Food Chemistry 293 (2019) 57–65

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

A novel niosome formulation for encapsulation of anthocyanins and T

modelling intestinal transport
Melike Fidan-Yardimcia, Seref Akayb, Fatemeh Sharific,d, Canan Sevimli-Gure, Gaye Ongena,
⁎
Ozlem Yesil-Celiktasa,c,
a
Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova, Izmir, Turkey
b
Department of Genetics & Bioengineering, Faculty of Engineering, Gumushane University, 29100 Gumushane, Turkey
c
Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge 02139, MA, USA
d
Mechanical Engineering Department, Faculty of Engineering, Sharif University of Technology, Tehran, Iran
e
Department of Biology, Biotechnology Discipline, Science and Art Faculty, Kocaeli University, 41380 Izmit, Kocaeli, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: The bioavailability of drugs can be improved by regulating the structural properties, particularly lipoid systems,
Niosome such as niosomes, can increase cellular uptake. Herein, we optimized double emulsion and niosomal formula-
Double emulsion tions for encapsulating anthocyanin-rich black carrot extract. Nanoparticles obtained by selected formulation
Anthocyanin were characterized in terms of morphology, particle size, drug encapsulation efficiency, in vitro release and
Neuro 2A
cytotoxicity. The optimum conditions for niosomal formulation were elicited as 30 mg of cholesterol, 150 mg of
In vitro release profile
Tween 20 and feeding time of 1 min at a stirring rate of 900 rpm yielding the lowest average particle size of
130 nm. In vitro release data showed the majority of the encapsulated anthocyanins were released at the end of
10 h. A mathematical model was developed to estimate the absorption of anthocyanins released from niosomes
and cytotoxicity was assessed against neuroblastoma. Overall, these findings suggest that niosomal vesicles
might be suitable delivery systems for anthocyanins.

1. Introduction
Advancements in material and pharmaceutical sciences have led to
the design of novel drug delivery systems overcoming the drawbacks of
conventional dosage forms by increasing bioavailability, thereby re-
ducing side effects for the ultimate aim of increased patient compliance.
Adsorption and release of drugs mainly depend on the structural
properties of the host-matrix (Zhang et al., 2017) and can be regulated
to maximize cellular uptake. Vesicular systems, such as niosomes
(Basiri, Rajabzadeh, & Bostan, 2017), liposomes (Rasoulianboroujeni
et al., 2017), transferosomes (Marín, Alemán, Sánchez-Faure, Montero,
& Gómez-Guillén, 2018) and pharmacosomes (Wu, Jiang, & Meng,
2015), have attracted considerable interest. Niosomes produced by non-
ionic surfactants have various advantages such as high biocompat-
ibility, low toxicity, immune system activation and suitability for tar-
geted drug delivery components (Ojeda et al., 2016). A large number of
non-ionic surfactants are reported to form vesicles by self-assembly of
the amphiphiles in aqueous media, which results in formation of closed
bilayer structures where hydrophobic drugs can be encapsulated within
the bilayers, whereas hydrophilic counterparts are in the aqueous
spaces of the vesicle (Imran et al., 2016). Niosomes are also reported to
inhibit P-glycoprotein, therefore increasing bioavailability of some an-
ticancer drugs (Mahale, Thakkar, Mali, Walunj, & Chaudhari, 2012).
Anthocyanins are one of the most notable compounds among flavo-
noids, possessing a wide range of biological activities, including, but
not limited to, antioxidant (Abdel-Aal, Hucl, & Rabalski, 2018), anti-
inflammatory (Wu, Yin, Zhang, Long, & Zheng, 2016), anticancer
(Sevimli-Gur, Cetin, Akay, Gulce-Iz, & Yesil-Celiktas, 2013) and anti-
diabetic activities (Ghosh & Konishi, 2007). Among these, the effects of
anthocyanins particularly on cancer, diabetes and cardiovascular dis-
eases are encouraging (Wallace, Slavin, & Frankenfeld, 2016). Although
profound therapeutic benefits exist, anthocyanins have not been widely
used as health-promoting agents due to their instability during storage
and processing (Guan & Zhong, 2015). Because of the sensitivity against
heat, light, metal ions, pH, glucose and ascorbic acid present in the
environment (Mazza & Brouillard, 1990), stabilization is an essential
step for the therapeutic use. However, it is not possible to keep an-
thocyanins stabilized, once isolated from plant cells, without adapting
protective measures (Frank, Reichardt, Shu, & Engel, 2012). In addi-
tion, bioavailability of these compounds are very low due to low

