Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245

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Colloids and Surfaces B: Biointerfaces

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

Nanostructured lipid carriers for improved oral delivery and

prolonged antihyperlipidemic effect of simvastatin
Heba A. Fathi a , Ayat Allam b , Mahmoud Elsabahy a,b,c,d,∗ , Gihan Fetih a,b ,
Mahmoud El-Badry a,b
a
Assiut International Center of Nanomedicine, Al-Rajhy Liver Hospital, Assiut University, Assiut 71515, Egypt
b
Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71515, Egypt
c
Laboratory for Synthetic-Biologic Interactions, Department of Chemistry, Texas A&M University, College Station, TX, USA
d
Misr University for Science and Technology, 6th of October City, Egypt

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

Article history: The purpose of the current study is to develop nanostructured lipid carriers (NLCs) for the delivery of
Received 18 September 2017 the antihyperlipidemic drug simvastatin (SIM) to increase its extremely low oral bioavailability (<5%)
Received in revised form and prolong its antihyperlipidemic effect. NLCs were prepared via emulsification-solvent evaporation
17 November 2017
technique followed by ultrasonication, and the effect of composition of the nanocarriers on the particle
Accepted 27 November 2017
size, size distribution, surface charge, entrapment efficiency, drug release kinetics and physical stability
Available online 28 November 2017
was extensively studied. NLCs exhibited nanosized (<200 nm) spherical morphologies with narrow size
distribution and high drug entrapment efficiency (>75%), sustained drug release pattern, and negative
Keywords:
Nanostructured lipid carriers
surface charge (zeta potential of −35–40 mV) that imparts sufficient electrostatic physical stability. When
Simvastatin tested in vivo, SIM-NLCs of the optimal composition demonstrated improved and prolonged reduction
Hyperlipidemia in the total cholesterol and non-high density lipoprotein cholesterol levels, as compared to the drug
Drug delivery suspension. After oral administration of a single dose of SIM-NLC, 4-fold increase in bioavailability was
Nanoparticles observed, as compared to the SIM suspension. Hence, NLCs might provide efficient nanodevices for the
management of hyperlipidemia and promising drug delivery systems to enhance SIM oral bioavailability.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Elevated blood cholesterol level (i.e. hypercholesterolemia)
leads to development and progression of atherosclerosis, and con-
sequently cardiovascular diseases, and it is estimated to result in 2.6
million deaths (4.5% of total mortality) globally [1]. Successful treat-
ment of hypercholesterolemia substantially reduces morbidity and
mortality from cardiovascular diseases [2,3]. Statins have the ability
to retard the accelerated atherosclerosis in hyperlipidemic indi-
viduals. Simvastatin (SIM) is one of the statins family, which is
considered as the first-line treatment of hypercholesterolemia,
dyslipidemia and coronary heart diseases [4]. SIM is a potent
inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA)
reductase which converts HMG-CoA into mevalonate, a precur-
sor in cholesterol synthesis. The biopharmaceutical classification
system classifies SIM as class 2 drug (i.e., low aqueous solubility
∗ Corresponding author at: Assiut International Center of Nanomedicine, Al-Rajhy
Liver Hospital, Assiut University, Assiut 71515, Egypt.
E-mail address: mahmoud.elsabahy@chem.tamu.edu (M. Elsabahy).
and high permeability) with short plasma half-life and variable
absorption [5]. SIM is exposed to extensive first pass metabolism
by cytochrome P3A (CYP3A) in the intestinal guts and the liver
[6]. The oral bioavailability of SIM in its intact form is ca. 5% due
to the slow dissolution rate in the intestinal fluid and significant
first-pass metabolism [7]. Various approaches have been utilized
to enhance the bioavailability of SIM, such as self microemulsi-
fication [8], nanoemulsification [9], nanoparticles [10], solid lipid
nanoparticles [11], nanocrystals [12] and nanosuspensions [13].
Nanocarriers including biodegradable polymeric nanoparticles
[14], polymeric micelles [15], nanocrystals [16], nanosuspensions
[17] and lipid colloidal nanocarriers have received significant con-
sideration as delivery systems for several drugs. Lipid nanocarriers
composed of natural or synthetic lipids have the advantage of
controlling drug release with high biocompatibility. Solid lipid
nanoparticles (SLNs) have emerged in the early nineties as an alter-
native carrier system to the existing traditional lipid carriers due
to the feasibility of large scale production, low toxicity and avail-
ability of their excipients [18]. However, the solid lipid matrix of
SLNs results in the formation of a relatively perfect crystal lat-
tice leaving a limited space for drug incorporation, which limits

