Artificial Cells, Nanomedicine, and Biotechnology

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ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20

Intranasal delivery of quercetin-loaded

mucoadhesive nanoemulsion for treatment of
cerebral ischaemia

Niyaz Ahmad , Rizwan Ahmad, Atta Abbas Naqvi, Md Aftab Alam,

Mohammad Ashafaq, Rehan Abdur Rub & Farhan Jalees Ahmad

To cite this article: Niyaz Ahmad , Rizwan Ahmad, Atta Abbas Naqvi, Md Aftab Alam, Mohammad
Ashafaq, Rehan Abdur Rub & Farhan Jalees Ahmad (2017): Intranasal delivery of quercetin-loaded
mucoadhesive nanoemulsion for treatment of cerebral ischaemia, Artificial Cells, Nanomedicine,
and Biotechnology, DOI: 10.1080/21691401.2017.1337024

To link to this article: http://dx.doi.org/10.1080/21691401.2017.1337024

Published online: 12 Jun 2017.

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017
https://doi.org/10.1080/21691401.2017.1337024

Intranasal delivery of quercetin-loaded mucoadhesive nanoemulsion for

treatment of cerebral ischaemia
Niyaz Ahmada,b , Rizwan Ahmadc, Atta Abbas Naqvid, Md Aftab Alame, Mohammad Ashafaqf,
Rehan Abdur Rubg and Farhan Jalees Ahmadg
a
Department of Pharmaceutics, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University (Formerly University of Dammam),
Dammam, Kingdom of Saudi Arabia; bDepartment of Pharmaceutical Chemistry, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal
University (Formerly University of Dammam), Dammam, Kingdom of Saudi Arabia; cDepartment of Natural Products and Alternative
Medicine, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University (Formerly University of Dammam), Dammam, Kingdom of
Saudi Arabia; dDepartment of Pharmacy Practice, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University (Formerly University
of Dammam), Dammam, Kingdom of Saudi Arabia; eDepartment of Pharmaceutics, School of Medical and Allied Sciences, Galgotias
University, Greater Noida, India; fNeuroscience and Toxicology Unit, College of Pharmacy, Jazan University, Jazan, Saudi Arabia;
g
Nanomedicine Lab, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India

ABSTRACT ARTICLE HISTORY

Background: Quercetin (QUR), as an antioxidant flavonoid, exhibits potential role in the amelioration of Received 13 February 2017
cerebral ischaemia; however, poor solubility as well as oral absorption results low serum and tissue lev- Accepted 22 May 2017
els for this drug.
Purpose of the study: To enhance bioavailability, this study aims to prepare QUR nanoemulsions and
KEYWORDS
administer via non-invasive nasal route in order to evaluate the drug targeting in brain. Quercetin; cerebral
Methods: Quercetin mucoadhesive nanoemulsion (QMNE) was prepared (ionic gelation method) and ischaemia; UPLC-ESI-Q-TOF-
optimized using various parameters, that is, particle size, entrapment efficiency, zeta potential and ex MS/MS method validation;
vivo permeation study. mucoadhesive nanoemul-
Results: The results observed for optimized QMNE were as follows: mean globule size (91.63 ± 4.36 nm), sion; brain
zeta potential (
17.26 ± 1.04 mV), drug content (99.84 ± 0.34%) and viscosity (121 ± 13 cp). To evaluate pharmacokinetics and
the extent of bioavailability for QMNE via post-intranasal (i.n.) administration, Ultra performance liquid pharmacodynamics
chromatography-mass spectroscopy (UPLC-ESI-Q-TOF-MS/MS)-based bioanalytical method was developed
and validated for pharmacokinetics, biodistribution, brain-targeting efficiency (9333.33 ± 39.39%) and
brain drug-targeting potential (2181.83 ± 5.69%) which revealed enhanced QUR brain bioavailability as
compared to intravenous administration (i.v.). Furthermore, improved neurobehavioral activity (locomotor
and grip strength), histopathology and reduced infarction volume effects were observed in middle cere-
bral artery occlusion (MCAO)–induced cerebral ischemic rats model after i.n. administration of QMNE.
Conclusion: This study supports a significant role for QMNE in terms of high brain-targeting potential
and formulation efficiency due to ease of access and effective targeting in brain.

