Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

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Colloids and Surfaces B: Biointerfaces

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

Formation mechanism of monodisperse, low molecular weight chitosan

nanoparticles by ionic gelation technique
Wen Fan a,b,1 , Wei Yan b,1 , Zushun Xu b , Hong Ni a,
a
b

School of Life Science, Hubei University, Wuhan 430062, China

Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan 430062, China

a r t i c l e

i n f o

Article history:
Received 1 September 2011
Received in revised form
16 September 2011
Accepted 19 September 2011
Available online 2 October 2011
Keywords:
Low molecular weight chitosan
Nanoparticles
Monodisperse
Ionic cross-linking
Drug carrier

a b s t r a c t
Chitosan nanoparticles have been extensively studied for drug and gene delivery. In this paper, monodisperse, low molecular weight (LMW) chitosan nanoparticles were prepared by a novel method based on
ionic gelation using sodium tripolyphosphate (TPP) as cross-linking agent. The objective of this study was
to solve the problem of preparation of chitosan/TPP nanoparticles with high degree of monodispersity and
stability, and investigate the effect of various parameters on the formation of LMW chitosan/TPP nanoparticles. It was found that the particle size distribution of the nanoparticles could be signicantly narrowed
by a combination of decreasing the concentration of acetic acid and reducing the ambient temperature
during cross-linking process. The optimized nanoparticles exhibited a mean hydrodynamic diameter of
138 nm with a polydispersity index (PDI) of 0.026 and a zeta potential of +35 mV, the nanoparticles had
good storage stability at room temperature up to at least 20 days.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Chitosan is a linear polysaccharide composed of randomly
distributed -(1-4)-linked d-glucosamine and N-acetyl-dglucosamine. Due to the advantageous biological properties
of chitosan, such as relative non-toxicity, biocompatibility,
biodegradability, cationic properties, bioadhesive characteristics
and permeability-enhancing properties, chitosan-based particles
have been extensively studied for delivery of anti-cancer agents,
therapeutic proteins, genes, antigens, and so on [13]. In recent
years, low molecular weight (LMW) chitosan nanoparticles have
shown great potential in the applications of drug delivery and
non-viral vector for gene delivery [46]. This is because, compared
with high molecular weight (HMW) chitosan, LMW chitosan shows
better solubility, biocompatibility, bioactivity, biodegradability
and even less toxicity [79]. Furthermore, many studies have
emphasized the signicance of size and revealed the advantages
of nanoparticles over the microspheres [10].
Among variety of methods developed to prepare chitosan
nanoparticles, ionic gelation technique has attracted considerable
attention due to this process is non-toxic, organic solvent free, convenient and controllable [11]. Ionic gelation technique is based

on the ionic interactions between the positively charged primary

amino groups of chitosan and the negatively charged groups of
polyanion, such as sodium tripolyphosphate (TPP), which is the
most extensively used ion cross-linking agent due to its non-toxic
and multivalent properties [12]. This physical cross-linking process
not only avoids the use of chemical cross-linking agents and emulsifying agents which are often toxic to organisms, but also prevents
the possibility of damage to drugs, particularly biological agents
[13].
However, chitosan/TPP nanoparticles prepared by conventional
methods usually have a wide particle size distribution and poor
stability, which limit their usefulness in certain applications. Up to
now, it is still a challenge to prepare chitosan/TPP nanoparticles
with high degree of monodispersity and stability by a convenient and effective method. Thus, in this study, we choose to use
LMW chitosan with high degree of deacetylation, and focus on the
reproducible preparation of chitosan/TPP nanoparticles with those
desirable characteristics, which wished to promote the development of chitosan/TPP nanoparticles in the applications of drug and
gene delivery.
2. Materials and methods
2.1. Materials

Corresponding author. Tel.: +86 27 88661237; fax: +86 27 88661571.

E-mail address: nihongcs@gmail.com (H. Ni).
1
These two authors contributed equally to this work.
0927-7765/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2011.09.042

Low molecular weight (LMW) water-soluble chitosan (viscosity

35 cps, deacetylation degree 91.5%) derived from crab shell, was

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

purchased from Jinhu Crust Product Co., Ltd., China. Sodium

tripolyphosphate (TPP), glacial acetic acid, sodium hydroxide
and all other chemicals were analytical grade, purchased from
Sinopharm Chemical Reagent Co., Ltd., China. Ultrapure water was
used throughout this study.

