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Iranian Polymer Journal
20 (5), 2011, 445-456
alginate;
drug delivery system;burst release;
ionic gelation;
tripolyphosphate.
(*) To whom correspondence to be addressed.
E-mail: [email protected]
A B S T R A C T
Key Words:
Reinforcement of Chitosan Nanoparticles Obtained
by an Ionic Cross-linking Process
Morteza Hasanzadeh Kafshgari, Mohammad Khorram*, Mobina Khodadoost,
and Sahar Khavari
Department of Chemical Engineering, University of Sistan and Baluchestan,
P.O. Box: 98161/164, Zahedan, Iran
Received 22 February 2010; accepted 27 November 2010
The reduction in burst release of a new drug nanocarrier system was achieved by
ionic gelation of nanoparticles made of chitosan and tripolyphosphate. Capability
of calcium alginate for reinforcement of the chitosan-tripolyphosphate nano-
particle matrix was studied. Bovine serum albumin (BSA) was loaded into nanoparticles
as a model drug. The final particle size, polydispersity index, entrapment efficiency and
the release rate of BSA were optimized in relation to variations in parameters such as
sodium alginate concentration, theoretical loading of BSA and degree of deacetylation
of chitosan. It was found that increases in sodium alginate concentration resulted in
smaller particle size, higher polydispersity index, lower entrapment efficiency and lower
release rate of BSA. The same parametric changes were observed for the theoretical
loading of BSA, with the exception of drug release which was followed by higher rate.
Nanoparticles of chitosan-tripolyphosphate with a high degree of deacetylation led to
production of smaller particle size, higher polydispersity index and entrapment
efficiency, and a faster drug-release profile. The experimental data of this study
revealed that burst release decreased significantly in reinforced chitosan-tripoly-
phosphate nanoparticles.
INTRODUCTION
Application of chitosan, a cationicpolysaccharide, has been exten-
sively investigated in various fields
of applications, such as biotechno-
logy [1], as a pharmaceutical
excipient in oral drug formulations
[2], waste water treatment [3], cos-
metics [4] and food science [5].
These broad fields of applications
are due to chitosan's unique set of
properties. Favourable biologicalproperties [6], low toxicity [7],
biodegradability [8], proper
mucoadhesive properties [9], abs-
orption enhancer across intestinalepithelium and mucosal sites for
drugs, peptides and proteins [10],
metal complexation [11], antimi-
crobial activity [12], good homo-
static properties [13], and accept-
able anti-cancerous properties [14]
are the most important set of prop-
erties. Among all applications of
chitosan drug delivery systems are
widely recognized as a promisingresearch field [15]. Tablets, micro-
spheres, microgels and nano-
particles are the different forms
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of chitosan-based drug delivery devices that have
been studied. In the past decade, chitosan nano-
particles have been extensively investigated because
they can control drug release rate, prolong the
duration of therapeutic effectiveness, and deliverdrugs to their specific sites in the body [16]. Chitosan
nanoparticles exhibit superior activity that can be
attributed to their small and quantum size
effect [17].
Common methods to prepare chitosan nano-
particles are ionic gelation, coacervation or precipi-
tation, emulsion-droplet coalescence, reverse
micellar, and self-assembly chemical modification
[14]. The ionic gelation process is commonly used to
prepare chitosan nanoparticles because it is a verysimple and mild method [18]. This process can be
performed either by chemical or physical cross-
linking. It is worth noting that chitosan has a high
density of amine groups in its backbone and the
amine groups are protonized to form -NH3+ in acidic
solution. These positively charged groups in chitosan
can be chemically cross-linked with dialdehydes such
as glutaraldehyde [19] and ethylene glycol diglycidyl
ether [20], or physically cross-linked with multivalent
anions derived from sodium tripolyphosphate (TPP)
[21], citrate [22,23] and sulphate [24]. Both
glutaraldehyde and ethylene glycol diglycidyl ether
are toxic and can cause irritation to mucosal
membranes [25]. Physically cross-linked chitosan
gels have been used in drug delivery systems due to
their enhanced biocompatibility over chemically
cross-linked chitosan [26].
