Liposome-in-alginate systems for encapsulation of natural

antioxidants
a

Isailovi Bojana , Djordjevi Verica , Nedovi Viktor and Bugarski Branko

a
b

Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, Serbia
Department of Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade, Nemanjina 6, Serbia

ABSTRACT
Natural antioxidants are usually very sensitive compounds and it is necessary to protect them from the
light and oxygen in order to preserve their stability and activity. Convenient way to achieve this is to
encapsulate antioxidants in proper matrix system. In this work resveratrol is used as a model antioxidant
substance. The aim of this study was to encapsulate resveratrol in a complex system liposome-in-alginate
in order to achieve its prolonged release, particularly in water based medium. Proliposome method was
applied to produce liposomes with encapsulated resveratrol. These liposomes were incorporated within
calcium alginate matrix of micro-sized beads (between 360-670 m) which were produced using
electrostatic extrusion technique. These systems provided high encapsulation efficiency of resveratrol (8395%). It was also shown that resveratrol release was extended (compared to release from simple systems
such as liposomes or hydrogels). The release started after approximately 30 minutes and lasted for about
5h. The liposome-in-alginate microbeads provided diffusion resistance between 1.99106 s/m and 2.80106
s/m, depending on alginate viscosity (low or medium) and alginate concentration (1% to 2.5% w/v).
Liposomeinalginate systems are shown to be convenient for encapsulation of resveratrol and have great
potential for protection and prolonged release of natural antioxidants.

Introduction

Nowadays natural antioxidants are desirable in different food products since they preserve flavour and colour of
the food and prevent vitamin destruction. It is also well known that antioxidants have strong impact on health;
hence, food with addition of natural antioxidants is often demanded by the market. Still, natural antioxidants are
very sensitive; therefore, there is a need for their protection. Encapsulation is proposed as one of convenient
method for that (Shi , Rao, & Yu, 2007). It was already explained that, both, liposomes and polymers can
protect antioxidants from the light and oxygen and preserve their stability and activity to some extent (Taylor,
Davidson, Bruce, & Weiss, 2005; Belak-Cvitanovi, et al., 2011). In order to achieve prolonged release of the
antioxidants, in this study we developed a complex systems based on liposomes incorporated within a polymer
matrix. Non-toxicity, biocompatibility, thermal and chemical stability (Stojanovic, et al., 2012) are the main
features, on account of which the alginate is selected for a polymer. Resveratrol (RSV) was used as a model
antioxidant substance. It is phytoalexin found in several plants and it is also well known compound of red wines
(Lu & Serrero, 1999). According to literature, proliposome method appeared to be a convenient way to produce
liposomes with encapsulated RSV (Isailovic, et al., 2013). The aim of this paper is to determine mass transfer
resistance provided by the membranes (liposomal and calcium alginate).

2
2.1

Material and Methods

Preparation of liposome with encapsulated RSV

Phospholipon 90G (P90G) (Natterman Phospholipids Germany) was the major component used in
liposome formulation and it is more than 90% phosphatidilcholine (PC). The trans-resveratrol standard (RSV,
>99% pure) was obtained from ChromaDex (Irvine, CA, USA) and it was used as a model antioxidant
substance. For preparation of liposome with encapsulated RSV proliposome method was used as Perrett and coworkers described (1991). In brief 2g P90G and 0,1g RSV was added to ethanol (96%) and stirred until the
P90G was dissolved. In the mixture heated to 60 0C for a few minutes after the addition of a small portion of
water. Then the mixture was cooled down to ambient temperature and 100 ml distilled water was added into
system as aqueous phase. The obtained suspension was stirred for 1 h at 800 rpm.
2.2

Preparation of liposome-in-alginate systems

InsideFood Symposium, 9-12 April 2013, Leuven, Belgium

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Low viscosity Na-alginate (LV) and medium viscosity Na-alginate (MV) were purchased from Sigma Aldrich (Germany) and were used for preparation of liposome-in-alginate systems. Low viscosity Na-alginate
was dissolved in distilled water in three different concentrations and after the addition of liposome suspensions,
the final alginate concentrations of the solutions were 1%, 1.5% and 2.5% w/v (LV 1%, LV 1.5% and LV
2.5%). Three more samples were prepared by dissolving medium viscosity Na-alginate in water and after
mixing with liposome suspensions, the final alginate concentrations were 1% and 1.5% w/v (MV 1% and MV
1.5%) (Strasdat & Bunjes, 2013; Dai, Wang, Zhao, Li, & Wang, 2006).The solutions were extruded through a
blunt stainless steel needle (23 G) at a constant flow rate of 25.2 mL h 1, by a syringe pump (Razel Scientific
Instruments, Stamford, CT, USA). The extrusion was done under an applied electric field between the positively
charged needle and grounded collecting solution (distance 3.0 cm). The potential difference was controlled by a
high voltage unit (Model 30R; Bertan Associates, Inc., New York, USA) and kept at a constant voltage of 6.3
kV. Collecting solution contained 50 mL of 2% w/v Ca-chloride. Solution droplets formed spherical insoluble
hydrogel liposome-in-alginate systems (microbeads), after ions exchange. The liposome-in-alginate systems
were left in the cross-linking solution for 30 min and then used for further analysis (Bugarski, et al., 1994).
2.3

