Colloids and Surfaces B: Biointerfaces 205 (2021) 111838

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Colloids and Surfaces B: Biointerfaces

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

Systematically designed chitosan-coated solid lipid nanoparticles of ferulic

acid for effective management of Alzheimer’s disease: A
preclinical evidence
Sumant Saini a, Teenu Sharma a, Atul Jain b, Harmanjot Kaur a, O.P. Katare a,
Bhupinder Singh a, b, *
a
University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh, 160014, India
b
UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites (Biomedical Sciences), Panjab University, Chandigarh, 160014, India

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

Keywords: Ferulic acid (FA) is a ubiquitous natural plant bioactive with distinctive promise in neurodegenerative disorders.
Chitosan However, its therapeutic efficacy gets compromised owing to its poor aqueous solubility, inadequate perme­
Lipid nanoparticles ability across lipophilic barriers, and extensive first-pass metabolism. The current studies, therefore, were un­
Neurodegenerative disease
dertaken to systematically develop chitosan-coated solid lipid nanoparticles (SLNs) using QbD paradigms for
Intranasal delivery
improved efficacy of FA in the management of Alzheimer’s disease (AD). SLNs of FA were formulated employing
Quality by Design (QbD)
Blood-brain barrier Compritol as lipid and polysorbate 80 as surfactant and optimised using a 32 Central Composite Design (CCD).
The optimized formulation, surface-coated with chitosan using ionic gelation, exhibited particle size of 185 nm,
entrapment efficiency of 51.2 % and zeta potential of 12.4 mV. FTIR and DSC studies verified the compatibility of
FA with formulation excipients, PXRD construed significant loss of drug crystallinity, while FESEM depicted
existence of uniform spherical nanoparticles with little aggregation. Notable improvement in ex vivo mucoad­
hesion and permeation studies using goat nasal mucosa, coupled with extension in in vitro drug release, was
obtained with SLNs. Substantial improvement with SLNs in cognitive ability through the reduction in escape
latency time during behavioural studies, together with significant improvement in various biochemical param­
eters and body weight gain was observed in AD-induced rats. Histopathological images of different rat organs
showed no perceptible change(s) in tissue morphology. Overall, these preclinical findings successfully demon­
strate improved anti-AD efficacy, superior nasal mucoadhesion and permeation, extended drug release, improved
patient compliance potential, safety and robustness of the developed lipidic nanoconstructs of FA through
intranasal route.

1. Introduction 16 million by 2050 [4].

Alzheimer’s disease (AD), one of the most prevalent neurodegener­
ative disorders, affects almost 3% of the world’s population [1]. Char­
acterized by serious but gradual diminution in cognitive functions,
incidences of AD get markedly augmented among the aged population of
over 65 years, thereby posing a huge health and economic burden on the
society [2]. Neuronal degeneration in AD results in substantial loss of
memory, communication skills, judgment and reasoning [3]. Albeit the
prevalence of AD has been rising all across, the number of people
inflicted with AD in the USA alone is expected to range between 11 and
Among the myriad treatment strategies currently adopted for man­
agement of AD, the primary ones include cholinesterase inhibitors and
NMDA receptor antagonists. A multitude of factors, such as inadequate
and inconsistent drug oral bioavailability, owing to their low aqueous
solubility, poor permeability across the biological membranes and high
first-pass metabolism, together with poor brain penetration and reten­
tion, need for repeated dosing, and high frequency of side effects, tend to
limit the optimum usage of such drugs [5,6]. This calls for an exploration
of newer therapeutic strategies for the effective management of AD [7,
8].

* Corresponding author at: University Institute of Pharmaceutical Sciences, National UGC Centre for Excellence in NanoBiomedical Applications, Panjab Uni­
versity, Chandigarh, 160 014, India.
E-mail addresses: bsbhoop@yahoo.com, bsbhoop@pu.ac.in (B. Singh).

https://doi.org/10.1016/j.colsurfb.2021.111838
Received 21 January 2021; Received in revised form 28 April 2021; Accepted 8 May 2021
Available online 14 May 2021
0927-7765/© 2021 Published by Elsevier B.V.
S. Saini et al.
Various plant bioactives, in this regard, have been documented to
possess anti-AD potential, which include, quercetin [9], morin [10],
apigenin [11], kaempferol [12], rutin [13], ferulic acid (FA) [14],
berberine [15], curcumin [16] and resveratrol [17]. FA, 3-hydroxy
cinnamic acid, one of such promising bioactive explored, is abun­
dantly found in various fruits and vegetables [18]. It is reported to
possess antioxidant, anticancer, cardioprotective and neuroprotective
potential [19,20]. However, the therapeutic efficacy of FA gets thwarted
by its poor aqueous solubility (i.e., soluble only in hot water) and
inability to traverse the lipophilic barriers (with log P of 0.78), it being a
potentially BCS class IV molecule [21–23]. Moreover, extensive
first-pass metabolism of FA further diminishes its efficacy [24].
Nanostructured systems have demonstrated stellar drug delivery
merits like high drug loading, enhanced stability and extended disso­
lution profiles. The lipidic nanocarriers, further, come with several ad­
vantages like controlled release, improved permeability across
biological barriers, excellent biocompatibility, improved drug stability,
high encapsulation, ease to scalability, sterilizability, and augmented
oral bioavailability of bioactive compounds [25,26]. Solid lipid nano­
particles (SLNs), in this regard, are considered promising lipidic nano­
carriers. However, these are easily distinguishable by the immune
system, due to their hydrophobicity and surface negativity, which
determine their opsonization and subsequent clearance. Surface func­
tionalization of nanoparticles with polymers like chitosan, therefore,
induces a positive charge, decreases opsonization and enhances their
residence time [27–29]. Various routes of administration have been
experimented for delivery of the therapeutic agents to the brain. Intra­
nasal administration, in this regard, has proved to be a quite promising
route, as it not only bypasses the blood-brain barrier (BBB) and delivers
drug molecules directly to the brain via olfactory and transgeminal
pathways, but also circumnavigates the hepatic first-effect, resulting in
rapid onset of drug action [30,31]. Chitosan, in this context, is consid­
ered an ideal polymer owing to its promising mucoadhesive character­
istics, high biocompatibility and non-toxic, and above all, its ability to
induce reversible opening of tight junctions in the nasal epithelial cells
[32,33]. Chitosan coating of SLNs is generally carried out using elec­
trogelation technique owing to its myriad benefits like uniform coating,
high entrapment efficiency (being water-based), safety and
cost-effectiveness [34,35].
