Journal of Microencapsulation, March 2005; 22(2): 179–192
Encapsulation of vitamin C in tripolyphosphate
cross-linked chitosan microspheres by spray drying
K. G. H. DESAI1 & H. J. PARK1,2
1
2
Graduate School of Biotechnology, Korea University, Seoul, South Korea
Clemson University, Clemson, SC, USA
(Received 26 June 2004; accepted 22 October 2004)
Abstract
This paper describes vitamin C-encapsulated chitosan microspheres cross-linked with tripolyphosphate (TPP) using a new process prepared by spray drying intended for oral delivery of vitamin C.
Thus, prepared microspheres were evaluated by loading efficiency, particles size analysis, scanning
electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared
(FTIR) spectroscopy, X-ray diffraction (XRD), zeta potential and in vitro release studies. The microspheres so prepared had a good sphericity and shape but varied with the volume of cross-linking agent
solution added. They were positively charged. The mean particle size ranged from 6.1–9.0 mm. The
size, shape, encapsulation efficiency, zeta potential and release rate were influenced by the volume
of cross-linking agent. With the increasing amount of cross-linking agent, both the particle size and
release rate were increased. Encapsulation efficiency decreased from 45.05–58.30% with the increasing
amount of TPP solution from 10–30 ml. FTIR spectroscopy study showed that the vitamin C was
found to be stable after encapsulation. XRD studies revealed that vitamin C is dispersed at the molecular level in the TPP-chitosan matrix. Well-defined change in the surface morphology was observed
with the varying volume of TPP. The sphericity of chitosan microspheres was lost at higher volume of
cross-linking agent. The release of vitamin C from these microspheres was sustained and affected by
the volume of cross-linking agent added. The release of vitamin C from TPP-chitosan microspheres
followed Fick’s law of diffusion.
Keywords: Vitamin C, chitosan, microspheres, TPP, spray drying, controlled release, cross-linked
microspheres
Introduction
Microencapsulation is defined as a technology of packaging solids, liquids or gaseous
materials in miniature, sealed capsules that can release their contents at controlled rates
under specific conditions (Benita 1996). The microencapsulation technology has been used
by the food industry for more than 60 years. In a broad sense, encapsulation technology
in food processing includes the coating of minute particles of ingredients (e.g. acidulants,
Correspondence: H. J. Park, Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-Ku,
Seoul-136-701, South Korea. Tel: 82-2-3290-3450. Fax: 82-2-953-5892. E-mail: [email protected]
ISSN 0265-2048 print/ISSN 1464-5246 online # 2005 Taylor & Francis Group Ltd
DOI: 10.1080/02652040400026533
180
K. G. H. Desai & H. J. Park
fats and flavours) as well as whole ingredients (e.g. raisins, nuts and confectionary products),
which may be accomplished by microencapsulation and macro-coating techniques,
respectively (Kirby 1991). With regard to microencapsulation of active agents, in earlier
publications, several new encapsulation devices have been successfully demonstrated
(Kim et al. 2002; Ko et al. 2002; Chen et al. 2003; Lee et al. 2003a). On the other hand, in
the food and pharmaceutical industries, biologically active food ingredients are encapsulated
for a variety of reasons including protection from volatilization during storage, protection
from undesirable interactions with other food components, minimization of flavour
interactions or light induced deteriorative reactions, controlled release applications and
protection against atmospheric conditions. In a recent study, extending the shelf-life of
minimally processed apples with edible coatings and anti-browning agents has also been
demonstrated (Lee et al. 2003b).
Vitamin C, a representative water soluble vitamin, has a variety of biological, pharmaceutical and dermatological functions. Vitamin C is widely used in various types of foods as
a vitamin supplement and as an anti-oxidant. This is an important anti-oxidant that may
reduce the risk of cancer by neutralizing reactive oxygen species or other free radicals that
can damage DNA (Jacobs et al. 2001). The prevention and treatment of cancer considers
different mechanisms of vitamin C activity, such as
(a) Enhancement of the immune system by increased lymphocyte production (Vohra
and Khan 1990)
(b) Stimulation of collagen formation necessary for ‘walling off’ tumours (Shklar and
Schwartz 1996)
(c) Inhibition of hyaluronidase (Shklar and Schwartz 1996)
(d) Inhibition of oncogenic micro-organisms (Zhang and Wakisaka 1997)
(e) Correction of an ascorbate deficiency, often seen in cancer patients (Dyke and
Craven 1994)
(f) Enhancement of the effect of certain chemotherapy drugs (Kurbacher et al. 1996)
(g) Reduction of the toxicity of other chemotherapeutic agents (i.e. doxorubicin)
(Shimpo et al. 1991; Kurbacher et al. 1996)
(h) Prevention of cellular free radical damage (Flagg et al. 1995), and
(i) Neutralization of carcinogenic substances (Block 1991).
