Treatment of a multiple sclerosis animal model by a novel nanodrop formulation of a natural antioxidant

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO), denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis (EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique polyunsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that while EAE-induced mice treated with natural PSO presented some reduction in disease burden, this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of specific size nanodroplets at much lower concentrations of the oil. Pathological examinations revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of lipids in the brains of the affected animals, which are hallmarks of this severe neurological disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may be considered for the treatment of patients suffering from demyelinating diseases. On the mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both demyelination and neurodegeneration.

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Open Access Full Text Article
http://dx.doi.org/10.2147/IJN.S92704
Treatment of a multiple sclerosis animal model
by a novel nanodrop formulation of a natural
antioxidant
Orli Binyamin1,*
Liraz Larush2,*
Kati Frid1
Guy Keller1
Yael Friedman-Levi1
Haim Ovadia1
Oded Abramsky1
Shlomo Magdassi2
Ruth Gabizon1
1Department of Neurology, The
Agnes Ginges Center of Human
Neurogenetics, Hadassah University
Hospital, 2Casali Institute of
Chemistry, The Hebrew University of
Jerusalem, Jerusalem, Israel
*These authors contributed equally
to this work
Abstract: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous
system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative
stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO),
denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis
(EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique poly-
unsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that
while EAE-induced mice treated with natural PSO presented some reduction in disease burden,
this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of
specific size nanodroplets at much lower concentrations of the oil. Pathological examinations
revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of
lipids in the brains of the affected animals, which are hallmarks of this severe neurological
disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may
be considered for the treatment of patients suffering from demyelinating diseases. On the
mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both
demyelination and neurodegeneration.
Keywords: nanodrops, PSO, EAE, oxidative stress, neurodegeneration
Introduction
Sensitivity to oxidative stress is a common pathological feature in all neurodegenerative
diseases.1 In fact, most of the key disease proteins forming aggregates in such condi-
tions are oxidized,2,3 and in prion diseases, oxidation of Met residues in PrP Helix3
precedes its acquisition of protease resistance.4 Also brain lipids are oxidized in these
and other brain diseases,5,6 suggesting that lipid oxidation may play an important role
in the pathogenesis of neurodegenerative diseases.7–9 Oxidized phospholipids gener-
ate compounds, such as 4-oxo-2-nonenal and acrolein, which are predominantly toxic
to brain cells.10 Recent evidence suggests that, in addition to autoimmune pathways,
the neurological damage in demyelinating diseases such as multiple sclerosis (MS)11
may also be of neurodegenerative nature.12–14 Indeed, oxidation of brain proteins
was shown to occur both in MS and in experimental autoimmune encephalomyelitis
(EAE),15 a well-studied animal model of MS. Most importantly, lipids in MS plaques
are oxidized, as demonstrated by immunostaining with an antibody against oxidized
phospholipids.6,16
We have recently shown that administration of a nanodroplet formulation of
pomegranate seed oil (PSO), denominated Nano-PSO, can significantly reduce the
oxidation of lipids in a transgenic mouse model of genetic prion disease.17 In the
Correspondence: Ruth Gabizon
Department of Neurology, The Agnes
Ginges Center for Human Neurogenetics,
Hadassah University Hospital, Jerusalem
91120, Israel
Tel +972 2 677 3744
Fax +972 2 642 9441
Email gabizonr@hadassah.org.il
Journal name: International Journal of Nanomedicine
Article Designation: Original Research
Year: 2015
Volume: 10
Running head verso: Binyamin et al
Running head recto: Treatment of a multiple sclerosis animal model
DOI: http://dx.doi.org/10.2147/IJN.S92704
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TgMHu2ME199K mice, reduction in lipid oxidation, as well
as reduced neuronal death, occurred concomitantly with a
significant delay in disease onset, constituting a proof of
concept that an advanced formulation of a natural antioxidant
may fight neurodegeneration. No apparent side effects were
observed in the time frame (6–8 months) and doses of Nano-
PSO administered to the mice in these experiments.
PSO comprises a unique component named punicic acid
(PA), a polyunsaturated fatty acid also considered as one of
the strongest natural antioxidants.18 The oil-in-water (O/W)
nanoemulsion of PSO denominated as Nano-PSO19 may
increase the bioability and activity of PSO. This approach,
as is the case for delivery systems, such as phospholipid
micelles or nanodroplets,20–22 may allow the distribution of
the oil components to organs other than the liver, thereby
enabling a longer circulation that may increase the levels of
PA available to pass the blood–brain barrier (BBB). Indeed,
similar unsaturated fatty acids, such as linoleic acid, were
shown to readily cross the BBB.23–25 PA is present only in
PSO (60%–80%) and Trichosanthes kirilowii (40%)26 and
was effective in protecting tissue lipid profiles in inflam-
matory disease models.27 PSO’s lack of toxicity and partial
bioavailability was already established in humans.26 An addi-
tional antioxidant, β-sitosterol, which was demonstrated to
accumulate in the plasma membrane of brain cells,28 is pres-
ent in PSO at significantly higher concentrations as compared
to oils from other plants,29 indicating PSO may constitute a
natural compound with stronger antioxidant activities than
its individual components.