The current studies, accordingly, report our successful endeavours to
deliver FA across BBB using chitosan-coated SLNs. The lipidic nano­
constructs were explored vis-a-vis pure FA employing a battery of in vitro
evaluation studies like particle size, zeta potential, entrapment effi­
ciency and drug release; diverse spectroscopic, calorimetric and X-ray
diffractometric studies; ex vivo mucoadhesive strength and permeability
studies on goat nasal mucosa; and finally, for their anti-Alzheimer effi­
cacy and safety potential through variegated in vivo investigations like
behavioural, biochemical, brain level and histopathological studies in
the disease-induced rats. Nanostructured systems were systematically
designed using Quality by Design (QbD) approach, encompassing initial
risk assessment, factor screening and optimization using response sur­
face mapping (RSM).
2. Materials and methods
2.1. Materials
Compritol 888 ATO, Transcutol and Gelucire 39/01 were obtained ex
gratis from M/s Gattefosse, France. Dynasan 114, Imwitor 960 K and
Precirol ATO were obtained ex gratis from M/s Cremer Oleo GmbH & Co,
Germany. FA, Tween 20, Tween 40, Tween 80, Span 80, Brij 35, sodium
triphosphate (STPP) and poloxamer 188 were purchased from M/s SRL
Chemicals, India. Stearic acid and glyceryl monostearate (GMS) were
purchased from M/s SD Fine Chemicals, India and Fischer Scientific,
India, respectively. Chitosan (medium grade) was purchased from M/s
HiMedia Laboratories Pvt Ltd, India. All the solvents and chemicals
2
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
used, throughout the current studies, were of analytical grades(s).
2.2. Methods
2.2.1. Selection of lipids and surfactants for formulation
Solubility studies on FA were conducted in various lipids, viz.,
Compritol, stearic acid, Dynasan, GMS, Inwitor and Precirol, and sur­
factants, viz., Transcutol, Tween 40, Tween 80, Span 80, Gelucire 39/01,
Brij and poloxamer 188, to select apt excipients for the formulation of
SLNs. Briefly, the lipids were heated to a temperature just above their
respective melting points, using a thermostatically-controlled water
bath (Riviera Glass Pvt. Ltd., Mumbai, India). An excess of FA was added
to each flask and shaken overnight. The samples were then withdrawn,
centrifuged (10 min) and the concentration of FA was determined in the
supernatant, following apt dilution, using a UV visible spectrophotom­
eter (Jenway-6315, Cole-Parmer, Staffordshire, UK) at 322 nm.
2.2.2. Preparation of SLNs
The SLNs were prepared using hot homogenization technique [36].
The lipid (Compritol) was heated above its melting point (i.e., 70–75
◦
C). FA, dissolved in ethanol, was added to it until it turned transparent.
The aqueous phase, comprising of water and Tween 80, was mixed to
obtain a clear solution and heated. Under isothermal conditions, the
lipidic phase was added drop-wise to the aqueous phase, employing a
high-speed homogenizer (IKA® T18 digital Ultra Turrax®, Washington,
USA) until a clear homogenous emulsion was obtained. The dispersion
was probe-sonicated (Misonix Sonicator® 3000, Farmingdale, NY, USA)
and then stirred on a magnetic stirrer (Remi, Mumbai, India) at 2000
rpm for 1 h to allow solvent diffusion from the lipid phase [37,38]. The
dispersion was then lyophilized (Alpha, 2–4 LD plus, Martin Christ,
Osterode am Harz, Germany) and stored in glass vials until further use
[39,40].
2.2.3. Systematic development of SLNs
2.2.3.1. Formulation objectives. According to the established QbD par­
adigms, a quality target product profile (QTPP) was outlined enumer­
ating variegated desirable quality traits of FA-loaded SLNs to achieve
maximal efficacy for therapeutic management of AD, as delineated in
Table S1. Appropriate justifications envisioned for the selection of
various quality attributes (CQAs) are outlined in Table S2.
2.2.3.2. Risk estimation studies. As elementary risk(s) assessment is
pivotal during systematic development of SLNs, an Ishikawa fishbone
diagram was conceptualized highlighting cause-effect relationships be­
tween key material and process attributes with various CQAs, using
Minitab® 17 software (Minitab, LLC, USA). Further, a risk estimation
matrix (REM) was established, classifying these input factors among
low, medium, and high-risk parameters, based upon the risk(s) allied
with each factor [37,39,41].
2.2.3.3. Factor screening studies. For selecting “influential few factors”
from a plethora of variables, a 7-factors, 8 run Taguchi design (TgD),
operating at low (-1) and high (+1) levels, was employed. Pareto and
half-normal plots, thus obtained, were analysed to discern significant
variables using Design-Expert® 12 software (StatEase Inc., Minneapolis,
USA). Table S3 illustrates the design matrix for all the prepared for­
mulations investigated, along with their factor levels [41].
2.2.3.4. Systematic optimization of SLNs. Statistically significant factors,
selected from screening studies, were optimized using a Central Com­
posite Design (CCD) for 2 factors, accounting for 13 experimental trials,
including quintuplicate runs around the centre point (0,0), as illustrated
in Table 1. The model fitness was analysed by computing the magnitudes
of the probability of type I error (i.e., p-value) and correlation coefficient
S. Saini et al. Colloids and Surfaces B: Biointerfaces 205 (2021) 111838

Table 1 temperature (◦ C) [51].

