1-s2.0-S0378517315300892-main

International Journal of Pharmaceutics 493 (2015) 347–356

Contents lists available at ScienceDirect

International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Novel self-assembled nano-tubular mixed micelles of Pluronics P123,
Pluronic F127 and phosphatidylcholine for oral delivery of nimodipine:
In vitro characterization, ex vivo transport and in vivo pharmacokinetic
studies
Emad B. Basalious* , Rehab N. Shamma
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-aini street, Cairo 1156, Egypt

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 16 May 2015
Received in revised form 25 July 2015
Accepted 28 July 2015
Available online 1 August 2015

Subarachnoid hemorrhage (SAH) is a major cause of death in patients suffering from stroke. Nimodipine
(NM) is the only FDA-approved drug for treating SAH-induced vasospasm. However, NM suffers from
poor oral bioavailability (5–13%) due to its low aqueous solubility, extensive first pass metabolism and
short elimination half-life (1–2 h). The objective of this study was to develop NM-loaded Pluronic/
phosphatidylcholine/polysorbate 80 mixed micelles (PPPMM) that can solubilize NM in aqueous media
even after dilution, prolong its circulation time, improve its bioavailability and eventually help in
targeting it to the brain tissue. PPPMM formulations were prepared using the thin film hydration
technique, and evaluated for drug payload, solubilization efficiency (SE), micellar size, zeta potential,
transmission electron microscopy (TEM) and ex vivo transport through rat intestine. The selected NMloaded PPPMM, containing PC to Pluronics1 molar ratio of 75:25, showed a drug payload, SE, micellar size
and zeta potential of 1.06
0.03 mg/mL, 99.2
2.01%, 571.5
11.87 nm and 31.2
0.06 mv, respectively.
The selected formulation had a much larger hydrophobic core volume for solubilization of NM and
exhibited the highest NM transport. TEM micrographs illustrated the formation of highly flexible nanotubular mixed micelles (NTMM). The in vivo pharmacokinetic study showed greater bioavailability of NM
in plasma (232%) and brain (208%) of rats from NM-loaded PPPMM compared to that of the drug solution
due to the efficiency of flexible NTMM to enhance absorption of NM from the intestinal mucosa. The
significant increase in drug solubility, enhanced drug absorption and the long circulation time of the
NTMM could be promising to improve oral and parenteral delivery of NM.
ã 2015 Elsevier B.V. All rights reserved.

Keywords:
Nimodipine
Nano-tubular mixed micelles
Pluronics
Phosphatidylcholine
Thermodynamic stability of micelles
Subarachnoid hemorrhage

1. Introduction
Subarachnoid hemorrhage (SAH) is a serious, life-threatening
type of stroke where cerebral vasospasm remains a serious
complication and a major cause of death and disability in these
patients. Nimodipine (NM) is a dihydropyridine calcium antagonist
with therapeutic indications for cerebrovascular spasm, stroke and
migraine (Gelmers, 1985; Langley and Sorkin, 1989). Recently, NM
has been shown to be effective in ameliorating memory
degeneration and preventing senile dementia in the old age
(Pantoni et al., 2000; Zhang, 1993). NM is also used for cerebral
malaria, cerebral vasospasm, acute ischemic stroke, and migraines
(Ahmed et al., 2000; Cabrales et al., 2010; Togha et al., 2012; Wolf

* Corresponding author.
E-mail addresses: [email protected],
[email protected] (E.B. Basalious).
http://dx.doi.org/10.1016/j.ijpharm.2015.07.075
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

et al., 2010). NM is the only FDA-approved drug for treating SAHinduced vasospasm. The oral administration of NM has several
limitations and disadvantages. NM belongs to Class II of the
Biopharmaceutical Classification System (BCS) with the typical
characteristics of high permeability and poor solubility (Fu et al.,
2013). The substantial factors that limit its oral bioavailability (5–
13%) and clinical efficacy are the very low aqueous solubility
(3.86 mg/mL) and the extensive first pass metabolism in the liver
(Soliman et al., 2010; Sun et al., 2008). NM was also found to have a
very short half-life (1–2 h) with subsequent need for frequent
dosing (every 4 h) (Langley and Sorkin, 1989).
Among several drug carriers currently under investigation for
improved drug absorption and efficacy, nanocarriers hold the
greatest promise (Soliman et al., 2010). Inclusion of poorly soluble
drugs into polymeric mixed micelles has been found to be very
attractive concept for solubilization and bioavailability enhancement (Wei et al., 2009; Zhao et al., 2012). Polymeric micelles are

