Chemical Engineering Journal 264 (2015) 134145

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Chemical Engineering Journal

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

Coreshell-type organogelalginate hybrid microparticles: A controlled

delivery vehicle
Sai S. Sagiri a, Vinay K. Singh a, Indranil Banerjee a, Krishna Pramanik a, Piyali Basak b, Kunal Pal a,
a
Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela 769008, India
b
School of Bioscience & Engineering, Jadavpur University, Kolkata 700032, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 An alternative approach has been

proposed to synthesize core
(organogel)shell (alginate) type
hybrid microparticles.
 Encapsulation of organogel within the
microparticles was thoroughly
checked by microscopic, XRD and DSC
studies.
 The microparticles enhanced the drug
encapsulation efciency by
preventing the leaching of internal
phase.
 The developed microparticles hold
promise for the sustained release of
bioactive agents.

a r t i c l e i n f o a b s t r a c t

Article history: The work reports the synthesis and characterization of novel biopolymeric coreshell microparticles for
Received 5 June 2014 controlled delivery applications. Alginate (shell) microparticles were prepared by encapsulating semi-
Received in revised form 2 November 2014 solid organogel (core) using the ionotropic gelation method. Confocal and phase contrast microscopic
Accepted 6 November 2014
studies showed the existence of distinct shell material around the organogel core. The presence of organ-
Available online 11 November 2014
ogel as the core material of the microparticle was conrmed by X-ray diffraction and differential scanning
calorimetric analyses. Entrapment of the organogel within the microparticles improved the drug encap-
Keywords:
sulation efciency by preventing the leaching of the internal phase. In vitro drug release studies showed
Alginate
Coreshell microparticles
sustained release of ciprooxacin from the organogel containing microparticles. The drug loaded micro-
Controlled drug delivery particles were found to have signicant antimicrobial activity when tested against Escherichia coli. The
Leaching microparticles were found to be mucoadhesive in nature. Further, the biocompatibility of the micropar-
Organogel ticles was tested by hemocompatibility and cytocompatibility studies. The preliminary results suggested
Stearic acid that the synthesized coreshell type hybrid microparticles hold promise as carrier for controlled delivery
applications.
2014 Elsevier B.V. All rights reserved.

Abbreviations: CFX, ciprooxacin; DEE, drug encapsulation efciency; DSC, differential scanning calorimeter; FTIR, Fourier transform infrared spectroscope; IP, Indian
Pharmacopeia; LDL, low density lipid; MSes, sesame oil containing microparticles; MSesC, sesame oil and ciprooxacin containing microparticles; MSoy, soy bean oil
containing microparticles; MSoyC, soy bean oil and ciprooxacin containing microparticles; MOG1, OG1 containing microparticles; MOG1C, OG1C containing microparticles;
MOG2, OG2 containing microparticles; MOG2C, OG2C containing microparticles; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density;
OG1, stearic acid based sesame oil organogel; OG1C, stearic acid based sesame oil organogel with ciprooxacin; OG2, stearic acid based soy bean oil organogel; OG2C, stearic
acid based soy bean oil organogel with ciprooxacin; PBS, phosphate buffer saline; SA, stearic acid; Ses, sesame oil; SI, supplementary information; Soy, soy bean oil; WAXS,
wide angle X-ray scattering; XRD, X-ray diffraction.
Corresponding author. Mobile: +91 8763366085.
E-mail addresses: pal.kunal@yahoo.com, kpal.nitrkl@gmail.com (K. Pal).

