Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73

Modulation of surface charge, particle size and morphological properties

of chitosan–TPP nanoparticles intended for gene delivery
Quan Gan a,∗ , Tao Wang a , Colette Cochrane a , Paul McCarron b
a School of Chemical Engineering, Queen’s University Belfast, Belfast BT9 5AG, UK
b School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, UK

Received 14 March 2005; received in revised form 6 June 2005; accepted 8 June 2005

Abstract

This work investigates the polyanion initiated gelation process in fabricating chitosan–TPP (tripolyphosphate) nanoparticles in the size
range of 100–250 nm intended to be used as carriers for the delivery of gene or protein macromolecules. It demonstrates that ionic gelation
of cationic chitosan molecules offers a flexible and easily controllable process for systematically and predictably manipulating particle size
and surface charge which are important properties in determining gene transfection efficacy if the nanoparticles are used as non-viral vectors
for gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, chitosan to
TPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface charge, and
tendency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The chitosan–TPP
nanoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticles was found
to be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of polyhedrons
(e.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process. This study
demonstrates that systematic design and modulation of the surface charge and particle size of chitosan–TPP nanoparticles can be readily
achieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Chitosan nanoparticles; Nanoparticle surface charge; Nanoparticle morphology; Ionic gelation; Gene delivery

1. Introduction Furthermore, there is evidence demonstrating that cationic

Chitosan is a non-toxic biodegradable polycationic poly-
mer with low immunogenicity. It has been extensively
investigated for formulating carrier and delivery systems
for therapeutic macrosolutes, particularly genes and protein
molecules primarily because positively charged chitosan can
be easily complexed with negatively charged DNAs and pro-
teins [1,2]. Chitosan can effectively bind DNA and protect
it from nuclease degradation [3,4]. It has advantages of not
necessitating sonication and organic solvents for its prepara-
tion, therefore minimizing possible damage to DNA during
the complexation process.
∗ Corresponding author. Tel.: +44 2890 274463; fax: +44 2890 381753.
E-mail address: q.gan@qub.ac.uk (Q. Gan).
polymers play an important role in both membrane adhesion
[5] and lysosomal escape [6] of the encapsulated DNA, pro-
viding a potential explanation for the superiority of polymer-
mediated gene transfer relative to naked DNA administration
in many applications. These hybrid DNA–chitosan systems
can be classified into two categories which differ in their
mechanism of formation and morphology: complexes and
nanospheres.
Gentle mixing, followed by incubation, of chitosan
and DNA solutions generated ‘broad’ distributions of
chitosan–DNA particulate complexes with mean sizes
between 100 and 600 nm, depending on the molecular
weight of the chitosan. Since particle formation was elicited
solely by the tropism of the two oppositely charged macro-
molecules for one another, these particles were termed