https://doi.org/10.1016/j.colsurfb.2017.11.064
0927-7765/© 2017 Elsevier B.V. All rights reserved.
 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
the loading capacity and leads to drug leakage during storage
[19]. Nanostructured lipid carriers (NLCs), the second-generation
of lipid nanoparticles, are composed of a mixture of spatially dif-
ferent lipid molecules, i.e., a solid lipid is blended with a liquid
lipid. The addition of liquid lipid distorts the formation of per-
fect lipid crystals, and, thus increasing drug loading capacity, and
decreasing particle size and risk of gelation and drug leakage dur-
ing storage [20,21]. Additionally, NLCs promote oral absorption
of encapsulated drugs via selective uptake through the lymphatic
route or the payer’s patches [22]. NLCs have been previously uti-
lized for enhancing the oral bioavailability of various drugs [23,24].
Moreover, SIM-NLCs have been reported earlier to improve SIM
amount of drug entrapped into the nanoparticles
Entrapment efficiency (%) =
amount of drug initially added
oral bioavailability, although pharmacodynamic profile was not
studied [7]. Pharmacokinetic parameters are not sufficient to pre-
dict the pharmacodynamic activity of statins [25]. In the current
study, SIM-NLCs are prepared via emulsification-solvent evapo-
ration technique followed by ultrasonication, and the effect of
composition on the physicochemical characteristics of the formu-
lations has been extensively studied. In vitro release kinetics and
stability of SIM-NLCs were also evaluated. In vivo pharmacokinetics
and pharmacodynamics of SIM-NLCs have been investigated in nor-
mal rats and poloxamer-induced hyperlipidemic Wister rat model,
respectively, to evaluate the ability of the developed nanocarriers
to improve the oral bioavailability and antihyperlipidemic activity
of the drug.
2. Materials and methods
2.1. Materials
Stearic acid was purchased from El-Nasr Chemicals Co. (Abu Zaa-
bal, Egypt). Oleic acid was purchased from Alpha Chemicals (Cairo,
Egypt). Simvastatin was purchased from El-Ryad Pharma (El-Ryad,
KSA). Pluronic F-68 was provided by BASF (Luwigshafen, Germany).
Lecithin was purchased from Sigma-Aldrich Co. (St. Louis, MO).
Other chemicals and reagents were of analytical grades.
2.2. Preparation of simvastatin-nanostructured lipid carriers
(SIM-NLCs)
SIM-NLCs were prepared via emulsification-solvent evapora-
tion technique followed by ultrasonication as reported earlier with
slight modifications [26]. Briefly, certain amount of SIM, stearic
acid, oleic acid, and lecithin were dissolved in chloroform and
heated at 70 ◦ C. Aqueous phase containing the surfactant (Pluronic
F-68) in 10 mL of deionized water was heated to 70 ◦ C. The aqueous
phase was then added to the lipid phase under stirring at 1200 rpm
and 70 ◦ C, and mixed for 5 min. The obtained pre-emulsion was
sonicated by probe-type sonicator (Cole-Parmer, Vernon Hills, IL)
for 7 min (40 W). Then, the dispersions were cooled down to the
room temperature under agitation at 300 rpm for 1 h to obtain the
aqueous NLC dispersions. Purification of SIM-NLCs was performed
via dialysis into 250 mL of double distilled water and stirred at
300 rpm for 24 h, to remove the free drug. The dialysis water was
replaced with fresh double distilled water after 12 h. The concen-
tration of the drug was adjusted to ensure that the purification
process is carried out under sink conditions (i.e. the dialysis vol-
ume exceeds the volume required to solubilize the total amount
of the drug). Eight different formulations of different compositions
were prepared (F1-F8, Supplementary information, Table S1). The
237
effects of oleic acid-to-stearic acid ratio and surfactant (Pluronic
F-68) concentration on the physicochemical characteristics of the
formulations were evaluated.
2.3. Characterizations of the prepared SIM-NLCs
2.3.1. Determination of the entrapment efficiency
NLCs containing SIM were separated from the free drug via dial-
ysis. The drug was then released from the NLCs samples by addition
of sufficient volume of methanol. The drug content was assessed
spectrophotometrically at 238 nm using UV–vis spectrophotome-
ter (Shimadzu Seisakusho, Ltd., Kyoto, Japan). The entrapment
efficiency (EE, %) was then calculated according to Eq. (1):
× 100 (1)
2.3.2. Particle size and zeta potential
Mean particle size (nm) and polydispersity index (PDI) of the
prepared formulations were measured by dynamic light scattering
using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire,
UK) equipped with a backscattered light detector operating at 173◦ .
The zeta potentials (mV) were measured by laser Doppler anemom-
etry using the same Malvern Zetasizer Nanoseries instrument. All
samples were diluted ten times with double distilled water prior
to measurements. All the measurements were performed at 25 ◦ C
in triplicates.
2.3.3. In vitro release study
In vitro release of SIM from the NLCs was studied under sink
conditions using the dialysis method [5]. The dialysis bag (molec-
ular weight cut off: 12–14 kDa) was soaked in distilled water for
12 h before use. Nanocarriers (equivalent to 1 mg of the drug) were
placed into the dialysis bag and immersed in 100 mL of phosphate-
buffered saline (PBS, pH 7.4) in a beaker. The beaker was placed
in a thermostatic shaker (Gesellschaft für Labortechnik mbH, Burg-
wedel, Germany) at 37 ◦ C and 100 rpm. Five milliliter aliquots were
withdrawn at pre-determined time intervals (0.5, 1, 2, 4, 6, 8 and
