Vol.2, No.10, 1061-1065 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.210131
Preparation and characterization of genipin-cross-linked
chitosan microparticles by water-in-oil emulsion solvent
diffusion method
Jesada Karnchanajindanun, Mangkorn Srisa-ard, Prasong Srihanam, Yodthong Baimark*
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahasarakham University,
Mahasarakham, Thailand; *Corresponding Author:
Received 27 July 2010; revised 30 August 2010; accepted 5 September 2010.
ABSTRACT
Chitosan (CS) microparticles with and without
cross-linking were prepared by a water-in-oil
emulsion solvent diffusion method without any
surfactants. Aqueous CS solution and ethyl ace-
tate were used as water and oil phases, respec-
tively. Genipin was used as a cross-linker. Influ-
ences of genipin ratios and cross-linking times
on CS microparticle characteristics were inves-
tigated. Non-cross-linked and cross-linked CS
microparticles were spherical in shape and rough
in surface. Microparticle matrices showed po-
rous structures. Surface roughness, mean par-
ticle sizes and bulk density of CS microparticles
increased and their dissolutions in acetic acid
solution decreased when genipin ratio and cross-
linking time increased.
Keywords: Chitosan microparticles; Porous
structure; Genipin; Cross-linking; Morphology
1. INTRODUCTION
Chitosan is a copolymer of 2-glucosamine and N-
acetyl-2-glucosamine prepared by alkaline deacetylation
of chitin that has received great attention for its possible
uses in medical, pharmaceutical and metal ion treatment
applications because of its biodegradability, biocompati-
bility and high concentration of amine functional groups
[1-4]. Chitosan microparticles have usually been fabri-
cated by precipitation, spray drying and water-in-oil (W /
O) emulsification-cross-linking methods [5]. The current
study describes an alternative method for preparation of
cross-linked and non-cross-linked CS microparticles by
the W / O emulsion solvent diffusion method.
Genipin was selected as a biocompatible cross-linker.
Genipin is a natural water-soluble bi-functional cross-
linker. It is obtained from geniposide, a component of
traditional Chinese medicine and is isolated from the
fruits of the plant, Gardenial jasminoides Ellis [6].
Genipin is a fully biocompatible reagent about 10,000
times less cytotoxic than glutaraldehyde [7]. The CS
device matrices were successfully cross-linked with ge-
nipin [8-10].
In this work, a novel approach to the preparation of
genipin-cross-linked chitosan microparticles by the sim-
ple W / O emulsion solvent diffusion method is reported.
Cross-linked chitosan microparticles were solidified and
formed after diffusion out of water from emulsion drop-
lets of chiosan solution to external continuous phase,
ethyl acetate. The influences of cross-linker ratio and
cross-linking time on CS microparticle characteristics
including morphology, particle size, dissolution and bulk
density were investigated and discussed.
2. EXPERIMENTAL
2.1. Materials
Chitosan with degree of de-acetylation and average
molecular weight of 90% and 100 kDa, respectively was
purchased from Seafresh Chitosan Lab Co., Ltd. (Thai-
land). Genipin (Challenge Bioproducts Co. Ltd., Taiwan)
and analytical grade ethyl acetate (Lab Scan) were used
without further purification.
2.2. Preparation of Chitosan Microparticles
Chitosan solution with 1% w / v was prepared by us-
ing a 2% (v / v) acetic acid aqueous solution as a solvent.
Chitosan microparticles were prepared by the water-
in-oil emulsion solvent diffusion method. The 0.5 mL of
0.5% w / v chitosan solution was added drop-wise to 200
mL of ethyl acetate with a stirring speed of 900 rpm for
1 h. The beaker was tightly sealed with aluminum foil
during the emulsification-diffusion process to prevent
ethyl acetate evaporation. The chitosan microparticles
suspended in ethyl acetate were collected by centrifuga-

J. Karnchanajindanun et al. / Natural Science 2 (2010) 1061-1065
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tion before drying in a vacuum oven at room tempera-
ture for 6 h.
Genipin-cross-linked chitosan microparticles were pro-
duced by the same method. The chitosan and genipin
solutions were mixed together under constant stirring at
room temperature for cross-linking before microparticle
preparation. The final chitosan concentration was 0.5%
w / v after cross-linking. The different genipin concen-
trations (5%, 10% and 20% w / w) and cross-linking
times (1.5, 3 and 6 h) were investigated.
2.3. Characterization of Chitosan
Microparticles
Morphology of the chitosan microparticles was de-
termined by scanning electron microscopy (SEM) using
a JEOL JSM-6460LV SEM. The microparticles were
coated with gold for enhancing conductivity before scan.
Mean microparicle size and the coefficient of variation
(CV) were calculated for each on SEM images by count-
ing a minimum of 100 particles using smile view soft-
ware (version 1.02). The CV value was calculated from
the following equation:
CV =
p
D