348

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

nanocarriers based on amphiphilic block copolymers of hydrophilic and hydrophobic chains that self-assemble in water above
the critical micelle concentration (CMC) (Abdelbary and Tadros,
2013). Pluronic mixed micelles have a core–shell structure which
enables the system to incorporate poorly soluble drugs. These
systems exhibit many advantages such as targeting ability, long
circulation and easy production. In addition, they can also inhibit
P-gp enhancing drug absorption (Chiappetta and Sosnik, 2007).
However, the low stability of these polymeric micelles upon
dilution in the bloodstream or gastrointestinal fluids and the
consequent drug precipitation circumvent their use in drug
delivery.
Pluronics1 P123/F127 mixed micelles (PMM) are considered to
be kinetically stable due to the stabilization effect of long
polyethylene oxide (PEO) chains of hydrophilic F127 blended with
P123 in micelles which might prevent the stacking of cylindrical
aggregates formed by the long polypropylene oxide (PPO) chains of
P123 (Wei et al., 2009). However, these micelles will finally
separate since they are thermodynamically unstable. It was
assumed that more hydrophobic mixed micelles display lower
CMC and concentrations remain above those values even after high
dilution (Chiappetta and Sosnik, 2007). A small concentration of
vegetable oil was introduced into Pluronic solutions to decrease
micelle degradation upon dilution while not compromising the
drug loading capacity of oil-stabilized micelles (Rapoport, 1999).
The ability of mixed micelle formulation to keep the drug in
solubilized form after dilution in the GI tract is a key factor in
bioavailability enhancement rather than solubility of the drug in
the formulation itself. Therefore, it seems necessary to develop
new hydrophobic thermodynamically stabilized PMM that could
resist precipitation upon dilution through incorporation of
phosphatidylcholine (PC) in micelle structure.
Herein, NM-loaded Pluronic/PC/polysorbate 80 mixed micelles
(PPPMM) were developed after incorporation of PC and Polysorbate 80 as examples of hydrophobic and hydrophilic molecules,
respectively in an attempt to increase the thermodynamic and
kinetic stabilities of these micelles. PC has the ability to enhance
oral absorption and bioavailability through improving portal blood
absorption and lymphatic delivery. (Marczylo et al., 2007; Mourao
et al., 2005; Shanmugam et al., 2011; Sugawara et al., 2001).
Moreover, it was reported that polysorbate 80 acts also as pglycoprotein and/or CYP450 enzymes inhibitors decreasing the
intestinal efflux and drug biotransformation (Basalious et al.,
2010). It was assumed that the incorporation of PC would increase
the thermodynamic stability of the micelles due to the tight
hydrophobic interactions with hydrophobic PPO blocks and the
consequent reduction of CMC. Increasing the hydrophobic
character of these copolymers favors also the transition of
morphology of micellar systems in aqueous solutions from
spherical into worm-like (Khimani et al., 2012). To the best of
our knowledge, no attempt has been reported to increase the

solubilizing efficiency of PMM through incorporation of PC in the
micellar structure.
The objective of this study was to develop NM-loaded PPPMM
that can solubilize NM in aqueous media at clinically relevant
concentrations even after dilution, prolong its circulation time,
reduce its frequency of administration and eventually target it to
the brain tissue. Investigations have been carried out to elucidate
the formation of spherical and tubular micelles and studying the
effect of the different morphologies on drug permeation. NMloaded PPPMM were evaluated for drug payload, solubilization
efficiency (SE), micellar size, zeta potential and ex vivo transport
through intestinal membranes of rats. The in vivo pharmacokinetic
behavior of the optimum PPPMM formulation in plasma and brain
tissue was compared to NM solution following oral administration
to rats.
2. Materials and methods
2.1. Materials
NM and amlodipine besylate (IS) were kindly donated by
Marcyrl for pharmaceutical industries (Cairo, Egypt). Difunctional
block copolymers of ethylene oxide/propylene oxide [Pluronic1
F127 and Pluronic1 P123] were purchased from Sigma chemicals
company (St. Louis, USA). L-a-Phosphatidylcholine (PC) from
soybean was purchased from MP Biomedicals (Santa Ana,
California, USA). Spectra/Pore1 dialysis membrane (12,000–
14,000 molecular weight cut off) was purchased from Spectrum
Laboratories Inc. (CA, USA). Disodium hydrogen phosphate was
procured from Merck (Darmstadt, Germany). Ethanol and Polysorbate 80 were from El-Nasr Chemical Co. (Cairo, Egypt). All the
materials were used as received without any further modifications.
2.2. Preparation of nimodipine-loaded Pluronic/PC/polysorbate
80 mixed micelles (PPPMM)
NM (10 mg), phosphatidylcholine (33, 66, and 100 mg) and
Pluronic1 (F127 and P123) mixture in the ratio 2:1 (100, and
200 mg) were accurately weighed and dissolved in ethanol (10 mL)
in a round-bottom flask. Polysorbate 80 (20% of the phosphatidylcholine content) was dissolved in the solution. The solvent was
slowly evaporated at 60  C under reduced pressure using a rotary
evaporator (Buchi R-110 Rotavapor, Flawil, Switzerland) revolving
at 120 rpm for 1 h until a thin dry film was formed on the inner wall
of the flask. The dried film was treated with distilled water (9 mL)
and the flask was allowed to revolve at a fixed hydration
temperature of 60  C for 30 min under normal pressure. The
mixture was then sonicated for 1 min and the volume was adjusted
into 10 mL at room temperature (25  C) to obtain nanocarrier
dispersions. For comparative purpose, liposomes containing
100 mg of phosphatidylcholine and PMM containing 200 mg

Table 1
Composition of nimodipine-loaded PPPMM, PMM and liposome and their characterization values.
Formula

Total Pluronics mixture
(mg)

PC
(mg)

PC: Pluronics molar
ratio

Tween 80
(mg)

Payload (mg/
mL)

SE (%)

PPPMM-1
PPPMM-2
PPPMM-3
PPPMM-4
PPPMM-5
PPPMM-6
Liposome
PMM

100
100
100
200
200
200
–
200

33
66
100
33
66
100
100
–

75:25
85:15
90:10
60:40
75:25
80:20
–
–

6.6
13.2
20
6.6
13.2
20
–
–

1.10
0.01
0.95
0.02
1.05
0.03
0.97
0.01
1.06
0.03
1.08
0.05
0.97
0.04
0.98
0.05

93.7
2.31
665.2
12.56
92.5
1.48
550.1
14.12
94.1
1.97
458.6
9.08
98.6
1.99
549.4
8.47
99.2
2.01
571.5
11.87
97.4
2.09
761.1
15.89
81.8
2.35
359.4
11.24
87.9
2.87 123.56
4.56

PS (nm)

PDI

Zeta potential (mV)

0.55
0.09
0.55
0.08
0.53
1.02
0.79
1.10
0.43
0.06
0.35
0.03
0.24
0.01
0.21
0.02

23.1
0.09
22.3
0.05
25.6
0.04
24.4
0.05
31.2
0.06
30.5
0.04
28.4
0.04
2.3
0.01

All formulations contained 10 mg NM.
PC: phosphatidylcholine, SE: solubilization efficiency, PDI: polydispersity index, PS: particle size, PMM: Pluronic p123/F127 mixed micelles.

