Design of multicomponent indomethacin-paracetamol and famotidine loaded nanoparticles for sustained and effective anti-inflammatory therapy

Article abstract of DOI:10.1002/ddr.21768

Indomethacin is one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that are widely prescribed drug for pain and inflammation. However, its notoriety of causing gastrointestinal effect, low water solubility, and its short half-life would affect patient compliance and its oral absorption and accordingly justify the need to develop a formula with a controlled and sustained release manner in combination with anti-ulcer drugs. Herein, we synthesized indomethacin-paracetamol co-drug loaded in nanoemulsion and encapsulated in famotiditine loaded polycaprolactone (PCL) nanoparticles. The synthesis of the co-drug was achieved by the formation of a hydrolyzable ester between the indomethacin and paracetamol. The synthesized co-drug was preloading in nanoemulsion (Co-NE), which encapsulated into famotidine PCL nanoparticles utilizing the nanoprecipitation approach. The developed nanosystem showed hydrodynamic size less than 200 nm and the zeta potential value above ?30 mV. TEM images confirmed the morphological structure of the formed nanoemulsion and the loaded PCL nanoparticles. Stability studies revealed that the developed nanosystem was stable at different temperatures and pHs over 1 month. Moreover, improvement of the solubilities of these three drugs leading to have a controlled-release multicomponent system of both co-drug and famotidine over 3 days. This multicomponent nanoparticle might be a potential platform to overcome the obstacles of NSAIDs, synergize drugs with different mechanisms of actions by co-encapsulating a small-sized nanoemulsion into PCL nanoparticles for reaching the goal of effective anti-inflammatory therapy.

Full text of DOI:10.1002/ddr.21768

Received: 15 July 2020
DOI: 10.1002/ddr.21768
Revised: 22 October 2020
Accepted: 23 November 2020
R E S E A R C H A R T I C L E
Design of multicomponent indomethacin-paracetamol and
famotidine loaded nanoparticles for sustained and effective
anti-inflammatory therapy
Mohyeddin Assali
| Nihal Zohud
Department of Pharmacy, Faculty of Medicine
and Health Sciences, An-Najah National
University, Nablus, Palestine
Abstract
Indomethacin is one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that are
widely prescribed drug for pain and inflammation. However, its notoriety of causing
gastrointestinal effect, low water solubility, and its short half-life would affect patient
compliance and its oral absorption and accordingly justify the need to develop a for-
mula with a controlled and sustained release manner in combination with anti-ulcer
drugs. Herein, we synthesized indomethacin-paracetamol co-drug loaded in nano-
emulsion and encapsulated in famotiditine loaded polycaprolactone (PCL)
nanoparticles. The synthesis of the co-drug was achieved by the formation of a
hydrolyzable ester between the indomethacin and paracetamol. The synthesized co-
drug was preloading in nanoemulsion (Co-NE), which encapsulated into famotidine
PCL nanoparticles utilizing the nanoprecipitation approach. The developed nano-
system showed hydrodynamic size less than 200 nm and the zeta potential value
above −30 mV. TEM images confirmed the morphological structure of the formed
nanoemulsion and the loaded PCL nanoparticles. Stability studies revealed that the
developed nanosystem was stable at different temperatures and pHs over 1 month.
Moreover, improvement of the solubilities of these three drugs leading to have a
controlled-release multicomponent system of both co-drug and famotidine over
3 days. This multicomponent nanoparticle might be a potential platform to overcome
the obstacles of NSAIDs, synergize drugs with different mechanisms of actions by
co-encapsulating a small-sized nanoemulsion into PCL nanoparticles for reaching the
goal of effective anti-inflammatory therapy.
Correspondence
Mohyeddin Assali, Department of Pharmacy,
Faculty of Medicine and Health Sciences,
An-Najah National University, Nablus,
Palestine.
Email: m.d.assali@najah.edu
K E Y W O R D S
D-limonene, esterase, kinetic release study, nanoemulsion, polycaprolactone nanoparticles,
sustained release
1
|
INTRODUCTION
(Ricciotti & FitzGerald, 2011). Inflammation is an essential status lead-
ing to the elimination of insulting factors and rejuvenation of tissue
Inflammation is a nonspecific, natural, and cellular response to multi-
ple stimuli like infection, injury, irritation, and microorganisms promot-
ing the release of different protective chemicals (Laroux, 2004).
Among these chemicals are prostaglandins (PGs) which are hormone-
like mediators that play a crucial role in the inflammatory processes
structure and physiological function (Nathan, 2002). Nonsteroidal
anti-inflammatory drugs (NSAIDs) are among the most commonly pre-
scribed and consumed drugs for pain and inflammation worldwide
(Badri et al., 2016; Hochberg et al., 2012). They act by blocking the
prostaglandin synthesis through the inhibition of cyclooxygenase
Drug Dev Res. 2020;1–10.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, LLC.
1

2
ASSALI AND ZOHUD
(COX) enzymes which is responsible for the desired anti-inflammatory
activity and the unwanted gastrointestinal effects (Higuchi
et al., 2009; Sostres et al., 2010). For various chronic diseases such as
rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis,
NSAIDs are often prescribed using high doses which often cause
many side-effects and poor patient compliance. Therefore, those
drugs require strict monitoring, especially in patients with renal and
cardiovascular diseases (Amadio Jr et al., 1997).
encapsulated into PCL nanoparticles containing FAM using
nanoprecipitation technique as shown in Scheme 1. We chose PCL as
it is considered a biodegradable polymer with exceptional rheological
and viscoelastic properties, high drug permeability, and long stability
(Guarino et al., 2017; Nair
& Laurencin, 2007; Woodruff &
Hutmacher, 2010). The in vitro release profiles of co-drug and FAM in
the developed multidrug nano delivery system were investigated in
addition to stability and biocompatibility studies on cells were
executed.