http://dx.doi.org/10.1016/j.cej.2014.11.032
1385-8947/ 2014 Elsevier B.V. All rights reserved.
 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
1. Introduction
Over the last decade, biopolymer based delivery systems have
gained considerable importance in the eld of controlled drug
delivery. This may be associated with the easy availability and
low cost of the biopolymers. Additionally, in general, biopolymers
have been found to be cytocompatible with most of the mamma-
lian cells. But biopolymers possess some disadvantages. The short-
comings associated with the biopolymeric microparticles include
lower drug encapsulation efciency, leaching of the active ingredi-
ents and quick release of drugs from the delivery vehicles. In gen-
eral, leaching of the internal phase undone the advantages (e.g.
taste masking, protection of the gastrointestinal tract from irrita-
tion causing drugs and labile drugs) of encapsulation. The drug
encapsulation efciency may be increased by enhancing the load-
ing of the drug within the biopolymeric matrices [12]. In such a
case, the excess amount of drug will be retained within the body.
Use of excess amounts of drug in the body may lead to severe com-
plications [3]. Polymeric coreshell microparticles have been
designed to solve the aforementioned problems [4]. Coreshell
microparticles provide effective encapsulation of the bioactive
agents within polymer/polymer complexes.
Gelation of the oil results in the formation of organogels and
may be achieved using organogelators [56]. Fatty acyl group of
compounds are commonly used organogelators and can immobi-
lize many organic solvents and vegetable oils [7]. Stearic acid
(SA), an 18 carbon fatty acid was used as the organogelator to syn-
thesize organogels. Oral consumption of stearic acid has been
reported to lower the LDL cholesterol [8]. Edible grade sesame oil
(Ses) and soybean oil (Soy) were used as the apolar phases. These
oils have antioxidant properties which may improve the shelf-life
of the formulations. These health benets of microparticulate com-
ponents will help in improving the efciency of the formulations.
Ciprooxacin (CFX) was chosen as the model drug to study the
drug release kinetics from the developed microparticles. CFX is a
2nd generation uoroquinolone antibiotic with broad spectrum
of activity against bacterial infections viz., urinary tract, gastroin-
testinal, abdomen and respiratory infections [910]. This drug
has been recommended to treat the infections especially caused
by Gram negative bacteria. In this regard, antimicrobial efciency
of the developed formulations was tested against Escherichia coli.
Since attempts were made to design sustained release dosage
forms, ciprooxacin base was chosen. Along with the benets, cip-
rooxacin therapy includes nausea (2.5% of patients) and vomiting
(1% of patients) [11]. These problems can be overcome by taste
masking of the antibiotic via the developed microparticles.
It was hypothesized that the gelation of the core will help in
preventing the leaching of the internal phase (containing bioactive
agents), a common phenomenon with the oil containing coreshell
microparticles [12]. To achieve this, Siqueira-Moura et al. have syn-
thesized gelled oil particles using polyethleneimine as the stabi-
lizer [13]. In the present study, an alternative approach has been
proposed to solve the problem of leaching and to improve the drug
encapsulation efciency. Biopolymeric coreshell microparticles
whose internal phase (core) contains semi-solid organogels were
synthesized. Alginate was chosen as the representative shell
material as it has been reported to form porous microparticles
[14]. The inuence of organogels on the physico-chemical, thermal
and leaching properties of the microparticles has been studied in-
depth. Sesame oil and soybean oil containing microparticles were
used as the controls. Sustained release of ciprooxacin was
checked by in vitro drug release studies and nally antimicrobial
efciency of the developed formulations was also tested.
135
2. Materials and methods
2.1. Materials
Sodium alginate, stearic acid, calcium carbonate, calcium chlo-
ride (fused), Span 80, Tween 80 and dialysis tubing (Molecular
weight cut-off 12 kDa) were purchased from Himedia, Mumbai,
India. Glacial acetic acid was purchased from MERCK, Mumbai,
India. Rened sesame oil (Tilsona, Recon oil industries Pvt. Ltd.,
Mumbai, India), soy bean oil (Saffola, Marico Pvt. Ltd., Jalgaon,
India) and sunower oil (Fortune sunlite, Adani Wilmar Ltd., Guj-
arat, India) were purchased from the local market. Fresh blood,
stomach and small intestine of goat were collected from the local
butcher shop. Double distilled water was used throughout the
study. All the studies were conducted in triplicates.
2.2. Preparation of organogels
Two types of stearic acid organogels were synthesized (Table 1).
The concentration of the stearic acid was kept at 22% (w/w). Ses-
ame oil and soybean oil were used as the liquid apolar phases.
The organogels were prepared by heatcool method as per the
reported literature [15]. In short, accurately weighed stearic acid
and vegetable oil mixture was heated in a water-bath maintained
at 70 C for 15 min to form a clear homogenous solution. The solu-
tion was incubated at room-temperature (25 C) to form organogel.
During the preparation of drug loaded organogels, ciprooxacin
(0.5% w/w), was dispersed in the homogeneous gelatoroil mixture
and mixed at 500 rpm. The solution kept stirring until the molten
mixture get solidied at room-temperature.
2.3. Preparation of microparticles
Calcium alginate microparticles were prepared by modied
internal gelation method [1617]. Briey, 0.5 g of sodium alginate
was dissolved in 20 ml of water by stirring (500 rpm) for 20 min
using an overhead stirrer. 0.25 g of calcium carbonate was dis-
persed in the alginate solution. 0.5 g of span 80 and 2.5 g of organ-
ogel (internal phase) were added and homogenized (500 rpm) for
20 min. The homogeneous mixture, so formed, was slowly added
drop-wise into 50 ml of ice cold sunower oil (external oil phase)
and homogenized (300 rpm) for 3 min. Thereafter, acidied oil
(5.0 ml of sunower oil + 1.0 ml of glacial acetic acid) was added
and homogenized for 5.0 min. Acidication resulted in the forma-
tion of ionotropically crosslinked microparticles. The microparti-
cles were washed with 300 ml of 0.1 M calcium chloride solution
and water containing 1.0% (w/v) Tween 80. Sesame oil and soy
bean oil containing microparticles were synthesized in the similar
manner using vegetable oils as the internal phase. Vegetable oil