Design matrix as per a 2-factor Central Composite Design for optimization of the
prepared SLNs formulations of ferulic acid, along with employed factor levels in 2.2.4.5. Powder X-ray diffraction (PXRD). PXRD studies were conduct­
coded and actual form. ed for lyophilized SLNs and cSLNs using X’Pert PRO® diffractometer
Run A: Lipid B: Surfactant system (PANalytical, Netherlands) employing Cu-Ka radiation at 1.5406
1 0 1 Å at an accelerating voltage of 45 kV. Samples, packed in aluminum pan,
2 1 1 scanned between 4 and 50◦ values of 2θ [52,53].
3 1 0
4 0 0
5 1 − 1
2.2.4.6. Drug entrapment efficiency (EE). The EE of coated and uncoated
6 0 0 SLNs was measured as per a method reported previously [36]. Aliquots
7 0 − 1 (2 mL) were centrifuged (ThermoFisher Scientific, Legend™ XTR Sor­
8 − 1 − 1 vall™ USA) at 12,000 rpm (16128xg) for 30 min at 4 ◦ C. The superna­
9 − 1 0
tant was then pipetted, filtered and suitably diluted for the
10 0 0
11 0 0 quantification of unentrapped FA using UV–vis spectrophotometer at
12 0 0 322 nm. The amount of entrapped FA was calculated using Eq. 1.
13 − 1 `1
Total amount of FA added − Free FA in supernatant
Factors Low(-1) Medium(0) High(+1) EE = × 100 (1)
Total amount of FA added
Lipid (mg) 150 275 400
Surfactant (g) 1.50 2.75 4.00 2.2.4.7. Drug release studies. Dialysis sac technique was used to study
the in vitro release behaviour of FA from the formulations for a period of
(i.e., R). Factor-response relationship(s) were graphically represented as 48 h, vis-à-vis pure drug suspended in sodium carboxymethyl cellulose
3-D response surfaces and corresponding 2-D contour plots for each of solution (0.25 %, w/v). The dissolution was performed in phosphate
the CQAs [42]. buffer pH 6.5 (50 mL) at 37 ± 0.5 ◦ C, 100 rpm. SLNs (1 mg of FA), were
placed in the dialysis sac (Molecular weight cut-off of 12 kDa), and
sample aliquots (2 mL) were removed at pre-scheduled time-intervals.
2.2.3.5. Coating of optimized SLNs. The optimized SLNs dispersion was
An equal volume of fresh dissolution medium was replenished and the
further coated with chitosan employing ionic gelation technique [29,35,
dissolution data were corrected for losses of drug and/or drug release
43,44]. Briefly, SLNs was added drop-wise into an equal volume (10 mL)
volume occurred during sampling, and cumulative percent of FA release
of chitosan solution (0.175 %, w/v) under stirring (25 ◦ C, 20 min).
was computed likewise [54].
Subsequently, ionic crosslinker, STPP (4.5 mL of 0.5 %, w/v), was added
drop-wise and stirred further at 600 rpm for 30 min, to ensure uniform
coating. Chitosan-coated SLNs (cSLNs) dispersion was lyophilized and 2.2.4.8. Determination of mucoadhesive strength. Goat nasal mucosa was
stored, until further use [28,33,34,45,46]. The chitosan coating effi­ procured from a local slaughter-house (Nayagaon, Chandigarh). The
ciency in the current studies was determined as per a previously re­ mucosa was washed with normal saline solution to remove adherent
ported method [47]. connective tissues and stored in simulated nasal fluid (SNF), pH 6.5, till
tested for its mucoadhesive strength, for at least 12 h, as per a method
2.2.4. Characterization of optimized and cSLNs reported previously [55,56] on TA-XT plus Texture Analyser (Stable
Micro Systems, Godalming, UK) in an adhesive test mode.
2.2.4.1. Particle size and zeta potential. Mean values of particle size
(Snm) and zeta potential (ZP) for optimized SLNs and cSLNs were 2.2.4.9. Ex vivo permeability studies. Ex vivo nasal mucosal permeation
determined by dynamic light scattering at an angle of 90◦ employing was determined on Franz diffusion cell (PermGear Inc., Pennsylvania,
Zetasizer® (NANO ZS-90, Malvern, Worcestershire, UK). Prior to the USA) [55]. SLNs and cSLNs, dispersed in 1 mL of SNF, pH 6.5 (5 mL),
analysis, the samples were suitable diluted [48]. were added to donor compartment, which was fitted and clamped onto
the receptor compartment containing fresh SNF. Periodically, the sam­
ples were withdrawn and suitably replaced with fresh medium.
2.2.4.2. Field emission scanning electron microscopy (FESEM). The sur­
Isothermal conditions and physiological temperature were maintained
face topology of the SLNs was studied using FESEM (8010, Hitachi,
through a water jacket system. The samples were quantified using an
Japan). The sample was analysed at a working gap of 7.2 mm, and im­
HPLC method, previously reported by us [57].
ages were obtained at a suitable accelerating voltage of 5.0 kV [49].
FA permeating per unit surface area (A/S) was plotted against time
(t). The flux (J) was computed from the graph’s slope for all the com­
2.2.4.3. Fourier-transform infrared spectroscopy (FTIR). The drug-
binations using Eq. 2 [58,59].
excipient interactions were investigated using FTIR analysis. The sam­
ples for FTIR analysis was prepared by finely grinding the sample with A Pcoff Atot h2
= [t − ] (2)
KBr which was then compressed into pellets. The spectra were recorded S V 6D
on an FTIR spectrophotometer (Perkin Elmer, MA, USA), and were then
interpreted for changes in the spectral peaks [50]. Where, Atot, V, h and D depict the quantity of FA, volume of donor cell,
thickness of dialysis membrane, and diffusion coefficient, respectively.
