Drug Development and Industrial Pharmacy

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Formulation and optimization of intranasal

nanolipid carriers of pioglitazone for the
repurposing in Alzheimer’s disease using Box-
Behnken design

Gifty M. Jojo, Gowthamarajan Kuppusamy, Anindita De & V. V. S. Narayan

Reddy Karri

To cite this article: Gifty M. Jojo, Gowthamarajan Kuppusamy, Anindita De & V. V. S. Narayan
Reddy Karri (2019): Formulation and optimization of intranasal nanolipid carriers of pioglitazone
for the repurposing in Alzheimer’s disease using Box-Behnken design, Drug Development and
Industrial Pharmacy, DOI: 10.1080/03639045.2019.1593439

To link to this article: https://doi.org/10.1080/03639045.2019.1593439

Published online: 28 Mar 2019.

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DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY
https://doi.org/10.1080/03639045.2019.1593439

RESEARCH ARTICLE

Formulation and optimization of intranasal nanolipid carriers of pioglitazone

for the repurposing in Alzheimer’s disease using Box-Behnken design
Gifty M. Jojo, Gowthamarajan Kuppusamy, Anindita De and V. V. S. Narayan Reddy Karri
Department of Pharmaceutics, JSS College of Pharmacy, Ootacamund, JSS Academy of Higher Education and Research, Mysuru, India

ABSTRACT ARTICLE HISTORY

Growing evidence suggest that Alzheimer’s disease (AD), the most common cause of dementia among Received 14 October 2018
the elderly is a metabolic disorder associated with impaired brain insulin signaling. Hence, the diabetic Revised 4 March 2019
drug can be a therapeutic option for the management AD. The researches in this area are ongoing and Accepted 4 March 2019
Pioglitazone (PIO) is one of the most investigated diabetic drug in AD. Eventhough PIO treatment was
KEYWORDS
found to improve AD significantly in the preclinical models, the poor blood-brain barrier (BBB) permeabil- Intranasal route;
ity and serious peripheral side effects limited its success in the clinical trials. The objective of the present pioglitazone; nanolipid
study was to formulate and optimize intranasal (IN) nano lipid carriers (NLC) of PIO for its targeted deliv- carriers; Box-Behnken
ery to the brain. A Box-Behnken design was employed to optimize the effect of three independent varia- design; Alzheimer’s disease
bles on two dependent variables. The optimized formulation had a particle size (PS) of 211.4 ± 3.54 nm
and zeta potential of (ZP) of 14.9 ± 1.09 mv. The polydispersibility index (PDI) and entrapment efficiency
(EE) was found to be 0.257 ± 0.108 and 70.18 ± 4.5% respectively. Storage stability studies performed has
confirmed the stability of NLCs at 4
C and 25
C. The in-vitro drug release study has exhibited a sustained
release of drug from the NLC. The formulation was observed to improve the nasal permeability of PIO
ex-vivo significantly. Toxicity studies were performed to confirm the safety of formulation for the in-vivo
administration. In-vivo biodistribution study in rats has shown a direct transport of drug from the nose to
brain from the IN-NLC.

Introduction improvement in cognition, oxidative stress and cerebral glucose

Alzheimer’s disease (AD), the most common cause of dementia
among the elderly is an age-related progressive neurodegenera-
tive disorder [1]. Until now the exact cause of the disease is
unknown and there is no medicine that prevents or stop the dis-
ease progressing [2]. Growing evidence suggested that AD is a
metabolic disorder associated with impaired brain insulin signal-
ing; hence also called as type 3 (diabetes mellitus) DM. Insulin
resistance/deficiency in the brain was identified as the key factor
behind the AD development and this endogenous brain impair-
ment can occur even in the absence of type 2 DM [3,4]. These
findings opened the door towards the extensive researches in the
area of “repurposing of diabetic drugs for AD management” since
it will take around 14 years and a cost of about US Dollar 2.6 bil-
lion to bring a new molecule to the market approval. The repur-
posed drug already passed many of the initial studies can bypass
much of the early time and cost [5].
Among the diabetic drugs, PIO, a PPARc agonist class of insulin
sensitizer is one of the most investigated in AD [6]. In addition to
improving the brain insulin signaling, PIO was found to have dir-
ect role in controlling multiple targets involved in AD [7–10]. One
of the earliest study conducted in Mesencephalic neuron-microglia
mixed culture has reported a noticeable reduction of oxidative
stress via the activation of PI3K/Akt pathway and the down-
regulation of p38 mitogen-activated protein kinase (p38 MAPK)
pathway with PIO treatment [11]. Later, an invivo investigation in
streptozotocin induced-memory impaired rats was observed with
utilization with PIO treatment [12]. Further exploration of PIO to
the animal model of AD has shown improvement in various
biomarkers such as Ab (Amyloid beta) plaques, oxidative stress,
neuroinflammation and NFT (neurofibrillary tangles) formation
[13–16]. The drug was reported to normalize the hyperactivation
of cyclin-dependent kinase 5 (Cdk5) in AD models [10]. Most
recently, a combination of intranasal leptin with oral PIO in a
mouse model of AD was found to improve the spatial memory
deficits, Ab deposit, synapse loss and neocortical glial response
[17]. However, the later clinical trials conducted with usual dia-
betic doses of PIO has shown only limited success. The primary
factor that has limited the therapeutic success of PIO at the clin-
ical stage was the inadequate concentration of drug reaching the
brain. Evidences says that PIO permeate BBB poorly [13,18,19] and
is also a substrate for the efflux transporters present at the BBB
[20–23]. Higher concentration of the substrate drug are required
to overcome the transporter activity to achieve a therapeutic con-
centration at the site of action and hence to execute the max-
imum therapeutic response [24]. For that reason, all the preclinical
trials were conducted with doses higher than the usual diabetic
dose. However, higher doses in human volunteers are restricted
due to the serious-dose dependent peripheral side effects of PIO
such bladder cancer and peripheral edema [25].
The present situation enriches the scope of novel techniques
for efficiently delivering PIO to the brain. Even though different
techniques are available for targeting the brain, intranasal route

