International Journal of Pharmaceutics 607 (2021) 121050

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International Journal of Pharmaceutics

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

Nose-to-brain delivery of amisulpride-loaded lipid-based poloxamer-gellan

gum nanoemulgel: In vitro and in vivo pharmacological studies
Dnyandev Gadhave a, b, Shrikant Tupe a, Amol Tagalpallewar a, c, Bapi Gorain d,
Hira Choudhury e, Chandrakant Kokare a, *
a
Department of Pharmaceutics, Sinhgad Technical Education Society’s, Sinhgad Institute of Pharmacy (Affiliated to Savitribai Phule Pune University), Narhe, Pune
411041, Maharashtra, India
b
Department of Pharmaceutics, HSBPVTS, GOI, College of Pharmacy (Affiliated to Savitribai Phule Pune University), Kashti, Ahmednagar 414701, Maharashtra, India
c
School of Pharmacy, Department of Pharmaceutics, MIT World Peace University, Pune 411038, Maharashtra, India
d
School of Pharmacy, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor 47500, Malaysia
e
Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, 57000 Kuala Lumpur, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Unfavorable side effects of available antipsychotics limit the use of conventional delivery systems, where limited
Intranasal nanoemulgel exposure of the drugs to the systemic circulation could reduce the associated risks. The potential of intranasal
Gellan gum delivery is gaining interest to treat brain disorders by delivering the drugs directly to the brain circumventing the
Box-Behnken design
tight junctions of the blood–brain barrier with limited systemic exposure of the entrapped therapeutic. Therefore,
Amisulpride
Agranulocytosis
the present research was aimed to fabricate, optimize and investigate the therapeutic efficacy of amisulpride
Brain-targeting potential (AMS)-loaded intranasal in situ nanoemulgel (AMS-NG) in the treatment of schizophrenia. In this context, AMS
Antipsychotic activity nanoemulsion (AMS-NE) was prepared by employing aqueous-titration method and optimized using Box-
Behnken statistical design. The optimized nanoemulsion was subjected to evaluation of globule size, trans­
mittance, zeta potential, and mucoadhesive strength, which were found to be 92.15 nm, 99.57%, − 18.22 mV,
and 8.90 g, respectively. The AMS-NE was converted to AMS-NG using poloxamer 407 and gellan gum. Following
pharmacokinetic evaluation in Wistar rats, the brain Cmax for intranasal AMS-NG was found to be 1.48-folds and
3.39-folds higher when compared to intranasal AMS-NE and intravenous AMS-NE, respectively. Moreover,
behavioral investigations of developed formulations were devoid of any extrapyramidal side effects in the
experimental model. Finally, outcomes of the in vivo hematological study confirmed that intranasal adminis­
tration of formulation for 28 days did not alter leukocytes and agranulocytes count. In conclusion, the promising
results of the developed and optimized intranasal AMS-NG could provide a novel platform for the effective and
safe delivery of AMS in schizophrenic patients.

1. Introduction revealed that second-generation antipsychotics are considerably better

Schizophrenia is a complex psychiatric disorder associated with
remarkable dysfunctions in the patient’s behavior, emotions, percep­
tions, and bradyphrenia that principally affecting a person’s lifestyle
and may develop suicidal tendencies (Cui et al., 2020; Gadhave et al.,
2018). A steep surge in the number of elderly patients associated with
schizophrenia had been observed during the past decade (Gadhave
et al., 2019a). Based on the current standard treatment options of
schizophrenia, first-generation antipsychotics have been proven effec­
tive against positive symptoms of psychosis. However, recent reports
in improving quality of life of patients (Gründer et al., 2016). AMS is a
USFDA approved second-generation atypical antipsychotic, which has
been reported for its activity against positive and negative symptoms of
schizophrenia because of its moderation on two receptors, dopamine
(D2) and serotonin (5HT2A) present in CNS (El Assasy et al., 2019;
Gamal et al., 2017). However, recent research revealed that the long-
term therapy of AMS results in lethal hematological toxicities, such as
agranulocytosis or leukopenia in schizophrenic patients (Gadhave et al.,
2019a; Pickard et al., 2016; Woon et al., 2018). Additionally, AMS is
known to produce extrapyramidal symptoms (EPS) (Maatallah et al.,

* Corresponding author.
E-mail addresses: bapi.gorain@taylors.edu.my (B. Gorain), chandrakantkokare.siop@sinhgad.edu (C. Kokare).

https://doi.org/10.1016/j.ijpharm.2021.121050
Received 6 April 2021; Received in revised form 16 July 2021; Accepted 23 August 2021
Available online 25 August 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.
D. Gadhave et al.
2017). Conventionally, AMS is available as extended-release tablets,
with an oral bioavailability of only 48% (El Assasy et al., 2019; Gamal
et al., 2017). Alternatively, parenteral administration could enhance its
systemic availability, however, it would potentiate the undesired he­
matological toxicities. Hence, there has always been a demand for the
enhancement in the design and development of suitable drug delivery
systems for the accurate, effective, and safe delivery of the therapeutics
to the targeted site through a non-invasive route of administration
(Gadhave et al., 2019b; Kozlovskaya et al., 2014; Pardeshi and Bel­
gamwar, 2013).
Delivery of drugs to the brain has always been a challenge, where the
tight junctions of endothelial cells in the CNS acts as a biological barrier
that safely regulates the movement of molecules into the brain (Kumb­
har et al., 2020). Consequently, conventional drug delivery systems and
routes of administration used in the treatment of CNS disorders have
several pros and cons (Chatterjee et al., 2019). Among the various routes
of administration, the intranasal route has gained enormous attention in
current research utilized for BBB infiltration. In addition, a few research
studies in this field have already progressed towards product commer­
cialization across different countries (Chatterjee et al., 2019). Nose-to-
brain delivery is utilized as a non-invasive route of administration, of­
fering direct brain access for the centrally acting therapeutics, as well as
the potential of avoiding BBB and eliminating the hepatic metabolism of
administered therapeutics (Banks, 2012; Choudhury et al., 2017a;
Hosny and Hassan, 2014). Interestingly, the intranasal cavity offers two
distinctive pathways for transportation of the drug viz., olfactory and
trigeminal pathways, granting direct access to the brain (Pund et al.,
2013). Recently, many researchers have proven the effectiveness of in
vivo intranasal administration of therapeutics in direct nose-to-brain
targeting via the olfactory nerve pathway (Gadhave et al., 2019b; Gad­
have et al., 2018; Kumbhar et al., 2020). Despite these benefits, the
major limitation of intranasal delivery is the faster elimination of the
applied formulations due to nasal mucociliary clearance, which serves
as a natural physiological defense (Gorain et al., 2020). As a conse­
quence of this physiological defense mechanism, there would be a
decrease in nasal localization time of the applied formulation, thereby
retarding its absorption and transportation from nose to brain. Incor­
poration of mucoadhesive polymers such as gellan gum, chitosan,
poloxamer, carbopol, etc., in nasal formulations could improve the
localization time of the therapeutics at the nasal region and enhance
permeation of the entrapped therapeutics to the brain (Chin et al., 2021;
Hosny and Hassan, 2014; Karavasili and Fatouros, 2016). Gellan gum is
a natural anionic hetero-polysaccharide obtained by aerobic fermenta­
tion of Sphingomonas elodea (Bali and Salve, 2020). Being free from
living microorganisms (e.g., Escherichia coli, Pseudomonas aeruginosa,
Salmonella spp, etc.), gellan gum is recommended as safe for pharma­
ceutical and human use (Das and Giri, 2020; Gadhave et al., 2021). It
possesses ideal characteristics of gel formation (ion-sensitive in situ
gelling) in aqueous environment in the presence of cations (mono or
divalent), which are also usually found in nasal secretions (Bali and
Salve, 2020). Additionally, poloxamer 407 is generally employed to
formulate thermosensitive in situ gels to promote viscosity and
mucoadhesiveness of the formulation (Yu et al., 2018). Many re­
searchers have used the temperature-sensitive gelling system for the
intranasal delivery of therapeutic agents. This system remains in a liquid
state at room temperature and turns into gel at systemic temperature
(34.5 ± 0.5 ◦ C) (Fouad et al., 2013; Yu et al., 2018). In the present work,
an approach was made to fabricate an intranasal lipid-based nano­
emulgel (NG) system of AMS for effective and safe delivery to the brain.
NGs are comprised of three-dimensional cross-linked polymers that
show swelling behavior in the aqueous environment in vivo (Chatterjee
et al., 2019; Choudhury et al., 2017b; Kumbhar et al., 2020). A combi­
nation of gellan gum and poloxamer 407 in an NG formulation repre­
sents an ideal composition to improve intranasal retention of the
nanocarrier and augments penetration of the therapeutics to the brain
(Pich and Richtering, 2012). Thus, extensive evaluations were carried
2
International Journal of Pharmaceutics 607 (2021) 121050
out for the judicious selection of the formulation components, followed
by optimization by Box-Behnken statistical design of the prepared
formulation and final characterization of formulations. Estimation of ex
vivo permeation, nasal ciliotoxicity, in vivo brain and plasma pharma­
cokinetics, animal behavior, and hematological studies of the developed
formulation were conducted to confirm the safety and efficacy of AMS in
the treatment of schizophrenia.
2. Materials and methods
2.1. Materials
Amisulpride was received as a gift sample from Swapnroop Drugs
and Pharmaceuticals (Aurangabad, India). Gellan gum was purchased
from Chemkart (Mumbai, India). Labrasol, Maisine CC, and Transcutol
HP were received from Gattefosse (Mumbai, India) as gift samples. In
situ gelling agent (Poloxamer 407) was obtained from Yarrow Chemical
Products (Mumbai, India). Tweens were collected from BASF, Corpo­
ration (Mumbai, India).
2.2. Screening of oil, surfactant and co-surfactant for nanoemulsion
formulation
A saturation solubility procedure was used to screen the appropriate
oil, surfactant, and co-surfactant for developing nanoemulsion (NE) of
AMS (Kokare et al., 2020). Therefore, the oils (Maisine CC, Peceol, Oleic
acid, Sunflower oil, Isopropyl myristate, Castor oil, and Labrafac), sur­
factants (Labrasol, Tween 20, Tween 80 and Cremophor RH 40), and co-
surfactants (Transcutol HP, Polyethylene glycol 600, Propylene glycol,
and Ethanol) were screened to determine the solubility of AMS via
orbital shaker (Kytos, Germany) at ambient temperature for 72 h. An
excess quantity of AMS was incorporated in 10 mL of every vehicle in
glass vials; thereafter, the aforementioned blends were centrifuged at
10,000 rpm for 30 min using a centrifuge instrument (Remi Laboratory
instruments). The resultant supernatant was filtered through 0.45 µm
membrane filter and diluted with methanol. Finally, the samples were
analyzed at a wavelength of 274 nm using a UV–visible spectropho­
tometer (V-630, Jasco, Japan).
2.3. Development of pseudo-ternary phase diagrams
Phase diagrams were constructed to determine the NE area and the
experiment was performed via water titration technique. Surfactant and
co-surfactant were blended in predefined composition ratios (2:1, 1:1,
and 1:2). The phase diagrams were constructed using the data obtained
from preparing clear dispersions of specific Smix (combination of sur­
factant and co-surfactant) with the selected oil ratio (9:1, 8:2, 7:3, 6:4,
5:5, 4:6, 3:7, 2:8, and 1:9, v/v) (Agarwal et al., 2018) and gradual
addition of a desired quantity of water with constant stirring. The
pseudo-ternary phase diagrams, consisting of oil, Smix, and water as the
three axes, were constructed using software (CHEMIX School 7.00).
2.4. Preparation of AMS-loaded nanoemulsion
AMS-loaded NEs (AMS-NE) were formulated using low energy
emulsification approach, aqueous-titration technique. Initially, different
proportions of the components were selected from the pseudo-ternary
phase diagram in order to formulate the blank NE. The measured
amount of AMS was added to the selected oil (Maisine CC) and stirred
until complete solubilization was accomplished. The combination of
surfactant and co-surfactant (Labrasol: Transcutol HP:: 2:1) was added
to the drug solubilized oil phase at different ratios. Thereafter, drop-wise
addition of water into the oil-Smix mixtures was done with continuous
stirring for 15 min to get clear, homogeneous and transparent NEs
(Gamal et al., 2017; Gorain et al., 2014). Finally, clear or lightly bluish
NEs were examined for globule size and zeta potential using size
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