2.2. Preparation of LMW chitosan/TPP nanoparticles

LMW chitosan nanoparticles were prepared according to
a modied method of Calvo et al. [14], based on the ionic
gelation of chitosan with TPP anions. Based on an optimization procedure designed by us, a number of parameters were
investigated by changing one parameter while keeping the
others constant. These varying parameters included concentration of chitosan solution, concentration of TPP solution,
temperature of chitosan solution, pH of chitosan solution, mass
ratio of chitosan to TPP, concentration of acetic acid solution,
ambient temperature during cross-linking process and stirring
speed.
The optimization procedure was as follows: LMW chitosan was
dissolved in an aqueous solution of acetic acid to form a 0.5 mg/mL
chitosan solution. The concentration of acetic acid was 0.4 times
(0.2 mg/mL) that of chitosan. The chitosan solution was stirred
overnight at room temperature using a magnet stirrer. The pH
of the resulting solution was around 3.6 and this was adjusted
to 4.74.8 using 20 wt% aqueous sodium hydroxide solution. The
chitosan solution was then passed through a syringe lter (pore
size 0.45 m, Millipore, USA) to remove residues of insoluble
particles. TPP was dissolved in ultrapure water at a concentration of 0.5 mg/mL and also passed through a syringe lter (pore
size 0.22 m, Millipore, USA). To prepare chitosan nanoparticles,
a magnetic stirrer was placed in a chest freezer, in which the
ambient temperature was controlled at 24 C, temperature uctuations and ow of cold air should be avoided as much as possible.
Ten milliliters of chitosan solution in a 25 mL round-bottom ask
was preheated in a water bath at 60 C for 10 min, the ask was
then placed on the magnetic stirrer stirring at 700 rpm, 3.0 mL
of 24 C TPP solution was quickly added to the chitosan solution with a plastic Pasteur pipette. The reaction was carried out
for 10 min and the resulting suspension was subjected to further
analysis.

2.3. Characterization and morphology of LMW chitosan/TPP

nanoparticles
The z-average particle size, particle size distribution and polydispersity index (PDI) of the chitosan/TPP nanoparticles were
measured at 25 C by dynamic light scattering (DLS) on a high
performance particle sizer (HPPS-5001, Malvern, UK). The zeta
potential values of the nanoparticles were performed using a Zetasizer 3000 HAS (Malvern, UK). The samples were left standing
overnight at room temperature for stabilization and were then
inverted several times before the measurements. Mean values were
obtained from the analysis of three different batches, each of them
measured three times.
The morphological characteristics of the nanoparticles were
examined using a high resolution transmission electron microscope (TEM, Tecnai G20, FEI, Netherlands). A droplet of suspension
was placed on a carbon lm-covered copper grid (200 mesh)
without being stained. Five minutes later, the excess liquid was
removed by touching the edge of the copper grid with a piece of
lter paper. The sample was then air-dried before observation by
TEM.