CONTACT Gifty M. Jojo giftymjojo@gmail.com Department of Pharmaceutics, JSS College of Pharmacy, Ootacamund, JSS Academy of Higher Education and
Research, Mysuru, India; Gowthamarajan Kuppusamy gowthamsang@jssuni.edu.in Department of Pharmaceutics, JSS College of Pharmacy, Ootacamund, JSS
Academy of Higher Education and Research, Mysuru, India
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 G. M. JOJO ET AL.
(IN) of administration came to the front as it is the noninvasive-
direct route to the brain that bypasses BBB. It is possible to
achieve a significantly high concentration of the drug in the brain
from a lower dose via this direct route [26–29]. Formulation
aspects also have seen to play crucial role in determining the bio-
availability of intranasally administered drug. Among the conven-
tional and novel formulation investigated for nose to brain
targeting, nanoformulations are considered to be the most prom-
ising strategy [30,31]. Most recently, a Silva-Abreu et al. have
investigated the permeability of PLGA nanoparticles loaded with
PIO across various mucosal tissues such as buccal, sublingual,
nasal, and intestinal mucosa. The nasal mucosa was appeared to
be an attractive and noninvasive pathway for delivering PIO to
the brain in this preliminary study [32]. However, the study has
limited by the absence of in-vivo evidence and toxicity evalua-
tions. Hence, further studies are necessary to confirm the potential
applicability of IN route for the repurposing of PIO in a biological
system. Also, the toxicity studies have to be performed to confirm
the safety of IN formulations.
Present study has used a lipid-based nanoformulation called
nanolipid carriers (NLC) system to deliver PIO to the brain via IN
route since comparing the polymeric nanocarriers, lipid nanocar-
riers are more biocompatible, safe and easy to scale up [33]. The
high entrapment efficacy, stability and property to accommodate
both lipophilic and hydrophilic molecules made the NLC superior
over other lipid nanocarriers. NLCs has shown better results for the
purpose of targeted drug delivery and nowadays are more consid-
ered for brain targeting [34]. In the present NLC formulation, cap-
mul MCM and tripalmitin were used as liquid and solid lipid
respectively. The surfactant system consisted of a combination of
tween80 and pluronic F68. In order to enhance the bioavailability
of the formulation from the nasal mucosa, the positive charge
inducer stearyl amine (SA) was incorporated. Nanoformulations
with positive zeta potential (ZP) forms mucoadhesion by interact-
ing with negatively charged sialic acid group present on the muco-
sal surface; thereby overcome the mucociliary drainage of the
administered formulation [35]. Optimization of the final formula-
tion was carried out using a Design of experiments (DoE) based
approach. DoE is a systematic and scientific approach to study and
optimize the effect of various independent variables on the
dependent variables of a formulation. In comparison to the con-
ventional methods, DoE allows the effects of several factors to be
assessed by a fewer number of trials [36]. Response surface meth-
odology (RSM) is one of the techniques used by the DoE software
to estimate the main effects and their interactions. There are two
RSM methods called central composite design (CCD) and Box-
Behnken design. In the present work, a Box-Behnken design was
employed since it requires a fewer number of trials to optimize the
formulation parameters compared to the CCD. Thus the design
overcomes the limitations of the conventional methods that are
time-consuming, expensive and laborious [37]. Hence, the frame of
the present work would be using a Box-Behnken design to design
NLC of PIO to further enhance the brain uptake of PIO in-vivo.
Material and methods
Materials
The active ingredient Pioglitazone (PIO) Solid lipid tripalmitin,
were was purchased from BMR Pharma and chemicals, Hyderabad,
India; whereas the liquid lipid MCM was obtained as a gift sample
from Subhash chemicals, Maharashtra, India. The surfactant such
as tween 80 and pluronic F68 obtain from SD fine chemicals,
Bengaluru, Karnataka, India. HPLC-grade methanol, acetonitrile
and ammonium acetate were purchased from Merck, Mumbai,
India. Irbesartan (98.06%) was purchased from NeuconPharma Pvt.
Ltd, Goa, India. membrane filter was purchased from HiMedia,
India. 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (M-5655), Dulbecco’s Modified Eagle Medium (DMEM) and
Dimethyl sulphoxide (DMSO) were purchased from Sigma Aldrich,
USA. Fatal bovine serum (FBS), L-glutamine and sodium bicarbon-
ate were purchased from Merck, Germany.
Methods
Selection of lipids
Solubility study was carried out according to the method reported
previously. Briefly, an excess amount of drug was added to 2 mL
of the oil contained in the 5 mL centrifuge tube and mixed using
vortex mixer (Yorco Instruments, Delhi, India). Then an isothermal
shaker at 25 ± 0.5
C (IKAVR KS400i, Germany) was used to con-
stantly agitate the centrifuge tubes for 72 h in order to reach
equilibrium. The equilibrated samples then transferred to centrifu-
gation machine (Remi, India), where they were centrifuged at
10,000 rpm for 15 min, filtered through a 0.22 mm membrane filter
and the supernatants were analyzed spectrophotometrically using
“reverse-phase high-performance liquid chromatography” (RP-
HPLC) [38,39].The partition coefficient (Pc) study was conducted
for selecting the solid lipid. In this study, 50 mg of the drug was
added to the mixture of with 1 g of solid lipid and 10 mL of dis-
tilled water and the whole components were heated together at
a temperature above 70
C in a test tube. The melted mixture
was then mixed properly using a vortex mixer for 30 min. The
solidification of the melted lipid was prevented by frequent
exposure to the heated water bath and vortex mixer. The mixture
was then centrifuged at 2000 rpm for 30 min at 25
C to separate
the lipid. The aqueous portion obtained was analyzed using
HPLC. The Pc was calculated using the equation
Pc ¼ ðDiDwÞ=Dw
Whereas, Di is the initial amount of drug taken (50 mg) and
Dw is the amount of drug in the aqueous phase [39,40].
Selection of surfactants
Surfactants were selected based on their ability to produce least
particle size (PS) and poly dispensability index (PDI). Surfactants
such as tween 80, pluronic F68, solutol HS15 and hydrogenate dm
Phosphatidylcholine (at a concentration of 0.8%) were investigated
for their effect on PS and PDI [40].
Preparation of nano lipid carriers
The NLC was prepared using microemulsion method. The oil
phase contains the accurately weighed quantities of tripalmitin
(70 mg), MCM (30 mg) and stearyl amine (4.3 mg) were heated to
75
C. PIO (10 mg) was added to the melted lipids under continu-
ous stirring to get a clear phase. The aqueous phase (20 mL) con-
tains the surfactants was heated to the same temperature and
added to the oil phase dropwise under magnetic stirring at an
rpm of 350 for 15 min to get a clear phase. NLC dispersion was
obtained by dispersing the warm o/w microemulsion dropwise
into ice-cold distilled water (50 mL) (external phase) maintained at
(3–4
C) in a beaker under continuous stirring at an rpm of 10,000
for 20 min. Later the formulation was centrifuged at 10,000 rpm
for 10 min and supernatant was discarded. The NLC pellets were