2.2.4.4. Differential scanning calorimetry (DSC). DSC analysis of FA and Herein, PcoffAtot/V, delineates the slope (J), while, h2/6D, denotes the
its blend with each of the excipients was carried out by triturating them abscissa intercept, furnishing the value of lag time.
with a pestle in mortar. Samples, 3–5 mg each, were weighed in a
Tzero™ aluminum pan, which was heated at a rate of 10 ◦ C/min. Three 2.2.4.10. Stability studies. The lyophilized formulations, packed in air-
runs were conducted for each sample with nitrogen flow of 20 mL/min. tight glass vials were evaluated for any significant alteration(s) in
The eventual thermograms of FA and physical mixture were analysed drug assay and other CQAs at 5 ± 2 ◦ C, 25 ± 2 ◦ C and 60 ± 5% RH, 40 ±
using Star and Universal, while that of SLNs using DSC-TA Q20 (all by 2 ◦ C and 75 ± 5% RH (ICH guidelines Q1(R2)). The formulation was
M/s TA instruments, New Castle, DE). Appropriate scans were recorded, evaluated for a storage period of 3 months, at monthly intervals, for
and the corresponding plots were obtained between heat flow (W/g) and various parameters to establish stability of surface-modified colloidal

3
S. Saini et al.
system, viz., % assay, particle size, EE, ZP, % release and f2 values.
2.2.4.11. In vivo studies in rats. To investigate the in vivo efficacy of
optimized SLNs, the formulations were tested on Wistar rats (180− 200
g), as per the protocols approved by Panjab University Institutional
Animal Ethics Committee (vide Letter No. PU/45/99/CPCSEA/IAEC/
2019/242). Animals were obtained from Central Animal House of Uni­
versity, housed in animal cages, and acclimatized for 7 days with a 12 h
day-and-night cycle (27 ± 3 ◦ C and 55 ± 5% RH), before the conduct of
studies. Rats were fed with standard diet along with drinking water, ad
libitum.
2.2.4.11.1. Drug treatment and dosing schedule. For pharmacody­
namic evaluation of FA and its formulations, animals were distributed
randomly into 8 groups, each comprising of three rats, as described in
Table S4. AD was induced in rats by intracerebroventricular (ICV) in­
jection of streptozocin (STZ) [60,61]. The drug was administered via
oral and intranasal route. All the experimental studies were carried out
for 28 days (STZ); 3 mg/mL, FA; 80 mg/kg.
2.2.4.11.2. Induction of AD. Animals were anaesthetized with
intraperitoneally injected ketamine (80 mg/kg) and xylazine (10 mg/
kg) on a stereotaxic apparatus. Following midline sagittal incision in rat
skull, holes were drilled for Hamilton syringe into the lateral ventricle
(0.8 mm on the coronal axis (C), 1.5 mm on sagittal axis (S), and 3.6 mm
horizontal axis (H); from the zero-reference point (Bregma)). Artificial
cerebrospinal fluid and/or STZ were infused into the rat brain [62]. The
incision was then sutured, dental fixer and neosporin were applied. Rats
were allowed access to water and food ad libitum.
2.2.4.11.3. Behavioural studies. 2.2.4.11.3.1. Spatial navigation and
learning
Improvement in the cognitive ability of AD-induced rats after treat­
ment with developed SLNs was analysed using previously reported
behavioural tests [63]. Morris water maze (MWM) studies were con­
ducted to assess animal behaviour in each group. A large circular pool,
with dimensions of 150 × 45 × 30 cm, was filled with water. The pool
was partitioned into 4 equal quadrants, and a target quadrant was
selected. A submerged platform was placed in the pool, and the rats were
trained in the acquisition phase at intervals of 30 s. Acquisition index or
escape latency time (ELT) was noted to locate the hidden platform in
water maze. Successively, the probe trial was performed wherein the
spatial memory was consolidated by removing the platform 24 h after
the last training session, and the time spent by the subject in target
quadrant (TSTQ) was determined.
2.2.4.11.3.2. Biochemical assessment
Various biochemical markers were estimated as per standard pro­
tocols in supernatant, viz., lipid peroxidation (LPO) [64] and the levels
of glutathione (GSH) [65], nitrite [66], protein [67], superoxide dis­
mutase (SOD) [68], and acetylcholinesterase (AChEs) [69]. Brains har­
vested after euthanasia (cervical dislocation) were rinsed with chilled
isotonic saline and homogenized. The homogenates were centrifuged at
10,000 rpm (11,200xg) for 10 min at 4 ◦ C. The data were analysed using
one-way ANOVA at p < 0.05 to infer any statistically significant change
in the biochemical parameters among various treatment groups.
2.2.4.11.3.3. Estimation of brain levels
Rats were randomly divided into 3 treatment groups (Table 2), each
with 3 animals. The supernatant (2 mL; as per the procedure mentioned
in Section 2.2.4.11.3.2.) was mixed with 2 mL of acetonitrile [70]. The
solution was then centrifuged at 10,000 rpm (11,200 x g) for 5 min, and
the supernatant was analysed by our previously reported HPLC method
[57].
Table 2
2.2.4.11.3.4. Histopathological studies
Various organs like brain, lung, heart, kidney and liver were isolated,
fixed in formalin and subjected to histopathological examinations. Tis­
sue specimens were fixed in paraffin block, sectioned, sliced (5 μm
thick), stained with hematoxylin and eosin, and observed under optical
4
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
Table 2
Constitution of various animal treatment groups for pharmacodynamic studies
of ferulic acid and its SLNs formulations in rat (n = 3).
GROUP TREATMENT DESCRIPTION
1 Naïve Animals receiving normal saline orally.
2 Control /sham Animals injected with bilaterally
intracerebroventricular (ICV)-artificial
cerebrospinal fluid
3 Positive control /test Animals injected with ICV-STZ bilaterally
4 FA Animals injected with ICV-STZ bilaterally along
with administration of pure drug suspension,
intranasally
5 FA SLN i.n. Animals injected with ICV-STZ bilaterally along
with administration of optimized formulation,
intranasally
6 CS-coated optimized ICV-STZ bilaterally along with administration of
FA SLNs i.n. chitosan-coated optimized SLNs of FA,
intranasally
7 FA suspension p.o. ICV-STZ bilaterally and FA administered peroral
suspension in 0.25 %w/v sodium
carboxymethylcellulose solution
8 CS-coated optimized ICV-STZ bilaterally along with administration of
FA SLNs p.o. optimized SLNs formulation perorally
microscopical [71].