containing microparticles were used as the controls. Ciprooxacin
containing microparticles were also synthesized in the similar
manner, in which drug containing organogels were used.
2.4. Viscosity studies
The apparent viscosity of the primary emulsions used for the
preparation of the microparticles was analyzed using a rotational
cone and plate viscometer (BOHLIN VISCO-88, Malvern, U.K.). The
cone angle and diameter are 5.4 and 30 mm, respectively. A gap
of 0.15 mm was maintained between the cone and the plate
throughout the study. The viscosity of the emulsions was mea-
sured under variable shear rates ranging from 13 s1 to 95 s1 at
room temperature [18].
136 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
Table 1
Composition of the organogels.
Sample Stearic acid Sesame oil Soy bean oil Ciprooxacin
(% w/w) (% w/w) (% w/w) (% w/w)
OG1 22.0 88.0
OG2 22.0 88.0
OG1C 22.0 87.5 0.5
OG2C 22.0 87.5 0.5
In addition to the viscometric studies, backward extrusion stud-
ies were also conducted to check the viscosity of the primary emul-
sions [19]. A dynamic texture analyzer (TA HD-plus, Stable
Microsystems, U.K) was used to perform the backward extrusion
studies. A cylindrical probe with at bottom (diameter: 30 mm)
was moved in uniaxial direction at a speed of 1 mm/s into the
emulsions up to 20 mm and returned to its original position with
the same speed.
2.5. Microscopy and size distribution analysis
The microstructure of the microparticles was studied by an
upright bright eld microscope (LEICA-DM 750 equipped with
ICC 50-HD camera, Germany), confocal laser scanning microscope
(FV1000, Olympus, USA) and scanning electron microscope (JEOL,
JSM-6390, Japan). Freshly prepared microparticles were used for
the bright eld microscopy. For confocal imaging, 0.1% (w/w) urol
yellow 088 dye in vegetable oils was used for the preparation of
the microparticles. The chromophore (urol yellow) was excited
at 405 nm and the uorescence at 515 nm was observed [2021].
For scanning electron microscopy, dried microparticles were sput-
ter coated with platinum prior to the analysis. The size distribution
analysis was performed using NI Vision 2010 software (National
Instruments, USA). For the analysis, >1000 microparticles were
considered to reduce the sampling error [22].
2.6. Leaching and swelling studies
Leaching studies were conducted as per the reported literature
[17,23]. In brief, 0.5 g of the microparticles were placed on a lter
paper (#1Whatmann, Sartorius, Germany) and incubated at
37 0.5 C for 1.0 h.
The quantication of the leached internal phase and the swell-
ing of the microparticles was carried out as per the reported liter-
ature [12]. Briey, 0.1 g (W1) of the microparticles was soaked in
1.0 g (W2) of acidic buffer (pH 1.2) or basic buffer (pH 7.4) for 1.0 h
at 37 0.5 C in microcentrifuge tubes. The tubes were subse-
quently centrifuged at 10,000 rpm for 2 min (MC-02, SPINWIN,
Tarsons, India). The pellet (W3) and the supernatant (W4) were
weighed separately and then dried at 55 C for 48 h. Subsequently,
the dried pellet (W5) and supernatant (W6) were weighed again.
The swelling power of the microparticles was calculated as:
W3
Swelling power
W5
The percentage of leaching from the microparticles was calcu-
lated as:
W6
% leaching  100
W1
2.7. Drug encapsulation efciency
Accurately weighed 1.0 g of the dried microparticles was tritu-
rated for 5 min in 10 ml of acetone. The whole mixture was trans-
ferred to a 50 ml volumetric ask and sealed using glass stopper.
The mixture was then equilibrated in the presence of acetone for
6.0 h at 37 C to achieve drug equilibration. Thereafter, the mixture
was ltered through a lter paper (#1Whatmann) and the ltrate
was analyzed at 274 nm using UVvisible spectrophotometer
(UV-3200, LabIndia, India). The drug encapsulation efciency was
calculated by the following equation and was represented as %
DEE (% drug encapsulation efciency).
Practical loading
% DEE  100 3
Theoritical loading
2.8. Molecular characterization studies
The physicochemical properties of the microparticles were ana-
lyzed by XRD (X-ray diffraction) studies and FTIR (Fourier trans-
form infrared) spectroscopy. Wide angle X-ray scattering (WAXS;
Panalytical, X-Pert Pro; Almelo, Netherlands) studies were per-
formed using dried microparticles in the 2h range of 5 to 50 at
2 /min. The X-ray source was CuKa and was operated at 35 kV
and 20 mA. FTIR studies were performed using ALPHA-E spectro-
photometer (Bruker, Germany) operated in the ATR (attenuated
total reection) mode in the wave number range of 4000 to
500 cm1. An average of 24 scans was conducted prior to the nal
spectrum [24].
2.9. Thermal studies
Differential scanning calorimeter (DSC-200 F3 MAIA, Netzsch,
Germany) was used to study the thermal properties of the dried
microparticles. 1015 mg of the dried microparticles were
weighed in pure aluminum pan and hermetically sealed with
pierced aluminum lid. The analysis was conducted under inert N2
atmosphere in the temperature range of 20 C to 150 C at 2 C/
min [25].
2.10. Biocompatibility and mucoadhesivity studies
Hemocompatibility of the microparticles was evaluated using
goat blood as per the ASTM standard protocol described earlier
[15,26]. Positive and negative controls were 0.1 M HCl and normal
saline (0.9% NaCl), respectively. % hemolysis in the presence of
microparticles was calculated using Eq. (4).
ODtest  ODnegative
%Hemolysis  100 4
ODpositive  ODnegative
where,
ODtest = optical density for the test sample.
ODpositive = optical density for the positive control.
ODnegative = optical density for the negative control.
The cytocompatibility of the microparticles was determined
against MG63 osteoblast cells. In short, 1 g of the microparticles
was put into the dialysis tubing and was subsequently dipped into
1 25 ml of phosphate buffer saline (PBS). 200 ll of the leachate was
added to a well of a 96 well plate. The plate was previously seeded
with 1  104 cells/well and subsequently incubated (37 C, 5% car-
bon dioxide) for 12 h to allow adherence of the cells. After the addi-
tion of the leachate, the plate was further incubated for 48 h. After
2 incubation, the viability of the cells was assessed using MTT assay
[27].
Mucoadhesivity studies were conducted using dried micropar-
ticles by in vitro wash off method [28]. Fresh goat stomach and
intestine (ileum) was cut and adhered to glass slide using acrylate
adhesive such that the inner lumen was exposed. The microparti-
cles were placed on the exposed mucosal surface and a 50 g weight
was put over the microparticles for 5 min to promote adhesion.
 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