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

Pluronic1 (F127 and P123) mixture in the ratio 2:1 were prepared
according to the thin film hydration technique. Table 1 depicts the
composition of the prepared formulations. The selected PPPMM
formulation was frozen at 20  C in presence of 5% w/v mannitol as
a cryoprotectant. Then, samples were lyophilized at 45  C and
pressure of 7  102 mbar for 24 h (Novalyphe-NL 500; Savant
Instruments Corp., USA).
2.3. Characterization of NM-loaded PPPMM
2.3.1. Determination of particle size (PS), polydispersity index (PDI)
and zeta potential
Size and size distribution of NM-loaded PPPMM were determined by the dynamic light scattering method using a Malvern
Mastersizer (DLS, Zetasizer Nano ZS, Malvern instruments,
Malvern, UK). The samples were diluted with distilled filtered
water before measurement until being translucent. Additionally,
polydispersity index (PDI) was measured to assess the particle size
distribution. Finally, zeta potential (ZP) of the diluted samples,
having pH ranged between 5.5 and 6.5, were analyzed for
evaluation of their physical stability. Three samples for each
formula were used for size determination and the average
values
SD were calculated.
2.3.2. Determination of drug payload and solubilization efficiency (SE)
Exact quantity of NM-loaded PPPMM dispersion was dissolved
in ethyl alcohol. Filtration was performed using a 0.45 mm filter. A
2 mL aliquot of filtrate was injected in triplicate into UPLC column
(BEH, C18, 1.7 mm, 2.1, 50 mm). Chromatography was performed
using a Waters Alliance system with a UV detector at 238 nm. The
column temperature was 25  C and the flow rate was 0.25 mL/min.
The mobile phase composed of 10% methanol, 70% acetonitrile and
20% buffer (10 mL glacial acetic acid in 1 L water). The drug payload
was calculated as the milligrams of NM per milliliter of PPPMM
dispersion.
The SE was determined after storing the prepared NM loaded
PPPMM dispersion at 5
3  C for 24 h for possible drug precipitation. A sample of NM loaded PPPMM dispersion was filtered
through 0.45 mm membrane filter to separate the crystallized NM.
The filtrate was assayed for NM content using the same previously
mentioned UPLC method. The SE was calculated as the ratio
between the drug amount in the filtered sample and that added in
the formulation process. Determinations were done in triplicates
for three independent samples of each formula and the average
values
SD were calculated.
2.3.3. Ex vivo transport study
On the day of the experiment, Wistar rats (200
20 g) were
anesthetized and a midline abdominal incision was made and the
entire length of the intestine was removed. The excised intestine
was flushed with cold saline to remove any intestinal contents and
cut into segments (4 cm long). The study performed in this section
was approved by Research Ethics Committee (REC), Faculty of
Pharmacy, Cairo University. The Ex vivo transport studies of NM
from PPPMM were performed in a USP dissolution apparatus.
Accurate volumes of drug-loaded PPPMM dispersion (containing
the equivalent to 2 mg of NM) were placed in double open-sided
glass cylindrical tubes (2.5 cm in diameter and 5 cm in length, with
area = 4.9 cm2) tightly covered from one side with rat intestine. The
loaded cylindrical tubes were attached from the second side to the
shafts of the USP dissolution tester apparatus. This assembly
represents the donor compartment (Fouad et al., 2013). The shafts
rotated at a speed of 50 rpm in 250 mL of 0.5% sodium lauryl sulfate
solution (receptor compartment) at 37  C (FDA dissolution
methods). Aliquots were taken at different time intervals and
replaced instantly by equal amount of fresh medium in order to

349

maintain the same volume. The samples were analyzed for drug
content using the same UPLC method previously mentioned. The
cumulative amount of drug permeated through the membrane per
unit area (mg/cm2) was plotted as a function of time (h) for each
formulation. Experiments were repeated three times and the
results were expressed as the mean values
SD.
2.3.4. Morphological examination of PPPMM by transmission electron
microscopy (TEM)
The morphologic examination of the selected formulations was
performed by transmission electron microscopy (TEM) operating
at 80 kV (model JEM-1230, Jeol, Tokyo, Japan). One drop of the
diluted nanocarrier dispersion was deposited on the surface of a
carbon coated copper grid, negatively stained with 2% phosphotungstic acid then allowed to dry at room temperature for 10 min
for investigation by TEM.
2.3.5. Differential scanning calorimetry (DSC)
The thermal analysis of NM powder, physical mixture (PM) and
lyophilized selected formulation was determined using Shimadzu
differential scanning calorimeter (DSC-50, Kyoto, Japan). Approximately 4 mg of each sample was heated in aluminum pans in a
temperature range of 30–300  C at a heating rate of 10  C/min
under inert nitrogen flow (25 mL/min).
2.3.6. Powder X-ray diffraction (XRD)
Diffraction patterns of NM powder, physical mixture and
lyophilized selected formulation were determined in a Scintag
X-ray diffractometer (USA) using Cu Ka radiation with a nickel
filter, a voltage of 45 kV, and a current of 40 mA.
2.3.7. In vivo pharmacokinetics (PK) of NM loaded PPPMM in Wistar
rats
2.3.7.1. Study design. The in vivo study was carried out to
determine the pharmacokinetics of NM in the plasma and brain
after oral administration of PPPMM dispersion compared to oral
solution. The protocol of this study was reviewed and approved by
the research ethics committee (REC) at Faculty of Pharmacy, Cairo
University (Cairo, Egypt). NM was administered as a single oral
dose of 4 mg/kg using oral gavage (Wang et al., 2006). The study
was done using Wister albino rats (200–250 g). Before initiation of
the experiment, the animals were fasted for 10 h with free access to
water.
2.3.7.2. Plasma PK study. Twenty four Wister male albino rats
weighing 200–250 g were divided into two groups, each
comprising 12 rats, before the experiment. Group 1 received
oral NM solution prepared (1 mg/mL) by dissolving NM in a solvent
mixture composed of alcohol/PEG400/water in a weight ratio of
7.5:8:8. Group 2 received oral NM-loaded selected PPPMM. Blood
samples (1 mL) were obtained from the retro-orbital plexus of
rats by a well-trained practitioner and single sample was obtained
from each individual eye. The samples were collected into
heparinized tubes prior dosing and at 15, 30, 60, 120, 180 and
240 min after administration. Plasma was harvested immediately
by 10 min of centrifugation at 6000 rpm and stored at 80  C until
analysis. The animals were sacrificed by cervical dislocation at the
end of the experiment.
2.3.7.3. Brain PK study. Thirty six Wister male albino rats weighing
200–250 g were divided into two groups, each comprising 18 rats,
before the experiment. Group 1 received oral NM solution (1 mg/
mL) and group 2 received oral NM-loaded selected PPPMM. At
different time intervals (0.5, 1, 2, 3, 4 and 6 h after administration),
the animals were decapitated, their skulls were cut open and the