24 h), and replaced immediately with equal volumes of fresh PBS to
maintain the same volume. The release of free SIM (SIM suspended
in PBS) was examined as a control. The withdrawn aliquot at each
time interval was analyzed for the amount of SIM spectrophoto-
metrically at a wavelength of 238 nm. The cumulative amount of
drug release was calculated as a function of time [27]. The release
kinetics were analyzed by curve fitting to different kinetic models
of zero order, first order, Higuchi and Korsemeyer-Peppas model
[28–30].
2.3.4. Stability studies
Samples of the prepared formulations were stored in sealed
glass vials at room temperature or at 4 ◦ C for 30 days. Samples from
each batch were withdrawn at definite time intervals and evaluated
for physical appearance, particle size and zeta potential. In addition,
further experiments were performed to study the changes of parti-
cle characteristics (size, PDI, zeta potential) for the selected optimal
formulation (SIM-NLCs, F4) in media of different pHs (1.2, 6.8, 7 and
7.4). The particle characteristics were assessed immediately after
mixing with the buffers (t0 ) and 2 h after the incubation (t2 ).
2.3.5. The particle morphology
Morphology of the formed particles was studied by transmis-
sion electron microscopy (TEM). Samples were prepared by placing
a drop of SIM-NLCs onto a 400-mesh copper grid coated with a car-
bon film, and left for air drying. TEM images were then observed at
an acceleration voltage of 80 kV by TEM (JEM 100 CX11, Japan). The
surface morphology of SIM-NLCs was visualized by scanning elec-
238 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
tron microscopy (SEM, JSM-5400 LV; JEOL, Japan). Samples were
prepared by placing a droplet onto an aluminum specimen stub,
dried overnight, and sputter-coated with gold prior to imaging. An
acceleration voltage of 15 kV was utilized for SEM visualization.
2.3.6. Fourier transform-infrared spectroscopy
Prior to characterizations by the Fourier transform-infrared
(FT-IR) spectroscopy, differential scanning calorimetry and x-ray
diffractometry, SIM-NLCs were frozen at – 80 ◦ C overnight prior
to the lyophilization process. Freeze-drying was performed using
Alpha 2–4 LD plus freeze-dryer (Martin Christ GmbH, Osterode,
Germany) and the operating conditions were set at a temperature
of − 60 ◦ C and a pressure of 0.011 mbar. The FT-IR spectra of the
selected formulations (F4 and F6), corresponding physical mix-
tures and the individual solid components were recorded using
FT-IR spectrophotometer (IR-470; Shimadzu, Kyoto, Japan). Sam-
ples were mixed with potassium bromide (spectroscopic grade)
and compressed into disks using hydraulic press before scanning
from 4000 to 500 cm−1 .
2.3.7. Differential scanning calorimetry
The thermal behaviors of the selected formulations (F4 and F6),
corresponding physical mixtures and the individual solid com-
ponents were investigated by differential scanning calorimetry
(DSC-60; Shimadzu Corporation, Tokyo, Japan). Samples of 4–7 mg
were sealed in aluminum pans and heated over a temperature range
of 40–200 ◦ C at a constant rate of 10 ◦ C/min. Thermal analysis data
were recorded using a TA 50I PC system with Shimadzu software
programs. Indium standard was used to calibrate the DSC temper-
ature and enthalpy scale. N2 was used as purging gas at a rate of
50 mL/min. An empty sealed aluminum pan was used as a reference.
2.3.8. X-ray diffraction
X-ray diffraction (XRD) patterns were studied to identify the
crystal form of SIM dispersed in the lipid matrix. XRD patterns of the
selected formulations (F4 and F6), corresponding physical mixtures
and the individual solid components were recorded by Philips X-ray
diffractometer model PW 1710 (Philips, Amsterdam, Netherlands)
with CuK ␣ radiation ( = 1.5405 Å), at a voltage of 40 kV and 30 mA
current. Samples were scanned at room temperature from 2␪ = 5◦
to 2␪ = 50◦ with a step of 0.06◦ /min.
2.4. In vivo studies
In vivo experiments were carried out to study the pharmaco-
dynamics and pharmacokinetics of SIM-NLCs as compared to SIM
suspension after oral administration in rats.
2.4.1. Experimental animals
The research protocol was reviewed and approved by the Insti-
tutional Animal Ethical Committee of the Faculty of Pharmacy,
Assiut University, and it adheres to the Guide for the Care and Use of
Laboratory Animals, 8th Edition, National Academies Press, Wash-
ington, DC. Male Wister rats (200–250 g) were obtained from the
Animal House at the Assiut University Faculty of Medicine. Rats
were allowed to acclimatize to the experimental conditions of tem-
perature and humidity one week prior to the experiments. The rats
were fed a standard rat pellet diet and allowed free access to water.
The rats were maintained at a temperature of 25 ± 2 ◦ C with a 12 h
dark/light cycles throughout the study.
2.4.2. Pharmacodynamic study of SIM-NLCs
The anti-cholesterolemic effects of different SIM-NLCs and SIM
suspension were studied using poloxamer-induced hyperlipidemic
rat model [31,32]. Poloxamer is a non-ionic surfactant that induces
hyperlipidemia via indirect stimulation of HMG-CoA reductase
enzyme, which is involved in cholesterol biosynthesis [19–21].
Twenty rats were divided into five groups, four animals in each
group, namely, normal group receiving normal saline, hyperlipi-
demic control group receiving water, standard group receiving
free drug in a form of suspension, and two test groups receiving