The arrangements with stomach and intestine were kept vertically
in the USP (United States Pharmacopeia) disintegration baskets (EI
tablet disintegration apparatus, India) containing 900 ml of acidic
(pH 1.2) and basic (pH 7.4) buffers, respectively. The retention time
of the microparticles on the mucosal surfaces was checked over a
period of 36 h.
2.11. In vitro drug delivery studies
In vitro drug delivery studies were carried out in the buffers of
pH 1.2 (gastric pH) and 7.4 (intestinal pH). Buffers were prepared
as per the Indian Pharmacopeia (IP) protocols. 0.5 g of the micro-
particles were put in the dialysis bags and submerged in 50 ml of
buffer (37 C, 100 rpm). The dissolution medium was replaced with
fresh buffer every 1.0 h for 24 h. The samples were analyzed at
274 nm using UVvisible spectrophotometer.
2.12. Antimicrobial studies
The antimicrobial efcacy of the microparticles was determined
against E. coli (NCIM 5051) (Gram negative bacterium). The antimi-
crobial study was carried out by well diffusion method. 100 ll of
bacterial suspension containing 2  105 CFU/ml was spread evenly
on the autoclaved nutrient agar medium. Two wells with a diame-
ter of 9.0 mm were made in each plate using a stainless steel borer.
The microparticles without ciprooxacin were used as the control.
The petri plates were incubated in orbital shaker incubator at 37 C
for 12 h. The zone of inhibition was measured using a ruler at the
end of the study.
To establish the minimum inhibitory concentration of the drug,
microparticles with different concentrations of drug were loaded
in E. coli (inoculum size: 2x105 CFU/ml) nutrient agar plates. Load-
ing of bacteria was carried out depending on the intestinal bacte-
rial load. In general, intestinal bacterial load varies from 105 to
108 bacteria per ml of uid [29]. It has been reported earlier that
the sensitivity of the microorganisms is higher during the expo-
nential growth phase, hence the E. coli suspension used in this
study was selected from the exponential growth phase [3031].
The concentration at which zone of inhibition appeared was
regarded as the minimum inhibitory concentration of ciprooxacin
from the developed formulations.
2.13. Statistical analysis
The differences in the data was statistically analyzed by one-
way ANOVA using Minitab 14.0 (trial version) and MS ofce excel
2007 software.
3. Results and discussion
3.1. Preparation of organogels
Stearic acid was dissolved in vegetable oils (sesame oil and soy-
bean oil) at 70 C and subsequently cooled to room-temperature
(25 C). During cooling, the clear homogeneous mixture became
turbid and organogels were formed. Formation of organogels was
conrmed by inverted tube method [32]. Both the organogels were
smooth, semi-solid and opaque. OG1 (sesame oil organogel) and
OG2 (soybean oil organogel) were yellow and white in color,
respectively (Fig. 1). The difference in their color can be attributed
to the color of the vegetable oil used. There was no change in the
apparent properties of the organogels after the addition of 0.5%
(w/w) ciprooxacin. The organogels (with or without ciprooxa-
cin) were stable for 12 + months at room-temperature. There was
neither any change in the physical properties (e.g. syneresis) nor
137
any change in the visual appearance (e.g. color, microbial contam-
ination) of the organogels.
3.2. Preparation of microparticles
The addition of organogels to the alginate solution increased the
viscosity of the alginate solution. The change in apparent viscosity
of the primary emulsions was checked by performing viscosity
analysis (Fig. 2). The viscosity study indicated an initial increase
in the apparent viscosity of the emulsions followed by either
decrease (organogels containing emulsions) or plateau region (oil
containing emulsions) as the shear rate was increased. Primary
emulsion of OG2 showed the highest apparent viscosity, followed
by OG1, Ses and Soy, respectively. Similar results were also
observed from the backward extrusion studies (Fig. S1 in SI). The
slope of the backward extrusion curve during the forward move-
ment of the probe corresponds to the index of viscosity [19].
Addition of the cooled primary emulsion to the ice-cold sun-
ower oil (external oil phase) resulted in the formation of internal
phase-in-water-in-oil type of multiple emulsion. Acidication of
the multiple emulsion resulted in the solidication of the alginate
layer by crosslinking with calcium ions [14]. This resulted in the
encapsulation of the internal phase (vegetable oil or organogel).
The alginate microparticles, so formed, were further cured by
treating with 0.5 M aqueous calcium chloride solution and washed
with 1% (w/w) Tween 80 containing water. Then the microparticles
were dried at 45 C for overnight and subsequently stored at 4 C
for further analysis. Stable microparticles were also formed when
ciprooxacin containing internal phase was encapsulated. Drug
encapsulation efciency of the organogel containing microparticles
was much higher as compared to the vegetable oil containing
microparticles (Table 2). This may be explained by the increased
viscosity of the organogels which decreased the diffusion of the
drug molecules (evident from the in vitro drug release studies).
This prevented the loss of the drug from the microparticles during
the preparation and washing stages.
3.3. Microscopy
The structural analysis of the microparticles was investigated in
detail by different microscopic techniques. Size distribution analy-
sis of the microparticles was carried out using bright eld micro-
graphs (Fig. 3ad). Micrographs suggested the formation of
spherical microparticles. Size distribution analysis suggested a
broad size distribution prole for the vegetable oil containing
microparticles, whereas, organogel containing microparticles
showed a narrow size distribution prole (Fig. S1 in SI). The aver-
age diameter (Davg) of the microparticles was calculated as the size
of the 50% of the total microparticles population (shown as dotted
lines in Fig. S1b in SI). The average diameter of MOG1 and MOG2
was higher than MSes and MSoy. Increase in the size of the
MOG1 and MOG2 was due to the higher viscosity of the primary
emulsions. Due to this reason, large droplets (internal phase-in-
alginate solution) of primary emulsion were formed during the for-
mation of the multiple emulsions when organogels were used as
the internal phase. Bright eld micrographs showed translucent
MSes and MSoy and opaque MOG1 and MOG2. The opaque internal
structures of MOG1 and MOG2 may be attributed to the semi-solid
nature of the organogels at room-temperature.
