Colloids and Surfaces A: Physicochem. Eng.

Aspects 465 (2015) 137146

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Physicochemical and morphological properties of size-controlled

chitosantripolyphosphate nanoparticles
John Antoniou a , Fei Liu a , Hamid Majeed a , Jing Qi a , Wallace Yokoyama b , Fang Zhong a,
a
Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122,
PR China
b
Processed Foods Research Unit, Western Regional Research Center, ARS, USDA, Albany, CA 94710, United States

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

Chitosantripolyphosphate nanopar

ticles were prepared through ioniccrosslinking.

Control of particle size by selecting
appropriate conditions.
Inuence of initial pH and salinity
of chitosan solution on nanoparticles
formation.
Separation of aggregates from
nanoparticles by centrifugation.
Ultra-sonication reduced particle size
but caused fragmentation on their
structure.

a r t i c l e

i n f o

Article history:
Received 17 June 2014
Received in revised form 9 October 2014
Accepted 23 October 2014
Available online 30 October 2014
Keywords:
Chitosan nanoparticles
Ionic crosslinking
Ionic strength
Particle size
Morphology
Ultra-sonication

a b s t r a c t
Chitosantripolyphosphate nanoparticles have been extensively studied during the last decade because
of their numerous applications. In this study, we describe conditions to optimize chitosan nanoparticles
as potential nano-llers in edible lms. The ionic cross-linking between the cationic amino groups on the
chitosan (CS) chain and the anionic phosphate groups of sodium tripolyphosphate (TPP) was veried via
FTIR. Particle size, polydispersity index (PDI) and surface -potential were controlled by chitosans Mw
and concentration, CS:TPP mass ratio, and external conditions such as pH and salinity of the initial chitosan solution. Post-processing methods such as centrifugation and ultra-sonication were used to further
control particle size. We show that particle size can be controlled by selecting appropriate conditions.
Particles with sizes below 120 nm were produced at different CS:TPP mass ratios depending on the CS
concentration. Dilute NaCl was the optimal solution ionic composition that decreased the size by 25% and
also resulted in a narrow particle size distribution. We show using UVvis spectrophotometry that particles of different size, separated by centrifugation had different phosphorus content. Ultra-sonication can
be used to reduce the size by 50% but long time caused fragmentation of the nanoparticles. Transmission
electron microscopy (TEM) revealed the differences in the morphology of chitosan nanoparticles under
various fabrication conditions.
2014 Elsevier B.V. All rights reserved.

1. Introduction

Corresponding author. Tel.: +86 510 85328307.

E-mail address: fzhong@jiangnan.edu.cn (F. Zhong).
http://dx.doi.org/10.1016/j.colsurfa.2014.10.040
0927-7757/ 2014 Elsevier B.V. All rights reserved.

Chitosan (CS) is obtained by the deacetylation of the naturally

occurring polysaccharide chitin, one of the most abundant biopolymers in nature. It is composed of -(1-4)-linked d-glucosamine and
N-acetyl-d-glucosamine units [1]. The degree of deacetylation (DD)

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J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