Vitamin C also prevents the adverse effects, increases the effects of and decreases
resistance to chemotherapeutic agents (Shils et al. 1999). With respect to colorectal
cancer, vitamin C has been shown to inhibit this type of cancer in rodents (Logue and
Frommer 1980). The use of vitamin C supplements could substantially reduce the risks
of colorectal cancer. However, vitamin C is very unstable to air, moisture, light, heat,
oxygen and base, it easily decomposes into biologically inactive compounds such as
2,3-diketo-L-gulonic acid, oxalic acid, L-threonic acid, L-xylonic acid and L-lyxonic acid
(Machlin 2001). Therefore, in order to overcome the associated drawbacks, microencapsulated vitamin C was considered (Trindade and Grosso 2000; Uddin et al. 2001).
Chitosan is a hydrophilic, biocompatible and biodegradable polysaccharide of low
toxicity. In recent years, it has been used for development of oral drug delivery systems
(Hejazi and Amiji 2003). It is also a well-known dietary food additive (Muzzarelli and
Vincenzi 1997). Small chitosan microspheres (<10 mm) were also developed for the
site-specific delivery of anti-cancer agents, such as oxantrazole (Hassan et al. 1992) and
5-fluorouracil (Ohya et al. 1993). Recently, it was found that chitosan is degraded by the
microflora that are available in the colon (Tozaki et al. 1997). As a result, this polymer
could be used for the preparation of colon-specific delivery systems.
Preparation of new encapsulation devices for vitamin C
181
A spray drying technique has been used to produce dry powders, granules or agglomerates
from drug-excipient solutions and suspensions (Wang and Wang 2002). The spray drying
process is widely used in food and pharmaceutical industries. This technique can be
used either to both heat resistant or heat sensitive drugs or to both water soluble and
water insoluble drugs or to both hydrophilic or hydrophobic polymers (Fu et al. 2001).
In addition, it is a one-stage continuous process, easy to scale-up and only slightly
dependent upon solubility of drug and polymer (Masters 1991). The particle size of the
microparticles prepared by the spray drying method ranged from microns to several tens
of microns and had a relatively narrow distribution (Masters 1991). The microparticles
prepared by spray drying can be used as oral dosage forms (dry powders, granules or
agglomerates), targeted systems to organs and tissues, long acting parenteral biodegradable
systems (He et al. 1999) and nasal powders (Illum et al. 1994). Thorough investigation has
also been carried out on the preparation parameters of chitosan microspheres by spray
drying (He et al. 1999), including polymer concentration, inlet temperature, feed rate,
compressed air flow rate and aspirator rate.
The aim of the present study is to encapsulate vitamin C in cross-linked chitosan
microspheres by spray drying for the delivery of vitamin C via an oral route. The encapsulation method involves the use of TPP as a cross-linking agent for the first time by spray drying
method. TPP is a non-toxic cross-linking agent preferred over other toxic cross-linking
agents (formaldehyde or gluteraldehyde). In this study, the prepared microspheres were
mainly characterized by particles size, loading efficiency, SEM, TEM, FTIR, zeta potential,
XRD and in vitro release studies.
Materials and methods
Materials
Vitamin C (99.5% purity) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).
Chitosan (medium molecular weight) was purchased from Sigma-Aldrich Chemie
(Steinheim, Germany). All other chemicals were of analytical grade and used as received.
Ultrapure water (Millipore, USA) water was used throughout the study.
Methods
Determination of molecular weight of chitosan. The average molecular weight of chitosan was
determined by batch mode method using multi-angle laser light scattering (MALLS)
photometer according to Chen and Tsaih (1998). The average molecular weight of chitosan
was found to be 1.336 106.
Determination of degree of deacetylation of chitosan. The % N-deacetylation of chitosan
was determined by a 1NMR spectroscopy method (Hirai et al. 1991; Lavertu et al. 2003).
The degree of deacetylation of chitosan was found to be 82.10%.