350

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

brain tissue was removed, rinsed with saline twice and blotted
with filter paper. The total brain tissue samples were taken,
homogenized with 1 mL of saline, and then transferred into tubes.
Homogenized brain samples were stored at 80  C until assayed.

409.10 ! 238.14 for IS. Mass Lynx software version 4.1 was used
to control all parameters of UPLC and MS. The lower and upper
limits of quantification of NM in plasma and brain samples were
5 to 200 ng/mL and 0.5 to 100 ng/mL, respectively.

2.3.7.4. Samples preparation. A volume of 50 mL of IS solution in
methanol (250 ng/mL) was added to 200 mL plasma samples or
500 mL homogenized brain samples then the samples were
vortexed for 30 s, followed by the addition of 200 mL 1 M NaOH.
The samples were further vortexed for 30 s. Extraction was applied
by the addition of 3 mL of mixture of diethyl ether:n-hexane (1:1 v/
v) followed by vortex for one minute, then samples were
centrifuged at 3000 rpm for 10 min A volume of 2.5 mL of the
upper organic layer were transferred accurately into another dry
clean tube. The organic layer was evaporated at 45  C, using
Eppendorf sample concentrator (Eppendorf, Germany) till dryness.
Residue was reconstituted in 150 mL mobile phase, and a volume of
2 mL from the reconstituted sample was injected into the UPLCMS/MS system.

2.3.7.6. Pharmacokinetic and statistical analysis. Plasma and brain
concentration–time
data
of
NM
was
analyzed
by
noncompartmental pharmacokinetic models using Kinetica1
software
version
5
(Thermo
Fisher
Scientific
Inc.).
Concentration–time profiles were plotted and the peak
concentration Cmax (ng/mL) and the necessary time Tmax (h) to
attain Cmax were obtained directly from it. The area under the drug
concentration–time curve (AUC) was calculated by the trapezoidal
method. The terminal elimination rate constant was calculated by
linear regression of the terminal portion of the natural logarithm of
the concentration and the elimination half-life was calculated. The
pharmacokinetic data obtained from different treatments were
analyzed for statistical significance by one-way analysis of variance
(ANOVA) adopting Kinetica1 software version 5 (Thermo Fisher
Scientific Inc.). The non-parametric test (Kruskal Wallis test) was
used to compare the tmax for test and reference.

2.3.7.5. Chromatographic conditions. Plasma and brain samples
were analyzed for NM adopting a sensitive, selective and accurate
LC-MS/MS method, developed and validated before the study
using UPLC-MS/MS system. Quantitative analysis was performed
on a Waters Acquity UPLC H-Class-Xevo TQD system (MA, USA)
interfaced with a Waters Quattro Premier XE triple quadrupole
mass spectrometer and equipped with electrospray ionization
operated in the positive ionization mode. Chromatographic
separation of analytes was carried out on ACQuity UPLC HSS C18
(50  2.1 mm, 1.8 mm) column. The isocratic mobile phase
composed of acetonitrile—0.1% formic acid (85: 15, v/v) and was
delivered at a flow rate of 0.25 mL/min. The column was
maintained at 35  C and the pressure of the system was
6500 psi. The source dependent parameters maintained for both
the analytes and internal standards (ISs) were: cone gas flow, 50 L/
h; desolvation gas flow, 800 L/h; capillary voltage, 3.5 kV, source
temperature, 120  C; desolvation temperature, 350  C. The
optimum values for compound dependent parameters like cone
voltage and collision energy were set at 25 V and 10 eV for NM and
30 V and 12 eV for IS, respectively. The mass transition ion pair,
performed in the multiple-reaction monitoring (MRM) mode, of m/
z
419.24 ! 343.09
was
followed
for
NM
and
m/z

Fig. 1. The ex vivo transport profiles of NM from PPPMM-5, PMM and liposomes
through rat intestine compared to that from aqueous NM suspension.