Non-toxicity and quick gelling ability of TPP are
the important properties that make it a favourable
cross-linker for ionic gelation of chitosan [14]. In
addition, TPP has been also recognized as an
acceptable food additive by the US Food and Drug
Administration [27]. Moreover, the process of ionic
gelation of chitosan with TPP as a cross-linker is
feasible for the scale-up of entrapment in a particle
processing operation [28]. Chitosan nanoparticles
prepared by TPP as an anionic cross-linker are
homogeneous, and possess positive surface charge
that make them suitable for mucosal adhesion
applications [14].
The properties of ionically cross-linked chitosanare influenced by electrostatic interactions between
the anionic cross-linker and chitosan. This interaction
depends on the variables such as: anionic molecular
structure, its charge density and molecular concen-
tration, pH of chitosan solution, and physical
properties of chitosan, i.e., molecular weight anddegree of deacetylation (DDA) [25]. Several studies
have investigated the effect of these variables on the
properties of ionically produced chitosan, its drug
entrapment efficiency and drug release behaviour
[25].
Advantages of protein delivery systems based on
chitosan-TPP nanoparticles are reported in many
research works performed in the past few years [29].
A shortcoming of chitosan-based nanoparticles as
protein release system is that such particles release30-70% of the protein within 3-6 h placed in release
environment. This is due to the mechanism of burst
release that has been considered as a slow release in
many studies [30].
Burst release management is a major challenge in
the development of drug delivery systems because it
may lead to inefficient delivery and significant
toxicity hazards. The degree of burst release general-
ly depends upon the nature of the polymer, drug
molecular structure and its molecular weight, poly-
mer/drug ratio, relative affinities of the drug and
polymer and the aqueous phase [31].
Many methods, including blending with other
polymers, grafting with monomers, varying the
chemistry of the polymer [32], adding excipients into
polymer phase [33], employing new polymers [34],
encapsulating particulate forms of the drugs into
microparticles, preparation of novel biopolymer/
inorganic material composites [35] and spray/
freezing [36] have been applied to improve the burst
release properties of the polymeric carriers.
Polymer coating is another burst release control
method. Tavakol et al. [37] have studied this method
for N,O-carboxymethyl chitosan (NOCC) beads
coated with chitosan. They produced beads of NOCC
and alginate with ionotropic gelation method and
then, coated the beads with chitosan. The effect of
coating and drying methods on the swelling and
release behaviour of the prepared beads has been
investigated. The results of this work indicate that
burst release has not been observed in chitosancoated beads. Using additional barrier layers is
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naturally an efficient method to manipulate drug
release profiles and reduce the burst release, though it
is inherently an expensive method due to the
additional materials employed and difficulties
associated with controlling the barriers quality. Chenet al. [30] have reported nanoparticles based on
N-trimethyl chitosan chloride (TMC) and TPP. They
used BSA as a model protein entrapped into the
particles with sodium alginate as a modifier of TMC
nanoparticles. They showed that burst release of BSA
can be reduced from 60% to 45%.
The main objective of this work was to reduce the
burst release of BSA-loaded chitosan nanoparticles
prepared by a one-step simple method. Very few
studies have focused on high loadings (10-15%) ofproteins in micro/nanospheres with low burst release.
Due to the nature of BSA, an optimum BSA delivery
system must undergo burst release as low as possible
while keeping drug loading as high as possible. The
design of an optimum system is the main objective in
this study. Comparison of the release test results of the
current work with other similar works clearly shows
higher efficiency of BSA-loaded chitosan nano-
particles by the method developed in this study. While
this work and the study carried out by Chen et al. [30]
are both using ionic gelation method, two important
differences are to be taken into account. Chen et al.
[30] usedN-trimethyl chitosan as a polymeric carrier
without any ionotropic cross-linker. In this work
chitosan is used as a polymeric carrier and CaCl2 as
an ionotropic cross-linker to further reinforce the
chitosan/alginate composite matrix. In order to
decrease burst release, chitosan nanoparticles with
two different degrees of deacetylation were modified
using calcium alginate, and the effects of sodium
alginate concentration on particle size, entrapment
efficiency and BSA release profile were investigated.
EXPERIMENTAL
Materials
Medium molecular weight chitosan (Fluka, The
Netherlands) was used as an encapsulating polymer.
According to its specification analysis, the molecular
weight was 400 kDa. The degree of deacetylation wasabout 83% as determined by potentiometric titration.