redispersed in Millipore water and centrifuged again to remove
both surfactant and then lyophilized [40,41]. Lyophilization was
carried out using the method developed and optimized in our
laboratory. In brief, 5% w/v of sucrose was added to the NLC as
cryoprotectant. The NLC dispersion was frozen in aqueous cryo-
protectant solution for 24 h at –20
C. Samples then transferred to
the freeze drier (Christ, Alpha 2-4LD plus, Germany) operated at
40
C and pressure of 0.001 bars for 72 h. The freeze-dried sample
was stored for further investigations [40].
Box-Behnken design
Preliminary trials were conducted to investigate the effect of vari-
ous independent variables on the dependent variables. The ratio
of the solid lipid to liquid lipid, stirring speed and stirring time
was fixed from the preliminary runs. A three-factor Box-Behnken
design was employed to optimize the effect of three independent
variables such as total surfactant (X1), percentage of tween 80 in
the surfactant mixture (X2), and the concentration of SA (X3) on
the dependent variables such as particle size (PS, Y1) and zeta
potential (ZP, Y2). Design expert software (Design-Expert 10.0.8
(64-bit) Installer- trial version) was used to analyze the data where
analysis of variance (ANOVA) was conducted to evaluate the sig-
nificance of each factor and their interactions. The upper and
lower limits selected is given in Table 1.
Evaluation of NLC
Particle size, zeta potential, and polydispersibility index. Particle
size (PS), zeta potential (ZP), and polydispersibility index (PDI)
were determined using Malvern Zetasizer system at 25
C. Double
distilled water was used as a dilution medium.
Entrapment efficiency and loading capacity. The entrapment effi-
ciency was determined using RP-HPLC technique according to the
previously published method. The supernatant containing the
unentrapped drug was analyzed with RP-HPLC equipped with a
UV detector at 254 nm (Shimadzu). C18 HPLC column was used as
stationary phase and 10 Mm acetate buffer and acetonitrile (70:30
ratio) used as the mobile phase at a flow rate of 0.8 mL/min [42].
Differential scanning calorimetry (DSC). The DSC study using DSC
Q200 (TA instrument, USA) was performed to investigate the inter-
action between the selected components at a heating rate of
10
C in the range 20–200
C under the nitrogen purge of 50 mL/
min. Samples of 2–8 mg of the pure drug, pure lipid, the physical
mixture of drug and tripalmitin and final formulation were taken
separately in standard aluminum pans for performing the investi-
gation. An empty pan was used as the reference [40].
In-vitro drug release study. In-vitro drug release study was per-
formed using dialysis bad method. The bag having a pore size of
Table 1. Box-Behnken design for optimization of PIO loaded NLC.
Levels
Independent variables 1 0 þ1
Percentage of Total surfactant (TS) 0.4 0.6 0.8
Percentage of tween 80 in surfactant 50 70 90
mixture (% of tween80)
Amount of stearyl amine (SA)(mg) 2.5 3.75 7 at 37
C in a humidified 5% CO2 incubator (NBS Eppendorf,
Dependent variables Constraints
Particle size (PS) (nm) Minimize
Zeta potential (ZP)(mv) Maximize
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3
2.4 nm and molecular weight cut off 12,000–14,000 (Dialysis mem-
brane-150) was used and is activated by soaking in distilled water
for 24 h before use. PIO solution and redisposed drug loaded NLC
(equivalent to 10 mg of PIO) were placed in the bags separately
and sealed at both the ends. The bags were placed in baskets
(USP Dissolution apparatus Type LabIndia, Mumbai) and immersed
in 200 mL of SNF pH 6.4 containing 1% SLS maintained at
37 ± 0.5
C and stirred at 100 rpm. A sample of 5 mL was taken out
from the dissolution vessel at 0, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h,
8 h, 16 h, 24 h 36 h, and 42 h duration and 5 mL fresh buffer were
added every time to maintain the sink condition. Samples were
analyzed using HPLC [40].
Ex-vivo permeation study. The ex-vivo permeation study was per-
formed using Franz diffusion cell with a surface area of 1.79 cm2
and volume of 25 mL (Kovai Glass Works, Coimbatore, India) across
freshly excised sheep nasal mucosa. The fresh excised nasal
mucosa collected from the slaughterhouse in phosphate buffer
saline pH 6.4 was made free from adhered tissues and mounted
between the donor and receptor compartment of the Franz diffu-
sion cell, with mucosal side facing the donor compartment. The
receptor chamber was filled with 20 mL of simulated nasal fluid
(SNF) pH 6.4 containing 1% SLS as the diffusion medium. The
mucosal tissue was allowed to stabilize and stirred under the dif-
fusion medium for 15 min on a magnetic stirrer. The diffusion cell
was thermostated at 37 ± 0.5
C. After 15 min, solutions from both
the compartments were removed, and the receptor compartment
was filled with fresh medium. The diffusion cell was placed in the
diffusion apparatus and the pure drug solution (containing 10 mg
of drug) and lyophilized PIO-NLC reconstituted with SNF (equiva-
lent to 10 mg of PIO) were placed over the donor compartment
under magnetic stirring at an rpm of 600. From the receptor com-
partment, 0.5 mL of media was withdrawn at time intervals 1 h,
2 h, 3 h, 4 h, 5 h, and 6 h and same volume was replaced with fresh
diffusion media maintained at 37 ± 0.5
C at each time of sample
withdrawal. The samples were filtered through 0.45 lm nylon
filter paper and analyzed for the PIO content using RP-HPLC
method [40].
Toxicity studies. Nasal ciliotoxicity study. Nasal ciliotoxicity study
was carried out using the freshly isolated sheep nasal mucosa col-
lected from the slaughterhouse in a phosphate buffered saline
(PBS) pH 6.4. The isolated mucosa divided into different pieces
and each piece was treated with 2 mL of PIO solution in PBS pH
6.4 (1 mg/mL), blank NLC, lyophilized drug loaded NLC (equivalent
to 1 mg/mL of PIO), PBS pH 6.4 (as a negative control) and isopro-
pyl alcohol (IPA) (as a positive control), respectively for 2 h. After
treatment, all the samples were washed with distilled water and
were preserved with 10% formalin until further analysis. Each sam-
ple was sectioned and stained with H and E. The mucosa was
then dissected out, and the mucociliary was examined on an
optical microscope by a pathologist [40].
Cytotoxicity study. Cytotoxicity of the PIO-NLC and the pure drug
was investigated on SHSY5Y (Neuroblastoma) cells lines using MTT
assay method. The cell line was cultured in 25 cm2 tissue culture
flask with DMEM supplemented with 10%) and the antibiotic solu-
tion containing: Penicillin (100 mg/mL), Streptomycin (100 mg/mL),
and Amphotericin B (2.5 mg/mL). Cultured cell lines were kept
Germany). Two days old confluent monolayer of cells were trypsi-
nized and the cells were suspended in 10% growth medium,
100 mL cell suspension (5  104 cells/well) was seeded in 96 well
4 G. M. JOJO ET AL.
tissue culture plate and incubated at 37
C in a humidified 5%
CO2 incubator.
MTT of 15 mg was reconstituted in 3 mL PBS until completely
dissolved and sterilized by filter sterilization. Samples solutions of
2 mg, 4 mg, 6 mg, 8 mg, and 10 mg of pure drug and PIO-NLC dis-
solved in 500 mL of 5% DMEM and filtered through 0.22 mm
Millipore syringe filter to ensure the sterility. After 24 h of the
incubation period, the sample content in wells was removed and
30 mL of reconstituted MTT solution was added to all test and cell
control wells, the plate was gently shaken well, then incubated at
37
C in a humidified 5% CO2 incubator for 4 h. After the incuba-
tion period, the supernatant was removed and 100 mL of MTT
solubilization solution ((Dimethyl sulphoxide (DMSO))) was added
and the wells were mixed gently by pipetting up and down in
order to solubilize the formazan crystals. The absorbance values
were measured by using microplate reader at a wavelength of
540 nm [43].
The percentage of growth inhibition was calculated using the
formula:
Mean optical density of sample  100
% of viability
Meanoptical densityof control group
Stability study. The storage stability of the PIO-NLC was investi-
gated by storing the optimized formulation in dark room for
90 days at temperatures 4 ± 1
C and 25 ± 1
C. Samples were with-
drawn (1 mL) at 30th, 60th, and 90th days and analyzed for PS,
ZP, PDI, and EE [39,40].
In-vivo biodistribution study. LCMS method development for esti-
mation of PIO in the biological samples. Irbesartan (99.44%) used
as an internal standard (IS). In brief, extraction of PIO from the
sample was done by mixing 50 mL aliquot of Serum/Brain homo-
zine sample with 25 mL of the IS working solution (1000 ng/mL of
irbesartan). To this, 200 mL of 5% formic acid was added after vor-
tex mixing for 10 s. The sample mixture was loaded onto a Strata-
X 33 mm polymeric sorbent cartridge (30 mg/1 mL) that was pre-
conditioned with 1 mL of methanol followed by of 1 mL water.
The extraction cartridge was washed with 1 mL of 5% formic
acid followed by 1 mL of water. PIO was eluted with 1 mL of the
mobile phase. Aliquot of 10 mL of the extract was injected into
the LC-MS/MS system. Analytes were determined using electro-
spray ionization in a triple quadrupole mass spectrometer. The
system (Shimadzu, Kyoto, Japan) consisting of a Waters, X-terra,
C18 column (100  4.6 mm, 5 mm; Waters Inc), a binary LC-
20ADprominence pump, an autosampler (SIL-HTc) and a solvent
degasser (DGU-20 A3) was used for the study. Aliquots of the
processed samples (10 mL) were injected into the column, which
was kept at ambient temperature. The elution system used was
0.1% aqueous formic acid: acetonitrile (40:60) at a flow rate of
0.6 mL/min. Quantification was achieved with MS-MS detection in
positive ion mode for both the analytes and the internal standard
using an MDS Sciex API-4000 Q-trap mass spectrometer (Foster
City, CA, USA) equipped with a Turboionspray interface at 500
C.
The ion spray voltage was set at 5500 V. The source parameters
viz. the nebulizer gas, curtain gas and collision gas were set at 4,
12 and 12 psi, respectively. Detection of the ions was carried out
in the multiple-reaction monitoring mode (MRM), by monitoring
the transition pairs of m/z 356.99 precursor ion to the m/z 133.92
for pioglitazone and m/z 429.2 precursor ion to the m/z 207.1 for
the IS.
Design of the in-vivo biodistribution study. The biodistribution
study was performed using male Wistar rats with approval from
the institutional animal ethical committee (IAEC), J.S.S College of
Pharmacy, Udhagamandalam, India (Proposal no.JSSCP/IAEC/PhD/
Ph.Ceutics/02/2017–18). The animals were divided into 3 groups
(n ¼ 3 for each group). Group I received PIO-NLC (IN), group II
received PIO solution (IN) and Group III received PIO solution (IV)
at a dose equivalent to 1 mg/kg of PIO. The rats were lightly anes-
thetized by exhaling diethyl ether before nasal administration. IN
administration was carried out using micropipette attached to
low-density polyethene tube having 0.1 mm internal diameter.
Animals (n ¼ 3) were sacrificed by cervical dislocation and blood
samples of approximately 0.25 mL were collected by cardiac punc-
ture after 30 min of administration. Blood samples were trans-
ferred to anticoagulant tubes and centrifuged at 3000 rpm for
15 min. After centrifugation, the plasma obtained was stored at
–20
C until analysis. The brain samples collected were rinsed with
saline solution and blotted with filter paper to remove the blood
taint and blood vessels. Brain samples were homogenized in PBS
(pH 7.4) to determine the amount of drug in the brain tissue. The
homogenate was centrifuged at 6000 rpm for 15 min at 4
C and
the supernatant was collected and frozen at -20
C until further
analysis by LCMS method [40].
Results and discussion
Selection of lipids
The solubility of PIO in different oils is shown in Figure 1. Among
the different oils investigated, capmul MCM was selected for the
formulation of NLC since PIO has shown the highest solubility of
1021 ± 12.5 mg/mL in the capmul MCM. Selection of the oil is very
much important as the EE is highly dependent on the solubility of
the drug in the selected lipid. Even if the drug is more soluble in
the liquid lipid than in the solid lipid, the drug may precipitate
out to the aqueous media in-vivo if the petition coefficient is low
[39]. Therefore, it is equally important to conduct the partition
coefficient study with different solid lipids. Among the various
solid lipid, PIO has shown the highest partition coefficient value in
tripalmitin. The result of the partition coefficient study is shown in
Figure 2.
Selection of surfactants
Among the various surfactants investigated, least PS and PDI were
obtained with tween 80 and pluronic F68. Combination of both
surfactants has shown to reduce the PS and PDI further. The
details of this study is given in Table 2.
Box-Behnken design
It was identified from the initial trials that the independent varia-
bles such as the total surfactant (X1), percentage of tween 80 in
the surfactant mixture (X2), and the concentration of SA (X3) are
the independent variables significantly influencing the dependent
variables such as particle size (Y1) and zeta potential (Y2). Box-
Behnken design required 17 runs with 5 center points per block
was developed by the Doe software to optimize the effect of three
independent variables on two dependent variables. All the formu-
lations ware made triplicate and analyzed for PS, ZP, PDI, and EE.
The PDI of all formulations were within the limit; that indicates a
homogenous distribution of particles within the formulation. The
EE of the 17 formulations wares seen to be comparatively higher
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 5