analyzer (Nanophox, Sympatech, Germany), and zeta meter (Delsa Nano 2.7. Preparation of AMS-loaded poloxamer-gellan gum in situ gel (AMS-
C, USA), respectively. NG)

2.5. Thermodynamic stability of amisulpride nanoemulsion The optimized AMS-loaded NE (1 mg/mL) was first developed in
accordance with results obtained from the QbD based optimization to
The thermodynamic stability of the developed nanoformulation was yield desired globule size, PDI, and % transmittance. Later, the NE
assessed by applying different thermodynamic conditions (Gorain et al., formulation was converted to NG using poloxamer 407 and gellan gum
2014; Kumbhar et al., 2020). Firstly, the centrifugation was conducted that were dispersed separately in deionized water. Furthermore, the
at 10,000 rpm for 20 min and nanoformulations were examined for combination of poloxamer 407 and gellan gum produced optimal gelling
instability. Subsequently, the formulations, which were stable after strength under physical conditions. Moreover, studies have reported
centrifugation, were exposed to three cycles of freeze–thaw study at a that gellan gum prevents drug dissolution in the nostril as well as res­
temperature of − 20 ◦ C and +40 ◦ C using a deep freezer (RQV-200 plus, piratory region and prolongs the availability of the drug at the site of
Remi, Mumbai) for freezing and temperature cabinet (LAB-HOSP Cor­ administration (olfactory region) (Wavikar and Vavia, 2015). Hence, the
poration, Mumbai) for heating. Finally, the formulations were examined combination of these two polymers was selected. Consequently, polox­
for physical instability, such as creaming, phase separation, and amer aqueous dispersion was then incorporated in the NE replacing the
instrumentally for globule size determination. required aqueous phase with a constant stirring at +4 ◦ C. Thereafter, the
aqueous dispersion of gellan gum was gradually incorporated in the
2.6. Box–Behnken experimental design resulting mixture, to get final in situ gel formulation containing 17% and
0.3% (w/v) poloxamer 407 and gellan gum, respectively. This combi­
Box–Behnken statistical design was employed for the optimization of nation of poloxamer 407 and gellan gum was selected due to its ability to
developed NE, where the Design–Expert software provided seventeen form a gel-like structure under physiological conditions (Wavikar and
theoretical runs having three independent variables at respective three Vavia, 2015). Finally, the formulation was equilibrated through gentle
levels, low (− 1), medium (0), and high (+1) (Table 1) (Akrawi et al., magnetic stirring to obtain clear and homogenous AMS-NG formulation
2020; Gadhave et al., 2019b). Based on the recommendation provided (1 mg/mL) and refrigerated at +4 ◦ C (Jelkmann et al., 2020) until
by the software, formulations were fabricated by the method described further use. Prepared NG formulation was further characterized as
in Section 2.4 and subsequently analyzed for thermodynamic stability. depicted below.
Based on the statistical design, interaction of three formulation factors,
the composition of oil (A), Smix (B) and water (C), on dependent vari­ 2.8. Characterization of the AMS-loaded optimized formulations
ables such as globule size (Y1), PDI (Y2) and % transmittance (Y3) of the
NE were assessed (Table 1). The software provided formula of the 2.8.1. Globule Size, PDI and zeta potential
quadratic model was depicted in equation (1). The NG was diluted (10 times) with deionized water and droplet size
and PDI were analyzed using the size analyzer (Nanophox, Sympatech,
Y = b0 + b1 A + b2 B + b3 C + b12 AB + b13 AC + b23 BC + b11 A2 + b22 B2 + b33 C2 Germany). The potential difference between the stationary layer of the
(1) dispersed droplets and the medium, i.e. the surface charge between the
optimized NE and NG droplets and the subsequent medium was
where the responses were expressed by Y, the regression coefficients by measured using a zeta meter (Delsa Nano C, USA) (Kokare et al., 2020).
b1 to b33, the independent variables by A, B, and C, and the intercept was
expressed by b0. 2.8.2. Percent transmission measurement
The % transmittance of optimized formulations was measured using
Table 1 UV–visible spectroscopy at λmax of 650 nm. Before the analysis, opti­
The experimental design layout created for 17 batches of amisulpride nano­ mized formulations were diluted in proportions of 1:1 and 1:5 with
emulsion by Box-Behnken statistical design.
deionized water, wherein the deionized water was used as a blank (with
Batch Coded values Responses a transmittance of 100%) (Kumar et al., 2008).
A B C Y1 Y2 Y3
(Oil) (Smix) (Water) (Globule (PDI) (Transmittance) 2.8.3. Entrapment efficiency (%)
mg mg mg Size) nm % The entrapment efficiency (% EE) of AMS in NE and NG formulations
F1 0 − 1 − 1 203.17 0.76 97.08 was analyzed by adding 1 mL of the formulation in 10 mL of methanol,
F2 0 0 0 174.21 0.64 97.47 subsequently centrifuged at 10,000 rpm for 30 min. The supernatant
F3 1 1 0 387.44 0.89 94.35 was collected and further dilutions were made using methanol. Finally,
F4 0 0 0 157.38 0.61 97.58
F5 0 − 1 1 149.25 0.51 96.95
the dilutions were analyzed using a validated RP-HPLC system (Pandey
F6 − 1 0 − 1 98.87 0.58 98.95 et al., 2018).
F7 0 0 0 176.31 0.68 96.97
F8 1 0 1 248.59 0.82 96.34 2.8.4. Refractive index measurement
F9 0 0 0 184.66 0.67 97.47
The refractive index of the formulations was measured using Digital
F10 0 1 1 149.37 0.59 97.29
F11 − 1 − 1 0 110.12 0.59 98.31 Abbe’s refractometer (Atago Co Ltd., Tokyo, Japan) in the presence of
F12 − 1 0 1 95.47 0.57 98.86 visible light.
F13 − 1 1 0 92.15 0.46 99.57
F14 0 1 − 1 134.48 0.63 97.83 2.8.5. Viscosity measurement
F15 1 − 1 0 230.12 0.86 92.31
F16 0 0 0 178.24 0.64 97.36
Rheological properties of the optimized formulations were evaluated
F17 1 0 − 1 223.98 0.81 95.42 through Brookfield viscometer (Oswal’s Scientific PES/MCOP), with
number-21 spindle. The rotation was slowly increased from 5 to 100
Independent Variables Level used, actual coded
rpm. For the viscosity analysis, an appropriate quantity of sample was
Low (− 1) Medium (0) High (+1)
tested at the temperature of 25 ◦ C and 34 ◦ C (Buyana et al., 2020).
A = Oil (%) 4 6 8
B = Smix (%) 40 45 50 2.8.6. Ex vivo mucoadhesive force
C = Water (%) 42 49 56
Intranasal localization of optimized formulation was evaluated