CONTACT Niyaz Ahmad nanhussain@uod.edu.sa, niyazpharma@gmail.com Department of Pharmaceutics and Pharmaceutical Chemistry, College of Clinical
Pharmacy, Imam Abdulrahman Bin Faisal University (Formerly University of Dammam), P.O. Box 1982, Dammam–31441, Kingdom of Saudi Arabia
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
2 N. AHMAD ET AL.
Introduction
Ischaemic stroke, reason of lasting disability, results due to
cerebral blood flow reduction and restriction to major brain
artery. The cascade of reactions in the shape of protein,
nucleic acid and lipid oxidation in brain results ischaemic
stroke and disturbed metabolic process, that is, decreased
ATP production, abnormal release of excitatory amino acids,
intracellular calcium accumulation and free radical formation
hence neuronal cell death. The disease become more severe
as soon as oxidative stress is implicated in ischaemia–reperfu-
sion injury [1–3], as it leads to surplus reactive oxygen species
(ROS) production.
Oxidative stress (an imbalance between reactive oxygen
species (ROS) and their quenching by the antioxidant sys-
tem) is suggested as a dominant factor in the pathogenesis
of ischemic/reperfusion injury, as reported [4,5]. In order to
evaluate deleterious mechanisms involved in ischaemia, ani-
mal models have been treated with different antioxidants
(experimental model of neurodegeneration) whereby a sup-
portive mechanism was established for antioxidants, that is
to boost the defence system against neurodegeneration
[6,7].
Quercetin, commonly found in fruits and vegetables, has
been reported with a potential antioxidant and free radical
scavenging, anti-inflammatory, anti-ischaemic and as anti-
coagulant property [8,9]. In addition, neuroprotective effects
with improved morphological as well as functional activity
have been observed for QUR in various brain injury models
of ischaemia as reported [10,11].
Despite the efficacy in treatment of cerebral ischaemia,
QUR due to poor solubility and absorption along with rapid
metabolism and quick elimination poses the drawback of low
serum and tissue concentrations [12,13]. In order to overcome
the drawback, different strategies in terms of formulations
and route of administration have been applied. To bypass
blood–brain barrier (BBB) and hence effectively target the
brain in order to treat neurological disorders [14] is among
the interesting areas of research nowadays. Intranasal (i.n.)
administration of QUR in this regard is an effective way to
bypass BBB and achieve a high concentration of drug via
increased supply into cortex, hippocampus and caudate-puta-
men as compared to i.v. route [15]. Due to need of develop-
ing a suitable formulation for enhanced bioavailability, more
attention is gained by lipid-based formulation whereby an
enhanced drug uptake in CNS was observed [2,16]. To over-
come the aforementioned drawbacks, this study investigated
a formulated QMNE through i.n. route in middle cerebral
artery occlusion-induced (MCAO) focal cerebral ischaemia in
Wistar rats. The nanoemulsions with small particle size
(10–200 nm) showed more stability in suspension [17]. In add-
ition, nanoemulsions with small oil droplets size, transparent
appearance along with weak light-scattering properties,
favour them to be incorporated in an optically transparent
product without adversely affecting their clarity [18]. Current
research focus to formulate, characterize and target CNS by
quercetin-loaded mucoadhesive nanoemulsion (QMNE) in
order to treat the cerebral ischaemia. In addition, the most
important parameter, that is, targeting efficiency of drug in
the brain is also calculated herewith.
Experimental
Materials and methods
Quercetin (>95%) and Rutin (RxBiosciences, 18908 Bonanza
way, Gaithersburg, MD20879), MS-grade ammonium acetate
and ammonium formate from Fluka analytical (Sigma-Aldrich,
the Netherlands), whereas formic acid (purity >98%) was
obtained from Fluka analytical (Steinheim, Germany).
Deionized water was purified using Milli-Q water purification
system (Millipore, Bedford, MA). Oleic acid, PEG 400 and
Tween 80 samples were provided by Jubilant Formulation
Research Department (Noida, India). HPLC-MS grade
Acetonitrile and Methanol (purity¼ 99.9%) was purchased
from Sigma Aldrich (Steinheim, Germany).
Preparation of nanoemulsion
Titration method was used to prepare QNE using oil (oleic
acid), cosurfactant (PEG 400), surfactant (Tween80/labrasol/
Transcutol HP) and purified water (continuous phase). Briefly,
oil phase mixed with Smix, that is, oil:Smix (0–3:3–0), was taken
in various ratios (1–9:9–1), titrated with purified water and
added dropwise to internal phase (drug loaded) with con-
stant stirring. Pseudo ternary-phase diagrams were con-
structed to evaluate the clear nanoemulsion.
Optimization of nanoemulsion
Formulation of QUR containing different oil (%) compositions
(2.5%, 5% and 7.5% v/v), surfactant-cosurfactant mixture
(20%, 30%, 40% and 50% v/v) and water with different pro-
portions were evaluated for globular size (G), zeta potential
(Z), percentage transmittance (%T) and dilution characteristic.
The final composition of NE was optimized based on GS, ZP,
%T and dilution characteristics.
Characterization of emulsions
The droplet size, PDI zeta potential, SEM and TEM of the
investigated samples were obtained (in triplicate) by calculat-
ing the average of thirteen (13) measurements at an angle of
173  C as method reported by Ahmad et al. [2].
Ex vivo permeability study
Ex vivo permeability method was adopted by Ahmad et al.
[3].
In vivo study
For in vivo pharmacokinetic, biodistribution and pharmaco-
dynamics studies proper approval from Animal Ethical
Committee, Jamia Hamdard (New Delhi, India), was taken
which complies with National Guidelines on the Care and Use