⁎
Corresponding author at: Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornoval, Izmir, Turkey.
E-mail address: ozlem.yesil.celiktas@ege.edu.tr (O. Yesil-Celiktas).

https://doi.org/10.1016/j.foodchem.2019.04.086
Received 2 November 2018; Received in revised form 31 March 2019; Accepted 24 April 2019
Available online 25 April 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
M. Fidan-Yardimci, et al.
absorption and fast metabolization in the body (McGhie & Walton,
2007). Hence, the best strategy is to adopt an encapsulation technique
to overcome these obstacles while maximizing cellular uptake. There-
fore, we hypothesized that if lipoidal carriers are formulated and op-
timized for encapsulation of anthocyanins yielding particles at nano
sizes, then a comparatively fast release can be achieved in the in-
testines. In this study, an anthocyanin-rich black carrot extract (BCE)
was encapsulated with double emulsion solvent evaporation and nio-
some methods. Selected encapsulation formulation was characterized in
terms of particle size distribution, morphology, thermal analysis (DSC),
encapsulation efficiency, in vitro release studies and cytotoxic activities.
Additionally, intestinal transport is modelled to estimate absorption.
2. Materials and methods
2.1. Materials
Black carrots (Daucus carota L. spp.) obtained from the local market
(Izmir, Turkey) were washed in water, crushed and then dried at 30 °C.
Dried parts were homogenized in a blender and stored at 4 °C.
Anthocyanins from black carrots were obtained by successive extraction
where dried samples weighing 1 g were extracted ultrasonically
(Everest Cleanex-401) with 10 ml water for 60 min at 50 °C (Yesil-
Celiktas, Otto, Gruener, & Parlar, 2009). The extraction was performed
until a colorless extract was obtained, all extract phases were combined,
subsequently filtered, lyophilized and stored at −20 °C before use.
Polycaprolactone (PCL) was purchased from Sigma (USA), whereas
Tween 20 was from Merck (Darmstadt, Germany). The HPLC-grade
dichloromethane and diethyl ether were purchased from Merck
(Darmstadt, Germany). All other chemicals were of analytical grade
purity. Cyanidin-3-glucoside (Assay (HPLC) 98%) was from Phytolab
(Vestenbergsgreuth, Germany).
2.2. Preparation of particles by double emulsion method
Anthocyanin rich extract loaded particles were produced by double
emulsion (w/o/w) solvent evaporation method, using dichloromethane
as an organic solvent (oily phase) and polycaprolactone PCL as a
coating material. In brief, anthocyanin rich extract in defined amount
was mixed with distilled water and was emulsified by using homo-
genizer for 1 min with 7 ml of dichloromethane containing PCL. The
first emulsion (w/o) was administered dropwise into 20 ml of distilled
water containing surfactant (NaCMC, Tween 20 or PVA) in external
aqueous phase under stirring with a homogenizer to produce a double
w/o/w emulsion. This emulsion was stirred mechanically at room
temperature until organic solvent completely evaporated.
2.3. Preparation of particles by niosome method
Niosomes were prepared by ether injection method. In this method,
cholesterol and non-ionic surfactant (Tween 20) were weighed accu-
rately and dissolved in 10 ml of diethyl ether. The resulting solution
was taken by a syringe and injected slowly through a needle into 5 ml of
hydrating solution phosphate buffer (pH 7.4) containing anthocyanin
rich extract in the aqueous phase and the solution was stirred on
magnetic stirrer by maintaining the temperature at 60 °C. As the lipid
solution was injected slowly into the aqueous phase, vaporization of
diethyl ether resulted in the formation of niosomes.
2.4. Characterization
2.4.1. Particle size analysis
The mean particle size and polydispersity index of the anthocyanin