Alginic acid as its sodium salt (medium viscosity,
3500 cps, 2% (w/v) aqueous solution at 25C from
Sigma-Aldrich, The Netherlands) was used as a rein-
forcing polymeric agent. Sodium tripolyphosphate as
ionic cross-linker was supplied by Sigma-Aldrich.Calcium chloride was provided by Merck Chemical
Co., Germany, and used as ionotropic cross-linking
agent. As a model protein molecule for entrapment
and control release studies, BSA was obtained from
Merck Chemical Co., Germany. The other chemicals
were analytical grade reagents and used as received.
Purification of Chitosan
The chitosan was dissolved in 2% (v/v) acetic acid
solution which was filtered to remove the residues ofinsoluble particles. A two-molar solution of NaOH
was added to the filtrate to obtain purified chitosan in
the form white precipitate. The precipitated chitosan
was washed thoroughly using double distilled water,
and then dried at 40C for 48 h. Further deacetylation
of chitosan occurred during the purification process.
Determination of Chitosan Degree of Deacetylation
The degree of deacetylation numerical values of the
unpurified and purified chitosan samples were
determined by potentiometric titration of chitosan
(25 mL of 0.2 M HCl) against 0.1 M NaOH using the
following equation [38]:
(1)
where, V is the volume of alkali consumed in the
titration of amino groups present in the chitosan
samples, CNaOH is the concentration of sodium
hydroxide used for titration, MChitosan is the mass of
chitosan sample, and 16 and 0.0994 are the molecular
weight and theoretical molar mass of the amino
groups, respectively.
Preparation of Chitosan Nanoparticles
Chitosan nanoparticles were prepared on the basis of
ionic gelation of chitosan in presence of TPP.
Unpurified and purified medium molecular weight
chitosan were dissolved in 1% (v/v) acetic acid
solution at the final concentration of 0.5% (w/v). Two
different concentrations of 20% and 50% (w/w) BSA,based on chitosan and alginate, were added each into
447Iranian Polymer Journal / Volume 20 Number 5 (2011)
Reinforcement of Chitosan Nanoparticles Obtained ...Hasanzadeh Kafshgari M et al.
0994.0
1610(%)
3
=
Chitosan
NaOH
M
CVDDA
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a separate chitosan solution prior to nanoparticles
formation. The pH of chitosan-BSA solution was
adjusted to 5.9 by 2 N NaOH solution. Then, 10 mL
of TPP solution (0.5% w/v) prepared in double
distilled water was added into the chitosan-BSAsolution (50 mL) through an insulin syringe needle at
the speed of 60 mL/h under magnetic stirring at room
temperature. The pH of chitosan-BSA solution
reached 5.5 after adding TPP solution. The procedure
described so far results in producing unreinforced
chitosan nanoparticles.
Reinforced chitosan nanoparticles were prepared
by the same procedure, except that various amounts
of sodium alginate (10% and 20% w/v) were
dissolved in the TPP solution before adding thechitosan-BSA solution. Chitosan nanoparticles were
formed upon adding the TPP solution to chitosan-
BSA solution and mixing. After ten minutes of
mixing these two solutions, 0.9 g calcium chloride
was added to the suspension which was stirred
for 20 min. Finally, the nanoparticles were
isolated by centrifugation at 6000 rpm for 45 min.
The supernatant was stored for further analysis.
The precipitate was suspended in double distilled
water, centrifuged again and dried at 37C. The
dried chitosan nanoparticles were suspended
in milli-Q distilled water for characterization tests
where appropriate; or directly used for in vitro
Table 1. Formulations of the prepared chitosan-TPP
samples loaded with BSA.
release experiments.
Formulations of all chitosan-TPP samples that
were used to investigate the effect of BSA loading,
alginate concentration, and degree of deacetylation
on nanoparticles and in vitro release profiles arepresented in Table 1.
Characterization Tests of Nanoparticles
The chemical interactions between chitosan,
alginate, TPP and BSA were established through
FTIR spectrometry. FTIR Spectra of chitosan, TPP
and BSA prepared nanoparticles were recorded on a
460 Plus Jasco Inc FTIR spectrophotometer
(Maryland, USA) within KBr pellets. The instrument
was operated with resolution of 4 cm-1
andfrequency range of 400-4000 cm-1.
