Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297

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Colloids and Surfaces B: Biointerfaces

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

Encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-in-water

emulsion and ionic gelation processes
Rangrong Yoksan a,b,∗ , Jatesuda Jirawutthiwongchai b , Kridsada Arpo b
a
Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b
Division of Physico-Chemical Processing Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand

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

Article history: The encapsulation of ascorbyl palmitate (AP) in chitosan particles was carried out by droplet formation via
Received 8 July 2009 an oil-in-water emulsion, followed by droplet solidification via ionic gelation using sodium triphosphate
Received in revised form 13 October 2009 pentabasic (TPP) as a cross-linking agent. The success of AP encapsulation was confirmed by FT-IR, UV–vis
Accepted 11 November 2009
spectrophotometry, TGA, and XRD techniques. The obtained AP-loaded chitosan particles were spherical
Available online 18 November 2009
in shape with an average diameter of 30–100 nm as observed by SEM and TEM. Loading capacity (LC)
and encapsulation efficiency (EE) of AP in the nanoparticles were about 8–20% and 39–77%, respectively,
Keywords:
when the initial AP concentration was in the range of 25–150% (w/w) of chitosan. Augmentation of the
Chitosan
Ascorbyl palmitate
initial AP concentration led to an increase of LC and a reduction of EE. The amount of AP released from
Encapsulation the nanoparticles in ethanol and tris buffer (pH∼8.0) increased with increasing LC and decreasing TPP
Nanoparticles concentration.
Emulsion © 2009 Elsevier B.V. All rights reserved.
Ionic cross-linking

1. Introduction increasing the degree to which those compounds become available

For decades, ascorbyl palmitate (AP), a fat-soluble ester form of
vitamin C, has been used as a source of vitamin C and as an antiox-
idant for foods, pharmaceuticals, and cosmetics [1,2]. Although AP
is more stable than ascorbic acid, the low chemical stability and
water insolubility limit its utilizations.
Encapsulation potentially can protect active molecules from
degradation by direct exposure to severe environments, e.g., light,
oxygen, chemicals, etc. In other words, encapsulation can reduce
the loss of activity of the active compounds. An encapsulant, or
shell, frequently plays an important role as a carrier for delivery of
the molecules to the target organs. In addition, the shell performs a
release mechanism to control the diffusion level of active molecules
under specific conditions, resulting in prolonged activities of these
molecules. A few materials such as lipids [3–6], poly(d,l-lactide) [7],
and poly(d,l-lactide-co-glycolide) [7] have been used as encapsu-
lants for AP in the forms of microemulsions [3], liposomes [3,5,8],
solid lipid nanoparticles [3], nanostructured lipid carriers [4,6] and
nanoparticles [7]. In addition, the nano-level encapsulation would
likely enhance the bioavailability of lipophilic compounds, thus
∗ Corresponding author at: Department of Packaging and Materials Technology,
Faculty of Agro-Industry, Kasetsart University, 50 Paholyothin Rd., Ladyao, Jatujak,
Bangkok 10900, Thailand. Tel.: +66 2 5625097; fax: +66 2 5625092.
E-mail address: rangrong.y@ku.ac.th (R. Yoksan).
to the target tissues.
Chitosan, a natural copolymer of N-acetyl-d-glucosamine and d-
glucosamine units, is one of the polysaccharides potentially most
suitable as carriers for a large number of active compounds [9–16].
Amino groups along chitosan backbones allow solubility in dilute
organic acid solutions, ionic cross-linking, and chemical modifica-
tion of the biopolymer to form gels, beads, films, particles, etc. In
addition to its biodegradability, biocompatibility and non-toxicity,
chitosan has received much attention in the development of micro-
and nanoencapsulation systems [9–16].
The formation of chitosan particles by ionic gelation or polyionic
coacervation has been reported for the delivery systems of various
active molecules, e.g., proteins [10,15], hydrophilic and hydropho-
bic drugs [9,11,13,16], and vitamins [12,14,17]. Although vitamin
C has been incorporated into chitosan–tripolyphosphate particles
by spray-drying [12,14,17], the encapsulation of its fat-soluble
derivative, AP, by chitosan has not been reported. Spray-drying
is a convenient process to produce particles encapsulating active
compounds; however, particles obtained are of micro-level size.