Indomethacin (IND) is considered one of the most potent non-
selective NSAIDs owning very effective antipyretic, analgesic, and
anti-inflammatory activities (Yeh, 1985). It is chemically classified as a
derivative of indoleacetic acid (Fritsche et al., 2001) with low water
solubility and a short half-life of 4–5 h, causing multiple daily doses
prescription (Clarysse et al., 2009; Ziltener et al., 2010). It is most typi-
cally used for the treatment of inflammation resulting from rheuma-
toid diseases. The nonselective inhibitory activity of COX enzymes
and the undesirable physical property (poor solubility) are responsible
for the gastrointestinal (GI) irritation side effects (Alsaidan et al., 1998;
Saeedi et al., 2011). Furthermore, it can cause CNS disruption espe-
cially a frequent headache (Lucas, 2016). Therefore, various studies
were reported to reduce the mentioned side effects through indo-
methacin encapsulation or conjugation in proper nano-vehicles
(Giacomelli et al., 2007; Tan et al., 2018).
2
|
MATERIALS AND METHODS
| Reagents and materials
2.1
PCL, D-limonene were purchased from Sigma-Aldrich Company. Poly-
oxyethylene cetyl ether (POE) and polyvinyl alcohol (PVA) were pur-
chased from CS Company, New Zealand. Sorbitan monooleate (Span®
80) was purchased from Alfa Aesar, UK. Polyoxyethylene sorbitan
monooleate (Tween® 80) was purchased from Arcos organics. Ace-
tone, methanol (MeOH), dichloromethane (DCM), and isopropyl alco-
hol were purchased from C.S. Company, Haifa. Disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium carbonate, and
sodium bicarbonate were purchased from CS company, Haifa. Spec-
tra/Por® four dialysis membranes (12–14 KD MWCO, 25 mm flat
width, 100 ft length) were purchased from Spectrum Laboratories,
Inc. Porcine liver esterase (PLE) was purchased from Sigma-Aldrich.
Dulbecco's free Ca+2 phosphate-buffered saline and L-glutamine solu-
tion, Pen-Strep Solution were purchased from Biological Industries,
Jerusalem. RPMI was purchased from Manassas VA, Trypsin–EDTA
solution 1X, fetal Bovin Serum, trypan blue solution, and MTS kit were
purchased from Promega.
Several studies showed combining NSAIDs with paracetamol
(PAR) results in synergistic analgesia and decreases the required dose
which would reduce the raised adverse effects (Ong et al., 2010). Fur-
thermore, famotidine (FAM) is widely used for the treatment of acid-
related GI conditions, and it was found to be effective in preventing
gastric injury caused by Indomethacin, even at the lowest dose (Naito
et al., 2008). FAM is a competitive inhibitor of histamine H2-receptor
that blocks histamine actions by binding to the histaminic receptor
located on the parietal cells of the stomach resulting in inhibition of
gastric secretion (Berlin et al., 1986; Talke & Solanki, 1993). FAM
improves ulcer healing and reverses the GI side effects of IND by
inhibiting the secretion of gastric acid and also by increasing collagen
secretions (Hassan et al., 1997; Perez-Aisa et al., 2003).
2.2
|
Techniques and instruments
On this point, combination therapy through combining multiple
treatments with various mechanisms is a highly effective strategy in
the treatment of various diseases like cancer, inflammatory diseases,
HIV, and others (Baek et al., 2017; Baek & Cho, 2015; Coradini
et al., 2015; Freeling et al., 2014). To achieve this approach, different
drug delivery systems based on suitable nanomaterials and capable of
behaving combinations of various therapies have been successfully
explored (Gadde, 2015). Therefore, we designed multidrug delivery
systems that allow the sustained release of IND-PAR co-drug and
FAM encapsulated in polycaprolactone (PCL) nanoparticles.
Dynamic light scattering (DLS) and zeta potential analysis were mea-
sured on NanoBrook Omni (Brookhaven Instruments). High-
performance liquid chromatography with binary HPLC pump and
Herein, a co-drug of indomethacin and paracetamol (IND-PAR)
was synthesized and encapsulated in fruit flavor nanoemulsion
(NE) containing limonene as it masks the unpleasant bitter taste and
promotes gastric ulcer healing by increasing mucus production and
enhances the solubility and bioavailability of the co-drug (de Souza
et al., 2019; Gursoy & Benita, 2004; Mohsin et al., 2009; Moraes
et al., 2013). The co-drug loaded nanoemulsion was then
SCHEME 1 The general scheme demonstrates the chemical
structure of the co-drug and the general approach of the study

ASSALI AND ZOHUD
3
waters 2298 photodiode Array Detector (Waters 1525) were utilized
to quantify drugs. Transition electron microscope (TEM) images were
taken at 60 kV using Morgagni 286 transmission microscope (FEI
Company, Eindhoven, Netherlands). Esco cell culture CO2 incubator
was used to incubate the cell line. Unilab microplate reader 6000 was
utilized in the cell viability test to read the plate.
HPLC analytical method was developed and validated to measure
the loaded amount of drugs according to our recently published
method (Assali et al., 2020).
After that, the percentage of the encapsulation efficiency (EE) for
FAM and co-drug was calculated using the following equation.
EEð%Þ = ðweight of drug−loaded in the nanoparticlesÞ=
ðweight of drug initially usedÞ × 100%:
2.3
|
Synthesis of IND-PAR co-drug
The synthesis and characterization of IND-PAR co-drug have been
developed and published recently (Assali et al., 2020).
2.6
|
In vitro release studies
2.6.1
|
In vitro release of co-drug and FAM from
2.4
|
Preparation of nanoemulsion
PCL NPs without esterase enzyme
The method of nanoemulsion preparation was developed using the
ultrasonication method (Li & Chiang, 2012a). In brief, the organic
phase consists of 140 mg of sorbitan monolaurate (Span® 80) and
750 mg of D-limonene which was sonicated for 5 min. After that, it
was added dropwise under mild stirring to the aqueous phase that
consists of 360 mg of tween 80 and 8.75 ml of milli-Q water. The
resultant flocculated emulsion was mildly stirred for 15 min and soni-
cated for 10 min to obtain a blank nanoemulsion. To load the co-drug
in the nanoemulsion, 2 mg of the co-drug was added into the organic
phase and following the same mentioned procedure to obtain co-drug
encapsulated in nanoemulsion (Co-NE). Finally, the hydrodynamic
size, polydispersity index (PDI), and zeta potential were measured.