the selected formulations (F4 and F6). The drug suspension was
prepared by suspending 10 mg of SIM in 10 mL of distilled water
containing 0.5% w/v sodium carboxymethyl cellulose. The rats were
fasted overnight prior to the study with free access to water. Hyper-
lipidemia was induced by a single intraperitoneal injection of 1 g/kg
poloxamer F-127 solution (20% w/v). The rats were orally dosed
with multiple doses of drug suspension and formulations after
12 h of the poloxamer injection for three days (dose = 5 mg/kg/day).
Blood samples were withdrawn at 0, 12, 36 and 60 h.
For further investigation of the sustained antihyperlipidemic
activity of SIM-NLCs in comparison to SIM-suspension, pharmaco-
dynamic study during the first 24 h after single oral administration
of the different treatments was also examined. Eight rats were
divided into two groups (four rats in each group) for the oral admin-
istration of a single dose of the selected SIM-NLCs to one group and
the drug suspension to the second group (dose = 5 mg/kg). Multi-
ple blood samples were collected before drug administration and
2-, 6-, 12- and 24 h following drug administration. Blood samples
were allowed to clot for 15–30 min at room temperature, and serum
was separated by centrifugation at 3000 rpm for 10 min (Sigma
Laborzentrifugen GmbH, Osterode am Harz, Germany). The serum
samples were then frozen at − 20 ◦ C and stored for further analysis
[33].
Blood serum was analyzed for total cholesterol (TC) and high
density lipoprotein cholesterol (HDL-C) levels. TC levels were
determined by enzymatic, colorimetric assay, and HDL-C levels
were measured by homogeneous enzymatic colorimetric assay
on Roche/Hitachi cobas c 311 analyzer (Roche Ltd. Mannheim,
Germany). The non-HDL-cholesterol (non-HDL-C) concentrations
(i.e. cholesterol concentrations in the lower density lipoproteins
(chylomicrons, low and very low density lipoproteins)) were cal-
culated by subtracting HDL-C from the TC. Moreover, the% of initial
TC level and% of initial non-HDL-C level were also calculated at each
time point according to Eqs. (2) and (3):
Measured TC level
% of initial TC level = × 100 (2)
Baseline TC level
Measured non − HDL − C level
% of initial non − HDL − C level = × 100 (3)
Baseline non − HDL − C level
2.4.3. Pharmacokinetic study
Pharmacokinetic study was performed to investigate the ability
of NLCs to improve SIM oral bioavailability. The rats were randomly
divided into two groups (three animals per group) and received a
single oral dose of SIM (20 mg/kg) in the form of free drug sus-
pension for the first group and SIM-NLCs for the second group.
Although this dose is higher than the clinically used dose, phar-
macokinetic studies in rodents indicate that higher statin doses
(as compared to human) are required to achieve similar effec-
tive concentrations [13,14]. Hence, we have selected the dose of
20 mg/kg/day, which is consistent with doses that have been used
in other reported pharmacokinetic studies for SIM [15]. Also, a
higher dose (20 mg/kg) was used in the pharmacokinetic studies,
as compared to the pharmacodynamic studies (5 mg/kg), to allow
for the detection of low concentrations of the drug in the plasma
(on the nanogram scale). All formulations were freshly prepared.
All rats were fasted overnight prior to the administration of the
formulations and were fed again 4 h after treatments. Blood sam-
ples (0.5 mL) were obtained via vein puncture from the caudal vein
at designated time points (1, 2, 4, 8, 12 and 24 h after adminis-
tration) and were transferred into heparinized tubes. The plasma
 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
samples were separated by centrifugation at 3000 rpm for 10 min.
The plasma samples were then stored frozen at −20 ◦ C for further
analysis.
The SIM concentrations in plasma were analyzed using a
reversed phase-HPLC as described previously, with some modifi-
cations [34]. The HPLC method was performed on a Dionex Ultimat
3000 UHPLC system (Thermo Scientific, Waltham, MA) equipped
with a HPG-3200 RS pump, a DAD-3000 RS detector, a WPS-
3000TRS analytical autosampler, and a Hypersil BDS C18 analytical
column (dimensions of 150 mm × 4.6 mm ID × 5 ␮m). The mobile
phase composed of acetonitrile:deionized water (65:35 v/v) and
was adjusted to pH 3.5 by phosphoric acid. The flow rate was set
at 1.0 mL/min, the injection volume was 20 ␮L and elute was ana-
lyzed with DAD detector set at a wavelength of 239 nm. Acetonitrile
(1 mL) was added to the plasma sample (200 ␮L) to precipitate the
plasma proteins, and the samples were then vortexed and cen-
trifuged at 4000 rpm for 15 min [35]. The supernatant was collected,
filtered through 0.45 ␮m syringe filter (Millipore, Billerica, MA),
and analyzed using the HPLC. A calibration curve of SIM in the
plasma was constructed using blank plasma spiked with standard
SIM solutions to obtain a concentration range of 0.01–10 ␮g/mL.
The spiked plasma samples were then subjected to the same extrac-
tion procedure described previously.
The pharmacokinetic parameters were calculated by fitting the
plasma concentration–time data to a suitable model using Win
Nonlin Professional Edition software version 2.0 (Science Consult-
ing, Apex, NC). The area under the plasma concentration-time curve
from zero to infinity (AUC0-∞ ) was calculated using the trapezoidal
rule method. The maximum concentration (Cmax ), time to reach
the maximum concentration (Tmax ), elimination half-life (t½ ), elim-