3. Results and discussion

3.1. Effect of the concentration of chitosan solution or TPP
solution
The characteristics of the chitosan/TPP particles prepared with
different concentrations of chitosan or TPP were studied. The
results indicated that the particle size increased with increasing
the concentration of either chitosan or TPP. Calvo et al. [14] found
that the formation of chitosan/TPP nanoparticles was only possible
for some specic concentrations of chitosan and TPP. This fact was
also veried in our study that in order to avoid the formation of any
micro-particles, the concentration of chitosan or TPP needed to be
below 1.5 mg/mL and 1.0 mg/mL, respectively. In these concentration ranges, it seemed that the concentration of chitosan or TPP had
little effect on the monodispersity of the nanoparticles, since their
PDI values were all below 0.05.
It is known that under acidic conditions, there is electrostatic
repulsion between chitosan molecules due to the protonated amino
groups of chitosan, meanwhile, there also exist interchain hydrogen
bonding interactions between chitosan molecules. Below a certain concentration of LMW chitosan (2.0 mg/mL as reported), the
intermolecular hydrogen bonding attraction and the intermolecular electrostatic repulsion are in equilibrium [15]. Therefore, in
this concentration range, as chitosan concentration increases, chitosan molecules approach each other with a limit, leading to a
limited increase in intermolecular cross-linking, thus larger but still
nanoscale particles are formed. Above this concentration, microparticles are easily formed probably due to the stronger hydrogen
bonding interactions leading to plenty of chitosan molecules
involved in the cross-linking of a single particle. The formation
of micro-particles usually leads to a occulent precipitate due to
the electrostatic repulsion between particles is not sufcient to
maintain the stability of these large particles.
It was also found that chitosan at low concentration could form
stable nanoparticles even at a low mass ratio of chitosan to TPP,
while chitosan at higher concentration could only form stable
nanoparticles at a higher mass ratio of chitosan to TPP. For example,
when the concentration of TPP was xed at 0.5 mg/mL, a chitosan
concentration of 0.5 mg/mL could form stable nanoparticles at a
mass ratio of 3.3:1, while a chitosan concentration of 1.0 mg/mL
would form aggregates at this mass ratio. To explain this phenomenon, it is inferred that as chitosan concentration decreases,
the intermolecular distance increases, thus leading to a decrease in
intermolecular cross-linking between chitosan molecules while an
increase in cross-linking density between chitosan and TPP, namely
an increase in the ratio of moles of TPP to the moles of chitosan
repeating units [13]. This can be utilized to prepare chitosan/TPP
nanoparticles with smaller size, since an appropriate increase in
the mass ratio is conducive to reduce the particle size, which will
be discussed in detail in Section 3.2.
3.2. Effect of the mass ratio of chitosan to TPP
Fig. 1 shows the effect of the mass ratio of chitosan to TPP on
the particle size and zeta potential by adding different volumes of
TPP to 10.0 mL of chitosan solution. With increasing TPP volume
from 2.5 mL to 3.5 mL (mass ratio from 4.0:1 to 2.9:1), the particle size rst gradually decreased from 172 nm to 133 nm and then
increased dramatically to 237 nm. The zeta potential decreased
almost linearly from +39 mV to +26 mV due to the neutralization
of protonated amino groups by TPP anions. Within this mass ratio
range, it seemed that the mass ratio was not the crucial parameter
for monodispersity since the resulting PDI values were all below
0.06. However, a TPP volume of 3.6 mL would lead to a occulent
precipitate.

W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

Fig. 1. Effect of the mass ratio of chitosan to TPP on particle size and zeta potential
by adding different volumes of TPP to 10.0 mL of chitosan solution.

The large positive charge density due to the high degree of

deacetylation and protonation makes chitosan molecules have a
large number of potential cross-linking sites. When TPP volume
was below 2.5 mL, the reaction solution would be a clear solution
without visible opalescence, indicating that the TPP volume was
inadequate to lead to the formation of a cross-linked structure of
chitosan. As TPP volume increased from 2.5 mL to 3.3 mL, the particle size decreased due to increased cross-linking density between
chitosan and TPP. As TPP volume continued to increase from 3.3 mL
to 3.5 mL, it can be inferred that chitosan molecules were almost
fully cross-linked and the excess TPP would lead to more chitosan
molecules involved in the formation of a single nanoparticle, resulting in larger particle size. When TPP volume was 3.6 mL, the low
surface charge density of the particles was no longer able to maintain the stability of these large particles during stirring, resulting in
the precipitation of particles.

3.3. Effect of the pH of chitosan solution

The effect of the pH of chitosan solution on the formation of
chitosan/TPP particles was investigated by adjusting the pH from
3.6 to 5.5 (3.6, 4.0, 4.2, 4.5, 4.7, 5.0, 5.2, 5.5). The results indicated
that the critical mass ratio of chitosan to TPP for the formation of
an opalescent suspension decreased with the pH decreasing. When
the pH was below 4.5, it was not easy to produce nanoparticles with
unimodal particle size distribution, while when the pH was above
5.2, micro-particles were unavoidable present in the suspension.
Chitosan is a weak polyelectrolyte with a pKa around 6.5, the
protonation degree of chitosan is mainly controlled by solution pH.
Shu and Zhu [12] have studied the relationship between the pH
of chitosan solution and the protonation degree of chitosan (the
deacetylation degree and viscosity average molecular weight of
the reported chitosan were 86% and 460,000, respectively). Their
results show that as the pH of chitosan solution increased from
4.7 to 8, the protonation degree of chitosan decreased rapidly from
100% to 0%, indicating that there exists a critical pH above which
chitosan starts to be deprotonated. Meanwhile, the charge number and ionic species of TPP are affected by solution pH. In original
TPP solution (pH 9.7), the concentration of tripolyphosphoric ions
(P3 O10 5 and HP3 O10 4 ) is high but the concentration of hydroxide
ions is also present. The hydroxide ions or tripolyphosphoric ions in
the medium can competitively react ionically with the protonated
amino groups of chitosan by deprotonation or ionic cross-linking,
respectively [12,16]. Moreover, the hydroxide ions are supposed