3
D. Gadhave et al.
through a Texture analyzer (Brookfield Engineering Labs). Fresh sheep
nasal mucosa was obtained from the local slaughterhouse and washed
with a simulated nasal electrolyte solution (SNES). Clean mucosa was
spread over the test holder of the instrument (Gadhave et al., 2021;
Gadhave and Kokare, 2019). Before analysis, the cylindrical probe was
immersed in a beaker containing AMS-NE and AMS-NG formulation for
10 s to form a thin layer of the respective formulation around it.
Thereafter, the probe was touched the mucous surface with a
compressive energy of 0.5 N for 60 s and separated at a speed of 1 mm/s
including a triggered force of 3 g. The strength needed for detachment of
the contact between mucosa and nanoformulation containing probe was
screened by the Texture Pro CT V1.3 Build 15 software.
2.9. In vitro release pattern of AMS from the optimized formulations
Release of AMS from the optimized AMS-NE and AMS-NG formula­
tions was compared with AMS-Suspension across the dialysis membrane
(molecular weight cut off: 12,000–14,000 D). The suspension was
fabricated by the addition of required amount of AMS (1 mg/mL) to the
mixture of hydroxypropyl methylcellulose (HPMC, 2%) and Tween 80
(4%) with continuous trituration using mortar and pestle. Thereafter,
the fabricated NE, NG and suspension formulations were investigated
through vertical Franz diffusion cell (15 mL) at 34.5 ± 0.5 ◦ C for 8 h with
continuous stirring using magnetic beads (Chin et al., 2021), where
SNES was used as the release medium for the study. Prior to the inves­
tigation, the activated dialysis membrane was dipped into SNES and
fully stabilized for 15 min. Respective formulations (1 mL each) were
uniformly spread in the donor compartment and SNES in the acceptor
compartments were stirred using magnetic stirrers. Aliquots (0.1 mL)
were collected from the acceptor compartments every 1 h and refilled
with the same quantity of fresh SNES. Furthermore, the samples were
analyzed for the determination of cumulative drug release using the
developed RP-HPLC method with appropriate dilutions using the mobile
phase. Finally, the release data obtained for each formulation was
evaluated for the release kinetic models.
2.10. Ex vivo drug permeation study
Ex vivo permeation study was conducted to investigate the perme­
ation potency of AMS from AMS-NE, AMS-NG, and AMS-Suspension
across the nasal mucosa (Pathak et al., 2014). Freshly isolated sheep
nasal mucosa was stored in SNES (pH 6.4) at 37 ◦ C ± 0.5 ◦ C to preserve
the viability of the cells. The adhesive tissues and unwanted debris were
removed cautiously without damaging the mucosa and a clean mucosal
membrane was used further for the study. Franz diffusion cell (15 mL
volume) was used for the ex vivo permeation study. The mucosa of
suitable size (3.14 cm2) was placed above the acceptor compartment,
facing the donor compartment. The mucosa was completely equilibrated
with the SNES before experimentation. For the experimental analysis, 1
mL formulations were uniformly spread upon the nasal mucosa in the
donor compartments. At proposed time intervals (1.0, 2.0, 3.0, 4.0, 5.0,
6.0, 7.0, and 8.0 h), 0.1 mL of aliquots were withdrawn from the
acceptor compartments, which were filtered through 0.45 μm mem­
brane filter and analyzed using previously developed RP-HPLC method
(Chin et al., 2021). Permeation coefficient (Papp) and flux (Jss) of
selected formulations were estimated using the following equations (2)
and (3).
Slope
P= × VD (2)
S
Jss = P × CD (3)
where Papp, Jss, VD, CD, and S represent permeation coefficient, flux,
volume of donor compartment (mg/mL), concentration of AMS in donor
compartment, and effective surface area of nasal mucosa, respectively.
4
International Journal of Pharmaceutics 607 (2021) 121050
2.11. In vivo animal experiments
Male Wistar rats weighed 200–250 g were purchased from the Na­
tional Institute of Biosciences Pune (India) and housed at standard
laboratory conditions (25 ± 2 ◦ C and 55 ± 5% RH) a week prior to the
study. The experimental protocols for in vivo pharmacokinetics, bio-
distribution, animal-behavioral and toxicological studies were
approved by the animal ethical committee (No. 1697/PO/Re/S/13/
CPCSEA/2020/05).
2.11.1. Pharmacokinetic study
For the pharmacokinetic study, the animals were divided into three
groups (n = 15 in each group):
Group I: Intranasal administration of AMS-NE (1 mg/kg body
weight)
Group II: Intranasal administration of AMS-NG (1 mg/kg body
weight)
Group III: Intravenous administration of AMS-NE (1 mg/kg body
weight)
The dose of AMS was calculated from the human dose based on body
surface area (Kumbhar et al., 2020). For the group III animals, AMS-NE
was administered intravenously via tail vein. Before intranasal delivery
in Group I and II animals, the animals were slightly anesthetized using
anesthetic ether. After anesthesia, 40 µL of AMS-NE and 40 µL of AMS-
NG (20 µL/nostril) were administered to the respective group of animals
with the help of an 18/20 polyethylene cannula attached to a micropi­
pette. Consequently, 0.3–0.5 mL blood samples were collected in the
microcentrifuge tubes (precoated with heparin to prevent clotting) at
pre-determined time points (0.5, 2.0, 4.0, 8.0, and 24.0 h) through retro-
orbital plexus (Kaur et al., 2020). Subsequently, blood samples were
centrifuged at 4000 rpm for 10 min to obtain a clear supernatant which
was separated using micropipette. Simultaneously, for the brain kinetic
study, three animals from every group at each time point were sacrificed
and the brains of rats were isolated, then washed with normal saline and
blotted. These collected plasma and brain samples were stored at − 70 ◦ C
until further analysis.
2.11.2. Analysis of AMS in brain and blood plasma samples
Chromatographic separation of AMS was achieved by Shimadzu
Prominence (Kyoto, Japan) using Kromosil C18 (4.6 × 250 mm) column
with a mobile phase consisting of methanol: 0.2% formic acid (70:30), at
a flow-rate of 0.5 mL/min. The chromatographic detection was per­
formed at the wavelength of 274 nm by SPD-M− 20A variable wave­
length photodiode array (PDA) detector. Amisulpride-d5 was utilized as
an internal standard for the bio-analysis of AMS in brain and plasma
samples. For the analysis of AMS quantity in brain samples, brain tissue
was homogenized with normal saline using tissue homogenizer, and
homogenates were utilized for drug concentration analysis. A 10 μL of
(50 μg/mL) internal standard and 400 μL of methanol was added to
previously measured volume of 100 μL (each treated plasma and brain
homogenate), wherein methanol acted as protein precipitating agent.
These samples were then mixed in a vortex for 15 min and centrifuged at
4000 rpm for 10 min. The supernatant was collected, filtered, and a
measured aliquot (20 μL) of the filtrate was injected into the RP-HPLC
for AMS analysis. All pharmacokinetic parameters including maximum
brain or plasma concentration (Cmax), area under the curve (AUC), and
time to obtain the maximum brain or plasma concentration (Tmax), were
analyzed through WinNonlin 8.1 software (Bali and Salve, 2020;
Kumbhar et al., 2020). The targeting factors, such as drug targeting
index (DTI), targeting efficiency (% DTE), and direct transport per­
centage (% DTP) were evaluated for intranasal administrations using
AUC values of AMS in brain and plasma following the equations (4) to
(7).
D. Gadhave et al.
[AUCBrain /AUCBlood ].Intranasal
DTI =
[AUCBrain /AUCBlood ].Intravenous
[AUCBrain /AUCBlood ].Intranasal
%DTE = × 100
[AUCBrain /AUCBlood ].Intravenous
[BIntranasal − Bx. ]
%DTP = × 100
[BIntranasal ]
Bx. =
[BIntranasal. ]
× PIntranasal
[PIntravenous. ]
where
BIntranasal = AUC0- 24 of brain resulting through intranasal
administration.
BIntravenous = AUC0- 24 of brain resulting through intravenous
administration.
PIntranasal = AUC0-24 of plasma resulting through intranasal
administration.
PIntravenous = AUC0-24 of plasma resulting through intravenous
administration.
2.12. Animal behavioral studies
The preliminary in vivo efficacy of AMS nanoformulations were
examined by catalepsy test, locomotor activity, and paw testing. Male
Wistar rats were divided into four different groups (n = 6), e.g., control,
vehicle (blank NE) treatment, AMS-NE treatment, and AMS-NG treat­
ment. In the positive control group, the animals were treated with a 3
mg/kg dose of haloperidol. The dose of AMS nanoformulations was set
at 1 mg/kg body weight for animal studies. One week prior to the study,
all animals were acclimatized and kept separately in polypropylene
cages at standard temperature and humidity conditions. Commonly
available medicines for schizophrenia showed their side effects after two
to three weeks of administration, hence a 28 days treatment protocol
was selected for each treatment group to examine the catalepsy, induced
locomotor activity, and paw testing. Observations were made and re­
ported on the 1st, 7th, 14th, and 28th days of administration to
acknowledge the consistency in treatment (Kumbhar et al., 2020).
2.12.1. Catalepsy screening
Catalepsy screening was performed to examine the EPS related to the
intranasal treatment of the developed nanoformulations. This experi­
ment was carried out by placing the forelimbs of an individual animal on
a 10 cm height of a horizontal bar and hind-limbs rested on the platform
(Bricker et al., 2014). Duration of time that animals remained in atypical
position, was considered for the evaluation of EPS. All the observations
were made on the 1st, 7th, 14th, and 28th days post intranasal
treatment.
2.12.2. Induced locomotor activity test
Locomotor activity was tested using a digital actophotometer
(Mevtex) for 10 min. This test was performed to investigate the antag­
onistic effect of AMS on dopamine receptors. Herein, Carbidopa (2.5
mg/kg) and L-dopa (10 mg/kg) were intraperitoneally injected in the
experimental animals to induce hyperactivity. Additionally, separate
groups of animals were treated intranasally with fabricated AMS-NE and
AMS-NG. The antagonistic efficacy of administered formulations was
measured by the number of locomotor counts in various groups and
comparing them with the control and vehicle groups (Kumbhar et al.,
2020).
2.12.3. Paw test
The paw test conducted on a perspex platform, 30 cm × 30 cm × 20
cm (side × width × height) with four holes, bifurcating into two holes
International Journal of Pharmaceutics 607 (2021) 121050
(diameter 4 cm) for forelimbs and the other two holes (diameter 5 cm)
(4)
for the hindlimbs (Kumbhar et al., 2020). The purpose of this test was to
demonstrate the therapeutic effects and EPS associated with the use of
the fabricated intranasal formulations of AMS. The time required to
(5)
withdraw the forelimbs is called forelimb retraction time (FRT). Simi­
larly, for hindlimbs, the time required is called hindlimb retraction time
(HRT). The HRT is associated with EPS that can be attributed to the
(6)
effect of treated drugs, whereas the antipsychotic effect of the treatment
could be correlated by the FRT. In this experiment, a duration of 60 s was
(7) fixed for estimating HRT and FRT for the experimental groups and the
readings were reported in triplicates with an interval of 5 min. The
average time required for animals treated with nanoformulations was
compared with control and vehicle group animals to determine the
outcome of the paw test.
2.13. In vivo safety assessment
The male Wistar rats weighed 200–250 g were divided into seven
experimental groups with six animals in each.
Group I: Control group (untreated)
Group II: Intranasal AMS-NG treated animals (low dose- 1 mg/kg)
Group III: Intranasal AMS-NG treated animals (medium dose- 2.5
mg/kg)
Group IV: Intranasal AMS-NG treated animals (high dose- 5 mg/kg)
Group V: Intravenous AMS-NE treated animals (low dose- 1 mg/kg)
Group VI: Intravenous AMS-NE treated animals (medium dose- 2.5
mg/kg)
Group VII: Intravenous AMS-NE treated animals (high dose- 5 mg/
kg)
The treatments were administered daily to the above-mentioned
groups of animals for 28 days. The administration of NG and NE in
medium and high dose groups was given for several intervals in a day to
maintain the volume of administration to 20 µL/nostril/administration.
Finally, the AMS-induced agranulocytosis was evaluated using the
collected blood samples. Simultaneously, the animals were screened for
any hematological toxicity, gross pathology, and histopathological
complications due to the treatments (Pokharkar et al., 2017).
2.13.1. Evaluation of formulation-induced agranulocytosis
Blood samples were collected via retro-orbital plexus from the anes­
thetized animals in anticoagulant (EDTA) coated tubes. The hemato­
logical investigation of the blood samples was performed using auto-
analyzer (URIT 2900 Vet Plus UK). The obtained results were
compared amongst individual groups of animals to establish the rela­
tionship of the treatments.
2.13.2. Gross pathology
Visual inspection of the animals was made during the entire 28-day
experimental protocol and observations were made from deviation in
the appearance of normal healthy animals vs. treated animals. De­
viations in appearances and behavior were noted for unfavorable signs,
such as blinking, nasal bleeding, eye irritation, nasal secretions, ery­
thema, nostril irritation, as well as abnormal moments inside or outside
the cage prior and post intranasal administration. Any signs and symp­
toms of toxicity and mortality amongst animal groups during the
experimental period were later correlated with the previous observa­
tions (Gadhave et al., 2021).
2.13.3. Histopathological evaluation
The isolated nasal mucosal sections were placed in a 10% formalin
solution. Before this investigation, each section was washed for removal
of any connective tissue. Isolated sections were stained with hematox­
ylin and eosin solution. Finally, the sections were assessed for necrosis
and structural damage using a light microscope (Salunke and Patil,
5
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

