Sains Malaysiana 43(2)(2014): 203–209

Investigation of the Gelation Mechanism between Amino Acid Surfactant Based

Microemulsion and Kappa-Carrageenan Gel Network
(Kajian tentang Mekanisme Penggelan antara Mikroemulsi Berdasarkan Surfaktan
Amino-Asid dan Rangkaian Gel Kappa-Carrageenan)

NASIMA AKTER*, SHAHIDAN RADIMAN, FAIZAL MOHAMED, NAZARUDDIN BIN RAMLY,

EDY GIRI RACHMAN PUTRA & ARI SULISTYO RINI

ABSTRACT
Kappa-carrageenan is one form of necessary hydrocolloid. Hydrocolloids are macromolecular materials, which swell
upon absorption of water; in some cases, forming a stiff gel in the presence of additives. This property is very important
to suspend nanocarriers into gel network, which provide them long time stability at a varying temperature range. In
this work, we prepared microemulsion and trapped these particles inside the kappa-carrageenan gel network. The
microemulsion was composed of sodium N-lauroylsarcosinate hydrate (SNLS), oleic acid and deionized water. The
purpose of this study was to immobilize them into the gel network, giving longer shelf life at a range of temperatures for
oral drug delivery. Morphological properties were investigated by transmission electron microscope (TEM), dynamic
light scattering (DLS) and Fourier transform infrared (FTIR) spectra. The TEM results showed that microemulsions are
trapped in the gel network, and the diameter of the microemulsions are below 100 nm, which is comparable with the DLS
results. The important functional groups of kappa-carrageenan and microemulsion were shown from the FTIR result of
the complex microemulsion gel. These results confirmed the interaction between SNLS based microemulsion and kappa-
carrageenan gel.

Keywords: Gel; kappa-carrageenan; microemulsion

ABSTRAK
Kappa-carrageenan adalah sejenis hidrokoloid. Hidrokoloid merupakan bahan makromolekul yang mengembang apabila
menyerap air; dalam kes tertentu boleh membentuk gel yang tegar dengan penambahan bahan aditif. Sifat ini sangat
penting bagi mengapungkan nanopembawa ke dalam rangkaian gel dan memberikan kestabilan jangka panjang pada
julat suhu yang luas. Dalam kajian ini, kami menyediakan mikroemulsi yang diperangkap ke dalam rangkaian gel kappa-
carrageenan. Mikroemulsi mempunyai komposisi daripada sodium –N-lauroylsarcosinat hidrat (SNLS), asid oleik dan air
nyahion. Tujuan kajian ini adalah untuk memegunkan mikroemulsi ke dalam rangkaian gel bagi memberikan masa simpan
yang lama pada julat suhu penghantaran dadah secara oral. Sifat morfologi telah ditentukan dengan menggunakan
mikroskop elektron transmisi (TEM), serakan cahaya dinamik (DLS) dan spektrum inframerah penjelmaan Fourier (FTIR).
Hasil TEM menunjukkan bahawa mikroemulsi terperangkap dalam rangkaian gel dengan diameter mikroemulsi kurang
daripada 100 nm tekal dengan ukuran DLS. Kumpulan berfungsi utama kappa-carrageenan dan mikroemulsi juga dikenal
pasti melalui pencirian spektroskopi-FTIR dalam kompleks gel mikroemulsi. Hasil kajian membuktikan bahawa interaksi
yang kuat wujud antara mikroemulsi berdasarkan SNLS dan kappa-carrageenan gel.

Kata kunci: Gel; kappa-carrageenan; mikroemulsi

INTRODUCTION
galactent, mostly used for the industrial applications due
There is a pressing need to use poly-electrolytes in nano- to their unique physico-chemical properties. It is a high
level to create new biocompatible nanocarriers. These molecular weight polysaccharide with 15 to 40% of ester-
materials in gel form play an important role as suspending sulfate content (Fennema 2002), formed by alternate units
medium and thus provide comparatively long term stability of D-galactose and 3.6 anhydro-galactose (3.6-AG) joined
to those nanoparticles. The complex product can be by α-1, 3 and β-1, 4 -glycosidic linkage. On the other
easily adapted in the biological environment due to their hand, microemulsions are fine oil-in-water or water-in-
biocompatible property. A large number of hydrocolloids oil droplets or biphasic dispersions, having droplet size
can produce such bio-poly-electrolytes. ranging from 20–300 nm. These size ranges enable them
This work deals on the use of one form of poly- as excellent drug delivery vehicle. By immobilizing them
electrolyte ‘kappa-carrageenan’, to prepare a microemulsion into the carbohydrate gel network, these types of carrier
gel. Kappa-carrageenan in its natural origin is a sulfated can be used as an oral drug delivery vehicle.
204