In this work, we describe the clinical and pathological
effects of Nano-PSO in the treatment of EAE-induced mice.
We show that while administration of large doses of PSO in
food can reduce disease burden in the EAE mice, Nano-PSO
exerts a wider effect at a much lower dose. We also estab-
lished an optimal size for the activity of PSO lipid droplets
in this clinical model. Concomitant with decreased disease
burden, pathological examination of brain sections from
Nano-PSO-treated mice also revealed reduced demyelination
and almost eradication of brain lipid oxidation. Our results
therefore reinforce the notion that lipid oxidation is an impor-
tant factor in demyelinating diseases and show that advanced
formulations of natural antioxidants may be both safe and
efficient in the long-term treatment of diseases such as MS.
Materials and methods
Animal experiments
All animal experiments were conducted under the guidelines
and supervision of the Hebrew University Ethical Committee,
which approved the methods employed in this project (Permit
Number: MD-13-13772-5).
Induction of EAE
Induction of myelin oligodendrocyte glycoprotein (MOG)
EAE was done as previously described.30,31 Shortly, 6- to
8-week-old female C57BL/6 mice were immunized with an
emulsion containing 200 μg of MOG35–55 (70% purified; syn-
thesized at Hebrew University, Jerusalem, Israel) in saline and
an equal volume of complete Freund’s adjuvant containing
5 mg/mL H37RA (Difco Laboratories, Detroit, MI, USA). The
inoculum (0.2 mL) was injected subcutaneously into right and
left flanks. One hundred nanograms of pertussis toxin (List
Biological Labs, Campbell, CA, USA) in 0.1 mL saline was
also injected intraperitoneally on day 0 and 48 hours later.
EAE scoring system
Mice were observed daily for the appearance of neurological
symptoms, which were scored as follows: 0, asymptomatic;
1, partial loss of tail tonicity; 1.5, limp tail; 2, hind limb
weakness (right reflex); 3, ataxia; 4, early paralysis; 5, full
paralysis; and 6, moribund or dead.
Preparation of PSO-enriched food
One kilogram of mouse-pelleted food (Harlan, Teklad) was
dissolved in water and subsequently supplemented with 25 or
75 mL of PSO (Flavex, Rehlingen, Germany). The mixture
was next reassembled and dehydrated as pellets.
Preparation of O/W pomegranate oil
nanoemulsion by sonication
A nanoemulsion with 10.8% oil fraction was prepared as
follows: 1.56 g of pomegranate oil, 0.65 g of Tween 80, and
0.39 g of glyceryl monooleate were mixed by a magnetic
stirrer for 20 minutes. Approximately 2.5 g of the aforemen-
tioned mixture was mixed with 0.277 g of glycerol using a
magnetic stirrer for 15 minutes. Then, 2 g of the glycerol
mixture was added drop wise to 8 g of deionized water. A
crude white emulsion was obtained. At the second stage, this
crude emulsion was sonicated using a horn sonicator (model
Vibra-Cell; Sonics & Materials, Inc., USA) for 10 minutes at
750 W. The samples were cooled in an ice water bath during
the sonication process. A bluish emulsion was obtained.
Preparation of 30 nm self-emulsifying
nanodroplets
A mixture comprising 26.3% PSO, 31.57% Tween 80, and
42.13% Cremophor RH 40 was mixed using a magnetic stirrer

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Treatment of a multiple sclerosis animal model
for 20 minutes. Then, 10 μL of the mixture was added to 3 mL
of deionized water. After vortexing for 30–60 seconds, an
emulsion with nanosized droplets was obtained. The size of
the O/W nanoemulsion droplets was 101 nm; Z average, peak
1: 30 nm, 92.5%; peak 2: 334 nm, 7.1%; peak 3: 4,796 nm,
0.4%; and polydispersity index (Pdi): 0.403.
Thiobarbituric acid reactive substances
measurement
Lipid peroxidation was evaluated by measuring levels of
malonaldehyde (MDA). A total of 0.15 g of brain tissue was
homogenized in 1.35 mL lysis buffer containing 1% deion-
ized Triton X-100 in 25 mM Tris–HCl pH 7.5, 150 mM NaCl,
and 5 mM ethylenediaminetetraacetic acid and centrifuged
at 3,000 rpm for 15 minutes at 4°C. Then, 100 μL of the
supernatant was added to 50 μL of 8.1% sodium dodecyl
sulfate, 20 μL of 20% acetic acid pH 3.5, 10 μL of 1.33%
thiobarbituric acid, and 120 μL deionized distilled water
and incubated for an hour at 95°C. After cooling, 300 μL of
n-butanol:pyridine (15:1) was added, and the mixture was
centrifuged at 10,000 rpm for 5 minutes at room temperature.
