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Research Article
Chitosan Nanoparticles of Gamma-Oryzanol: Formulation, Optimization,
and In vivo Evaluation of Anti-hyperlipidemic Activity
Tejal Rawal,
1
Neha Mishra,
1
Abhishek Jha,
1
Apurva Bhatt,
1
Rajeev K. Tyagi,
2,3
Shital Panchal,
1
and Shital Butani
1,4
Received 25 December 2017; accepted 19 March 2018
Abstract. The elevated blood levels of cholesterol and low-density lipoproteins result in
hyperlipidemia. The available expensive prophylactic treatments are kindred with severe side
effects. Therefore, we fabricated the polymeric nanoparticles of gamma-oryzanol to achieving
the improved efficacy of drug. The nanoparticles were prepared by ionic gelation method and
optimized using 2
3
full factorial design taking drug/polymer ratio (X
1
), polymer/cross linking
agent ratio (X
2
), and stirring speed (X
3
) as independent variables. The average particle size,
percentage entrapment efficiency, and in vitro drug release at 2, 12, and 24 h were selected as
response parameters. The factorial batches were statistically analyzed and optimized. The
optimized nanoparticles were characterized with respect to particle size (141 nm) and zeta
potential (+ 6.45 mV). Results obtained with the prepared and characterized formulation
showed 83% mucoadhesion towards the intestinal mucosa. The in vitro findings were
complemented well by in vivo anti-hyperlipidemic activity of developed formulation carried
out in Swiss albino mouse model. The in vivo studies showed improved atherogenic index,
malondialdehyde, and superoxide dismutase levels in poloxamer-407-induced hyperlipidemic
animals when treated with oryzanol and gamma-oryzanol nanoformulation. Based on our
findings, we believe that chitosan-mediated delivery of gamma-oryzanol nanoparticles might
prove better in terms of anti-hyperlipidemic therapeutics.
KEY WORDS: gamma-oryzanol; chitosan; nanoparticles; poloxamer-407; atorvastatin.
INTRODUCTION
The changes in the lifestyle lead to the prevalence of
hyperlipidemia. The hyperlipidemia is the presence of
abnormal levels of lipids in blood and marked by an elevated
cholesterol low-density lipoprotein (LDL) and low or unal-
tered high-density lipoproteins (HDL) levels in the blood [1].
Hyperlipidemia is treated by various classes of marketed
drugs, but the expensive treatment and side effects associated
with the long-term treatment are the major pitfalls [2].
Therefore, safe and cost-effective formulations are inevitably
required, and molecules which are generally recognized as
safe (GRAS) with no side effects might be good alternatives
to allopathic treatments [3]. The rice bran oil is considered as
a healthy vegetable oil [4], and gamma-oryzanol is a mixture
of ferulic acid esters of phytosterol and tri-terpene alcohols.
This is a group of compounds present in the rice bran oil
comprising anti-oxidant, anti-hyperlipidemic, immuno-modu-
latory, and anti-cancer activities [5,6]. The gamma-oryzanol
has low bioavailability majorly due to poor aqueous solubility
[7]. Moreover, poor physicochemical nature is one of the
reasons for the failure of many drugs to enter the clinics [8].
There has been lot of work being done for the
development of novel drug delivery systems (NDDS) which
are seen better in terms of targeting, improved bioavailabil-
ity, and reduced adverse effects over the conventional
therapy [9]. Owing to the poor water solubility of gamma-
oryzanol, Ghaderi et al. [10] prepared ethyl cellulose-based
gamma-oryzanol nanoparticles in order to improvise the
stability and functional properties of liquid foodstuffs
containing gamma-oryzanol. Seetapan et al. [11] fabricated
gamma-oryzanol solid lipid nanoparticles and assessed their
rheological behavior under different temperature conditions.
Many researchers are working on the development of
different formulations of gamma-oryzanol. Viriyaroj A.
et al. [12] developed gamma-oryzanol liposomes composed
of phosphatidylcholine and cholesterol and investigated
their physicochemical properties and antioxidant activity
for the cosmetic applications. The niosomal formulation of
1
Department of Pharmaceutical Technology, Institute of Pharmacy,
Nirma University, Ahmedabad, Gujarat 382481, India.
2
Institute of Science, Nirma University, Ahmedabad, Guajrat 382481,
India.
3
Department of Periodontics, College of Dental Medicine, Georgia
Regents University, Augusta, Georgia 30912, USA.
4
To whom correspondence should be addressed. (e–mail:
shital.butani@nirmauni.ac.in)
AAPS PharmSciTech ( #2018)
DOI: 10.1208/s12249-018-1001-8
1530-9932/18/0000-0001/0 #2018 American Association of Pharmaceutical Scientists
gamma-oryzanol by Manosroi et al. [13] was prepared using
cholesterol and Tween 61 to enhance the transdermal
absorption. Sapino et al. [14] incorporated gamma-
oryzanol in β-cyclodextrin nanosponges in order to enhance
its stability and effectiveness. The nanoparticulate systems
are advantageous with respect to their stability over other
systems [15]. The systems such as liposomes and niosomes
get easily cleared by the reticulo-endothelial systems which
reduce their bioavailability. Therefore, nanoparticulate
systems are reportedly seen to evade the reticulo-
endothelial systems for bettering the bioavailability of
gamma-oryzanol [16].
Chitosan is a natural polymer widely used due to its
salient features of biodegradability, biocompatibility, low
toxicity, anti-bacterial, and anti-microbial properties [17,
18]. The mucoadhesive properties of chitosan help in the
fabrication of nanoparticles due to its cationic behavior. The
negatively charged intestinal mucosa will manifest a greater
uptake for the oppositely charged chitosan nanoparticles.
The hydrophobic polymers may lead to fast elimination of
gamma-oryzanol by reticulo-endothelial system (RES), and
therefore the use of hydrophilic polymeric carriers might
serve as a boon in the effective delivery of the drug.
Therefore, the chitosan is chosen as suitable polymer
candidate for the fabrication of nanoparticles of gamma-
oryzanol in order to protect the molecule from the RES
system to increase its bioavailability to achieving good anti-
hyperlipidemic activity.
Present work encompasses the formulation of chitosan
nanoparticles of gamma-oryzanol to improve its bioavail-
ability and efficacy. The nanoparticles were freeze-dried in
powder form and evaluated for their anti-hyperlipidemic
activity in an animal model. In the end, potency of drug-
loaded chitosan nanoparticles was compared with plain
gamma-oryzanol and standard anti-hyperlipidemic drug,
atorvastatin.
MATERIALS AND METHODS
Materials
Sodium tripolyphosphate (TPP), Tween 80, sodium
sulfate, and sodium metabisulfite were procured from the
Central drug house, New Delhi. Glutaraldehyde was
obtained from S.D. Fine Chemical Ltd., Mumbai. Atorva-
statin tablets (5 mg) were procured from Troikaa Pharma-
ceuticals Limited, India. Gamma-oryzanol was purchased
from Tokyo Chemical Company. Poloxamer-407 (Pluronic
RF-127) was obtained from Sigma-Aldrich, USA. Diagnos-
tic kits for lipid profile were procured from Lab Care
Diagnostics Pvt. Ltd., India. Diagnostic kits for activated
partial thromboplastin time (APTT) and plasma prothrom-
bin time (PT) were obtained from Diagnostic Stago, France.