For biological application, amino acid based bio corner in the ternary phase diagram. The phase separation
surfactants are considered to be more suitable due to their of this region was determined by visual appearance of the
bio-origin, less-toxicity, biodegradability, cheaper rate sample. The phase, which was stable for several months,
and above all, their excellent physicochemical properties. was selected for the preparation of microemulsion gel. In
These molecular properties enable them to self assemble this experiment the point at 90 wt. % water with SNLS:
into fascinating structures (Akter et al. 2013; Infante oleic acid wt. % ratio 4:1 was selected as the stable point
et al. 2004). An amino acid based surfactant, Sodium for microemulsion formation. No attempts were made to
N-lauroylsarcosinate hydrate (SNLS), was used to prepare identify the other regions of the phase diagram in detail
microemulsion. It is reported that SNLS has very rich and these have been described in terms of their visual
morphologies to self assemble into different aggregate appearance. The phase diagram is shown in Figure 1.
structures. The carboxylate and amide functional groups The total concentration of each sample was divided into
are highly active sites for hydrogen bonding formation. two parts, i.e. 50% (3 mL) of the sample was prepared as
Besides, chemical richness, it represents superior skin gel and 50% (3 mL) as microemulsion and mixed together
with sonication at 80ºC. After preparation, each sample
permeability (Karande et al. 2007). The SNLS based
nanocarrier was found to be stable at very low pH and
was checked through cross polarizers. No birefringence
physiological temperature (Ghosh & Dey 2011). These are
was observed between cross polarizers. The prepared
very important characteristics for targeted drug delivery.
microemulsion sample was transparent and isotropic. For
The already established fascinating properties of SNLS
based systems motivated us to prepare microemulsion gel direct evidence, the samples were characterized by TEM
to use in drug delivery field. following negative staining method.
Our objective was to prepare a stable drug delivery
vehicle. Before using in clinical purpose, a systematic PREPARATION OF KAPPA-CARRAGEENAN BASED
study should be done to confirm the morphology of MICROEMULSION GEL
the nanocarriers. With this intention, we examine the Carrageenan (0.2 g/L) was dissolved in 30 mM KCl
mechanism of immobilization of microemulsion inside solution and heated above 80ºC for 2 h to dissolve the
the kappa-carrageenan gel network. Formation of carrageenan completely. Finally, both the microemulsion
microemulsion was confirmed by visual inspection of and kappa-carrageenan gel ware mixed in a bath sonicator
the sample, using cross polarizing filters, transmission for 10 min above 80ºC to get the microemulsions trapped
electron microscope (TEM), dynamic light scattering (DLS) in gel network.
and Fourier transform infrared spectra (FTIR). All of these
characterizations confirmed the presence of microemulsion
TRANSMISSION ELECTRON MICROSCOPIC
droplets trapped inside the gel network. (TEM) CHARACTERIZATION
Observation of the kappa-carrageenan gel and
EXPERIMENTAL DETAILS microemulsion gel was examined using TEM (Philips-
CM12). To preserve their structure, gels were placed on
MATERIALS the carbon grid very carefully and left for partial drying to
allow the gel structures to adhere onto the carbon grid. The
excess gel was removed by filter paper from the opposite
Kappa-carrageenan was purchased from Fluka-chimica
(USA). Sodium N-lauroylsarcosinate hydrate was purchased
direction. A drop of 3% uranyl acetate solution was added
from Toshima chemical industry (TCI), Japan. Potassium
to the grid and left for 10 s. Again, the excess solution was
chloride (KCl) and Oleic acid was purchased from Fluka
removed washing the grid thrice. Each time the liquid was
absorbed with filter paper, the sample was dried in the
(Buchs, Switzerland). All samples were prepared with
deionized water (Milli-Q filtered, 18.2 MΩ/cm2 resistivity).
air. The sample was then characterized under an electron
microscope at an accelerating voltage of 100 kV.
PREPARATION OF MICROEMULSION
SNLS was used as surfactant and oleic acid was used as oil MEASUREMENT OF THE PARTICLES
phase. Phase diagram was determined at 25ºC by stepwise
The size distribution on microemulsion droplets was
addition of water to the mixture of oil and surfactant. After
determined with a Zetasizer Nano ZS (Malvern Instruments,
each addition, the samples were mixed with gentle hand
Herrenberg, Germany) after equilibration. Z-average
shaking and then centrifuged. The occurrence of phase
particle sizes were measured at the scattering angle of 90º
separation was observed and the single phase sample was
at 25°C using DLS combined with Malvern’s DTS software
selected for the preparation of kappa-carrageenan based
(v.5.02). The Fourier transform infrared (FTIR) spectra were
microemulsions.
recorded using a Perkin Elmer Spectrum BX FTIR system
over the region of 4000-400 cm-1 of the sample solution.
PHASE DIAGRAM DETERMINATION Prior to the assay, liquid samples were prepared by placing
Approximately 10 samples of each 3 mL were prepared to a few drops of the solution between two sodium chloride
cover a narrow range of compositions in the surfactant rich (NaCl) salt plates.
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FIGURE1. Ternary phase diagram of a SNLS/oleic acid/water system, (a) represents the phase diagram of the
whole system with 100% of each component and (b) represents the partial phase diagram of the system