Preparation of vitamin C-encapsulated chitosan microspheres. The flow diagram of preparation
process of vitamin C-encapsulated chitosan microspheres by spray drying is shown in
Figure 1. The required volume (usually 250 ml) of 1% w/v chitosan solution was prepared
using aqueous acetic acid solution (1% w/v). Required amount of vitamin C (2.5 gms) was
dissolved in 10 ml of ultrapure water (Millipore, USA). The vitamin C solution was added
to aqueous chitosan solution and homogenized at 8000 rpm for 10 min using Young Ji HMZ
182
K. G. H. Desai & H. J. Park
Vitamin C
solution (1% w/v)
Chitosan solution
(1% w/v)
Homogenizing (8000 rpm, 10 min)
Vitamin C and chitosan mixture
10 ml
1% w/v TPP
20 ml
Homogenizing (8000 rpm, 20 min)
30 ml
Spray drying
Inlet temperature 175°C
Pump rate 3 ml/min
Compressed air flow 10 l/min
Vitamin C-encapsulated chitosan microspheres
Figure 1. Procedure of preparation of vitamin C-encapsulated chitosan microspheres by spray drying.
20DN stirrer (Hana Instruments). Various amounts of a 1% w/v aqueous solution of TPP
(10–30 ml) were added dropwise under constant stirring at 8000 rpm for 20 min using
Young Ji HMZ 20DN stirrer (Hana Instruments). TPP was used as a cross-linking agent.
Spray drying was then performed using a SD-04 spray drier (Lab Plant, UK), with
a standard 0.5 mm nozzle. When the liquid was fed to the nozzle with peristaltic pump,
atomization occurred by the force of the compressed air, disrupting the liquid into small
droplets. The droplets, together with hot air, were blown into a chamber where the solvent
in the droplets was evaporated and discharged out through an exhaust tube. The dry
product was then collected in a collection bottle. Spray drying conditions, the inlet
temperature, spray flow and compressed spray air flow (represented as the volume of the
air input) were set at 175 C, 3 ml min1, 10 l min1, respectively.
Loading efficiency. Twenty-five milligrams of vitamin C-encapsulated chitosan microspheres were dissolved in 100 ml of 0.1 N HCl. The solution was passed through 0.2 filter
(Millipore, USA) and then vitamin C content was assayed by measuring the absorbance
at 244 nm (max of vitamin C in 0.1 N HCL) after suitable dilution using a UV
spectrophotometer (Shimadzu 1601PC, Japan). Experiments were performed in triplicate
(n ¼ 3) and loading efficiencies were calculated using equation (1).
Loading efficiencyð%Þ ¼
Calculated vitamin C concentration
100
Theoretical vitamin C concentration
ð1Þ
Measurement of particles size. Chitosan microspheres prepared by spray drying exhibited
quick swelling in liquid medium and, hence, sizes could not be determined using a laser
diffraction technique in a particle size analyser. Therefore, the particle size was determined
Preparation of new encapsulation devices for vitamin C
183
by microscopy. Briefly, 5 mg of chitosan microspheres were taken on a glass slide and sizes
of 100 particles were measured (n ¼ 3) by using a biological microscope (Olympus, Japan).
Surface morphology and shape. The surface morphology of vitamin C-encapsulated chitosan
microspheres was examined by means of an Hitachi (Japan) scanning electron microscope.
The powders were previously fixed on a brass stub using double-sided adhesive tape and
then were made electrically conductive by coating, in a vacuum, with a thin layer of platinum
(3–5 nm) for 100 s and at 30 W. The pictures were taken at an excitation voltage of 15 kv
and a magnification of 18, 6, 8 or 3 k. The shape of chitosan microspheres (non-cross-linked
and cross-linked) was examined by TEM. The sample was added on a formvar coated grid.
The shape of the microspheres were observed using a Philips TECNAI 12 transmission
electron microscope (The Netherlands) at an accelerating voltage of 120 kV.
FTIR spectroscopy. FTIR spectra of pure vitamin C, placebo chitosan microspheres and
vitamin C-encapsulated chitosan microspheres were obtained by using a FTIR spectrometer-430 (Jasco, Japan). The samples (vitamin C or placebo chitosan microspheres or
vitamin C-encapsulated chitosan microspheres) were previously ground and mixed
thoroughly with potassium bromide, an infrared transparent matrix, at 1:5 (sample: KBr)
ratio, respectively. The KBr discs were prepared by compressing the powders, under force
of 5 t for 5 min in a hydraulic press. Thirty scans were obtained at a resolution of 2 cm1,
from 4500–400 cm1.