The measurements were taken at 25ꢀC and using 90ꢀ of the angle
scattering. The ζ-potential measured using the phase analysis light
scattering (PALS) technique.
Ten milligram of freeze-dried samples Co-NE-FAM@PCL
nanoparticles were dissolved in 3 ml freshly prepared phosphate
buffer saline (PBS) at pH 7.4 and then transferred into a dialysis bag
(donor compartment). This bag was firmly closed, then was placed into
40 ml of PBS at pH 7.4 (receptor compartment), fully immersed, and
gently stirred for more than 3 days at 37ꢀC. Aliquots of 1 ml from
each receptor compartment were taken and replaced directly with
1 ml freshly prepared PBS pH 7.4 at determined periods. These sam-
ples were analyzed using HPLC and percentage release of co-drug
and FAM were calculated along the time.
2.6.2
|
In vitro hydrolysis of co-drug loaded in
nanoemulsion and PCL nanoparticles
The synthesized co-drug was exposed to esterase enzyme to study its
hydrolysis to its parent drugs (IND & PAR). One milligram of co-drug
was incubated in 10 ml PBS containing 2 mg of esterase enzyme at
37ꢀC for 1 h (Assali et al., 2016; Assali et al., 2019; Assali, Zaid,
Abdallah, et al., 2017). For the in vitro hydrolysis of co-drug in NE or
PCL nanoparticles, the same procedure was followed by adding 1 ml
of Co-NE or Co-NE-FAM@PCL nanoparticles into the same afore-
mentioned media and gently stirred for 5 h. At different time intervals,
aliquots of 1 ml were obtained and replaced with equal volumes of
fresh BPS to mimic the sink conditions, filtered using a 45 μm syringe
filter, and then analyzed by HPLC. Percentage hydrolysis and percent-
age conversion of co-drug were determined using the developed
HPLC validated method.
2.5
|
Preparation of polymeric nanoparticles
Nanoprecipitation technique was used for the preparation of PCL
nanoparticles (Assali, Shawahna, Shareef, & Alhimony, 2018b; Assali,
Zaid, Bani-Odeh, et al., 2017). Briefly, the organic component consists
of 25 mg of PCL and 5 mg POE dissolved in 5 ml acetone which was
added drop-wisely to the aqueous component consisting of 3 g of 1%
PVA and 7 ml of Milli-Q water under mild stirring. The resulting sus-
pension was stirred mildly for 30 min and then, the solvent (acetone)
evaporated by rotary evaporator. After that, a 0.45 μm pore size
membrane syringe filter was used to filtrate the solution to get a uni-
form particle size. Purification of the formed nanoparticles was per-
formed by centrifugation at 15000 rpm for 10 min and washed three
times with Milli-Q water to remove any residual of PVA. The obtained
nanoparticles were dissolved in 2 ml milli-Q water and stored in a
refrigerator. Finally, effective diameter, PDI, and zeta potential were
determined using DLS.
2.7
|
Drug release kinetics
To assess the release mechanism of both co-drug and FAM from the
designed and formulated nanosystems, different kinetic models were
applied using the DDsolver program which helps to get satisfied dis-
solution data modeling and analysis. A comparison between each
model and each nanosystem was based on the linear regression
To prepare PCL nanoparticles loaded with the Co-NE and FAM,
1 ml of the well-characterized Co-NE and 2 mg FAM were added to the
aqueous phase and following the same procedure as mentioned above.

4
ASSALI AND ZOHUD
(R2adjusted) (P. Costa & Lobo, 2001), the akaike information criterion
(AIC) (Akaike, 1974b), and the model selection criterion (MSC). Data
analysis was performed using the Excel add-in DDSolver program and
2.9.3
|
Cell biocompatibility test
HeLa cells and 3 T3 fibroblasts were seeded in 96-well plates and
then incubated with 100 μl per well CGM supplemented with differ-
ent concentrations of the tested Co-NE-FAM@PCL nanoparticles for
24 h. After that, they were incubated for 2 h at 37ꢀC and 5% CO2
with 20 μl per well of MTS reagent. Lastly, the absorbance of each
concentration was determined by a plate reader at a wavelength of
490 nm. The percentage of viability was calculated by the following
equation:
the best-fitted models were taken based on the higher R2
and
adjusted
the lower AIC and MSC values more than 2.0.
2.8
|
Stability studies at different temperatures
and pH buffers
2.8.1
|
Stability studies at different temperature
%viability = ðabsof treated cells−absof blankÞ=
ðabsof control cells−absof blankÞ x100%
The stability of Co-NE-FAM@PCL nanoparticles was tested at differ-
ent temperatures (4–8, 25, and 40ꢀC). One milliliter of nanoparticles
was stored at the required temperature and aliquots were taken at
specified time intervals for ꢁ1 month. Each aliquot was tested by
DLS, polydispersity, and zeta potential analysis.
3
|
RESULTS AND DISCUSSION
3.1
|
Synthesis of the co-drug and preparation of
2.8.2
|
Stability studies using different buffers
PCL nanoparticles
The stability of Co-NE-FAM@PCL nanoparticles was determined at
two different buffers (acetate pH = 4.2 and carbonate pH = 9.4) by
diluting 1.5 ml of nanoparticles with 1.5 ml of each buffer, and ali-
quots were taken for DLS and zeta potential measurements during
1 month.
Indomethacin is one of the potent anti-inflammatory drugs that inhibit
COX enzymes but induces gastric ulcer and has a short half-life.
Herein, we developed a new multicomponent nano-drug delivery sys-
tem composed of IND-PAR co-drug nanoemulsion encapsulated in
FAM PCL nanoparticles to reduce the required dose, side effects, and
provide a sustained release profile to improve patient compliance. In
this regard, we have recently synthesized a co-drug of IND-PAR con-
nected through a well-known hydrolyzable ester bond that can easily
be converted to the parent drugs in the presence of esterase enzyme
as shown in Scheme 2 (Assali et al., 2020).