Absorbance of the organic phase was measured using spectro-
photometer at λ=532 nm. The amount of thiobarbituric acid
reactive substances (TBARS) was determined according to
a standard calibration curve generated from MDA.
Cryo-TEM
The nanodroplets were imaged using a transmission electron
microscope Tecnai G2Spirit Twin T-12. The microscope
was equipped with an FEI 4k Eagle CCD camera. Sample
preparation was done using Vitrobot Mark IV (FEI).
Dynamic light scattering
Size measurements of the droplets were performed with
a Zetasizer Nano S (Malvern Instruments, Malvern, UK)
in triplicates after dilution of the emulsion in water. The
size of the coarse white emulsion droplets was in the range
of few microns. The average size of the droplets of the
O/W nanoemulsion used in these experiments was 30 and
180 nm.
Pathological examinations
Histological evaluations were performed on paraffin-
embedded sections of brain samples. Sections were stained
with Luxol fast blue/periodic acid Schiff (EMD Millipore,
Billerica, MA, USA) to assess demyelination. Paraffin-
embedded sections of brains were used for immunohis-
tochemistry with EO6 an antibody against oxidized lipids.
Consecutive section was stained with hematoxylin and eosin
to recognize infiltrates.
Materials
Most chemicals were from Sigma-Aldrich Co. (St Louis,
MO, USA). PSO was from Flavex, and EO6 was from Avanti
Polar Lipids, Alabaster, AL, USA.
Statistical studies
Analyses of EAE score graphs were performed with the
Microsoft Excel software (2010). Graphs represent the mean
and respective standard error of clinical scores of groups of
mice. The differences between experimental groups were
assessed by one-way analysis of variance followed by the
paired two-tailed Student’s t-test. Also the statistical analysis
of TBARS experiments was done in the same way.
Quantification of pathology immunostaining was per-
formed by measuring the stain-positive area in different fields
at a magnification ×40. Stained pixels were measured using
image pro analyzer 3D software, Media Cybernetics.
Results
Low levels of Nano-PSO signicantly
reduced disease burden in EAE-induced
mice
Nano-PSO droplets were prepared as described in the
“Materials and methods” section and characterized by
dynamic light scattering measurement of droplet size as
well as by Cryo-TEM imaging (Figure 1A and B). Unless
stipulated otherwise, mice suffering from EAE were treated
with Nano-PSO droplets with an average size of 180 nm.
To induce EAE, groups of C57Bl naïve female mice
were immunized with an emulsion comprising MOG35–55
peptide and complete Freund’s adjuvant, resulting in an
acute neurological paralytic disease followed by partial
remission.32,33 Following the induction, mice were scored
daily for disease signs as described in the “Materials and
methods” section. Figure 2A shows results from an experi-
ment in which EAE-induced mice were fed from the day
of induction either with normal chow (untreated) or with
chow to which increasing concentrations of PSO (see levels
of oil in graph legend) were added per 3 g of chow, which
is the average daily consumption of food by each mouse.
The figure shows that daily doses of 100–300 μL of PSO
were effective in reducing disease burden in the EAE
mice (60% of the highest score in the EAE untreated mice
for the 100 μL PSO dose and 36% for the 300 μL dose;
P,0.05 for PSO-treated groups versus untreated groups).

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Binyamin et al
Lower levels of PSO were not effective against the disease.
While this constitutes a proof of principle that high levels
of PSO can have an anti-EAE effect, such levels of oil, in
particular when converted to human doses (4–12 mL of oil/d),
are difficult to consume on a daily basis.
To overcome this limitation, we next tested the α-EAE
activity of Nano-PSO at different concentrations. As opposed
to the continuous consumption of PSO in food, Nano-PSO
was administered once a day by gavage (diluted into 150 μL
of water). Figure 2B shows that a 10% dose of the lowest
active concentration of PSO in food mostly abolished dis-
ease presentation when administered as Nano-PSO (dashed
line) (P,0.05). Moreover, even lower levels of Nano-PSO
(0.8 μL/d) were effective against EAE. This indicates that
Nano-PSO is a much more effective formulation than the
natural oil in the treatment of EAE, as was the case for
the delay of disease onset of genetic CJD in a transgenic
model.17
Figure 2 Nano-PSO as an α-EAE agent.
Notes: Mice were induced for EAE and treated from day 1 of the induction either with PSO or with Nano-PSO. (A) Designated EAE-induced groups were fed either with
normal mouse chow (untreated group; n=10) or with chow enriched with PSO at the concentration in which 3 g (daily intake) comprises the levels designated in the gure
insert: 100 (n=10) or 300 μL (n=8) PSO. P,0.05 for all PSO-treated groups versus the untreated group. (B) Designated EAE-induced groups were either left untreated or
treated (by gavage) with 150 μL solution comprising 0.2, 0.8 (n=6), or 10 μL (n=7) PSO in the form of Nano-PSO. P,0.05 for 0.8 and 10 μL PSO-treated group versus the
untreated group.
Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune encephalomyelitis.
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Abbreviations: DLS, dynamic light scattering; PSO, pomegranate seed oil; cryo-TEM, cryogenic transmission electron microscope.