Zeta potential. Microspheres concentrations 0.3% w/v was made by dispersing microspheres in KCl solution (pH 7). The zeta potential of microspheres was recorded using laser
doppler anemometry (Malvern Zetasizer, UK). Each sample was measured in triplicate.
XRD. X-ray powder diffraction patterns of pure vitamin C, placebo chitosan microspheres
and vitamin C-encapsulated chitosan microspheres were obtained at room temperature
using a Philips X’Pert MPD diffractometer (Philips, The Netherlands), with Co as anode
material and graphite monochromator, operated at a voltage of 40 kV. The samples were
analysed in the 2 angle range of 2–60 and the process parameters were set as: scan step size
of 0.025 (2), scan step time of 1.25 s and time of acquisition of 1 h.
In vitro release studies. The in vitro release of vitamin C from chitosan microspheres was
determined using a dissolution apparatus (TW-SM, Wooju Scientific, Co., Korea). In order
to suspend the microspheres in the dissolution medium, microspheres equivalent to 25 mg
of vitamin C were taken into a cellulose dialysis bag (previously soaked in dissolution
medium for 3 h) containing 3 ml of dissolution medium and tied to the paddle. The in vitro
release studies of vitamin C were carried out at a paddle rotation of 100 rpm in 500 ml
of phosphate buffer (pH 7.4 and 25 C). In order to facilitate a compatible environment for
the released vitamin C, the temperature of the dissolution medium was maintained
at 25 0.1 C until the end of the study. An aliquot of the release medium (5 ml) was
withdrawn through a sampling syringe attached with 0.2 mm filter at pre-determined time
intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and an equivalent amount of fresh dissolution medium
which was pre-warmed at 25 C was replaced. Collected samples were then analysed for
vitamin C content by measuring the absorbance at 265 nm using a UV spectrophotometer
(Shimadzu 1601PC, Japan). In vitro release studies were performed in triplicates (n ¼ 3)
in identical manner.
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K. G. H. Desai & H. J. Park
Results and discussion
Preparation of vitamin C-encapsulated microspheres
Microencapsulation of food ingredients such as vitamins, aroma compounds, fats, oils,
minerals, essential oils, etc., enables forming a protective film (wall system) around droplets
or particles of the microencapsulated (core) material. The wall system is designed to protect
a sensitive core material from factors that may cause its deterioration. The wall can also be
designed to allow controlled or sustained core release under specific conditions covering
a wide range of applications (Shahidi and Han 1993). Although many microencapsulation
techniques have been developed, spray drying is the most commonly used in the food and
pharmaceutical industries (Huang et al. 2002).
The spray drying process begins as pre-heated solution of core and coating materials is
pumped to the atomizer. The atomizer increases the surface area of the solution by creating
a fine mist. The mist is sprayed into a chamber of air heated to a temperature above
the vaporization temperature of the solution’s solvent. As the mist contacts the hot air,
the solvent (water) vaporizes. The rate of solvent vaporization is dependent upon the
solution flow rate, temperature of solution, flow rate of air, temperature of air, size of
the mist droplets and total solid concentration. The vaporized solvent and dried vitamin
C-encapsulated particles are then removed from the chamber. A cyclone separates the
entrained particles from the humid air. The dry particles are forced to the bottom of
the cyclone separator and the air is expelled to the atmosphere or goes to a scrubber.
Chitosan microspheres loaded with cimetidine, famotidine and nizatidine have been
prepared by spray drying and effects of manufacturing parameters (inlet temperature,
pump rate and compressed air flow rate) on the characteristics of the resulting microspheres
has also been studied and reported by He et al. (1999).
In the present study, TPP-chitosan microspheres were prepared by spray drying for the
delivery of vitamin C by the oral route. Non-cross-linked and TPP cross-linked vitamin
C-encapsulated chitosan microspheres were prepared. Non-cross-linked chitosan microspheres cannot be kept suspended in water because of swelling and dissolution (He et al.
1999). In order to prepare the stabilized vitamin C-encapsulated chitosan microspheres,
non-toxic cross-linking agent (TPP) was used to solidify the chitosan microspheres.