Confocal images of MSes and MSoy showed the presence of oil
globules (green in color) in different sizes (Fig. 3e and f). On the
other hand, MOG1 and MOG2 have shown globular structures of
uniform sizes of the internal phase (Fig. 3g and h). The difference
can be attributed to the structuring of the oils by the gelator (stea-
ric acid) molecules in MOG1 and MOG2 [33]. Some of the oil glob-
ules in MSes and MSoy seemed to be closer to the alginate
138 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
Fig. 1. Preparation of organogels: (a) OG1 and (b) OG2.
Fig. 2. Viscosity proles of the microparticles.
boundary layer. This kind of microstructure may promote leaching
of the internal phase oil. On the other hand, MOG1 and MOG2
showed globular structures uniformly surrounded by an alginate
layer. This kind of encapsulated structure is preferred (indicated
by an arrow) to prevent the leaching so as to enhance the encapsu-
lation efciency. Similar kind of observations were found when
microparticles were viewed under phase contrast and scanning
electron microscopes (Fig. S2 in SI).
3.4. Leaching studies
The results of the leaching studies (lter paper method) indi-
cated that the MSes and MSoy showed extensive leaching of the
internal phase (marked by the formation of a dark zone) from the
dried microparticles against the MOG1 and MOG2 (Fig. 4ad). The
absence of leaching from the organogel containing microparticles
can be attributed to the gelation of the vegetable oils using stearic
acid; due to which apparent viscosity of the oils was increased
(explained in Section 3.2). The semisolid nature of the internal
phase might have prevented the leaching of the vegetable oil due
to the immobilization of the oil within the brous matrix of stearic
acid.
The leaching of the internal phase was quantied by incubating
the microparticles under gastric (pH 1.2) and intestinal (pH 7.4) pH
buffers (Fig. 4e and f). Similar leaching proles were observed
under both the conditions indicating that the change in the pH
did not affect the leaching properties. The studies indicated higher
leaching of the internal phase in the vegetable oil containing
microparticles (>25%) as compared to the organogel containing
microparticles which showed much lower leaching (<5%). The
results were signicantly different (p < 0.05) and supported the
observations of the qualitative leaching studies.
The swelling power of the microparticles was in the range of
2.53.5 in gastric buffer and 2.02.7 in intestinal buffer. Though
not much difference was observed in the swelling power of the
microparticles, organogel containing microparticles have shown
slightly higher swelling power (Fig. 4e and f). This may be accounted
to the ability of the fatty acid organogels to accommodate water
 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
Table 2
Composition, % DEE and index of viscosity of primary emulsions of microparticles.
Sample Internal phase Ciprooxacin (% w/w)
MSes Sesame oil
MSoy Soybean oil
MOG1 OG1
MOG2 OG2
MSesC Sesame oil + CFX 0.5
MSoyC Soybean oil + CFX 0.5
MOG1C OG1C 0.5
MOG2C OG2C 0.5
Fig. 3. Bright eld microscopic images of: (a) MSes, (b) MSoy, (c) MOG1, (d) MOG2; and Confocal images of: (e) MSes, (f) MSoy, (g) MOG1, (h) MOG2.
within their architecture [34]. But the back pressure created by the
alginate layer on to the internal phase might have been a limiting
factor for the organogels to accommodate higher proportions of
water molecules within them.
3.5. Molecular characterization studies
X-ray diffraction analysis of sodium alginate, stearic acid,
organogels (OG1, OG2) and the developed microparticles was per-
formed (Fig. 5a and b). Presence of vegetable oil in MSes and MSoy
changed the diffraction pattern of sodium alginate (Na-Alg) to
show a single broad peak. Similarly, the organogels showed amor-
phous behavior as compared to the nascent stearic acid. Amor-
phous behavior was evident from the hump-like structures in
OG1 and OG2. The fatty acyl molecules in the vegetable oils were
responsible for the appearance of the humps at 20 2h [35]. Even
though, the organogels were amorphous in nature the crystalline
peaks of stearic acid were observed. These peaks were also found
in the diffractograms of MOG1 and MOG2. The crystalline peaks
in MOG1 and MOG2 were due to the presence of stearic acid.
The crystalline peaks of the stearic acid were conserved and
retained at almost the same 2h (Braggs angle) in MOG1
and MOG2. This suggested the presence of stearic acid without
any changes in the physical nature of the organogels [35]. Although
the peaks of stearic acid were conserved in MOG1 and MOG2, their
intensities were poor when compared to stearic acid and OG1 and
OG2, respectively. Presence of alginate layer around the organogels
might have reduced the peak intensity in MOG1 and MOG2. This
suggested that the organogels were encapsulated within the
139
Index of viscosity of the primary emulsions (mPa s) % DEE
2.826
4.522
5.652
5.935
48 2.8
45 3.2
77 2.9
82 2.5
microparticles without any major alterations in the organogel
structure. X-ray diffraction analysis also suggested the formation
of core (organogel)shell (alginate) type microparticles.
Fig. 5b shows the interplanar spacings [hkl] of the peaks and
their corresponding d-values have been listed in the Table 3. In
general, the intensities of all the peaks of the organogels were
decreased after encapsulation, but the effect was more pronounced
in long spacing peaks. The major changes in the pattern of the
peaks revealed that the order of the molecular layers was signi-
cantly altered due to the presence of the other components of
the microparticles [35]. The results indicated an increase in the
amorphous nature of the microparticles associated with the major
variations in the d-value of the long spacing peaks. There were neg-
ligible changes in the d-values of the short spacing peaks in micro-
particles as compared to the organogels.
Along with the decrease in the peak intensity and changes in d-
value; crystallite size (D) of the stearic acid was also changed when
the organogels were incorporated within the microparticles
(Table 3). The crystallite size was calculated based on the [1 1 0]
crystal plane of stearic acid at 2h 21.37. This crystal plane was
chosen due to the highest peak intensity in the diffractograms of
the microparticles and stearic acid. The crystal size was calculated
using DebyeScherrer equation [36].
Kk
D 5
b cos h
where K is the DebyeScherrer constant or shape factor, 0.89 (as
shape of the crystallite is unknown); k is the CuKa wavelength
140 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145