Chitosan (degree of deacetylation ratio of 75–85%) and
dialysis bags (MWCO 12–14 kDa) were obtained from
Himedia, Laboratory Pvt. Ltd., India. All other chemicals
and reagents used were of analytical grade.
Pre-formulation Studies
Drug-Excipient Compatibility Studies by Fourier Transform
Infrared Spectroscopy
The compatibility studies were carried out by FTIR
spectroscopy (Jasco FT-IR 6100, Japan). All the samples were
stored for 1 month at room temperature and analyzed further
for drug excipient compatibility. FTIR spectra of pure drug
and physical mixture of drug and excipient were studied by
making KBr mixture pellet. The absorption peaks of gamma-
oryzanol, excipients, and combination of gamma-oryzanol
and excipients were obtained at different wave numbers.
Formulation Development
Preparation of Chitosan Nanoparticles
The chitosan nanoparticles can be prepared by several
methods such as thermal/emulsion cross-linking, ionic gela-
tion, coacervation precipitation, and coalescence.The ionic
gelation method is the simplest and a viable one. Sodium
tripolyphosphate (TPP), a polyanion, interacts with positively
charged chitosan via electrostatic forces. This reversible
physical cross-linking via electrostatic interaction avoids the
toxicity of several reagents. The oppositely charged particles
undergo complexation and precipitated out to form spherical
particles [19]. The nanoparticles were formulated via ionic
gelation method, and chitosan was dissolved in an aqueous
solution containing acetic acid and Tween 80. TPP and drug
were dissolved in water and added dropwise into chitosan
solution with stirring. The nanoparticles were separated by
centrifugation at 15,000 rpm for 15 min at 10 °C. The particles
were suspended in phosphate buffer (PBS) pH 7.4 and
analyzed [20–23].
Preliminary Batches
Many preliminary batches were prepared to evaluate the
effect of various formulation parameters, including drug to
polymer ratio (1:1, 1:1.5, and 1:2), polymer to TPP ratio (2:1,
4:1, 6:1, and 8:1), concentration of acetic acid solution (1, 2, 4,
and 8%), concentration of Tween 80 (0.25, 0.5, and 0.75%),
as well as process parameters such as stirring speed (200, 400,
and 600 rpm), the size of syringe needle (18G, 22G) on the
particle size, entrapment efficiency, drug loading, in vitro drug
release, and permeation.
Optimization of Nanoparticles
A systematic optimization of the polymeric nanoparticles
is required to understand the interaction amongst various
parameters using suitable experimental designs. The data of
the preliminary trials was used to select the high-risk factors
for the optimization. Full factorial design (2
3
) was applied for
the optimization of various formulations using drug/polymer
ratio (X
1
), polymer/cross-linking agent ratio (X
2
), and stirring
speed (X
3
) as the independent variables and particle size, %
entrapment efficiency, and in vitro drug release at 2, 12, and
Rawal et al.
24 h were taken as dependent variables. Eight batches were
prepared and evaluated extensively (Table I). The design
expert 7.0.0 software (StatEase Inc., USA) was used for data
treatment and design space generation. The suspension of
selected batch was centrifuged (BIO LAB BL 150 R) at
10,000 rpm, and pellets were collected. The pellets were dried
using 20% w/vlactose as cryo-protectant using freeze-dryer
(DELVAC) for 24 h over the temperature range of −40 °C to
20 °C for 1 h. The freeze-dried powder was then sealed,
stored in a refrigerator, and evaluated.
Characterization of Nanoparticles
Particle Size Distribution
The average particle size of nanoparticles was deter-
mined by differential laser scanning (DLS microtrac software)
taking PBS pH 7.4 as background. The particle size of the
selected batch was measured using zetasizer (ZS 200,
Malvern Instruments, UK), and experiments were done in
triplicate. The average values were employed for the
calculation of response surfaces [24].
Entrapment Efficiency
The supernatant was collected at the final stage of
separation by centrifugation during the formulation of
nanoparticles. The supernatant was analyzed for the estima-
tion of the amount of free drug present using UV spectro-
photometer (UV 1800 Shimadzu, ScientificInstruments,
Japan) at 327 nm. Amount of the entrapped drug was
calculated using the following formula [25].
Entrapment efficiency %ðÞ¼
initial drug−free drugðÞ
initial drug 100 ð1Þ
In vitro Drug Release Studies
The drug release was measured using the method
reported elsewhere [26]withslightmodifications. The
nanoparticles equivalent to 100 mg of gamma-oryzanol were
placed in a dialysis bag (Himedia; molecular weight cutoff
12,000–14,000 Da) prepared using cellophane membrane
previously soaked in PBS pH 7.4 for 24 h. The dialysis bag
was kept in 100 ml of intestinal media (PBS pH 7.4) at 37 ±
0.5 °C with continuous stirring at 100 rpm. Subsequently, a
series of 2 ml solutions were withdrawn at specific time
intervals and replaced with the same volume of PBS (pH 7.4),
and absorbance was measured at 327 nm using UV-Visible
spectrophotometer. The drug release was also performed
using PBS in the presence of 20% ethanol to determine the
effect of pH and alcohol.
Measurement of Zeta Potential
The zeta potential of the selected batch was measured
using Malvern Zetasizer ZS 200 at 25 ± 0.5 °C. All the
samples were measured in triplicate. [27]
Morphological Analysis by Scanning Electron Microscopy
The surface morphological characteristics of gamma-
oryzanol-loaded chitosan nanoparticle batch were carried
out using a scanning electron microscope (SEM LEO 1530,
Oberkochen, Germany). The method used was plasma
deposition having a gold coating unit to make the sample
surface conductive to scanning electron beam. SEM of
samples was carried out at high vacuum with specimen
working distance of 6.3 mm and an accelerating voltage of
5kV[28].
Mucoadhesion Studies
Freshly cut, 2-cm-long piece of rat intestinal mucosa was
taken and tied onto the glass slide with the help of rubber
bands. Fifty milligrams of nanoparticles of the selected batch
was accurately weighed and transferred onto mucosa. It was
kept under controlled humidity for 10 min to allow the
anionic mucosa to interact with cationic chitosan. Then, the
slide was hanged at an angle of 45° under the burette tip and
Table I. Composition and Evaluation of the Optimization Batches
Batch no. X
1a
(drug/polymer)
X
2a
(polymer/TPP)
X
3a
(stirring speed)
PS (nm) PDI % EE % In vitro drug release
2h 12h 24h
B1 −1−1−1 216.0 ± 0.1 0.218 ± 0.02 43.5 ± 3.1 12.6 ± 1.1 40.2 ± 0.9 57.0 ± 3.1
B2 1 −1−1 168.4 ± 0.2 0.333 ± 0.03 26.0 ± 0.9 10.4 ± 2.3 38.4 ± 0.8 55.9 ± 0.6
B3 −11−1 180.2 ± 1.1 0.246 ± 0.01 72.5 ± 0.1 28 ± 0.1 68.0 ± 1.4 77.7 ± 0.9
B4 1 1 −1 186.7 ± 8.1 0.261 ± 0.01 68.2 ± 3.6 24 ± 0.1 67.8 ± 1.6 75.6 ± 1.6
B5 −1−1 1 141.6 ± 2.6 0.382 ± 0.02 65.5 ± 0.4 13.2 ± 1.2 43.4 ± 2.1 59.6 ± 5.1
B6 1 −1 1 99.10 ± 0.4 0.515 ± 0.04 39.5 ± 0.1 10.9 ± 0.4 39.7 ± 0.3 56.7 ± 2.3
B7 −1 1 1 317.0 ± 0.1 0.342 ± 0.01 75.4 ± 0.6 29.7 ± 0.3 69.4 ± 0.1 79.8 ± 0.1
B8 1 1 1 333.0 ± 1.2 0.521 ± 0.02 69.3 ± 1.1 26.0 ± 0.9 67.9 ± 0.5 76.8 ± 0.4
PS: Particle size, PDI: Polydispersity Index, % EE :% entrapment efficiency
a
Transformed values for X
1
,X
2
, and X
3
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
PBS (pH 7.4) was allowed to flow at the rate of 2 ml/min.