2016). Transcutol HP is a commonly used component in pharmaceutical

2.14. Statistical analysis
All collected data are shown in the format as mean ± SD. A student t-
test was applied to assess statistical significance in the animal testing
procedures. The outcomes of pharmacological and hematological testing
data were considered statistically significant if *p ≤ 0.05. Statistical
analysis was performed using GraphPad Prism version 5.00 software.
3. Results and discussion
Therapeutic applications of available antipsychotic agents are asso­
ciated with certain life-threatening adverse events such as EPS, agran­
ulocytosis, leukopenia, and other hematological toxicities (Gadhave
et al., 2019a). On the other hand, application of the antipsychotic agents
are limited due to low solubility and bioavailability. In the present
study, an attempt has been made to develop AMS-NG formulation for
direct nose-to-brain delivery which could help to overcome the associ­
ated limitations thereby reducing the peripheral exposure of the
administered drugs via direct delivery to the brain. Nose-to-brain drug
delivery approach is based on two important physicochemical properties
of the medicinal agent, molecular weight and lipophilicity. AMS is a BCS
class II drug, i.e., highly lipophilic with log p-value of 1.06 and suitable
molecular mass (369.479 Da) (Maatallah et al., 2017; Pickard et al.,
2016).
3.1. Developmental approach of AMS nanoemulsion
The selection of the formulation components is a crucial step while
developing any NE formulation. Thus, all the essential components for
the development of NE formulation were selected through a saturation
solubility study. For the development of a novel drug delivery system in
the current research, excipients were selected based on the solubility of
AMS in various GRAS (Generally Recognised as Safe) approved oils,
surfactants, and co-surfactants. Amongst the oils, Maisine CC showed
the highest solubility for AMS (105 ± 1.52 mg/mL) (Fig. 1). Similarly,
around 140 ± 2.54 mg/mL of AMS was dissolved in Labrasol (Fig. 1).
Alternatively, Transcutol HP had shown the solubility of 160 ± 3.01 mg/
mL for AMS (Fig. 1). Therefore, Maisine CC, Labrasol and Transcutol HP
were selected as oil, surfactant, and co-surfactant, respectively for the
development of nanoemulsion formulation. Labrasol is a non-ionic
surfactant with an HLB value of 14, and it is commonly used in the
nanoparticulate drug delivery system to enhance the permeability of
drugs across the biological barriers (Sigward et al., 2013). Additionally,
200
Solubility (mg/mL)
products which is devoid of any genotoxicity, sub-chronic and chronic
toxicity, as evidenced from the available reports (Sullivan et al., 2014).
Therefore, these components were preferred for the development of NE
formulation.
In the next step of formulation development, the NE regions were
measured based on obtained data from pseudo-ternary phase studies.
Enhancing the nanoformulation fluidity is essential to permeate the drug
from the hydrophobic section of the surfactant into the oil phase. This
phenomenon effectively decreases the interfacial tension between the
oil and aqueous phase. The role of the co-surfactant is to further increase
the fluidity of the formulation to form a stabilized coating of surfactant
encapsulating the oil globules. Furthermore, the surfactants and co-
surfactants (Smix) were employed in different proportions, such as 2:1,
1:1, and 1:2, to create the pseudo-ternary phase diagrams and observe
the maximum NE regions. The diagram was constructed using CHEMIX
School (7.00) software and the largest NE region was observed for 2:1
Smix ratio (Fig. 2). Therefore, it was be inferred that the formulations
containing 2:1 ratio of Smix would be able to produce a clear, uniform
and stable NE formulation as compared to other Smix ratios. Hence, a 2:1
Smix ratio was selected for further development of AMS-loaded NE
formulations.
Different trial batches of NE formulations were prepared by blending
4–8% of oil phase (Maisine CC), 40–50% of Smix (Labrasol: Transcutol
HP at 2:1 ratio) and 42–56% of the aqueous phase using aqueous
titration method with constant stirring (Choudhury et al., 2014). The
quality of fabricated NE was examined by their globule size, PDI, and
thermodynamic stability studies. The stability of all developed NEs was
conducted by centrifugation and freeze–thaw cycle techniques. The
findings from centrifugation and freeze–thaw studies of the developed
NEs did not reveal any signs of creaming, phase separation, sedimen­
tation or drug precipitation, assuring excellent thermodynamic stability
of the prepared formulations (Ragelle et al., 2012).
3.2. Optimization of AMS-loaded nanoformulations
Droplet size of NE could be controlled by monitoring the concen­
tration of oil, surfactant and co-surfactant in the composition, where an
increase in oil percentage has been reported to increase in droplet size.
Alternatively, an increase in surfactant concentration is associated with
decrease in droplet size (Fig. 3(a)). Furthermore, decrease in the size of
the dispersed droplets increases the oil-in-water interfacial area, thus the
absorption and permeation of the entrapped drug from the NE could be
improved through the biological membranes (Choudhury et al., 2017a).
Concurrently, decrease in droplet size of the NE increases the stability of
the formulation through decreasing coalescence and flocculation of the
dispersed phase, which might be due to a significant reduction in
attractive forces between the dispersed droplets. Thus, optimization of
the composition to obtain a uniform/monodisperse droplet size with low
PDI dictates a stable NE with a narrower size distribution of dispersed

150
droplets (Marzuki et al., 2019). PDI value represents the ranges of
droplet sizes in the formulation. PDI ranging from 0.08 to 0.7 yields the
100 best results, whereas >0.7 PDI represents a broader range of droplet size
distribution (Asmawati et al., 2014; Danaei et al., 2018). Percentage of
50
oil and surfactant in formulation affects PDI values of NEs (Fig. 3(b)).
Based on the literature, the spectrophotometric determination of %
transmittance of the fabricated nanoemulsion revealed its nature,
0 whether the formulation consisting of coarse or fine droplets. This na­
ture of NE is highly dependent on its composition, wherein increase in
to e

gl 0
Et ol
La fac

ee ol

l
le cu 40
yr il

br l
ro ow id

op lyc HP
Pe C

Is Su ic l

0
t

no
La r oi

0
80
le o

lm ro

C ista

re Tw n 2

yc
C

en l 6
Tw as
op nf ac
O ce

hy ns H
a

ha
g l
op een
ne

oil and surfactant concentrations in formulation would result in a

e

o
ne to
br

R
as
si

e
ly Tr or
l
ai

decrease or increase in % transmittance, respectively (Fig. 3(c))

M

py

yl
a
m

(Choudhury et al., 2014). Thus, optimization of the formulation pa­

Pr
C

et

rameters is of high importance when we are planning to fabricate a

Po

Oil Surfactant Co-surfactant stable, homogenous, and transparent NE formulation.