of typical commercial chitosan is usually between 70% and 95%,

and the Mw between 10 and 1000 kDa [2]. Chitosan is an ingestible
food ingredient that is biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional [3]. In an
acidic environment CS is positively charged and soluble due to
protonation of the amino groups of glucosamine. Chitosan nanoparticle (CSNPs) formation is based on the simple and mild technique
of ionotropic gelation where the CS protonated amino groups
are cross-linked by inter- and intramolecular bonds by multivalent polyanions. Sodium tripolyphosphate (TPP) is a very popular
polyanion because it is non-toxic and forms gels with desirable
properties [46].
CSNPs because of their biodegradability, low toxicity and biocompatibility have been frequently developed as a delivery system
for bioactive compounds such as, drugs [712], proteins [1316],
avonoids [17], essential oils [18,19], and genes [5,20]. The formation conditions of the nanoparticle in these studies were examined
in relation with the encapsulation efciency and in tandem with
the release prole or antimicrobial activity. Particles have been
prepared in the size range of 50 to 300 nm with encapsulation efciency reaching 95% showing that ionotropic gelation is an efcient
method for CSNPs preparation. CSNPs have been reported to have
broad antimicrobial properties. Their antibacterial activity can be
higher than that of bulk chitosan due to their higher surface area
and charge density [21,22].
However, there have been limited reports on the use of the
nanoparticles as a modier of mechanical and physicochemical
properties of biopolymer lms. The present study investigated
the physical characteristics of CSNPs for use as a potential nanocomposite modier. CSNPs have been incorporated in edible or
biodegradable lms [4,2325] and demonstrated very good dispersability in biopolymers and showed potential antimicrobial
activity in the lms [26]. Nanoparticles improve mechanical and
barrier properties by occupying the empty spaces within the lm
matrix which induces the collapse of the pores [27]. The purpose of
this study was to optimize CSNPs intended as bio-nanocomposites
for lm applications and focused on the fabrication of compact
nano-sized particles with narrow polydispersity index (PDI). The
behavior of the particle as a function of size, PDI and surface charge
(-potential) was examined by changing key parameters such as,
CS molecular weight (Mw), CS concentration, CS:TPP mass ratio and
external conditions such as, pH and salinity of the initial CS solution.
In processes where a mixture of nanoparticles and aggregates were
formed, centrifugation and ultra-sonication were applied to isolate
nanoparticles from large particles and aggregates. Transmission
electron microscopy (TEM) was used in order to observe morphological changes in the structure for various fabrication processes.
The conditions for the production of narrow PDI nanoparticles and
of a specic size range would be useful for improved edible and
biodegradable lm production.
2. Materials and methods
2.1. Materials
Chitosan of Mw 50 and 100 kDa and DD of 90% derived from
crab shells were obtained from Golden-Shell Biochemical Co., Ltd.
(Hangzhou, China). Sodium tripolyphosphate was purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other
reagents were all commercially available and used as received.
Distilled water was used in all experiments.
2.2. Preparation of CSNPs
CSNPs were produced according to the procedure rst reported
by [13], based on the ionic gelation of CS with TPP anions. CS

(0.5 mg/mL) was dissolved in 1% (v/v) aqueous acetic acid and the
pH was adjusted using 10 M NaOH. A 0.7 mg/mL aqueous solution
of TPP was added drop-wise to the CS solution under vigorous magnetic stirring at room temperature. CSNPs formed spontaneously. In
order to use different mass ratios of CS:TPP and CS:NaCl, the initial
concentration of CS was kept constant while the volume of TPP and
NaCl added was varied. All samples were prepared in triplicate and
immediately subjected to further analysis, centrifuged at 10,000 g
for 30 min at room temperature in order to separate the nanoparticles from large particles or aggregates or freeze dried to obtain
powder samples. After centrifugation sediment and supernatant
were separated by carefully removing the supernatant layer.

2.3. Characterization of CSNPs

The measurements of particle diameter, PDI and -potential
of nanoparticles were performed at 25 C on a Zetasizer Nano-ZS
(Malvern Instruments, Worcestershire, UK) on the basis of dynamic
light scattering (DLS). The pH of the particle suspensions was measured at room temperature using a Mettler Toledo EL20 (Shanghai,
China).
The morphological characteristics of nanoparticles were examined by a high-performance digital imaging TEM machine (JEOL
H-7650, Hitachi High-Technologies Corp., Tokyo, Japan). One drop
of the nanoparticle solution was spread onto a carbon-coated copper grid and stained with 2% (w/v) phosphotungstic acid. After
drying at room temperature the samples were placed for TEM analysis using accelerating voltage of 100 kV.
FTIR spectra of CS, TPP and CSNPs were determined using an
infrared spectrometer (FTIR) (Thermo Fisher Scientic Inc., Nicolet iS10, USA). The spectra were obtained in the range of 500 and
4000 cm1 , using 16 scans at a resolution of 2 cm1 . Powdered
CSNPs were obtained by freeze drying and the samples were prepared using KBr to form pellets.