The mean particle size and size distribution of
nanoparticles were determined by dynamic light
scattering (DLS) using a Malvern Zetasizer Nano-
ZS-ZEN 1600 (Malvern Instruments Co., UK). The
analysis was carried out at a scattering angle of 90 at
a temperature of 25C using nanoparticles dispersed
in de-ionized distilled water. Every sample measure-
ment was repeated 10 times. Particle size distribution
of the nanoparticles is reported as a polydispersity
index (PDI). The morphology of dried nanoparticles
was observed by transmission electron microscopy
(TEM). For TEM analysis, about 3 mL ethanol was
added to the prepared chitosan nanoparticles, and
dispersed in Sonicator (Power Sonic 405, Hwashin
Technology Co., Korea) for 5 min, and one droplet of
the nanoparticles solution was dripped onto copper
grids. The samples were dried by natural air flow
and then analyzed using TEM without any further
treatment or coating.
To determine the entrapment efficiency (EE) of
BSA into nanoparticles, the amount of free BSA in
supernatant was analyzed with UV-Vis spectro-
photometer at 595 nm using the Bradford protein
assay. The supernatant of non-loaded chitosan (DDA:
83.5% or 96.4%) nanoparticle suspension was used
as a blank. The BSA entrapment efficiency (EE) was
calculated using the following equation:
(2)
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inBSAfreeaddedBSAtotalEE = [((%)
100]/) addedBSAtotaltsupernatan
Sample Alginate concentration
(w/v%)
Theoretical
loading (w/w%)
DDA
(%)
III
III
IV
V
VI
VII
VIII
IX
X
XI
XII
0.00.1
0.2
0.0
0.1
0.2
0.0
0.1
0.2
0.0
0.1
0.2
2020
20
50
50
50
20
20
20
50
50
50
83.5483.54
83.54
83.54
83.54
83.54
96.38
96.38
96.38
96.38
96.38
96.38
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In Vitro Drug Release
The dried BSA-loaded nanoparticles were resuspend-
ed in 25 mL of 0.1 M phosphate buffer solution (PBS,
pH 7.4), then incubated at 37C while stirred at
100 rpm. At specific time intervals, 0.5 mL of thesample was removed and replaced with fresh PBS.
The withdrawn samples were centrifuged at
6000 rpm for 5 min. The BSA released from the
nanoparticles and present in the supernatant was
measured by Bradford protein assay. The blank
sample consisted of non-loaded chitosan nano-
particles resuspended in PBS. Triplicate samples were
analyzed for each measurment.
RESULTS AND DISCUSSION
Chitosan TPP nanoparticles are formed by ionic
cross-linking (ionic gelation) of oppositely charged
ions of chitosan and TPP. However, the preparation of
reinforced chitosan-TPP nanoparticles has been
attributed to the three following phenomena: (a) ionic
gelation technique between positively charged
chitosan and negatively charged TPP and BSA, (b)
electrostatic interaction between the positively
charged -NH3+ of chitosan and negatively charged
-COO- of alginate, and (c) ionotropic gelation process
between negatively charged alginate and calcium ion
(Ca+2) [39]. In this study, calcium ion is used as a
cross-linking agent to strengthen and stabilize the
particles.
The ratio between chitosan and TPP is critical and
does control the size and PDI of the nanoparticles. It
is shown that by increasing the chitosan/TPP ratio,
smaller nanoparticles can be produced [16]. TPP is a
polyfunctional cross-linking agent and can create five
ionic cross-linking points with amino groups of
chitosan. Thus, in this study the ratio of 5/1 (w/w) was
selected as a suitable chitosan/TPP ratio [16].
The ionic interaction between chitosan and TPP is
dependent on the solution pH. This is due to the
variation in ionization degree of chitosan and TPP.
Other researchers [25] showed that a pH range 3-6
was a suitable range for ionic gelation of chitosan
with TPP. The pH of 5.9 is selected in the present
work because BSA (PI = 4.8) is negatively charged atthis pH and electrostatic interaction between the
negatively charged BSA and positively charged
chitosan causes greater entrapment of BSA into the
nanoparticles.
FTIR was used to confirm the incorporation of
calcium alginate into the chitosan matrix and loadingof BSA in the prepared nanoparticles. The FTIR
spectra of chitosan, alginate, BSA, BSA-loaded
chitosan nanoparticle (sample IV) and BSA-loaded
chitosan-alginate nanoparticles (sample VI) are
presented in Figure 1.
Three characteristics absorption bands observed in
chitosan (spectrum a in Figure 1), at 3413, 1672 and
1317 cm-1, as in the given order are due to the N-H,
amide I and amide III groups present in chitosan.