loaded particles were evaluated using Malvern Zetasizer Nano-ZS
(Malvern Inst. Ltd., UK). All experiments were done in triplicate.
58
Food Chemistry 293 (2019) 57–65
2.4.2. Encapsulation efficiency
Encapsulation efficiency was determined by an ultracentrifugation
method. The freshly prepared nanoparticles were centrifuged at
20,000 rpm for 30 min, supernatant was separated from the pellet and
analyzed by a pH differentiation method for anthocyanin content. The
latter, when subtracted from the total anthocyanin amount, gave the
entrapped anthocyanin. Encapsulation efficiency was expressed as the
ratio of actual and theoretical anthocyanin loaded. The analyses were
carried out in triplicate.
2.4.3. Differential scanning calorimeters (DSC) analysis
Thermal properties of the particles and anthocyanin extract were
studied by differential scanning calorimetry DSC (Perkin Elmer, model
DSC 8000). Approximately 5 mg of samples were compressed and
loaded onto standard aluminium pans. The samples were purged with
pure dry nitrogen at a flow rate of 50 ml/min. The analysis was carried
out at a temperature heating rate of 10 °C/min. and a temperature
range of 20–200 °C.
2.4.4. Scanning electron microscopy (SEM) analysis
Scanning electron microscopy was used to investigate the mor-
phology of anthocyanin loaded particles. SEM photographs were taken
using a scanning electron microscope, FEI Quanta 250 FEG, at the re-
quired magnification at room temperature. Photographs were taken at
suitable magnification.
2.5. In vitro release studies
The dialysis membrane method was used to determine the amount
of the drug molecules diffused from the encapsulated particles. Release
of anthocyanin from the nanoparticles was determined through a cel-
lulose acetate membrane. The nanoparticle preparation was taken in a
dialysis membrane (cut-off molecular weight: 12000–14000), which
performed as a donor compartment. Dialysis membrane containing
appropriate volume of nanoparticle dispersion was placed in a beaker
containing 50 ml of phosphate buffer saline of pH 7.4, which worked as
a receptor compartment. The beaker was placed on a shaker, and mixed
at 37 ± 0.5 °C and 50 rpm. Aliquots of dialysate were taken at pre-
determined times and replenished immediately with the same volume
of fresh phosphate buffer. The withdrawn samples were analyzed by U-
HPLC to determine the release of anthocyanin content. The fraction of
anthocyanin release at specific time points was determined by com-
paring the released anthocyanin content with the total content of an-
thocyanin entrapped in particles.
2.6. U-HPLC analysis
Chromatographic separations were performed on an Ace C-18
150 mm × 4.0 mm × 5 μm column (Advanced Chromatography
Technologies Ltd, Aberdeen, Scotland) using an Accela U-HPLC system
equipped with PDA detector, auto sampler, quaternary pump and
column oven (Thermo Fisher Scientific Inc., Massachusetts, USA). A
mobile phase consisting of 0.01% TFA in water (solvent A) and acet-
onitrile (solvent B) was used. Conditions were as follows: 0–4 min, 15%
B; 7 min, 19% B; 18 min, 24% B; 20–22 min, 95% B; followed by a 3 min
equilibration period. The flow rate was 500 µl/min and peaks were
detected at 520 nm. A linear fit equation of the calibration curve was
obtained as y = 138348x–967,705 for cyanidin-3-glucoside (x: con-
centration (µg/ml), y: peak area). The correlation coefficient (R2) was
determined as 0.9998. The results are expressed as cyanidin-3-glucoside
equivalent.
2.7. Modelling intestinal transport to estimate absorption
In the transport model, the initial concentration of the anthocyanin
inside the lumen is considered to be equivalent to the release profile,
M. Fidan-Yardimci, et al. Food Chemistry 293 (2019) 57–65