2.4. Statistical analysis
The data obtained from different formulations were analyzed
for statistical significance by one-way analysis of variance
(ANOVA) adopting SPSS statistics program (version 16, SPSS Inc.,
Chicago, USA) followed by post hoc multiple comparisons using
the least square difference (LSD). Differences were considered to
be significant at p  0.05.
3. Results and discussion
3.1. Preliminary studies and formulation of mixed micelles of different
morphologies
Vain attempts to solubilize NM were performed by preparation
of several Pluronic P123/F127 mixed micelles (PMM). The total
amount of Pluronics1 relative to NM reached up to 1:36 (w/w) and
Pluronic P123 and F127 were used in ratios of 1:2, 1:1 and 2:1 (w/
w). All formulations showed precipitation of NM crystals after
storage for one week at 5
3  C (data not shown). Pluronic micellar
systems were reported to have certain drawbacks such as the
formation of aggregates with a large particle size, the low stability
and the possible reversion to the phase separated state (Abdelbary
and Tadros, 2013). It was reported that the hydrophilic long PEOshell of the micelles formed by F127 and P123 had a protective
effect on the micelle dispersion. However, the mixed micelles were
still in a dynamic state and presented only temporary stability (Wei
et al., 2009). The low stability of solubilized micellar system may
be also due to the highly hydrophobic nature of NM molecule
which makes its full accommodation in the hydrophobic core of
the micelles practically difficult. Thus, our objective was to
increase the thermodynamic and kinetic stabilities of PMM
through incorporation of PC and polysorbate 80, respectively. It
was reported that Pluronics1 can self-assemble into spherical and
non-spherical micelles in dilute aqueous solutions (Park et al.,
2015). Amphiphilic block copolymers generally self-assemble in
dilute aqueous solution into spherical micelles and/or tubular
micelles based on the relative weight fraction of hydrophilic and
hydrophobic blocks. Spherical micelles form spontaneously when
the hydrophilic block such as PEO is the largest block by mass. By
decreasing the weight fraction of the PEO block to just less than
about 50%, hydration and swelling of the corona imparts just
enough curvature to the copolymer assembly that tubular micelles
that may reach up to microns in length and similar in diameter to

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

351

Fig. 2. Transmission electron microscopy (TEM) of PPPMM-5 (a) and PPPMM-1 (b) showing the tubular morphologies of the micelles.

the spheres are the predominant morphology (Cai et al., 2007). The
aggregation characteristics of Pluronics1 in water are modified
quite significantly in the presence of additives that have a strong
influence on their solubilization characteristics. Increasing the
hydrophobic character of these copolymers, for example through
the incorporation of PC in the structure of PMM, favors micelle
formation by reducing the critical micellar concentration (CMC),
critical micellar temperature (CMT), and the sphere-to-rod
transition temperature of their aqueous solutions (Khimani
et al., 2012).

3.2. Drug payload and solubilization efficiency (SE)
Table 1 shows the drug payload and SE of PPPMM, Liposomes and
PMM. The drug payload of all NM-loaded PPPMM ranged from 0.95 to
1.1 mg/mL. Their SE after storage at 5
3  C for 24 h ranged from
92.5
1.48% to 99.2
2.01%. The accommodation of NM in the core of
micelles was very efficient as demonstrated by the high values of SE
especially for PPPMM containing 200 mg total Pluronics1. Incorporation of PC would increase the thermodynamic stability of the
micelles due to the tight hydrophobic interactions with hydrophobic
PPO blocks and the consequent reduction of CMC. On the other hand,
incorporation of polysorbate 80 would increase the kinetic stability
of the micelles due to the steric hindrance that minimize micelle
aggregation. The relatively low SE of systems containing 100 mg total
Pluronics1 resulted from the release of NM from PPPMM and the
formation of crystals of NM in the external aqueous phase. The very
low SE of liposomes and PMM (81.8
2.35% and 87.9
2.87,
respectively) confirmed the inability of both systems to fully
accommodate NM in their structure leading to drug precipitation
after storage at 5
3  C for 24 h.
3.3. Particle size distribution and zeta potential

Fig. 3. The ex vivo transport profiles of NM from the different formulations of
Pluronic/PC/polysorbate 80 mixed micelles (PPPMM) through rat intestine
compared to liposomes.

As shown in Table 1, NM-loaded PPPMM have mean particle
diameters ranging from 458.6
9.08 to 761.1
15.89 nm. The PS of
liposomes and PMM were 359.4
11.24 and 123.56
4.56 nm,
respectively. Incorporation of PC and polysorbate 80 in the
structure of PPPMM significantly increased their particle size
(p < 0.05). The polydispersity index (PDI) is a dimensionless
number expressing the particle size distribution of the investigated system. The closer the polydispersity value to zero, the more
homogenous are the particle population. Contrary to liposomes
and PMM, the PDI of all PPPMM formulations show a wide range of
values starting from 0.35 to 0.79, thus indicating a wide size
distribution of the measured dispersion (Essa et al., 2002). The
presence of different morphologies such as spherical and worm-

352

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

Fig. 4. Schematic illustration for the different micellar morphologies formed by Pluronics P123/F127 and their mixture with phosphatidylcholine (PC).

like (tubular) micelles could explain the wide distribution of
particle size of Pluronic dispersion containing PC.
The zeta potential was measured and found to be in values
ranging from 22.3
0.05 to 31.2
0.06 mV. Values more than

20.0 suggested good stability of the prepared systems with low
probability of aggregation and particle growth (Shanmugam et al.,
2011).
3.4. Ex vivo transport study
To investigate the impact of different morphologies of the
nanocarrier systems on the drug transport, ex vivo transport from
PPPMM-5, PMM and liposomes through rat intestine compared to
that from aqueous NM suspension (1 mg/mL in 1% HPMC solution)
was performed and illustrated in Fig. 1. Exact volume of each
formulation equivalent to 2 mg NM was placed on rat intestine
attached to a cylindrical tube having surface area 4.9 cm2. Amongst
the formulations tested, NM-loaded PPPMM showed the highest
cumulative amount of NM permeated after 20 h (110.25
12.3 mcg/
cm2) followed by PMM (102.35
9.81 mcg/cm2), the liposomes
(56.97
6.37 mcg/cm2) and finally the aqueous suspension
(10.23
5.53 mcg/cm2). The poor water solubility of NM is the
main reason for its low transport through intestinal membrane.
Incorporation of poorly water soluble drugs into liposomes caused
substantial enhancement in absorption and bioavailability. Liposomes may provide increased solubility of their load and protection
from the hostile environment in the gastrointestinal tract.
Moreover, the similarity between liposomal lipid bilayers and
biological membranes and the relatively small size of liposomes