Figure 1. Solubility of PIO in different oils showing highest solubility in MCM.

Table 3. Characterization of NLC.

Formulation X1 X2 X3 PS (nm) ZP (MV) PDI EE (%)
F1 0.6 90 5 264 ± 3.81 6.8 ± 1.53 0.052 ± 0.005 64.51 ± 5.21
F2 0.6 70 3.75 213 ± 3.52 14.5 ± 3.06 0.098 ± 0.004 70.54 ± 4.9
F3 0.6 50 2.5 242 ± 4.85 4.9 ± 2.54 0.141 ± 0.012 66.28 ± 4.8
F4 0.4 50 3.75 342 ± 0.98 10.2 ± 1.85 0.304 ± 0.006 70.25 ± 3.9
F5 0.4 90 3.75 310 ± 3.54 5.6 ± 2.54 0.275 ± 0.184 65.54 ± 4.5
F6 0.4 70 2.5 274 ± 4.04 10.3 ± 1.75 0.498 ± 0.047 73.25 ± 5.6
F7 0.6 70 3.75 209 ± 3.52 15.2 ± 3.06 0.098 ± 0.008 70.54 ± 3.4
F8 0.8 70 5 243 ± 2.94 10.5 ± 1.95 0.035 ± 0.024 68.25 ± 4.9
F9 0.6 70 3.75 210 ± 3.52 13.9 ± 3.06 0.098 ± 0.008 70.54 ± 3.4
F10 0.6 50 5 264 ± 4.84 10.1 ± 2.56 0.065 ± 0.005 69.85 ± 6.1
Figure 2. Partition coefficient study of PIO in different solid lipids showing the F11 0.6 70 3.75 216 ± 3.52 13.5 ± 3.06 0.098 ± 0.008 70.54 ± 3.4
highest value in tripalmitin. F12 0.4 70 5 320 ± 2.54 14.9 ± 1.98 0.298 ± 0.006 68.45 ± 3.9
F13 0.8 70 2.5 198 ± 5.84 4.8 ± 2.51 0.059 ± 0.145 73.89 ± 5.4
F14 0.8 90 3.75 238 ± 2.45 3 ± 1.58 0.028 ± 0.157 71.58 ± 6.1
Table 2. PS and PDI with different surfactants. F15 0.8 50 3.75 241 ± 4.11 5.4 ± 2.54 0.128 ± 0.014 68.24 ± 5.8
Surfactant PS (nm) PDI aF16 0.6 90 2.5 224 ± 5.89 3.4 ± 2.47 0.058 ± 0.014 70.24 ± 4.7
F17 0.6 70 3.75 217 ± 3.52 14.5 ± 3.06 0.098 ± 0.008 70.54 ± 3.4
Tween 80 243 0.184
Pluronic F68 256 0.187
Solutol HS15 and 276 0.248
Hydrogenate dm Phosphatidylcholine 304 0.276 The final quadratic equation generated by the software for PS
Tween 80þ pluronic F68 218 0.134 and ZP in terms of coded factors is shown below. The positive
sign in the equation indicates a synergistic effect and negative
sign indicate an antagonistic effect.
due to the lipophilic nature of the drug. The results of the formula-
tion characterization are given in Table 3. PS ¼ þ213:0040:75  A6:63  B þ 19:13  C þ 7:25  AB
Three tests namely the sequential model sum of squares, lack of
fit tests and model summary statistics were used by the software þ 40:00  A2 þ 29:75  B2
for suggesting the best fitting model for the current data. The ZP ¼ þ14:322:16  A1:48  B þ 2:36  C0:2:22  A2
Prob > F value of p < 0.0001, higher R squared value, the higher
sum of square, lower standard deviation and lower PRESS values  6:05  B21:97  C2
suggested a quadratic model for both responses. Analysis of vari-
ance (ANOVA) was applied to validate the polynomial equations Perturbation graphs were plotted to find out the factors for
and the model terms were considered significant when Prob > what the response is most sensitive. A steep slope or curvature
F < 0.0500 and not significant Prob > F > 0.1000. The ANOVA results indicates that the response is sensitive to factor whereas the rela-
for the response surface quadratic model of PS and ZP is given in tively flat line indicates insensitivity towards the response. In the
Table 4. It was observed form the ANOVA test results that the case of PS, factor A has shown a steep curvature, factor B has
model terms A, B, C, AB, A2, B2 are significant for the PS. While A, shown slight but noticeable curvature and factor C has shown a
B, C, A2, B2, C2 are the significant model terms for the ZP. The lack relatively steep slope. Hence, the response PS was very much sen-
of fit p values > 0.0500 indicates that the lack of fit is not significant sitive to change in factor A and factor C. In case of ZP all the
for both PS and ZP. The value of the coefficient of correlation R2 for three factors have shown steep curvatures. Hence, changes in the
PS was found to be 0.9915, indicating a good fit. The predicted R2 three selected factors could influence the ZP significantly. The per-
value of 0.8899 has seen to be in reasonable agreement with the turbation graphs for PS and ZP were illustrated in Figure 3.
adjusted R2 of 0.9807. Similarly, the R2 value of 0.9890 obtained The relationship of the independent variables with the depend-
indicate a good fit for the ZP. The predicted R2 value of 0.9018 was ent variables was further represented by the 3 D graphs. The 3 D
in close agreement with the adjusted R2 of 0.9748 [39]. graphs for PS and ZP are illustrated by Figure 4.
6 G. M. JOJO ET AL.