2.4. Determination of phosphorus in CSNPs

The determination was based on the method reported by [28]
with modications. Powdered samples of CSNPs were prepared by
freeze drying. The samples were weighed and placed into Kjeldahl
asks and digested by a mixture of sulfuric acid, perchloric acid and
nitric acid. The hydrolyzed solution was cooled to room temperature and transferred to 100 mL asks by rinsing the Kjeldahl asks
several times with distilled water. 2 mL of the obtained solutions
were transferred into 20 mL tubes and 2 mL solution of ammonium
molybdate, 1 mL solution of sodium sulte and 1 mL hydroquinone
were added and mixed. At last, distilled water was added to reach
the mark of the tube and left 30 min at room temperature to ensure
colour development. The same procedures were used for blanks.
Monopotassium phosphate (KH2 PO4 ) was used as the standard.
For the P determination in both standard and working solutions
the absorbance was determined by absorption at 660 nm using a
UV-1600 Spectrophotometer (Shanghai, China). All samples were
analyzed in triplicate and the content of phosphorus was calculated
using the following equation:
X=

M  V 
1
1
M

V2

100

(1)

X = the content of phosphorus in the sample (mg/100 g); M1 = the

content of phosphorus in the sample solution (from the standard
curve or regression equation (mg)); V1 = the total volume of sample
digestion liquid (mL); V2 = the volume of sample digestion liquid for
determination (mL); M = the weight of the sample (g).

J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

139

2.5. Ultra-sonication
Ultra-sonication was performed based on the method reported
by Floris, et al. [29] with modications. Ultra-sonic treatments were
administered using an ultra-sonic probe with diameter of 20 mm
and a 500 W high-intensity ultra-sonic processor (VCX 500, Sonics and Materials Inc., USA) operating at 20 kHz. The samples were
placed in 50 mL beakers and ultra-sonicated under discontinuous
mode in ice bath in order to avoid temperature increase. The ultrasonic radiation was interrupted by breaks where the time fraction
of both ultrasonic radiation and the break was 0.5 s. The probe was
immersed 2 cm into the sample and ultra-sonicated over a period
of 12 min.
2.6. Statistical analysis
OriginPro 8 SR4 (Version 8.0, Northampton, USA) and SPSS
Statistics software (Version 17.0, SPSS Inc. Chicago, IL) were used to
analyze the resulting data. Data were initially evaluated by analysis
of variance (ANOVA), and signicant results were further analyzed
using Duncans multiple range tests (P < 0.05) to compare the mean
values.
3. Results and discussion
CSNPs were prepared by the ionic interaction between the positively charged amino groups (NH+
) of chitosan and the negatively
3
charged phosphate groups (P3 O5
10 ) of TPP. Several parameters can
be varied during the fabrication of the nanoparticle to vary the size,
-potential and PDI.

3.1. Fourier transform infrared (FTIR) spectroscopy

FTIR spectroscopy was used in order to conrm the linkage
between phosphoric and ammonium ions of TPP and CS, respectively, in the nanoparticles (Fig. 1). The major peaks in the IR spectra
appeared in two regions, 37002800 cm1 and 1700400 cm1 .
In the chitosan spectra, the broad band at 3423 cm1 is a result
of O H stretching vibration associated with free, inter, and intramolecular bound hydroxyl groups. In the same region, the peaks
of N H stretching from primary amine and type II amide are
overlapped. The peaks in the region 30002800 cm1 are typical
C H vibration [30]. Asymmetric stretch of C O C in glycosidic
linkage is found at around 1154 cm1 and the broad peak at
1075 cm1 indicates C O stretching vibration [31]. In the TPP

Fig. 1. Infrared spectra of, (

) TPP, (

) chitosan and (

) chitosan nanoparticles.

spectrum two intense absorption bands were found. The peaks in

the range of 12201080 cm1 can be attributed to P O stretching
and at 899 cm1 to P O along with P O P [32].
With incorporation of TPP in chitosan the peak of 3421 cm1
becomes wider indicating that the hydrogen bonding is enhanced.
Also, In the TPP spectra the peak at 1212 cm1 which indicates P O
stretch, appeared in the CSNPs spectra at 1218 cm1 as a result of
the cross-linking between CS and TPP. Finally, in the spectra of the
nanoparticles the peaks for N H bending vibration of amine I at
1598 cm1 and the amide II carbonyl stretch at 1654 cm1 shifted
to 1568 cm1 and 1630 cm1 , respectively. These results indicate
the interaction between amino groups of chitosan and phosphate
groups of TPP. We can conclude that the appearing of these peaks
is an indication of nanoparticle formation and that the inter- and
intra-molecular actions are enhanced in chitosan nanoparticles
[4,33,34].
3.2. Effect of CS Mw and CS concentration
CS concentration is perhaps the most important determinant of
particle size. Fig. 2 shows the effect of concentration of two different sized CS, 50 and 100 kDa, on particle size and PDI at a constant

Fig. 2. Effect of CS concentration on (a) particle size and (b) PDI of chitosan nanoparticles for CS with Mw (

) 50 kDa and (

) 100 kDa. (CS:TPP mass ratio = 5:1, pH = 4.8).