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Treatment of a multiple sclerosis animal model
Figure 3 Individual α-EAE activity of Nano-PSO ingredients.
Notes: Mice were induced for EAE and treated from day 1 of induction (by gavage)
with the reagents described in the insert of the gure (n=7 for each of the groups).
Nano-PSO was administrated at a dose of 2 μL PSO per 150 μL solution. Mice were
scored daily for EAE signs for 2 additional weeks. P,0.05 for the results in the Nano-
PSO group versus all others.
Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune
encephalomyelitis.
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Anti-EAE activity of Nano-PSO
components
As stated in the “Introduction” section, the main compo-
nent of PSO is PA, a 18:3 polyunsaturated fatty acid. This
is also the main or only compound that differentiates PSO
from other vegetable oils,29 in particular soybean oil. To test
whether PA is indeed the Nano-PSO component that exerts
most of the α-EAE effect, EAE-induced mice were treated
from the day of the induction with a comparable Nano-Soya
formulation, as well as with a mixture of the emulsifying
agents. Figure 3 shows that only Nano-PSO (2 μL PSO/d)
exerted a beneficial clinical effect on the EAE-induced mice
(P,0.05), while groups treated with Nano-Soya or the sur-
factants alone behave similarly in disease pattern and score
to the untreated EAE mice.
Treatment as compared to prevention in
EAE-induced mice
Next, we tested whether Nano-PSO can reduce neurological
damage already inflicted by EAE induction, as opposed to
prevent/delay EAE onset. To this effect, we compared the
clinical effect of Nano-PSO administration from the day of
induction (10 μL PSO/d) to that of the same dose adminis-
tered from day 7 postinduction (graph results from day 10
postinduction). It is well established that at this time point of
EAE induction, activated immune cells are already formed
and infiltrated into the central nervous system (CNS).34
Figure 4 shows that Nano-PSO administration exerts a benefi-
cial effect at both time points (P,0.05); however, while the
early treatment shows both delay in disease onset and reduced
disease burden, the latter treatment only shows reduced
scores but a similar kinetics of disease presentation as the
untreated mice. This is a very encouraging result, indicating
Nano-PSO could be beneficial to humans already suffering
from early signs of demyelinating diseases such as MS.
Effect of size of Nano-PSO droplet on its
α-EAE activity
Since the rational for using PSO nanodroplets in these experi-
ments lies in the possibility that such entities, as opposed to
the large drops of natural PSO, may escape the liver trap on
their first passage, we next asked whether the size of such
droplets is important. All batches of Nano-PSO in the previ-
ous experiments comprised droplets of 180 nm (Figure 1)
since this size of nanoparticles was shown to be successful
for other brain conditions.35,36 We now compared the α-EAE
activity of the 180 nm nanodrops with that of a Nano-PSO
formulation in which the average diameter of the droplets
is close to 30 nm, a size used for the targeting of RNA and
drugs containing lipid droplets to peripheral organs, such as
liver37 or lungs.38 To prepare these 30 nm droplets formula-
tion, a self-emulsifying system was developed according
Figure 4 Nano-PSO in the prevention and treatment of EAE.
Notes: Mice induced for EAE were administered Nano-PSO in two different start
points. As shown in the insert, while one group of induced mice was left untreated
(n=8), a second group was treated with Nano-PSO from day 1 of the induction (n=6)
and a third group from day 7 of the induction (n=7). Mice were scored daily for EAE
signs for 2 additional weeks. P,0.05 for both Nano-PSO treatments.
Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune
encephalomyelitis.
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to the procedure described in the “Materials and methods”
section and in patent No. 14/523,408.39 PSO nanodroplets
of both sizes were administered to EAE-induced mice from
day 1 of the induction at a dose of 10 μL of PSO per day.
Figure 5 shows that the formulation comprising the lower
size droplets had no beneficial effect in EAE-induced mice,
as opposed to the strong response of the sick mice to the
180 nm formulation (P,0.05). The 30 nm formulation was
even less active than the 10 μL dose of PSO in its natural
form (Figure 1). It is possible that lower size nanodroplets
may be absorbed by peripheral organs soon after ingestion
and have no opportunity to pass the BBB from the blood
and generate a beneficial effect in the CNS. Also, smaller
droplets may be more prone to oxidation than the larger ones.
Whether small Nano-PSO droplets can or cannot generate
a beneficial effect in other clinical settings outside the brain
remains to be established.
Treatment of EAE-induced mice with
Nano-PSO inhibits demyelination and
oxidation of brain lipids
To test if the lower clinical scores resulting from Nano-PSO
treatment of EAE-induced mice are consistent with reduced
appearance of EAE pathological markers, we looked in the
brains of treated and untreated mice for several parameters,
such as infiltration of immune cells, demyelination, and
lipid oxidation. Figure 6 shows that while immune infil-
trates can be detected in both treated and untreated EAE
brains (sections E and G), Nano-PSO administration sig-
nificantly reduced lipid oxidation levels (sections A and B
and enlarged in D and F), as detected by the EO6 antibody
staining in both treated and untreated brains. As opposed to
EAE mice, no EO6 immunostaining (section C) or immune
infiltrates (not shown) could be observed in naïve mice.