Table 4. ANOVA for response surface quadratic model of measured responses.

Sum of squares df F value Prof > F p-Value
Source Y1 Y2 Y1 Y2 Y1 Y2 Y1 Y2
Model 28266.99 308.08 9 9 91.13 69.75 <0.0001 <0.0001
A-TS 13284.50 37.41 1 1 385.46 76.23 <0.0001 <0.0001
B-% OF TWEEN80 351.13 17.40 1 1 10.19 35.46 0.0152 0.0006
C-SA 2926.13 44.65 1 1 84.90 90.98 <0.0001 <0.0001
AB 210.25 1.21 1 1 6.10 2.47 0.0428 0.1604
AC 0.25 0.30 1 1 7.254E-003 0.62 0.9345 0.4581
BC 81.00 0.81 1 1 2.35 1.65 0.1691 0.2398
A2 6736.84 20.80 1 1 195.47 42.38 <0.0001 0.0003
B2 3726.58 153.99 1 1 108.13 313.76 <0.0001 <0.0001
C2 139.21 16.38 1 1 4.04 33.38 0.0844 0.0007
Lack of Fit 191.25 1.75 3 3 5.10 1.38 0.0747 0.3699
Residuals 241.25 3.44 7 7
Pure error 15.00 1.69 4 4
Cor Total 28508.24 311.52 16 16
R2 value Predicted R2 Adjusted R2
Y1 0.9915 0.8899 0.9807
Y2 0.9890 0.9018 0.9748

Figure 3. Perturbation graphs showing the sensitivity of responses towards the selected independent variables. PS is found to be more sensitive for change in varia-
bles A and C whereas the ZP is very much sensitive to all the three independent variables selected.

Figure 4. 3D plots.

Effect of independent variables on dependent variables

the oil globules into the smaller size. Also, the orientation of sur-
The independent variable “A” was found to have an antagonistic factant molecules on the particle surface stabilize newly formed
effect on PS. The increase in the concentration of surfactant nanoparticles and thus prevent its coalescence [44,45]. However, it
decreases the surface tension that helps the further breakdown of has seen from the preliminary studies that further increase in
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7

surfactant above 0.8%, in fact, increased the PS. The possible

explanation is that at higher concentration micelle formation takes
place in the external phase instead of the orientation of surfactant
molecules on the particle surface. This will increase the local
osmotic pressure and thereby some of the external phase mole-
cules move towards the nanoparticles surface and deplete the
continuous phase between the nanoparticles. Finally, the aggrega-
tion of oil drops take place and increase the particle size [46]. It is
evident from previous investigations [47–49] and our own prelim-
inary studies that blending of surfactants produce particles smaller
than the individual surfactants can do. Blending can enhance the
flexibility of the surfactant layer and also enhance the ability to
partition at higher levels into the oil-water interphase; both stabil-
izes the newly formed oil globules [50]. In the present study, a
combination of tween 80 and pluronic F68 at a ratio of 70:30 ratio
has shown to produce the smallest particles. As observed by the
previous investigators [51,52], in the present work also the SA has
shown a direct relationship with PS.
The intensity of positive ZP was found to decline with the
increase in factor “A”. This could be due to the increment in Figure 5. PS and ZP of optimized formulation.
tween 80 since it is evident from previous studies and our own
studies that use of tween 80 impart a negative ZP to the nanocar-
the nanocarriers and the drug is in the amorphous form rather
riers [53–55]. Replacement of a part of the tween 80 with the non-
than in a crystalline form [40].
ionic surfactant pluronic F68 was found to reduce not only the
particle size but also the intensity of the negative ZP. Among the
different ratios, highest ZP was achieved with 70:30 ratio of tween In-vitro drug release study
80: pluronic F68. In agreement with previous studies [56–58], fac-
tor “C” has a synergistic effect on ZP. The ZP has seen to increase The drug PIO is having limited solubility in water with log p
significantly with an increase in the concentration of SA. values of 3.4 [58]. Therefore 1%, SLS was used in the dissolution
Hence, the tween 80 that minimize the PS impart a negative media to maintain sink conditions [39]. The NLC formulation has
zeta potential; while the positive charge inducer SA increase the shown a biphasic release pattern; that is an initial fast release
PS. The replacement of a portion of the total tween80 with non- followed by a sustained release. The initial fast release from the
ionic surfactant pluronic F68 reduced the intensity of the negative NLC was contributed by a certain quantity of PIO that got
zeta potential as well as the PS. The design expert software was adsorb on nanocarrier surface. Also, the difference in melting
used to optimize the level of these three factors so that NLC with point of solid and liquid lipid results accumulation of certain
minimum PS and maximum ZP can be formulated. liquid lipid on the outer shell of NLC. The drug entrapped in the
liquid lipid deposited on the outer shell release rapidly [59,60],
while the portion of the drug accumulated in the core get
Optimization of formulation release slowly by the diffusion from the matrix or by the erosion
of the matrix system [61]. It has seen from the further analysis
The criteria for the optimized formulation has set in the software
that the release pattern form NLC is fitted with Higuchi’s equa-
in such a way that the resulting NLC should be of the minimum
tion that means the release is diffusion controlled. The “n” value
particle size and maximum zeta potential. The model has pre-
obtained from the Korsmeyer’s Peppas was found to be 0.16
dicted NLC formulation having PS of 208.926 nm and ZP of
12.55at 0.72% of A, 67.91% of B and 4.33 mg of C. To validate the that indicate the Fickian diffusion [62]. The graph showing the
experimental model, the predicted formulation has made triplicate cumulative amount of the drug released vs. time is illustrated in
and the mean observed values of PS and ZP were found to be Figure 7.
211.4 ± 3.54 nm 14.9 ± 1.09 mv respectively. The observed values
have shown a close agreement with the predicted values that Ex-vivo permeation studies
means the current model is significant in optimizing the formula-
tion parameters. The Zetasizer reports showing the PS and ZP of The permeation rate of PIO-NLC was found to be higher as com-
the optimized formulation is illustrated in Figure 5. pared to the PIO solution. At the end of the sixth hour,
1789 ± 4.50 mg/cm2 of PIO was found to permeate across the tis-
sue from the NLC with the flux of 8.609 mg/cm2/min and the per-
DSC study meability coefficient of 0.00086 cm2/min. While the amount of
The DSC thermograms of PIO, Tripalmitin, physical mixture of PIO drug permeated from the solution at this time interval was
and tripalmitin and the PIO-NLC are shown in Figure 6. A sharp 989 ± 3.58 mg/cm2 with the flux of 4.733 mg/cm2/min and the per-
endothermic peak indicating the crystalline nature of the com- meability coefficient of 0.00047 cm2/min.
pound has been observed for the PIO and tripalmitin at 165.01
C
and 72.68
C respectively. Appearance of same peaks in the phys-
Toxicity studies
ical mixture indicate no interaction between the two components.
Whereas, the PIO-NLC has shown an endothermic peak corre- Nasal ciliotoxicity study
sponding to tripalmitin at 66.54
C and the drug peak was absent. Treatment of nasal mucosa with IPA, the positive control resulted
Absence of drug peak indicate complete entrapment of PIO inside in extensive mucosal damage, shrinkage and loss of epithelial cells
8 G. M. JOJO ET AL.
Figure 6. DSC study results showing no interaction between formulation components.
Figure 7. In-vitro drug release study showing sustained release of PIO from the NLC.
and cilia. While no changes have been observed with the tissues
treated with PBS pH 6.4, blank NLC, lyophilized PIO-NLC or PIO
solution. The result shows that the developed formulation as well
as the PIO solution are safe for nasal administration. The histo-
pathological sections of nasal mucosa treated with different sam-
ples are illustrated in Figure 8.
Cytotoxicity study
Since the various formulation parameters like surface charge, the
concentration of surfactants etc can induce toxicity to the neu-
rons, it is highly recommended to conduct the cytotoxicity study
of the nanoformulations. The percentage cell viability and LC50
values were calculated to indicate the cytotoxicity of the samples.
The percentage cell viability results have shown in Figure 9. The
LC50 values calculated using ED50 PLUS V1.0 Software was found
to be 16.626 mg/mL and 17.3874 mg/mL for the pure drug and PIO-
NLC respectively. The absence of any significant changes in the
investigated parameters of PIO-NLC and the pure drug has con-
firmed the neuronal safety of developed formulation.
Storage stability study
In the present work, the surface charge was limited below þ20mv
as the high cationic charge is neurotoxic [63–65]. Since the surface
charge is one of the factors influencing the stability of formulation
significantly, it is necessary to examine the storage stability of final
formulation. Result of storage stability study is shown in Table 5.
It has seen that there were no significant changes in the investi-
gated parameters of the PIO-NLC (p < 0.05) at the investigated
temperatures. While a slight but non-significant changes have