The seventeen AMS-NE formulations (Table 1) were formulated as
Fig. 1. Solubility of AMS in oils, surfactants and co-surfactants. described under the methodology section and optimized the interaction

6
D. Gadhave et al.
Fig. 2. Pseudo-ternary phase diagrams of Smix ratios (a) 1:2, (b) 1:1 and (c) 2:1.
of the three formulation factors on droplet size, PDI, and % trans­
mittance. The experimental values for dependent variables, such as
globule size (Y1), PDI (Y2), and % transmittance (Y3) for the seventeen
formulations, were found to be in close agreement with predicted values
(Table 1 of the supplementary data). The percent error was found to be
less than 5%, suggesting that the experimental values were accurately
matched with the predicted values (Fig. 3(d)-(f)). Percent error was
helpful for the measurement of accuracy about generated equations.
Further, the statistical result of the interaction between three inde­
pendent variables, oil phase, Smix, and aqueous phase on droplet size is
presented in Table 2 of the Supplementary Data. The model F-value of
6.74, 8.66, and 7.80 for the interaction of independent variables on
globule size, PDI, and % transmittance, respectively indicted that the
quadratic model is significant for all three dependent variables. In terms
of the significance of the model terms, model term A (changes of oil %)
have shown a significant effect (p < 0.05) on all the dependent variables,
whereas model terms B and C did not exhibit any significant effect.
Adequate precision values 10.163, 9.280, 10.705 for globule size, PDI,
and % transmittance, respectively represented an adequate signal with
quadratic model for all the dependent variables. Therefore, the model
can be used to navigate the design space.
Additionally, the significant effect of model term A on all three
dependent variables was strengthened by the highest coefficient values
of model term A for globule size (+86.70), PDI (+0.1475), and %
transmittance (− 2.16) in the generated polynomial equations on the
interaction between independent variable on globule size (equation (8)),
PDI (equation (9)) and % transmittance (equation (10)).
7
International Journal of Pharmaceutics 607 (2021) 121050
Y1 (globuleSize) = + 174.16 + 86.70*A + 8.84*B − 2.23*C + 43.84*AB
+ 7.00*AC + 17.20*BC + 19.22*A2 + 11.56*B2 − 26.66*C2
(8)
Y2 (PDI) = + 0.6480 + 0.1475*A − 0.0187*B − 0.0363*C + 0.0400*AB
+ 0.0050*AC + 0.0525*BC + 0.0622*A2 − 0.0102*B2 − 0.0153*C2
(9)
Y3 (%Transmittance)=+97.37− 2.16*A+0.5487*B+0.0200*C+0.1950*AB
+0.2525*AC0.1025*BC− 0.565*A2 − 0.6700*B2
+0.5875*C2
(10)
Further, the highest coefficient values of model term A in equation
(8), (9) and (10) are in agreement with the 3D surface plots (Fig. 3(a)-
(c)). The negative coefficient value of A on % transmittance indicated
that an increase in oil % has resulted in decrease in % transmittance.
Moreover, a decrease in % transmittance represents the increase in
droplet size. Therefore, if it correlates, an increase in oil % could lead
increase in droplet size of the NE formulation, which is in agreement
with the positive coefficient value (+86.70) of model term A in equation
(8).
NE with a large globule size could show retarded drug absorption
and permeation. According to statistical analysis, the final optimized NE
formulation consisted of the oil (4%), Smix (50%) and water (46%) and
further this composition showed compliance with optimum
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

Fig. 3. Three-dimensional surface response plots showing effect of oil, Smix, water on (a) globule size, (b) polydispersity index, (c) % T and linear correlation plots
between the actual and predicted values for (d) globule size, (e) polydispersity index, (f) % T.

requirements of globule size (92.15 ± 0.42 nm), PDI (0.46 ± 0.03) and
% T (99.57 ± 0.6%). Thus, all responses were appropriately correlated
with the NE system and this formulation was selected for further studies.
3.3. Transformation of nanoemulsion (NE) to nanoemulgel (NG)
Temperature-sensitive conversion of poloxamer 407 from sol-to-gel
plays a crucial role in the formation of in situ gel at physiological
temperature. Therefore, conversion of gel into the nasal environment
following intranasal application might improve the mucoadhesive
properties of the formulation that helps to increase nasal residence time.
This leads to prolonged absorption of the entrapped drug adhered to the
site of administration (Chin et al., 2021; Gorain et al., 2020). Alterna­
tively, the natural gelling agent (gellan gum) has possessed the char­
acteristics to form gel due to its ion-sensitive gelation properties (Swain
et al., 2019). The rapid sol-to-gel transition of the gellan gum containing

8
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

Table 2 The research findings from the initial trial batches confirmed that
Ex vivo permeation of amisulpride permeated through the sheep nasal mucosa poloxamer 407 produces a gel of adequate consistency at 17% concen­
(concentration of AMS formulations in the donor compartment [CD = mg] and tration. Moreover, the formulation also showed a quick sol–gel transi­
volume of formulation in the donor compartment [VD = mL]. tion. The required mucoadhesive force for separating the formulation
Formulations Jss (µg/cm2/ Papp (cm/h × AMS permeated (µg/ from the mucosal membrane was improved by the addition of a 0.3%
h) 103) cm2) concentration of gellan gum (Shah et al., 2017). If the concentration of
AMS-NE 46.010 ± 1.05 46.01 ± 0.16 713.65 ± 12.84 poloxamer 407 and gellan gum was increased further than the selected
AMS-NG 57.701 ± 1.75 57.70 ± 0.65 954.61 ± 9.52 concentration, proportionate increase in the hardness of gel was
AMS- 14.140 ± 1.07 14.14 ± 0.24 324.42 ± 8.42 observed, thereby, the administration of nanoformulation would not be
Suspension
feasible. Simultaneously, the reduced concentration of mucoadhesive
Jss: Steady-state flux, Papp: Permeability coefficient, AMS permeated: Cumulative and thermoresponsive polymers than the selected concentration was
AMS permeated through sheep nasal mucosa for 8 h. found to reduce the mucoadhesive force and gel-forming potential,
which is pointing towards the loss of medication from the nasal cavity
NE formulation would be facilitated an intranasal administration due to due to mucociliary clearance. Hence the selected composition (17%) of
the presence of cationic ions in the nasal secretions, such as Ca++ ions. gelling and (0.3%) mucoadhesive agents comply with the experimental
The presence of Ca++ ions directly interacts with the gellan gum expectations. The combination of temperature-sensitive with the ion-
resulting in gel formation (Gadhave et al., 2021; Shah et al., 2017; Swain sensitive gelling agents would provide an augmented in situ gelation
et al., 2019). of the sol–gel type formulation that will simultaneously reduce the

(a) (b)

(c) (d)

(e) AMS-NE AMS-NG AMS-NG (f) 12

120 * **
Mucoadhesive force (g)

8
80
Viscosity (cP)

4
40

*
0
0
E

G
N

N
C

C
o

S-

S-
M
25

25

34

M
A

Temperature at formulation tested Optimized formulations

Fig. 4. Characterization of particle size: (a) AMS-NE, (b) AMS-NG, zeta potential: (c) AMS-NE, (d) AMS-NG. Investigation of viscosity (e) and ex vivo mucoadhesive
strength (f) of AMS-NE and AMS-NG formulations. Values are expressed as the mean ± SD (n = 3), **p < 0.01, *p < 0.05 compared to AMS-NE formulation.

9
D. Gadhave et al.
gelation time of formulation following intranasal administration.
Thereby, it prevents therapeutic loss due to the mucociliary clearance
from the nasal cavity. The gelation time depends on the amount of in situ
gelling agents, physiological temperature, and concentration of cations
present in the nasal cavity (Gadhave et al., 2021). The recorded sol–gel
transition time was 4–6 s for developed optimized nanoformulation,
which could further overcome the barrier of intranasal administration.
3.4. Physicochemical characterization of the optimized nanoemulgel
formulation
3.4.1. Globule size, PDI and zeta potential
The size of the scattered globules in the prepared NE/NG system
signifies the droplet size, where the globule size of the optimized AMS-
NE and AMS-NG formulations were found to be 92.15 ± 0.42 nm (Fig. 4
(a)) and 106.11 ± 0.26 nm (Fig. 4(b)), respectively. It is considered that
the nanodroplets of the optimized formulation should have a wide sur­
face area possessing the potential to bypass BBB via intranasal route to
efficiently transport the therapeutics into the CNS. Moreover, the uni­
form distribution of the nanodroplets of the formulation was examined
by evaluating PDI. The PDI values of the optimized AMS-NE and AMS-
NG formulations were found to be 0.46 ± 0.03 and 0.51 ± 0.01,
respectively, which are less than 0.7, indicating homogeneity of the
dispersed droplets in the formulation. The zeta potential of the opti­
mized AMS-NE and AMS-NG formulations were recorded as − 18.22 ±
1.5 mV (Fig. 4(c)) and − 16.01 ± 2.3 mV (Fig. 4(d)), respectively. The
zeta potential of the developed system can be attributed to the presence
of Maisine (Du et al., 2010) as Maisine (glyceryl monolinoleate) consists
of linolenic acid and oleic acid contributing to the negative zeta po­
tential of the system (Abd El-Rehim et al., 2013; Vitorino et al., 2020).
As well as the composition of oil, pH of the system, and electrolytes
present within the aqueous system can also contribute to the negative
charge on the dispersed nanodroplets (Kokare et al., 2020).
3.4.2. Measurement of % transmittance and entrapment efficiency (%)
Optical clarity and transparency of developed nanoformulations
were characterized by the % transmittance study. Transmittance of the
AMS-NE and AMS-NG were found to be 99.57 ± 0.6% and 98.47 ± 1.3%,
respectively. The appearance of AMS-NE was clear with a bluish shade,
whereas the AMS-NG appeared clear and transparent. These findings
confirm the clarity and transparency of the developed nano­
formulations. The solubilization of the drug in the desired quantity of
lipid phase provides maximum entrapment of the loaded drug in the
formulation. In this context, all the batches of AMS-NE formulation (F1-
F17) showed the % EE within the range of 93.11 ± 0.5% to 99.11 ±
1.8%. Finally, the % EE of the optimized AMS-NE and AMS-NG formu­
lations were found to be 99.01 ± 0.86% and 98.93 ± 0.48%, respec­
tively. Due to its highest encapsulation capacity, NE is a highly desirable
carrier system for the delivery of therapeutics.
3.4.3. Determination of refractive index and rheological behavior
The refractive index of nanoemulsion was used to determine the
clarity and nature of the formulation. A larger globule size interferes
with the light passes through formulation resulting higher refractive
index of nanoemulsion. Whereas, the nanosized droplets in nano­
emulsion show a lower refractive index. Refractive index of the opti­
mized AMS-NE and AMS-NG formulations was found to be 1.360 ± 0.15
and 1.380 ± 0.21, respectively, which was similar to the water (1.330 ±
0.27) and closer to the refractive index of Maisine CC (1.363). Therefore,
the findings of the refractive index study confirmed that there were no
interactions between nanoemulsion components and AMS. Although,
these optical values were found to be directly proportional to the oil
content of the formulations, however, the outcomes of the present study
revealed that the developed nanoformulations are clear and isotropic in
nature (Yeo et al., 2021).
The rheological behavior of the optimized formulations is very
10
International Journal of Pharmaceutics 607 (2021) 121050
crucial for the determination of fluidity of formulations intended for
intranasal administration. In due course of this experiment, the AMS-NG
formulation showed significantly higher viscosity (12.6 ± 1.7 cP; *p <
0.05) at room temperature as compared to AMS-NE (8.69 ± 0.3 cP),
which might be due to the presence of polymers in the NG composition.
Based on observations, it is evident that higher viscosity could help the
AMS-NG to maintain low fluidity and adherence to the nasal mucosa.
Adherence was also attributed to the gelling capacity of AMS-NG in
presence of extracellular ions in the nasal mucosa, depicting and con­
firming ionic gelation behavior (Fig. 4(e)). Subsequently, at the nasal
temperature, the viscosity of the formulation was enhanced to 106.5 ±
2.8 cP (Fig. 4(e)). These findings confirmed that the formulation was less
viscous at room temperature whereas, it showed higher viscosity at
physiological temperature. This sol–gel transition occurred due to the
presence of a temperature-sensitive in situ gelling agent (poloxamer
407) in the formulation. From the outcomes of the study, it could be
inferred that the presence of poloxamer and gellan gum could enhance
the viscosity of nanoformulation at nasal physiology; where this con­
version of AMS-NG could counteract the mucociliary clearance.
3.4.4. Ex vivo mucoadhesive potential
Ciliary clearance is a defense strategy of the nasal cavity. Hence, the
liquid formulations are washed out within a short period from the nostril
after application. Thereby, the inappropriate localization time is the
principal barrier for the absorption of therapeutics through the intra­
nasal route to the brain. In due course, the AMS-NE formulation depicted
lower mucoadhesive force 1.24 ± 0.2 g, which showed higher fluidity
and less viscosity compared to the corresponding NG formulation. The
AMS-NG showed a mucoadhesive force of 8.90 ± 0.6 g at nasal tem­
perature, which was significantly higher as compared to AMS-NE (**p <
0.01). In situ gelling polymers help to enhance the mucoadhesion of
applied ASM-NG formulation in the nasal environment (Fig. 4(f)),
hence, the localization time could be extended. An increase in locali­
zation time will thereby cause the gel formulation to remain adhered for
a longer time at the site of absorption. Therefore, the outcomes could aid
in enhancing the delivery of the entrapped drug to the desired site
demonstrating superior activity of AMS-NG in overcoming the limita­
tions related to mucociliary clearance.
3.5. In vitro AMS release profile
This study was aimed to investigate the drug release behavior of the
fabricated nanoformulations relative to AMS-Suspension. Cumulative
release of AMS from the developed AMS-NE was 99.99 ± 1.82% within
4 h, whereas the release from AMS-NG and the AMS-Suspension were
found 98.96 ± 2.45% and 61.54 ± 1.84%, respectively within the time-
frame of 8 h (Fig. 5(a)). The experimental outcomes of this study
revealed that the NE formulation of the drug showed rapid release than
that of the AMS-NG formulation. It could be due to the presence of the
polymers in AMS-NG formulation, resulting in a sustained release profile
(Shah et al., 2017). Rapid release of AMS from the nanoemulsion might
be due to increased surface area of the fabricated nanodroplets, which
permeated easily through the dialysis membranes. Furthermore, the
droplet size of AMS-NE and AMS-NG were found to be <110 nm, which
helps in enhancing the solubility of AMS. Whereas, the incomplete
release of the drug from the AMS-Suspension could be due to the solu­
bility issue of the drug, where the coarse particles of suspension retard
the rate of AMS release through the pores of the dialysis membranes.
As determined by the kinetic models, the highest regression coeffi­
cient (R2, 0.996) of AMS-NG was found in Hixson Crowell cube root
model (Table 3 in the Supplementary Data); hence, this model was
selected as a best-fit model. Additionally, this model shows the rate of
release of drugs from developed formulations depending on the change
in the diameter of the droplet and surface area. The findings of the
release study depict that AMS-NG shows enhanced sustained-release
effect within 8 h as compared to AMS-NE. The AMS-NE showed an
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