After 3 h, a number of nanoparticles which did not adhere to
the mucosal surface were collected on Whatman filter paper
and weighed. The percentage of mucoadhesion was calcu-
lated using following formula [28,29].
%Mucoadhesion ¼w1−w2ðÞ
w1100 ð2Þ
where
w1 weight of gamma-oryzanol-loaded chitosan nanopar-
ticles taken initially.
w2 weight of gamma-oryzanol-loaded chitosan nanopar-
ticles collected on filter paper.
Stability Studies of Formulation
The stability is defined as the extent to which a product
remains within specified limits throughout its period of
storage and use. The selected batch of nanosuspension and
freeze-dried formulation was stored as per ICH guidelines at
25° ±2 °C and 60 ± 5% RH and at refrigerated condition (5 ±
3 °C) in a closed vial for 3 months and characterized with
respect to particle size and entrapment efficiency [30].
Animal Studies
In vivo Anti-hyperlipidemic Activity
The animal protocol was approved by the Institutional
Animal Ethics Committee (Protocol no. IP/PCEU/MPH/14-1/
010). The Swiss albino male mice weighing 60–40 g were
obtained from the Institute of Pharmacy, Nirma University’s
animal house. All animals were treated in accordance with
the guidelines of the committee for CPCSEA.
Study Design
Swiss albino mice (n= 30) were randomized into follow-
ing groups:
(a) Normal control (NC): administered with saline for
3 days
(b) Normal induced (P-407): administered with saline for
3 days and on the 3rd day, poloxamer-407 (500 mg/
kg) was injected intraperitoneally
(c) Standard group (STD): administered with aqueous
suspension of atorvastatin (2 mg/kg/day, orally) for
all 3 days and on the 3rd day, animals were given
poloxamer-407 (500 mg/kg) intraperitoneally
(d) Gamma-oryzanol powder-treated group (OZ): admin-
istered with an aqueous suspension ofgamma-oryzanol
powder (100 mg/kg/day, orally in 0.5% CMC) for
3 days and on the 3rd day, animals were treated with
poloxamer-407 (500 mg/kg) intraperitoneally
(e) Gamma-oryzanol-loaded chitosan nanoparticle-
treated group (OZF): administered with an aqueous
suspension of drug-loaded freeze-dried powder of
nanoparticles (100 mg/kg/day, orally) for 3 days and
on the 3rd day, animals were treated with poloxamer-
407 (500 mg/kg, i.p.).
The blood was collected after 24 h of poloxamer-407
treatment, and animals were euthanized to extract the livers
[31–34].
Assessment of Various Biochemical Parameters
Determination of Body Weight
Hyperlipidemia is defined as the elevations in the
fasting total cholesterol levels in the body [1]. Hence, it
becomes imperative to determine the body weight to
determine anti-hyperlipidemic activity of formulation. The
body weights were determined before and after the therapy.
Lipid Profile
The lipid profile is to quantify the body lipids and
atherogenic index (AI) to define the cardiac risk [35]. The
increased lipid levels and AI represent the hyperlipidemic
conditions. It therefore becomes inevitable to study the
said effects in order to evaluate the anti-hyperlipidemic
activity. The blood was collected by puncture of retro-
orbital plexus of mice and allowed to stand for 10 min
without the addition of anti-coagulant followed by centri-
fugation at 4000 rpm for 10 min. The collected serum was
stored at 4 °C, and total cholesterol (TC), triglycerides
(TG), and high-density lipoprotein (HDL) were estimated
by procedure following the manufacturer’s recommenda-
tions (Lab Care Diagnostics, India). The very low-density
lipoprotein (VLDL) and low-density lipoprotein (LDL)
were determined using Friedewald’s formula.
VLDL−cholesterol ¼TG
5ð3Þ
LDL−C¼TC−HDL−VLDLðÞ ð4Þ
In the AI, HDL ratio was measured using the following
equation:
Atherogenic index AIðÞ¼
TC−HDLðÞ
HDL ð5Þ
HDL ratio ¼HDL−C
TC ð6Þ
The LDL-C/HDL-C ratio was calculated as well.
Rawal et al.
Blood Coagulation Parameters
Patients with hyperlipidemia exhibit elevated serum
cholesterol levels and have a high risk of thrombosis.
Thrombosis is a condition which may result in either local
coagulation or blood clotting in the circulatory system
pathway. Thus, this necessitates the study of blood coagula-
tion [36]. The APTT and PT are the coagulation assays widely
used to diagnose the general state of coagulation system [37].
The APTT is a general coagulation screening test which
measures the time required to produce fibrin from initiation
of intrinsic pathways. The APTT measurement involves the
recalcification of plasma in the presence of a standardized
amount of coagulation factor XII activator (kaolin) and
cephalin (platelet substitute). PT is a screening test of
extrinsic pathways (coagulation factors II, V, VII, and X).
The PT measurement involves the use of calcium thrombo-
plastin to induce plasma coagulation. The plasma was
collected by addition of anti-coagulant, sodium citrate, in
the blood followed by centrifugation at 4000 rpm for 10 min
and stored at 4 °C. APTT and PT were measured in the
plasma using commercially available diagnostic kits (C.K.
PREST®, Diagnostica Stago, France).
Oxidative Stress Parameters
The isolated liver was washed by ice-cold buffer; tissues
were minced and homogenized in ice-cold PBS with 25
strokes of a tight Teflon pestle of glass homogenizer (Remi
Motors Pvt. Ltd.) at 2500 rpm. The clear supernatant was
taken to estimate catalase (CAT), superoxide dismutase
(SOD), and reduced glutathione (GSH) levels, and tissue
homogenate were used to estimate malondialdehyde (MDA)
levels. All parameters were measured following the proce-
dures as reported elsewhere [38].
Histopathology of Carotid Artery
The heart removed from anesthetized animals was
washed by physiological saline and fixed with 10% formalin.
After opening the right atrium, the heart was immediately
transferred to 10% formalin for 48 h for fixation. Next, the
left coronary artery was dissected from its proximal part and
processed by liquid paraffin for sectioning. The sections were
stained by hematoxylin-eosin stain and observed under light
microscope.
Statistical Analysis
Each experiment was performed in triplicates. The
statistical significance was calculated using ANOVA. The
mean difference was considered significant (p< 0.05).
RESULTS AND DISCUSSION
Hyperlipidemia is a major health issue and established as
one of the major reasons for cerebrovascular diseases,
coronary artery diseases, and peripheral vascular diseases.
Lipid accumulation promotes endothelial injury due to the
formation of Oxi-LDL and initiates atherosclerotic events.