ination rate constant (Kel ), mean residence time (MRT), clearance
(CL) and volume of distribution (Vd ) were calculated. The relative
bioavailability (Fr , %) of SIM-NLCs after oral administration was
calculated according to Eq. (4):
(AUC)SIM−NLCs
Relative bioavailability (Fr, %) = × 100
(AUC)SIM suspension
2.5. Statistical analysis
Experiments were performed in triplicates unless otherwise
indicated. Statistical significance for differences between exper-
imental groups was assessed by one-way analysis of variance
or two-sided Student’s t-test for pairwise comparison (Graph-
Pad Prism 6.0, GraphPad, San Diego, CA). Differences between
means were considered statistically non-significant for p values
>0.05. The differences were considered as statistically significant
for 0.05> p ≥ 0.01, and highly significant for p < 0.01.
3. Results and discussion
3.1. Preparation and characterizations of the SIM-NLCs
SIM-NLCs were prepared via emulsification-solvent evapora-
tion technique followed by ultrasonication. The effects of oleic
acid-to-stearic acid ratio and Pluronic F-68 concentration were
evaluated for further optimization of the formulations (Supplemen-
tary Information, Table S1). Since oleic acid concentration affects
the viscosity inside NLCs, different formulations were prepared
using constant Pluronic F-68 concentration (1.5% w/v) and various
oleic acid-to-stearic acid ratios (15, 30, 50, 70 and 80 wt% of oleic
acid/total lipid), and coded as F1, F2, F3, F4 and F5, respectively.
On the other hand, the surfactant at the aqueous/organic interface
governs the effectiveness of the emulsification process and sta-
bilization of the oil nanodroplets during the solvent evaporation
process. Therefore, Pluronic F-68 was used at different concentra-
239
tions of 1.5, 1, 3 and 5% w/v (coded as F4, F6, F7 and F8, respectively)
while maintaining the oleic acid-to-stearic acid ratio constant. Eval-
uation of the prepared NLCs was carried out by measuring the
particle size, PDI, zeta potential and entrapment efficiency.
Table S2 (Supplementary Information) shows the particle size,
PDI and zeta potential of the freshly prepared NLCs at different oleic
acid and surfactant concentrations. All formulations were in the
nanosized range, and demonstrated a highly significant decrease
(p < 0.001) in both the particle size and PDI upon increasing oleic
acid concentration from 15% to 70%. The decrease in size is due
to the difference in viscosity between the liquid lipid (oleic acid)
and solid lipid (stearic acid) [19,36]. The high oleic acid content
reduced the viscosity inside the NLCs, and, thus reducing the inter-
facial tension and afford the formation of smaller particles with
homogeneous size distribution and smooth surface [36,37]. On the
contrary, there was an increase in particle size upon increasing the
concentration of solid lipid (stearic acid) due to the increased vis-
cosity and interfacial tension, which is consistent with previously
reported data [37,38]. The small particle size and high negative
zeta potential values of NLCs indicate the stability of the formed
colloidal dispersion. No change in the zeta potential values was
observed upon increasing the oleic acid content of nanoparticles,
which is consistent with previously reported data for clobetasol-
loaded NLCs [36].
Regarding the effect of surfactant concentration, highly sig-
nificant reduction (p < 0.001) in particle size and increase in the
zeta potential values (p < 0.001) were observed upon increasing the
surfactant concentration from 1 to 5% w/v (Supplementary informa-
tion, Table S2). The reduction of particle size might be attributed to
the decreased interfacial tension between the lipid matrix and the
aqueous phase upon increasing the surfactant concentration. The
nonionic nature of the surfactant (Pluronic F-68) provides steric
stabilization, and, thus preventing aggregation of the particles,
and consequently preventing the coalescence of the droplets and
preserving the physical stability of the lipid nanoparticles. Since
(4) Pluronic F-68 is nonionic, the increase of zeta potential values that
is observed upon increasing the surfactant concentration can be
attributed to the coating layers of the surfactant that shield the
negative surface charge of stearic acid [39,40]. It was observed that
there was no significant change in the PDI of NLCs upon increasing
surfactant concentration from 1 to 3% (p > 0.05). On the other hand,
further increase of surfactant concentration to 5% led to a highly
significant increase (p < 0.01) of PDI of NLCs from 0.18 to 0.30.
3.2. Entrapment efficiency
The effect of concentrations of oleic acid and Pluronic F-68 on
the EE of the drug in the prepared NLCs was also studied (Sup-
plementary information, Table S2). Increasing the concentration of
oleic acid from 15 to 80 wt% resulted in a highly significant increase
(p < 0.001) in the EE of the drug into the NLCs from 40.44 ± 2.7 to
78.40 ± 2.9%, respectively. The resulted improve in EE upon increas-
ing oleic acid content is in agreement with a previous study, which
reported that incorporation of liquid lipids into solid lipids led to a
massive crystal order disturbance that left enough space to accom-
modate drug molecules, thus leading to improved drug entrapment
efficiency [36]. On the other hand, no significant change (p > 0.05) in
EE was detected upon increasing surfactant concentration from 1 to
3%. Moreover, further increase of surfactant concentration to 5% led