Nano-PSO administration also reduced demyelination lev-
els, as can be seen by comparing Luxol fast blue staining
for myelin in both brains and spinal cords of untreated, EAE
and naïve mice (section H–J). Interestingly, while the EO6
antibody recognized in the untreated mice both a diffuse
and a light staining as well as focal points stained intensely
(section D), the latter one reminiscent of the plaques in the
patients with MS,6 in the Nano-PSO-treated EAE brains,
only the light and diffuse pattern of immunostaining was
apparent (section F). Quantification of the levels of EO6
stain was performed by measuring the positive area in six
different fields at a magnification ×40. Stained pixels were
measured using image pro analyzer 3D software, Media
Cybernetics (“Materials and methods” section). The quan-
tification results show that while for EAE-treated sections
the percentage of positive area was 1.11±0.08, for the
nontreated samples, it was 5.17±1.13 and for the wild-type
brains 0.07±0.03.
Interestingly, there was no colocalization between infil-
trates and the oxidated plaques in the untreated EAE brains
(compare sections D and E), indicating that while infiltration
of activated immune cells may induce demyelination via oxi-
dation of focal points, inhibition of such oxidation even in the
presence of infiltrates may ameliorate disease signs (compare
F and G). As we have shown previously,17 EO6 does not
recognize any forms in naïve mice suggesting that oxidized
phospholipids are a feature of brain disease (section C).
Administration of Nano-PSO reduced
MDA levels in EAE-induced mice brains
MDA is a product of lipid peroxidation, and its levels can
be empirically measured by the TBARS.40 It was recently
shown to be increased in blood and saliva of MS patients.41
To establish whether MDA levels are elevated in the brains
of EAE-induced mice and whether such elevation can be
alleviated by Nano-PSO treatment, we subjected brain
samples from six wild type, five EAE, and three treated
EAE mice to the TBARS test (“Materials and methods”
section). Figure 7 shows that while MDA levels are elevated
significantly in the brains of EAE-induced mice (P,0.001
between naïve and untreated EAE brains), these are reduced
almost to the levels of naïve brains following Nano-PSO
Figure 5 Small Nano-PSO particles are inactive against EAE.
Notes: Mice induced for EAE were treated with Nano-PSO in different droplet
sizes. As shown in the insert, while one group of induced mice was left untreated
(n=8), a second group was treated with 180 nm droplets of Nano-PSO (n=7), and a
third group with 30 nm Nano-PSO droplets (n=7). P,0.05 for the untreated group
versus the group treated with 180 nm droplets of Nano-PSO.
Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune
encephalomyelitis.
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7171
Treatment of a multiple sclerosis animal model
treatment (P,0.663 between naïve and EAE-treated brains).
These results, together with the results from Figure 6 also
demonstrating reduced levels of oxidized phospholipids in
Nano-PSO-treated EAE mice, strongly suggest that Nano-
PSO may reduce lipid oxidation.
Discussion
We have shown here that, as is the case for some dietary
unsaturated lipids,42 administration of PSO comprising high
levels of PA, a polyunsaturated fatty acid, can be clinically
beneficial to mice induced for EAE, a model of MS. How-
ever, while the oil in its natural form could only reduce
disease burden significantly when given at very high doses,
a superior clinical effect was achieved when 1% of these
PSO levels were administrated to the EAE-induced mice
in the form of emulsified nanodroplets, denominated Nano-
PSO. Nano-PSO was also beneficial to the EAE mice when
treatment commenced close to disease manifestation (day 7)
and not only when administered concomitant with disease
induction. This suggests that our reagent may be effective not
Figure 6 Pathological markers of EAE in Nano-PSO treated and untreated mice.
Notes: Nano-PSO treated and untreated mice were sacriced 3 weeks after induction of EAE, and their formalin-xed, parafn-embedded brain sections as well as those
of age-matched naïve mice (C and J) were stained by mAb EO6 (A–D and F), H&E (E and G), and LFB/PAS (brains and spinal cords) (H–J). (D) and (F) represent an
enlargement of the squares in (A) and (B); (E) and (G) are serial sections of (D) and (F), respectively. Arrows in (D–G) indicate immune inltrates. Arrows in (H) represent
demyelinated areas.
Abbreviations: EAE, experimental autoimmune encephalomyelitis; PSO, pomegranate seed oil; mAb, monoclonal antibody; H&E, hematoxylin and eosin; LFB, Luxol fast
blue; PAS, periodic acid Schiff.
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International Journal of Nanomedicine 2015:10
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Dovepress
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7172
Binyamin et al
only for disease prevention but also for abrogation of disease
progression. We have also demonstrated that the size of the
nanodroplets is important for their therapeutic effect since
30 nm droplets of PSO were inactive in this clinical setting
as opposed to those approximately 200 nm size. Indeed, the
small droplets may succumb to disruption by biosurfactants
such as bile salts at a different rate than larger droplets,43,44
which may remain intact for longer periods of time, and
subsequently cross the BBB.