According to Yuan et al. [40] the peak of 3413 cm-1
corresponds to stretching vibration of N-H in pure
chitosan. It is noticeable that this peak has shifted to
lower wavenumbers centered at 3395 cm-1 and it is
broader and stronger in chitosan particles (samples IV
and VI, spectra d and e in Figure 1). This is an
evidence of molecular interaction between these
Figure 1. FTIR Spectra of: (a) chitosan, (b) alginate, (c)
BSA, (d) BSA-loaded chitosan nanoparticle (sample IV) and(e) BSA-loaded chitosan-alginate nanoparticle (sample VI).
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groups and sodium tripolyphosphate and also due to
hydrogen bonding involvement. Primary amines also
show sharp peak between 3500 and 3400 cm-1 which
could be attributed to the asymmetric and symmetric
stretching of the N-H bonds [40]. The peak in purechitosan and chitosan particles appears broad in this
region due to the contribution of O-H stretching peaks
and hydrogen bonding [41].
FTIR Spectra of BSA showed peaks around 1655,
1534 and 3309 cm-1, reflecting the acetylamino I,
acetylamino II and (NH2) groups, respectively [42].
Acetylamino I at 1655 cm-1 and acetylamino II at
1534 cm-1 in BSA (spectrum c in Figure 1) overlapped
1672 cm-1 of amide I and 1597 cm-1 in chitosan, so
more intensive peaks appeared for the BSA-loadedchitosan nanoparticles (sample IV, spectrum d in
Figure 1).
The characteristic peaks of sodium alginate
included O-H at 3397 cm-1, COO- (asymmetric) at
1617, COO- (symmetric) at 1429 and 1028 cm-1 for
C-O-C stretching. For the BSA-loaded chitosan
nanoparticle of sample IV, the stretching vibration of
-OH and -NH2 at 3392 cm-1 became broader.
Spectrum e in Figure 1 is an indication of intra-
molecular and intermolecular hydrogen bonds which
were formed and enhanced between chitosan and
alginate molecules [43].
The mean particle size, PDI and EE of the
prepared samples are presented in Table 2. PDI is a
Table 2. Mean particle size and entrapment efficiency of
chitosan-TPP nanoparticles loaded with BSA.
measure of homogeneity in dispersed systems and
ranges from 0 to 1. Homogeneous dispersion has PDI
value close to zero while PDI values greater than 0.3
suggest high heterogeneity [44]. Except the three
samples (samples I, IV and XII) other prepared
nanoparticles have PDI values lower than 0.3
(Table 2).
Morphological studies of nanoparticles were
conducted by TEM. The TEM images such as those
presented in Figure 2 indicate that nanoparticles are
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450 Iranian Polymer Journal / Volume 20 Number 5 (2011)
Sample Mean particle
size (nm)
PDI Entrapment
efficiency (%)
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
396
116
459
615
255
459
295
255
190
459
220
122
0.162
0.572
0.188
0.179
0.201
0.226
0.075
0.123
0.631
0.274
0.261
0.523
70.54
71.55
75.14
60.97
59.48
55.4
84.55
75.21
71.12
80.74
69.16
68.56
(a) (b)
Figure 2. TEM Photographs of samples: (a) IV and (b) VI.
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not spherical in shape. The size of nanoparticles
based on TEM was smaller than the size determined
by DLS. This can be attributed to the dehydration of
the nanoparticles in the process of sample preparation
required for TEM. It is worth noting that DLS givesthe size of swollen particles. Swelling ratio of some
selected samples of the nanoparticles was measured
according to the method proposed by Denkbas et al.
[45].
About 0.050 g of the nanoparticles was inserted
into a tube with a diameter of 5 mm with a height of
100 mm. The level of the beads in the tube was
marked before filling it with 1 mL of distilled water
and the tube was left for 24 h. After 24 h, the height
of the beads in the solution was measured. Thepercentage of swelling was calculated based on the
following equation:
(3)
where S is the percentage of swelling, ht is the height
of swollen beads (cm) at specific time t and ho is the
initial height of the beads (cm).
The obtained results for samples IV, V and VI
based on three independent measurements for eachsample were 119.3% 20.3, 223% 67 and 146.3%
18.8, respectively.
Effect of Alginate Concentration on
Physicochemical Properties of Nanoparticles
As can be seen in Table 2, mean particle size
decreases as sodium alginate concentration increases.