23

Fig. 2. Effect of the temperature of chitosan solution on particle size.

to preferentially bind to the protonated amino groups due to their

higher mobility [17].
Thus, it is inferred that as TPP solution is mixed with chitosan
solution, when the pH of chitosan solution is below the critical pH,
the hydroxide ions in TPP solution are partially neutralized by the
hydrogen ions in chitosan solution. The remaining hydroxide ions
(if any) have little effect on the protonation degree of chitosan and
the chitosan maintains its fully protonated state. Then the protonated amino groups are linked via tripolyphosphoric ions in spite of
the strong intramolecular electrostatic repulsion. When the pH of
chitosan solution is around the critical pH, the remaining hydroxide ions may have an apparent effect on the protonation degree of
chitosan and the chitosan becomes less extended, which favors the
formation of a particle structure. When the pH of chitosan solution
is above the critical pH, the chitosan is already less protonated and
the remaining hydroxide ions will cause a signicant decrease in
the protonation degree of chitosan and the potential capacity of chitosan to form cross-linking with tripolyphosphoric ions is supposed
to decrease.

3.4. Effect of the temperature of chitosan solution

The effect of the temperature of chitosan solution on the particle
size was studied. As shown in Fig. 2, the particle size displayed a
clear tendency to diminish when the temperature was increased
from 10 C to 60 C, while when the temperature was above 60 C,
the particle size was slightly decreased. When the temperature was
10 C, the PDI value of the resulting particles was 0.112, while when
the temperature was between 25 C and 70 C, the resulting PDI
values were all below 0.05.
It was reported that the intrinsic viscosity of chitosan solution decreases linearly with increasing solution temperature. This
is because as solution temperature increases, the ratio of radius
of gyration of chitosan is decreased and the hydrogen-bonded
hydration water of chitosan is reduced, resulting in an increase
in chitosan chain exibility and a decrease in specic volume
of chitosan molecule, respectively [18]. Both these effects will
probably facilitate the approaching of chitosan molecules and
promote the formation of a compact structure during crosslinking process, resulting in a decrease in particle size. However,
when the temperature is above 60 C, above effects probably tend to a limit, and thus the decrease in particle size is
negligible.

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

Table 1
The characteristics of chitosan/TPP particles prepared at different ambient temperatures.
Ambient temperature ( C)
1625
1015
04
a

Mean particle size range (nm)

Particle size distributiona

PDI

150160
135150
130140

Bimodal
Broad unimodal
Narrow unimodal

>0.2
0.10.15
<0.05

Particle size distribution by intensity.

3.5. Effect of the ambient temperature during cross-linking

process
Table 1 shows the characteristics of chitosan/TPP particles
prepared at different ambient temperatures. From the results, it
seems that low ambient temperature is conducive to improve the
monodispersity of the particles.
Generally, opalescence can be observed within seconds after the
mixing of chitosan and TPP, indicating that the ionic cross-linking
is a rapid reaction. Moreover, after adding a cold solution of TPP
to a hot solution of chitosan, the suspension at different ambient
temperature has a similar sudden drop in temperature (Fig. 3AC).
So it can be assumed that the inuence of ambient temperature
on the formation of chitosan/TPP particles mainly takes effect after
the initial formation of the particles. It is clear that as the temperature of chitosan solution decreases, there are an increase in
specic volume of chitosan molecule and a decrease in chitosan
chain exibility. At low ambient temperature, the suspension has a
faster cooling rate (Fig. 3AC), causing the hydrogen bond interactions between the polar groups of chitosan and water molecules
increase rapidly, resulting in the nanoparticles surrounded by a
hydration layer in time after the formation of the particles, and
thus the probability of collision between particles during stirring
is expected to decrease. Meanwhile, low ambient temperature also
causes the chitosan molecule to become stiff faster, which should
serve to gradually stabilize the structure of the particles. In addition, it was found that a low ambient temperature but with a ow
of cold air would not help to improve the monodispersity. This is
probably caused by the temperature uctuations inside the uid
(Fig. 3D).
3.6. Effect of the concentration of acetic acid
The commonly used concentration of acetic acid used to dissolve
chitosan is 1.5 times that of the concentration of chitosan, or even