which shows the concentration of the anthocyanin versus time inside convection diffusion equation. The Galerkin method was used to ap-
the lumen of the intestine. Part of the anthocyanin inside the small proximate the nonlinear partial differential governing equations with a
intestine is absorbed or metabolized either by the intestine or by the system of ordinary differential equations.
mircobiota. Considering aforementioned factors, the rate equation for
anthocyanin inside the small intestine can be obtained via 2.8. Determination of cytotoxicity
Clumen = (CF − Cs ) − CL − intestine (1)
2.8.1. Cancer cell lines
C
⎡ ⎛ Vmax P I The cancer cell lines MCF-7 and MDA-MB–231 (human, breast,
dCi C ⎞
= K × Clumen + Q × ⎛CB − I ⎞ − ⎜ ⎟ ⎢⎜ i:B
⎟⎟ adenocarcinoma), Saos-2 (human, osteosarcoma), A-549 (human, al-
dt ⎝ Pi:B ⎠ ⎢ ⎜ Km + CI veolar adenocarcinoma), Neuro 2A (Mus musculus, neuroblastoma) and
⎝ Pi : B ⎠metab
⎣
VERO (African green monkey kidney) normal cell line were obtained
CI
⎛ Vmax Pi : B ⎞ ⎤ from the American Cell Culture Collection (ATCC Mannassas, VA),
+ ⎜⎜ ⎟⎟ ⎥
C
Km + P I ⎥ whereas NA2A (human, neuroblastoma) was from HUKUK (Foot &
⎝ i:B ⎠microb⎦ (2) Mouth Disease Institute, Cell Bank, Turkey). The cells were cultured in
RPMI 1640 or DMEM-Ham’s F12 supplemented with 10% fetal bovine
C
CI = ⎛ i ⎞
⎜ ⎟ serum, L-glutamine, (2 mmol/l), penicillin (100 U/ml), streptomycin
⎝ Vi ⎠ (3) (100 µG/ml) and were maintained at 37 °C in a humidified atmosphere
where Clumen, Cs and CL-intestine are anthocyanin concentration in lumen, with 5% CO2.
stomach and large intestine, respectively. CF is the concentration of
anthocyanin from ingested food, Ci is the concentration of anthocyanin 2.8.2. Cytotoxic activity of niosomes
in intestine, CB is the concentration in blood, K is the absorption rate The cytotoxic activities of niosomes on various cancer cells were
constant, Km Michaelis-constant, Pi:B is partition coefficient of antho- measured by MTT assay. Cells in exponential growth phase were placed
cyanin between intestine and blood, Q is the blood flow rate in intestine in 96-wells plates to make 6000 cells/wells and sample solutions were
and Vi is the volume of intestine. Since anthocyanin is encapsulated added at concentrations ranging from 100 to 6.25 µg/ml. Subsequently,
inside the particles, absorption in mouth and stomach are neglected. the cells were incubated for 48 h. Negative control was treated with
Also, absorbed concentration of anthocyanin in the large intestine is 0.1% methanol. Cell proliferation was determined by adding 0.5 mg/ml
neglected for the sake of simplicity. Therefore, Eq. (1) can be rewriten per well, prepared as a sterile stock solution of 5 mg/ml in
as follows: Dulbecco’s–phosphate buffered saline (Gibco, USA), diluted 1:10 with
medium prior to use. Medium was removed 4 h later and blue formazan
Clumen = CF (4)
crystals were dissolved in 200 µl 100% dimethyl sulfoxate per well.
Governing unsteady partial differential equation, specifically con- Additionally, time-dependent cell viabilities of the extract, empty and
vection-diffusion equation along with ordinary differential equation loaded (6.25 µg/ml) noisome particles were quantitatively assessed by
were solved using a two-dimensional finite element method. The MTT assay at 24, 48, 72, 96 h post exposure to Neuro2A cells. The
overall distribution of ACN is obtained using unsteady mass transfer absorbance was measured at 570–690 nm by a microplate reader
equation, known as convection- diffusion equation: (Versamax, Tunable Microplate Reader, USA). The data were obtained
from three independent assays and cytotoxicity was determined ac-
∂Ci →
+ ∇ ·N = Ri cording to the percent cell viability.
∂t (5)