significantly facilitates permeation (Chen et al., 2009). Several
researchers have studied the underlying mechanism for the
facilitated oral absorption by liposomes. It was assumed that
physiological bile salts can interact with phospholipids in the
liposomal vesicles inducing a vesicle–micelle transition to form
mixed micelles that play important roles in enhancing absorption of
poorly water-soluble drugs. The resultant mixed micelles have been
shown to function as excellent vehicles for poorly water-soluble
drug molecules and one of the most important mesophases before
absorption (Andrieux et al., 2009; Chen et al., 2009; Hildebrand
et al., 2003). Therefore, transferosomes containing bile salts as edge
activators can readily transform into mixed micelles in the
gastrointestinal environment, thus enhancing membrane penetration. This conclusion could explain why the spherical PMM showed
about 2 folds increase of NM transport compared to NM-loaded
liposomes (Fig. 1). PMM is more capable to keep NM in solubilized
form during the transport study than liposomes as previously
confirmed by the higher values of SE (Table 1). The highest
enhancement of NM transport through rat intestine was achieved
by PPPMM-5. Incorporation of PC in the structure of Pluronic
micelles increases the thermodynamic stability of the micelles
preventing NM crystallization during the transport study and favors
the conversion of micelles from spherical into tubular morphologies. As shown in Fig. 2a, TEM micrograph of PPPMM-5 illustrates
the worm-like or tubular morphology of the micelles. The length
and diameter of the nanotubular micelles are in the nano range. The
flexible morphology of the nano-tubular mixed micelles (NTMM)
could explain the high enhancement of NM transport through rat
intestine (Giddi et al., 2007). It was reported that the flexibility of

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

tubular micelles allow them to penetrate nanoporous gels where
100 nm sized vesicles cannot enter (Kim et al., 2005). The results of
transport study confirmed that the morphology of micelles affected
drug transport through biological membranes where the efficiency
for drug transport could be ranked as follows; nano-tubular mixed
micelles (NTMM)  spherical PMM > vesicles (liposomes).
Fig. 3 illustrates the ex vivo transport of NM from the different
formulations of Pluronic/PC/polysorbate 80 mixed micelles
(PPPMM) through rat intestine. Amongst the formulations tested,
liposomes and PPPMM-3 showed the lowest cumulative amount of
NM permeated after 24 h (61.30
14.23 and 53.51
12.52 mcg/
cm2, respectively) followed by PPPMM-6 and PPPMM-2
(73.54
15.61 and 79.61
4.95 mcg/cm2, respectively), and
PPPMM-4 (91.73
16.89 mcg/cm2) and finally PPPMM-5 and
PPPMM-1 (112.73
19.42 and 118.47
22.49 mcg/cm2, respectively). There is no significant difference between the permeation
profiles of NM from liposomes and PPPMM-3 through rat intestine
(P < 0.05). The similarity of the morphology of the nanocarrier of
both formulations could explain why they gave the same lowest
drug transport rate through membranes. As shown in Table 1,
PPPMM-3 had the highest PC content relative to Pluronics1
(90:10 molar ratio). Thus, the vesicular liposomal shape is the main
morphology of this formulation. As PC content decreased from
0.9 into 0.85 and 0.8 mole fraction (PPPMM-2 and PPPMM-6,
respectively), the drug transport slightly increased which may be
due to the formation of some spherical Pluronic mixed micelles in
addition to the drug loaded vesicles. PPPMM-4, containing 0.6
mole fraction of PC, showed a slight increase of drug transport
which indicated the formation of large proportion of spherical
Pluronic micelles due to the large content of Pluronic. PPPMM5 and PPPMM-1, surprisingly containing the same molar ratio of PC
to Pluronics1 (75:25), gave the highest NM transport due to the
formation of highly flexible nanotubular mixed micelles (NTMM).
The tubular morphologies of PPPMM-5 and PPPMM-1 were
elucidated by the TEM micrographs shown in Fig. 2a and b.
The concept of critical packing parameter (CPP) for surfactant
systems by Israelachvili could be applied to predict and explain the
change of morphology as being driven by a change in the
spontaneous curvature of the system (Arleth et al., 2005; Rupp
et al., 2010). When surfactants aggregate with each other, they tend
to form monolayers that have curvature allowing the most efficient
packing of the molecules. Spherical micelles are formed when CPP
is less than 1/3, wormlike micelles when CPP is between 1/3 and 1/
2, vesicles when CPP is between 1/2 and 1, and lamellar or bilayer
structure when CPP is 1 (Chu et al., 2013). As illustrated in Fig. 4,
Pluronics1 P123/F127 form spherical or nearly spherical mixed
micelles in dilute aqueous solution, and consequently have a CPP
near 1/3. PC has two alkyl chains and therefore a higher CPP (close
to unity). As a result, it tends to form vesicle structures. Mixtures of
the two types of molecules in optimum PC: Pluronics1 ratio
(75:25 molar ratio) should give rise to CPP ranging from 1/3 to 1/2.
This would indeed result in wormlike or tubular morphology of the
micelles as confirmed by TEM.
As shown in Table 1, PPPMM-5 showed significantly higher
solubilization efficiency than PPPMM-1 although both formed nanotubular mixed micelles (NTMM). The higher content of Pluronics1
and PC in PPPMM-5 formulations could explain the high SE. The PPO
of Pluronics1 and the alkyl chains of PC are assembled in a
hydrophobic core. Thus, the tubular micelles of PPPMM-5 have a
much larger core volume for encapsulation. It was reported that
higher paclitaxel loading was possible by worm-like micelles (Cai
et al., 2007). This hydrophobic core is surrounded by a hydrophilic
shell consisting of PC polar head groups and hydrophilic PEO of
Pluronics1 and polysorbate (Fig 4). Due to the optimum SE and the
higher permeation properties, PPPMM-5 formulation was selected
for further investigation and in vivo PK study.