TPP is a non-toxic cross-linking agent preferred over other cross-linking agents such as
formaldehyde or gluteraldehyde which are toxic in nature (Lim et al. 1997). It has been
reported that chitosan forms gels with the gentle and non-toxic multivalent counterions,
TPP (Lim et al. 1997). The ionic interaction between the positively charged amino
groups and negatively charged counterion, TPP, were used to prepare microspheres
by spray drying. The anionic counterion, TPP, can form either inter-molecular or intramolecular linkages; this is responsible for the successful formation of the cross-linked
chitosan microspheres (Lim et al. 1997). Effect of volume of cross-linking agent
(10–30 ml) on the properties of chitosan microspheres was studied. Based on preliminary
solution investigations, inlet temperature, spray flow and compressed spray air flow were
set at 175 C, 3 ml min1 and 10 l min1, respectively.
Properties of vitamin C-encapsulated chitosan microspheres
Yield and encapsulation efficiency are both key aspects that must be considered in
microencapsulation. The yield of microspheres by spray drying depends upon the
experimental conditions (inlet temperature, flow rate and compressed air flow). In the
present experimental conditions, yield of vitamin C-encapsulated chitosan microspheres
Preparation of new encapsulation devices for vitamin C
185
Table I. Mean particle size, yield, encapsulation efficiency, zeta potential and time of cumulative release of
cross-linked chitosan microspheres prepared by spray drying process.
Formulation
code
NCPM
F1
F2
F3
Volume
of 1% w/v
TPP (ml)
Mean particle
size (mm)*
Yield
(%)
Encapsulation
efficiency (%)*
Zeta
potential
(mV)*
T50
(min)
T75
(min)
T90
(min)
—
10
20
30
6.1 1.4
8.0 1.0
8.2 0.9
9.0 1.1
60.15
62.83
61.17
61.10
—
58.30 1.27
53.41 1.44
45.05 1.74
8.2 1.3
52.4 3.5
44.3 2.8
33.7 4.2
—
180
180
120
—
300
300
240
—
NA
NA
360
*Mean SD n ¼ 3; T50, T75 and T90: the time of cumulative release % of 50, 75 and 90, respectively. NA: not
attained; TPP: tripolyphosphate; NCPM: non-cross-linked placebo microspheres.
did not vary much with varying volume of cross-linking agent (Table I). The encapsulation
efficiencies (Table I) ranged from 45.05–58.30%. As the volume of cross-linking agent
solution increased, the encapsulation efficiency decreases. This can be attributed to the
surface irregularities of the chitosan microspheres observed with a higher volume of crosslinking agent. Damaged microspheres with surface irregularities, fragmentation or holes are
likely to cause the loss of a substantial amount of vitamin C during the spray
microencapsulation process. It was reported that surface irregularities observed with poly
(D,L-lactide) microspheres also decreased the encapsulation efficiency of chlorambucil (Fu
et al. 2001). The mean particle size of chitosan microspheres ranged from 6.1–9.0 mm and
increased slightly with the increasing volume of cross-linking agent. The microspheres were
positively charged. The zeta potential of the microspheres varied with the volume of crosslinking agent added. For instance, non-cross-linked chitosan microspheres had a zeta
potential of 8.2 mV. The zeta potential of the chitosan microspheres cross-linked with TPP
was higher than that of non-cross-linked microspheres. However, the zeta potential of
microspheres decreased with increasing volume of TPP solution.
Surface morphology and shape
The morphology and shape of the vitamin C-encapsulated chitosan microspheres
were examined by SEM and TEM, respectively. The sphericity of the microspheres was
good, even for non-cross-linked microspheres. SEM pictures of chitosan microspheres
(non-cross-linked and cross-linked with TPP) are shown in Figure 2. The non-cross-linked
chitosan microspheres had spherical shape with wrinkled surface (Figure 2(a)). For the
stabilized microspheres, surface morphology varied with the volume of cross-linking agent
(TPP) added. For instance, microspheres cross-linked with 10 ml TPP solution exhibited
a smooth surface (Figure 2(b)). In the case of microspheres cross-linked with 20 ml TPP
solution, although microspheres exhibited a smooth surface but also had depressed surface
morphology (Figure 2(c)). The sphericity of chitosan microspheres was lost when
microspheres were cross-linked with 30 ml TPP solution. As a result, microspheres
with holes could be formed (Figure 2(d)). The shape of the chitosan microspheres (noncross-linked and cross-linked with TPP) observed by TEM are shown in Figure 3. It can be
seen that the shape of non-cross-linked microspheres was spherical (Figure 3(a)).