of Laboratory Animals. Wistar rats (300–400 g, 8–10 weeks
old) were kept in an environmentally controlled room (tem-
perature: 25 ± 2  C, humidity: 60 ± 5%, 12-h dark/light cycle)
for at least one (1) week before the experiments. Animals
were fed on a standard pelleted diet and water (ad libitum).
Bioanalytical method development and validation by
UPLC/ESI-quadrupole-time-of-flight-MS/MS
Waters ACQUITY UPLCTM system (Waters Corp., MA) with
specifications; binary solvent system of delivery, autosampler,
column manager as well as tunable MS detector (Synapt MS;
Waters Corp., Manchester, UK), whereas Waters ACQUITY
UPLCTM BEH C-18 column with dimensions, that is,
2.1  100 mm; particle size 1.7 lm was used for chromato-
graphic separation. The gradient system used for mobile
phase was; acetonitrile (90%):2 mM ammonium acetate
(10%):formic acid (0.01%) v/v/v, with a flow rate of
0.2 mL/min and injection volume of 10 lL. The US Food and
Drug Administration, [19] guidelines were followed for the
validation of developed bioanalytical method, whereas
regression equation (weighting factor for IS concentration
i.e. 1/x2) was applied to obtain the best fit relationship for
the concentration-detector response.
Pharmacokinetics, biodistribution, brain-targeting
efficiency and brain drug-targeting potential
For pharmacokinetics and biodistribution studies, rats were
divided into seven (07  03 ¼ 21) groups. Briefly, QUR solution
and QMNE (10 mg/kg body weight) were administered separ-
ately, that is, i.v. and i.n., blood samples at determined times
of 0.5, 1, 2, 4, 8, 12 and 24 h of pre-treatment were collected
and successively analysed according to the method described
in section “Bioanalytical method development and validation
by UPLC/ESI-Q-TOF-MS/MS” in order to obtain data for Cmax,
t1/2 and AUC (0–t) calculation. For biodistribution study, total
21 animals (3  7) were dosed according to the method
described in pharmacokinetic study. Animals (n ¼ 3) were sac-
rificed for lungs, plasma and brain homogenates followed by
successive analysis as per method “bioanalytical method
development and validation” in order to obtain data for
%DTE (brain-targeting efficiency) and %DTP (brain drug-tar-
geting potential) calculation using the formulae [20]:
% DTE ¼ ðAUCbrain =AUCbloodÞi:n: =ðAUCbrain =AUCblood Þi:v:  100
% DTP ¼ ðBin
Bx Þ=Bin  100
where Bx ¼ (Biv/Piv)Pin, Bx¼area under the curve (AUC) for
brain fraction (contributed by systemic circulation through
BBB after i.n. administration); Biv and Piv, whereas Bin and Pin
are brain and blood AUC0–24 after i.v. and i.n. administration,
respectively.
Pharmacodynamic study in cerebral ischaemia
For pharmacodynamics studies; rats were divided into seven
(07  06 ¼ 42) groups as; Sham operated (Group I, i.e. con-
trol), Sham þ Nanoemulsion (Group II, i.e. without drug and
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3
placebo:substantial control), MCAO induced (Group III), MCAO
induced þ QUR solution (10 mg/kg body weight equivalent)
(Group IV) treated with i.v. and i.n. route, separately and
QMNE (Group V, i.e. treated with i.v. and i.n. route, separ-
ately). MCAO was induced via intraluminal filament according
to the method adopted from Longa et al. [21]. The animals
(after a reperfusion period of 22 h) were observed for behav-
ioural activity (locomotor and grip strength) successively fol-
lowed by sacrifice in order to obtain brain and lungs for
antioxidant activity evaluation as well as histopathological
studies.
Locomotor activity (closed field activity monitoring) and
grip strength
The Lannert and Hoyer [22] method, utilizing a digital photo-
actometer was adopted to determine spontaneous locomotor
activity. The method described by Ahmad et al. [16], using
grip strength meter was followed. For grip strength activity;
animal front paw was placed on rigid meter and moved till
the grasping is released. The force, as shown on screen (kilo-
gram), exerted by animal is recorded.
Histopathological studies
The brain from individual group of animals were removed
and kept in a fresh buffer containing 30% sucrose as
described; Ahmad et al. [14]. In detail; removed brain were
sliced into coronal sections (12 lm) on a cryostat (Leica,
Nußloch, Heidelberg, Germany) and 10 sections (cortex
region) were mounted on a glass slide for eosin and haema-
toxylin staining.
Infarct volume analysis
The method was adopted from Khan et al. [23] where the
infarct volume was determined as; average infarct area on
both sides  section thickness, whereas for total brain infarct
volume calculation; add infarct volume (from each section)
and fix for oedema.
Statistical analysis
The results were analysed and expressed as mean ± standard
error of mean (SEM). Student’s t-test was also applied for
difference between unpaired observations using ANOVA
(p values <.05).
Results
Preparation and characterization of nanoemulsion
Selection of nanoemulsion formulations
Skin and mucosal irritation have been reported [24] with the
use of more amount of surfactant and hence, determination
and optimum concentration of surfactant in a formulation is
very essential. To focus the mentioned concept, pseudoter-
nary phase diagrams were selected during the study in order
4 N. AHMAD ET AL.

to select formulations with desired properties, that is, the

Drug content (%)

99.84 ± 0.34
98.44 ± 1.01
97.16 ± 0.74
96.39 ± 0.83
amount of oil phase which completely solubilize the drug
and accommodate an optimum quantity of Smix and distilled
water. In addition, formulation factors such as maximum oil
solubility, globular size, stress stability and thermodynamic
stability studies were also considered as contributing factors

0.22 ± 0.01
0.23 ± 0.01
0.30 ± 0.06
0.39 ± 0.08
during final selection of the formulation for in vitro perme-

PDI
ation studies.
Due to lipophilic nature of QUR, an appropriate solvent
with maximum solubilizing property was necessary, hence dif-

Viscosity (cp)
ferent oils, surfactant and cosurfactant were evaluated.

121 ± 13
128 ± 16
137 ± 17
139 ± 23
For oil selection; oleic acid was found the most suitable
due to added advantages, that is, maximum QUR solubility
(199.4 mg/mL) and powerful transmembrane delivery enhan-
cer as it forms domains hence increases intracellular lipid bar-

5.6 ± 0.27
5.7 ± 0.28
5.7 ± 0.39
6.4 ± 0.41
pH
riers fluidity in the stratum corneum [25]. For surfactant and
cosurfactant; QUR revealed maximum solubility in Tween 80,
PEG 400, labrasol, carbitol, Transcutol HP, Cremophore EL,

Thermodynamic
PEG 200, tween 20, Span 20 and ethyl alcohol. The selection

stability
was based mainly on the basis of HLB values, drug solubility,
safety as well as stability profile. Non-ionic surfactants are

Pass
Pass
Pass
Pass
well known for their highly stable and non-toxic nature [26]
and hence, use of non-ionic surfactant for NE formulation is

Transmittancea
on raise nowadays. Based on the maximum nanoemulsion

98.06 ± 0.39
99.3 ± 0.44
99.0 ± 0.47
99.1 ± 0.39
(%)±SEM
region, seen with the Surfactant/cosurfactant, Tween-80/PEG-
400 strived for the selection of QNE formulation.