(a) 120 AMS-NE AMS-NG AMS-Suspension significant increase (*p < 0.05) in Papp and Jss with AMS nano­
formulations was noted when compared to AMS-Suspension. The Jss of
100 AMS-NE, AMS-NG, and AMS-Suspension was found to be 46.01 ± 1.05
Cumulative AMS release (%)

µg/cm2/h and 57.70 ± 1.75 µg/cm2/h and 14.14 ± 1.07 µg/cm2/h,

80 respectively (Table 2). Concurrently, permeation rate of AMS from AMS-
Suspension is limited due to suspended drug particles in the formulation.
60
The highest Jss of the NG confirmed that the surfactant in the presence of
mucoadhesive agents enhances the rate of drug permeation. The main
40
reason for higher permeation from in situ gel formulation has remark­
20 able features of surfactant with natural polymers such as alteration or
disruption into the tight junction of a barrier (Wavikar et al., 2017).
0 Permeation improving property of surfactants in presence of mucoad­
0 2 4 6 8 10 hesive polymers resulting in the opening of pores of a barrier. Finally,
Time (h)
the AMS-NG confirmed higher AMS permeation compared to the AMS-
(b) NE and AMS-Suspension; hence, it could be considered as an effective
carrier for the AMS delivery.

3.7. In vivo pharmacokinetic study

To determine the pharmacokinetic profile, AMS-NG and AMS-NE

Fig. 5. a) Cumulative drug release study of AMS-NE, AMS-NG and AMS-
Suspension over 8 h through a dialysis membrane, b) ex vivo permeation
study of AMS-NE, AMS-NG and AMS-Suspension over 8 h by a sheep nasal
mucosa. Values are expressed as the mean ± SD (n = 3).
immediate release effect, thereby limiting the transport of released drug
to the brain. Whereas, the complete and sustained release of AMS from
the AMS-NG could be beneficial for prolonged absorption through
intranasal delivery as compared to other formulations (*p < 0.05).
3.6. Ex vivo permeation study
A natural membrane displays more accurate and actual drug
permeation properties. Anatomy of the human nasal mucosa is similar to
the sheep (Agu and Ugwoke, 2007; Kumbhar et al., 2020); hence, the
sheep’s nasal mucosa was chosen to perform ex vivo permeation. The
excised nasal mucosa was immediately stored in SNES to maintain the
viability of mucosa. This study was intended to examine the effect of
formulation characteristics on drug permeation profile. The cumulative
permeation of drug from AMS-NE, AMS-NG and AMS-Suspension was
found to be 713.65 ± 12.84 µg/cm2, 954.61 ± 9.52 µg/cm2 and 324.42
± 8.42 µg/cm2, respectively, after 8 h. As shown in Fig. 5(b), AMS-NE
and AMS-Suspension indicate 71.36 ± 1.97%, and 32.44 ± 2.51% of
cumulative drug permeation, respectively, after 8 h. However, AMS-NG
depicted 95.46 ± 2.83% drug permeation, which was significantly
higher than the other two formulations (**p < 0.01). Subsequently, a
were delivered through the intranasal and intravenous route in male
Wistar rats. Mean plasma and brain drug concentration versus time
profile following intranasal and intravenous administration of AMS
nanoformulations are represented in Fig. 6(a) and (b), respectively.
These figures revealed that the intranasal administration of AMS-NG and
AMS-NE formulations possessed a higher brain concentration when
compared to the intravenous administration of AMS-NE. The peak time
following administration of NE was shorter than that of NG, which in­
dicates an unexpected release profile for both formulations. From the
pharmacokinetic data, as displayed in Table 3, it was observed that
intranasal AMS-NG attains maximum brain concentration of AMS and
least plasma concentration, while it showed vice-versa in case of intra­
venously administered AMS-NE. The brain concentration of AMS upon
intranasal administration of AMS nanoformulations was found to
significantly higher (*p < 0.05) when compared to the brain concen­
trations in animals with intravenous AMS-NE. The pharmacokinetic data
displayed that the intranasal delivery of AMS-NG and AMS-NE formu­
lations directly transported AMS to the brain might be via trigeminal or
olfactory pathway. Whereas, NG formulation contained mucoadhesive
and gelling polymers, which helped the formulation to last longer in the
nasal cavity. Furthermore, the sustained release of the formulation at the
site of application was allowed to increase the duration of transportation
of AMS to the brain. Alternatively, the NE formulation is free from
mucoadhesive and gelling agents thereby, the formulation is prone to
mucociliary clearance. Hence, higher mucoadhesion due to gelling
capability of the in situ gel leads to longer residence at the site of
application with lesser mucociliary clearance of AMS-NG than AMS-NE.
This portrayed in higher brain exposure of the drug with intranasal
AMS-NG when compared to intranasal AMS-NE formulation (Gadhave
et al., 2021; Md et al., 2013).
Further, DTI, % DTE, and % DTP were used to evaluate the direct
nose-to-brain delivery of the drug. Here, AMS-NG displayed the
maximum values for DTI, % DTE, and % DTP as depicted in Table 3.
When compared, the % DTE of the formulation was enhanced from
314.08% for the intranasal AMS-NE to 1821.7% for the AMS-NG. The

Table 3
Results of pharmacokinetics parameters, DTI, % DTE , % DTP, and AUC0-∞ of AMS-loaded formulations through intranasal and intravenous administration in rats (n =
15, mean ± SD).
Formulations Brain Plasma Brain targeting parameters

Cmax (ng/mL) Tmax (h) AUC0-∞ (h.ng/mL) Cmax (ng/mL) Tmax (h) AUC0-∞ (h.ng/mL) % DTE DTI % DTP

AMS-NG (Intranasal) 220.92 ± 22.41 2.00 2598.62 ± 218.41 38.60 ± 5.70 2.00 293.50 ± 24.62 1821.72 18.21 275.09
AMS-NE (Intranasal) 148.63 ± 14.23 2.00 1414.84 ± 126.93 120.94 ± 11.34 2.00 926.84 ± 81.74 314.08 3.140 76.13
AMS-NE (Intravenous) 65.01 ± 6.50 2.00 769.34 ± 62.82 246.63 ± 24.72 0.0 1581.73 ± 138.42 – – –

DTI: Drug targeting index, DTE (%): Drug targeting efficiency, DTP (%): Direct transport percentage, AUC: Area under curve.