0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2005.06.001
66 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
‘complexes’. The simplicity of chitosan–DNA complexes is
both an advantage and a drawback. Though such complexes
are extremely easy to synthesize, the fact remains that their
transfection efficacy is significantly below that of cationic
liposomes in vitro and viral vectors in vivo. Aside from
N/P ratio and chitosan molecular weight, there remain
few parameters in the synthesis protocol which can be
modulated in an effort to augment transfection. To address
this issue, investigators sought to develop more sophisticated
DNA-loaded chitosan nanoparticles.
The application of DNA–chitosan nanoparticles has
advanced in vitro DNA transfection research, and data have
been accumulating that shows their usefulness for gene
delivery [7,8]. The therapeutic efficacy of the nanoparticles
could be due to their ability to protect the therapeutic agent
from degradation due to lysosomal enzymes.
Due to their sub-cellular and sub-micron size,
chitosan–TPP nanoparticles can penetrate deep into tissues
through fine capillaries, cross the fenestration present in the
epithelial lining (e.g., liver) [9]. This allows efficient delivery
of therapeutic agents to target sites in the body. Also, by
modulating nanoparticles characteristics, such as enzymatic
degradation rate, size and surface charge density, one can
control the release of a therapeutic agent from nanoparticles
to achieve desired therapeutic level in target tissue for
required duration for optimal therapeutic efficacy [6].
The major drawback associated with using chitosan as
non-viral gene delivery system is the relatively low transfec-
tion rate in comparison to viral vectors, even though the later
has its own limitations in patient safety, difficulty in scale-
up production, and possible toxicity, immune responses, and
inflammatory responses. It is understood that transfection
efficacy of cationic polymers depends primarily on: (i) parti-
cle size, which determines their intracellular uptake, different
pathways of their uptake, intracellular trafficking and sorting
into different intracellular compartments, and (ii) the inten-
sity of particle surface charge which influence their ability to
efficiently condensate DNA and interact with cell.
Stable and reproducible chitosan nanoparticles were in
early days formulated via chemical cross-linking in water-
in-oil emulsion system for entraping and delivering drugs
[10]. However, the negative effects of cross-linking agents,
e.g., glutaraldehyde, on cell viability and the integrity of
macromolecular drugs led to the development of prepara-
tion method under mild conditions. Preparation methods by
ionically cross-linking cationic chitosan with specific polyan-
ions were particularly successful as, aside from its com-
plexation with negatively charged polymers, chitosan has
the ability to gel spontaneously on contact with multivalent
polyanions due to the formation of inter- and intramolecular
cross-linkage mediated by these polyanions. Among some
polyanions investigated, tripolyphosphate (TPP) is the most
popular because of its non-toxic property and quick gelling
ability. The chitosan–TPP nano system exhibits some attrac-
tive features which render them promising carriers for the
delivery of macromolecules. These features include forma-
tion under mild conditions; homogeneous and adjustable size
and a positive surface charge that can be easily modulated
and a great capacity for the association of peptides, proteins,
oligonucleotides, and plasmids [11].
Therefore, ability to control and modulate the properties
of chitosan–TPP nanoparticles, most importantly particle size
and density of surface charge, is central in determining gene
transfection efficiency. It is important that these characteris-
tic properties be predictably produced and easily modulated
in a flexible and reliable nano fabrication process with high
yield and particle stability. It is therefore the focus of this
paper to report on how systematically manipulating process-
ing parameters in the TPP initiated chitosan gelation to obtain
predictable and optimal nanoparticle properties for desired
applications in relation to gene/protein delivery.
2. Materials and experimental methods
2.1. Materials
Three different molecular weight chitosan, derived from
crab shell, in the form of fibrils flakes were obtained
from Sigma–Aldrich [Catalogue No. LMW 448869, MMW
448877, HMW 419419]. The degree of deacetylation for the
low molecular weight chitosan (LMW Chitosan), medium
molecular weight chitosan (MWM chitosan) and high molec-
ular weight chitosan (HMW chitosan) is 86.6%, 84.7%,
and 82.5%, respectively. Sodium Tripolyphosphate was pur-
chased from Sigma–Aldrich Chemical Co. Ltd. All other
reagents used were of analytical grade.
2.2. Purification of chitosan
Since medical applications of animal derived biomateri-
als entail an inherent risk of protein contamination which has
in recent years aroused great awareness and anxiety among
the public, drug companies, and the industry regulators, it
is of utmost importance to ensure that chitosan intended for
medical applications is of the highest purity and free of pro-
tein contamination. It is therefore decided to further purify
the purchased chitosan materials and examine whether there
are changes in chemical as well as physical properties before
and after purification. The origin and purity of purchased chi-