On the mechanistic side, we have shown here that Nano-
PSO can inhibit both demyelination and lipid oxidation in
EAE brains even in the presence of immune infiltrates in the
CNS. These results indicate that while oxidized lipids may
not feature in the initial activation of immune cells, they
may well play a central role in the subsequent demyelina-
tion, as previously suggested.45 Since oxidized lipoproteins
are neurotoxic and have proinflammatory properties, lipid
peroxidation products could be involved in demyelination
and axonal injury in MS.46,47 In addition, the activity of
Nano-PSO, at least at the particle size shown to be benefi-
cial for EAE, may be mostly confined to the CNS, thereby
interfering with brain oxidation features and demyelination
and less with inhibition of infiltration. Owing to the lack of
toxicity of these reagents, Nano-PSO may be a good choice
for individuals at initial stages of demyelinating diseases
such as MS. At later stages, Nano-PSO may be used in com-
bination with advanced MS treatments such as Natalizumab
(Tysabri), which reduces the migration of lymphocytes to
the CNS by binding to the α4 integrin very late antigen,48 or
in combination of other antioxidant formulations.49 In addi-
tion to demyelinating diseases, oxidative stress is also an
important feature of most neurodegenerative conditions.6,14
Indeed, the EO6 antibody shown here to stain plaques in
the untreated EAE brains as it was shown to mark human
MS plaques6 also reacts extensively with brain slices from
TgMHu2ME199K mice, a mouse model of genetic prion
disease,17 suggesting that lipid oxidation may be a general
feature of neurodegeneration. Most importantly, Nano-PSO
was also beneficial in the prevention and treatment of genetic
prion disease in the TgMHu2ME199K model, indicating that
reagents that can prevent lipid oxidation may be beneficial for
an array of neurodegenerative diseases. A significant number
of natural antioxidants are ubiquitously present in a healthy
human diet. Many of them, such as sulforaphane from broc-
coli, curcumin, and epigallocatechin gallate from green tea,
were recognized for their neuroprotective properties in cells
and tested in appropriate animal models.50–52 However, their
in vivo activity was limited by the subpharmacological doses
presented in food, their poor bioavailability to humans, rapid
chemical degradation, and reduced distribution to different
organs in the body, in particular, the CNS. In this work, and
after PSO by itself was found to be clinically active in its
natural form, we made an effort to overcome such limitations
by tailoring a more active formulation in the form of Nano-
PSO. The results presented in this work indeed indicate that
natural compounds can be formulated into active drugs.
Acknowledgments
This work was funded by a grant from the Agnes Ginges
Center for Human Neurogenetics and from a gift from
the Darmoni family. We thank Dr Y Levi-Kalisman from
the Nanocenter of the Hebrew University for her help in the
cryo-TEM studies.
Disclosure
Based on the results described in this manuscript, we are in
the process of developing a commercial venture to test Nano-
PSO in patients. The authors report no conflicts of interest
in this work.
References
1. Butterfield DA, Kanski J. Brain protein oxidation in age-related neu-
rodegenerative disorders that are associated with aggregated proteins.
Mech Ageing Dev. 2001;122(9):945–962.
2. Aguzzi A, O’Connor T. Protein aggregation diseases: pathogenicity and
therapeutic perspectives. Nat Rev Drug Discov. 2010;9(3):237–248.
3. Morales R, Green KM, Soto C. Cross currents in protein misfolding
disorders: interactions and therapy. CNS Neurol Disord Drug Targets.
2009;8(5):363–371.
4. Canello T, Frid K, Gabizon R, et al. Oxidation of Helix-3 methionines
precedes the formation of PK resistant PrP. PLoS Pathog. 2010;6(7):
e1000977.
Figure 7 TBARS levels in EAE brains.
Notes: Samples from naïve, as well as treated and untreated EAE brains were
subjected to the TBARS test.
Abbreviations: TBARS, thiobarbituric acid reactive substances; EAE, experimental
autoimmune encephalomyelitis; MDA, malonaldehyde.
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1DwYH









7UHDWHG($(8QWUHDWHG($(
0'$
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International Journal of Nanomedicine 2015:10 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7173
Treatment of a multiple sclerosis animal model
5. Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neu-
rodegenerative diseases: a review of upstream and downstream antioxi-
dant therapeutic options. Curr Neuropharmacol. 2009;7(1):65–74.
6. Haider L, Fischer MT, Frischer JM, et al. Oxidative damage in multiple
sclerosis lesions. Brain. 2011;134(pt 7):1914–1924.
7. Perluigi M, Coccia R, Butterfield DA. 4-Hydroxy-2-nonenal, a reactive
product of lipid peroxidation, and neurodegenerative diseases: a toxic
combination illuminated by redox proteomics studies. Antioxid Redox
Signal. 2012;17(11):1590–1609.