to a highly significant decrease (p < 0.01) in EE from 75.77 ± 4.9 to
54.58 ± 3.7%, which might be attributed to the hydrophobic nature
of the drug that tends to diffuse out from the oil nanodroplets
and solubilize in the micelles formed in the aqueous phase upon
increasing the surfactant concentration [41]. Drug loading did not
240 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
Fig. 1. In vitro release profiles of simvastatin from nanostructured lipid carriers, as compared to the drug suspension: (A) Different oleic acid concentrations and (B) Different
surfactant (Pluronic F-68) concentrations. Data are presented as means ± SD (n = 3).
increase the size of NLCs, which is consistent with a previous report
[42].
3.3. In vitro drug release
The release profiles of SIM from the lipid nanocarriers of vari-
ous compositions were studied over 24 h (Fig. 1). The drug release
patterns demonstrated initial burst release followed by sustained
release at a constant rate over 24 h. The initial rapid release may
be attributed to the fast release of drug entrapped near the sur-
face of the nanoparticles. The sustained release of drug after the
initial burst release could be related to the lipophilic nature of SIM
that is entrapped deeply in the core matrix of the NLCs. SIM in
the core of the nanoparticles has a longer diffusion path to reach
the surface, as compared to the drug entrapped near the surface
[43]. Faster drug release patterns were observed upon increasing
the concentrations of oleic acid (Fig. 1A) and the surfactant con-
centration (Fig. 1B). This enhanced drug release from the NLCs
might be attributed to the significant decrease in particle size, and
thus increasing the specific surface area and, subsequently, the
release rate of the drug [36]. Table S3 (Supplementary informa-
tion) summarizes the release kinetics and correlation coefficients
(R2 ) calculated for the tested formulations. The in vitro release data
indicated that the release of SIM from NLC formulations followed
first order kinetics except for F1 (15 wt% OA) and F2 (30 wt% OA),
which were fitted to a diffusion–controlled mechanism (Higuchi
model). Korsmeyer-Peppas was utilized to provide further informa-
tion on drug release mechanisms, whether it is Fickian (diffusion),
non-Fickian (anomalous), or erosion-mediated (zero order) release.
The values of the diffusional release exponents for all formulations
were <0.5 except for F6 (1% w/v Pluronic F-68), which indicates that
Fickian mechanism is the dominant mechanism that controls the
drug release from the nanoparticles.
3.4. Stability studies
The stability of the various NLC formulations was monitored at
4 ◦ C and at room temperature for 30 days to assess their long-term
stability (Supplementary Information, Table S4). The visual obser-
vation of all the prepared NLCs did not indicate any sign of gelation,
creaming, color change or particle aggregation either at 4 ◦ C or at
room temperature. After 4 weeks, it could be noticed that there
were no significant differences (p > 0.05) in either particle size or
zeta potential values for all the formulations except for F5, F7 and
F8, which had a significant increase (p < 0.001) in the mean particle
size after 30 days of storage. Among all NLC formulations, formula-
tions F4 and F6 were selected for further investigations due to their
small particle sizes, higher entrapment efficiencies, slower in vitro
cumulative drug release, higher stability upon storage, and their
lower surfactant contents. Moreover, neither F4 nor F6 exhibited
any significant change (p > 0.05) in the EE after 30 days of stor-
age either at 4 ◦ C (F4: 70.64 ± 7.6%, F6: 66.30 ± 8.2%) or at room
temperature (F4: 68.62 ± 6.61%, F6: 51.30 ± 15%).
The effect of pH on the particle size, PDI and zeta potential was
also studied (Supplementary information, Tables S5 and S6). At low
pH, there was no significant change (p > 0.05) in size and PDI of the
NLCs in comparison to the original sample (NLCs in water), which
may be attributed to the steric stabilization effect of the nonionic
surfactant (Pluronic F-68) [12]. At pH 7.4, there was a slight increase
in particle size, which is consistent with earlier studies [12,13]. The
zeta potential of the SIM-NLCs reached 1.27 ± 1.67 mV when the pH
was decreased to 1.2, which might be attributed to protonation of
the anionic groups of lecithin and oleic acid, thereby reducing the
net negative charge [14].
3.5. Morphology of the SIM-NLCs
Fig. 2A shows the transmission electron micrograph of SIM-
NLCs. The lipid nanocarriers appeared as spherical or elliptical
vesicles with smooth surfaces. There were several dark spots on
the spherical structures of the NLCs, which might be attributed to
the liquid lipids sticking on the surface of the lipid nanoparticles
[44,45]. The average particle size was ca. 169.4 ± 33.9 nm (n = 20),
which is in agreement with the DLS results (Fig. 2C). Scanning elec-
tron micrograph clearly demonstrates the spherical shape of the
prepared NLCs with characteristic smooth surface (Fig. 2B).
3.6. Fourier transform-infrared spectroscopy
The FT-IR spectra of the pure drug, the selected formulations (F4
and F6), physical mixtures, and the individual solid components,
were recorded (Fig. 3). The spectrum of pure SIM drug showed
characteristic peaks at 3012 cm−1 (C CH), 1698.9 cm−1 (aromatic
C O), 3551.4 cm−1 (aliphatic O-H) and 1466.7 cm−1 (benzene ring).
 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245 241