Therefore, dyslipidemia and thrombosis along with oxidative
stress play a vital role in this whole process. Despite the
availability of various first-line drugs such as statins and
fibrates, the adverse effects limit their use and beget the need
to explore the use of various novel herbal nutrients. The
gamma-oryzanol, extracted from rice bran oil, shows the
potential of improving serum lipid profile and decreases
coronary risk factors. Therefore, chitosan nanoparticles of
gamma-oryzanol were prepared in order to augment its
bioavailability and good anti-hyperlipidemic activity.
Drug-Excipient Compatibility Study
The characteristic peaks of gamma-oryzanol were iden-
tified with pure drug and combination of drug-excipient
spectrum by FTIR after the storage for 1 month at room
temperature. FTIR spectra of gamma-oryzanol and its
mixture with chitosan and TPP (Fig. 1) show the presence
of identical peaks (2954.00, 1602.00, 3298.68, and 1689.34/cm)
in both gamma-oryzanol and its mixture with chitosan and
TPP (2950.55, 1606.41, 3299.61, and 1689/cm) showing their
compatibility. The peaks represent the following groups
present in gamma-oryzanol: 2954/cm of alkanes (C-H
stretching), 1602/cm of aromatic hydrocarbons (C=C
stretching), 3298.68/cm of alcohol (intermolecular H bonded
OH stretching), and 1689.34/cm of ester (C=O stretching
enolic) groups.
Formulation of Nanoparticles
Preliminary Trials
Of all the factors studied, the drug to polymer ratio, the
polymer to TPP ratio, and stirring speed were identified as
important factors and selected for optimization. The concen-
tration of acetic acid, the concentration of Tween 80, and size
of the syringe needle were fixed at 4%, 0.5%, and 22 gauge,
respectively. In all, eight batches were prepared at two levels
of each factor: drug/polymer, polymer/TPP, and stirring
speed.
Optimization of Nanoparticles
The 2
3
full factorial design was applied for the
optimization of the nanoparticles. Eight batches were
prepared using drug/polymer (X
1
), polymer/cross-linking
agent ratio (X
2
), and stirring speed (X
3
) as independent
variables (Table I).
Characterization of Nanoparticles
Particle Size
Nanoparticle size plays a crucial role in determining their
penetration throughout the intestinal mucosal layers. Red-
head et al. [39] reported penetration of nanoparticles of
100 nm size through the sub-mucosal layers of a rat intestinal
loop model than that seen with the microparticles which got
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
localized in the epithelial linings. As reported elsewhere [40,
41], nanoparticles within size range 200–400 nm were seen
absorbed by the intestinal lining through macrophage uptake.
Hence, the particles with the size range within 100–400 nm
were taken into consideration for proper absorption across
the intestinal mucosa. All the batches showed particle size in
the range of 99.1 ± 0.4 to 333 ± 1.2 nm [42]. Thus, it can be
said that the fabricated nanoparticles might get easily
absorbed through the intestinal linings for facilitated uptake
via macrophages. The polydispersity index (PDI) values
represent the average uniformity of a particle solution. Large
PDI values represent the large size distribution of the
particles. The PDI values ranged between 0.218 ± 0.02 and
0.521 ± 0.02. PDI, an important determinant of particle size
distribution as well as the stability of the nanoparticles,
presented well below 0.3 at lower speeds. However, at higher
speeds, the nanoparticles became polydisperse with PDI
values greater than 0.3 [42].
The polynomial equation obtained for the two-level
three-factor factorial design using Design Expert 7.0.0
software for the particle size was as follows:
YPS ¼2:025−8:45X1þ48:97X2−17:425X3þ14:075X1X2
þ53:35X2X3þ1:825X1X3ð7Þ
The equation indicates that drug/polymer ratio had a
negative impact on the particle size. This may be explained by
the fact that the reduced chitosan concentration led to the less
cross-linking leading to small matrix formation. This in turn
may have driven the smaller particle size, whereas the
polymer/TPP and the stirring speed had a positive impact
on the particle size. The particle size was increased with
increasing polymer/TPP ratio. This is probably due to
increased cross-linking prompts large matrix formation be-
cause of increase in the polymer concentration. Further, the
increase in the stirring speed leads to the decrease in the
particle size due to higher shear forces.
Entrapment Efficiency
The entrapment efficiency is an important parameter to
determine whether the drug is getting entrapped within the
nanoparticles or not. The polynomial equation obtained for
the entrapment efficiency was as follows:
Y%EE ¼57:48−6:73X1þ13:86X2þ4:93X3
þ4:13X1X2−3:93X2X3−1:28X1X3ð8Þ
The drug to polymer ratio had a negative impact on the
entrapment efficiency due to the decreased entrapment of the
drug within the same polymer concentration matrix upon
increasing the drug concentration. On the contrary, polymer/
TPP ratio and stirring speed leave a positive impact. This
could be explained due to the presence of the greater amount
of polymer leading to complete entrapment of drug. More-
over, increased stirring speed led to the formation of compact
nanoparticles resulting into the greater drug entrapment.
Fig. 1. Overlay FTIR spectra of gamma-oryzanol and its mixture with the excipients (chitosan and TPP)
showing compatibility of gamma-oryzanol with chitosan and TPP
Rawal et al.
In vitro Drug Release
The polynomial equation to calculate the in vitro drug
release at 2, 12, and 24 h:
Y2h¼19:35−1:525X1þ7:57X2þ0:6X3−0:4X1X2
þ0:325X2X3þ0:025X1X3ð9Þ
Y12h¼54:35−0:9X1þ13:925X2þ0:75X3þ0:475X1X2
þ0:375X2X3þ0:04X1X3ð10Þ
Y24h¼67:38−1:13X1þ10:08X2
þ0:83X3−0:13X1X2−0:01X2X3−0:33X1X3ð11Þ
The coefficient values calculated by the equation of
all three factors reveal that the selected factors did not
show any significant effect on the drug release. The Food
and Drug Administration (FDA) has enforced the testing
of oral dosage forms in release media containing ethanol.
The FDA has mentioned that the potential interaction
between the oral formulation and alcohol may result in
the impairment of the formulation and dose dumping.
Hence, it becomes essential to carry out the in vitro
release studies in the presence of alcohol. However, no
dose dumping effect was observed in any of our batches
on the inclusion of 20% ethanol in PBS pH 7.4 [43].
In order to obtain the overlay plot, the particle size
between 100 and 200 nm and maximum drug entrapment
were selected as constraints. The desirability graph
showed the desirability of 0.922 for the composition of
Fig. 2. Contour plots and the overlay plot showing design space (range of desired product)
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
batch B5 and was selected as the Boptimized batch.^The
contour plots and overlay plot (Fig. 2) showed the yellow
acceptable region, within which the formulated batches
would fulfill the criteria. To identify the significance of the
effects and their interactions, ANOVA was run for each
parameter. The ANOVA analysis indicates that the
estimated responses are well described by the model, as
evinced by pand R
2
values. The significant pvalues (<
0.05) for the particle size and the entrapment efficiency
were 0.0135 and 0.0120, and R
2
values were 0.999 for both
the responses. However, the pvalue was greater than 0.05
(R
2
=0.997) in case of in vitro drug release, thus showing
the insignificant effect of the factors on the response
variables.
Zeta Potential of the Optimized Batch
Zeta potential of batch B5 was + 6.45 mV indicating
that the nanoparticles have a little cationic charge due to
the presence of chitosan that would help in the better
interaction of these nanoparticles to that with anionic
intestinal linings. However, the lower positive value of
zeta potential might result in lower repulsive forces
between particles which may lead to the agglomeration.
