Escolar Documentos
Profissional Documentos
Cultura Documentos
Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
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
Article history:
Received 8 July 2009
Received in revised form 13 October 2009
Accepted 11 November 2009
Available online 18 November 2009
Keywords:
Chitosan
Ascorbyl palmitate
Encapsulation
Nanoparticles
Emulsion
Ionic cross-linking
a b s t r a c t
The encapsulation of ascorbyl palmitate (AP) in chitosan particles was carried out by droplet formation via
an oil-in-water emulsion, followed by droplet solidication via ionic gelation using sodium triphosphate
pentabasic (TPP) as a cross-linking agent. The success of AP encapsulation was conrmed by FT-IR, UVvis
spectrophotometry, TGA, and XRD techniques. The obtained AP-loaded chitosan particles were spherical
in shape with an average diameter of 30100 nm as observed by SEM and TEM. Loading capacity (LC)
and encapsulation efciency (EE) of AP in the nanoparticles were about 820% and 3977%, respectively,
when the initial AP concentration was in the range of 25150% (w/w) of chitosan. Augmentation of the
initial AP concentration led to an increase of LC and a reduction of EE. The amount of AP released from
the nanoparticles in ethanol and tris buffer (pH8.0) increased with increasing LC and decreasing TPP
concentration.
2009 Elsevier B.V. All rights reserved.
1. Introduction
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 antioxidant 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 specic conditions, resulting in prolonged activities of these
molecules. A few materials such as lipids [36], poly(d,l-lactide) [7],
and poly(d,l-lactide-co-glycolide) [7] have been used as encapsulants 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
293
Fig. 1. FT-IR spectra of (a) chitosan akes, (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. UVvis absorption spectra were recorded on a Thermo Spectronic Helios Gamma
spectrophotometer (Thermo Scientic, Waltham MA, USA) with a
scan speed of 60 nm/min over a wavelength range of 200400 nm
(max = 247 nm).
2.4. Determination of loading capacity and encapsulation
efciency of AP
The content of AP loaded in chitosan nanoparticles was determined by TGA/DTG (derivative thermal gravimetric) technique.
The amount of loaded AP per 100 g of sampleloading capacity (LC) and the amount of loaded AP per 100 g of initial AP (in
feed)encapsulation efciency (EE) were thus calculated from Eqs.
(1) and (2), respectively:
%LC =
%EE =
weight of loaded AP
weight of sample
weight of loaded AP
weight of initial AP
100
(1)
100
(2)
294
Fig. 2. UVvis absorption spectra of supernatant obtained from immersion of different 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 cm1 , and new peaks appeared around
12301160 cm1 (PO and P O), implying the complex formation 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 comparison with the FT-IR spectrum of chitosan particles, the addition of AP
resulted in a markedly increased intensity of the peak at 1732 cm1 ,
indicating an increase in the content of ester groups, which might
come from AP molecules (Fig. 1c).
The success of AP encapsulation was also conrmed by UVvis
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 UVvis
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 246248 nm (Fig. 2be), 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 246248 nm increased with increasing initial AP content (Fig. 2be). This reected 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.
295
Fig. 4. XRD patterns of (a) chitosan, (b) chitosan particles; and (c) AP-loaded chitosan particles with CTS to AP weight ratio of 1:1.00.
diameters of aggregate particles and/or hydrated individual particles. The results reected 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 investigate 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 200400 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 individual particles with diameters ranging from 60 to 100 nm were
observed (Fig. 6c and d). In addition, TEM micrographs conrmed
the spherical shape and nanosize structure of individual chitosan
and AP-loaded chitosan particles, and the aggregation of those individual particles (Fig. 7). Chitosan particle sizes were in a range
of 2550 nm (Fig. 7a), while AP-loaded chitosan particles were
3060 nm (Fig. 7b).
3.3. Loading capacity and encapsulation efciency of AP
Although spectrophotometry and high performance liquid chromatography (HPLC) are effective techniques to determine the
Fig. 6. SEM micrographs at 15 kV of (a) chitosan particles (10,000) and (b)(d) APloaded 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).
CTS:AP
TPP (%)
1:0.25
1:0.50
1:1.00
1:1.50
0.5
0.5
0.5
0.5
a
b
LCa (%)
EEb (%)
8.46
8.45
13.87
19.78
76.67
68.78
43.27
38.91
296
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).
Fig. 8. In vitro release proles of AP in ethanol at ambient temperature from APloaded 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 mechanism 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 electrostatic 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 interaction 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.
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).