Fig. 2. Transmission electron micrograph (A), scanning electron micrograph (B) and Intensity-, volume- and number-averaged hydrodynamic diameter histograms of
the SIM-NLCs (F4) (C). The intensity-, volume- and number-averaged hydrodynamic diameters of SIM-NLCs (F4) demonstrated on Fig. 2C are 185.2, 175.9 and 120.9 nm,
respectively.

peared, shifted or replaced by the peak of stearic acid, and thus

indicating the drug entrapment in the lipid matrix [47].

3.7. Differential scanning calorimetry

DSC thermograms of the pure drug, the selected formula-

Fig. 3. FT-IR spectra of the pure drug (SIM) (A), stearic acid (B), Pluronic F-68 (C),
physical mixture (SIM/stearic acid) (D), physical mixture (SIM/Pluronic F-68) (E), F4
(F) and F6 (G).
This pattern is in agreement with previously recorded spectrum
for the same drug [46]. The characteristic peaks of the drug were
also observed in the FT-IR spectrum of the physical mixture of pure
drug/Pluronic F-68 due to the absence of predominant chemical
interactions between the drug and the surfactant. However, in the
spectrum of SIM-NLCs, the characteristic peaks of the drug disap-
tions (F4 and F6), physical mixtures, and solid components, were
recorded (Fig. 4I). The thermogram of the pure SIM showed a sharp
single melting peak at ca. 138.5 ◦ C, which corresponds to the melt-
ing of the crystalline drug [7,11,34]. In the physical mixture of SIM
and stearic acid, the characteristic peak of the drug disappeared
with slight increase in the onset temperature of the stearic acid
(62.2 ◦ C). The disappearance of the drug characteristic peak can
be ascribed to solubilization of the drug within the lipid matrix
[47,48]. DSC thermograms of F4 and F6 showed a decrease in the
onset of melting peak of the used lipid and disappearance of the
characteristic melting peak of the drug, which may be attributed
to drug solubilization within the lipid matrix in amorphous and/or
molecularly dispersed form [49]. On the other hand, the decrease
in the melting temperature of the used lipid might be due to the
interactions between the solid lipid and the liquid lipid and the sur-
factant. Also, it may be attributed to the small particle size of the
prepared NLCs, as a result of the kelvin effect, which is described by
the Thomson equation. According to this equation, small isolated
242 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
Fig. 4. DSC analysis (I) and X-ray diffraction patterns (II) of the pure drug (SIM) (A),
stearic acid (B), Pluronic F-68 (C), physical mixture (SIM/stearic acid) (D), physical
mixture (SIM/Pluronic F-68) (E), F4 (F) and F6 (G).
particles (SIM-NLCs) would melt at a lower temperature than the
bulk material (stearic acid) [38].
3.8. X-ray diffraction
X-ray diffractograms of the pure drug, F4, F6, physical mixtures,
and the individual solid components are presented in Fig. 4II. The
crystalline nature of pure SIM was demonstrated upon detection
of the diffraction peaks in the XDR pattern. These characteristic
diffraction peaks of the pure drug were also detected on the XDR
pattern of the physical mixtures, however, they disappeared in the
diffractogram of SIM-NLCs, which clearly confirm that SIM did not
crystallize in the lipid matrix. The crystalline nature of the pure
lipid and surfactant was also confirmed by the diffraction peaks
observed in the XDR. However the disappearance of the character-
istic peaks of the surfactant on diffractogram of SIM-NLCs indicates
that the crystallinity of Pluronic F-68 is lost when it is adsorbed on
the stearic acid nanoparticles, due to the change in the surfactant
conformation [47].
3.9. Pharmacodynamic study of SIM-NLCs
Hypercholesterolemia is usually managed by lowering the
serum concentration of TC and low density lipoprotein choles-
terol (LDL-C) or non-HDL-C. Non-HDL-C contains more atherogenic
cholesterol than LDL-C, and is considered as a more accurate
measurement of the total amount of atherogenic particles in the
circulation [50]. Moreover, recent investigations indicate that non-
HDL-C ≥ LDL-C is a predictor of coronary artery diseases [51]. SIM is
a potent lipid-lowering drug that suppresses the formation of bad
cholesterol in the liver through inhibiting the HMG-CoA reductase
enzyme, thus resulting in substantial decrease in mortality from
cardiovascular diseases. For statins, the pharmacokinetic param-
eters are not sufficient to predict the pharmacodynamic activity
[52]. This issue has been adequately discussed in a previous study
which reported that plasma concentrations of atorvastatin did not
correlate with the reduction in LDL-C [25]. Therefore, the pharma-
codynamics and pharmacokinetics of the selected SIM-NLCs and
the drug suspension were evaluated.
After 12h of poloxamer injection into the rats, there was ca. 5-
fold increase in the TC level of the hyperlipidemic control group
and the different treatment groups as compared to normal group
(data not shown). The rats were orally dosed with multiple doses
of drug suspension and formulations after 12 h of the poloxamer
injection for three days (dose = 5 mg/kg/day). Percentages of the
initial (before drug administration) TC and non-HDL-C levels were
estimated to evaluate the efficacy of the treatment in each group.
Percentages of initial TC and non-HDL-C levels after oral admin-
istration of the SIM-NLCs (F4 and F6) and SIM suspension are
presented on Fig. 5I. The TC level after 12 h was significantly higher
(p < 0.001) in the control group as compared to the treated groups,
which indicate the ability of the SIM-NLCs and the drug suspension
to prevent the increase in TC levels (Fig. 5IA). After 36 h of the treat-
ment, there was a further increase in the TC levels of the control
rats, and rats treated with the SIM suspension and SIM-NLCs-F6.
Interestingly, in the case of F4 formulation, the TC significantly
decreased (p < 0.001). After 60h of the treatment, F4 reduced the
TC level to a highly significant level (p < 0.01), as compared to F6
and the drug suspension. Similar significant decrease (p < 0.001)
in the non-HDL-C level was also observed for F4-treated group, as
compared to F6 and the SIM suspension-treated groups (Fig. 5IB).
The greater lipid lowering activity of the F4 (1.5% w/v Pluronic F-
68) as compared to F6 (1% w/v Pluronic F-68) might be attributed
to the higher Pluronic F-68 concentration of F4. The presence of
Pluronic F-68 might enhance the absorption of NLCs, due to its
effect on the intestinal epithelial permeability [53]. Furthermore,
F4 has smaller particle size that might increase the absorption and
dissolution rate, and thus improving the drug bioavailability [34].
For further investigation of the sustained antihyperlipidemic
effect of the SIM-NLCs in comparison to the SIM suspension, single
dose pharmacodynamic study was evaluated and the percentages
of the initial TC and non-HDL-C levels were measured (Fig. 5II).
After 2 h of the treatment, the TC and non-HDL-C levels were sig-
nificantly higher (p < 0.001) in the SIM suspension-treated group,
as compared to the SIM-NLCs-treated group (F4). Moreover, the
enhanced efficacy of the F4 remained detectable during the 24 h of
the treatment period. On the contrary, SIM suspension resulted in
a detectable increase in TC and non-HDL-C levels above the base-
line level, and this increase was continued during the 24 h of the
treatment. The sustained antihyperlipidemic activity of the SIM-
NLCs might lower the dose and frequency of administration, and
thus improving patient tolerability and decreasing side effects of
the drug.
3.10. Pharmacokinetic study
Selected SIM-NLCs (F4) and SIM suspension were orally admin-
istrated as a single dose to male Wistar rats. SIM plasma
concentrations were measured using a previously reported HPLC
method, with some modifications [34]. SIM was completely sep-
arated as a sharp peak at a retention time of ca. 8.2 min without
any interfering peaks. SIM plasma concentrations after oral admin-
istration of SIM-NLCs and the drug suspension are presented on
Fig. 6. The plasma concentration-time data were subjected to a non-
compartmental analysis to determine the mean pharmacokinetic
parameters (Supplementary information, Table S7).
The SIM plasma concentrations were remarkably higher after
administration of SIM-NLCs, as compared to those observed for
SIM suspension. The Cmax for SIM-NLCs was significant higher than
 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245 243