Figure 8. Histopathological section of nasal mucosa treated with different samples showing no toxicity with PIO-NLC and PIO solution.
Figure 9. Percentage of cell viability vs. concentration graphs of PIO-NLC and
pure drug.
Figure 10. In-vivo biodistribution study results. Administration of IN NLC was
observed to improve the concentration of PIO reaching the brain significantly.
been observed with the PS and EE at 25
C. Hence the formulation
is found to be stable at 4
C and 25
C.
In-vivo biodistribution studies
With the present LCMS method, linearity was established for the
range of concentrations 10 – 5000 ng/mL with a coefficient of
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 9
correlation (r) of 0.9999. The lower limit of quantification (LOQ)
was identifiable and reproducible at 10 ng/mL. The method has
been successfully applied to estimation of PIO in serum and brain
homogenized samples. Biodistribution study has shown that the
IN-NLC formulation significantly improved the amount of PIO
reaching the brain. A concentration of 1.64 mg/mL of PIO has
reached the brain at the end of 30th min from the PIO-NLC. While
only 0.38 mg/mL and 0.88 mg/mL of the drug has seen to reach the
brain from the IV solution and IN solution respectively.
It was observed from the ex-vivo permeation study that the
NLC formulation has improved permeability of PIO across the
nasal mucosa significantly. Hence, an enhanced bioavailability of
drug in the brain as well as in the serum could be expected in-
vivo. Eventhough the IN-NLC has improved the concentration of
PIO reaching the brain to a great extent, the serum drug concen-
tration obtained was almost same for both IN-NLC and IN solution
(Figure 8). The degree of plasma protein binding of the PIO might
have played the major role here. In normal cases, PIO is exten-
sively protein bound (>99%) in human serum [66]. The protein
bound PIO retained in the blood as it is unable to cross the bio-
logical membrane due to the macro size [67]. Whereas, the
entrapment of drug inside the nanocarrier restrains the degree of
protein binding. In addition, the small size and improved lipophi-
licity of the NLC offers a faster distribution of the drug to various
organs including the brain from the blood [68]. The enhanced vol-
ume of distribution of PIO-NLC compared to the drug solution
might have narrowed the plasma drug concentration in the for-
mulation-treated group.
The bioavailability of the drug in the brain from a nose to
brain-targeted drug delivery system is dependent on several fac-
tors including characteristics of the drug, formulation parameters,
physiological properties and also the position of the head. In the
present study, all the IN administration were given in supine pos-
ition with head at 90
; as the supine position with 70
and 90
is
the most favorable position for nose brain transport [29]. The
maximum exposure of the formulation to the olfactory region of
the nose at this position significantly enhanced the bioavailability
10 G. M. JOJO ET AL.

Table 5. Storage stability study results.

Particle size (nm) Zeta potential (mv) Polydispersibility index Entrapment efficiency (%)







Days 4 C 25 C 4 C 25 C 4 C 25 C 4
C 25
C
0 days 211.4 ± 3.54 211.4 ± 3.54 14.9 ± 1.09 14.9 ± 1.09 0.198 ± .14 0.198 ± .142 72.89 ± 8.4 72.89 ± 8.41
30 days 212 ± 2.15 213 ± 4.64 14.48 ± 2.01 14.21 ± 2.9 0.199 ± .85 0.210 ± .25 71.93 ± 5.4 71.08 ± 5.21
60 days 212 ± 3.25 216 ± 2.45 13.98 ± 1.95 13.51 ± 1.06 0.204 ± .25 0.235 ± .35 71.20 ± 2.2 69.94 ± 6.01
90 days 214 ± 2.14 219 ± 1.98 13.51 ± 1.4 13.15 ± 2.04 0.214 ± .15 0.289 ± .27 70.84 ± 3.46 68.87 ± 5.04

in the brain. In the present experiment, the concentration of the Acknowledgment

drug reaching brain and blood from the IN solution was further
influenced by the mucociliary clearance as well as by the efflux
transporters present at the mucosal surface [69]. On other hands,
the electrostatic interaction formed by the NLC with the nasal
mucosa minimized the drug loss via mucociliary clearance. Also,
the unique property of nanocarriers to escape the efflux transport-
ers present on the mucosal surface further improved the bioavail-
ability from the PIO-NLC [70].
The brain/plasma ratio was calculated for all the treated
groups. Ratios for IN-NLC, IN-PIO solution and IV-PIO solution
were found to be 1.6, 0.84, and 0.15 respectively. A ratio >1 indi-
cates the direct nose to brain transport [71]. Direct transport of
drug to the brain from the IN-NLC has significantly improved the
concentration of PIO reaching the brain in the present experi-
ment. The current results shows the possibility of achieving a
therapeutic concentration of PIO in the brain from lower doses via
administration of PIO-NLC intranasally. So that the risk of develop-
ment of bladder cancer and peripheral edema associated with
high dose of PIO probably get minimize [25]. The results of the
invivo biodistribution study are illustrated in Figure 10.
The authors would like to mention one thing here that in the
current in-vivo study concentration of the drug in the brain is
being expressed in mg/mL of brain homogenize; while many of
the other researchers have used the unit of mg/g of tissue to
express the biodistribution of samples in various organs. This is
because our major focus was on the brain/plasma ratio to investi-
gate the possible direct nose to brain transport of PIO from the
IN-NLC. Since the plasma concentration can be expressed only in
mg/mL, we have used the same unit to express the brain concen-
tration to avoid further errors.
Conclusion
PIO loaded NLC formulation was successfully prepared and opti-
mized using Box-Behnken design. The optimized nanoformulation
has a PS of 211.4 ± 3.54 nm and ZP of 14.9 ± 1.09mv. The EE and
PDI were found to be 70.18 ± 4.5 and 0.257 ± 0.108 respectively
and the final formulation is observed to be stable at 4
C and
25
C. The in-vitro drug release study conducted has exhibited a
sustained release of PIO from the NLC. The formulation was found
to improve the permeability of PIO across the nasal mucosa sig-
nificantly ex-vivo. Toxicity study performed has confirmed the
safety of the developed formulation for in-vivo administration.
Direct nose to brain transport of the drug was observed from the
IN-NLC that has significantly improved the concentration of the
drug reaching the brain in-vivo. Thus, the present work demon-
strated the utility of IN-NLC for the repurposing of PIO in the
management of AD.
Disclosure statement
The authors report no conflict of interest.
The authors would like to thank the Department of Science and
Technology–Fund for Improvement of Science and Technology
Infrastructure in Universities and Higher Educational Institutions
(DST_FIST), New Delhi for their infrastructure support to
our department.
Funding
This work was financially supported by the University Grants
Commission (UGC), Government of India, under the scheme of the
UGC-MANF Fellowship. The author, Ms Gifty M Jojo wishes to
express his gratitude to the University Grants Commission (UGC),
New Delhi, India for the award of the UGC-MANF Fellowship
[2016–17/MANF-2015–17-KER-54943].
References
[1] Saxena U. Bioenergetics breakdown in Alzheimer’s disease:
targets for new therapies. Int J Physiol Pathophysiol
Pharmacol. 2011;3:133–139.
[2] Folch J, Petrov D, Ettcheto M, et al. Current research thera-
peutic strategies for Alzheimer’s disease treatment. Neural
Plast. 2016;2016:1.
[3] Watson GS, Craft S. The role of insulin resistance in the
pathogenesis of Alzheimer’s disease: implications for treat-
ment. CNS Drugs. 2003;17:27–45.
[4] de la Monte SM, Wands JR. Alzheimer’s disease is Type 3
diabetes-evidence reviewed. J Diabetes Sci Technol. 2008;2:
1101–1113.
[5] Mullin R. Cost to develop new pharmaceutical drug now
exceeds $2.5B. Sci Am. 2014;24.
[6] Hu SH, Jiang T, Yang SS, et al. Pioglitazone ameliorates
intracerebral insulin resistance and tau-protein hyperphos-
phorylation in rats with type 2 diabetes. Exp Clin
Endocrinol Diabetes. 2013;121:220–224.
[7] Sastre M, Dewachter I, Rossner S, et al. Nonsteroidal anti-
inflammatory drugs repress beta-secretase gene promoter
activity by the activation of PPARgamma. Proc Natl Acad
Sci USA. 2006;103:443–448.
[8] Camacho IE, Serneels L, Spittaels K, et al. Peroxisome prolif-
erator-activated receptor gamma induces a clearance
mechanism forthe amyloid-beta peptide. J Neurosci. 2004;
24:10908–10917.
[9] Bogacka I, Xie H, Bray GA, et al. Pioglitazone induces mito-
chondrial biogenesis in human subcutaneous adipose tis-
sue in vivo. Diabetes. 2005;54:1392–1399.
[10] Chen J, Li S, Sun W, et al. Anti-diabetes drug pioglitazone
ameliorates synaptic defects in ad transgenic mice by
inhibiting cyclin-dependent kinase5 activity. PloS One.
2015;10:e0123864.
[11] Xing B, Xin T, Hunter RL, et al. Pioglitazone inhibition of
lipopolysaccharide-induced nitric oxide synthase is