2.9
|
Cell biocompatibility tests
| Cell line
2.9.1
The synthesized IND-PAR co-drug was then encapsulated in a
small-sized D-limonene flavored nanoemulsion (NE) to improve the
water solubility of the co-drug and provide a pleasant flavor to the
formula. The nanoemulsion was composed of two nonionic and bio-
compatible surfactants TWEEN 80 and SPAN 80 that can mix easily
and form a stable o/w nanoemulsion (Koroleva et al., 2018). More-
over, various studies showed the addition of D-limonene provides
higher stability, better taste, odor, and decreases the particle size of
the formed o/w nanoemulsion (Li & Chiang, 2012a). Interestingly,
D-limonene promotes gastric ulcer healing by increasing mucus pro-
duction and displays anti-inflammatory activity (de Souza et al., 2019;
Moraes et al., 2013).
The cytotoxicity of the Co-NE-FAM@PCL nanoparticles was investi-
gated on HeLa cells and 3 T3 fibroblasts.
2.9.2
|
Cell culture
The cells were cultured in T-175 cell culture flasks supplemented with
cell culture growth medium (CGM) composed of RPMI basal medium
supplemented with L-glutamine (1%), FBS (10%), and penicillin/strep-
tomycin (1%). The cells were kept in a standard cell culture incubator
at 5% CO2, 37ꢀC, and 99% humidity.
For sub-culturing, the medium was suctioned and washed with an
excess of Ca2+-free PBS. After that, the cells were incubated with
0.025% trypsin for up to 5 min in the cell culture incubator until suffi-
cient cells detached from the flask. Then trypsin was inactivated by
CGM, the cell suspension was collected and the viable cell count was
determined using trypan blue stain before adjusting the cell concen-
tration to 50.000 cell/ml. Finally, the cells were seeded in a 96-well
plate as 5000 cells/well. The cells were left to adhere and accommo-
date overnight before running any test.
The resulting yellowish o/w nanoemulsion was characterized by
TEM to study the size and the morphology of the formed nano-
emulsion. Figure 1 showed the formation of the spherical shape of the
nanoemulsion with a size of ꢁ2 nm in diameter. The formed transpar-
ent solution of the micelle structure represents ultra-small micelles
formation that is dispersed in the aqueous phase. The very small size
of the formed micelle is due to the addition of the oil-soluble
D-limonene that could easily homogenize the formed aggregates of
the surfactants and facilitate the diffusion of the surfactant in the

ASSALI AND ZOHUD
5
SCHEME 2 Synthetic scheme of indomethacin-paracetamol co-drug
TABLE 1 Characterization of blank and co-NEs
Blank NE (blank)
Co-NE
2.33
Hydrodynamic size (nm)
Polydispersity index
Zeta potential (mV)
1.64
0.162
−38.02
0.272
−47.77
Once the Co-NE was successfully prepared and characterized, it
was encapsulated into PCL nanoparticles using the nanoprecipitation
method developed previously in our research group (Assali, Shawahna,
Dayyeh, Shareef,
& Alhimony, 2018a; Assali, Zaid, Bani-Odeh,
et al., 2017). The developed nanoparticles were decorated with poly-
ethylene oxide which acts as a masking agent against opsonization for
the drug transporter and accordingly prevents phagocytosis by mono-
nuclear phagocyte system (MPS) (Torchilin & Trubetskoy, 1995). Upon
the preparation of the PCL nanoparticles, the Co-NE and FAM were
added to the organic layer to form Co-NE-FAM@PCL nanoparticles.
The prepared nanoparticles were characterized by TEM, DLS, and zeta
potential analysis as shown in Figure 2, and Table 2. Figure 2 shows
the TEM image of blank PCL nanoparticles obtaining large and
rounded nanoparticles with a diameter range between 150 and
200 nm. However, Figure 2b shows the TEM image of Co-NE-
FAM@PCL nanoparticles demonstrating the formation of spherical
nanoparticles with a double layer of nanoemulsion surrounded by the
PCL nanoparticles and a diameter size in the range of 170–210 nm.
Table 2 shows the hydrodynamic diameters, PDI, and zeta poten-
tial data for the blank and loaded PCL nanoparticles. The values
showed the formation of nanoparticles with a hydrodynamic diameter
between 170 and 200 nm, monodisperse nanoparticles as the values
of PDI less than 0.3, and high stable solutions with zeta potential
values above −25 mV.
FIGURE 1 TEM image showed the morphology of the formed
nanoemulsion
aqueous phase (Aziz et al., 2019; Chang et al., 2013; Chang &
McClements, 2014; Kuznetsova et al., 2020). Moreover, the utilization
of the ultrasonication has additional assistance to ease the formation
of ultrasmall micellar structure (Li & Chiang, 2012). Moreover, the
formed nanoemulsion was further characterized by DLS and zeta
potential analyzer to determine the hydrodynamic diameter size, poly-
dispersity index (PDI), and zeta potential as shown in Table 1. The
small particle size of the formed NE was obtained in the range of
1.5–2.5 nm. Zeta potential values demonstrate the net charge on the
surface of nanomaterials; enhance their physical state in liquids and
accordingly their interactions with biological systems. Zeta potential
values were higher than
25 mV considered highly stable
nanomaterials (Bhattacharjee, 2016). In our case, we obtained
nanoemulsions with zeta potential values were above −35 mV which
indicates the formation of high stable nanoemulsions. It was noticed
that the size was slightly increased in the co-drug loaded nano-
emulsion as a consequence of co-drug loading with a loading capacity
of 100%. Therefore, we formed a small-sized, monodisperse, stable,
and monodisperse fruit-flavor Co-NE which allowed for greater
absorption due to small-sized droplets with greater surface area
(Jaiswal et al., 2015).
In order to determine the loading capacity of co-drug and FAM,
and the in vitro release profiles, a validated HPLC method was devel-
oped and recently published (Assali et al., 2020). From the developed
calibration curves and applying the encapsulation efficiency
(EE) equation, we obtained high values EE of 100% and 85% for co-
drug and FAM, respectively, which demonstrated the effectiveness of
PCL in encapsulating Co-NE and FAM.