In addition, the use of high temperature (e.g., 170 ◦ C) during the
microparticle preparation might induce the degradation of the
active compounds.
The present work thus focuses on the fabrication of AP-loaded
chitosan nanoparticles by a two-step process: emulsion formation
and ionic gelation. This process is unique because it not only pro-
vides very tiny particles or nanoparticles, but also avoids the use of

0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2009.11.007
 R. Yoksan et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297
high temperature. We also clarified the successful encapsulation by
Fourier transform infrared (FT-IR) and ultraviolet–visible (UV–vis)
spectrophotometry, thermal gravimetry analysis (TGA), and X-ray
diffraction (XRD) techniques, and determined the shape and size of
the particles by scanning electron microscopy (SEM) and transmis-
sion electron microscopy (TEM). In addition, the release of AP from
chitosan nanoparticles was investigated.
2. Materials and methods
2.1. Materials
Chitosan (deacetylation degree of 0.95 and molecular weight of
∼700,000 Da) was purchased from the Seafresh Chitosan (Lab) Co.,
Ltd. (Bangkok, Thailand). Ascorbyl palmitate (AP), sodium triphos-
phate pentabasic (TPP), Tween 60 and Span 60 were supplied by
Fluka Chemika (Buchs, Switzerland). Acetic acid was obtained from
Merck (Darmstadt, Germany). Soybean oil (Jade Brand) was pur-
chased from Lam Soon Public Co., Ltd. (Bangkok, Thailand). All
chemicals were used as received without further purification.
2.2. Preparation of AP-loaded chitosan nanoparticles
AP-loaded chitosan nanoparticles were prepared according to a
method modified from the ones described by Ko et al. [9] and Ajun
et al. [16]. However, soybean oil was used instead of CH2 Cl2 for
preparation of the oil phase in order to avoid toxicity of the chemi-
cal residue. Briefly, aqueous and oil phase solutions were produced.
Chitosan solution (1.5% (w/v)) was prepared by agitating chitosan
in an aqueous acetic acid solution (1% (v/v)) at ambient tempera-
ture (ca. 25–28 ◦ C) overnight. Tween 60 (0.45 g) was subsequently
added to the solution (40 mL) and stirred at ambient temperature
until the mixture was homogeneous. Soybean oil (10 mL) and Span
60 (0.05 g) were mixed at 50 ◦ C for 2 h and then cooled to ambi-
ent temperature. AP was added to the oil mixture and agitated to
achieve a homogeneous oil phase solution.
The oil–AP fraction (10 mL) was gradually dropped into the
aqueous chitosan solution (40 mL) during homogenization at a
speed of 16,000 rpm for 2 min to obtain an oil-in-water emulsion.
TPP solution (0.5% (w/v), 40 mL) was then slowly dropped into
the agitated emulsion. Agitation was continuously performed for
30 min. The formed particles were collected by centrifugation at
10,000 × g for 15 min at 20 ◦ C, and subsequently washed several
times with Tween 60 solution (0.1% (v/v)) and water. The particles
were dried at ambient temperature under reduced pressure and
stored in dry condition at 25 ◦ C. Weight ratios of chitosan to AP
(CTS:AP) of 1:0, 1:0.25, 1:0.50, 1:1.00 and 1:1.50 were used for the
present study.
2.3. Characterization of nanoparticles
FT-IR spectra were obtained by using a Thermo Nicolet Nexus
670 spectrometer (Thermo Electron Corp., Madison WI, USA) with
32 scans at a resolution of 4 cm−1 over a wavenumber range of
4000–400 cm−1 . XRD patterns were recorded over a 2 range of
5–50◦ by a JEOL JDX-3530 (JEOL Ltd., Tokyo, Japan) with a step angle
of 0.04 ◦ C/min. A Mettler-Toledo TGA/SDTA 851e thermogravimet-
ric analyzer (Columbus OH, USA) was used, with a N2 flow rate of
60 mL/min and a heating rate of 10 ◦ C/min from 30 to 600 ◦ C. The Z-
average diameter of samples was determined at 20 ◦ C by a Malvern
Zetasizer (model 3600, Malvern Instruments Ltd., Worcestershire,
UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm
with a fixed scattering angle of 90◦ . SEM analysis of the products
was carried out using a JEOL JSM LV-5600 at an operating voltage
of 15 kV. Transmission electron micrographs were observed by a
293
Fig. 1. FT-IR spectra of (a) chitosan flakes, (b) chitosan particles and (c) AP-loaded
chitosan particles with CTS to AP weight ratio of 1:1.50.