where N is the flux vector, 3. Results and discussion

→ →
N = −D ∇ Ci + Ci →
u (6)
denoting the diffusion and convection terms of the mass balance
equation. D is diffusion coefficient and u is velocity vector, whereas R is
composed of release and absorption terms of ACN. The schematic of the
radially oriented intestinal crypt and villus units is shown in Fig. S1. It
is assumed that the lumen surface of intestine is covered with crypt-
villus units. Appropriate initial and boundary conditions for ordinary
differential equation and convection diffusion were applied based on
the release profile of ACN and physiological condition of intestine. As
ACN is released in the intestine, the concentration of ACN inside the
luminal part is similar to the ACN released. Hence, the distribution of
ACN inside the lumen and crypt-villus unites is obtained by solving the
3.1. Preparation of particles by double emulsion method
Double emulsion studies were carried out by employing Taguchi
method to define the most influential process parameters for producing
particles with diameters of about 100 nm. The design involved seven
factors at two levels, which were feeding time, temperature, mixing
speed, PCL, anthocyanin and Tween 20 amounts (Table 1). The analysis
of variance (ANOVA) indicated that the fitted model represented the
experimental data well (R2 = 0.8534) and the model was statistically
significant (p < 0.05). Mixing speed, PCL amount and feeding time
were found to be significant (p < 0.05), whereas the other four factors
were not statistically significant (p > 0.05) for particle size (STable 1).