 100 (1)
where
 is a standard deviation, and Dp is a mean micropar-
ticle diameter measured from SEM images. Lower CV val-
ues indicate high microparticle monodispersity in size.
Percentage of dissolution of the chitosan microparticles
was investigated by shaking 50 mg of the chitosan micro-
particles in 1.5 mL of 2% w / v acetic acid aqueous solu-
tion at room temperature for 24 h. The residue microparti-
cles were recovered by centrifugation before drying in a
vacuum oven at 50C until its weight remained constant.
The percentage of dissolution of the chitosan microparti-
cles was then calculated by following the equation. Each
average percentage of dissolution value was calculated
from a mean of three measurements (see Eq. 2 ).
where initial and remaining CS microparticles are the
weights of chitosan microparticles before and after dis-
solution test, respectively.
Bulk density of the chitosan microparticles was meas-
ured by gas displacement method using an ultrapyc-
nometer 1000 (Quantachrom, USA) under helium gas.
Each average density value was calculated from a mean
of five determinations.
3. RESULTS AND DISCUSSIONS
Here, the water-in-oil (W / O) emulsion solvent diffu-
sion method without any surfactant was used to prepare
chitosan (CS) microparticles. The maximum water solu-
bility in ethyl acetate is 3.30% (CAS No. 141-78-6). Then
the polymer particles should form, if the less than 3.3
mL of aqueous polymer solution (W phase) was added
drop-wise into 100 mL of ethyl acetate (O phase) with
stirring. The water in dispersed emulsion droplets of CS
solution diffused out to the continuous phase, ethyl ace-
tate. It was found in our cases that the CS microparticles
were successfully prepared using this method. In our
preliminary test, the magnetic stirring speed of 900 rpm
is found the most appropriate for microparticle prepara-
tion. The almost aqueous CS solution could not be bro-
ken to form uniform droplets when the stirring speed
was lower than 900 rpm. Meanwhile, almost all particles
were stuck at the wall of glassware during emulsifica-
tion-diffusion process when higher stirring speed than
900 rpm was applied. The chitosan microparticles could
not be completely solidified when the stirring time was
shorter than 1 h. The CS aggregates were found when
higher 0.5 mL of CS solution was used due to its high
viscosity of CS solution.
3.1. Morphology and Sizes of CS
Microparticles
Figure 1 shows SEM images of non-cross-linked and
cross-linked CS microparticles prepared with different
genipin ratios. They were nearly spherical in shape sug-
gested that the genipin ratios did not effect on the parti-
cle shape. Surfaces of these CS microparticles are illus-
trated in Figure 2. The non-cross-linked CS microparti-
cles in Figure 2(a) showed rough surfaces. This may
occur from diffusion out of water from dispersed drop-
lets of CS solution to continuous ethyl acetate phase
during particle solidification. The surface roughness
increased with the genipin ratio as shown in Figures 2(b)
-2(d). The results may be explained that the cross- link-
ing can increase viscosity of CS solution. The diffusion
out of water from cross-linked CS solution droplets was
difficult.
The cross-linked CS microparticles prepared with dif-
ferent cross-linking times also showed the spherical-like
shape, as illustrated in Figures 3(b)-3(d). This indicates
that cross-linking times did not effect on the particle
shape. The CS microparticles prepared with longer cross-
linking time showed rougher surface than the shorter
cross-linking time, as shown in Figure 4. This may be
due to increasing of the viscosity of CS solution when
the cross-linking time increased.
% dissolution = 100
(mg) clesmicroparti CS initial
](mg) clesmicroparti CS remaining(mg) clesmicroparti CS [initial  (2)

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Figure 1. SEM micrographs of (a) non-cross-linked chitosan
microparticles and cross-linked chitosan microparticles with
genipin ratios of (b) 5%, (c) 10% and (d) 20% w/w for
cross-linking time of 6 h. All bars = 100 m.
Figure 2. Expanded SEM micrographs of surfaces of (a) non-
cross-linked chitosan microparticles and cross-linked chitosan
microparticles with genipin ratios of (b) 5%, (c) 10% and (d)
20% w/w for cross-linking time of 6 h. All bars = 5 m.
Internal morphology of the CS microparticle matrix
was examined through their broken surfaces (Figure 5).
It can be seen that the microparticle matrices contained
porous structure, resembling a sponge. These sponge-like
particles were presumably created by rapid solidification
of the chitosan matrix during diffusion out of the water
from emulsion droplets. However, the porous structures
were completely covered with a continuous outer parti-
cle surface. The matrix of cross-linked CS micro-parti-
cles was denser than that of the non-cross-linked micro-
particles. As example of which is shown in Figure 5(b)
for the cross-linked CS microparticles prepared with 20%
w/w genipin ratio compared with the non-cross-linked CS
microparticles in Figure 5(a).
Figure 3. SEM micrographs of (a) non-cross-linked chitosan
microparticles and cross-linked chitosan microparticles with
cross-linking times of (b) 1.5, (c) 3 and (d) 6 h for genipin
ratio of 20% w/w. All bars = 50 m.
Figure 4. Expanded SEM micrographs of surfaces of (a) non-
cross-linked chitosan microparticles and cross-linked chitosan
microparticles with cross-linking times of (b) 1.5, (c) 3 and (d)
6 h for genipin ratio of 20% w/w. All bars = 5 m.
Mean particle sizes of the CS microparticles were de-
termined from several SEM images instead of scattering
method (suspension in water) because of the partial
swelling and dissolution of microparticles. The mean par-
ticle sizes and CV of the CS microparticles are summa-
rized in Table 1. It was found that the particle sizes in-
creased when the genipin ratio and the cross-linking time
were increased. The CV for every sample is below 30%
indicated the CS microparticles with low dispersity in
size were formed. The results suggest that the CV of mi-
croparticles did not appear to affect the genipin ratio and
cross-linking time.
3.2. Dissolution of CS Microparticles
Dissolution behavior of the CS microparticles indi-