tosan material depends on its source, season, and conditions
of the chemical deacetylation process, which may vary across
different suppliers. Further purification process is crucial to
ensure that the starting chitosan material for nanoparticle
fabrication possesses the highest purity and integrity. In this
work, purchased chitosan materials were subjected to a vig-
orous purification process which involved mixing the solid
chitosan flakes in 1 M NaOH solution, allowing 1 g of chi-
tosan for 10 ml NaOH solution. This solid–liquid mixture was
heated and continuously stirred for 2 h at 70 ◦ C, and then fil-
tered using a Buchner funnel. Chitosan was insoluble in the
caustic solution, and the recovered flakes were washed thor-
oughly and dried at 40 ◦ C for 12 h.
 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
The NaOH treated chitosan flakes were dissolved in 0.1 M
acetic acid solution which was filtered using a filter paper
to remove residues of insoluble particles. One molar NaOH
solution was used to adjust pH value of the filtrate to pH 8.0,
resulting in purified chitosan in the form of white precipi-
tates. The precipitated chitosan was washed thoroughly using
deionized water, and the product was vacuum-dried at room
temperature for 24 h. The dried samples were used for FT-IR
analysis and preparation of the chitosan–TPP nanoparticles.
2.3. Preparation of chitosan–TPP nanoparticles
Chitosan solutions of different concentration and molecu-
lar weight were prepared by dissolving purified chitosan with
sonication in 1% (w/v) acetic acid solution until the solution
was transparent. Once dissolved, the chitosan solution was
diluted with deionized water to produce chitosan solutions
of different concentrations at 0.05%, 0.10%, 0.15%, 0.20%,
0.25%, and 0.30% (weight/volume). Tripolyphosphate was
dissolved in deionized water at the concentration 0.7 mg/ml.
The chitosan solution was flush mixed with an equal vol-
ume of TPP solution and the formation of chitosan–TPP
nanoparticles started spontaneously via the TPP initiated
ionic gelation mechanism. The nanoparticles were formed
at selected chitosan to TPP weight ratios of 3:1, 4:1, 5:1, 6:1
and 7:1. The nanoparticle suspensions were gently stirred for
60 min at room temperature before being subjected to further
analysis and applications.
2.4. FT-IR
In FT-IR analysis of both purified and raw chitosan sam-
ples, transmittance spectra were obtained using a Perkin-
Elmer FT-IR spectrometer (SPECTRUM 1000) fitted with
an attenuated total reflectance mode (ATR) cell. The equip-
ment was positioned in a laboratory maintained at 25 ± 1 ◦ C.
A small chitosan sample (7.0–9.0 mg) was placed on a NaCl
plate and subjected to light within the infrared spectrum. The
instrument operated with a resolution of 4 cm−1 and 128
scans were collected for each sample. The IR absorbency
scans were analysed between 700 and 4000 cm−1 for changes
in the intensity of the sample peaks.
2.5. Measurement of size and zeta potential of
chitosan–TPP nanoparticles
Measurement of physical size, zeta potential and polydis-
persity (size distribution) of the chitosan–TPP nanoparticles
were performed using a 3000HSA Zetasizer (Malvern Instru-
ments, England).
2.6. Morphology observation
The morphological characteristics of the nanoparticles
were examined using a high resolution TEM (Transmission
Electron Microscope) machine (Tecnai F-20, Phillips Co.,
67
Netherlands). One drop of dilute chitosan–TPP nanoparti-
cles solution was syringe placed on a carbon film 300 mesh
copper grid, allowing to sit until air-dried. The sample was
stained with 1 M uranyl acetate solution for 5 s at 7 ◦ C before
viewing on the TEM.
3. Results and discussion
3.1. Purification and characterisation of supplied
chitosan material
FT-IR was used to identify if there were variations in
chemical functional groups present at the surface of chi-
tosan samples of different molecular weight, and to deter-
mine variations among purified and unpurified chitosan sam-
ples. By comparing the characteristic transmittance spec-
trum of different chitosan samples, it is possible to ascertain
changes in the constituent surface functional groups (e.g.,
NH2 , CH2 NH) during the purification process, an indi-
cation of removal of impurities from the purchased chitosan
material.
Fig. 1 compares the transmittance spectrum of purified
HMW chitosan with the original supplied materials. The con-
trasting difference in the spectrum evidently demonstrates
the changes in surface chemistry of the original supplied chi-
tosan material after purification, indicating possible removal
of impurities, such as protein molecules and pigments. No
attempts were made in this paper to identify what partic-
ular chemical bonds are associated with the spectrum peaks
which require additional verification beyond the scope of this
study. The variation in the transmittance spectrum for LMW
and MMW chitosan samples was much less pronounced than
the HMW chitosan before and after purification. Fig. 2 com-
pares the FT-IR transmittance spectrum of purified chitosan
samples of different molecular weight. The figure reveals
variations in corresponding peak values of the same func-
tional groups among different molecular weight samples, but
not significant variations in presence of the functional groups
themselves.
Fig. 1. Representative FT-IR transmittance spectra of purified and unpurified
HMW chitosan samples.
68 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73