JEOL JEM-1220 at an accelerate voltage of 80 kV. UV–vis absorp-
tion spectra were recorded on a Thermo Spectronic Helios Gamma
spectrophotometer (Thermo Scientific, Waltham MA, USA) with a
scan speed of 60 nm/min over a wavelength range of 200–400 nm
(max = 247 nm).
2.4. Determination of loading capacity and encapsulation
efficiency of AP
The content of AP loaded in chitosan nanoparticles was deter-
mined by TGA/DTG (derivative thermal gravimetric) technique.
The amount of loaded AP per 100 g of sample—loading capac-
ity (LC) and the amount of loaded AP per 100 g of initial AP (in
feed)—encapsulation efficiency (EE) were thus calculated from Eqs.
(1) and (2), respectively:
 weight of loaded AP 
%LC = × 100 (1)
weight of sample
 weight of loaded AP 
%EE = × 100 (2)
weight of initial AP
2.5. Study on in vitro release of AP from chitosan nanoparticles
Ethanol and tris buffer (pH ∼8.4) were used as model media
for an in vitro AP release study. Wet samples (10 mg) and media
(1.2 mL) were placed in a microtube and incubated at ambient
temperature. At sampling time, the incubated mixture was cen-
trifuged and 100 ␮L of supernatant was collected. Evaluation of the
amount of AP released was determined using a spectrophotome-
ter at a wavelength of 247 nm. An equal volume of fresh media was
then replaced in the mixture, and the same procedure was repeated
for the subsequent sampling. These in vitro release studies were
performed in triplicate for each of the samples.
3. Results and discussion
3.1. Characteristics of AP-loaded chitosan nanoparticles
AP-loaded chitosan particles were prepared by a two-step pro-
cess. The first step involved the formation of oil droplets (including
AP) by an oil-in-water emulsion. The second step was the solid-
ification of the formed droplets by an ionic gelation of chitosan,
enveloping the oil droplets, with TPP.
FT-IR spectra of the obtained particles are presented in Fig. 1.
In general, chitosan flakes show characteristic peaks at 3382
(–OH and –NH2 stretching), 2886–2854 (–CH stretching), 1634
(amide I), 1565 (amide II), 1062 (C–O–C) and 887 (pyranose ring)
(Fig. 1a). For chitosan particles, the peak of amide II (–NH2 bending)
294 R. Yoksan et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297
Fig. 2. UV–vis absorption spectra of supernatant obtained from immersion of dif-
ferent samples in ethanol (0.0083 mg/mL) at ambient temperature for 150 min: (a)
chitosan particles and (b)–(e) AP-loaded chitosan particles with different CTS to AP
weight ratios: (b) 1:0.25, (c) 1:0.50, (d) 1:1.00 and (e) 1:1.50.
shifted from 1565 to 1550 cm−1 , and new peaks appeared around
1230–1160 cm−1 (P–O and P O), implying the complex forma-
tion via electrostatic interaction between phosphoric groups and
ammonium ions (Fig. 1b). This result was similar to ones reported
by Xu and Du [10], and Bhumkar and Pokharkar [18]. In compari-
son with the FT-IR spectrum of chitosan particles, the addition of AP
resulted in a markedly increased intensity of the peak at 1732 cm−1 ,
indicating an increase in the content of ester groups, which might
come from AP molecules (Fig. 1c).
The success of AP encapsulation was also confirmed by UV–vis
spectrophotometry. Since ethanol is a good solvent for AP, it was
used as a release medium. After dispersing chitosan and AP-loaded
chitosan particles in ethanol at ambient temperature for 150 min,
the supernatants were collected and characterized by a UV–vis
spectrophotometer. Chitosan (CTS) particles showed no absorption
bands at wavelengths ranging from 220 to 300 nm (Fig. 2a), whereas
AP-loaded chitosan particles gave a maximum absorption peak at
a wavelength of ∼246–248 nm (Fig. 2b–e), corresponding to that
of AP (data not shown). This implied that some content of AP was
released or diffused out from chitosan particles. In other words,
the encapsulation of AP in chitosan particles was accomplished.