Encapsulation efficiency

The sample of collecting solution after cross-linking was used for spctrophotometrical determination of
the amount of RSV in order to calculate encapsulation efficiency (EE%). The difference between the amount of
RSV detected in collecting solution and the amount of RSV used for preparation of liposome-in-alginate
systems is equivalent to the amount of encapsulated RSV. The EE is calculated as shown in equation 1:
EE%=(ms-mc)/ms 100

(1)

where ms is the amount of RSV used for preparation of liposome-in-alginate microbeads and mc is the amount
of RSV detected in collecting solution after cross linking.
2.4

Characterisation of liposome-in-alginate systems

Size of the liposome-in-alginate microbeads was observed using an optical microscope

Imager A1 (Carl Zeiss, Jena, Germany).
2.5

Axio

In vitro release studies and determination of mass transfer resistance of the systems

The release studies were performed using Jacketed Franz diffusion cell (donation of PermeGear, Inc.,
USA). This cell has two compartments which are separated with acetate-cellulose membrane (pore size of 0.45
m). The donor compartment is used for sample placing (~1.2 g of liposome-in-alginate microbeads). The
receptor compartment had a fixed volume of 20 ml. It was filled with medium (ethanol 50%) and mixed using
magnetic steering (Klimundova, Satinsky, Sklenarova , & Solich, 2006). The samples were taken in appropriate
time intervals during 6 hours. The concentration of RSV was detected using spectrophotometer at 306 nm.
The results obtained by release studies were benefit for determination of diffusion coefficients and
resistance to mass transfer of RSV from liposome-in-alginate microbeads. The diffusion coefficients were
calculated using Ficks second law (Pjanovi, et al., 2010):
0
0
CD CR
ln
D t
CD CR

(2)

where CD and CR are concentrations of RSV in donor and receptor compartments at time t, and CD0 and CR0 are
concentrations at time t=0. D is the diffusion coefficient and is a geometrical constant.
The overall mass transfer resistance can be calculated using equation 3

(3)
D
where is membrane (acetate-cellulose membrane plus layer of microbeads) thickness and D is diffusion
coefficient. This overall resistance is cumulative resistance of the acetate-cellulose membrane and the resistance
provided by liposome membranes and alginate matrix. In order to calculate the resistance stemming from
liposome membranes and alginate matrix, the resistance of synthetic membrane (earlier determined using RSV
solution of the same concentration) was subtracted from the overall mass transfer resistance.

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3
3.1

Results
Encapsulation efficiency and the average diameter

RSV loaded liposomes were prepared using proliposme method. Electrostatic extrusion technique was
used for production of liposome-in-alginate microbeads and it was performed under the same conditions for all
samples as it is described in section 2.2. With this technique, it was possible to process low viscosity Naalginate up to 2.5% w/v and medium viscosity Na-alginate up to 1.5% w/v. The results of encapsulation
efficiency (EE%) and average diameter of the microbeads are presented in Table 1.
Table 1. The influence of alginate viscosity and concentration on encapsulation efficiency of RSV and average diameter of
microbeads.

3.2

Sample (w/v)

EE%

LV 1.0%
LV 1.5%
LV 2.5%
MV 1.0%
MV 1.5%

83.90
90.42
94.36
94.27
95.06

Average
diameter (m)
360.98
446.49
671.36
381.66
583.26

The release studies

The release studies were performed in Franz diffusion cell and presented in figure (Fig. 1) by plotting
release percentage against time.

1,6

1% MV w/v
1,5% MV w/v
1% LV w/v
1,5% LV w/v
2,5% LV w/v

1,4
1,2

0,8

C/CD (%)

1,0

0,6
0,4
0,2
0,0

50

100

150

200

250

300

350

t (time)

Fig 1: RSV release curve for liposome-in-alginate microbeads

Diffusion coefficients and mas transfer resistances

0
0
CD CR
ln

After plotting CD CR against time (figures are not presented) diffusion coefficient D for each of the

samples was calculated from the slope of the linear part of the curve and the results are presented in Table 2.
Table 2. Diffusion coefficients for different liposome-in-alginate systems and membrane thickness of the Frantz cell

Sample (w/v)
, mm
LV 1.0%
2.60
LV 1.5%
2.93
LV 2.5%
2.26
MV 1.0%
2.54
MV 1.5%
2.42
The mass transfer resistances stemming from lipid and alginate
in section 1.5 and the results are presented in Figure 2 .