Table 1. Final compositions and characterization of quercetin nanoemulsion (QNE) selected for in vitro permeation studies.
More than 71 nanoemulsions were prepared based on dif-
ferent concentration ratio of Tween20:PEG400 and

Zeta potential

17.26 ± 1.04

19.48 ± 1.06

13.12 ± 0.79

19.64 ± 1.08
Labrasol:PEG400 whereby a QNE was finalized for In-vitro per-

(mV)±SEM
meation studies (Table 1).
The selected nanoemulsions (QNE5, QNE33, QNE51 and
QNE61) were prepared and further characterized for drug
content, pH, globule size and distribution, zeta potential and
161.96 ± 14.67
Globule size

90.32 ± 4.36
112.38 ± 7.64
145.64 ± 9.45
viscosity (Table 1). The results observed were as follows:
(nm)±SEM

Quercetin content {QNE5 (99.84%), QNE33 (98.44%), QNE51

(97.16%) and QNE61 (98.39%)}, pH (within the nasal pH
range, i.e. 5.6–6.4, globule size range (90.32–161.96 nm), zeta
potential (
13.12 to
19.64), whereas polydispersibility indi-
AQ (%)
44
40
50
40

ces found were {QNE5 (0.22), QNE33 (0.23), QNE51 (0.30) and
QNE61 (0.39). Hereby, it was concluded that the nanoemul-
sions attained a monodisperse stable system and is able to
Smix (%)
38
34
35
40

effectively deliver the drug owing to large surface area.

Oil (%)

Preparation of quercetin mucoadhesive nanoemulsion

18
26
15
20

(QMNE)
Mucoadhesive nanoemulsions were prepared by the addition
Values are represented as mean ± SD, n ¼ 3
Formulation

of mucoadhesive polymer such as chitosan (showing max-

33
51
61
5

imum strength, selection of mucoadhesive agent) in order to

QNE
QNE
QNE
QNE

make the nanoemulsion optically clear [27]. For the prepar-

ation of mucoadhesive nanoemulsions, initially, a minimum
(Tween80: PEG400)
(Tween80: PEG400)
(Labrasol: PEG400)
(Labrasol: PEG400)

volume of the external phase was used to solubilize the drug

followed by the addition of required amount of polymer solu-
tion to make a final concentration. QMNE was prepared as
described in the section “QUR nanoemulsion preparation”,
whereas chitosan was added in a concentration of 0.25%,
2:1
3:1
1:3
1:2
System

0.50%, 1.0% w/v with continuous stirring for 30 min. The

Smix
Smix
Smix
Smix

preparation stages for nanoemulsion and mucoadhesion

a

Figure 1. Dynamic light-scattering techniques for determining the particle size distribution of quercetin mucoadhesive nanoemulsion globule size (A), zeta potential
(B), scanning electron microscopy (SEM) (C), and transmission electron microscopy (TEM) (D) images of quercetin mucoadhesive nanoemulsion.
involved the addition of rhodamine (Rd) in order to see the
effects of colour changes for result calculation [14].
Ex vivo permeation studies
In order to compare the release of drug, ex vivo permeation
studies were performed across nasal mucosa for QUR (same
drug quantity) nanoemulsions, that is, QNE5, QNE33, QNE51
and QNE61) as shown (Table 1). Ex vivo permeation profile
observed for QNE across nasal mucosa was as; highest for
QNE5, intermediate for QNE33 and 61 whereas lowest for
QNE51 (Figure 3(A)). The nasal permeation profile with signifi-
cant difference was observed for QNE5 when compared to
QNE51 (p < .05) as shown in Figure 3(A). The significant differ-
ence in quercetin permeation between nanoemulsion formu-
lations was probably due to the mean size of internal phase
droplets, which were significantly smaller in nanoemulsions,
whereas the maximum permeation by QNE5 may be attrib-
uted due to lowest droplet size and less viscosity as com-
pared to remaining nanoemulsions.
Hence, based on the aforementioned results along with ex
vivo permeation study (Table 1 and Figure 3(A)), QNE5 was
selected for further pharmacokinetic and pharmacodynamic
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5
study. In addition, globule size (91.63 nm), PDI (0.224) and
zeta potential (
17.26 mV) as shown (Figure 1(A,B)) were con-
firmed by TEM (Figure 1(D)) and SEM (Figure 1(C)),
respectively.
Qualitative nasal mucosa retention studies of
nanoformulations using CLSM
In order to observe disposition pattern for nanoemulsions in
the nasal mucosa, cross section of goat nasal mucosa was
examined by CLSM. Briefly; confocal images of different cross
sections were washed with buffer and exposed to Rd-nanoe-
mulsion for qualitative assessment whereby an intense red
coloured fluorescent areas (located in between and inside the
mucosal cells) were observed. QMNE exhibited more intense
red-coloured area as compared to QNE due to mucoadhesive
nature as shown in Figure 2.
The in vitro diffusion study, with an aim to assess the drug
release profile, was performed on excised goat mucosa using
a biological membrane simulating the actual in vivo barrier to
drug permeation. The results, for % cumulative drug perme-
ated across nasal mucosa from QNE and QMNE, calculated as
shown (Figure 3(A,B), respectively) revealed the following
6 N. AHMAD ET AL.

Figure 2. CLSM images of quercetin non-mucoadhesive nanoemulsion (QNE) and quercetin mucoadhesive nanoemulsion (QMNE).

Figure 3. (A) Ex vivo permeation profile of quercetin non-mucoadhesive nanoemulsion (QNE) from TNE5, TNE33, TNE51 and TNE 61. (B) ex vivo permeation profile of
quercetin mucoadhesive nanoemulsion (QMNE) from QNE5, QNE (0.25%), QNE (0.50%) and QNE (1.0%).