Fig. 4. Leaching of vegetable oils from (a) MSes, (b) MSoy, (c) MOG1 and (d) MOG2; and Swelling power and % leaching of microparticles in (e) gastric and (f) intestinal pH
buffers.

i.e., 0.154 nm; b is the FWHM (full width half maximum) of the dif-
fraction peak in radians and h is the Braggs diffraction angle.
Crystallite size of stearic acid was found to be higher when it
was in pure form, followed by encapsulated organogels and
organogels, respectively. This suggested that the stearic acid in
microparticles was structurally closer to its native form than in
the organogels. Increase in the crystallite size also suggested an
increase in the crystallinity of the stearic acid in microparticles
than in the organogels. This was supported by the FWHM values
which showed that the FWHM values were lower in the organogel
encapsulated microparticles as compared to organogels (Table 3).
The FTIR spectra of the microparticles showed the characteristic
peaks associated with the alginate (Fig. 5c and d). The observed
characteristic peaks of alginate include COO (1620 and
1410 cm1) and COH stretching vibrations. COH stretching
vibrations exist as OH stretching vibrations at 3450 cm1, CO
stretching vibrations of secondary and tertiary alcohols at 1110
and 1150 cm1, respectively. The characteristic peaks of stearic
acid were observed at 1710 and 940 cm1. The peak at
1710 cm1 overlapped with the COO vibrations of the alginate.
In drug containing microparticles, peaks corresponding to cipro-
oxacin (1610 and 1260 cm1, due to phenolic groups conjugated
to COO and CF bond, respectively) were observed apart from
the peaks associated with the alginate and stearic acid molecules.
There was no shift in the peaks of ciprooxacin indicating that
the drug molecules were present in their native state.
 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145 141