associated with altered activity of p38 MAP kinase and
PI3K/Akt. J Neuroinflammation. 2008;5:4.
[12] Pathan AR, Viswanad B, Sonkusare SK, et al. Chronic admin-
istration of pioglitazone attenuates intracerebroventricular
streptozotocin induced-memory impairment in rats. Life Sci.
2006;79:2209–2216.
[13] Jiang LY, Su-Su T, Wang XY, et al. PPARc agonist pioglita-
zone reverses memory impairment and biochemical
changes in a mouse model of type 2 diabetes mellitus. CNS
Neurosci Ther. 2012;18:659–666.
[14] Li-ping L, Tian-hua Y, Li-ying J, et al. Pioglitazone amelio-
rates memory deficits in streptozotocin-induced diabetic
mice by reducing brain b-amyloid through PPARc activa-
tion. Acta Pharmacol Sin. 2013;34:455–463.
[15] Yin Q-Q, Pei J-J, Xu S, et al. Pioglitazone improves cognitive
function via increasing insulin sensitivity and strengthening
antioxidant defense system in fructose-drinking insulin
resistance rats. PLoS One. 2013;8:e59313.
[16] Yang S, Chen Z, Cao M, et al. Pioglitazone ameliorates
Ab42 deposition in rats with diet-induced insulin resistance
associated with AKT/GSK3b activation. Mol Med Rep. 2017;
15:2588–2594.
[17] Fernandez-Martos CM, Atkinson RAK, Chuah MI, et al.
Combination treatment with leptin and pioglitazone in a
mouse model of Alzheimer’s disease. Alzheimer’s Dement.
2017;3:92–106.
[18] Maeshiba Y, Kiyota Y, Yamashita K, et al. Disposition of the
new antidiabetic agent pioglitazone in rats, dogs, and mon-
keys. Arzneimittelforschung. 1997;47:29–35.
[19] Landreth G, Jiang Q, Mandrekar S, et al. PPARgamma ago-
nists as therapeutics for the treatment of Alzheimer’s dis-
ease. Neurotherapeutics. 2008;5:481–489.
[20] Tornio A, Niemi M, Neuvonen PJ, et al. Trimethoprim and
the CYP2C83 allele have opposite effects on the pharma-
cokinetics of pioglitazone. Drug Metab Dispos. 2008;36:
73–80.
[21] Nozawa T, Sugiura S, Nakajima M, et al. Involvement of
organic anion transporting polypeptides in the transport of
troglitazone sulfate: implications for understanding troglita-
zone hepatotoxicity. Drug Metab Dispos. 2004;32:291–294.
[22] Kirchheiner J, Roots I, Goldammer M, et al. Effect of genetic
polymorphisms in cytochrome p450 (CYP) 2C9 and CYP2C8
on the pharmacokinetics of oral antidiabetic drugs: clinical
relevance. Clin Pharmacokinet. 2005;44:1209–1225.
[23] Chang KL, Pee HN, Yang S, et al. Influence of drug trans-
porters and stereoselectivity on the brain penetration of
pioglitazone as a potential medicine against Alzheimer’s
disease. Sci Rep. 2015;5:9000.
[24] Jojo GM, Kuppusamy G. Scope of new formulation
approaches in the repurposing of pioglitazone for the man-
agement of Alzheimer’s disease. J Clin Pharm Ther. [cited
2019 Feb 9]. DOI:10.1111/jcpt.12808.
[25] Tuccori M, Filion KB, Yin H, et al. Pioglitazone use and risk
of bladder cancer: population based cohort study. BMJ.
2016;352:i1541.
[26] Dhuria SV, Hanson LR, Frey WH. 2nd. Intranasal delivery to
the central nervous system: mechanisms and experimental
considerations. J Pharm Sci. 2010;99:1654–1673.
[27] Thorne RG, Pronk G, Frey WH. Delivery of insulin-like
growth factor-1 to the brain and spinal cord along olfactory
and trigeminal pathways following intrantasal administra-
tion: a noninvasive method for bypassing the blood-brain
barrier. Soc Neurosci Abstract. 2000;26:1365.
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 11
[28] Hanson LR, Frey WH. II. Intranasal delivery bypasses the
blood-brain barrier to target therapeutic agents to the cen-
tral nervous system and treat neurodegenerative disease.
BMC Neurosci. 2008;9:S5.
[29] Woensel MV, Wauthoz N, Rosiere R, et al. Formulations for
intranasal delivery of pharmacological agents to combat
brain disease: a new opportunity to tackle GBM?. Cancers.
2013;5:1020–1048.
[30] Ong WY, Shalini SM, Costantino L. Nose-to-brain drug deliv-
ery by nanoparticles in the treatment of neurological disor-
ders. Curr Med Chem. 2014;21:4247–4256.
[31] Fonseca-Santos B, Gremi~ao MPD, Chorilli M.
Nanotechnology-based drug delivery systems for the treat-
ment of Alzheimer’s disease. Int J Nanomed. 2015;10:
4981–5003.
[32] Silva-Abreu M, Espinoza L, Halbaut L, et al. Comparative
study of ex vivo transmucosal permeation of pioglitazone
nanoparticles for the treatment of Alzheimer’s disease.
Polymers. 2018;10:316.
[33] Mukherjee S, Ray S, Thakur RS. Solid lipid nanoparticles: a
modern formulation approach in drug delivery system.
Indian J Pharm Sci. 2009;71:349–358.
[34] Ghasemiyeh P, Mohammadi-Samani S. Solid lipid nanopar-
ticles and nanostructured lipid carriers as novel drug deliv-
ery systems: applications, advantages and disadvantages.
Res Pharm Sci. 2018;13:288–303.
[35] Ozsoy Y, Gungor S, Cevher E. Nasal delivery of high
molecular weight drugs. Molecules. 2009;14:3754–3779.
ttir BS, Gudnason PI, Thorsteinsson F, et al.
[36] Snorrado
Experimental design for optimizing drug release from sili-
cone elastomer matrix and investigation of transdermal
drug delivery. Eur J Pharm Sci. 2011;42:559–567.
[37] Ranade SS, Thiagarajan P. Selection of a design for
response surface. IOP Conf Ser: Mater Sci Eng. 2017;263:
022043.
[38] Klien W, Kordel W, Weiss M, et al. Updation of the OECD
test guideline 107 partition coefficient n-octanol water
OECD library intercomparison test on the HPLC method.
Chemosphere. 1988;17:361–386.
[39] Barauh UK, Kuppusamy G, Ravisankar V, et al. Optimization
of chloquin loaded nanostructured lipid carriers using Box-
Behnken design and its antimalarial efficacy. J DrugTarget.
2017;21:01.
[40] Jain K, Sood S, Gowthamarajan K. Optimization of arte-
mether-loaded NLC for intranasal delivery using central
composite design. Drug Deliv. 2015;22:940–954.
[41] Joshi M, Patravale V. Formulation and evaluation of