Similar observations can be made for microspheres cross-linked with 10 and 20 ml TPP
solution (Figures 3(b) and 3(c)). In the case of microspheres cross-linked with 30 ml TPP
solution, sphericity was lost (Figure 3(d)) which was also evidenced from the SEM picture
(Figure 2(d)).
186
K. G. H. Desai & H. J. Park
A
B
C
D
Figure 2. Scanning electron micrographic photographs of placebo non-cross-linked chitosan
microspheres (A), cross-linked with 10 ml TPP (B), cross-linked with 20 ml TPP (C) and crosslinked with 30 ml TPP (D) solution.
FTIR
The FTIR spectrogram of pure vitamin C, placebo chitosan microspheres and vitamin Cencapsulated chitosan microspheres are presented in Figure 4. The characteristic peaks
of pure vitamin C were obtained at 1027, 1120, 1141, 1321, 1673, 3031, 3411 and
3525 cm1 (Figure 4(a)). The peaks at 1141, 1321 and 1673 cm1 are assigned due to
stretching and bending vibrations of C¼O and OH groups present in vitamin C molecules.
The spectra of vitamin C-encapsulated chitosan microspheres also showed these
characteristic peaks (shown by arrows) confirming the stability of vitamin C after encapsulation. On the other hand, placebo chitosan microspheres showed comparatively broader
peaks at 1072, 1384, 1560 and 3426 cm1 (Figure 4(b)), which resulted in the appearance
of superimposed peaks of vitamin C and chitosan at that region in the vitamin Cencapsulated chitosan microspheres spectra (Figure 4(c)).
XRD
The XRD studies always help to understand the nature of core material (crystalline or
amorphous) in the polymeric matrix (Palmieri et al. 2001). The X-ray diffractograms of pure
Preparation of new encapsulation devices for vitamin C
A
B
C
D
187
Figure 3. Transmission electron microscopy images of placebo non-cross-linked chitosan microspheres (A), cross-linked with 10 ml TPP (B), cross-linked with 20 ml TPP (C) and cross-linked with
30 ml TPP (D) solution.
vitamin C, placebo chitosan microspheres and vitamin C-encapsulated chitosan microspheres are presented in Figure 5. From Figure 5(a), it is evident that vitamin C exhibited
characteristic crystalline peaks at 2 of 10.3, 14.09, 17.3, 25.24, 40.29, 48.19 and 54.3,
indicating the presence of crystalline vitamin C. Placebo chitosan microspheres did not
exhibit any crystalline peaks under the present experimental conditions (Figure 5(b)). The
characteristic crystalline peaks of vitamin C disappeared after encapsulation in cross-linked
chitosan microspheres (Figure 5(c), (d) and (e)), but instead only placebo microspheres
pattern was obtained. This indicates that vitamin C is dispersed at the molecular level in
the TPP-chitosan matrix and, hence, no crystals were found in the vitamin C-encapsulated
microspheres.
In vitro release
Controlled release of food ingredients at the right place and the right time is a key
functionality that can be provided by microencapsulation. A timely and targeted release
188
K. G. H. Desai & H. J. Park
Transmittance (%)
C
B
A
3900
3400
2900
2400
1900
1400
900
400
Wave length (CM−1)
Figure 4. FTIR spectra of (A) pure vitamin C, (B) placebo chitosan microspheres and (C) vitamin Cencapsulated chitosan microspheres.
E
D
C
B
A
10
20
30
40
50
60
2θ
Figure 5. XRD spectra of pure vitamin C (A), placebo chitosan microspheres (B) and vitamin Cencapsulated chitosan microspheres cross-linked with 10 ml TPP (C), 20 ml TPP (D) and 30 ml TPP
(E) solution.
Preparation of new encapsulation devices for vitamin C
189
improves the effectiveness of food additives, broadens the application range of food
ingredients and ensures optimal dosage, thereby improving the cost effectiveness for the
food manufacturer (Augustin et al. 2001). Reactive, sensitive or volatile additives (vitamins,
cultures, flavours, etc.) can be turned into stable ingredients through microencapsulation
(Augustin et al. 2001). With carefully fine-tuned controlled release properties, microencapsulation is no longer just an added value technique, but the source of totally new
ingredients with matchless properties.