Table 1
Mean sizes of anthocyanin loaded particles prepared by double emulsion solvent evaporation method.
Run A: feeding time B: mixing time C: temp. D: mixing speed E: PCL amount F: anthocya nin conc. G: Tween 20 Response: mean size PDI
(sec) (min) (°C) (rpm) (mg) (µg/ml) conc. (%) (nm)

1 30 5 4 17,000 30 6 5 252.4 0.207

2 5 5 25 17,000 60 6 0.5 523.8 0.268
3 5 1.5 4 17,000 60 18 5 331.9 0.256
4 30 1.5 25 8000 60 6 5 772.1 0.686
5 30 1.5 25 17,000 30 18 0.5 420.3 0.405
6 5 1.5 4 8000 30 6 0.5 351.2 0.679
7 30 5 4 8000 60 18 0.5 1003 0.990
8 5 5 25 8000 30 18 5 462.1 0.683

59
M. Fidan-Yardimci, et al. Food Chemistry 293 (2019) 57–65

Particle size decreased as the mixing speed increased in the double Table 3
emulsion solution, which is in agreement with the existing studies re- Mean size of anthocyanin loaded niosomes.
porting better mixing of the emulsion and uniform particle size dis- Run A: cholesterol B: Tween 20 C: feeding Response: mean
tributions (Mukherjee, Ray, & Thakur, 2009; Rosca, Watari, & Uo, amount (mg) amount (mg) time (min) size (nm)
2004). When the volume of one of the emulsion phases were kept as low
1 30 400 3 162.3
as 20 ml, increasing the amount of PCL resulted in an increase in the
2 40 300 2 260.9
particle size, which was also in agreement with the literature (Hussein, 3 50 200 3 143.3
Fakhru’l-Razi, & Abdullah, 2013). Another drawback of high PCL 4 50 200 1 210.8
amount was the aggregating manner of the particles during evaporation 5 40 300 0.3 201.4
of DCM from the emulsion, which could potentially be the reason of 6 30 400 1 187
7 40 300 3.7 233.6
larger particle sizes.
8 50 400 3 295.1
Feeding time was also a critical process parameter, which involved 9 23.2 300 2 166.3
the addition rate of the second phase to the first emulsion under stir- 10 40 300 2 333.5
ring. After formation of the initial phase, the first emulsion was slowly 11 40 468.2 2 154.4
12 40 300 2 245.4
added to the second phase. When the feeding time was prolonged, the
13 40 131.8 2 130.4
stability of the emulsion was observed to be altered, which resulted in 14 50 400 1 216.1
particles combining with each other and forming larger particles. 15 56.8 300 2 277.6
Therefore, the homogeneous first emulsion obtained with a high stir- 16 30 200 1 130
ring speed was injected as rapid as 5 s into the second phase. The 17 30 200 3 163.9
polydispersity index (PDI) values of double emulsion PCL particles were
in the range of 0.207–0.990, which were comparatively high indicating
surface was probably due to Tween 20 providing a large hydrophilic
poor uniformity in particle size distribution (Natarajan, Krithica,
head group (Waddad et al., 2013). Near to neutral or slightly low zeta
Madhan, & Sehgal, 2011) which was obviously undesirable. Further-
potentials were expected as a nonionic surfactant was used, which al-
more, the particles were not stable after 24 h at 4 °C based on ob-
lowed the particles to suspend for a long time without aggregating and
servations of aggregation and adhering to each other. As the en-
interfering with each other. Indeed, niosomal dispersion with zeta po-
countered problems might have affected encapsulation efficiency and
tential of ± 30 mV is reported to have long term stability (Müller,
release kinetics, another technique was investigated overcome these
Jacobs, & Kayser, 2001).
problems.
The next step was to ascertain the optimum values of feeding time,
cholesterol and Tween 20 amounts elicited as significant by Taguchi
3.2. Preparation of particles by niosome method and optimization method. For this purpose, a Central Composite Design (CCD) was ap-
plied (Table 3) where the fitted model represented the experimental
Similarly, Taguchi design was used to determine the most sig- data well (R2 = 0.7698) and the model was statistically significant
nificant process parameters for noisome formation among seven dif- (p < 0.05). Cholesterol, Tween 20 amounts and interactions of these
ferent parameters affecting particle size, which were cholesterol, Tween three parameters were found to be significant, whereas feeding time,
20, anthocyanin amounts, feeding time, ultrasonication, ultrasonic alone was statistically not significant (STable 3). Decreasing the
probe and temperature (Table 2). Apart from these, various stirring amounts of cholesterol and the surfactant resulted in lower size and PDI
rates between 900 and 13500 rpm were tested to elicit the condition values where the minimum particle size was obtained at ranges of
yielding the minimum particle size. As stirring rate increased, the 200–250 mg and 30–35 mg, respectively (Fig. 1a). Cholesterol is a
particle size increased as well, probably due to rapid evaporation of crucial component affecting many properties of lipid bilayers, playing
ether, resulting in aggregation of cholesterol and Tween 20. Conse- an important role in particle formation, affecting the stability and
quently, a stirring rate of 900 rpm or even lower rates would favour permeability of vesicular structures (Fathalla, Abdel-Mageed, Abdel-
formation of smaller sized particles. The fitted model from the analysis Hamid, & Ahmed, 2014). Generally, lower PDI and lower mean sizes of
of variance represented the experimental data well (R2 = 0.9608) and particles were obtained by using low amounts of cholesterol (Ravaghi,
the model was statistically significant (p < 0.05). Among the seven Razavi, Mousavi, Sinico, & Fadda, 2016), which is in agreement with
process parameters, feeding time, cholesterol and Tween 20 amounts the obtained results. Cell membrane like structures tend to grow when
were statistically significant (p < 0.05) (STable 2). high amounts of surfactant and stabilizing agents are added to the
The sizes of the niosome particles were between 100 and 200 nm formulation, leading to increased particle sizes (Kumar & Rajeshwarrao,
while the PDI values were in the range of 0.2–0.3, much lower than the 2011). Apart from the stability of the vesicular structures, the stability
values obtained for double emulsion PCL particles indicating that of anthocyanins are of prime importance as well. The non-ionic sur-
homogeneous size distributions were achieved. Sample 5 had the best factant, Tween 20 is reported to provide a stabilization effect on an-
size distribution with an average particle size of 145.7 nm and a zeta thocyanins (MohdMaidin, Oruna-Concha, & Jauregi, 2019). In this
potential value of −3.31 mV. The negative charge of the niosome study, the volume of water phase was kept constant in all experiments,