6
ASSALI AND ZOHUD
FIGURE 2 TEM image of
(a) Blank PCL nanoparticles;
(b) Co-NE-FAM@PCL
nanoparticles
TABLE 2 Characterization of PLGA and PCL nanosystems
co-drug HPLC peak with a concomitant increase of IND-PAR HPLC
peaks, and this conversion was quantified by the developed equa-
tions. As seen in Figure 3, more than 80% of the co-drug was
converted to IND and PAR after almost 6 h of incubation which con-
firms the successful conversion and hydrolysis of ester bond to the
active pharmacological drugs.
Blank PCL NPs
171.0
Co-NE-FAM@PCL NPs
Hydrodynamic size (nm)
PDI
196.4
0.216
−39.42
0.074
Zeta potential (mV)
−27.19
3.2.2
|
In vitro release of co-drug and FAM
3.2
|
In vitro release studies
without PLE
The developed HPLC analytical method was utilized to investigate the
in vitro conversion of co-drug to its parents' drugs (IND-PAR) at nano-
emulsion (Co-NE), and co-drug in PCL nanoparticles (Co-NE-
FAM@PCL) in the presence of esterase enzyme (1 U/ml) in PBS
(pH 7.4) 37ꢀC.
The in vitro release profiles of co-drug and FAM were investigated
using the dialysis membrane technique. It observed an initial burst of
about 22% of co-drug from the PCL nanoparticles especially in the
first 20 min followed by a sustained release of about 90% for co-drug
for more than 3 days as displayed in Figure 4 red line. However, the
release profile of FAM was about 40% after 20 min and followed by a
rapid increase of about 80% with a sustained release for more than
80 h, as can be observed in Figure 4 black line. Therefore, FAM was
firstly released from the shell of the PCL nanoparticles to provide a
gastro-protective effect followed the sustained release manner of the
co-drug from the nano-emulsion core of the PCL nanoparticles as can
be observed in Figure 4.
3.2.1
|
Percentage of conversion at Co-NE and Co-
NE-FAM@PCL
For the conversion of the co-drug to IND-PAR, we used esterase
enzyme through the incubation with PLE enzyme, a decrease in the
FIGURE 3 (a) % conversion of co-drug loaded in the nanoemulsion; (b) % conversion of co-drug loaded co-NE-FAM@PCL nanoparticles

ASSALI AND ZOHUD
7
was noted the best model of the FAM Weibull model which
suggested that FAM was released from the spherical polymeric matrix
with a sustained release manner. Interestingly, Weibull which is
known as stretched exponential is the most common model applied to
fit diffusion-controlled experimental data (Ignacio et al., 2017;
Kosmidis et al., 2003).
The most suitable model for co-drug was first order with maxi-
mum release fraction (Fmax) value 85.99% of co-drug, where the rate
was concentration-dependent and this result was in accordant with
co-drug hydrolysis in which first-order kinetic model was the most
suitable.
3.4
|
Stability studies
The stability of Co-NE-FAM@PCL nanoparticles was investigated by
varying the temperatures (4–8, 25, and 40ꢀC) and the pH (4.2 and
9.4). It was studied by analyzing the size of particles and the zeta
potential as a function of time. It was obvious from Table 4 that the
nanoparticles were stable at the three different temperatures and the
two buffer conditions as particle size and polydispersity changes are
within the acceptable limits and do not exceed a size above 220 nm
and polydispersity also less than 0.3 for all measurements and that
results were with the agreement that PCL polymer considered as a
long-term stable polymer (Woodruff & Hutmacher, 2010). Moreover,
zeta potential measurements were taken for the last readings and
confirm high stability for the whole period of the study (1 month)
where the values were above −30 mV.
FIGURE 4 % release of co-drug and FAM from co-NE-FAM@PCL
nanoparticles
TABLE 3 The most fitted models for both nanosystems
Drug
Kinetic model
Weibull
R2
AIC
MSC
2.51
2.98
adjusted
FAM
0.9756
0.9705
100.79
116.98
Co-drug
First-order with Fmax
3.3
|
Drug release kinetics
The kinetic models of the in vitro release were investigated. To dis-
criminate the most appropriate model, R2adjusted, the AIC, and the
MSC were determined (Akaike, 1974a; Handbook, 1995). The best
kinetic model should show the highest coefficient of determination
3.5
|
Cellular biocompatibility
It is very essential to determine the biocompatibility of the developed
nanoparticles. For that reason, we have investigated the cellular com-
patibility on HeLa cells and 3 T3 fibroblast cells. The viability test was
performed using MTS assay to determine the percentage of viability
of the cells upon their incubation with the developed nanosystem
after 24 h. As can be observed in Figure 5, both cell lines were incu-
bated with various concentrations (25–400 μg/ml) of the developed
nanosystem. The results showed excellent viability of both cell lines in
comparison to the control without any statistical difference in any
tested concentration which confirms the biocompatibility of the
developed Co-NE-FAM@PCL nanoparticles.
R2
(Costa & Lobo, 2001), the lowest Akaike Information (AIC)
adjusted
(F. Costa et al., 2003), and a value of more than two of MSC (Koizumi
et al., 2001; Mayer et al., 1999).
The results for the curve-fitting studies revealed that FAM
release from PCL nanoparticles could be best described by the
Weibull model whereas first-order with Fmax would be the best for
co-drug as seen below in Table 3. As can be observed, the R2 for FAM
and co-drug was above 0.97, AIC values were the lowest, and MSC
values were above two for both drugs as represented in Table 3. It
TABLE 4 Stability study of co-NE-
FAM@PCL NPs at different temperatures
and buffers
Temperature (ꢀC)
Buffers
4–8
25
50
Acetate
209.38
0.100
Carbonate
214.77
0.128
Hydrodynamic size (nm) on the first day.
189.29
0.190
229.60
0.061
−30.48
178.03
0.176
181.19
0.196
−32.31
222.13
0.134
214.71
0.181
−33.53
Polydispersity index on the first day.