Fig. 3. Particle size and zeta potential of the optimized batch
Rawal et al.
The particle size and zeta potential of the optimized batch
are shown in Fig. 3.
Morphological Analysis by Scanning Electron Microscopy
SEM analysis (Fig. 4) showed the spherical shape of
spherical gamma-oryzanol-loaded nanoparticles. The polydis-
persity index value of the optimized batch (0.281 ± 0.01)
revealed the uniform distribution of nanoparticles.
Mucoadhesion Studies
Chitosan is well-known polymer for its mucoadhesive
properties. The higher the mucoadhesion, the better is the
effect. Sakloetsakun et al. [44] reported 44% of
mucoadhesion of the chitosan nanoparticles to the intestinal
mucosa. Bhosale et al. [45] investigated 67.3% mucoadhesion
of poly (lactic-co-glycolic acid) nanoparticles to the intestinal
mucosa. Our chitosan nanoparticle formulation exhibited a
high mucoadhesion of 83 ± 0.02%. The high mucoadhesion
value reveals good adherence property of prepared nanopar-
ticles to the intestinal mucosa which would help drug
absorption from the gastrointestinal (GI) tract.
Stability Studies
Freeze-drying has been reported as an effective method
by many researchers for converting liquid formulation into
solid to increase its shelf-life [46].Batch B5 after freeze-drying
showed similar results of zeta potential, particle size, and
entrapment(Table II). However, the freeze-dried product was
stable when stored in a tightly closed vial in refrigerated
condition for 3 months whereas the liquid suspension showed
an increase in particle size due to agglomeration. The stability
study should be further continued to label storage condition
and expiry date on the product.
Animal Studies
In vivo Anti-hyperlipidemic Study
The hyperlipidemia has been estimated by the various
models developed: high cholesterol diet-induced hyperlipid-
emia, triton-induced hyperlipidemia, poloxamer-407-induced
hyperlipidemia. The intraperitoneal administration of
poloxamer-407 was seen better to induce hypertriglyc-
eridemia within 24 h. It deviates normal lipoprotein level
from its regular range by lipoprotein lipase inhibition and
upregulation of 3-hydroxy-3-methylgluteryl coenzyme A
reductase (HMG-CoA reductase), regulatory enzyme for
cholesterol biosynthesis pathway.
Determination of Body Weight
As P-407 is a hyperlipidemia-inducing agent [47], group
animals exhibited increased body weight. Moreover, OZ- and
OZF-treated animals exhibited comparatively normal body
weight. The estimation of the changes in body weight is
shown in Fig. 5.
Table II. Stability Studies of the Optimized Nanoparticle Batch Before and After Lyophilization
Condition Time Average particle
size (nm)
Drug entrapment
efficiency (%)
25 °C/60% RH Liquid nanoparticles Initial 141.6 ± 2.6 65.5 ± 0.4
1 month 242.8 ± 6.8 56.1 ± 3.6
3 months 466.0 ± 3.2 44.3 ± 2.1
Refrigerated condition (5 °C ± 3 °C) Initial 141.6 ± 2.6 65.5 ± 0.4
1 month 167.0 ± 3.9 62.8 ± 1.3
3 months 208.3 ± 3.2 49.6 ± 2.7
25 °C/60% RH Freeze-dried powder Initial 157.0 ± 2.1 65.5 ± 0.8
1 month 199.9 ± 0.8 64.2 ± 0.6
3 months 309.0 ± 2.4 53.2 ± 0.9
Refrigerated condition (5 °C ± 3 °C) Initial 157.0 ± 2.1 65.5 ± 0.8
1 month 159.2 ± 0.5 65.0 ± 0.3
3 months 168.0 ± 1.2 59.6 ± 1.2
Fig. 4. SEM image of the optimized design batch showing the size of
the nanoparticles
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
Lipid Profile
Hyperlipidemia is characterized by increased levels of
serum TC, TG, and LDL resulting in increased risk of
coronary heart disease. The LDL is bad cholesterol
whereas HDL is good cholesterol as it conveys extra
cholesterol to the liver for the removal from the body.
The following effects were observed in lipid profile: P-407
is reported to increase TC, TG, LDL, and VLDL and
reduce the HDL levels [48,49]. In our study, P-407 group
animals showed elevated TC, TG, VLDL, and LDL levels
by the upregulation of HMG-Co-A reductase enzyme and
decreased HDL levels, whereas STD, OZ, and OZF
groups showed a significant reduction in TC, TG, VLDL,
and LDL levels and significant elevation in HDL levels as
compared to that seen with the P-407-induced group.
However, the insignificant difference in lipid profile was
observed between OZ and OZF groups.
It has been reported that P-407 increases AI which is a
representative marker of atherosclerosis (plaque or lipid
deposition in aorta and liver) [50]. In our study, P-407 group
exhibited an elevation in AI which was seen decreased in
STD, OZ, and OZF animal group. The decrease in AI
represents reduced cardiac risk and therefore revealing the
good anti-hyperlipidemic activity of nanoparticle
formulation.
The low LDL/HDL ratio is an indicator of lower risk
for coronary diseases. P-407 group animals showed
elevated LDL/HDL ratio whereas OZ and OZF showed
asignificant decrease in LDL/HDL ratio as compared to
P-407-induced group and representing their good anti-
atherogenic property. However, again, the insignificant
difference was seen in the effects of OZ and OZF
(Table III;Fig.6). All these values were seen normalized
in STD, OZ, and OZF animals.
Blood Coagulation Parameters
The plasma levels of APTT and PT were measured as
the intrinsic, extrinsic, and common coagulation pathways.
APTT and PT were seen reduced in the P-407 group. OZ
and OZF animals showed elevated APTT and PT. OZF-
treated animals showed a significant increase in APTT and
PT when compared with all other groups. The pretreat-
ment with OZF prolonged the APTT and PT and showed
its positive effect on both intrinsic and extrinsic coagula-
tion pathways (Table IV)[51]. The elevations in the
APTT and PT values for an extended period of time
might be observed due to the sustained release of gamma-
oryzanol from the nanoparticles. The elevations in APTT
and PT present the reduced risk of thrombosis and
advocate the good anti-hyperlipidemic activity of the
nanoparticle formulation.
Oxidative Stress Parameters
The oxidative stress occurs when MDA is the by-
product of lipid peroxidation. MDA is an indicator for the
index of lipid peroxidation in liver homogenate. P-407 is
reported to aggravate oxidative damage and formation of
oxi-LDL [51]. The anti-oxidant enzymes (SOD, CAT, and
GSH) inhibit the MDA levels and reduce the risk of
atherogenesis. This study shows that the P-407 group
exhibited a marked rise in MDA levels and reduces the
levels of antioxidant enzymes. A significant reduction in
MDA levels and increase in SOD, CAT, and GSH levels
were seen in STD-, OZ-, and OZF-treated animals in
comparison to that seen with the P-407-induced group. A
significant rise in SOD was observed in OZF-treated
animals when compared with the OZ-treated animals
indicating a preventive effect of OZF against
atherothrombotic complications and oxidative stresses
(Fig. 7). The levels of CAT and GSH levels were the
same in both OZ and OZF animals.