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J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

Fig. 3. Effect of CS:TPP mass ratio on (a) particle size (

) and PDI (

) and (b) -potential of chitosan nanoparticles, (CS = 0.5 mg/mL, Mw = 100 kDa, DD = 90%, pH = 4.8).

CS:TPP mass ratio (5:1). The particle size increased about 100% and
almost linearly for concentrations ranging from 0.5 to 2.0 mg/mL
and remained constant for concentrations higher than 2.0 mg/mL.
Similar results were reported by Hu, et al. [17], Liu and Gao [35] and
Gan, et al. [5]. However, other studies have reported no noticeable
difference between particle sizes when CS of Mw 50 and 100 kDa
were used [5,17]. The different results between the studies might
be related to alterations of the CSNPs structure due to different
processing conditions such as mechanical shear during mixing and
temperature as well as the different source of CS [36]. Differences
in reactivity due to Mw might be expected since the molar concentration of the 50 kDa CS solution is twice as high as the 100 kDa
and its rate of diffusion is also expected to be higher. The PDI of the
particles had similar trend with the particle size (Fig. 2b). For CS
concentrations in the range of 0.5 to 1.5 mg/mL, the PDI of both CS
(50 kDa and 100 kDa) was almost constant but it increased signicantly (P < 0.05) for concentrations above 1.5 mg/mL. At higher CS
concentrations, unlike particle size, PDI of CS 100 kDa was found
to be higher than that of CS 50 kDa. It is possible that at these
high concentrations, the larger CS agglomerated more quickly thus
increasing the polydispersity of the particles.
3.3. Effect of CS:TPP mass ratio
CS:TPP mass ratio is also critical to control particle size and size
distribution. A wide range, 3:1 to 15:1 of CS:TPP mass ratios were
evaluated with constant CS concentration, 0.5 mg/mL (Fig. 3). The

Fig. 4. Visual observation of chitosan nanoparticle solutions at different CS:TPP

mass ratios (constant CS concentration at 0.5 mg/mL). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of
this article.)

particle size was greatest for the lowest CS:TPP mass ratio (3:1) and
decreased linearly to 6:1. For the same CS:TPP range, Hu, et al. [17]
reported that with increasing TPP concentration the suspension
became more turbid indicating the formation of aggregates when
superuous TPP connect individual nanoparticles. The -potential
of the particles at very low CS:TPP mass ratios was reduced significantly as expected (Fig. 3b). As can be observed from Fig. 4, at low
CS:TPP mass ratios the solution obtained an opalescent color which
faded as the mass ratio increased. At mass ratio 3:1 the aggregation is so intense that the particles precipitated immediately. The
superuous TPP linked residual positively charged groups of separate CSNP resulting in aggregation. At CS:TPP mass ratios ranging
from 6:1 to 9:1 the particle size was constant and below 130 nm.
However, at very high CS:TPP mass ratio (>9:1) the particle size
increased again while the -potential of the suspension remained
constant. The particle size of nanoparticles and aggregates in the
range of 9:1 to 15:1 CS:TPP mass ratio was analyzed further (Fig. 9)
where after centrifugal separation, nanoparticles with particle size

Fig. 5. Effect of initial CS solution pH on (a) particle size and (b) -potential of chitosan nanoparticles at two different CS concentrations, (
(CS Mw = 100 kDa, DD = 90%, CS:TPP mass ratio = 7:1).

) 0.5 and (

) 1.5 mg/mL

J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

Fig. 6. Inuence of NaCl concentration on (a) particle size (

mass ratio = 6:1, pH = 4.8).