The absorption intensity at ∼246–248 nm increased with increas-
ing initial AP content (Fig. 2b–e). This reflected that the amount
of AP released from chitosan particles depended on the initial AP
content (see Section 3.4, below).
TGA is a simple analytical technique to study the weight change
of a material as a function of temperature. Fig. 3A shows that
the weight of chitosan and AP-loaded chitosan particles decreases
Fig. 3. (A) TGA and (B) DTG thermograms of (a) AP, (b) chitosan particles and (c)–(d) AP-loaded chitosan particles with different CTS to AP weight ratios: (c) 1:1.00 and (d)
1:1.50.
with increasing the temperature from 100 to 600 ◦ C. The degree
of weight loss (slope) was different for different temperature
ranges and samples. AP possessed only one level of weight loss
(Fig. 3Aa), while chitosan and AP-loaded chitosan particles showed
two (Fig. 3Ab) and three levels of weight loss (Fig. 3Ac and d),
respectively. These weight losses reflected the degradation of each
component in the materials. The temperatures corresponding to
the maximum slopes of each weight change stage are clearly
observed when the first derivative of the TGA curve with respect
to temperature, the so-called derivative thermogravimetry (DTG)
thermogram, is plotted (Fig. 3B). The DTG thermogram thus reflects
the rate of weight change, or the change in sample weight over time
[d(w)/dt] on the y-axis. The temperatures which give the highest
rate of weight loss at each stage (i.e., peaks in the DTG thermogram)
are usually considered as degradation temperatures (Td ) of compo-
nents in the material. From DTG thermograms, chitosan particles
exhibited two-step degradation, at 235 and 377 ◦ C—which might
be the Td of free chitosan and of chitosan cross-linked with TPP,
respectively (Fig. 3Bb). By loading of AP, the particles showed new
Td at 277 ◦ C (Fig. 3Bc and d), which corresponded to the Td of AP
(Fig. 3Ba). The result suggested the successful loading of AP into
chitosan nanoparticles. The weight loss at this temperature was
then used to determine the content of loaded AP (see Section 3.3,
below).
The packing structures of the obtained particles were deter-
mined by XRD technique. Generally, chitosan shows two peaks at
2 of 11◦ and 20◦ (Fig. 4a). After ionic cross-linking with TPP, a
shift of peak positions, reduction of peak intensity, and broadness
of peaks were observed, reflecting the destruction of the native chi-
tosan packing structure (Fig. 4b). In addition, a new peak at 2 of 24◦
appeared for chitosan particles. These phenomena might be due to
a modification in the arrangement of molecules in the crystal lattice
induced by ionic interaction [18]. For AP-loaded chitosan particles,
the peak intensity at 2 of 12◦ decreased significantly, while the
peak at 2 of ∼20◦ was markedly sharp (Fig. 4c). This implied that
the incorporation of AP resulted in a change in the chitosan–TPP
packing structure.
3.2. Shape and size of AP-loaded chitosan nanoparticles
The diameters of chitosan and AP-loaded chitosan particles were
determined by dynamic light scattering (DLS) technique. Fig. 5
shows that chitosan particles possessed an average diameter of
∼2.5 ␮m. A submicron-level size was obtained for the AP-loaded
chitosan particles, i.e., 250–930 nm. The mean diameter of parti-
cles decreased with increasing the initial AP content. However, the
size measured by the DLS technique might be the hydrodynamic
 R. Yoksan et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297
Fig. 4. XRD patterns of (a) chitosan, (b) chitosan particles; and (c) AP-loaded chi-
tosan particles with CTS to AP weight ratio of 1:1.00.
diameters of aggregate particles and/or hydrated individual parti-
cles. The results reflected that the aggregation and/or swelling of
AP-loaded chitosan particles (in water) were lower than those of
chitosan particles. This might be a result of the hydrophobicity of
AP molecules entrapped inside/on the particles. In order to inves-
tigate the detailed shape and morphological information of the
individual particles, electron microscopy techniques were applied.