order for in vitro and ex vivo permeability for quercetin nano-
emulsion; QNE 5 > QNE 33 > QNE 51 > QNE 61.
Finally, QNE 5 is the best final quercetin mucoadhesive
nanoemulsion (QMNE) for pharmacokinetic and pharmacody-
namic study. QMNE shown in vitro release of NE and MNE
system in nasal mucosal membrane demonstrated prompt
and effective release with more than 60% of drug release
in 5 h.
Furthermore, in order to enhance the penetration of nano-
emulsion in the nasal cavity, chitosan was added. Chitosan as
a mucoadhesive agent increases the partition/permeation of
drug in the biological membranes via increased contact time
and aids in opening of the nasal tight junctions thus more
drug is accessed into the brain. Hence, the bioavailability and
therapeutic effects are potentiated due to addition of
chitosan.
Nanoemulsions and mucoadhesive nanoemulsions of quer-
cetin was successfully prepared by aqueous titration method.
NEs of QUR has small globule size (<100 nm) as confirmed
via TEM (Figure 1(D)) and negative zeta potential (Figure 1(B))
along with spherical surface as confirmed via SEM (Figure
1(C)). The pH (5.6–6.4) as well as viscosity (50–150 cps) of NEs
and MNEs were compatible with nasal fluid.
Bioanalytical method development and validation by
UPLC/ESI-Q-TOF-MS/MS
The bioanalytical method was developed and validated are
shown in Figure 4 (MS and MS/MS scans of analyte and IS,
respectively), whereas chromatograms shown in Figure 5 cor-
respond to blank brain homogenate [A], extracted brain hom-
ogenate quercetin (BHQ) [B], extracted lungs homogenate
quercetin (LHQ) [C], plasma-extracted quercetin (PLQ) [D],
extracted brain homogenate rutin (BHR) [E], plasma-extracted
rutin (PLR) [F]. The mean recovery (n ¼ 6) of QUR from plasma
and brain homogenates was >83% and the method found
was as r2 > 0.998, for PLQ and BHQ over the range of
1–2000 ng/mL for quercetin. Representative chromatograms
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 7

Figure 4. Mass spectrum of (A) quercetin parent ion (deprotonated precursor [M–H]- ions at m/z 301.04) and (B) Quercetin product ion (major fragmented product
ion at m/z 151.03) showing fragmentation transitions. Mass spectrum of, (C) Rutin (IS) precursor ion (deprotonated precursor [M-H]- ions at m/z 609.21 and (D) IS
product ion (major fragmented product ions at m/z 299.21) showing fragmentation transitions.

Figure 5. Typical chromatograms of blank brain homogenate (A), extracted brain homogenate quercetin (BHQ) (B), Extracted lungs homogenate quercetin (LHQ)
(C), plasma extracted quercetin (PLQ) (D), extracted brain homogenate rutin (BHR) (E), plasma extracted rutin (PLR) (F) (IS).
 8

Table 2. Precision and accuracy data of quercetin in different biomatrices

Intra-batch precision Inter-batch precision
Quality Theoretical
controls concentration Observed concentration Precisionb Observed concentration Precisionb
Biomatrix samples (ng/mL) or (ng/g) (ng/mL) or (ng/g)±SD. Accuracya (%) (%C.V.) (ng/mL) or (ng/g)±S.D. Accuracya (%) (%C.V.) Recoveryc (%)
Brain homogenate LLOQQC 1.01 0.97 ± 0.011 96.03 1.13 0.95 ± 0.017 94.05 1.79 86.15
LQC 2.90 2.75 ± 0.051 94.82 1.85 2.84 ± 0.058 97.93 2.04 84.12
MQC 800.00 792.30 ± 4.97 99.03 0.62 788.14 ± 3.841 98.51 0.49 83.04
N. AHMAD ET AL.

HQC 1600.00 1588.17 ± 4.09 99.26 0.26 1584.27 ± 5.011 99.02 0.32 82.97
Lungs homogenate LLOQQC 1.01 0.93 ± 0.019 92.07 2.04 0.94 ± 0.022 93.07 2.34 84.65
LQC 2.90 2.79 ± 0.042 96.20 1.50 2.79 ± 0.013 96.20 0.47 85.28
MQC 800.00 779.87 ± 4.99 97.48 0.64 777.99 ± 5.21 97.25 0.66 84.02
HQC 1600.00 1577.02 ± 6.01 98.56 0.38 1588.37 ± 2.97 99.27 0.19 85.36
Plasma LLOQQC 1.01 0.91 ± 0.041 90.09 4.50 0.91 ± 0.007 90.09 0.77 83.95
LQC 2.90 2.74 ± 0.055 94.48 2.00 2.79 ± 0.021 96.20 0.75 85.67
MQC 800.00 778.89 ± 4.098 97.36 0.53 781.29 ± 4.011 97.66 0.52 87.02
HQC 1600.00 1589.09 ± 3.98 99.32 0.25 1579.01 ± 5.214 98.69 0.33 85.68
Values (Mean ± SD) are derived from six replicates:
a
Accuracy (%) ¼ Mean value of [(mean observed concentration)/(theoretical concentration)]  100;
b
Precision (%): Coefficient of variance (percentage) ¼ standard deviation divided by mean concentration found 100;
c
Recovery (%) ¼ Mean value of (peak height (mV) obtained from extracted biological sample)/(peak height (mV) obtained from aqueous sample)  100.