Table III. Results of Coronary Risk Factors
Groups Atherogenic index (AI) HDL ratio LDL/HDL
NC 0.549 ± 0.09 0.66 ± 0.04 0.36 ± 0.09
P-407 9.432 ± 0.97 0.10 ± 0.01 7.18 ± 0.78
STD (2 mg/kg) 1.808 ± 0.13 0.36 ± 0.02 1.42 ± 0.12
OZ (100 mg/kg) 1.582 ± 0.14 0.39 ± 0.02 0.89 ± 0.11
OZF (100 mg/kg) 1.209 ± 0.44 0.45 ± 0.01 0.76 ± 0.04
Fig. 5. Comparative estimation of body weight of normal control and
the treatment groups. Each group contains six animals; values
expressed as mean ± SEM. NC normal control group, P-407
poloxamer-407-induced group, STD standard drug atorvastatin
(2 mg/kg), OZ gamma-oryzanol drug powder (175 mg/kg) in 0.5%
CMC, OZF gamma-oryzanol-loaded chitosan nanoparticles (175 mg/
kg); asterisk indicates significant difference from normal control
group; number sign indicates significant difference from P-407-
induced group (p< 0.05)
Rawal et al.
Histopathology of Coronary Artery
The reduction in the damage to endothelium and
lipid core was seen on the histological samples of the
carotid artery of animals treated with our prepared
formulation (Fig. 8). It can be clearly seen that P-407
induced thrombus formation in the artery due to lipid
accumulation and formation of reactive oxygen species
(Fig. 8b). The STD and OZ led to the reduced lipid
accumulation (Fig. 8c, d). The complete clearance of
accumulated lipids was observed with OZF-treated group
(Fig. 8e) and therefore indicating its beneficial effect
against thrombotic risk in the body.
CONCLUSION
In this study, we encapsulated gamma-oryzanol into
chitosan-TPP nanoparticles to develop a polymeric
nanocarrier with improved anti-hyperlipidemic activity.
The optimized nanoparticle formulation had a particle
size of 141.6 ± 2.6 nm falling well within the desired range
(100–200 nm). The formulation exhibited well in vivo
anti-hyperlipidemic activity similar to that of marketed
formulation (atorvastatin). Although insignificant differ-
ence was observed in the lipid profile exhibited by plain
gamma-oryzanol and gamma-oryzanol nanoparticles, the
nanoparticulate formulation manifested a prominent effect
on blood coagulation and oxidative stress parameters.
The histopathological examination of carotid artery also
revealed the complete clearance of accumulated lipids in
the artery via gamma-oryzanol nanoparticles in compari-
son to atorvastatin and plain gamma-oryzanol. Therefore,
it can be stated that chitosan-loaded gamma-oryzanol
Fig. 6. Comparative estimation of lipid profile (A: total cholesterol; B: triglyceride; C: very low-density lipoproteins; D : high-density
lipoprotein) of normal control and the treatment groups. Each group contains six animals; values expressed as mean ± SEM. NC normal
control group, P-407 poloxamer-407-induced group, STD standard drug atorvastatin (2 mg/kg), OZ gamma-oryzanol drug powder (175 mg/kg)
in 0.5% CMC, OZF gamma-oryzanol-loaded chitosan nanoparticles (175 mg/kg); asterisk indicates significant difference from normal control
group; number sign indicates significant difference from P-407-induced group (p< 0.05)
Table IV. Coagulation Parameters of All Animal Groups
Groups APTT (s) PT (s)
NC 20.33 ± 0.92 11.83 ± 0.48
P-407 (500 mg/kg) 18.65 ± 0.37 10.83 ± 0.60
STD (2 mg/kg) 25.98 ± 0.77 18.50 ± 0.76
OZ (100 mg/kg) 30.08 ± 0.73 22.17 ± 0.79
OZF (100 mg/kg) 32.00 ± 1.58 26.10 ± 1.12
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
Fig. 8. Histopathological images of the carotid artery of groups. aNormal control. bP-407 treated. cSTD treated. dOZ
(175 mg/kg) treated. eOZF (175 mg/kg) treated
Fig. 7. Comparative estimation of oxidative stress parameters (A: malondialdehyde; B: superoxide dismutase; C: catalase; D: glutathione) of
normal control and the treatment groups. Each group contains six animals; values expressed as mean ± SEM. NC normal control group, P-407
poloxamer-407-induced group, STD standard drug atorvastatin (2 mg/kg), OZ gamma-oryzanol drug powder (175 mg/kg) in 0.5% CMC, OZF
gamma-oryzanol-loaded chitosan nanoparticles (175 mg/kg); asterisk indicates significant difference from normal control group; number sign
indicates significant difference from P-407-induced group (p< 0.05)
Rawal et al.
nanoparticles can serve as a better carrier with improved
anti-hyperlipidemic activity.
ACKNOWLEDGEMENTS
The authors would like to thank Institute of Pharmacy,
Nirma University, Ahmedabad for providing us all the
facilities to carry out our research work.
COMPLIANCE WITH ETHICAL STANDARDS
The animal protocol was approved by the Institutional
Animal Ethics Committee (Protocol no. IP/PCEU/MPH/14-1/
010). All animals were treated in accordance with the
guidelines of the committee for CPCSEA.
Conflict of Interest The authors declare that they have no
conflict of interest.
REFERENCES
1. Nelson RH. Hyperlipidemia as a risk factor for cardiovascular
disease. Primary Care: Clinics in Office Practice [Internet]. 2013
[cited 2017 Aug 23];40:195–211. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0095454312000991
2. Baker WL, Tercius A, Anglade M, White CM, Coleman CI. A
meta-analysis evaluating the impact of chitosan on serum
lipids in hypercholesterolemic patients. Ann NutrMetab.
2009;55:368–74.
3. Zhang J, Zhang W, Mamadouba B, Xia W. A comparative study
on hypolipidemic activities of high and low molecular weight
chitosan in rats. Int J Biol Macromolecules [Internet]. 2012
[cited 2017 Nov 20];51:504–8. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0141813012002346
4. Zavoshy R, Noroozi M, Jahanihashemi H. Effect of low calorie
diet with rice bran oil on cardiovascular risk factors in
hyperlipidemic patients. J Res Med Sci. 2012;17:626–31.
5. Cicero AF, Gaddi A. Rice bran oil and gamma-oryzanol in the
treatment of hyperlipoproteinaemias and other conditions.
Phytother Res. 2001;15:277–89.
6. Ghatak SB, Panchal SJ. Anti-hyperlipidemic activity of
oryzanol, isolated from crude rice bran oil, on Triton WR-
1339-induced acute hyperlipidemia in rats. RevistaBrasileira de
Farmacognosia [Internet]. 2012 [cited 2017 Nov 20];22:642–8.
Ava i lab l e from : http://www.scielo.br/
scielo.php?script=sci_arttext&pid=S0102-
695X2012000300024&lng=en&nrm=iso&tlng=en
7. Cho J-Y, Lee HJ, Kim GA, Kim GD, Lee YS, Shin SC, et al.
Quantitative analyses of individual γ-Oryzanol (Steryl
Ferulates) in conventional and organic brown rice (Oryza sativa
L.). J Cereal Sci [Internet]. 2012 [cited 2017 Aug 23];55:337–43.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0733521012000173
8. Plapied L, Duhem N, des Rieux A, Préat V. Fate of polymeric
nanocarriers for oral drug delivery. Curr Opin Colloid Interface
Sci [Internet]. 2011 [cited 2017 Aug 23];16:228–37. Available
from: http://linkinghub.elsevier.com/retrieve/pii/
S1359029411000033
9. Letchford K, Burt H. A review of the formation and classifica-
tion of amphiphilic block copolymer nanoparticulate structures:
micelles, nanospheres, nanocapsules and polymersomes. Eur J
Pharm Biopharm [Internet]. 2007 [cited 2017 Aug 23];65:259–
69. Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0939641106003316
10. Ghaderi S, Ghanbarzadeh S, Mohammadhassani Z,
Hamishehkar H. Formulation of gammaoryzanol-loaded nano-
particles for potential application in fortifying food products.