D, m/s
9.3710-10
9.0410-10
6.6910-10
8.0310-10
7.3610-10
membranes were calculated as described

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Rlip+alg (s/m)

3,0x10

2,5x10

2,0x10

1,5x10

1,0x10

5,0x10

0,0
1% MV

1,5% MV

1% LV

1,5% LV

2,5% LV

Alginate concentration (w/v)

Fig 2: Mass transfer resistances of liposome-in-alginate systems

4
4.1

Discussion
Encapsulation efficiency and average diameter

The results of encapsulation efficiency are shown in Table 1. As seen, all liposome-in-alginate
microbeads were able to incorporate liposomes loading RSV in high yields (83-95%). As more dense the
alginate network is the more of RSV it encapsulates. Thus, the highest EE was achieved in microbeads made of
2.5% medium viscosity alginate (EE% 95.06%) and the lowest one was determined for microbeads produced
from 1% of low viscosity alginate solution (EE% 83.90%). According to previous studies as the alginate
network is less concentrated, larger pores are within and the surrounding membrane is less tick (Shapiro &
Cohen, 1997). Large pore structure of a network enables easy leakage of the encapsulated compound to an
external solution. The size of microbeads is also influenced by the concentration and type of alginate (Table 1).
Thus, the average diameter increased from ~360 to ~670 m with the increase in concentration from 1 to 2.5%
w/v for low viscosity alginate. The result is in agreement with previous studies on electrostatic extrusion in
which the size of droplets forming microbeads is related to viscosity of the polymer solution. Manojlovic and
co-workers (2006) have shown that the increase in viscosity of the polymer solution resulted in larger
microbeads with wider size distribution.
4.2

The release studies

The release studies were performed using Frantz diffusion cell and the release kinetics is presented in Figure 1.
The release started after approximately 30 minutes and diffusion equilibrium was reached after about 300
minutes. According to our previous studies, liposomes (the same formulations as here) per se were able to retain
RSV up to 3 to 5 minutes when release started, while the steady state was reached after 3.5 h (Isailovic, et al.,
2013). Obviously, alginate matrix of liposome-in-alginate microbeads provided extended release of RSV
compared to liposomes. Figure 1 also shows that diffusion rate is higher as the alginate is less concentrated.
Here, it should be emphasized that the rate of RSV release is also influenced by the membrane thickness
(membrane is composed of acetate-cellulose membrane plus layer of microbeads) presented in Table 2; thus,
microbeads made from medium viscosity alginate was not able to retain RSV better than microbeads with low
viscosity alginate of the same concentration (since values of membrane thickness are higher for samples with
MV than LV alginate). Since RSV has low solubility in aqueous surroundings such as the one in hydrogel, it
was released only up to 1.5 % (expressed on the base of the initial concentration of RSV in the donor
compartment of the Frantz cell).
4.3

Diffusion coefficients and mas transfer resistances

Ficks second law was used for determination of diffusion coefficients of RSV from liposome-in-alginate
microbeads and the values are given in Table 2. As shown the diffusion coefficients decrease with the increase
in alginate concentration for both types of alginate (LV and MV). As expected, the values are lower for samples
made of low viscosity alginate when compared with the microbeads produced with medium viscosity alginate of
the same concentration. Diffusion coefficients of the same order were reported by Pjanovi and co-workers who
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investigated encapsulation of lidocaine hydrochloride and dihydroquercetin in liposomes which were

incorporated within sodium polyacrylate and carbopol resin matrixes. For sake of comparison, the values are
one order of magnitude smaller than those which have been obtained in our recent report for diffusion
coefficient of RSV from free liposomes (Isailovic, et al., 2013). The release of active compound from any
system is directly affected by the mass transfer resistance provided by the membrane. Comparison of mass
transfer resistance provided by both, lipid membrane and alginate matrix cumulatively, for liposome-in-alginate
microbeads are given in Figure 2. As it was predictable, the mass transfer resistance increased with alginate
viscosity and alginate concentration increasing. Among all samples, the highest value was obtained for
liposome-in-alginate microbeads with 2.5% w/v low viscosity alginate (2.80106 s/m).

Conclusions

In this study RSV was efficiently encapsulated in liposomes-in-alginate microbeads. The increase in
alginate concentration and/or alginate viscosity led to an increase in size of microbeads and efficiency of
encapsulation. The system investigated here provided prolonged release of RSV, up to 5h, where diffusion rate
was affected by concentration of the alginate matrix (release is more rapid as the hydrogel concentration is
lower), but also by thickness of the Franz cell membrane. Finally, it was able to calculate resistance to mass
transfer imparted by encapsulation. The obtained values are of the order 10 6 s/m and they clearly show that mass
transfer resistance increases with alginate viscosity and alginate concentration increasing. Liposome-in-alginate
microbeads seem to be suitable for protection and prolonged release of natural antioxidants.
Acknowledgements
This work was supported by the Ministry of Education and Science, Republic of Serbia (Project No.
III46010) and COST action FA1001.
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