Hydrodynamic size (nm) after 1 month.
Polydispersity after 1 month.
206.33
0.152
206.07
0.187
Zeta potential after 1 month.
−45.22
−42.61

8
ASSALI AND ZOHUD
FIGURE 5 Viability assay of
co-NE-FAM@PCL nanoparticles
incubated with (a) HeLa cells;
(b) 3 T3 fibroblast
Amadio, P., Jr., Cummings, D. M., & Amadio, P. B. (1997). NSAIDs revisited:
Selection, monitoring, and safe use. Postgraduate Medicine, 101(2),
257–271.
4
|
CONCLUSION
Assali, M., Abualhasan, M., Zohud, N., & Ghazal, N. (2020). RP-HPLC
method development and validation of synthesized Codrug in combi-
nation with indomethacin, Paracetamol, and famotidine. International
Journal of Analytical Chemistry, 2020, 1–9. https://doi.org/10.1155/
2020/1894907.
In this study,
a multicomponent nanosystem was successfully
designed and obtained for effective anti-inflammatory therapy based
on nanoemulsion and PCL nanoparticles. This nanosystem was
achieved by loading the synthesized IND-PAR co-drug into a fruit-
flavor nanoemulsion followed by the encapsulation of the latter into
PCL nanoparticles containing FAM in its core. These nanosystems
were characterized using different techniques and their sustained
release manners and hydrolysis of co-drug were studied by a novel,
developed, and validated RP-HPLC analytical method. The developed
nanosystem showed high biocompatibility and had good stability at
different conditions. Subsequently, it is a promising platform for over-
coming the obstacles of NSAIDs and enhancing patient compliance
with their therapy.
Assali, M., Joulani, M., Awwad, R., Assad, M., Almasri, M., Kittana, N., &
Zaid, A. N. (2016). Facile synthesis of ciprofloxacin Prodrug analogues
to improve its water solubility and antibacterial activity. Chemistry
Select, 1(6), 1132–1135. https://doi.org/10.1002/slct.201600091.
Assali, M., Kittana, N., Qasem, S. A., Adas, R., Saleh, D., Arar, A., &
Zohud, O. (2019). Combretastatin A4-camptothecin micelles as combi-
nation therapy for effective anticancer activity. RSC Advances, 9(2),
1055–1061. https://doi.org/10.1039/c8ra08794f.
Assali, M., Shawahna, R., Dayyeh, S., Shareef, M., & Alhimony, I.-A. (2018).
Dexamethasone-diclofenac loaded polylactide nanoparticles: Prepara-
tion, release and anti-inflammatory activity. European Journal of Phar-
maceutical Sciences, 122, 179–184. https://doi.org/10.1016/j.ejps.
2018.07.012.
ACKNOWLEDGMENT
Assali, M., Zaid, A. N., Abdallah, F., Almasri, M., & Khayyat, R. (2017). Sin-
gle-walled carbon nanotubes-ciprofloxacin nanoantibiotic: Strategy to
improve ciprofloxacin antibacterial activity. International Journal of
Nanomedicine, 12, 6647–6659. https://doi.org/10.2147/ijn.s140625.
Assali, M., Zaid, A. N., Bani-Odeh, M., Faroun, M., Muzaffar, R., &
Sawalha, H. (2017). Preparation and characterization of carvedilol-
loaded poly (d, l) lactide nanoparticles/microparticles as a sustained-
release system. International Journal of Polymeric Materials and Poly-
meric Biomaterials, 66(14), 717–725.
Nihal Zohud acknowledges the Faculty of Graduate Studies at
An-Najah National University to facilitate the achievement of this
work. The authors thank Duaa Qattan at the University of Jordan for
her assistance in the TEM analysis.
CONFLICT OF INTEREST
Authors declare no conflict of interest.
Aziz, Z. A. A., Nasir, H. M., Ahmad, A., Setapar, S. H. M., Ahmad, H.,
Noor, M. H. M., Rafatullah, M., Khatoon, A., Kausar, M. A., Ahmad, I.,
Khan, S., al-Shaeri, M., & Ashraf, G. M. (2019). Enrichment of eucalyp-
tus oil nanoemulsion by micellar nanotechnology: Transdermal analge-
sic activity using hot plate test in rats' assay. Scientific Reports, 9(1),
13678. https://doi.org/10.1038/s41598-019-50134-y.
DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are included within
the article.
Badri, W., Miladi, K., Nazari, Q. A., Greige-Gerges, H., Fessi, H., &
Elaissari, A. (2016). Encapsulation of NSAIDs for inflammation man-
agement: Overview, progress, challenges and prospects. International
Journal of Pharmaceutics, 515(1–2), 757–773.
ORCID
Mohyeddin Assali
https://orcid.org/0000-0002-2286-9343
Baek, J.-S., & Cho, C.-W. (2015). Controlled release and reversal of
multidrug resistance by co-encapsulation of paclitaxel and verapamil in
solid lipid nanoparticles. International Journal of Pharmaceutics, 478(2),
617–624.
REFERENCES
Akaike, H. (1974). A new look at the statistical model identification. IEEE
Transactions on Automatic Control, 19(6), 716–723.
Alsaidan, S. M., Alsughayer, A. A., & Eshra, A. G. (1998). Improved dissolu-
tion rate of indomethacin by adsorbents. Drug Development and Indus-
trial Pharmacy, 24(4), 389–394.
Baek, J.-s., Yeo, E. W., Lee, Y. H., Tan, N. S., & Loo, S. C. J. (2017). Con-
trolled-release nanoencapsulating microcapsules to combat inflamma-
tory diseases. Drug Design, Development and Therapy, 11, 1707–1717.

ASSALI AND ZOHUD
9
Berlin, R. G., Clineschmidt, B. V., & Majka, J. A. (1986). Famotidine: An
appraisal of its mode of action and safety. The American Journal of
Medicine, 81(4), 8–12.
use of nonpharmacologic and pharmacologic therapies in osteoarthritis
of the hand, hip, and knee. Arthritis Care & Research, 64(4), 465–474.