Non-cross-linked chitosan microspheres did not maintain the form of spheres in water
and phosphate buffer pH 7.4. The microspheres swelled and dissolved. Non-cross-linked
chitosan microspheres were, therefore, unsuitable for the purpose of microspheres
controlled release systems (Genta et al. 1995). The release profile of vitamin C from
cross-linked chitosan microspheres is shown in Figure 6, along with pure vitamin C itself.
The dissolution of pure vitamin C in phosphate buffer pH 7.4 was rapid and 93% of
vitamin C dissoluted within 30 min. This is obvious because of greater solubility of
vitamin C in the aqueous medium. Since non-cross-linked chitosan microspheres system
is not suitable for controlled release, non-toxic cross-linking agent was used to modulate
the release behaviour of vitamin C from chitosan microspheres. Figure 6 also shows the
influence of the cross-linking agent on the release rate of vitamin C. It can be seen that
the release rate of vitamin C from TPP-chitosan microspheres increased with the increasing
volume of cross-linking agent solution. The increased release rate of vitamin C from TPPchitosan microspheres could be due to decreased sphericity of microspheres. As seen from
the SEM pictures (Figure 2), structure of the vitamin C-encapsulated chitosan microspheres
cross-linked with 10 ml TPP solution was dense (non-porous). As the volume of TPP
solution increased from 10–30 ml, the sphericity of vitamin C-encapsulated chitosan
microspheres was lost and resulted in porous microspheres (Figure 2(d)). The results of
120
Cumulative release (%)
100
80
60
40
20
0
0
1
2
3
4
5
6
Time (hr)
Figure 6. In vitro release profiles of pure vitamin C () and chitosan microspheres cross-linked with
10 ml TPP (g), 20 ml TPP (m) and 30 ml TPP (œ) solution.
190
K. G. H. Desai & H. J. Park
the present study demonstrated that the release rate of vitamin C was affected by the density
of the TPP-chitosan matrix (porous or non-porous microspheres). This is in agreement with
Mi et al. (1999a, b, c), where TPP-chitosan microparticles with pores structure exhibited
higher release rate of the drug. This porous structure (porous microsphere), i.e. low density
structure, is more degradable than the high density structure (non-porous microsphere),
therefore the release behaviour of vitamin C from chitosan microspheres cross-linked with
a higher volume of TPP solution (20 and 30 ml) was much faster than that of chitosan
microspheres cross-linked with 10 ml TPP solution. The time of 50, 75 and 90% cumulated
vitamin C release is presented in Table I. It was noted that time of 50, 75 and 90% cumulated vitamin C release was extended when the volume of TPP solution added decreases.
To analyse the release mechanism of the vitamin C from TPP-chitosan microspheres, the
release data obtained were fit to the Higuchi equation (equation 2):
Q ¼ kH t 1=2
ð2Þ
where Q is the amount of vitamin C release at time t, kH the Higuchi rate constant.
The dissolution data were plotted as the percentage of vitamin C release against the square
root of time (see Figure 7). Linearity was observed with the plots since the correlation
coefficient (R2) ranged from 0.991–0.9933. This indicates that the release of vitamin C from
TPP-chitosan microspheres followed Fick’s law of diffusion.
100
TPP-10 ml
y=45.75x−15.832
2
R = 0.991
Cumulative release (%)
80
TPP-20 ml
y = 43.879x −15.926
2
R = 0.9926
TPP-30 ml
60
y = 42.261x −16.293
2
R = 0.9933
40
20
0
0
0.5
1
1.5
2
2.5
Time (hr1/2)
Figure 7. Higuchi plot of vitamin C-encapsulated chitosan microspheres cross-linked with different
volume of 1% w/v TPP solution.
Preparation of new encapsulation devices for vitamin C
191
Conclusions
This paper describes the encapsulation of vitamin C in chitosan microspheres cross-linked
with TPP by a spray drying method. By using the suitable volume of TPP solution, stable
microspheres loaded with vitamin C could be prepared. Microspheres of different size, zeta
potential, loading efficiency and surface morphology could be obtained by varying
the volume of TPP solution. The particles size and encapsulation efficiency ranged
from 6.1–9 mm and 45.05–58.30%, respectively. The TPP-chitosan microspheres loaded
with vitamin C were spherical and had smooth surface. The vitamin C release from TPPchitosan microspheres was sustained and followed Fick’s law of diffusion.
Acknowledgements
This study was supported by a grant from the Korea University.
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