Table 3. Stability data of quercetin in different biomatrices

LQC(2.90 ng/mL or ng/g) MQC(800 ng/mL or ng/g) HQC (1600 ng/mL or ng/g)
Exposure condition Brain homogenate Lungs homogenate Plasma Brain homogenate Lungs homogenate Plasma Brain homogenate Lungs homogenate Plasma
Long-term stability; recovery (ng) after storage (280  C)
Previous day 2.83 ± 0.05 2.82 ± 0.027 2.76 ± 0.024 781.36 ± 3.01 780.02 ± 3.09 786.34 ± 4.36 1578.00 ± 2.80 1572.35 ± 3.01 1576.65 ± 3.01
30th Day 2.79 ± 0.06 (98.58%) 2.78 ± 0.039 (98.58%) 2.72 ± 0.078 (98.55%) 779.65 ± 4.69 (99.78%) 778.36 ± 4.02 (99.78%) 775.36 ± 3.64 (98.60 %) 1571.06 ± 2.81 (99.56%) 1569.35 ± 3.07 (99.80%) 1571.59 ± 3.00 (99.68%)
Freeze–thaw stress; recovery (ng) after freeze–thaw cycles (280–25  C)
Pre-cycle 2.84 ± 0.05 2.79 ± 0.048 2.84 ± 0.049 786.36 ± 3.68 788.96 ± 4.05 789.25 ± 3.02 1582.65 ± 2.33 1583.67 ± 3.59 1581.55 ± 2.99
First cycle 2.75 ± 0.05 (96.83%) 2.76 ± 0.033 (98.92%) 2.81 ± 0.031 (98.94%) 774.62 ± 3.65 (98.50%) 779.68 ± 3.67 (98.82%) 782.34 ± 3.68 (99.13%) 1581.30 ± 2.22 (99.91%) 1581.21 ± 2.09 (99.84%) 1580.10 ± 2.86 (99.24%)
Second cycle 2.74 ± 0.07 (96.47%) 2.76 ± 0.016 (98.20%) 2.77 ± 0.078 (97.53%) 772.35 ± 4.37 (98.22%) 779.36 ± 2.98 (98.78%) 781.38 ± 3.59 (99.00%) 1579.66 ± 2.07 (99.81%) 1574.39 ± 2.08 (99.41%) 1578.39 ± 3.01 (98.80%)
Third cycle 2.73 ± 0.09 (96.12%) 2.74 ± 0.039 (98.20%) 2.71 ± 0.007 (95.42%) 769.26 ± 2.61 (97.83%) 772.65 ± 3.65 (97.93%) 781.05 ± 3.82 (98.96%) 1577.37 ± 2.79 (99.67%) 1571.38 ± 2.03 (99.22%) 1572.66 ± 2.98 (99.44%)
Bench top stability; recovery (ng) at room temperature (25  C)
0h 2.82 ± 0.05 2.81 ± 0.039 2.78 ± 0.041 779.99 ± 3.85 780.39 ± 4.09 782.96 ± 3.65 1583.65 ± 2.64 1581.64 ± 2.05 1587.65 ± 2.36
24 h 2.80 ± 0.04 (99.29%) 2.77 ± 0.039 (98.57%) 2.73 ± 0.046 (98.20%) 780.55 ± 2.451 (99.29%) 778.64 ± 2.98 (99.41%) 773.68 ± 2.94 (97.65%) 1579.36 ± 2.56 (99.73%) 1578.69 ± 2.36 (99.82%) 1569.99 ± 2.48 (98.89%)
Post-processing stability; recovery (ng) after storage in auto sampler (4  C)
0h 2.81 ± 0.09 2.83 ± 0.028 2.78 ± 0.042 782.75 ± 2.98 783.65 ± 3.65 786.09 ± 3.08 1591.02 ± 2.56 1579.35 ± 2.38 1584.56 ± 2.03
4h 2.79 ± 0.08 (99.65%) 2.82 ± 0.043 (98.58%) 2.73 ± 0.036 (97.49%) 781.06 ± 2.531 (99.02%) 779.28 ± 3.65 (99.45%) 781.65 ± 2.56 (98.07 %) 1586.26 ± 2.05 (99.70 %) 1577.64 ± 3.04 (99.89%) 1574.36 ± 3.08 (99.36%)
Values (Mean ± SD) are derived from six replicates. Figures in parenthesis represent analyte concentration (%) relative to time zero. Theoretical contents; LQC: 2.9 ng/mL; MQC: 800 ng/mL; and HQC: 1600 ng/mL.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 9

of extracted blank plasma and blank brain homogenates forti-
fied with quercetin revealed selectivity of the method. To
summarize inter-day and intra-day precision and accuracy;
the intra-batch and inter-batch %CV for all the QC levels of
quercetin obtained for LH, BH and PL was between 0.25–4.50,
0.19–2.34, whereas intra-batch and inter-batch accuracy
found for all QC levels of quercetin was within the range of
90.09–99.32 and 90.09–99.27% for LH, BH and PL, respectively
(Table 2). Furthermore, the results regarding stability experi-
ments for quercetin (Table 3) revealed proper stability of
quercetin at all storage conditions, that is long-term, freeze-
thaw, bench-top and post-processing stability (Table 3) [14].
with i.n. QUR (10 mg/kg; p < .001) MCAO group exhibited spon-
taneous comparative activity. However, locomotor activity was
improved highly with QMNE (p < .001) and QUR (p < .05) solu-
tion at a dose of 10 mg/kg, as compared to MCAO group.
Effect on grip strength
A significant decrease in grip strength was observed in MCAO
group (p < .001) as compared to sham group, whereas rats
pre-treated with QUR- solution (p < .05) and QMNE (p < .001)
at a dose of 10 mg/kg (Figure 6(C)) showed more improvement
in grip strength compared to MCAO group significantly.

Pharmacokinetics, biodistribution, %DTE and %DTP Morphological changes

The pharmacokinetic parameters (Cmax, Tmax, t1/2, Ke and
AUC0–t) obtained after i.n. and i.v. administration of QMNE
compared with pure quercetin solution is presented in
Table 4. A significantly higher AUC0–t was observed after
QMNE administration compared to pure quercetin in all three
areas (brain, lungs and plasma). Brain/plasma ratio (Table 4)
revealed a higher brain concentration following i.n. adminis-
tration as compared to i.v. administration. In addition, %DTE,
%DTP and AUCi.n/AUCi.v. (Tables 4,5, Figure 6(A)) further sup-
ports the fact; i.n. administration results enhanced brain bio-
availability for developed QMNE.
The histopathological changes in neuron, after ischaemia–re-
perfusion injury, were examined through haematoxylin–eosin
staining. No pathological change was observed for normal
cells in Sham group sections, whereas MCAO group sections
showed a focus of brain damage with neuronal loss and the
presence of numerous vacuolated spaces as well as the
absence of intact neurons. The sections from MCAO þ QMNE
group showed partial neuronal loss with the presence of
intact neurons in between the vacuolated spaces in the corre-
sponding area. The neuronal abnormalities in the
MCAO þ QMNE group were ameliorated with QMNE treat-
ment as compared with the MCAO group animals (Figure 7).