Table 2
Mean sizes of anthocyanin loaded niosomes.
Run A: Cholesterol amount B: Tween 20 C: anthocyanin conc. D: feeding time E: ultrasonic bath F: ultrasonic G: Temp Response: mean PDI
(mg) amount (mg) (µg/ml) (min) (min) probe (°C) size (nm)

1 50 200 18 3 30 − 40 143.4 0.370

2 50 400 6 3 30 + 60 174 0.277
3 50 200 18 1 45 + 60 151.1 0.196
4 30 200 6 1 30 + 40 164.8 0.137
5 30 200 6 3 45 − 60 145.7 0.195
6 30 400 18 1 30 − 60 216.6 0.280
7 30 400 18 3 45 + 40 191.3 0.204
8 50 400 6 1 45 − 40 178.6 0.131

60
M. Fidan-Yardimci, et al.
Fig. 1. Three dimensional and contour response surface plots of particle size showing the effects of cholesterol and Tween 20 amount (a); the effect of feeding time
and cholesterol amount at constant Tween 20 amount (305 mg) (b).
so that formation of large vesicles with high amounts of Tween 20 and
cholesterol are associated with these parameters. Although feeding time
alone has not influenced the size, the minimum particle size was ob-
tained at 1 min (Fig. 1b). Based on the computed experimental results,
the optimum conditions were elicited as 30 mg of cholesterol, 150 mg of
Tween 20 and feeding time of 1 min at a stirring rate of 900 rpm,
yielding the lowest average particle size of 130 nm, which was ex-
perimentally validated as well. The surface topologies of empty and
loaded niosomal formulations were observed by SEM at 5000× mag-
nification (Fig. 2a&b). The micrographs provided crystal structure and
orientation. Some unevenness observed on the surfaces of the vesicles
may be due to drying under room temperature conditions. DSC studies
were performed to understand the nature and the physical state of the
extract and niosomes (Fig. 2c). The melting points for empty and loaded
niosomes were 130.03 °C and 172.36 °C, whereas the glass transition
temperatures were 153.62 °C and 151.04 °C, respectively. Two en-
dothermic peaks at 42 °C and 130 °C were exclusively observed in
thermograms of empty and loaded niosomes. The common peak at
61
Food Chemistry 293 (2019) 57–65
130 °C is associated with the cholesterol referring to the DSC thermo-
gram of cholesterol reported in a study focusing on paclitaxel loaded
niosomes (Sezgin-Bayindir & Yuksel, 2012), whereas the other peak is
probably related to Tween 20. Different endothermic peaks observed
for the extract and loaded niosomes indicated that the encapsulated
system was represented by a physical mixture of both compounds and a
chemical interaction has not occurred between the extract and the
niosomal system. An encapsulation efficiency of 40% was achieved
under the optimized conditions for niosomal formulation. Entrapment
efficiency was reported to decrease with increased cholesterol amounts
(Abdul Hasan Sathali & Rajalakshmi, 2010), which might compete with
the drug for packing space within the bilayer and limit the assembly of
the drug into the vesicle (Lingan, Sathali, Kumar, & Gokila, 2011).
Therefore, cholesterol amount was kept constant at 30 mg and addi-
tional experiments were planned to investigate the effect of surfactant
on entrapment efficiency. Lower and higher amounts of 100 mg and
200 mg of Tween 20 were tested where much lower efficiencies of 13
and 10% were noted, respectively. It is worth mentioning that not only
M. Fidan-Yardimci, et al.
Fig. 2. Scanning electron micrographs of anthocyanin loaded niosome particles (a); empty niosome particles (b) reported at 5000× magnification; DSC thermo-
graphs of anthocyanin loaded niosomes (LN), empty niosomes (EN) and black carrot extract (BCE) (c).
cholesterol and surfactant amounts play important roles in entrapment
efficiency, but also the hydrophilic or hydrophobic natures of the drug
molecules pose significant influences as well. In this study, the hydro-
philic nature of anthocyanins might have caused a comparatively low
encapsulation efficiency. Hydrophilic compounds encapsulated by
ether injection method are reported to exhibit lower loading efficiencies
of around 30–40% (Aggarwal, Garg, & Kaur, 2004) in comparison to
hydrophobic counterparts where 80–90% can be reached (Sundaresan,
Sravanthi, & Gowtham, 2012).
3.3. In vitro release profile and intestinal transport model of absorption
The release profile of anthocyanins from niosomal formulation was
investigated for a duration of 7 days (Fig. 3a). Subsequent to a burst
effect in the first half hour, which can generate the initial dose, ap-
proximately 90% of the anthocyanins were released at the end of 10 h.
62
Food Chemistry 293 (2019) 57–65
The remaining anthocyanins were totally released at the end of 5 days
and degradation was observed during the last 2 days. It is suggested to
change the amounts of cholesterol and surfactant in order to manip-
ulate the release rate of the drug molecule. For instance, increased
amount of cholesterol is reported to enhance the rigidity of the mem-
brane, thereby decreasing the efflux of the drug molecules from the
niosomal vesicles (Varshosaz, Pardakhty, Hajhashemi, & Najafabadi,
2003).
In addition to the in vitro release profile, a mathematical model was
developed to show the absorption of anthocyanins released from nio-
some particles. When anthocyanins are administered to the mouth and
passed through the gastrointestinal tract, absorption will occur in the
stomach and small intestine (Peng, Yang, Whitaker, Shangguan, &
Fang, 2016). However, very few drugs are absorbed through the sto-
mach due to the acidic environment (pH 1.5–3.5) and the small intes-
tine (duodenum, jejunum, and ileum) acts as the major site for drug
M. Fidan-Yardimci, et al. Food Chemistry 293 (2019) 57–65

Fig. 3. Release profiles of anthocyanin loaded niosome particles in pH 7.4 phosphate buffer at 37 °C. The results are the mean of three experiments ± S.D (a);
modelling of anthocyanin absorption inside the intestine villus, a schematic of the villus inside the small intestine (b), grid distribution inside the villus (c), ACN
distribution inside the villus (d), comparison of the simulated results with experimental data obtained by Steinert et al. (Steinert et al., 2008) (e); time-dependent
cytotoxicity of anthocyanin loaded niosome particles. [NC cell + medium (without nanoparticle), NC cell + medium (with nanoparticle), MN (anthocyanin loaded
niosome)] (f).

absorption (Shekhawat & Pokharkar, 2017). Furthermore, phospholi-
pids and lipoidal surfactants are stated not to be hydrolyzed by gastric
lipases, suggesting that vesicular systems, such as niosomes, might be
protected in the stomach (Tavano, Muzzalupo, Picci, & Cindio, 2014).
Therefore, equation for modelling small intestine is taken into
consideration, whereas absorption in the stomach is neglected when
formulating the transport model. The small intestine is about 3–5 m
long, which consists of many intestinal villi increasing the surface area
for absorption by diffusion, where nutrients are absorbed via blood
vessels connected to the villi (Celli, Ghanem, & Brooks, 2017).