141

b
) and PDI (

) and (b) -potential of chitosan nanoparticles (CS = 1.5 mg/mL, Mw = 100 kDa, DD = 90%, CS:TPP

ranging from 85 to 115 nm were produced. The wide range of TPP

concentrations that form particles is due to the fact that TPP can
create as many as ve ionic cross-links with amino groups of CS
resulting in particle and aggregate of particle formation [11].
3.4. The effect of initial CS solution pH
The pH of the initial CS solution is a very important environmental variable for the formation of CSNPs. Solubilization of CS, a weak
polybase, is enhanced by protonation of the NH2 group when dissolved in acetic medium. Since the formation of the nanoparticle
is driven by the ionic interaction between CS and TPP, it is reasonable to conclude that the ionic cross-linking among CS and TPP is
pH-dependent [8]. Fig. 5 shows the variation of particle size and potential at two different CS concentrations (0.5 and 1.5 mg/mL) at
a range of initial pH values of the CS solution. At both CS concentrations the -potential decreased almost linearly with increasing pH.
However, the particle size did not increase until the pH was above
5.5 at low CS concentration (0.5 mg/mL). At higher CS concentration (1.5 mg/mL) on the other hand, larger particles were formed
at low initial pH and the size was reduced as the pH increased to

4.5. The smallest size was reached at pH 4.5 to 5 and size increased
sharply above pH 5. Other researchers have reported that at pH
occurred during cross-linking
4.55 less neutralization of NH+
3
and ultimately reduced particle size [5,17]. At low initial pH values
the strong positively charged protonated -NH2 groups led to repulsions in the CS chain resulting in larger particles. When initial pH
was above 4.5, the ionization of CS was optimum for cross-linking
with TPP and as it increased above 5 and close to chitosans pKa
6.3, less protonation of the amino groups took place resulting in
agglomeration and therefore formation of large particles.

3.5. Effect of NaCl

Changes in the ionic strength of the CS chain may provide an
additional, ner and more-versatile control of the polymer/crosslinker interactions [37]. The inuence of ionic strength on particle
size, PDI and -potential of the nanoparticle was evaluated by
preparing CSNPs in saline. A monovalent salt (NaCl) at different
concentrations was used to dissolve the CS before cross-link with
the negatively charged TPP-anions.

Fig. 7. TEM micrographs of chitosan nanoparticles (a) with addition of NaCl (1.0 mg/mL) and (b) without. (CS = 1.5 mg/mL, Mw = 100 kDa, DD = 90%, CS:TPP mass ratio = 6:1,
pH = 4.8).

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Fig. 8. Size distribution by intensity for particles at different CS:TPP mass ratios (a) 6:1, (b) 9:1, (c) 12:1, (d) 15:1 (CS = 0.5 mg/mL, Mw = 100 kDa, DD = 90%, pH = 4.8).

Fig. 6 shows that the use of monovalent salt at very low concentration (0.5 mg/mL) during ionotropic gelation reduced the charge
of CS and yielded nanoparticles with narrow size distributions
and smaller particles (150 nm) decreasing the size by approximately 25%. However, as the NaCl concentration increased above
1.0 mg/mL the -potential was reduced even further and the size
increased linearly. The original particle size was restored when
the NaCl concentration was 3.0 mg/mL. With increasing NaCl concentration no signicant changes (P > 0.05) were observed in the
PDI of the particles. As TEM micrographs reveal in Fig. 7, in the
absence of NaCl, the nanoparticles attained a swollen conformation
due to the electrostatic repulsive forces between the protonated
amino groups and local excess of TPP led to rapid coagulation [38].
Conversely, in the presence of moderate NaCl concentrations, the
neutralization of the amino groups on the backbone of the CS kept
the newly formed particles in a more compact conformation and
nely dispersed. Similar to the effect of the initial pH, particles with
low mean diameter were only formed when the -potential value
was around 18 mV. Stronger or lower protonation of the amino

groups in the CS chain led to repulsion or agglomeration, respectively.

Table 1 shows the -potential of the particles at different stages
of the particle formation with addition of TPP and NaCl. Addition of NaCl (1.0 mg/mL) decreased the -potential signicantly
(P < 0.05) for all CS concentrations except the highest (3.0 mg/mL).
The most profound effect was exhibited for CS 0.5 mg/mL by

Table 1
The -potential (mV) for different CS concentrations at all the stages of the particle
formation (CS:TPP mass ratio = 6:1, NaCl = 1.0 mg/mL)*
CS concentration (mg/mL)
CS
CS + TPP
CS + NaCl
CS + NaCl + TPP

0.5
31.1
28.0
20.1
17.4

1.5

0.35
1.13b
0.35c
0.28d

26.6
23.7
16.5
16.1

3.0

0.28
1.98b
1.48c
0.57c

18.4
17.7
16.3
15.7

0.71a
0.99ab
0.21ab
1.06b

*
Values are given as mean standard deviation. Different superscript letters in
the columns indicate signicant difference (P < 0.05).