Nanostructured Lipid Carrier (NLC)-based gel of Valdecoxib.
Drug Dev Ind Pharm. 2006;32:C11–918.
[42] Srinivasulu D. Sastry BS, Omprakash G. Development and
validation of new RPHPLC method for determination of
Pioglitazone HCL in pharmaceutical dosage forms. Int J
Chem Res. 2010;1:18–20.
[43] Talarico LB, Zibetti RG, Faria PC, et al. Anti-herpes simplex
virus activity of sulfated galactans from the red seaweeds
Gymnogongrus griffithsiae and Cryptonemia crenulata. Int J
Biol Macromol. 2004;34:63–71.
[44] Das S, Ng WK, Tan RB. Are nanostructured lipid carriers
(NLCs) better than solid lipid nanoparticles (SLNs): develop-
ment, characterizations and comparative evaluations of clo-
trimazole-loaded SLNs and NLCs? Eur J Pharm Sci. 2012;47:
139–151.
[45] Rowe EL. Effect of emulsifier concentration and type on the
particle size distribution of emulsions. J Pharm Sci. 1965;54:
260–264.
[46] Wulff-Perez M, Torcello-Go mez A, Galvez-Ruız MJ, Martın
Rodrıguez A. Stability of emulsions for parenteral feeding:
preparation and characterization of o/w nanoemulsions
with natural oils and Pluronic f68 as surfactant. Food
Hydrocoll. 2009;23:1096–1102.
[47] Ebrahimi HA, Javadzadeh Y, Hamidi M, et al. Repaglinide-
loaded solid lipid nanoparticles: effect of using different
surfactants/stabilizers on physicochemical properties of
nanoparticles. Daru. 2015;23:46.
[48] Tan SW, Billa N, Roberts CR, et al. Surfactant effects on the
physical characteristics of Amphotericin B-containing nano-
structured lipid carriers. Colloids Surf A: Physicochem Eng
Asp. 2010;372:73–79.
[49] Fei H, Sanming L, Ran Y, et al. Effect of surfactants on the
formation and characterization of a new type of colloidal
drug delivery system: nanostructured lipid carriers. Colloids
Surf A: Physicochem Eng Asp. 2008;315:210–216.
[50] Li P, Ghosh A, Wagner RF, et al. Effect of combined use of
nonionic surfactant on formation of oil-in-water microemul-
sions. Int J Pharm. 2005;288:27–34.
[51] Pardeshi CV, Rajput PV, Belgamwar VS, et al. Novel surface
modified solid lipid nanoparticles as intranasal carriers for
ropinirole hydrochloride: application of factorial design
approach. Drug Deliv. 2013;20:47–56.
[52] Baviskar A, Hiremath S, Devanna N, et al. Influence of vari-
ous process variables and formulation excipients on the
engineering of sertaconazole solid lipid nanoparticles.
IOSRPHR. 2016;6:51–63.
[53] Saeedi M, Rafati MR, Morteza-Semnani K, et al. Evaluation
of effect of tween 80 on characteristics of tadalafil 0.1%
suspension. Mazums-PBR. 1:35–43.
[54] Tao X, Li Y, Hu Q, et al. Preparation and drug release study
of novel nanopharmaceuticals with polysorbate 80 surface
adsorption. doi:10.1155/2018/4718045
[55] Prabhakar K, Afzal SM, Surender G, et al. Tween 80 contain-
ing lipid nanoemulsion for delivery of indinavir to brain.
Acta Pharm Sin B. 2013;3:345.
[56] Reddy LH, Sharma RK, Chuttani K, et al. Etoposide-incorpo-
rated tripalmitin nanoparticles with different surface charge:
formulation, characterization, radiolabeling, and biodistribu-
tion studies. AAPS J. 2004;6:55–64.
[57] Bangham AD, Standish MM, Watkins JC. Diffusion of univa-
lent ions across the lamellae of swollen phospholipids. JMB.
1965;13:238–252.
[58] Kim DH, Termsarasab Cho HJ, et al. Preparation and charac-
terization of self-assembled nanoparticles based on low-
molecular-weight heparin and stearylamine conjugates for
controlled delivery of docetaxel. Int J Nanomed. 2014;9:
5711–5727.
[59] Hu F-Q, Jiang S-P, Du Y-Z, et al. Preparation and character-
ization of stearic acid nanostructured lipid carriers by solv-
ent diffusion method in an aqueous system. Colloids Surf B
Biointerfaces. 2005;45:167–173.
[60] Teeranachaideekul V, Souto EB, Junyaprasert VB, et al. Cetyl
palmitate-based NLC for topical delivery of Coenzyme
Q(10) – development, physicochemical characterization and
in vitro release studies. Eur J Pharm Biopharm. 2007;67:
141–148.
[61] M€ uhlen AZ, M€ uhlen EZ, Niehus H, et al. Atomic force
microscopy studies of solid lipid nanoparticles. Pharm Res.
1996;13:1411–1416.
[62] Ramteke KH, Dighe PA, Kharat AR, Patil SV. Mathematical
models of drug dissolution: a review. Sch Acad J Pharm.
2014;3:388–396.
[63] Li Y, Ju D. Chapter 12 – the application, neurotoxicity, and
related mechanism of cationic polymers. Neurotoxicity
Nanomater Nanomed. 2017:285–329. doi:10.1016/B978-0-
12-804598-5.00012-X.
[64] Vidal F, Vasquez P, Cayuman FR, et al. Prevention of synap-
tic alterations and neurotoxic effects of PAMAM dendrimers
by surface functionalization. Nanomaterials (Basel). 2017;
258:7.
[65] Gramowski A, Flossdorf J, Bhattacharya K, et al.
Nanoparticles induce changes of the electrical activity of
neuronal networks on microelectrode array neurochips.
Environ Health Perspect. 2010;118:1363–1369.
[66] https://www.drugbank.ca/salts/DBSALT000555. Last
accessed on 21/01/2019.
[67] https://www.cambridgemedchemconsulting.com/resources/
ADME/distribution.html. Last accessed on 21/01/2019.
[68] Arms L, Smith DW, Flynn J, et al. Advantages and limita-
tions of current techniques for analyzing the biodistribution
of nanoparticles. Front Pharmacol. 2018;9:802.
[69] Oliveira P, Fortuna A, Alves G, et al. Drug-metabolizing
enzymes and efflux transporters in nasal epithelium: influ-
ence on the bioavailability of intranasally administered
drugs. Curr Drug Metab. 2016;17:628–647.
[70] Hoosain FG, Choonara YE, Tomar LK, et al. Bypassing P-
glycoprotein drug efflux mechanisms: possible applications
in pharmacoresistant Schizophrenia therapy. Biomed Res
Int. 2015:2015;1.
[71] Yasir M, Singh Sara UV. Solid lipid nanoparticles for nose to
brain delivery of haloperidol: in vitro drug release and
pharmacokinetics evaluation. Acta Pharmaceutica Sin B.
2014;4:454–463.