Fig. 2. Representative FT-IR transmittance spectra of purified HMW, MMW

Fig. 3. Effect of chitosan concentration from 0.05% to 0.30% (w/v) on par-
and LMW chitosan samples.
ticles size with three different chitosan molecular weight. Chitosan to TPP
mass ratio = 5:1, T = 20 ± 1 ◦ C, pH 5.0.

The purification study demonstrates convincingly that fur-

ther purification of supplied chitosan materials is essential The effect of chitosan to TPP weight ratio on particle size
especially with high molecular weight chitosans. was also very prominent (Fig. 4), showing a linear increase of
size with increasing chitosan to TPP weight ratio within the
tested chitosan to TPP ratio range. These linear relationships
3.2. Modulation of particle size provide a simple processing window for manipulating and
optimising the nano size for intended applications.
Particle size is one of the most significant determinant
in mucosal and epithelial tissue uptake of nanoparticles and 3.3. Modulation of particle surface charge
in the intracellular trafficking of the particles [6]. Smaller
size nanoparticles (∼100 nm) demonstrated more than 3- Chitosan has a rigid crystalline structure through inter- and
fold greater arterial uptake compared to larger nanoparticles intra- molecular hydrogen bonding. Chitosan molecules in
(∼275 nm) in an ex vivo canine carotid artery model [12,13] aqueous solutions adopt extended conformation with a more
as the smaller nanoparticles were able to penetrate throughout flexible chain because of the electrostatic charge repulsion
the sub-mucosal layers while the larger size micron-particles between the chains. When chitosan and TPP were mixed with
were predominantly localized in the epithelial lining. Prabha each other in dilute acetic acid, they spontaneously formed
et al. [14] investigated the gene transfection levels of differ- compact nano complexes with an overall positive surface
ent size fractions of PLGA nanoparticles and found that the charge, and the density of the surface charge is reflected by
lower size nanoparticle fraction produced a 27-fold higher measured zeta potential values.
transfection in COS-7 cells and 4-fold higher transfection in
HEK 293 cells for the same dose of nanoparticles. These stud-
ies also suggested that uniform particle size distribution are
important to enhance the nanoparticle-mediated gene expres-
sion.
Chitosan’s ability of quick gelling on contact with polyan-
ions relies on the formation of inter- and intramolecular
cross-linkages mediated by these polyanions. Nanoparticles
are formed immediately upon mixing of TPP and chitosan
solutions as molecular linkages were formed between TPP
phosphates and chitosan amino groups. Size and size distri-
bution of the chitosan–TPP nanoparticles depend largely on
concentration, molecular weight, and conditions of mixing,
i.e., stirring or sonication.
Fig. 3 shows the effect of chitosan concentration on parti-
cles size at three different molecular weight. It demonstrates
that the size of HMW chitosan nanoparticles was mostly
affected by the increased chitosan solution concentration, and Fig. 4. Effect of chitosan to TPP mass ratio from 3:1 to 7:1 on parti-
the increase in size with concentration showed a linear rela- cles size with three different chitosan molecular weight. c = 0.50% (w/v),
tionship within the tested range. T = 20 ±1 ◦ C, pH 5.0.
 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
Fig. 5. Effect of chitosan to TPP mass ratio on particle zeta potential with
three different chitosan molecular weight. c = 0.50% (w/v), T = 20 ± 1 ◦ C,
pH 5.0.
It was reported that the ability of nanoparticles to escape
the endo-lysosomes was dependent on the surface charge of
the nanoparticles [15,16]. Nanoparticles which show transi-
tion in their surface charge from anionic at pH 7 to cationic
in the acidic endosomal pH (pH 4–5) were found to escape
the endosomal compartment whereas the nanoparticles which
remain negatively charged at pH 4–5 were retained mostly
in the endosomal compartment. Thus, by varying the surface
charge, one could potentially be able to direct the nanoparti-
cles either to lysosomes or to cytoplasm [6]. The efficacy of
nanoparticles as drug carriers is also closely related to their
interaction, predominantly influenced by surface charge, with
proteins and enzymes in different body fluids. Calvo et al. [17]
analysed the interaction phenomenon between lysozyme, a
positively charged enzyme that is highly concentrated in
mucosas, and poly-␧-caprolactone coated nanoparticles, and
found that the interaction of lysozyme with the nanoparticles
and their consequent degradation was highly dependent on
their surface charge.
The zeta potential of chitosan–TPP nanoparticles
increased linearly with increasing chitosan to TPP weight
ratio from 3:1 to 7:1 (Fig. 5). Again, this simple linear rela-
tionship could be easily explored for modulating the particle
surface charge density to facilitate the adhesion properties
and transport properties of the nanoparticles.
The effect of chitosan concentration on zeta potential was
also investigated at a fixed chitosan to TPP weight ratio of
5:1. The results in Fig. 6 show that, unlike the trend of particle
size increase with increasing chitosan concentration, the zeta
potential decreased with increasing chitosan concentration.
3.4. The effect of solution pH
Chitosan is a weak base polysaccharide, having an aver-
age amino group density of 0.837 per disaccharide unit [18],
and insoluble at neutral and alkaline pH values. In an acidic