J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

reducing -potential from 31.1 0.35 to 20.1 0.35 mV. As the CS

concentration increased the -potential of CS decreased therefore
the effect of the salt was less signicant (P > 0.05) since the positive charge was lower. These results are in agreement with the
ndings of Jonassen, et al. [38] and Huang and Lapitsky [37]. Addition of TPP-anions in both aqueous and saline systems decreased
the -potential further. The high NaCl concentration resulted in
decreased interactions between CS and TPP due to neutralization of
charge on the chitosan chains. As a result the use of saline increases
the concentration range of CS to prepare nanoparticles and a moderate concentration of a monovalent salt was found to enhance the
colloidal stability during microgel formation and lead to a narrow
particle size distribution.

143

3.6. Separation of CSNPs by centrifugation

The mass ratio of CS:TPP has a signicant effect on particle size
and we observed that for mass ratios above 9:1, two groups of particles with different size and intensity appeared (Fig. 8). One group of
particles had an average diameter below 100 nm and lower intensity and the second above 100 nm and higher intensity. The greater
the CS:TPP mass ratio the bigger the difference in size and intensity
among these two groups of particles. Centrifugation with different
speed and time was applied to the suspensions in order to separate
the <100 nm particles from the >100 nm particles and aggregates.
In our system centrifugation 10,000 g for 0.5 h was the optimum
conditions to separate the two groups.
As shown in Fig. 9 for CS:TPP mass ratios 6:19:1 both supernatant and sediment had the same particle size (<100 nm) and
PDI. For mass ratios above 9:1 the centrifugation isolated bigger
particles in the sediment while smaller particles remained in the
supernatant. However, at this range of CS:TPP mass ratio the PDI
of both supernatant and sediment increased rapidly. TEM micrographs in Fig. 10 showed that at CS:TPP mass ratios above 9:1 there
was a greater amount of unlinked CS in the suspension. The limited
amount of TPP was not enough to cross-link with all the CS in the
solution leaving a lot of CS in a diffused state. This unlinked CS
chains appeared as large particles in the suspended solution. The
centrifugation separated the CSNPs from the unlinked CS, isolating nanoparticles with very small size but with low yield which
decreased as the CS:TPP mass ratio increased (Fig. 9c).
3.7. Phosphorus element detection
In order to support our hypothesis that the bimodal size distribution is due to unlinked CS a series of analysis were conducted to
characterize the nanoparticles after separation by centrifugation at
10,000 g for 0.5 h. The TPP content of the sediment and supernatant fraction were determined by the analysis of phosphorus (P)
content. Only the CS:TPP range from 3:1 to 12:1 at high CS concentration (1.5 mg/mL) was analyzed because at higher mass ratios the
amount of TPP was insufcient to be detected accurately.
As shown in Fig. 11a the weight of the freeze dried sediment
decreased slightly and the weight of the supernatant increased as
the mass ratio increased. This difference in the weight at lower mass
ratios (3:19:1) indicates the existence of large and small particles
at each mass ratio. As the mass ratio increases, this difference in
weight between sediment and supernatant reduced until equilibration at 12:1 indicating a high constant molar ratio of CS to TPP
in the small supernatant particles. At low CS:TPP mass ratio only
a fraction of TPP is utilized since a high molar ratio of CS:TPP is
optimum.
The relation between TPP amount and particle size at different CS:TPP mass ratios is shown in Fig. 11b and c. At low CS:TPP
mass ratios (6:1) large particles (>200 nm) are formed and found
in the sediment after centrifugation. However, a small amount of

Fig. 9. The (a) particle size, (b) PDI and (c) yield of the ( ) sediment and ()
supernatant after separation of chitosan nanoparticles by centrifugation at different
CS:TPP mass ratios (CS = 0.5 mg/mL, Mw = 100 kDa, DD = 90%, pH = 4.8).

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J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

Fig. 10. TEM micrographs of chitosan nanoparticles at different CS:TPP mass ratios (a) 6:1 and (b) 12:1 (CS = 0.5 mg/mL, Mw = 100 kDa, DD = 90%, pH = 4.8).

nanoparticles with size below 150 nm remained in the supernatant.

