Preparation and characterization of genipin-cross-linked chitosan microparticles by water-in-oil emulsion solvent diffusion method

doi:10.4236/ns.2010.210131

Paper Menu >>

Journal Menu >>

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: [email protected].

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

1062

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 =



 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)

J. Karnchanajindanun et al. / Natural Science 2 (2010) 1061-1065

106

1063

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-

J. Karnchanajindanun et al. / Natural Science 2 (2010) 1061-1065

1064

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 (%)

24%

27%

110

25%

112

29%

1.5

28%

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).

J. Karnchanajindanun et al. / Natural Science 2 (2010) 1061-1065

106

1065

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.

REFERENCES

[1] Kumar, M.N.V.R., Muzzarelli, R.A.A., Muzzarelli, C.,

Sashiwa, H. and Domb, A.J. (2004) Chitosan chemistry

and pharmaceutical perspectives. Chemical Review, 104,

6017-6084.

[2] Muzzarelli, R.A.A. and Muzzarelli, C. (2005) Chitosan

chemistry: Relevance to the biomedical sciences. Advan-

ces in Polymer Science, 186, 151-209.

[3] Crini, G. (2005) Recent developments in polysaccharide-

based materials used as adsorbents in wastewater treat-

ment. Progress in Polymer Science, 30, 38-70.

[4] Learoyd, T.P., Burrows, J.L., French, E. and Seville, P.C.

(2008) Modified release of beclometasone dipropionate

from chitosan-based spray-dried respirable powders. Pow-

der Technology, 187, 231-238.

[5] Agnihotri, S.A., Mallikarjuna, N.N. and Aminabhavi, T.

M. (2004) Recent advances on chitosan-based micro- and

nanoparticles in drug delivery. Journal of Controlled Re-

lease, 100, 5-28.

[6] Mi, F.L., Shyu, S.S. and Peng, C.K. (2005) Characteriza-

tion of ring-opening Polymerization of genipin and pH-

dependent cross-linking reactions between chitosan and

genipin, Journal of Polymer Science, Part A: Polymer

Chemistry, 43, 1985-2000.

[7] Nishi, C., Nakajima, N. and Ikada, Y. (1995) In vitro

evaluation of cytotoxicity of diepoxy compounds used

for biomaterial modification. Journal of Biomedical Ma-

terial Research, 29, 829-834.

[8] Yuan, Y., Chesnutt, B.M., Utturkarr, G., Haggard, W.O.,

Yang, Y., Ong, J.L. and Bumgardner, J.D. (2007) The ef-

fect of cross-linking of chitosan microspheres with geni-

pin on protein release. Carbohydrate Polymers, 68, 561-

567.

[9] Silva, S.S., Motta, A., Rodrigues, M.T., Pinheiro, A.F.M.,

Gomes, M.E., Mano, J.F., Reis, R.L. and Migliaresi, C.

(2008) Novel genipin-cross-linked chitosan/silk fibroin

sponges for cartilage engineering strategies. Biomacromo-

lecules, 9, 2764-2774.

[10] Muzzarelli, R.A.A. (2009) Genipin-crosslinked chitosan

hydrogels as biomedical and pharmaceutical aids. Carbo-

hydrate Polymers, 77, 1-9.