Ignacio, M., Chubynsky, M. V., & Slater, G. W. (2017). Interpreting the
Weibull fitting parameters for diffusion-controlled release data.
Physica A: Statistical Mechanics and its Applications, 486, 486–496.
Jaiswal, M., Dudhe, R., & Sharma, P. (2015). Nanoemulsion: An advanced
mode of drug delivery system. 3 Biotech, 5(2), 123–127.
Koizumi, T., Ritthidej, G. C., & Phaechamud, T. (2001). Mechanistic model-
ing of drug release from chitosan coated tablets. Journal of Controlled
Release, 70(3), 277–284.
Bhattacharjee, S. (2016). DLS and zeta potential–what they are and what
they are not? Journal of Controlled Release, 235, 337–351.
Chang, Y., & McClements, D. J. (2014). Optimization of orange oil nano-
emulsion formation by isothermal low-energy methods: Influence of
the oil phase, surfactant, and temperature. Journal of Agricultural and
Food Chemistry, 62(10), 2306–2312. https://doi.org/10.1021/
jf500160y.
Chang, Y., McLandsborough, L., & McClements, D. J. (2013). Physicochem-
ical properties and antimicrobial efficacy of carvacrol nanoemulsions
formed by spontaneous emulsification. Journal of Agricultural and Food
Chemistry, 61(37), 8906–8913. https://doi.org/10.1021/jf402147p.
Clarysse, S., Psachoulias, D., Brouwers, J., Tack, J., Annaert, P.,
Koroleva, M., Nagovitsina, T., & Yurtov, E. (2018). Nanoemulsions stabi-
lized by non-ionic surfactants: Stability and degradation mechanisms.
Physical Chemistry Chemical Physics, 20(15), 10369–10377. https://
doi.org/10.1039/c7cp07626f.
Kosmidis, K., Argyrakis, P., & Macheras, P. (2003). A reappraisal of drug
release laws using Monte Carlo simulations: The prevalence of the
Weibull function. Pharmaceutical Research, 20(7), 988–995.
Kuznetsova, D. A., Gabdrakhmanov, D. R., Kuznetsov, D. M.,
Lukashenko, S. S., Sapunova, A. S., Voloshina, A. D., Nizameev, I. R.,
Kadirov, M. K., & Zakharova, L. Y. (2020). Biocompatible supramolecu-
lar systems based on novel cationic imidazolium- and urethane-
containing amphiphiles: Self-assembly and antimicrobial properties.
Journal of Molecular Liquids, 319, 114094. https://doi.org/10.1016/j.
molliq.2020.114094.
Duchateau, G., Reppas, C.,
& Augustijns, P. (2009). Postprandial
changes in solubilizing capacity of human intestinal fluids for BCS class
II drugs. Pharmaceutical Research, 26(6), 1456–1466.
Coradini, K., Friedrich, R. B., Fonseca, F. N., Vencato, M. S., Andrade, D. F.,
Oliveira, C. M., Battistel, A. P., Guterres, S. S., da Rocha, M. I. U. M.,
Pohlmann, A. R., & Beck, R. C. R. (2015). A novel approach to arthritis
treatment based on resveratrol and curcumin co-encapsulated in lipid-
core nanocapsules: in vivo studies. European Journal of Pharmaceutical
Sciences, 78, 163–170.
Costa, F., Sousa, J., Pais, A., & Formosinho, S. (2003). Comparison of disso-
lution profiles of ibuprofen pellets. Journal of Controlled Release, 89(2),
199–212.
Laroux, F. S. (2004). Mechanisms of inflammation: The good, the bad and
the ugly. Frontiers in Bioscience, 9, 3156–3162.
Li, P.-H., & Chiang, B.-H. (2012). Process optimization and stability of d-
limonene-in-water nanoemulsions prepared by ultrasonic emulsifica-
tion using response surface methodology. Ultrasonics Sonochemistry,
19(1), 192–197. https://doi.org/10.1016/j.ultsonch.2011.05.017.
Lucas, S. (2016). The pharmacology of indomethacin. Headache: The Jour-
nal of Head and Face Pain, 56(2), 436–446.
Costa, P., & Lobo, J. M. S. (2001). Modeling and comparison of dissolu-
tion profiles. European Journal of Pharmaceutical Sciences, 13(2),
123–133.
de Souza, M. C., Vieira, A. J., Beserra, F. P., Pellizzon, C. H.,
Nóbrega, R. H., & Rozza, A. L. (2019). Gastroprotective effect of limo-
nene in rats: Influence on oxidative stress, inflammation and gene
expression. Phytomedicine, 53, 37–42. https://doi.org/10.1016/j.
phymed.2018.09.027.
Mayer, B. X., Mensik, C., Krishnaswami, S., Derendorf, H., Eichler, H. G.,
Schmetterer, L.,
&
Wolzt, M. (1999). Pharmacokinetic-
pharmacodynamic profile of systemic nitric oxide-synthase inhibition
with L-NMMA in humans. British Journal of Clinical Pharmacology, 47
(5), 539–544.
Freeling, J. P., Koehn, J., Shu, C., Sun, J., & Ho, R. J. (2014). Long-acting
three-drug combination anti-HIV nanoparticles enhance drug expo-
sure in primate plasma and cells within lymph nodes and blood. AIDS
(London, England), 28(17), 2625–2627.
Fritsche, E., Baek, S. J., King, L. M., Zeldin, D. C., Eling, T. E., & Bell, D. A.
(2001). Functional characterization of cyclooxygenase-2 polymor-
phisms. Journal of Pharmacology and Experimental Therapeutics, 299(2),
468–476.
Gadde, S. (2015). Multi-drug delivery nanocarriers for combination ther-
apy. Medicinal Chemistry Communications, 6(11), 1916–1929.
Giacomelli, C., Schmidt, V., & Borsali, R. (2007). Nanocontainers formed by
self-assembly of poly(ethylene oxide)-b-poly(glycerol mono-
methacrylate)−drug conjugates. Macromolecules, 40(6), 2148–2157.
https://doi.org/10.1021/ma062562u.