Tabriz University of Medical Sciences; 2014 p.
11. Seetapan N, Bejrapha P, Srinuanchai W, Ruktanonchai UR.
Rheological and morphological characterizations on physical
stability of gamma-oryzanol-loaded solid lipid nanoparticles
(SLNs). Micron [Internet]. 2010 [cited 2018 Feb 6];41:51–8.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S096843280900122X
12. Viriyaroj A, Ngawhirunpat T, Sukma M, Akkaramongkolporn
P, Ruktanonchai U, Opanasopit P. Physicochemical properties
and antioxidant activity of gamma-oryzanol-loaded liposome
formulations for topical use. Pharm Dev Technol [Internet].
2009 [cited 2018 Feb 6];14:665–71. Available from: http://
www.tandfonline.com/doi/full/10.3109/10837450902911937,14,
665, 671
13. Manosroi A, Chutoprapat R, Abe M, Manosroi W, Manosroi J.
Transdermal absorption enhancement of rice bran bioactive
compounds entrapped in niosomes. AAPS Pharm Sci Tech
[Internet]. 2012 [cited 2018 Feb 6];13:323–35. Available from:
http://www.springerlink.com/index/10.1208/s12249-012-9751-1
14. Sapino S, Carlotti ME, Cavalli R, Ugazio E, Berlier G, Gastaldi
L, et al. Photochemical and antioxidant properties of gamma-
oryzanol in beta-cyclodextrin-based nanosponges. J Incl Phe-
nom Macrocycl Chem [Internet]. 2013 [cited 2018 Feb 6];75:69–
76. Available from: http://link.springer.com/10.1007/s10847-012-
0147-3
15. Gelperina S, Kisich K, Iseman MD, Heifets L. The potential
advantages of nanoparticle drug delivery systems in chemother-
apy of tuberculosis. Am J Respir Crit Care Med [Internet]. 2005
[cited 2018 Feb 6];172:1487–90. Available from: http://
www.atsjournals.org/doi/abs/10.1164/rccm.200504-613PP
16. Ghosh PK. Hydrophilic polymeric nanoparticles as drug car-
riers. Indian J Biochem Biophys. 2000;37:273–82. Available
from:http://nopr.niscair.res.in/bitstream/123456789/19833/1/
IJBB%2037%285%29%20273-282.pdf
17. QiL,XuZ,JiangX,HuC,ZouX.Preparationand
antibacterial activity of chitosan nanoparticles. Carbohydr Res
[Internet]. 2004 [cited 2018 Feb 6];339:2693–700. Available
from: http://linkinghub.elsevier.com/retrieve/pii/
S000862150400388X
18. Nagpal K, Singh SK, Mishra DN. Chitosan nanoparticles: a
promising system in novel drug delivery. Chem Pharm Bull.
2010;58:1423–30.
19. Fan W, Yan W, Xu Z, Ni H. Formation mechanism of
monodisperse, low molecular weight chitosan nanoparticles by
ionic gelation technique. Colloids Surf B: Biointerfaces [Inter-
net]. 2012 [cited 2017 Nov 20];90:21–7. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0927776511005650
20. Yadav R, Kumar D, Kumari A, Yadav SK. Encapsulation of
podophyllotoxin and etoposide in biodegradable poly-D,L-
lactide nanoparticles improved their anticancer activity. J
Microencapsul [Internet]. 2014 [cited 2017 Nov 20];31:211–9.
Available from: http://www.tandfonline.com/doi/full/10.3109/
02652048.2013.834988, 31, 211, 219
21. Rajitha P, Gopinath D, Biswas R, Sabitha M, Jayakumar R.
Chitosan nanoparticles in drug therapy of infectious and
inflammatory diseases. Expert Opin Drug Deliv [Internet].
2016 [cited 2017 Nov 20];13:1177–94. Available from: http://
www.tandfonline.com/doi/full/10.1080/17425247.2016.1178232,
13, 1177, 1194
22. Paolicelli P, de la Fuente M, Sánchez A, Seijo B, Alonso MJ.
Chitosan nanoparticles for drug delivery to the eye. Expert
Opin Drug Deliv [Internet]. 2009 [cited 2017 Nov 20];6:239–53.
Available from: http://www.tandfonline.com/doi/full/10.1517/
17425240902762818, 6, 239, 253
23. Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. Recent
advances on chitosan-based micro- and nanoparticles in drug
delivery. J Control Release [Internet]. 2004 [cited 2017
Formulation and Evaluation of Gamma-Oryzanol-Loaded Chitosan Nanoparticles
Nov 20];100:5–28. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0168365904003803
24. Bucci R, Magrì AD, Magrì AL, Marini F. Comparison of three
spectrophotometric methods for the determination of γ-
oryzanol in rice bran oil. Anal Bioanal Chem [Internet]. 2003
[cited 2017 Nov 20];375:1254–9. Available from: http://
link.springer.com/10.1007/s00216-002-1700-5, 375, 1254, 1259
25. Guan J, Cheng P, Huang SJ, Wu JM, Li ZH, You XD, et al.
Optimized preparation of levofloxacin-loaded chitosan nano-
particles by ionotropic gelation. Physics Procedia [Internet].
2011 [cited 2017 Nov 20];22:163–9. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S1875389211006808
26. Sahu BP, Das MK. Nanosuspension for enhancement of oral
bioavailability of felodipine. Appl Nanosci [Internet]. 2014
[cited 2017 Nov 20];4:189–97. Available from: http://
link.springer.com/10.1007/s13204-012-0188-3, 4, 189, 197
27. Dong Y, Ng WK, Hu J, Shen S, Tan RBH. A continuous and
highly effective static mixing process for antisolvent precipita-
tion of nanoparticles of poorly water-soluble drugs. Int J Pharm
[Internet]. 2010 [cited 2017 Nov 20];386:256–61. Available from:
http://linkinghub.elsevier.com/retrieve/pii/S0378517309008072
28. Rawal T, Parmar R, Tyagi RK, Butani S. Rifampicin loaded
chitosan nanoparticle dry powder presents an improved thera-
peutic approach for alveolar tuberculosis. Colloids Surf B:
Biointerfaces [Internet]. 2017 [cited 2017 Nov 29];154:321–30.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0927776517301625
29. Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous
soluble tetrazolium/formazan assay for cell growth assays in
culture. Cancer Commun. 1991;3:207–12.
30. Semete B, Booysen L, Lemmer Y, Kalombo L, Katata L,
Verschoor J, et al. In vivo evaluation of the biodistribution and
safety of PLGA nanoparticles as drug delivery systems.
Nanomedicine: Nanotechnol, Biol Med [Internet]. 2010 [cited
2017 Nov 20];6:662–71. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S1549963410000961
31. Araujo FB, Barbosa DS, Hsin CY, Maranhão RC, Abdalla DS.
Evaluation of oxidative stress in patients with hyperlipidemia.