11
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

(a) corresponding accumulation of the AMS to the brain via intranasal de­
300 livery was revealed through the DTI, wherein, the value of DTI greater
AMS-NG (Intranasal)
than 1 proves direct nose-to-brain transportation of the AMS (Wang
Conc. of AMS in blood (ng/mL)

250 AMS-NE (Intranasal) et al., 2008). Experimental findings with a DTI value of 18.21 after
AMS-NE (Intravenous) intranasal AMS-NG delivery represented a measurable enhancement of
200 brain targeting of AMS. The achieved pharmacokinetic parameters of
AMS-NG were in good accordance with the previously published
150
research on a similar approach (Kumbhar et al., 2020).
100
3.8. Animal behavior studies
50
3.8.1. Outcomes on cataleptic evaluation
0 Cataleptic investigation helps to determine EPS followed by admin­
0 2 4 6 8 10 12 14 16 18 20 22 24
istration of investigating formulations in the experimental animals. This
Time (h)
experiment was conducted to evaluate the time required for animals to
(b) remain in an atypical position. In this context, different groups of
250 AMS-NG (Intranasal) experimental rats were treated with haloperidol, AMS-NE, and AMS-NG
Conc. of AMS in brain (ng/mL)

AMS-NE (Intranasal) to identify any symptoms of EPS. The outcomes of the study on the 28th
200 AMS-NE (Intravenous) day are presented in Fig. 7(a). Wherein, the figure depicted that the
animals treated with AMS-NE and AMS-NG did not reflect any cataleptic
150 symptoms when compared to the animals in the control group. Simul­
taneously, animals in the haloperidol-treated group had revealed a sig­
100 nificant (**p < 0.01) increment in the cataleptic activity when compared
to the animals in the control group. The findings of all groups for the
50 catalepsy activity were found to be similar on 1st, 7th, and 14th day of
drug administration (Bricker et al., 2014; Dadhania et al., 2016). The
0 fast permeation and sustained-release effect of the AMS from the
0 2 4 6 8 10 12 14 16 18 20 22 24 developed formulations could be effective in decreasing the cataleptic
Time (h)
influence.
Fig. 6. The mean (a) blood, (b) brain concentration–time curve for AMS-NG
(Intranasal), AMS-NE (Intranasal) and AMS-NE (Intravenous) administration 3.8.2. Investigation of induced locomotor activity analysis
at 0.5, 2, 4, 8 and 24 h and it was found significant for intranasal administra­ Determination of locomotor activity was performed to assess the
tion. All values are presented as the mean ± SD (n = 15), *p < 0.05 vs. intra­ antagonistic effect of AMS on dopamine (D2) receptors when delivered
venous administration. via intranasal route. AMS is an active agent against positive and negative
signs of schizophrenia, due to the combined action on D2 and serotonin
(5HT2A) receptors (Wiley, 2008). In this investigation, laboratory ani­
mals were treated with carbidopa (2.5 mg/kg) and L-dopa (10 mg/kg),
wherein administration of both the drugs results in augmented

(a) 400 (b) 1000

** Control
** Control
Cataleptic response (sec)

**
Vehicle 800 Vehicle
Locomotor count

300
AMS-NE-treated AMS-NE-treated
(Count/sec)

600
AMS-NG-treated AMS-NG-treated
200
Haloperidol-treated 400
** ** **
**
** ** ** **
100 200

0
0
14

28
1

7
1

Time (h) Days

(c) 10 (d) 60
** ** ** Control Control
**
** **
Fore limb retraction

8 Vehicle Vehicle
Hind limb retraction

**
** **
** ** AMS-NE-treated 40 ** ** AMS-NE-treated
time (sec)

time (sec)

6 **
AMS-NG-treated ** ** AMS-NG-treated
4
20
2

0 0
14

28

14

28
1

Days Days

Fig. 7. In vivo animal behavioral studies: (a) cataleptic evaluation up to 21st days in control, vehicle, AMS-NE-treated, AMS-NG-treated and haloperidol-treated
groups, (b) locomotor activity, (c) forelimb retraction time and (d) hind-limb retraction time in the paw test during 1st, 7th, 14th and 28th days in control,
vehicle, AMS-NE-treated, AMS-NG-treated groups. All values are presented as the mean ± SD (n = 6), **p < 0.01 vs. control group.

12
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

dopaminergic effect thereby enhancing the locomotor activity in the 3.8.3. Assessment of paw test
animals. The AMS-NE and AMS-NG treated groups displayed a signifi­ The effects of intranasal delivery of AMS-NE and AMS-NG are
cant (**p < 0.01) reduction in locomotor activity than the animals in the depicted in Fig. 7(c) and (d). The findings from this investigation rein­
control group that proved the antagonistic action of AMS on D2 re­ forced that there were no significant alterations observed in FRT of the
ceptors. The outcomes of the locomotor activity after the 1st, 7th, 14th, AMS nanoformulation treated animals via intranasal route, which indi­
and 28th days are presented in Fig. 7(b). Moreover, the AMS-NG cated the absence of EPS. The extended HRT in the treatment group of
revealed a superior role in locomotor activity when compared to AMS- animals exhibited therapeutic efficacy of AMS via intranasal adminis­
NE. However, both the formulations were found to be significantly tration. The reduction of catalytic potential by the intranasal AMS-NG
(**p < 0.01) active against schizophrenia. The significant action on formulations can be correlated with the outcomes of FRT, conse­
locomotor activity could be due to better permeation of the drug from quently by the absence of EPS (Bricker et al., 2014; Möller, 2003). An­
the lipid nanocarrier in the presence of stimuli-sensitive polymers that imals in the AMS-NG treated group showed slightly less FRT than the
promisingly improve the safety of AMS in CNS disorders. group treated with AMS-NE. The lesser FRT could be attributed to the

(a) 100 Control

AMS-NG (Intranasal) 1 mg/kg
** AMS-NG (Intranasal) 2.5 mg/kg
** ** *
Differential leukocyte count

80 *
AMS-NG (Intranasal) 5 mg/kg
AMS-NE (Intravenous) 1 mg/kg
60 AMS-NE (Intravenous) 2.5 mg/kg
AMS-NE (Intravenous) 5 mg/kg

40

20 ** *
** ** ** *
** * **
*
** ** * ** *
0

)
)
)

)
)

(%
(%
(%

(%
L

(%
/μ
10 3

ils
es
ils

ls
es

hi
yt

ph
ph

yt

op
oc
(x

oc

no
tro

as
ph

on

si
C

eu

B
Eo
m

M
B

Ly
W

White blood cell parameters

(b) 100 Control

AMS-NG (Intranasal) 1 mg/kg
** ** AMS-NG (Intranasal) 2.5 mg/kg
Differential erythrocyte count

**
80 ** AMS-NG (Intranasal) 5 mg/kg
* AMS-NE (Intravenous) 1 mg/kg
**
AMS-NE (Intravenous) 2.5 mg/kg
60 AMS-NE (Intravenous) 5 mg/kg

**
**
40 **
**** *

** ** ** ** ** *
20
** ** ** **
**
*

0
L)
L)

g)
L)

/d
(p
/μ

(f
10 6

gm
H
V

C
C

(
M

c.
M
(x

on
C

C
B
R

H
C
M

Red blood cell parameters

Fig. 8. Effect of AMS-NG (Intranasal) and AMS-NE (Intravenous) formulations on hematological parameters for the different test groups: group I (control), group II
(treated with 1 mg/kg AMS-NG), group III (treated with 2.5 mg/kg AMS-NG), group IV (treated with 5 mg/kg AMS-NG), group V (treated with 1 mg/kg AMS-NE),
group VI (treated with 2.5 mg/kg AMS-NE), group VII (treated with 5 mg/kg AMS-NE) animals. (a) Differential leukocytes count and (b) differential erythrocytes
parameter counts. All values are expressed as the mean ± SD (n = 6), **p < 0.01,*p < 0.05 vs. control group.

13
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

sustained permeation of AMS from the in situ gel and the sustained
penetration due to the presence of gellan gum. Simultaneously, signifi­
cant (**p < 0.01) influence was observed in HRT upon AMS treatment
groups as compared to the control group. Similarly, the AMS-NG was
found to have superior effect than AMS-NE in all aspects. Therefore, it
could be inferred that the safety and efficacy of intranasal in situ gel of
AMS could be improved significantly.
3.9. In vivo safety studies
were found within the standard physiological range (Fig. 8(a)). Unfor­
tunately, mortality was observed in 2 out of 6 animals in the 5 mg/kg
AMS-NE treated group after 27th day of treatment, which can be
corroborated by a higher systemic exposure of AMS, which might have
resulted in hematological toxicities leading to death. However, gelling
agents in AMS-NG formulation prolonged the residence time of the
formulation in the nasal cavity with limited permeation of AMS into the
blood as compared to AMS-NE formulation (**p < 0.01). Moreover, the
intranasal delivery could decrease systemic exposure leading to
decreased toxicity.

In this work, regular visual inspection of the experimental animals
was carried for 28 days and evaluated the occurrence of toxicological
signs and symptoms associated with the administration of nano­
formulations. The experimental findings from 28 days of treatment
protocol are discussed in the following section.
3.9.1. Agranulocytosis and cytotoxic effect on hematology
The hematological analysis was conducted to discover the toxic
manifestations that may occur following intranasal administration of
AMS formulations. This study was performed to examine AMS-induced
hemolysis and agranulocytosis in experimental animals following 28
days of treatment. It is well known that the AMS is associated with
agranulocytosis and neutropenia upon direct exposure to the systemic
circulation (Bhavna et al., 2014). The differential leukocyte parameters
of the treated groups were found to be within the limit and are repre­
sented in Fig. 8(a). Fig. 8(b) represents the red blood cell parameters,
which are also found to be within the normal physiological range.
Table 4 represents all hematological parameters for intranasal AMS-NG
administration, which are within acceptable limits and did not display
any significant physiological changes. Moreover, WBC parameters and
RBC parameters for intravenous AMS-NE treated groups at low dose
3.9.2. Gross pathology
Outcomes from in vivo safety studies revealed and confirmed that the
animals did not exhibit any significant changes, such as nasal secretion,
unusual blinking, nasal bleeding, and eye irritation for animal groups
with intranasal administration when compared to the animals in control
group. Furthermore, respiratory difficulties and unusual behavior were
also not observed after intranasal administration. Hence, concluding
that the developed AMS-NG formulation is safe for intranasal adminis­
tration for the treatment of CNS disorder. The AMS-NE (intravenous)
treated at low dose (1 mg/kg) animal group could not exhibit any
abnormal activity during the study period, whereas animals in medium
dose (2.5 mg/kg) and high dose (5 mg/kg) groups exhibited toxic
manifestations, such as unusual behavior and decreased food and water
consumption. Besides, mortality was also reported in animals treated
with high dose AMS-NE (intravenous). Hence, the outcomes of the gross
pathology examination proved that the developed intranasal AMS-NG is
safe and effective for the long-term treatment of mental disorders.
3.9.3. In vivo nasal tissue toxicity assessment
Nasal ciliotoxicity was examined by isolating nasal mucosa of the