3. Results and discussion
3.1. Selection of formulation lipids and surfactants
Selection of lipids and surfactants was made based on their solubi­
lization potential for FA. As inferred from the solubility profiles of FA in
various lipids (Figure S1), the trend observed was Compritol 888ATO >
stearic acid > Dynasan 114 > Imwitor 960 K > Precirol ATO > glyceryl
monostearate, whereas the solubility of FA in various surfactants was
found to follow the order as Tween 80 > Gelucire 39/01 > Brij 35 >
Tween 20 > Tween 40 > poloxamer 188 > Transcutol > Span 80. Thus,
Compritol and Tween 80 were selected as the solid lipid and surfactant,
respectively, for subsequent development of SLNs.
3.2. Postulation of QTPP and CQAs
Table S1 outlines the QTPP, based on brainstorming and literature
search, enumerating various intended quality characteristics to achieve
desired efficacy and safety of FA-loaded SLNs. Table S2 delineates
suitable justifications for selecting various CQAs of the proposed
nanocarriers.
3.3. Risk estimation studies
An Ishikawa fishbone diagram, delineating various plausible factors
impacting the chosen CQAs, is portrayed in Figure S2. Further, a risk
estimation matrix (REM) (Figure S3) was constructed. As input vari­
ables like amount of lipid (mg), amount of surfactant (g), homogeniza­
tion time (min), homogenization speed (rpm), probe sonication time
(min), stirring speed (rpm), and suspension stirring time (h) were
associated with medium to high risk; these were further prioritized using
factor screening investigations.
3.4. Factor screening studies
Figure S4 portrays half-normal and Pareto charts obtained during
factor screening studies using TgD. TgD was selected for factor
screening, it being an efficient first-order experimental design, since
only the main effects were to be identified and studied [72]. Amounts of
lipid and surfactant were found to significantly (p < 0.05 each) impact
various CQAs; hence were chosen as the critical material attributes
(CMAs). Multiple linear regression analysis was performed using Eq. 3,
S. Saini et al.
wherein the main effects were only considered.
Y = β0 + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + β6X6 + β7X7 (3)
Here, β0 represents the intercept, and β1− 7 are the coefficients for linear
terms for factors X1− 7, respectively. As observed from the Pareto charts
and half-normal plots, the amounts of lipid and surfactant had the t
values higher than their respective threshold limits for all the CQAs
analysed, viz., EE, Snm and ZP.
3.5. Systematic optimization studies on SLNs
The two influential CMPs, viz., amounts of lipid and surfactant, were
Fig. 1. 3-D response surface plots and corresponding 2-D contour plots for various CQAs; Particle size (A,B); Zeta potential (C,D); Entrapment efficiency (E,F); and
Drug Release (G,H).
5
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
subsequently optimized using a CCD, as it could unearth intricate factor-
response relationship(s) as well as various possible factor interaction(s)
[72,73]. Data were fitted into a second-order quadratic model,
comprising of main effects, interaction effects and quadratic terms, as
represented in Eq. 4.
Y = β0 + β1X1 + β2X2 + β3X1X2 + β4X21 + β5X22 (4)
In the aforesaid equation, β0 represents the intercept, β 1 and β2 as the
coefficients of linear terms X1 and X2 respectively, β3 as the coefficient of
interaction term, and β4 and β5 as the coefficients of respective quadratic
terms X21 and X 22, respectively. Table S5 enlists the magnitudes of various
model coefficients, along with the ANOVA parameters.
S. Saini et al.
The 3-D RSM plot and the corresponding 2-D RSM plot for Snm (Fig. 1
a and b, respectively) revealed that at low levels of surfactant, Snm tends
to increase with increasing lipid levels. However, at high levels of sur­
factant, Snm values tend to decrease. These observations indicate the
presence of a sufficient amount of surfactant to reduce the interfacial
tension with increasing amounts of lipid, thus leading to improved sta­
bilization of the nanoparticulate dispersion. Also, at high levels of lipid,
it was observed that increasing the surfactant amount tended to reduce
Snm.
The 3-D RSM plot and the corresponding 2-D RSM plot for ZP, as
depicted in Fig. 1c and d, respectively, show that increasing the levels of
lipid leads to a rise in ZP values, followed by a decline in their values at
the higher levels. In contrast, at higher levels of lipid, on increasing the
amount of surfactant, ZP tends to decrease, potentially ascribable to the
non-ionic character of Tween 80 as the surfactant. However, at low
levels of lipid, this increasing trend in ZP is observed to be less
pronounced.
The 3-D RSM plot for % EE is depicted in Fig. 1e and f, respectively. It
shows a curvilinear interactive and declining trend in % EE values at
lower and higher levels, though an increasing trend is observed at their
intermediate levels.
Fig. 1 (g and h) illustrate the influence of the levels of lipid and
surfactant on the values of % RelFA. The 3-D RSM plot (Fig. 1 g) portrays
a linear descent in the magnitude of % RelFA at all the levels of lipid,
indicating sustained kind of drug release of the nanoconstructs. Analo­
gous information can also be deduced from the corresponding 2-D
contour plot (Fig. 1h). However, a rise in % RelFA is observed on
increasing Tween 80, apparently owing to its emulsification
characteristics.
RSM facilitated establishing relations(s) among the CMAs and CQAs,
chiefly using these 3-D and 2-D RSM plots. Design space, as a definitive
outcome of graphical optimization, demarcated the composition of the
optimum formulation of prepared nanocarriers [36,37].
3.5.1. Search for the optimum solution
Figure S5 portrays the overlay plot and location of the optimal
formulation within the design space. The optimized SLNs formulation
comprised of Compritol (180 mg) and Tween 80 (2.25 g), with Snm of
181.44 nm and ZP of -2.72 mV. The EE of optimized formulation was
found to be 38.66 %, while cumulative % RelFA at 12 h was 74.01 %.