In 9:1 the size of large and small nanoparticles in the two phases
are approximately 100 nm. In higher mass ratios (9:1) the amount
of the nanoparticle in the supernatant was slightly higher than that

in the sediment indicating the existence of even smaller nanoparticles in the solution (100 nm). It is concluded that for specic
CS:TPP mass ratios at a specic CS concentration the formation of
the nanoparticles is homogenous and the nanoparticles fabricated

Fig. 11. The (a) total weight, (b) P content and (c) particle size of the ( ) sediment and () supernatant after separation of chitosan nanoparticles by centrifugation at
different CS:TPP mass ratios, (CS = 1.5 mg/mL, Mw = 100 kDa, DD = 90%, pH = 4.8).

J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

Fig. 12. Effect of ultra-sonication duration at 500 W on (

Mw = 100 kDa, DD = 90%, pH = not adjusted).

) particle size and (

145

) PDI of chitosan nanoparticles at CS:TPP mass ratio (a) 4:1 and (b) 9:1, (CS = 1.5 mg/mL,

Fig. 13. TEM micrographs of chitosan nanoparticles with CS:TPP mass ratio of (a) 4:1 and (b) 9:1 after ultra-sonication at 500 W for 10 min (CS = 1.5 mg/mL, Mw = 100 kDa,
DD = 90%, pH = not adjusted).

were all of the same size. However, outside of this CS:TPP mass ratio
range a small amount of TPP is utilized that produces nano-scale
particles. In the case we studied, uniform particles were produced
when the CS concentration was 1.5 mg/mL at CS:TPP mass ratio
9:112:1 and for CS concentration 0.5 mg/mL that mass ratio range
shifted to 6:19:1. For CS concentration of 1.5 mg/mL the optimum P element content for homogenous particle formation was
calculated to be around 4 mg/g of sample weight.
3.8. Ultra-sonication
Fig. 12 shows the effect of ultra-sonication duration over a
period of 12 min on the particle size and polydispersity of CSNPs.
Two different CS:TPP mass ratios were tested in order to obtain
different particle sizes. The results showed that the increase in
sonication time decreased the particle mean diameter. Ultrasonication for 10 min decreased the size by almost 50% but only for
CSNPs with low mass ratio (4:1). At higher mass ratio (9:1) where
smaller particles were formed the sonication had no effect on the
size. Tang, et al. [39] reported before that larger nanoparticles were
more susceptible to ultra-sonication compared to smaller nanoparticles. The initial size reduction of the nanoparticles, up to 6 min,
can be attributed to the disruption of aggregations. However, by

increasing the duration of sonication the rate of the size reduction decreased and it was probably due to the degradation of the
polymer which resulted in relaxation of the nanoparticles [40]. It is
possible that extensive ultra-sonication can detach polymer parts
from the nanoparticles. This fact could also explain the increase
in PDI as sonication times increases. This can be observed in TEM
images in Fig. 13 where ultra-sonication caused fragmentation of
the nanoparticles thus deforming their spherical compact structure. Fig. 13b reveals that although ultra-sonication had no effect
on the size of small nanoparticles fabricated at CS:TPP mass ratio of
9:1, it caused severe fragmentation. Floris et al. [29] reported that
during ultra-sonication the degradation of the polymer is mainly
caused by cavitation effect.

4. Conclusion
In this work the conditions controlling the size of
chitosantripolyphosphate nanoparticle (CSNPs) by ionotropic
interactions are detailed. The size and dispersion (PDI) of CSNPs
is affected by chitosan Mw, concentration, CS:TPP mass ratio, pH
and salinity of the initial CS solution. Controlling these parameters results in controlled size and PDI of CSNP and provides an

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J. Antoniou et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 465 (2015) 137146

opportunity for manipulating and optimizing the nanoparticle for

intended food applications. Separation of aggregates by centrifugation from nanoparticles and analysis of phosphorus content
particles in sediment and ltrate claried the utilization of the
TPP anion in the nanoparticle formation during the ionic gelation.
Moderate use of ultra-sonication was able to reduce signicantly
the particle size by disrupting aggregations. However, extensive
ultra-sonication caused severe fragmentation on their structure.
Acknowledgements
This work was nancially supported by National 863 Program
2011BAD23B02, 2013AA102207, NSFC 31171686, 30901000, 111
project-B07029 and PCSIRT0627
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