353

3.5. Differential scanning calorimetrical studies (DSC)
DSC was performed for the lyophilized NM-loaded PPPMM-5 as
well as the corresponding physical mixture and NM plain powder
in order to evaluate the phase transformation of NM during the
formation of the tubular micelles. As illustrated in Fig. 5, the free
drug was characterized by a single, sharp melting endothermic
peak at 127  C corresponding to the melting point of NM which
confirms its crystallinity (Fu et al., 2013). The DSC pattern of the PM
showed the presence of the characteristic peak of pure drug,
although with a lower intensity (due to dilution) indicating that
the drug retained its crystallinity (Elsayed et al., 2014). In addition,
the DSC pattern of the PM displayed a sharp melting endothermic
peak at 56  C, corresponding to the melting point of Pluronic F127
(Abdelbary and Makhlouf, 2014; Abdelbary and Tadros, 2013), and
a smaller intensity endothermic peak at 33  C corresponding to the
melting point of the phospholipid. The thermal behavior of NMloaded PPPMM was distinct from that found for the PM. The
thermogram of the micellar system revealed complete disappearance of the peaks of the drug and phospholipid. Disappearance of
the characteristic endothermic peak of NM probably signifies that
NM was either entrapped inside PPPMM (Salama et al., 2012a) or
completely transformed from crystalline form to molecular state in
the surfactant mixture. Beside the disappearance of the drug and
phospholipid endothermic peaks, the thermogram of NM-loaded
PPPMM revealed broadening, decrease in the intensity, and
shifting in the Pluronic F127 peak from 56  C to 46.6  C. A new
sharp endothermic peak appeared at 167.5  C, corresponding to the
melting point of mannitol (Chalikwar et al., 2012; Elsayed et al.,

Fig. 5. DSC thermograms of the lyophilized optimized NM-loaded PPPMM-5 as well
as the corresponding physical mixture (PM) and NM plain.

354

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

highest intensity at 2u of 12.89 , 20.37, and 26.33 (Zu et al., 2014).
On the other hand, the diffractogram of the prepared PPPMM
showed a typical diffuse pattern with complete absence of the
numerous distinctive peaks of NM indicating that NM was
molecularly dispersed in the core of tubular micelles of PPPMM-5.
Crystallinity was assessed by comparing some representative
peak heights in the diffractogram of the NM-loaded PPPMM-5 with
those of a NM powder. The relationship used to calculate the
relative degree of crystallinity (RDC) was RDC = Isample/Idrug, where
Isample is the peak height of the sample under investigation and Idrug
is the peak height at the same angle for the drug (Shoukri et al.,
2009). Pure drug peak at 2u = 20.37 was used for calculating the
RDC. The calculated RDC value was found to be 0.21, and 0.05, for
the PM, and the PPPMM, respectively. The characteristic diffraction
peaks of NM were detected in the PM, whereas they disappeared in
the corresponding PPPMM.
3.7. In vivo pharmacokinetic studies

Fig. 6. X-ray diffraction of the lyophilized optimized NM-loaded PPPMM-5 as well
as the corresponding physical mixture (PM) and NM plain.

Pharmacokinetic parameters for NM in rat plasma and brain
were assessed using UPLC-MS/MS method in order to understand
the in vivo behavior of the NM-loaded PPPMM compared to the
drug solution. The UPLC-MS/MS assay was validated and showed
acceptable inter- and intra-day reproducibility. The lower limits of
NM quantification in plasma, and in brain were 5 and 0.5 ng/mL,
respectively. To quantify the extent of nano-tubular micelles
accumulation in the brain, brain tissues were assayed for NM
concentration. Figs. 7 and 8 denote the mean NM concentrations in
plasma and in brain of rats, respectively, after administration of
single oral dose of NM solution and NM-loaded PPPMM formulation and the corresponding pharmacokinetic parameters are
shown in Table 2.
3.7.1. Pharmacokinetics of NM in blood
The mean Cmax estimated in plasma from the drug solution and
the PPPMM were 20.54 and, 33.18 ng/mL, reached after times
(Tmax) 0.25 h and 0.94 h, respectively. The differences between the
two treatments for Cmax and tmax were found to be statistically
significantly different (p = 0.044 and 0.016, respectively).
The non-parametric test (Kruskal Wallis test) showed a
significant difference between Tmax of both treatments. The mean
AUC0t estimated from PPPMM (42.21 ng h/mL), which reflects the
total amount of drug absorbed over the 4 h time period, was

Fig. 7. The mean NM concentrations in plasma of rats after administration of single
oral dose (4 mg/kg) of NM solution and NM-loaded PPPMM formulation.

2014), which was used as a cryoprotectant during the lyophilization process.
3.6. Powder X-ray diffraction
In order to further examine the physical form of the drug in the
selected formulation, pure NM, PM, and the lyophilized NM-loaded
PPPMM-5, were investigated using powder X-ray diffraction. As
shown in Fig. 6, The diffractogram of NM revealed its crystalline
nature as indicated by three prominent diffraction peaks with the

Fig. 8. The mean NM concentrations in brain of rats after administration of single
oral dose (4 mg/kg) of NM solution and NM-loaded PPPMM formulation.