Pharmacodynamic study in cerebral ischaemia

Effect on locomotor activity
The spontaneous locomotor activity was observed as men-
tioned in the methodology section. In MCA-occluded rats, a sig-
nificant reduction in locomotor count was observed (p <.001)
as compared to sham group (Figure 6(B)), whereas pre-treated
Effect of quercetin on TTC stain and infarction volume
The fact that quercetin solution and QMNE have a protective
role in stroke was supported by the observations in MCAO
group where a significantly increased infarct volume was
seen as compared with sham. As shown in Figure 8(A), TTC
(2,3,5-triphenyltetrazolium chloride) staining of MCAO brain

Table 4. Pharmacokinetic studies of QMNE after i.n. and i.v. administration to rats at the dose of 10 mg/kg in brain, lungs and plasma (n ¼ 6, mean ± SD).
Formulation sdministration Samples Cmax(ng/mL g) Tmax t1/2 (h) Ke (h
1) AUC0-t (ng min/mL g)
QUR (i.n.) Brain 202.10 ± 11.27 2.00 21.98 ± 3.09 0.03154 ± 0.008 2540.60 ± 199.76
Lungs 24.66 ± 2.98 2.00 10.79 ± 1.93 0.06426 ± 0.0009 216.84 ± 22.63
Plasma 61.59 ± 3.92 1.0 6.60 ± 0.48 0.10506 ± 0.0061 453.02 ± 28.10
QUR (i.v.) Brain 109.86 ± 12.01 2.00 7.85 ± 0.39 0.08831 ± 0.0038 1075.80 ± 71.92
Lungs 21.65 ± 2.63 2.00 9.20 ± 0.78 0.07535 ± 0.0064 172.12 ± 11.79
Plasma 1852.49 ± 46.92 1.00 7.90 ± 0.36 0.08770 ± 0.0014 18718.37 ± 388.76
QMNE (i.n.) Brain 1788.68 ± 3.67 2.00 36.46 ± 11.63 0.01901 ± 0.00037 24893.11 ± 368.85
Lungs 93.22 ± 6.89 2.00 10.96 ± 4.10 0.06323 ± 0.00389 1129.52 ± 177.27
Plasma 404.22 ± 6.98 2.00 29.61 ± 0.34 0.02341 ± 0.00621 6350.80 ± 166.56
QMNE (i.v.) Brain 374.59 ± 19.60 2.00 36.68 ± 7.41 0.01890 ± 0.00289 4704.76 ± 188.56
Lungs 80.10 ± 6.78 2.00 11.07 ± 1.38 0.06264 ± 0.0028 929.75 ± 102.75
Plasma 1828.00 ± 24.87 1.00 22.48 ± 1.96 0.03083 ± 0.00378 24988.47 ± 235.67
QUR (i.n.) Brain/Plasma 3.28 2.00 3.33 0.30 5.60
QUR (i.v.) Brain/Plasma 0.06 2.00 0.99 1.00 0.06
QMNE (i.n.) Brain/Plasma 4.43 1.00 1.23 0.81 3.92
QMNE (i.v.) Brain/Plasma 0.20 2.00 1.63 0.61 0.19

Table 5. Drug-targeting efficiency and direct nose-to-brain transport following intranasal administration of different formulations.
Comparative bioavailabilitya (AUCi.n./AUCi.v.); (%)
Drug-targeting Direct nose-to-brain
Formulations efficiency (%DTE)a transport (%DTP)a Blood Brain
Quercetin solution 2063.63 ± 5.98 546.75 ± 1.05 2.42 ± 0.06 236.16 ± 2.38
QMNE 9333.33 ± 39.39 2181.83 ± 15.69 25.41 ± 2.08 529.10 ± 38.04
a
Parameters are derived using mean ± SEM values of six different estimations.
10 N. AHMAD ET AL.

Figure 6. Pharmacokinetic profiles of quercetin concentration in brain at different time intervals after administration of developed QMNE compared with pure quer-
cetin. B,C: Graph showing results of various groups treated with QUR on locomotor activity, grip strength in middle cerebral artery occluded (MCAO) rats. Data repre-
sented as mean ± SEM of six animals. Significance was determined as p < .001 when compared with sham group; #p < .05 and ###p < .001 when compared with
MCAO group.

sections showed reproducible and readily detectable lesions
in the areas that are supplied by the MCA (after 22 h of reper-
fusion). The images for brain sections stained with 0.1% TTC
as well as measurement of infarct volumes for MCAO,
MCAO þ QUR solution and MCAO þ QMNE group are shown
in Figure 8(B). According to observations; MCAO group devel-
oped significant lesion compared with sham group, QUR solu-
tion and QMNE pre-treatment significantly reduced the
infarct volume as compared with MCAO group; however,
MCAO þ QUR solution group and MCAO þ QMNE group
revealed a significant (p < .05, p < .01) reduction in tissue
damage as compared to MCAO group.
Discussion
Since last few years, the concept of nanotechnology, to
improve bioavailability and targeted delivery of pharmaco-
logically active drugs particularly lipophilic drugs to disease
site, is under consideration however still numerous challenges
and hurdles exist [28,29].
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 11

Figure 7. Effect of quercetin mucoadhesive nanoemulsion (QMNE) (10 mg/kg) administration on haematoxylin and eosin staining in the brain sections of the sham,
MCAO, and MCAO þ QMNE groups. (A) Cortical area of sham group animal showed uniform distribution of neurons. Normal neurons with the characteristic conical
outlines with no abnormal features are seen. (B) Tissues around infarcted area in the MCAO group show a focal area of vacuolation and neuronal loss. (C) the
MCAO þ QMNE group rats show partial neuronal loss. (D) Quantification of neuronal damage of sham, MCAO and MCAO þ QMNE groups. Original magnification
20 and scale bar ¼20 lm.