8. Adibhatla RM, Hatcher JF. Lipid oxidation and peroxidation in CNS
health and disease: from molecular mechanisms to therapeutic oppor-
tunities. Antioxid Redox Signal. 2010;12(1):125–169.
9. Reed TT. Lipid peroxidation and neurodegenerative disease. Free Radic
Biol Med. 2011;51(7):1302–1319.
10. Singh M, Dang TN, Arseneault M, Ramassamy C. Role of by-products
of lipid oxidation in Alzheimer’s disease brain: a focus on acrolein.
J Alzheimers Dis. 2010;21(3):741–756.
11. Lassmann H. Mechanisms of inflammation induced tissue injury in
multiple sclerosis. J Neurol Sci. 2008;274(1–2):45–47.
12. Ellwardt E, Zipp F. Molecular mechanisms linking neuroinflammation
and neurodegeneration in MS. Exp Neurol. 2014;262(pt A):8–17.
13. Herz J, Zipp F, Siffrin V. Neurodegeneration in autoimmune CNS
inflammation. Exp Neurol. 2010;225(1):9–17.
14. Witte ME, Bol JG, Gerritsen WH, et al. Parkinson’s disease-associated
parkin colocalizes with Alzheimer’s disease and multiple sclerosis brain
lesions. Neurobiol Dis. 2009;36(3):445–452.
15. Castegna A, Palmieri L, Spera I, et al. Oxidative stress and reduced glu-
tamine synthetase activity in the absence of inflammation in the cortex
of mice with experimental allergic encephalomyelitis. Neuroscience.
2011;185:97–105.
16. Palinski W, Hörkkö S, Miller E, et al. Cloning of monoclonal autoan-
tibodies to epitopes of oxidized lipoproteins from apolipoprotein
E-deficient mice. Demonstration of epitopes of oxidized low density
lipoprotein in human plasma. J Clin Invest. 1996;98(3):800–814.
17. Mizrahi M, Friedman-Levi Y, Larush L, et al. Pomegranate seed
oil nanoemulsions for the prevention and treatment of neurodegen-
erative diseases: the case of genetic CJD. Nanomedicine. 2014;10(6):
1353–1363.
18. Schubert SY, Lansky EP, Neeman I. Antioxidant and eicosanoid
enzyme inhibition properties of pomegranate seed oil and fermented
juice flavonoids. J Ethnopharmacol. 1999;66(1):11–17.
19. Sawant RR, Torchilin VP. Multifunctionality of lipid-core micelles
for drug delivery and tumour targeting. Mol Membr Biol. 2010;27(7):
232–246.
20. Merian J, Boisgard R, Decleves X, Theze B, Texier I, Tavitian B. Syn-
thetic lipid nanoparticles targeting steroid organs. J Nucl Med. 2013;
54(11):1996–2003.
21. Margulis-Goshen K, Magdassi S. Formation of simvastatin nanopar-
ticles from microemulsion. Nanomedicine. 2009;5(3):274–281.
22. Kim D, Park JH, Kweon DJ, Han GD. Bioavailability of nanoemulsified
conjugated linoleic acid for an antiobesity effect. Int J Nanomedicine.
2013;8:451–459.
23. Dhopeshwarkar GA, Mead JF. Uptake and transport of fatty acids into
the brain and the role of the blood–brain barrier system. Adv Lipid Res.
1973;11(0):109–142.
24. Spector R. Fatty acid transport through the blood–brain barrier.
J Neurochem. 1988;50(2):639–643.
25. Avellini L, Terracina L, Gaiti A. Linoleic acid passage through the blood–
brain barrier and a possible effect of age. Neurochem Res. 1994;19(2):
129–133.
26. Yuan G, Sinclair AJ, Xu C, Li D. Incorporation and metabolism of
punicic acid in healthy young humans. Mol Nutr Food Res. 2009;
53(10):1336–1342.
27. Saha SS, Ghosh M. Antioxidant effect of vegetable oils containing
conjugated linolenic acid isomers against induced tissue lipid peroxida-
tion and inflammation in rat model. Chem Biol Interact. 2011;190(2–3):
109–120.
28. Shi C, Wu F, Zhu XC, Xu J. Incorporation of beta-sitosterol into the
membrane increases resistance to oxidative stress and lipid peroxidation
via estrogen receptor-mediated PI3K/GSK3beta signaling. Biochim
Biophys Acta. 2013;1830(3):2538–2544.
29. Kaufman M, Wiesman Z. Pomegranate oil analysis with emphasis on
MALDI-TOF/MS triacylglycerol fingerprinting. J Agric Food Chem.
2007;55(25):10405–10413.
30. Ben-Nun A, Mendel I, Bakimer R, et al. The autoimmune reactivity to
myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is
potentially pathogenic: effect of copolymer 1 on MOG-induced disease.
J Neurol. 1996;243(4 suppl 1):S14–S22.