Fig. 6a shows the aggregation of chitosan particles. The individual
particles were spheres with an average size of 200–400 nm. For
AP-loaded chitosan particles, the aggregation of particles was also
visible, and those aggregates seemed to be melting and combining
with each other (Fig. 6b). This might have resulted from some AP
content surrounding the particle surface. However, rounded indi-
vidual particles with diameters ranging from 60 to 100 nm were
observed (Fig. 6c and d). In addition, TEM micrographs confirmed
the spherical shape and nanosize structure of individual chitosan
and AP-loaded chitosan particles, and the aggregation of those indi-
vidual particles (Fig. 7). Chitosan particle sizes were in a range
of 25–50 nm (Fig. 7a), while AP-loaded chitosan particles were
30–60 nm (Fig. 7b).
3.3. Loading capacity and encapsulation efficiency of AP
Although spectrophotometry and high performance liquid chro-
matography (HPLC) are effective techniques to determine the
Fig. 5. Z-average diameter of chitosan particles and AP-loaded chitosan particles
with different CTS to AP weight ratios. Data are the mean ± standard deviation
(n = 10).
295
Fig. 6. SEM micrographs at 15 kV of (a) chitosan particles (10,000×) and (b)–(d) AP-
loaded chitosan particles with different CTS to AP weight ratios: (b) 1:1.00 (1000×),
(c) 1:1.00 (10,000×) and (d) 1:1.50 (10,000×).
content of guest molecules loaded in the particles, the complete
destruction of particles and dissolution of guest molecules in the
medium should be considered. In our present research, AP-loaded
chitosan nanoparticles were successfully degraded by the method
reported by Wang et al. [13]; however AP did not dissolve in an
aqueous HCl solution. As a result, spectrophotometry and HPLC
techniques were not useful.
Information obtained from TGA thermograms was used to
determine the content of AP loaded in chitosan nanoparticles.
Fig. 3Ab shows that chitosan particles exhibit three-step weight loss
at temperatures below 150 ◦ C (loss of moisture), 180–310 ◦ C (loss
of chitosan), and above 310 ◦ C (loss of chitosan cross-linked with
TPP). By comparison with the TGA thermogram of chitosan par-
ticles, AP-loaded chitosan particles showed four-step weight loss
(Fig. 3Ac and d). This new range of weight loss appeared at temper-
atures ranging from 250 to 310 ◦ C, corresponding to the Td of AP.
The percent weight loss at this temperature range was thus used to
compute the amount of loaded AP (see Supplementary material).
The loading capacity (LC) and encapsulation efficiency (EE) were
then calculated using Equations (1) and (2), respectively, and tab-
ulated in Table 1. The LC of AP was in a range of 8–20% when the
initial AP varied from 25 to 150% (w/w) of chitosan. In addition,
the LC increased with an increase of the initial AP content. This
result was in agreement with the findings regarding the loading
of ammonium glycyrrhizinate or BSA into chitosan-TPP nanopar-
ticles, which have been reported by Wu et al. [11] and Xu and Du
Table 1
Loading capacity and encapsulation efficiency of AP in AP-loaded chitosan
nanoparticles.
Sample LCa (%) EEb (%)
CTS:AP TPP (%)
1:0.25 0.5 8.46 76.67
1:0.50 0.5 8.45 68.78
1:1.00 0.5 13.87 43.27
1:1.50 0.5 19.78 38.91
a
LC = (weight of loaded AP/weight of sample) × 100.
b
EE = (weight of loaded AP/weight of AP in feed) × 100.
296 R. Yoksan et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297
Fig. 7. TEM micrographs at 80 kV of (a) chitosan particles (100,000×) and (b) AP-loaded chitosan particles with CTS to AP weight ratio of 1:1.00 (100,000×).
[10], respectively. The EE was about 39–77%. This value decreased
with an increase of the initial AP content, a result which could be
due to the saturation of AP loading into chitosan nanoparticles.
3.4. In vitro release of AP from chitosan nanoparticles
The release profiles of AP from AP-loaded chitosan nanoparti-
cles were investigated in vitro at ambient condition for 5 h. The
media used were ethanol and tris buffer (pH∼8.0) because AP dis-
solved well in both solvents. The solubility of AP was restricted in
phosphate-buffered saline (PBS) (pH∼7.4) and citric acid/trisodium
citrate buffers (pH∼3.0); as a result, the amount of AP could not be
determined by a spectrophotometer.