63
M. Fidan-Yardimci, et al.
Parameters used in this simulation are given in STable 4. The inner
layer of the small intestine is covered with many villi. The geometry
used in this model is chosen based on intestinal units present on the
inner layer of small intestine (Fig. 3b). The assumed crypt length is
150 µm with inner diameter between 25 and 50 µm and the average
length of the villus length is considered as 350 µm (Jin, Thiagarajah, &
Verkman, 2013). The computational grid used in this simulation con-
sists of 4586 triangular domain elements (Fig. 3c) as the mathematical
model can provide cost effectiveness and mesh-independence respect to
concentration profiles with this number of elements. The concentration
distribution from high to low depicts the absorption profile of the an-
thocyanin inside the villus (Fig. 3d). In order to validate the proposed
model, the computed results are plotted against experimental in vitro
data of anthocyanin absorption amounts (Steinert, Ditscheid, Netzel, &
Jahreis, 2008). The computed results are in good agreement with the
experimental in vitro data (Fig. 3e) indicating the validity of the de-
veloped model. Considering the lipid nature of niosomes, the bioa-
vailability of drugs after oral administration is enhanced via a me-
chanism similar to the traditional lipid based carriers. Surfactants
increase the permeability of the intestinal membrane in a concentra-
tion-dependent manner, but not all non-ionic surfactants enhance the
absorption of the drug when the surfactants are present at concentra-
tions above their critical micelle concentration, just the contrary, toxic
effects are reported to be more pronounced above the critical micelle
concentrations. Studies with Caco-2 cell monolayers indicated non-
ionic surfactants increase permeability by the paracellular pathway,
primarily by widening tight junctions (Dimitrijevic, Lamandin,
Uchegbu, Shaw, Florence & 1997). In another study, the effects of
(+)-catechin and (−)-epigallocatechin gallate (EGCG) loaded nio-
somes on cellular uptake and transport across human intestinal Caco-2
cell monolayer were evaluated. The uptake studies revealed the in-
volvement of endocytosis in the transport process, where the inhibitor
of permeability glycoprotein and multidrug resistance-associated pro-
tein 2 were reported to increase the uptake amount of niosomes.
Compared with free catechin and EPCG, niosomal formulation sig-
nificantly enhanced drug absorption. Additionally, drug-loaded nio-
somes exhibited stronger stability and lower toxicity (Song et al., 2014).
Another hypothesis considers translymphatic absorption of intact nio-
somes, which can provide the additional advantage of bypassing the
presystemic disposition. Enhanced oral delivery of griseofulvin nio-
somes (Jadon, Gajbhiye, Jadon, Gajbhiye, Ganesh, 2009) and improved
intestinal absorption of furosemide niosomes (Sultan, El-Gizawy,
Osman, El Maghraby, 2016) were explained on the basis of translym-
phatic transport of niosomes.
3.4. Cytotoxic activities of anthocyanin loaded niosomes
The cytotoxic activity of anthocyanin loaded niosomes has been
investigated in MDA-MB-231, MCF7, Saos-2, A549, NA2A, Neuro 2A
and Vero cell lines with IC50 values of 237.45, 189.97, 147.74, 38.53,
24.52, 12.48 and 287.93 μg/ml, respectively. Neuro 2A, NA2A and
A549 cells turned out to be the most susceptible cell lines and no cy-
totoxicity was observed for the normal Vero cells. Further time de-
pendent cytotoxicity studies were carried out with anthocyanin loaded
noisomes against Neuro 2A as the most promising results were obtained
with that cell line. At the end of 24 h, cell viability was normalized as
100% for all groups NC [cell + medium (without nanoparticle)], NC
[cell + medium (with nanoparticle)], MN (anthocyanin loaded nio-
some). While cell viability was 74.3% at the end of 48 h for loaded
niosome, it was 237.4% for the negative control. Cell viabilities for
loaded niosomes were 27.9%, 23.5%, 15.2% and 14.5% at the end of
72 h, 96 h, 110 h and 134 h, respectively (Fig. 3f) while cells incubated
with empty noisome and without any nanoparticle proliferated and
reached 564.8% at 134 h. These results indicate that the cytotoxicity is
associated with the anthocyanins.
64
Food Chemistry 293 (2019) 57–65
4. Conclusion
Anthocyanin loaded niosomes were successfully fabricated. The
lowest particle size was 130 nm among various formulations tested. An
encapsulation efficiency of 40% was achieved under the optimized
conditions for niosomal formulation. Based on the in vitro release data,
approximately 90% of the anthocyanins were released at the end of
10 h and the remaining part was totally released at the end of 5 days. A
computational model was developed to estimate the absorption of an-
thocyanins released from niosomal formulation. As for cytotoxic effect,
viabilities of Neuro 2A cells incubated with anthocyanin loaded nio-
somes decreased significantly, whereas a proliferative trend was ob-
served when incubated with empty niosomes. This niosomal formula-
tion might be a suitable candidate to be utilized in biotechnological and
pharmaceutical applications.
Declaration of interests
None.
Acknowledgement
The research fund provided by the Scientific and Technical Research
Council of Turkey, TUBITAK (113M196) is highly appreciated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.04.086.
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