3.5.2. Coating of SLNs with chitosan
The chitosan coating efficiency of cSLNs was found to be 61.7 %,
quite in concurrence with literature [47].
3.6. Characterization studies on optimized SLNs and cSLNs
3.6.1. Particle size and zeta potential studies
As illustrated in Figure S6 (a and c), Snm of SLNs and cSLNs was
observed to be 182.6 and 184.9 nm, respectively, while the corre­
sponding values of ZP were -2.63 and 12.4 mV (Figure S6 (b and d)),
respectively. The charge on ZP values got altered from negative to
positive, following chitosan coating, apparently owing to cationic nature
of the polymer. Though ZP got augmented in magnitude considerably
with cSLNs, yet it was not adequate to prevent agglomeration; hence the
formulation was lyophilised [74,75].
3.6.2. FTIR studies
The vibrational frequency of the − OH group stretch of FA is observed
at 3437.48 cm− 1 (Figure S7A) of Supplementary Information). The
FTIR spectra of physical mixtures of FA with Compritol (Figure S7B),
Tween 80 (Figure S7C), individually, and with both Compritol and
Tween 80 (Figure S7D) depict no appreciable change in the charac­
teristic vibrational frequency of the –OH group stretch of FA, indicating
the absence of any interaction. The vibrational frequency of –C– –O
group stretch of FA was observed at 1689.26 cm− 1, delineating no shift
6
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
in the vibrational frequency of –C– – O group stretch in the FTIR spectra
of the physical mixture of FA with the selected excipients [76]. This
indicates that the selected excipients were quite compatible with FA and
can be employed, in combination, for further studies.
3.6.3. DSC studies
The DSC thermogram of FA depicts a single characteristic endo­
thermic peak at 176.58 ◦ C (Figure S8A). The thermograms of physical
mixtures of FA with Compritol (Figure S8B), Tween 80 (Figure S8C),
and Compritol and Tween 80 together (Figure S8D), portray no new
peak(s), along with an intact peak of FA, thus refuting any plausible
distinct interaction(s) between FA and chosen excipients. The physical
mixture(s) showed characteristic peaks of the drug, which were absent
in the DSC thermogram of formulation, plausibly attributable to the
entrapment of FA into the formulation (Figure S8E). The melting point
peaks were found to be broadened, reduced in intensity, and slightly
shifted from their actual melting points, ascribable to the lower
enthalapy and the plasticizing effect of the surfactants and co-
surfactants, respectively, in consonance with various literature reports
[77,78]. Further, the endothermic peak of FA in SLNs indicates the
presence of FA in stable crystalline form in the lipidic matrix of the
formulation.
3.6.4. PXRD studies
Figure S9 illustrates PXRD patterns of FA (a), lyophilized SLNs (b)
and cSLNs (c). FA exhibits sharp peaks at 2θ scattered angles of 9.04,
12.83, 15.64, 25.97, 26.43 and 29.48, which are characteristic of its
crystalline nature. However, these peaks were conspicuously absent in
lyophilized formulations (Figure S9(B and D)), vividly indicating the
loss of crystallinity in the corresponding lipidic nanoconstructs.
3.6.5. FESEM studies
The FESEM images of optimized SLNs (Figure S10 (a and b)) and
cSLNs (Figure S10 (c and d)) indicate their uniform spherical geometry
with minuscule aggregation. However, surface coating was found to
introduce irregularities on surface of SLNs, thereby increasing the
effective contact surface area, which could plausibly enhance its nasal
mucoadhesion.
3.6.6. Drug entrapment studies
The system exhibited EE of 38.92 and 51.18 % for uncoated and
coated optimized SLNs, respectively. Lower values of EE can be ascribed
to the hydrophilic nature of molecule and reduced entrapment of FA in
the lipidic coat [74]. Augmented EE values of cSLNs, however, may be
ascribed to the presence of chitosan coating, which could have pre­
vented leaching out of FA from the SLNs [79].
3.6.7. In vitro drug release studies
Fig. 2 portrays nearly sustained-release profile for the SLNs. Only 19
% of FA was observed to be released within 12 h in its naïve form,
whereas encapsulating FA in SLNs and subsequent chitosan coating
significantly improved its release to 72 % and 64 %, respectively, in 12
h. Drug release kinetic modelling using Korsmeyer-Peppas model fur­
nished the release exponent values of 0.392 and 0.454 for optimized
uncoated and coated SLNs, indicating that FA release followed Fickian
and quasi-Fickian mechanisms, respectively. Encapsulating FA in SLNs
improved in vitro dissolution performance, which is quite evident from
the enhanced rate and extent of dissolution. Chitosan coating impeded
dissolution profile vis-à-vis uncoated optimized SLNs, which may be
attributed to the formation of a barrier layer of chitosan around the
SLNs, thereby increasing the effective path length for FA to get released
into the dissolution medium vis-à-vis the uncoated SLNs dispersion.
Hence, the rate, as well as the extent of dissolution, was observed to be
comparatively lowered, following chitosan coating, indicating relatively
superior controlled release characteristics of cSLNs.
S. Saini et al.
Fig. 2. Mean % cumulative drug release profiles of uncoated optimized SLNs and chitosan-coated optimized SLNs, along with pure drug suspension (n = 3).
3.6.8. Ex vivo mucoadhesion studies
Fig. 3a depicts enhanced mucoadhesive strength of SLNs, on chitosan
coating. The results construe that the detachment force, indicative of
nasal mucoadhesive strength, increased from 6.88 to 8.55 N, after
coating. cSLNs are known to possess a positive surface charge, which
tends to facilitate their binding to the anionic mucosal membranes [80,
81]. Further, mucoadhesion is documented to decrease drug loss due to
mucociliary clearance [82], thus enhancing the availability of drug(s) at
the site of the absorptive surface for prolonged periods. The intranasal
application of mucoadhesive nanoparticles causes them to swell, on
coming in contact with the nasal mucosal secretions and adhere to the
Fig. 3. (a) Ex vivo mucoadhesive strength of (A) uncoated optimized SLNs; and
(B) chitosan-coated optimized SLNs. (b) Mean permeation coefficient of pre­
pared SLNs as compared to pure FA. Each crossbar indicates mean +1 SD (n
= 3).