E.B. Basalious, R.N. Shamma / International Journal of Pharmaceutics 493 (2015) 347–356

355

Table 2
The mean pharmacokinetic parameters of NM in plasma and brain of rats after oral administration of single dose of NM solution and NM-loaded PPPMM.
Plasma data

Brain data

Parameter

NM solution

NM-loaded PPPMM

Significance (p-value)

NM solution

NM-loaded PPPMM

Significance (p-value)

Cmax (ng/mL)
Tmax (h)
AUC0t (ng h/mL)
AUC01 (ng h/mL)
T1/2 (h)

20.53
4.18
0.25
0.00
18.14
7.24
21.68
7.31
0.95
0.28

33.18
15.64
0.94
0.71
42.21
12.85
69.55
8.20
2.42
0.59

0.044*
0.016*
0.0004*
6.7167e-005*
0.0221*

31.74
15.57
0.50
0.00
33.88
14.42
34.41
14.84
0.91
0.14

85.78
12.29
0.67
0.28
70.61
13.34
70.84
13.39
0.72
0.11

0.009*
0.373
0.031*
0.034*
0.140

Values are expressed as mean
SD, n = 6 for plasma and n = 3 for brain.

determined to be about 2.32 folds higher and statistically
significantly different (p = 0.0004) compared to the mean AUC0t
estimated from the drug solution (18.14 ng h/mL). The relative
bioavailability (frel) of PPPMM compared to the drug solution was
estimated to be approximately 232%. The improved bioavailability
of NM from PPPMMs confirmed the ability of highly flexible NTMM
to flow through the nanopores of the intestinal membrane.
Moreover, the presence of PC facilitates the lymphatic delivery of
NM bypassing the hepatic first-pass effect. According to Iwanaga
et al. (1997), orally administered PC vesicles accumulate at the
brush–border membrane of enterocytes, resulting in increased
drug concentration gradient across the intestinal epithelium. This
enables absorption of significant amount of the drug into the
systemic circulation, and enhances the drug bioavailability. Kimura
et al. also reported the uptake and transport of intact PC liposomes
across the small intestine following oral administration via gut
associated lymphoid tissue (Kimura, 1988). It was reported that the
oral absorption of nano-lipid based particles occurs via lymphatic
transport (Chalikwar et al., 2012; Patil-Gadhe et al., 2014; Singh
et al., 2014) through Peyer’s patches of the intestine which results
in bioavailability improvement (Basavaraj and Betageri, 2014;
Florence et al., 1995). The lymphatic transport increases drug
bioavailability as the intestinal lymph vessels travel directly into
the thoracic duct, then to the systemic circulation, hence bypassing the portal circulation (Porter and Charman, 2001; Ryan
et al., 2014). Moreover, presence of Pluronics1 further contributes
to bioavailability improvement. Pluronic micelles were reported to
have a P-gp efflux inhibition character, which results in enhancement of drug permeability and absorption to the systemic
circulation (Abdelbary and Makhlouf, 2014).
The mean NM t1/2 estimate from PPPMM (2.42 h) was
determined to be higher and statistically significantly different
(p = 0.0221) compared to the mean t1/2 estimate from the drug
solution (0.95 h). These results could indicate the ability of the
optimized nanotubular dispersion to achieve sustained plasma
profile of NM after oral administration when compared to the oral
drug solution containing the drug in solubilized form. These results
were in accordance with previous studies that reported the long
circulation time of worm-like micelles in the blood circulation,
which appears longer than any other synthetic particle, including
stealthy vesicles bearing the same length of PEO chains (Cai et al.,
2007; Giddi et al., 2007).
3.7.2. Pharmacokinetics of NM in brain
It was important to study the pharmacokinetic behavior of NM
in brain tissue since it is the target organ of NM. From Table 2, it is
obvious that the values of Cmax and AUC0t for the NM-loaded
PPPMM in brain tissue were significantly higher than those of the
oral NM solution, (p = 0.009, and 0.031, respectively). The value of
Cmax of NM in brain after administration of NM-loaded PPPMM was
2.7 times that of the NM solution. The AUC0t for NM in brain after
administration of NM-loaded PPPMM and NM-loaded solution
were 70.61 and 33.88 ng h/mL, respectively. The high values of Cmax
and AUC0t of the PPPMM compared to that of the solution might

be attributed to the long circulation time of the nano-tubular
micelles and the presence of Pluronics1, polysorbate 80 and PC in
the PPPMM which acts as a penetration enhancer and inhibitor of
P-gp in the blood brain barrier (BBB) (Salama et al., 2012b). Several
researchers studied the mechanism of liposome transport across
the blood brain barrier and reported that it might be lipidmediated free diffusion or lipid-mediated endocytosis (Shah et al.,
2013).
The elimination half-life (t1/2) of ND from the brain tissues
showed no significant differences between the NM-loaded PPPMM
and NM-loaded solution (p = 0.140). Following oral administration
of NM-loaded PPPMMs, the pharmacokinetic behavior of NM in
plasma showed a higher sustained presence compared to that in
brain. This could be due to the encapsulation of NM in the long
circulating NTMMs resulting in retarded clearance of NM from
plasma. On the other hand, free NM penetrated BBB and reached
the brain tissues in a non-encapsulated form resulting in high
levels of drug in the cerebral tissue followed by its rapid clearance
from the brain tissues.
Based on these results, it can be concluded that the greater
bioavailability obtained from NM-loaded PPPMM, which was
about 208% (brain tissue), and 232% (plasma) larger than that
measured after administration of the drug solution might be
attributed to efficiency of flexible nanotubular micelles to
solubilize and enhance absorption of NM from the intestinal
mucosa and their long circulation time. Moreover, the P-gp efflux
inhibition character of Pluronic and polysorbate enhanced the drug
permeability and absorption into the systemic circulation and
brain tissues.
4. Conclusions
In this study, PPPMMs were prepared by incorporation of PC
and polysorbate 80 in the structures of Pluronic P123/F127
micelles. The aqueous mixtures of Pluronics1 with CPP < 1/
3 and PC with CPP > 1/2 showed the formation of nano-tubular
mixed micelles (NTMMs). This finding opened the door for future
preparation of thermodynamically and kinetically stable NTMMs
for enhanced solubilization and absorption of poorly water soluble
drugs compared to liposomes and spherical micelles. The
encapsulation of NM into NTMMs significantly improved the
pharmacokinetic profiles of the drug in plasma and brain
compared to the drug solution. The significant increase in drug
solubility, enhanced drug absorption and the long circulation time
of the prepared tubular micelles could be promising to improve
oral and parenteral delivery of NM. Further studies for elucidating
the key formulation factors required for the preparation of
intravenous NTMM for sustained delivery of NM into the brain
tissue are presently investigated.
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