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Figure 5. Low-magnification (left column) and high-magnifi-
cation (right column) SEM micrographs of broken surfaces of
(a) non-cross-linked chitosan microparticles and (b) cross-linked
chitosan microparticles with 20% w/w genipin ratio for cross-
linking time of 6 h. Bars = 20 and 10 m for (a) and (b), re-
spectively in left column. Bars = 5 and 2 m for (a) and (b),
respectively in right column.
rectly related to the degree of cross-linking. The higher
dissolution of CS microparticles related to lower degree
of cross-linking. Figure 6 shows dissolution of the CS
microparticles in acetic acid solution for 24 h. The non-
cross-linked CS microparticles were completely dissolved.
This due to the weak acid solution such as acetic acid
solution is a good solvent for CS. The dissolution of mi
croparticles was decreased when the CS was cross-linked
and increasing the genipin ratio and cross-linking time.
The results suggest that the degree of cross-linking in-
creased as the genipin ratio and cross-linking time in-
creased, according to the literatures [8,9]. This is an im-
portant advantage for application in drug delivery with
controllable drug release rate. Thus the drug release rates
can be tailored by varying the degree of cross-linking.
3.3. Bulk Density of CS Microparticles
The porous structures of CS microparticel matrices
with and without cross-linking can be clearly determined
from their density values, as shown in Figure 7. It was
found that the density values increased with the genipin
Table 1. Conditions for preparing chitosan microparticles and their particle sizes.
Sample No.
Process parameter
1 2 3 4 5 6
Genipin ratio (% w/w)
Cross-linking time (h)
Mean particle size (m)
CV (%)
0
0
85
24%
5
6
98
27%
10
6
110
25%
20
6
112
29%
20
1.5
92
28%
20
3
104
29%
Figure 6. Dissolution of chitosan microparticles in 2% w / v
acetic acid solution for 24 h prepared with different genipin
ratios for cross-linking time of 6 h (above) and different cross-
linking times for genipin ratio of 20% w/w (bottom).
Figure 7. Bulk density of chitosan microparticles prepared with
different genipin ratios for cross-linking time of 6 h (above)
and different cross-linking times for genipin ratio of 20% w/w
(bottom).

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ratio and cross-linking time. This can be explained that
the CS molecules were closer together when the higher
genipin ratio and cross-linking time were used. There-
fore, the denser microparticles were obtained. The bulk
density change after cross-linking corresponded to the
microparticle matrices of the broken CS microparticles
from the SEM images in Figure 5.
4. CONCLUSIONS
Non-cross-linked and genipin-cross-linked chitosan
microparticles with spherical-like shapes have been suc-
cessfully prepared using the simple and rapid W / O emu-
lsion solvent diffusion method. The surface roughness of
microparticles increased with genipin ratio and cross-
linking time but the particle shape did not change. All
chitosan microparticle matricess contained porous struc-
tures. The cross-linked microparticles showed denser ma-
trices than that of non-cross-linked microparticles. The
mean particle sizes and bulk density of microparticles
slightly increased as increasing the genipin ratio and the
cross-linking time.
This simple W / O emulsion solvent diffusion method
is promising for the preparation of drug-loaded chitosan
microparticles with and without cross-linking, especially
water-soluble drugs. Drug release rates from microparti-
cles might be controlled by adjusting the genipin ratio
and/or cross-linking time.
5. ACKNOWLEDGEMENTS
This work was supported by Mahasarakham University (fiscal year
2011), the National Metal and Materials Technology Center (MTEC),
National Science and Technology Development Agency (NSTDA),
Ministry of Science and Technology, Thailand (MT-B-52-BMD-
68-180-G) and the Center of Excellence for Innovation in Chemistry
(PERCH-CIC), Commission on Higher Education, Ministry of Educa-
tion, Thailand.
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