7
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
epithelial mucosa, thus leading eventually to their increased residence
time in the nasal cavity [83].
3.6.9. Ex vivo permeation studies
Distinct augmentation in the extent of FA permeating through the
nasal mucosa was observed with uncoated optimized SLNs (i.e., 25.35
%) as well as after their being coated with chitosan (i.e., 35.49 %) vis-
à-vis 2.29 % of pure drug suspension, in the first 12 h. This remarkable
enhancement in drug permeation through nasal mucosa with SLNs could
be ascribed to their lipidic architectural constitution [84]. However, as
portrayed in Fig. 3b, drug permeation rate in case of uncoated optimized
formulation (permeation coefficient of 0.00046) was found to be rela­
tively higher than with coated optimized formulation (permeation co­
efficient of 0.00036), as more drug may leach out of the uncoated
system.
3.6.10. Stability studies
The values of assayed drug content, as well as of various physical and
dissolution parameters, studied as CQAs, along with the f2 values
thereof, observed during the 90-days stability studies are enumerated in
Table S6. The freeze-dried cSLNs stored at 25 ± 2 ◦ C and 40 ± 2 ◦ C
demonstrated significant rise in Snm and ZP, along with reduction in EE
values, after reconstitution. Increasing growth in the size of nano­
particulate systems with time and temperature is well-established. The
escalating trend of ZP values at 25 and 40 ◦ C, on the other hand, can
potentially be attributed to the quite spontaneous crystalline re-
orientation of the formulations during three months of storage, result­
ing eventually in shifting the electronic charge on the surface of the
particle(s) from the erstwhile negative to positive ones [57,61]. Storage
of nanoparticles at 40 ◦ C/75 % RH, likewise, resulted in a much higher
increase of the ZP values vis-à-vis with samples stored at 25 ◦ C/60 % RH,
corroborating their far lower stability at high temperatures. Further,
entrapment of FA was observed to decline at all the storage conditions
with time, though minimal influence was seen at the refrigerated con­
ditions, i.e., around 5 ◦ C. Drug expulsion from solid lipid matrix, owing
to crystallization of lipid, is already documented to cause reduction in
EE on storage at 25 ◦ C as well as at 40 ◦ C [84]. Marginal alterations in
drug content and other studied parameters, viz., %EE, globule size and
Q12h, along with miniscule variations in the f2 values, well within the
desirable range of 50 and 100; all corroborate the robustness of the
formulated SLNs under the refrigerated conditions, in consonance with
various literature reports on SLNs [36]. Accordingly, on the basis of the
S. Saini et al.
aforesaid observations, the refrigerated conditions can be vividly
construed as most favourable for the storage of SLNs [85].
3.6.11. In vivo studies
3.6.11.1. Behavioural studies. Animals administered with pure drug, i.n.
and orally, with ICV-STZ treatment, showed a notable decline in the
cognitive abilities as compared to sham group (p < 0.001 on Day 26 and
27). An insignificant change was observed in mean escape latency time
with pure drug (p > 0.05 on Days 25–27).
Meanwhile, treatment of uncoated and coated optimized SLNs i.n.,
along with ICV-STZ treatment, significantly improved cognitive per­
formance (i.e., shortened ELT) vis-à-vis pure FA i.n. (p < 0.001 each on
Day 27). Moreover, i.n. treatment with coated optimized SLNs vis-à-vis
uncoated ones, significantly reduced mean escape latency time was
observed on Day 27 (p < 0.001), indicating superior efficacy of the
former in improving cognitive performance. Similarly, animals treated
with uncoated SLNs orally, along with ICV-STZ treatment, showed sig­
nificant improvement in cognitive performance vis-à-vis pure drug oral
treatment on Day 27 (p < 0.001). Also, a significant difference was
noticed between uncoated and coated SLNs i.n. vis-à-vis uncoated SLNs
oral treatment (Day 27, p < 0.001).
Animals receiving pure drug i.n. and pure drug oral treatment also
failed to locate the submerged platform as compared to the sham group,
devoting significantly less TSTQ (p < 0.001) (Fig. 4). Significant dif­
ference was perceptible between i.n. administration on of prepared SLNs
vis-à-vis oral intake of prepared SLNs in TSTQ as well as % change in
TSTQ wrt positive control (p < 0.001). Nearly 4.32- to 4.93-folds
improvement in TSTQ was seen in the animals treated with uncoated
and coated optimized SLNs i.n., respectively, vis-à-vis pure drug. The
uncoated optimized SLNs on oral treatment, however, showed only
2.66-folds improvement in TSTQ vis-à-vis the corresponding pure drug
(Fig. 4). Overall, following administration of the formulated SLNs,
significantly improved the navigational abilities of the treated rats to
memorize the platform position thus ratifying enhanced anti-Alzheimer
potential of surface-coated SLNs vis-à-vis pure FA, both administered i.n.
(p < 0.001).
3.6.11.2. Biochemical estimation studies. Sham-treated rats did not
exhibit any notable change(s) in the biochemical levels in hippocampus
and cortex brain regions, vis-à-vis naïve group. However, single ICV-STZ
administration (positive control group) significantly raised the LPO
levels (p < 0.001), nitrite concentration (p < 0.001), protein levels (p <
Fig. 4. Effect on various treatments on time spent in the quadrant during the retention phase of Morris Maze test. Data expressed as mean + 1 SD (n = 3). Data
analysed using one-way ANOVA. ***p < 0.001; N.S. Not Significant.
8
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
0.001), and AChE activity (p < 0.001), while caused depletion of GSH
levels (p < 0.001) and SOD activity (p < 0.001), in hippocampus and
cortex, vis-à-vis sham group (Fig. 5). A 28-day i.n. administration
regimen of uncoated and coated optimized SLNs significantly attenuated
oxidative stress parameters (LPO, nitrite concentration, protein levels
and AChE activity, and restored GSH levels and SOD activity) vis-à-vis
positive control group. Further, i.n. administration of coated optimized
SLNs also showed distinct improvement in oxidative stress parameters as
compared to the uncoated optimized SLNs via i.n. treatment.