Atherosclerosis. 1995;117:61–71.
32. Acay A, Ulu MS, Ahsen A, Ozkececi G, Demir K, Ozuguz U,
et al. Atherogenic index as a predictor of atherosclerosis in
subjects with familial Mediterranean fever. Medicina [Internet].
2014 [cited 2017 Nov 20];50:329–33. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S1010660X14001153
33. Johnston TP, Zhou X. Oxidation of low-density lipoprotein cholesterol
following administration of poloxamer 407 to mice results from an
indirect effect: J Cardiovasc Pharmacol [Internet]. 2007 [cited 2017
Nov 20];49:246–52. Available from: http://content.wkhealth.com/
linkback/openurl?sid=WKPTLP:landingpage&an=00005344-
200704000-00009
34. Yasuda T, Johnston TP, Shinohara M, Inoue M, Ishida T. The
effect of poloxamer 407 on the functional properties of HDL in
mice: P-407 mouse model of dysfunctional HDL. J Pharm
Pharmacol [Internet]. 2012 [cited 2017 Nov 20];64:677–87.
Available from: http://doi.wiley.com/10.1111/j.2042-
7158.2011.01444.x
35. Swapnil SA, Anup BT, Ashok B, Ravishankar B, Shukla V.
Evaluation of anti-hyperlipidemic activity of LekhanaBasti in
albino rats. AYU (An International Quarterly Journal of
Research in Ayurveda) [Internet]. 2013 [cited 2018
Feb 9];34:220. Available from: http://www.ayujournal.org/
text.asp?2013/34/2/220/119687
36. Owens AP, Byrnes JR, Mackman N. Hyperlipidemia, tissue
factor, coagulation, and simvastatin. Trends in Cardiovascular
Medicine [Internet]. 2014 [cited 2018 Feb 9];24:95–8. Available
from: http://linkinghub.elsevier.com/retrieve/pii/
S105017381300114X
37. Kim J-A, Kim J-E, Song SH, Kim HK. Influence of blood lipids
on global coagulation test results. Annals of Laboratory
Medicine [Internet]. 2015 [cited 2018 Feb 9];35:15. Available
from: https://synapse.koreamed.org/DOIx.php?id=10.3343/
alm.2015.35.1.15, 35, 15, 21
38. Lundquist P, Artursson P. Oral absorption of peptides and
nanoparticles across the human intestine: opportunities, limita-
tions and studies in human tissues. Advanced Drug Delivery
Reviews [Internet]. 2016 [cited 2017 Nov 20];106:256–76.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0169409X16302277
39. Redhead HM, Davis SS, Illum L. Drug delivery in poly(lactide-co-
glycolide) nanoparticles surface modified with poloxamer 407 and
poloxamine 908: in vitro characterisation and in vivo evaluation. J
Control Release [Internet]. 2001 [cited 2018 Feb 9];70:353–63.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0168365900003679
40. Chen J, Liu C, Shan W, Xiao Z, Guo H, Huang Y. Enhanced
stability of oral insulin in targeted peptide ligand trimethyl
chitosan nanoparticles against trypsin. J Microencapsul [Inter-
net]. 2015 [cited 2018 Feb 6];32:632–41. Available from: http://
www.tandfonline.com/doi/full/10.3109/02652048.2015.1065920,
32, 632, 641
41. Fan T, Chen C, Guo H, Xu J, Zhang J, Zhu X, et al.Design
and evaluation of solid lipid nanoparticles modified with
peptide ligand for oral delivery of protein drugs. Eur J Pharm
Biopharm [Internet]. 2014 [cited 2018 Feb 6];88:518–28.
Ava ila b le fr om: http://linkinghub.elsevier.com/retrieve/pii/
S0939641114002033
42. Pathak L, Kanwal A, Agrawal Y. Curcumin loaded self
assembled lipid-biopolymer nanoparticles for functional food
applications. J Food Sci Technol [Internet]. 2015 [cited 2018
Feb 14];52:6143–56. Available from: http://link.springer.com/
10.1007/s13197-015-1742-2, 52, 6143, 6156
43. Gohel MC, Bariya SH. Fabrication of triple-layer matrix tablets
of venlafaxine hydrochloride using xanthan gum. AAPS Pharm
Sci Tech [Internet]. 2009 [cited 2018 Feb 14];10:624–30. Avail-
able from: http://www.springerlink.com/index/10.1208/s12249-
009-9244-z
44. Sakloetsakun D, Perera G, Hombach J, Millotti G, Bernkop-
Schnürch A. The impact of vehicles on the mucoadhesive
properties of orally administrated nanoparticles: a case study
with chitosan-4-thiobutylamidine conjugate. AAPS
PharmSciTech [Internet]. 2010 [cited 2018 Feb 7];11:1185–92.
Available from: http://www.springerlink.com/index/10.1208/
s12249-010-9479-8
45. Bhosale UV, Kusum Devi V, Jain N. Formulation and optimi-
zation of mucoadhesive nanodrug delivery system of acyclovir. J
Young Pharmacists [Internet]. 2011 [cited 2018 Feb 7];3:275–83.
Available from: http://www.jyoungpharm.org/article/559
46. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-
drying of nanoparticles: formulation, process and storage
considerations. Adv Drug Deliv Rev [Internet]. 2006 [cited
2017 Nov 20];58:1688–713. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0169409X06001840
47. Chaudhary HR, Brocks DR. The single dose poloxamer 407
model of hyperlipidemia; systemic effects on lipids assessed
using pharmacokinetic methods, and its effects on adipokines. J
Pharm Pharm Sci. 2013;16:65–73.
48. Sarker M, Mahmud ZA, Saha SK, Tithi NS, Ali MS, Bachar SC.
Anti hyperlipidemic activity of flowers of Punica granatum in
poloxamer-407 induced hyperlipidemic mice model.
Pharmacogn J [Internet]. 2012 [cited 2017 Nov 20];4:66–70.
Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0975357512800241
49. Korolenko T, Johnston TP, Dubrovina NI, Kisarova YA,
Zhanaeva SY, Cherkanova MS, et al. Effect of poloxamer 407
administration on the serum lipids profile, anxiety level and
protease activity in the heart and liver of mice. Interdisc Toxicol
[Internet]. 2013 [cited 2017 Nov 20];6. Available from: http://
www.degruyter.com/view/j/intox.2013.6.issue-1/intox-2013-0004/
intox-2013-0004.xml
50. Omari-Siaw E, Wang Q, Sun C, Gu Z, Zhu Y, Cao X, et al. Tissue
distribution and enhanced in vivo anti-hyperlipidemic-antioxidant
effects of perillaldehyde-loaded liposomal nanoformulation
against Poloxamer 407-induced hyperlipidemia. Int J Pharm
[Internet]. 2016 [cited 2017 Nov 20];513:68–77. Available from:
http://linkinghub.elsevier.com/retrieve/pii/S0378517316307839
51. Ghatak SB, Dhamecha PS, Bhadada SV, Panchal SJ. Investiga-
tion of the potential effects of metformin on atherothrombotic
risk factors in hyperlipidemic rats. Eur J Pharmacol [Internet].
2011 [cited 2017 Nov 20];659:213–23. Available from: http://
linkinghub.elsevier.com/retrieve/pii/S0014299911003414
Rawal et al.