31. Friedman-Levi Y, Ovadia H, Hoftberger R, et al. Fatal neurological
disease in scrapie-infected mice induced for experimental autoimmune
encephalomyelitis. J Virol. 2007;81(18):9942–9949.
32. Steinman L, Zamvil SS. Virtues and pitfalls of EAE for the develop-
ment of therapies for multiple sclerosis. Trends Immunol. 2005;26(11):
565–571.
33. Gold R, Linington C, Lassmann H. Understanding pathogenesis and
therapy of multiple sclerosis via animal models: 70 years of merits
and culprits in experimental autoimmune encephalomyelitis research.
Brain. 2006;129(pt 8):1953–1971.
34. Brown DA, Sawchenko PE. Time course and distribution of inflam-
matory and neurodegenerative events suggest structural bases for
the pathogenesis of experimental autoimmune encephalomyelitis.
J Comp Neurol. 2007;502(2):236–260.
35. Jose S, Anju SS, Cinu TA, Aleykutty NA, Thomas S, Souto EB. In vivo
pharmacokinetics and biodistribution of resveratrol-loaded solid lipid
nanoparticles for brain delivery. Int J Pharm. 2014;474(1–2):6–13.
36. Blasi P, Schoubben A, Traina G, et al. Lipid nanoparticles for brain
targeting III. Long-term stability and in vivo toxicity. Int J Pharm.
2013;454(1):316–323.
37. Chen S, Tam YY, Lin PJ, Leung AK, Tam YK, Cullis PR. Develop-
ment of lipid nanoparticle formulations of siRNA for hepatocyte gene
silencing following subcutaneous administration. J Control Release.
2014;196:106–112.
38. Wang P, Zhang L, Peng H, Li Y, Xiong J, Xu Z. The formulation and
delivery of curcumin with solid lipid nanoparticles for the treatment of
on non-small cell lung cancer both in vitro and in vivo. Mater Sci Eng
C Mater Biol Appl. 2013;33(8):4802–4808.
39. Gabizon R, Ovadia H, Abramsky O, Magdassi S, Larush L, inventors;
Hadasit Medical Ressearch Services And Development Ltd., Yissum
Research Development Company Of Tthe Hebrew University Of Jeru-
salem Ltd., assignees. Pomegranate Oil For Preventing And Treating
Neurodegenerative Diseases . United States patent US 20150044314
A1. 2015 Feb 12.
40. Guillen-Sans R, Guzman-Chozas M. The thiobarbituric acid (TBA) reac-
tion in foods: a review. Crit Rev Food Sci Nutr. 1998;38(4):315–330.
41. Karlik M, Valkovic P, Hancinova V, Krizova L, Tothova L, Celec P.
Markers of oxidative stress in plasma and saliva in patients with multiple
sclerosis. Clin Biochem. 2015;48(1–2):24–28.
42. Kong W, Yen JH, Ganea D. Docosahexaenoic acid prevents dendritic
cell maturation, inhibits antigen-specific Th1/Th17 differentiation and
suppresses experimental autoimmune encephalomyelitis. Brain Behav
Immun. 2013;25(5):872–882.
43. Zhang Z, Gao F, Jiang S, et al. Bile salts enhance the intestinal absorp-
tion of lipophilic drug loaded lipid nanocarriers: mechanism and effect
in rats. Int J Pharm. 2012;452(1–2):374–381.
44. Schubert R, Schmidt KH. Structural changes in vesicle membranes and
mixed micelles of various lipid compositions after binding of different
bile salts. Biochemistry. 1988;27(24):8787–8794.
45. Ljubisavljevic S. Oxidative stress and neurobiology of demyelination.
Mol Neurobiol. Epub 2014 Dec 11.
46. Ferretti G, Bacchetti T. Peroxidation of lipoproteins in multiple scle-
rosis. J Neurol Sci. 2011;311(1–2):92–97.
47. Leitinger N. The role of phospholipid oxidation products in inflamma-
tory and autoimmune diseases: evidence from animal models and in
humans. Subcell Biochem. 2008;49:325–350.

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48. Sellebjerg F, Sorensen PS. Therapeutic interference with leukocyte recir-
culation in multiple sclerosis. Eur J Neurol. 2015;22(3):434–442.
49. Kizelsztein P, Ovadia H, Garbuzenko O, Sigal A, Barenholz Y.
Pegylated nanoliposomes remote-loaded with the antioxidant tem-
pamine ameliorate experimental autoimmune encephalomyelitis.
J Neuroimmunol. 2009;213(1–2):20–25.
50. Han JM, Lee YJ, Lee SY, et al. Protective effect of sulforaphane
against dopaminergic cell death. J Pharmacol Exp Ther. 2007;321(1):
249–256.
51. Hatcher H, Planalp R, Cho J, Torti FM, Torti SV. Curcumin: from
ancient medicine to current clinical trials. Cell Mol Life Sci. 2008;
65(11):1631–1652.
52. Choi YT, Jung CH, Lee SR, et al. The green tea polyphenol (-)-
epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity
in cultured hippocampal neurons. Life Sci. 2001;70(5):603–614.