Table 4
Comparison of Hemogram/complete blood count parameters between control, AMS-NG (1 mg/kg, 2.5 mg/kg and 5 mg/kg) treated, AMS-NE (1 mg/kg, 2.5 mg/kg and
5 mg/kg) treated in male Wistar rats (Mean ± SD).
Sr. Hemogram Referral control AMS-NG AMS-NG AMS-NG AMS-NE AMS-NE AMS-NE
No. Parameters physiological (Intranasal) (Intranasal) (Intranasal) (Intravenous) (Intravenous) (Intravenous)
values treated (1 mg/ treated (2.5 treated (5 mg/ treated (1 mg/ treated (2.5 treated (5 mg/
kg) mg/kg) kg) kg) mg/kg) kg)

1. HGB (g/dL) 12–18 16.1 ± 0.22 14.0 ± 0.13 13.1 ± 0.27 12.1 ± 0.23 14.6 ± 0.26 14.5 ± 0.29 12.1 ± 0.35
2. HCT i.e. PCV 35–55 44.3 ± 0.14 39.4 ± 0.27 46.5 ± 0.18 45.6 ± 0.15 48.3 ± 0.24 35.9 ± 0.18 35.1 ± 0.14
(%)
3. RBC (x 106/ 6.5–9.5 9.2 ± 0.05 9.5 ± 0.03 9.1 ± 0.09 8.6 ± 0.09 9.4 ± 0.07 7.4 ± 0.08 6.8 ± 0.05
µL)
4. WBC (x 103/ 4.5–12.5 9.2 ± 0.26 10.8 ± 0.21 6.3 ± 0.23 6.5 ± 0.29 7.4 ± 0.14 5.2 ± 0.28 4.1 ± 0.26
µL)
5. MCV (fL) 55–78 68.7 ± 0.81 76.9 ± 0.68 62.6 ± 0.42 74.3 ± 0.24 72.4 ± 0.43 76.8 ± 0.89 67.9 ± 0.81
6. MCH (pg) 16–22 20.6 ± 0.38 19.2 ± 0.42 19.4 ± 0.95 18.0 ± 0.83 18.4 ± 0.58 18.8 ± 0.75 16.34 ± 0.38
7. MCHC (g/dL) 28–38 34.8 ± 0.18 32.9 ± 0.12 34.9 ± 0.07 36.9 ± 0.25 29.9 ± 0.24 31.6 ± 0.13 34.5 ± 0.18
8. Platelets 2–10.5 9.7 ± 0.21 6.8 ± 0.26 6.4 ± 0.14 7.2 ± 0.25 9.7 ± 0.05 4.8 ± 0.23 9.4 ± 0.21
(Lakhs/µL)
9. MPV (fL) 6–12 8.1 ± 0.18 11.4 ± 0.14 9.5 ± 0.10 9.1 ± 0.16 11.3 ± 0.18 6.4 ± 0.04 11.7 ± 0.18
70. Differential
leukocyte
count
i. 65–85 78.0 ± 0.62 71.0 ± 0.82 70.0 ± 0.51 69.0 ± 0.84 73.0 ± 0.79 66.0 ± 0.56 61.0 ± 0.62
Lymphocytes
ii. 11–22 18.0 ± 0.13 16.0 ± 0.19 13.0 ± 0.05 17.0 ± 0.22 12.0 ± 0.19 11.0 ± 0.33 9.0 ± 0.13
Neutrophils
iii. 0–3 2.0 ± 0.02 1.0 ± 0.01 1.0 ± 0.03 1.0 ± 0.02 1.0 ± 0.01 2.0 ± 0.02 1.0 ± 0.02
Monocytes
iv. 0–3 1.0 ± 0.03 2.0 ± 0.04 1.0 ± 0.02 1.0 ± 0.01 1.0 ± 0.03 1.0 ± 0.03 1.0 ± 0.03
Eosinophils
v. Basophils 0–1 0.0 ± 0.0 1.0 ± 0.01 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
11. RBC Normocytic Normocytic Normocytic Normocytic Normocytic Normocytic Normocytic Normocytic
morphology Normochromic Normochromic Normochromic Normochromic Normochromic Normochromic Normochromic Normochromic
12. WBC Normal Normal Normal Normal Normal Normal Normal Normal
morphology

HGB: Hemoglobin, HCT: Hematocrit, PCV: Packed cell volume, RBC: Total red blood cells count, WBC: Total white blood cells count, MCV: Mean corpuscular volume,
MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, MPV: Mean platelet volume.

14
D. Gadhave et al. International Journal of Pharmaceutics 607 (2021) 121050

Fig. 9. Histopathological examinations of nasal mucosal sections after 28 days exposures of (a) control mucosa, (b) AMS-NG (1 mg/kg), (c) AMS-NG (2.5 mg/kg), (d)
AMS-NG (5 mg/kg) and (e) Blank-NG in male Wistar rats.

AMS nanoformulations treated animals after 28 days. A careful exami­
nation was done by observing the signs of toxicities such as inflamma­
tion, structural alteration, or severity of toxicity according to
histological studies. The pathological micrographs of control, AMS-NG
treated and blank-NG treated nasal tissues have been presented in
Fig. 9, where 28 days of treatment with different formulations revealed
no observed variations in the cellular morphology. The histological re­
ports and pathological score have been represented in Table 5, which
conclude that the treated sections did not show any major changes,
except for few lacerations. Moreover, the control and treated sections
observed similar to each other. The total pathological score in the report
concludes that no abnormalities were identified in treated mucosa sec­
tions (Chall et al., 2015). Hence, the developed AMS-NG formulations
are safe and suitable for intranasal administration.
4. Conclusion
The NE formulations of AMS were developed by low energy emul­
sification method, where the optimized NE revealed desired globule size
with ideal zeta potential that proved the stability of the developed
nanoformulation. Conversion of NE to NG was achieved using natural
cationic in situ gelling and thermo-sensitive gelling polymers. AMS-
loaded nanocarriers attained higher drug entrapment potential, where
the presence of stimuli-responsive polymers facilitated mucoadhesion
and enhanced localization time in the nasal cavity. Furthermore, the
presence of polymers improved the in vitro release profile and ex vivo
permeation of AMS from the developed AMS-NG. Moreover, the pres­
ence of Labrasol (surfactant) and gellan gum in the formulation further
assisted in opening tight junctions of capillary endothelial cells and
augmented the penetration of therapeutics towards the brain. The
developed intranasal nanosystem intended to decrease the peripheral
exposure and associated toxicities showed enhanced therapeutic effi­
cacy which can be attributed to permeation of the drug towards the CNS
upon intranasal administration. The superior brain pharmacokinetics
profile of AMS was recorded with intranasal AMS-NG. Behavioral
studies revealed reduced EPS upon intranasal administration of AMS-
NG. The significant activity of developed nanoformulation on positive
locomotor activity and HRT in the paw test established the antipsychotic
efficacy of the intranasal AMS-NG in treated animals. Subsequently, in
vivo evaluation established the safety of the prepared AMS-NG against
schizophrenia with diminished risk of agranulocytosis and hematologi­
cal toxicities. Hence, it could be concluded that a non-invasive system of
AMS intranasal drug delivery cargo, AMS-NG, could provide the plat­
form for the safe and efficient transport of the therapeutic towards the

Table 5
Histopathological report of control, AMS-NG (1 mg/kg, 2.5 mg/kg and 5 mg/kg) treated and blank-NG treated nasal mucosa after 28 days of treatment in male Wistar
rats.
Sr. Treated groups Histopathological observations Overall pathology
No. score
Congestion and hemorrhages in nasal Hyperplasia of nasal Inflammation, and cellular infiltration in
mucosa mucosa nasal mucosa

1. Control NAD NAD NAD NAD

2. AMS-NG (Intranasal: 1 NAD NAD NAD NAD
mg/kg)
3. AMS-N (Intranasal: 2.5 NAD NAD Focal (+) NAD
mg/kg)
4. AMS-NG (Intranasal: 5 NAD Focal (+) NAD NAD
mg/kg)
5. Blank-NG treated NAD NAD NAD NAD

Overall score as- NAD: No abnormalities detected, Minimal changes: +, Mild changes: ++, Moderate changes: +++, Severe changes: ++++.

15
D. Gadhave et al.
CNS. However, clinical investigations are required to support the pre-
clinical findings on the safety and efficacy of AMS-NG in human
mental disorders.
5. Compliance with ethical standards
Animal studies: Approval for the in vivo safety study was obtained
from the institutional animal ethics committee of HSBPVT’S, GOI, Col­
lege of Pharmacy, Ahmednagar, India, regulated by CPCSEA (Protocol
No. 1697/PO/Re/S/13/CPCSEA/2020/05).
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Dnyandev Gadhave: Methodology, Investigation, Data curation,
Conceptualization, Formal analysis, Software, Writing – original draft,
Visualization, Validation, Writing – review & editing. Shrikant Tupe:
Methodology, Investigation, Data curation, Writing – original draft.
Amol Tagalpallewar: Methodology, Investigation, Data curation,
Writing – original draft. Bapi Gorain: Data curation, Visualization,
Writing – review & editing. Hira Choudhury: Data curation, Visuali­
zation, Writing – review & editing. Chandrakant Kokare: Conceptu­
alization, Supervision, Visualization, Validation, Project administration,
Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Authors would like to thanks, Swapnroop Drugs and Pharmaceuti­
cals, Aurangabad, India for providing the gift sample of AMS. Authors
also wish to thank the Gattefosse Mumbai, India Pvt. Ltd. for providing
the gift samples of required ingredients and excipients.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.121050.
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