Mohsin, K., Long, M. A., & Pouton, C. W. (2009). Design of lipid-based for-
mulations for oral administration of poorly water-soluble drugs: Pre-
cipitation of drug after dispersion of formulations in aqueous solution.
Journal of Pharmaceutical Sciences, 98(10), 3582–3595.
Moraes, T. M., Rozza, A. L., Kushima, H., Pellizzon, C. H., Rocha, L. R. M., &
Hiruma-Lima, C. A. (2013). Healing actions of essential oils from Citrus
aurantium and d-limonene in the gastric mucosa: The roles of VEGF,
PCNA, and COX-2 in cell proliferation. Journal of Medicinal Food, 16
(12), 1162–1167. https://doi.org/10.1089/jmf.2012.0259.
Nair, L. S., & Laurencin, C. T. (2007). Biodegradable polymers as biomate-
rials. Progress in Polymer Science, 32(8–9), 762–798.
Naito, Y., Iinuma, S., Yagi, N., Boku, Y., Imamoto, E., Takagi, T., Handa, O.,
Kokura, S., & Yoshikawa, T. (2008). Prevention of indomethacin-
induced gastric mucosal injury in helicobacter pylori-negative healthy
volunteers: A comparison study rebamipide vs famotidine. Journal of
Clinical Biochemistry and Nutrition, 43(1), 34–40.
Guarino, V., Gentile, G., Sorrentino, L., & Ambrosio, L. (2017). Poly-
caprolactone: Synthesis, properties, and applications. Encyclopedia of
Polymer Science and Technology, 1–36.
Gursoy, R. N., & Benita, S. (2004). Self-emulsifying drug delivery systems
(SEDDS) for improved oral delivery of lipophilic drugs. Biomedicine &
Pharmacotherapy, 58(3), 173–182.
Nathan, C. (2002). Points of control in inflammation. Nature, 420(6917),
846–852.
Hassan, M. A., Salem, M. S., Sueliman, M. S., & Najib, N. M. (1997). Charac-
terization of famotidine polymorphic forms. International Journal of
Pharmaceutics, 149(2), 227–232.
Higuchi, K., Umegaki, E., Watanabe, T., Yoda, Y., Morita, E., Murano, M.,
Tokioka, S., & Arakawa, T. (2009). Present status and strategy of
NSAIDs-induced small bowel injury. Journal of Gastroenterology, 44(9),
879–888.
Hochberg, M. C., Altman, R. D., April, K. T., Benkhalti, M., Guyatt, G.,
McGowan, J., Towheed, T., Welch, V., Wells, G., & Tugwell, P. (2012).
American College of Rheumatology 2012 recommendations for the
Ong, C. K., Seymour, R. A., Lirk, P., & Merry, A. F. (2010). Combining para-
cetamol (acetaminophen) with nonsteroidal antiinflammatory drugs: A
qualitative systematic review of analgesic efficacy for acute postoper-
ative pain. Anesthesia & Analgesia, 110(4), 1170–1179.
Perez-Aisa, A., Sopena, F., Arceiz, E., Ortego, J., Sainz, R., & Lanas, A.
(2003). Effect of exogenous administration of transforming growth
factor-beta and famotidine on the healing of duodenal ulcer under the
impact of indomethacin. Digestive and Liver Disease, 35(6), 397–403.
Ricciotti, E., & FitzGerald, G. A. (2011). Prostaglandins and inflammation.
Arteriosclerosis, Thrombosis, and Vascular Biology, 31(5), 986–1000.

10
ASSALI AND ZOHUD
Saeedi, M., Akbari, J., Morteza-Semnani, K., Enayati-Fard, R., Sar-Reshteh-
dar, S., & Soleymani, A. (2011). Enhancement of dissolution rate of
indomethacin: Using liquisolid compacts. Iranian journal of pharmaceu-
tical research: IJPR, 10(1), 25–34.
Sostres, C., Gargallo, C. J., Arroyo, M. T., & Lanas, A. (2010). Adverse
effects of non-steroidal anti-inflammatory drugs (NSAIDs, aspirin and
coxibs) on upper gastrointestinal tract. Best Practice & Research Clinical
Gastroenterology, 24(2), 121–132.
Yeh, K. C. (1985). Pharmacokinetic overview of indomethacin and
sustained-release indomethacin. The American Journal of Medicine, 79
(4), 3–12.
Zhang, Y., Huo, M., Zhou, J., Zou, A., Li, W., Yao, C., & Xie, S. (2010).
DDSolver: An add-in program for modeling and comparison of drug
dissolution profiles. The AAPS Journal, 12(3), 263–271. http://dx.doi.
org/10.1208/s12248-010-9185-1.
Ziltener, J.-L., Leal, S.,
& Fournier, P.-E. (2010). Non-steroidal anti-
Talke, P. O.,
&
Solanki, D. R. (1993). Dose-response study of oral
inflammatory drugs for athletes: An update. Annals of Physical and
Rehabilitation Medicine, 53(4), 278–288.
famotidine for reduction of gastric acidity and volume in outpatients
and inpatients. Anesthesia and Analgesia, 77(6), 1143-1148.
Tan, J., Deng, Z., Liu, G., Hu, J.,
& Liu, S. (2018). Anti-inflammatory
polymersomes of redox-responsive polyprodrug amphiphiles with
inflammation-triggered indomethacin release characteristics. Biomaterials,
178, 608–619. https://doi.org/10.1016/j.biomaterials.2018.03.035.
Torchilin, V. P., & Trubetskoy, V. S. (1995). Which polymers can make
nanoparticulate drug carriers long-circulating? Advanced Drug Delivery
Reviews, 16(2–3), 141–155.
How to cite this article: Assali M, Zohud N. Design of
multicomponent indomethacin-paracetamol and famotidine
loaded nanoparticles for sustained and effective anti-
inflammatory therapy. Drug Dev Res. 2020;1–10. https://doi.
org/10.1002/ddr.21768
Woodruff, M. A., & Hutmacher, D. W. (2010). The return of a forgotten
polymer—Polycaprolactone in the 21st century. Progress in Polymer Sci-
ence, 35(10), 1217–1256.