higher. As the LMW chitosan we used was readily soluble in ultrapure water, in order to investigate the effect of the concentration
of acetic acid on the formation of chitosan/TPP particles, ve acetic
acid concentrations (0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL
and 0.8 mg/mL) were used to prepare 0.5 mg/mL chitosan solutions,
respectively. The results are shown in Table 2.
Without addition of acetic acid, the chitosan was less protonated and stable nanoparticles suspension could only form at a
high mass ratio of chitosan to TPP (>4.0:1). For example, as the
mass ratio of chitosan to TPP was 5.0:1, the mean particle size, PDI
and peak width of the particles were 315.6 nm, 0.081 and 71.1 nm,
respectively. At an acetic acid concentration of 0.1 mg/mL, the particles exhibited a bimodal particle size distribution, centered around
164 nm and 850 nm, respectively. However, an acetic acid concentration of 0.2 mg/mL would lead to a unimodal and narrow particle
size distribution, indicating that an appropriate increase in the concentration of acetic acid is conducive to enhance the degree of
protonation of chitosan, which increases the potential capacity of
chitosan to form cross-linking with TPP.
When the acetic acid concentration continued to increase to
0.5 mg/mL and 0.8 mg/mL, comparisons of their particle size distributions (by intensity, by number and by volume, respectively)
are shown in Fig. 4. The results indicate that as the acetic acid concentration is increased from 0.2 mg/mL to 0.8 mg/mL, there are not
only more smaller particles formed, but also an increase in the number of larger particles, resulting in a decrease in the monodispersity
of the particles.
As the concentration of acetic acid is increased from 0.2 mg/mL
to 0.8 mg/mL, an increased amount of NaOH should be consumed
to neutralize the excess acid and adjust the chitosan solution to
pH 4.74.8 since this pH is conducive to the formation of chitosan/TPP nanoparticles with a unimodal size distribution, causing
an increase in the ionic strength of the chitosan solution. In dilute
solution, the intermolecular interaction forces are weak because
of the long distance between chitosan molecules, thus the conformation of the molecules is the most important parameter that
determines the physical properties [19]. In low ionic strength solution, the intramolecular electrostatic repulsion effect, also called
the third electroviscous effect dominates, resulting in the chitosan molecules exist in an extended conformation. However, as
the ionic strength increases, the concentration of counter-ions
is raised which screens the protonated amine groups, and thus

Table 2
Effect of the concentration of acetic acid on the characteristics of chitosan/TPP
particles.a

Fig. 3. Cooling curves of chitosan/TPP nanoparticles suspensions reaction at different ambient temperatures: (A) 25 C, (B) 15 C, (C) 4 C and (D) 4 C with a ow of cold
air. (The data were measured by a temperature probe inserted into the suspension,
sampling rate 1 s, resolution 0.06 C.)

Acetic acid (mg/mL)

Size (nm)

Peak widthb (nm)

PDI

0
0.1
0.2
0.5
0.8

Precipitation
196.0
138.1
138.4
137.6

Bimodal
25.4
51.3
67.2

0.204
0.026
0.083
0.124

a
The mass ratio of chitosan to TPP was 3.3:1 and the pH of chitosan solution
was adjusted to 4.74.8, except when the acetic acid concentration was 0 mg/mL, of
which pH remained unchanged.
b
The values were obtained from the particle size distribution by intensity and
were given by the HPPS.

W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

25

aggregation of particles into larger, resulting in a broader particle

size distribution. So it can be concluded that a high acetic acid concentration (or in other words, a high ionic strength) is not conducive
to a narrow particle size distribution.
3.7. Effect of the stirring speed
We had also investigated the effect of stirring speed on particle
size distribution. The results showed that the particle size distribution was signicantly narrowed by increasing stirring speed from
200 rpm to 800 rpm. However, although a continued increase in
stirring speed would lead to an even narrower main peak, there
was also an aggregate formed at around 1000 nm. This probably
because adequate stirring can accelerate the dispersion of TPP in
chitosan solution and the increased shear force is help to improve
the monodispersity, while intense stirring may destroy the repulsive force between particles and lead to aggregation of particles.
3.8. Morphology of LMW chitosan/TPP nanoparticles

Fig. 4. Particle size distributions (A) by intensity, (B) by number and (C) by volume
of chitosan/TPP nanoparticles prepared with different acetic acid concentrations.