By contrast, PDI of the prepared nanoparticles
increases as more sodium alginate was added to the
gelation system. In ionic gelation process, TPP as a
cross-linking agent forms hydrogen bond with free
amine groups of BSA and chitosan. Sodium alginate
competes with TPP to form electrostatic complex
with chitosan and BSA. While smaller mean particle
size can be attributed to strong inter-chain reactions
between chitosan and alginate, as more sodium
alginate causes local aggregation and increases PDI
[30]. In other words, the reduction in particle size
determined by DLS at swollen state in presence of
calcium alginate in the network is due to higher
degree of cross-linking of the whole network. Higher
concentration of sodium alginate results in reduced
entrapment efficiency (Table 2). This may be due to
competition of anionic alginate and BSA to form
interchain bonds with cationic chitosan.
BSA Loading in Relation to the Physicochemical
Properties of Nanoparticles
The general pattern which can be deduced from the
experimental data given in Table 2 is that, lowering
theoretical loading of BSA leads to formation of
smaller nanoparticles. BSA loading of nanoparticles
is based on two different phenomena such as
entrapment into particles and adsorption on the
particle surface. As mentioned earlier, BSA
molecules are negatively charged under the solution
conditions used in this study. BSA entrapment intoparticles is due to interactions between oppositely
charged chitosan and BSA, and formation of
hydrogen bonds between the TPP and BSA.
Additional adsorption of BSA on particle surface may
also occur [14] that leads to higher mean particle size.
PDI of the prepared nanoparticles increases with
theoretical BSA of higher loading. Also, the increase
of the particle size and PDI by increased BSA can be
attributed to the increased viscosity of the chitosan/
BSA solution and ionic interaction, respectively. Fewdata from Table 2 regarding the effect of BSA loading
on entrapment efficiency of nanoparticles are
presented in Figure 3. Sample preparation for
samples I and IV were the same except for the
theoretical BSA loading that was higher for sample
IV compared to sample I. The same sample prepara-
tion was applied to sample pair VIII and XI. It can be
Figure 3. Effect of theoretical BSA loading on entrapment
efficiency.
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=
o
ot
h
hhS
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concluded from Figure 3 that higher theoretical BSA
loading results in lowering entrapment efficiency. It is
worth noting that data from Table 2 other than those
presented in Figure 3 also support this conclusion.
The decrease of entrapment efficiency with initiallyincreased drug content is due to increased chemical
potential of the drug in chitosan/BSA solution. This
conclusion is in agreement with reported studies such
as the one carried out by Xu et al. [46] and it is in con-
trast to other research reports attesting the opposite
effect of drug loading on entrapment efficiency [14].
DDA in Relation to the Physicochemical Properties
of Nanoparticles
The effects of degree of deacetylation on particle sizeand entrapment efficiency of BSA were investigated
and the obtained experimental data are presented in
Table 2. The data show that samples which were
prepared with higher DDA chitosan (purified
chitosan, DDA 96.4%) have smaller mean particle
size than those prepared with unpurified chitosan
(DDA 83.5%).
In this study, purification process was conducted
by NaOH. It is anticipated that further deacetylation
of chitosan molecules may have occurred under
strong caustic condition. This means that more amine
groups are presented in purified chitosan of high
DDA, which can be transformed into -NH3+ at acidic
solution. The ionic gelation process is dependent on
the number of positively charged groups (-NH3+) left
on the dissolved chitosan molecules. Due to sufficient
positively charged groups, the process of nano-
particles production occurs more easily for chitosan
with high DDA leading to smaller particles [47].
Entrapment of negatively charged BSA molecules is
also occurring more efficiently in the presence of
positively charged amine groups. Sample preparation
for samples I and VII was the same except for the
DDA that was higher for sample VII compared to
sample I. The same sample preparation is applicable
to sample pairs II-VIII, III-IX, IV-X, V-XI and VI-
XII. The presented data in Table 2 for these pairs
reveal that entrapment efficiency of BSA increases at
higher DDA.