RESULTS AND DISCUSSION carrageenan is a long thin stranded polymer with each
strand being approximately 20 nm in diameter (Spagnuolo
MICROSCOPY et al. 2005); thus each junction zone may consist of at least
5-10 double helices. The double helices aggregate into
The structures of k-carrageenan gel and microemulsion gel
continuous three dimensional networks in the presence of
were examined by macroscopic appearance, cross polarizing
K+ ion, called gelation (Morris et al. 1980).
filters, TEM, DLS and FTIR. Schematic representation of the
Until now, no reports represented the microscopic
mechanism of gel formation is shown in Figure 2(a), 2(b),
image of individual helices of kappa-carrageenan gel
2(c) and their inverted conditions are shown in Figure
network. This indistinctness was attributed to the poor
2(d), 2(e), 2(f). Owing to the coil like structure, the sample
contrast or resolution of microscope (Borhstrom et al.
before mixing and after mixing at high temperature remain
1996; Piculell 1998). According to Borgstrom et al.
in the liquid phase which can flow like water. It is well
known that in the presence of monovalent cations gelation (1996), the contrast in the image depends either on the
of carrageenan causes in a two-step mechanism: Firstly, sulfate group in the carrageenan backbone or the density
the pairs of random coils associate into double helices and difference between the gel network and the continuous
secondly these helices aggregated into larger crystalline phase. To confirm the interaction of carrageenan with any
clusters (Anderson et al. 1968; Rees et al. 1969). system, besides microscope some other evidences are
Gelation property of the compounds was tested by needed. Based on the microscopic evidence image and
subsequent heating and cooling method. Upon cooling DLS data, Spagnuolo et al. (2005) reported interaction of
at room temperature and inversion of the vial, if the kappa-carrageenan with casein micelles.
solution did not flow out of the vial, the compound was In this experiment, after mixing microemulsion
considered to be a gelator of the corresponding solvent. with kappa-gel at 80°C, this gel transforms into a liquid
This result indicated the already established results of state (coil confirmation). These coils rearrange and
kappa-carrageenan gelation, which results in due to the accommodate with the microemulsion droplets and found
formation of contact ion pair between K+ and sulfate new equilibrium states in helical conformation after cooling
groups (Braudo et al. 1991). Moreover, the absence of down at room temperature. The single phase macroscopic
phase separation between kappa-carrageenan gel and appearance, micrograph of TEM, DLS data (Figure 3) and
microemulsion complex indicates that microemulsion FTIR spectroscopic results indicated that there was an
droplets suspended within gel matrix (Bourriot et al. 1999). interaction between SNLS and a bio-electrolyte will be
The inverted solution of the kappa-carrageenan gel and replaced by kappa-carrageenan. Although the treatment
microemulsion gel is shown in Figure 2(g). of the samples for TEM with uranyl acetate might have a
In TEM characterization, gel network was observed possibility to cross-link the microemulsion droplets and
at room temperature; the bundle of helical structures with kappa-carrageenan gel and might have resulted in bridging
diameters in the range of 200 nm could be observed in between neighboring microemulsions via gel network.
each side of the tetrahedral network of the gel. Kappa- This possibility was ruled out by the evidence of DLS data
206

FIGURE 2. Schematic representation of helical structure of twisted kappa-carrageenan linear chain (a) represents the coil
conformation of linear chains in hot water, (b) helical formation in hot solution, (c) aggregated helical structure upon cooling,
(d), (e), (f) represents the inverted samples of (a), (b), (c), (g) visual observation of kappa-carrageenan gel
and microemulsion gel in upside down condition and (h) TEM image of gel network