the intramolecular electrostatic repulsion force decreases, which

makes the molecules contracted [20].
Therefore, the increased acetic acid concentration indirectly
causes an increase in the ionic strength, the less extended chitosan molecules and the increased shielding effect of counter-ions
(CH3 COO ) make chitosan molecules have less cross-linking points
to be accessed by TPP. Meanwhile, chitosan molecules with a contracted worm like chain conformation entangle harder than those
with an extended conformation [15]. For these reasons, fewer chitosan molecules involved in the formation of a single nanoparticle
and thus a greater number of smaller nanoparticles are formed
(Fig. 5). On the other hand, the increased shielding effect will
decrease the electrostatic repulsion between particles, and moreover, the increased electrolyte ions will reduce the thickness of the
surface hydration layer of the particles [21], which facilitate the

The typical morphology of the chitosan/TPP nanoparticles prepared at optimal conditions is shown in Fig. 6. The nanoparticles
exhibited a spherical shape and had a narrow particle size distribution with size in the range of 3050 nm. The discrepancy in the
size of chitosan nanoparticles between DLS and TEM can be that
chitosan nanoparticles swell in aqueous media and DLS gives a
hydrodynamic diameter of nanoparticles, while TEM gives an actual
diameter of nanoparticles in dry state. The part of aggregation of
the chitosan/TPP nanoparticles is probably because that the hydrogen bonding interactions between chitosan nanoparticles gradually
become dominant in the drying process.
It also can be noticed that these nanoparticles have a deeper
color in the core and surface, indicating that these regions have
higher electron density distribution. As TPP contains phosphorus
element, which has a higher electron density than those elements
of chitosan, so it can be inferred that chitosan has higher degree of
cross-linking density with TPP in these regions.
3.9. Colloid stability
The colloidal stability of the chitosan/TPP nanoparticles prepared at optimal conditions was evaluated by storage at room
temperature (1020 C) for different periods of time. The results
showed that after stored at room temperature for 20 days, the liquid

Fig. 5. Schematic representation of ionic crosslinking reaction between chitosan and TPP in (A) low ionic strength solution and (B) high ionic strength solution.

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 2127

of chitosan/TPP particles. These effects probably also can serve to

enhance the mechanical strength of the particles, thus favor the
encapsulation and slow release of drug. At last, the formation mechanisms presented in this study may also be applied to prepare
monodisperse nanoparticles based on chitosan derivatives, such as
N-trimethyl chitosan and carboxymethyl chitosan, which also have
promising applications in biomedical elds [33,34].

Acknowledgment
The authors appreciate Jinzhao Wang and Qiwei Yang for their
assistance in this work.

References

Fig. 6. TEM image of chitosan/TPP nanoparticles without being stained.

was still a clear suspension with light blue opalescence and the
nanoparticles did not show any statistically signicant change in
particle size and its distribution during the investigation (data not
shown). It was suggested that chitosan/TPP nanogels behaved as
a metastable system, since they underwent spontaneous aggregation and disintegration under very mild conditions in a short
period of time [22]. Our results indicate that these monodisperse
chitosan/TPP nanoparticles have excellent storage stability, which
probably due to the low ionic strength environment, appropriate pH conditions (the nal pH of the suspension was around
5.4), together with the high cross-linking density, small size, narrow particle size distribution and high surface potential of the
particles [13,16,23]. Reducing the storage temperature to 4 C or
using freeze-drying with a cryoprotective agent is expected to further improve the long-term storage stability of the chitosan/TPP
nanoparticles [22,24,25].
4. Conclusions
Although the drug delivery system based on chitosan/TPP
nanoparticles has its own advantages, the polydispersity and
poor stability of the colloids seriously limit the efciency of
nanoparticle-mediated drug delivery. In a polydisperse system,
larger nanoparticles usually have higher drug loading capacity,
while smaller nanoparticles are expected to have higher efciency
of delivering drug to tissues or cells [26]. This contradiction means
that even if the drug carrier system has a high encapsulation efciency, the delivery efciency may be poor. Thus, through the novel
method developed by this study, which allows a highly repeatability of producing monodisperse, LMW chitosan/TPP nanoparticles,
it is expected to further improve the efciency of drug utilization, especially for highly size-dependent applications, such as gene
transfer or delivery of drug via mucosal routes [27,28]. In addition,
several attempts have been made to increase the encapsulation and
release period of drug, and the major problem encountered is the
initial burst release of drug [2931]. This is partly associated with
the low mechanical strength of the chitosan/TPP particles, and the
initial burst release can be reduced by enhancing the mechanical
strength of the particles [32]. It has been found in this study that
an increase in the temperature of chitosan solution and a decrease
in the ionic strength of chitosan solution are expected to promote
the formation of a compact and sufcient cross-linking structure

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