In Vitro Release StudyExperimental data on BSA release studies are
presented in Figure 4. The data show that both BSA
release rate and especially burst release are reduced
significantly with higher sodium alginate concentra-
tion used in preparation of nanoparticles. Sustained
drug release from the nanoparticles is important inmany fields of applications. BSA release follows a
biphasic pattern, characterized by initial burst release
followed by a period of slower release. According to
the presented data in Figure 4, fractional BSA
released for sample IV (chitosan-TPP nanoparticles
without alginate) and sample VI (reinforced chitosan-
TPP with alginate) in 30 min are 10.5% and 3.5%,
respectively. Also, these quantities in 6 h reach 14%
and 11.8% for samples IV and VI, respectively. In
addition, fractional drug released for sample IV is35.7% at 98 h while this quantity for sample VI is
26.8% at 98 h. These results prove that reinforcement
of chitosan-TPP nanoparticles with CaCl2 can
reduce both burst release and release rate of BSA.
Reinforcement of chitosan-TPP matrix with
calcium alginate may be considered the cause for
lower burst effect and slower release rate.
Modification of N-trimethyl chitosan chloride
(TMC)-TPP matrix by sodium alginate studied by
Chen et al. [30] also showed that sodium alginate
demonstrates the same result on burst effect and
release rate as calcium alginate in this study. The
amine groups in chitosan interact with TPP through
ionic bonding. The interaction between chitosan
matrix and alginate and ionotropic gelation of sodium
alginate with CaCl2 in reinforced nanoparticles may
Figure 4. Effect of sodium alginate concentration on BSA
release profile from samples: () IV, () VI and (S) XIII.Release medium: T = 37C0.2, pH 7.4.
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also contribute to enhanced cross-linking density of
the matrix. Increased cross-linking density may lead
to lower diffusion of BSA from the reinforced matrix
which consequently decreases the burst release and
rate of release. To investigate the effect of sodiumalginate on drug release profile, without its
conversion to calcium alginate network, a new sample
designated as XIII, was prepared. Preparation of
samples XIII and VI were the same except that CaCl2was not used in preparation of sample XIII. The
release profile of this sample is shown in Figure 4. As
can be seen, the burst release and release rate of BSA
is lower in sample VI compared to sample XIII.
Fractional BSA release profiles for two theoretical
BSA loadings, 20% and 50% corresponding toloading capacity of 4.13% 0.04 and 9.75% 0.32,
are presented in Figure 5. Release profiles show that
when loading capacity of BSA increases from 4.13%
0.04 to 9.75% 0.32, total BSA release at 6 h
increases from 7.5% to 15.8%. The nanoparticles
prepared with higher loading capacity of BSA have
greater overall release. At higher BSA loading, BSA
binding on chitosan molecule is weaker, and some of
the loaded BSA molecules are adsorbed onto the
surface of particles. Weak binding and adsorption of
BSA on the particle surfaces leads to higher release
rate. This result is in agreement with reports by other
researchers [14].
Effect of DDA on the release profile is shown in
Figure 6. The results show that BSA release rate and
burst release increase with higher DDA. As it was
Figure 5. Effect of theoretical BSA loading on BSA release
profile from samples: () IX and () XII. Release medium:T = 37C0.2, pH 7.4.
Figure 6. Effect of chitosan degree of deacetylation on BSA
release profile from samples: () VI and () XII. Release
medium: T = 37C0.2, pH 7.4.
stated above, higher DDA causes significant
reduction in mean particle size, thus increasing the
ratio of surface area to volume of nanoparticles, that
consequently increases the release rate and burst
release.
CONCLUSION
Reinforced chitosan-TPP nanoparticles loaded with
BSA were prepared by ionic gelation. Calcium
alginate was used as a reinforcing agent. Size of the
prepared nanoparticles ranged from 116 to 615 nm
with PDI lower than 0.3. TEM Analysis demonstrated
that nanoparticles were not spherical. At higher
sodium alginate concentration smaller mean particle
size was obtained. High concentration of sodium
alginate solution resulted in lower entrapment
efficiency. Higher degree of deacetylated chitosan
lowered particle size and improved the entrapment
efficiency. This study showed that reinforcement of
chitosan-TPP nanoparticles with calcium alginate sig-
nificantly lowered the burst release and consequently
the BSA release rate. Increased theoretical loading of
BSA and degree of deacetylation of chitosan led to
increased burst release and release rate.
ACKNOWLEDGEMENT
The authors are grateful to the financial supports from
Reinforcement of Chitosan Nanoparticles Obtained ...Hasanzadeh Kafshgari M et al.
Iranian Polymer Journal / Volume 20 Number 5 (2011) 453
http://www.sid.ir/7/29/2019 Iranian Journal Chitosan
10/12
the Vice-Chancellery for Research and Technology
Affairs of University of Sistan and Baluchestan.
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