that showed an increase in droplet diameter. A significant 1260-1210 cm-1 is an evidence of the presence of sulphate
increase in droplet size was observed than the initial droplet ester (S=O) on the protruding side chain.
size (35 nm). Moreover, key functional groups of both SNLS The bands at 1158 cm-1 and 1030-1010 cm-1 assigned
and kappa-carrageenan of microemulsion appeared in the to C-O and C-C stretching vibrations of pyranose ring
FTIR spectra. Therefore, we can conclude that the ribbon- common to all polysaccharides. In the anomeric (950-700
like micro structures seen (Figure 3(b)) around the surface cm-1) region kappa-carrageenan shows several bands. The
of the microemulsion were k-carrageenan gel network. This strong band at 922 cm-1 assigned to the presence of 3,
is well known that kappa-carrageenan demonstrates twisted 6-anhydro-galactose residue. Bands at 770, 736 and 696
ribbon confirmation due to the torsion effect forced to the cm-1 are assigned to the skeleton bending of pyranose ring.
molecule through the 3, 6 anhydro-D-galactose bridges. The bands at 848 cm-1 corresponds to galactose-4-sulphate
The symmetry is 3:1 and the periods are 24.6 and 26.0A° (Abad et al. 2003; Coimbra et al. 1998; Synytsya et al.
for the kappa and the iota form of carrageenan, respectively 2010; Tapia et al. 2004; Volery et al. 2004).
(Anderson et al. 1969; Rees 1969). IR spectrum of Kappa-carrageenan gel also showed
a broad asymmetric band in the 850 cm-1 region due to
FTIR SPECTRA OF KAPPA-CARRAGEENAN the presence of D-galactose-4-sulphate (Figure 4(b)).
AND ITS DERIVATIVES Furthermore, additional absorption spectrum also observed
The evidence of interaction between polyelectrolyte in the region of 930 cm-1 indicating the presence of 3,
and microemulsion was studied by FTIR spectroscopy. A 6-anhydrogalactose. The band at 1233-1206 cm-1 with
spectrum of kappa-carrageenan in the range of 4000-400 a shoulder at 1047 cm-1, corresponds to the ester sulfate
cm-1 is presented in Figure 4. The broad band at 3400-3200 stretching of kappa-carrageenan in the former case and the
cm-1 is due to hydrogen bonded stretching vibration of OH latter represents the presence of glycosidic linkage.
group with a shoulder centered at 2958 cm-1 (Figure 4(a)). Both microemulsion and microemulsion gel present
The two bands from 4000-2000 cm-1 are common to all a peak around 3500 cm-1. This is assigned to the typical
polysaccharide standards and seaweed samples (Gómez- OH stretching vibration of the carboxyl acid functional
Ordóñez et al. 2011). The characteristic strong band at group of oleic acid. Two bands at 2924 and 2854 cm-1 and
 207

(a)
(b)

(c) (d)

FIGURE3. TEM image microemulsions; (a) without kappa-carrageenan gel and (b) with gel in immobilized state.
The arrow of (b) indicates that a collapsed-ordered gel network in the presence of surfactant mesophases
(c) and (d) represents the size distribution of microemulsion droplets without and with kappa-gel
Transmitence %

Wavenumber (cm-1)

FIGURE 4. FTIR spectrums of (a) kappa-carrageenan, (b) kappa-carrageenan gel,

(c) microemulsion (ME) and (d) kappa-carrageenan based microemulsion gel
208

a very weak band at 2957 cm-1 appeared as shoulder on conversion. Our results evidenced that k-carrageenan
the OH stretching vibration. These bands can be attributed in the helical form interacts with the surface of the
to the asymmetric and symmetric stretch of CH2 and microemulsion; perhaps the gelation process interrupts the
asymmetric stretch of CH3, respectively. The elimination mobility of self-assembled structures by hydrogen bonding,
of these bands from SNLS and oleic acid and recurrence as which has strong electronegative atoms in the periphery of
weak band with decreasing frequency in microemulsion the monolayer. Based on these results, it can be concluded
phase can be attributed to the closely packed acylation of that k-carrageenan microemulsion can be used as a carrier
the hydrocarbon chains (Wu et al. 2004). for the drug in the future.
In comparison to kappa-carrageenan, the characteristic
peak at 4000-2000 cm-1 of carbohydrate appears at the ACKNOWLEDGEMENTS
same position with slight broadening in microemulsion gel.
This broadening at 3400 cm-1 is an evidence of stretching We would like to thank Universiti Kebangsaan Malaysia for
vibration of OH and hydrogen bonding between OH of oleic the financial support of this research work through the grant
acid, amino acid based headgroup of surfactant and also number UKM-MI-OUP-2011 (13-00-09-001), STGL-007-
with water. The absorption band appeared at 1632 cm-1 2010/11, UKM-OUP-2012-137 and UKMDIP-2012-32.
in both microemulsion and microemulsion gel, a group
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