Fig. 5. (a and b) X-ray diffractograms and (c and d) FTIR spectra of the raw materials and microparticles. MSes/MSesC: microparticles with sesame oil/sesame oil + CFX;
MSoy/MSoyC: microparticles with soy bean oil/soy bean oil + CFX; MOG1/MOG1C: microparticles with OG1/OG1C; MOG2/MOG2C: microparticles with OG2/OG2C; OG1:
sesame oil based organogel; OG2: soy bean oil based organogel.

Table 3
XRD analysis of the organogels and the microparticles.

Sample d-value of peaks () FWHM of 21.3 2h D (nm)

[0 0 3] or 6.8 2h [0 0 5] or 11.4 2h [1 1 0] or 21.3 2h [1 1 2] or 23.7 2h
SA 12.82 7.69 4.15 3.75 0.157 51.4
OG1 12.89 7.74 4.15 3.74 0.195 41.5
OG2 12.53 7.60 4.13 3.73 0.227 35.6
MOG1 11.83 4.05 3.66 0.162 49.8
MOG2 13.01 8.09 4.49 3.63 0.194 41.4

3.6. Thermal studies
MSes and MSoy showed a broad endothermic peak at 70 C
(Fig. 6a). This may be due to the evaporation of the bound water
from the alginate layer. Though dried microparticles were used for
the thermal analysis, 100% removal of water from the alginate
microparticles was not possible [37]. The organogels showed endo-
thermic peaks due to their melting at 44 C. The melting endo-
therms of the organogels were also observed in the thermograms
of the MOG1 and MOG2, suggesting the presence of organogels
within the microparticles. The differences in the melting tempera-
tures between OG1, OG2 and MOG1, MOG2, respectively were
found to be signicant (Fig. 6b). This indicates that encapsulation
has improved the thermal properties of the organogels. In addition
142 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145
to the melting peaks of the organogels, microparticles showed addi-
tional peaks at 70 C and 100 C. The peak at 70 C was due to
the evaporation of the bound water in the alginate layer, whereas,
the peak at 100 C was due to the evaporation of the water mole-
cules absorbed by the organogels (within the microparticles) during
microparticle preparation [38]. Literature suggests that it is quite
possible that the water molecules exist within the dynamic and
transient microstructures of the fatty acyl organogels [34]. Evapora-
tion of water over a range of 60120 C, suggested the involvement
of dipoledipole interactions amongst the OH groups of the water
molecules and the fatty acid molecules. There is also a high proba-
bility of dipoledipole interactions amongst the water molecules
and the alginate molecules [39].
Moisture content of the microparticles was calculated based on
the differences in the change in enthalpies during the evaporation
of water in the temperature range of 60120 C. To calculate the
moisture content, DSC of pure water was performed in the temper-
ature range of 30150 C (Fig. S3 in SI). The change in enthalpy of
pure water at 60120 C was found to be 2076 J/g. Change in
enthalpy was calculated by the area under the curve in the given
temperature range [40]. The change in enthalpies and moisture
content of the microparticles were given in Table S1 of SI. The
moisture content of the dried microparticles varied from 5% to
7.5%. MOG1 and MOG2 possess higher moisture content as com-
pared to MSes and MSoy. The organogels present in MOG1 and
MOG2 might have retained water molecules even after drying.
3.7. Biocompatibility and mucoadhesivity studies
The microparticles were found to be highly hemocompatible in
nature (% hemolysis <5.0 as per the ASTM protocol) (see Table S2 in
Fig. 6. (a) DSC curves of the microparticles and organogels, (b) melting point of organogels, (c) cell viability index and (d) mucoadhesion times of the microparticles.
SI). Subsequently, cytocompatibility of the microparticles was
checked using MG63 osteoblasts. MTT assay indicated that the rel-
ative proliferation of the cells in the presence of the leachants and
the control was statistically insignicant (p > 0.05) thereby sug-
gesting the cytocompatibility of the prepared microparticles
(Fig. 6c). In vitro cell cytocompatibility and hemocompatibility
studies have conrmed that the developed formulations were
biocompatible in nature and can be employed for the in vivo
applications.
In addition to the biocompatibility studies, mucoadhesivity of
the microparticles was checked using goat stomach and small intes-
tine. Microparticles did not show any mucoadhesivity towards goat
stomach mucosa. All the microparticles got separated from the goat
stomach soon after they were placed in gastric buffer. When placed
in intestinal buffer, the microparticles showed different adhesion
times (depending on the composition) from the mucosal surface
of the small intestine. Detaching times/mucoadhesion times were
found to be higher in MOG1 and MOG2 (Fig. 6d). In general,
mucoadhesion of biopolymers is due to the non-specic and non-
covalent interactions viz., primarily electrostatic interactions,
hydrogen and hydrophobic bonding [41]. Leaching of oil from MSes
and MSoy might have reduced these interactions between the algi-
nate layer and the mucosal surface. This has lead to the quick
detachment of MSes and MSoy as compared to MOG1 and MOG2.
Higher mucoadhesive nature is expected to promote the residence
time of MOG1 and MOG2 in the gastrointestinal tract and lead to
the lower dosage administration frequency. Statistical analysis sug-
gested that the mucoadhesion times varied signicantly when the
internal phase was changed from oil to organogel (p < 0.05). This
suggested an improved mucoadhesive efciency of MOG1 and
MOG2.
 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145 143