Figure 8. Effect of quercetin pre-treatment for 21 days on brain infarct size by TTC stain after middle cerebral artery occlusion for 2 h and reperfusion of 22 h. A.
Representative photographs of brain sections stained with 0.1% TTC, and measurement of infarct volumes of MCAO, MCAO þ QUR solution group and
MCAO þ QMNE group are presented (A). MCAO group produced a significant lesion over sham group. B. However, MCAO þ QUR solution group and MCAO þ QMNE
group showed a significant (p < .05, p < .01) reduction in tissue damage as compared to MCAO group (B).

Different strategies, for bioavailability enhancement, have
been employed so far in this regard such as topical route
application [30], oral route of application [12], in vitro toxicity
study [31] as well as the study of QUR nanoemulsion for sur-
face methodology determination [32]; however, the accumu-
lation of flavonoids in the brain due to blood–brain barrier
(BBB) restriction is still a controversial question [33]. Various
literature reports the less access of flavonoids in the brain
due to BBB [33], but on contrary free QUR has been found to
accumulate more in brain tissues and exert better antioxida-
tive effects [33].
Present study aimed to enhance bioavailability of flavo-
noids, that is, QUR via preparation of QMNE for intranasal
delivery using different oil, surfactants and cosurfactants.
12 N. AHMAD ET AL.
This is the first time study of its nature for the prepar-
ation of QMNE and its application via i.n. route. The driv-
ing concepts for the development of QMNE and its
application using i.n. route includes; the use of Oleic acid
i.e. a powerful transmembrane delivery enhancer due to
the formation of domains in stratum corneum whereby flu-
idity of intracellular lipid barriers increases [25], the protec-
tion of encapsulated drug from chemical, biological and
extracellular transport by P-gp efflux proteins hence
increasing CNS drug availability, small diameter of nanofor-
mulations leading to more transcellular transport of drugs
through olfactory neurons into the brain via endocytic
pathway [14].
When the Cmax and AUC of brain concentrations of QUR
(i.n.), QUR (i.v.) and QMNE (i.n.) were compared, the Cmax
(1788.68 ± 3.67 ng/mL g) and AUC (24893.11 ± 368.85 ng
min/mL g) of QMNE were found to be significantly higher
because of the direct transport of drug through olfactory
route by bypassing the BBB. In addition to this, another rea-
son is the mucoadhesive nature of CS that decreased the
mucociliary clearance, which under normal circumstances
rapidly clears the instilled formulation. When we compared
the AUC in brain of QMNE (i.n.) and QUR solution (i.v.), it
was found to be nearly 8.85 times higher with QMNE (i.n.),
whereas 9.8 times higher AUC was found with QUR solution
(i.n.). This result reveals that the drug uptake into the brain
from the nasal mucosa mainly occurs via two different path-
ways. One is the systemic pathway by which some of the
drug is absorbed into the systemic circulation and subse-
quently reaches the brain by crossing the BBB. The other is
the olfactory pathway by which the drug partly travels from
the nasal cavity to CSF and/or brain tissue [2,14]. The results
of the current study revealed an increase in % DTP values
of QMNE (i.n.) as compared to QUR solution, that is, from
546.75 ± 1.05 to 2181.83 ± 15.69% as well as bioavailability,
that is, 529.10 ± 38.04 and 236.16 ± 2.38, respectively, for
QMNE and QUR solution (Table 5) and hence confirming i.n.
route as a direct pathway for the delivery of drug from
nose-to-brain [34]. Our findings were in concordance with
the previous studies, that is, enhanced drug delivery of cur-
cumin to CSF and CNS after i.n. administration of PNIPAM-
NPs [16] and higher uptake of i.n. administered curcumin
from nanoformulations compared to solution form as
reported [14]. These findings could be related to the
mucoadhesive nature of nanoformulations, which prolong
the residence time in the nasal cavity, thus provides the
opportunity for transient opening of tight junctions leading
to enhanced permeation as well as sustained drug delivery
to the brain. These results support the concept; QMNE
administered i.n. transport the drug directly to the brain
and enhances the bioavailability as well as targeting in the
brain.
This study reports for the first time a sensitive UPLC-ESI-Q-
TOF-MS/MS method with more efficiency in terms of high
sensitivity (picogram level) as well as wide application in
terms of economy, validated method and shorter retention
time (<3.0 min.). Furthermore, the developed method was
validated and successfully applied for brain pharmacokinetics
as well as biodistribution studies.
Conclusions
Enhanced brain bioavailability at low dose i.n. QMNE, for the
effective treatment of cerebral ischaemia was established in
this study. It was observed that QMNE could bypass the BBB
and enters the brain preferentially, decreasing undesirable
systemic effects thus seems to be a promising strategy for
the treatment of cerebral ischaemia. UPLC-ESI-Q-TOF-MS/MS
based bioanalytical method was developed, validated and
successfully applied for the pharmacokinetic and biodistribu-
tion studies of developed QMNE. Furthermore, QMNE was
evaluated in MCAO-induced cerebral ischaemia rat’s model
for locomotor activity, grip strength and histopathological
examination with infarction volume whereby significant
results were observed. To conclude; QMNE is a novel, non-
invasive, effective and safe brain-targeted delivery system for
cerebral ischaemia treatment however more pre-clinical and
clinical studies may be performed to prepare a formulation
with low-risk/high-benefit ratio.
Acknowledgements
I (Dr. Niyaz Ahmad) am grateful to Prof. (Dr.) Farhan Jalees Ahmad for
the collaboration research study in between University of Dammam,
Dammam, Saudi Arabia and Jamia Hamdard (Hamdard University), New
Delhi, India.
Disclosure statement
No conflict of interests exists among authors and no grants were
received.
ORCID
Niyaz Ahmad http://orcid.org/0000-0003-1874-6006
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