Fig. 5. (I) Changes in cholesterol levels in rats after administration of simvastatin suspension and simvastatin-nanostructured lipid carriers (F4 and F6) over 3 days. (II)
Changes in the cholesterol levels in rats after single dose administration of simvastatin suspension and simvastatin-nanostructured lipid carriers (F4). (A) Total cholesterol
and (B) Non-high density lipoprotein-cholesterol. *p < 0.05 when compared to the drug suspension; **p < 0.001 when compared to the drug suspension. Data are presented
as means ± SD (n = 4).

the SIM suspension (89.86 ± 4.11 vs. 45.73 ± 12.15 ng/mL, p < 0.05).
After 24 h of oral administration of SIM–NLCs, the SIM plasma
concentration was 34.54 ± 1.52 ng/mL, whereas SIM suspension
resulted in a plasma concentration of 9.71 ± 1.55 ng/mL. The higher
plasma concentrations observed following the oral administra-
tion of SIM-NLCs as compared to the drug suspension reflect the
enhanced systemic absorption of SIM when incorporated into the
NLCs. Similar enhancement of systemic absorption of the same drug
was previously reported using other drug delivery systems such as
solid lipid nanocarriers [7] and self microemulsifying drug deliv-
ery system [54]. In the case of SIM-NLCs, the pre-systemic hepatic
effect can be largely avoided via absorption through the lymphatic
pathway [7]. Improved solubilization of SIM due to the transforma-
tion from crystalline to amorphous form is another reason for the
increased systemic absorption [55]. The other factors that facilitate
the absorption in the intestinal milieu are the high dispersibility of
244 H.A. Fathi et al. / Colloids and Surfaces B: Biointerfaces 162 (2018) 236–245
Fig. 6. Plasma concentration–time profiles of SIM after single oral dose (20 mg/kg)
of SIM suspensions and SIM-NLCs. Values are presented as means ± SD (n=3).
the NLCs and small particle size of the NLC formulation (<200 nm)
that resulted in a great increase in the surface area of the particles.
Furthermore, NLCs could protect the drug from enzymatic degrada-
tion by reducing the exposure of the drug to the intestinal bacteria,
as well as, protecting the drug from the enzymatic degradation dur-
ing the absorption process, and thus allowing for a longer residence
time in the gastrointestinal tract [56]. In general, improved phar-
macokinetic profile was observed when SIM was loaded into the
NLCs (Supplementary Information, Table S7). The SIM-NLCs exhib-
ited a delayed Tmax (2 h), as compared to that of SIM suspension
(1 h), which could be attributed to the slow and sustained release
of the drug from the NLCs. Longer elimination half-life of SIM-NLCs
(35.04 ± 2.86 h) was also observed as compared to the SIM sus-
pension (10.20 ± 0.47 h), which might aid in reducing the dose and
frequency of administration. The extent of SIM absorption from NLC
(AUC0-∞ ) was significantly higher (p < 0.001) than the AUC0-∞ of the
SIM suspension.
The oral bioavailability of SIM after its incorporation into
solid lipid nanoparticles was improved by 3.37-fold [5]. Similarly,
increase in SIM oral bioavailability from self microemulsifying drug
delivery system (1.5-fold increase) [54] and nanosuspension (2.2-
fold increase) [13] have been also reported previously. Our group
has utilized various strategies to improve stability and delivery effi-
ciency of several drugs [14,57–61]. In the current study, the relative
bioavailability of SIM-NLCs was 4-fold higher than for the drug sus-
pension, indicating significant enhancement of oral delivery and
bioavailability of SIM when incorporated into the NLCs. The rate and
extent of systemic absorption of SIM were significantly increased,
and thus improving the oral bioavailability of SIM. It is worth men-
tioning that no mortalities have been observed in the treatment
group and the hyperlipidemic control group in the in vivo experi-
ments.
4. Conclusions
In the present study, SIM-NLCs were successfully prepared and
evaluated for their abilities to enhance oral delivery of SIM as com-
pared to the drug suspension. Several in vitro characterizations of
SIM-NLCs were carried out. SIM-NLCs of small particle size, high
entrapment efficiency, sustained in vitro drug release, high stability
and low surfactant concentration were selected for further charac-
terizations. The in vivo pharmacodynamic study of the SIM-NLCs
demonstrated a sustained and greater lipid lowering activity as
compared to the free drug suspension. There was a 4-fold increase
in the oral bioavailability of SIM-NLCs in comparison to the SIM sus-
pension. The developed NLCs might provide efficient nanodevices
to enhance the oral delivery and prolong the therapeutic effect of
SIM, and thus improving the patient compliance by eliminating the
need for frequent dosing of the drug.
Conflicts of interest
The authors report no conflicts of interest in this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.colsurfb.2017.11.
064.
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