Fig. 7. Drug release studies: (a) CPDR proles and (b) KP kinetics of the microparticles and (cf) Antimicrobial efciency studies of the microparticles. MSes/MSesC:
microparticles with sesame oil/sesame oil + CFX; MSoy/MSoyC: microparticles with soy bean oil/soy bean oil + CFX; MOG1/MOG1C: microparticles with OG1/OG1C; MOG2/
MOG2C: microparticles with OG2/OG2C.

3.8. In vitro drug delivery studies
In vitro drug delivery studies were carried out in simulated gas-
trointestinal conditions by incubating the microparticles in gastric
buffer for 4 h and subsequently in intestinal buffer for 20 h
(Fig. 7a). The CPDR was calculated based on the encapsulated drug
(as calculated from the encapsulation efciency). Under acidic con-
ditions, the drug release prole from the microparticles suggested
concentration independent drug release. The release of the drug
was higher from MSesC and MSoyC as compared to MOG1C and
MOG2C. The release of the drug at the end of 4 h was 5%. Enteric
coated formulations are meant to prevent the release of the drugs
under gastric environment. A release of <10% of the drugs during
the gastric transit is acceptable. Once the microparticles were
transferred to the intestinal buffer (pH 7.4), signicant changes
in the drug release pattern was noticed. There was a logarithmic
increase in the rate of release of the drug. The rate of release of
the drugs was higher from MSesC and MSoyC as compared to
MOG1C and MOG2C, respectively. This might be associated with
the leaching of the oil from MSesC and MSoyC. The lower release
rate of the drug from MOG1C and MOG2C may be associated with
the lower degree of leaching of the internal phase combined with
144 S.S. Sagiri et al. / Chemical Engineering Journal 264 (2015) 134145

Table 4 and MOG2C. Antimicrobial studies indicated that the drug was
The drug release kinetics of the microparticles. releasing in its active state from the microparticles.
Sample Model SSR R2 SST
MSesC Zero order 0.01148 0.951 0.0121 4. Conclusion
Higuchi 0.007974 0.922 0.00865
Weibull 0.396 0.98 0.404
BL 3.44E05 0.996 3.46E05
Ionotropic gelation method was successfully employed to
KP 0.00663 0.994 0.00667 synthesize organogel entrapped coreshell (alginate) type
MSoyC Zero order 0.0071 0.966 0.00735
microparticles. The organogel entrapped microparticles showed
Higuchi 0.00952 0.903 0.01054 improved drug entrapment efciency by preventing the leaching
Weibull 0.1554 0.99 0.157 of the internal phase. The encapsulation of organogels was con-
BL 2.99E05 0.991 3.02E05 rmed by microscopic, XRD, FTIR and DSC studies. Sustained
KP 0.00219 0.997 0.0022
release of ciprooxacin from the hybrid microparticles was noticed
MOG1C Zero order 0.01185 0.794 0.015 and the release was super case-II diffusion transport mediated. The
Higuchi 0.00467 0.931 0.00502
developed hybrid microparticles seem to have huge potential in
Weibull 0.3892 0.95 0.4097
BL 4.25E05 0.954 4.46E05 controlled delivery applications.
KP 0.0084 0.989 0.0085
MOG2C Zero order 0.0067 0.919 0.0073 Acknowledgements
Higuchi 0.00405 0.915 0.00442
Weibull 0.22181 0.984 0.2282
The funds leveraged from the project (SR/FT/LS-171/2009),
BL 1.96E05 0.988 1.99E05
KP 0.0119 0.987 0.0121 sanctioned by the Science and Engineering Research Board (SERB),
Govt. of India are hereby acknowledged. Authors are thankful to
the National Institute of Technology-Rourkela (NIT-R) for provid-
ing the instrumental facilities. Mr. Sai S. Sagiri is thankful for the
the increase in the viscosity of the internal phase (which might scholarship provided by NIT-R for his doctoral studies.
have restricted the diffusion of the drugs). The preliminary results
suggested that the MOG1C and MOG2C may be tried as sustained
Appendix A. Supplementary data
drug delivery vehicles.
The drug release kinetics from the microparticles was estimated
Supplementary data associated with this article can be found, in
by tting the data in different models (Table 4) (see Fig. S4 in SI).
the online version, at http://dx.doi.org/10.1016/j.cej.2014.11.032.
The drug release from the microparticles followed the Bakers
Lonsdale (BL) and Korsmeyer-Peppas (KP) models. Following the
BL model suggest that the developed formulations may be References
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