editing. Shak-
ila B. Rizwan: Conceptualization, Data curation, Formal analysis,
Validation, Visualization, Funding acquisition, Supervision, Project
administration, Methodology, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
153
Journal of Colloid and Interface Science 605 (2022) 146–154
Acknowledgements
The present work was supported by funding from Health
Research Council of New Zealand (SBR). RP and YM were supported
by a Doctoral Scholarship from the University of Otago. Authors
would like to acknowledge Mr. Richard Easingwood from the Otago
Centre for Electron Microscopy for help with acquisition and anal-
ysis of the electron microscopy data. The SAXS studies were per-
formed on the SAXS/WAXS beamline at the Australian
Synchrotron (BJB).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcis.2021.07.070.
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