In a nutshell, there was a nearly 4.9-folds improvement in TSTQ, 3.9-
folds reduction in MDA levels, 4.4-folds elevation in GSH levels, 6.6-
folds reduction in nitrite levels, 4.5-folds reduction in protein levels,
4.1-folds reduction in AChE activity, 5.2-folds improvement in SOD
activity and 6.9-folds increase in brain levels of FA in animals with
intranasally administered coated SLNs vis-à-vis pure drug. This signifies
that the developed nanosystem substantially improved the brain access
of FA through i.n. route, by potentially circumnavigating BBB.
3.6.11.3. In vivo brain drug level estimation studies. As observed from
Fig. 6, significantly enhanced FA concentration was observed in brain
homogenates of animals treated intranasally with uncoated optimized
SLNs (5.42-folds) as well as with coated SLNs (6.91-folds) vis-à-vis in
those treated with the pure drug (p < 0.001 each). Improved trans­
cellular transport of FA from the SLNs via olfactory route to brain might
cause such augmentation. Surface coating of chitosan on SLNs furnished
remarkably higher brain levels, plausibly owing to extended nasal
retention time, opening of tight junctions of nasal epithelial cells, pos­
itive charge of chitosan and improved stability in the biological milieu of
the developed nanosystem [27]. The brain: plasma level ratio, following
i.n. administration, with cSLNs (i.e., 2.35), was also found to be
considerably higher compared to uncoated SLNs (i.e., 1.29) and pure FA
(i.e., 0.95) administered via i.n. route. The results vividly indicate
noteworthy contribution of SLNs in direct nose-to-brain transport of FA
by bypassing the BBB and ratifying the superior delivery of cationic
nanoparticles via nasal route.
3.6.11.3.1. Histopathological findings. Figure S11 portrays histo­
pathological images (at 100X magnification) of different rat organs, viz.,
brain, lung, heart, kidney and liver, treated with normal saline (i.e.,
naïve group) (Figure S11 A, C, E, G and I), and those with coated
optimized SLNs i.n. (Figure S11 B, D, F, H and J). Hippocampus (A and
B) exhibited no significant change. Bronchioles with lymphoid cells and
alveoli (Figure S11 C and D) are observed to be normal. Heart tissue
(Figure S11 E and F)) showed normal anatomy. Further, the glomeruli
S. Saini et al.
Fig. 5. Change in the levels of various biochemical parameters on formulation treatment. Data are expressed as mean +1 SD (n = 3). Data were analysed using one-
way ANOVA. ***p < 0.001; N.S. as Not Significant.
and tubules in kidneys (Figure S11 G and H), were found to be normal
too. Cell cords, portal tracts, central veins and Kupffer cells of liver
(Figure S11 I and J) portrayed normal anatomy. Overall, these findings
indicated no histopathological change(s) in all the vital organs studied
between both of the animal groups. Accordingly, the results establish the
biosafety and tolerability of the developed formulation.
4. Conclusions
A number of plant bioactives, including FA, hold enormous promise
in alleviating various neurodegenerative disorders but are marked with
several physicochemical, biopharmaceutical and biodistributional pit­
falls. The current research work was accordingly orchestrated; investi­
gating plausible improvement in the anti-Alzheimer’s potential of FA
using novel lipidic nanocarriers through nasal route. Novel mucoadhe­
sive SLNs of FA, surface-coated with chitosan, were formulated not only
9
Colloids and Surfaces B: Biointerfaces 205 (2021) 111838
to augment its access to the brain, but also to improve its oral
bioavailability by possible circumnavigation of hepatic first-pass effect
and prolongation of retention in the body. Application of rational QbD
paradigms, during drug delivery development, delineated various cause-
effect relationships, prioritized the selection of vital few influential
variables, embarked upon the demarcation of a robust optimized
formulation and finally validation of DoE application. Evaluation of
stability studies of SLNs formulations indicated that the refrigerated
storage conditions (i.e., around 5 ◦ C) were quite suitable for maintaining
the robustness of the nanocoutered formulations. in vivo pharmacody­
namic behavioural studies in rats, employing Morris water maze test, as
well as alteration in the animal body weight as a biomarker, demon­
strated markedly improved cognition with the coated optimized SLNs,
followed by uncoated SLNs vis-à-vis pure drug. Intranasal administration
also showed superior results vis-à-vis oral route. Estimation of apt
biochemical parameters in rat brain homogenates indicated markedly
S. Saini et al.
Fig. 6. Drug concentrations in brain and plasma of different animal groups. Data expressed are as mean + 1 SD (n = 3). Data analysed using one-way ANOVA. ***p <
0.001; **p < 0.01.
improved regulation of MDA, GSH, nitrite, protein levels, AChE and SOD
activity in the animals receiving uncoated and coated optimized SLNs
intranasally vis-à-vis the pure drug. FA intake via intranasal route was
also found to be much more beneficial in upregulating the biochemical
parameters vis-à-vis oral route treatment. Histopathological studies
established the safety and tolerability of the coated SLNs. The successful
outcomes of the present work on QbD-enabled chitosan-coated SLNs
using lipidic nanoconstructs of FA via nose-to-brain delivery with
distinctly superior pharmacodynamic performance in AD can also be
rationally extrapolated to other problematic drugs and bioactives
encountering similar biodistributional and biopharmaceutical issues.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The author (B.S.) gratefully appreciates the bestowed generosity of
M/s Stat-Ease Inc., USA, in gifting a perpetual and multiple annual
licenses of Design-Expert software to him and his research team as a
mark of recognition to his unmatched contribution in the application of
QbD paradigms in developing diverse novel and nanostructured drug
delivery systems. The first author (S.S.) acknowledges the requisite
funding provided by the University Grants Commission, New Delhi,
India, to him under Meritorious Research Fellowship scheme.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2021.111838.
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