The amount of AP released at different times was measured by
a spectrophotometer at a wavelength of ∼247 nm. The release pro-
files of AP in ethanol (Fig. 8) and tris buffer (Fig. 9) were similar.
There were two stages of AP release, depending on the release rate
(the slope of the release profile). For the initial stage (i.e., the first
hour for ethanol and the first 20 min for tris buffer), the release rate
Fig. 8. In vitro release profiles of AP in ethanol at ambient temperature from AP-
loaded chitosan particles prepared by using different TPP concentrations: (a)–(d)
0.5%, (e) 2% and (f) 4%; and different CTS to AP weight ratios: (a) 1:0.25, (b) 1:0.50,
(c) 1:1.00 and (d)–(f) 1:1.50. Data are the mean ± standard deviation (n = 3).
was rapid, especially in the case of tris buffer (Fig. 9). The mecha-
nism of AP release at this stage could be explained by the diffusion of
AP localized at the particle surface, which might be involved with
the concentration gradient. The diffusion of AP was enhanced at
high pH due to the deprotonation of chitosan; as a result, the elec-
trostatic interaction between ammonium ions on chitosan chains
and phosphoric groups of TPP molecules weakened or disappeared,
and AP was eventually released very quickly (Scheme 1). In the
second stage, the release rate was relatively slow, i.e., almost zero
(Figs. 8 and 9). This might be due to the inability of ethanol and
tris buffer to break or destroy the nanoparticles, resulting in no
additional release of AP at this stage.
The release of AP in ethanol reached a plateau within 1 h, while
reaching a plateau in tris buffer required a shorter time (20 min).
This might be a result of the weakening of electrostatic interac-
tion between the cationic material and anionic TPP, which caused
both faster release and shorter release periods, as mentioned above.
However, the amount of AP released in ethanol was higher than that
in tris buffer, which might be due to the superior solubility of AP in
ethanol.
Fig. 9. In vitro release profiles of AP in tris buffer at ambient temperature from
AP-loaded chitosan nanoparticles prepared by using CTS to AP weight ratio of
1:1.50, and different TPP concentrations: (a) 0.5%, (b) 2% and (c) 4%. Data are the
mean ± standard deviation (n = 3).
 R. Yoksan et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 292–297
Scheme 1. Schematic drawing representing the loss of cross-linked structure via electrostatic interaction between ammonium ions on chitosan chains and phosphoric groups
of TPP molecules (a) due to the deprotonation of chitosan in tris buffer (pH ∼ 8) (b).
The amount of AP released in ethanol was influenced by the
amount of AP entrapped. The high LC provided a fast release rate
and a concomitantly high amount of released AP (Fig. 8a-d). This
might be explained by a wider AP concentration gap between
the polymeric particles and the release medium, which caused a
higher diffusion rate [10]. The concentration of TPP also affected
the amount of released AP. AP was more easily released when TPP
concentration was low (Figs. 8d–f and 9). This could be a result of
low density structure, which is in agreement with previous studies
[9,14,19].
4. Conclusion
Ascorbyl palmitate (AP)-loaded chitosan nanoparticles were
prepared by droplet formation via an oil-in-water emulsion,
followed by droplet solidification via ionic gelation using
tripolyphosphate (TPP) as a cross-linking agent. The obtained AP-
loaded chitosan nanoparticles were spherical, with an average size
of 60–100 nm as observed by SEM, and 30–60 nm by TEM. The load-
ing capacity (LC) and encapsulation efficiency (EE) of AP in the
nanoparticles was about 8–20% and 39–77%, respectively, when
the weight ratio of AP to chitosan was in the range of 0.25–1.50. By
increasing the weight ratio of AP to chitosan, LC increased, while
EE decreased. The release of AP in ethanol or tris buffer (pH∼8.0)
via the diffusion mechanism was completed within 1 h (ethanol)
and 20 min (tris buffer). The amount of released AP increased with
increasing LC and with decreasing TPP concentration.
Acknowledgements
This work has been supported by the Thailand Research Fund
(IRPUS/I250B02002); the Faculty of Agro-Industry, Kasetsart Uni-
297
versity, Thailand (AI-01-50); and the International Laboratories
Corp., Ltd., Thailand.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.colsurfb.2009.11.007.
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