69
Fig. 6. Effect of chitosan concentration on particle zeta potential at three
different chitosan molecular weight. Chitosan to TPP mass ratio = 5:1,
T = 20 ± 1 ◦ C, pH 5.0.
medium, the amine groups will be positively charged, con-
ferring to the polysaccharide a high charge density [19].
Therefore, the surface charge density of chitosan molecules
is strongly dependent on solution pH [20,21], and the ionic
cross-linking process for the formation of chitosan–TPP
nanoparticles is pH-responsive, providing opportunities to
modulate the formulation and properties of the chitosan–TPP
nanoparticles.
To study the effects of changing environmental pH val-
ues on nanoparticle size and zeta potential, chitosan–TPP
nanoparticles formulated with MMW chitosan at fixed con-
centration of 0.15% (w/v) and different chitosan to TPP mass
ratio between 4:1 and 7:1 were examined at varying chitosan
solution pH values. The variations in particle size and zeta
potential with chitosan solution pH are shown separately in
Fig. 7A and B. The nanoparticles formed at solution pH 5.5
had a smaller size but a higher particle zeta potential. Fig. 7B
also demonstrated an interesting trough in zeta potential val-
ues at pH 5.0.
The surface charge reversal of nanoparticles in the acidic
solution of endo-lysosomes is proposed as the mechanism
responsible for the endo-lysosomal escape of the nanoparti-
cles into cytoplasmic compartment for effective release and
gene expression [22]. Surface charge reversal occurs when
protons or hydronium ions from bulk solution are transferred
to nanoparticle surface under acidic conditions, resulting an
increased surface charge density and zeta potential, which
would allow stronger electrostatic interactions between the
nanoparticles and tissue cells, leading to localized destabili-
sation of the cell membrane and escape of nanoparticles into
cytoplasmic compartment.
Low molecular weight chitosan–TPP nanoparticles pro-
duced at solution pH 5.5 were tested for their size and zeta
potential response to changing pH values of the residing
solution medium by simply adjusting the solution pH to a pre-
determined value by titration with either 1 M NaOH or 1 M
HCl solutions. Fig. 8A and B showed that both measured par-
70 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
Fig. 7. (A) Effect of solution pH on MMW chitosan–TPP particle size at dif-
ferent chitosan to TPP mass ratio. c = 0.15% (w/v), T = 20 ± 1 ◦ C. (B) Effect
of solution pH value on MMW chitosan–TPP nanoparticles zeta potential.
c = 0.15% (w/v), T = 20 ± 1 ◦ C.
ticle size and zeta potential are very sensitive to the changing
pH values of the residing aqueous environment, indicating the
surface density of protonised amino groups and the degree
of protonisation are reversibly responsive to changing solu-
tion pH values. The increase in measured average particle size
could be caused mainly by particle aggregation when solution
pH value increased, rather than by further growth of the indi-
vidual particle size after initial formation. The sharp increase
in size at pH > 6.0 suggests that the degree of protonisation
at surface of the particles were reduced, decreasing elec-
trostatic repulsion between the particles thereby increasing
the probability of particle aggregation. The idea of depro-
tonisation of the particle surface was supported by results
presented in Fig. 8B which shows a continual decrease in
the positive zeta potential well before the pH value reached
pH 6.0. Fig. 8B also shows that an isoelectric point for
the chitosan–TPP nanoparticles is located at around pH 9.0.
Gelling of the nanoparticle colloidal system could easily
occur when the overall particle surface charge is neutral at
the isoelectric point. The gelling mechanism in relation to
swinging pH values has been investigated for producing smart
responsive nanoparticle systems for targeted drug delivery
[20,21].
Fig. 8. (A) Responsive particle size change in relation to changing resid-
ing solution pH values from 3.2 to 12.2. LMW chitosan–TPP nanoparticles
produced at conditions c = 0.50% (w/v), chitosan to TPP mass ratio = 5:1,
T = 20 ± 1 ◦ C, pH 5.5. (B) Responsive particle zeta potential change in
relation to changing residing solution pH values from 3.2 to 12.2. LMW
chitosan–TPP nanoparticles produced at conditions c = 0.50% (w/v), chi-
tosan to TPP mass ratio = 5:1, T = 20 ± 1 ◦ C, pH 5.5.
3.5. Stability of the chitosan–TPP nanoparticle system
The chitosan–TPP nanoparticle colloidal system is ther-
modynamically unstable, especially at unfavourable solu-
tion pH conditions and at high particle concentrations,
because of high surface energy associated with the nano
scale dimensions. Fig. 9 shows size growth kinetics of MMW
chitosan–TPP particles at a dilute chitosan concentration
0.15% (w/v). Ionic gelation and growth of the chitosan–TPP
nanoparticles were completed within the first 60 min with
subsequently slight increase in particle size over the next
24 h. No apparent aggregation of particles was observed
during this period at constant temperature and solution
pH.
However when the initial chitosan concentration was
increased over and above a critical concentration, large aggre-
gates formed instantaneously. The large aggregates were
observed using the TEM imaging technique (Section 3.6),
 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73 71

and the critical aggregation concentration was studied using

Zetasizer measurement which showed a drastic size increase
accompanied by a sudden large reduction in zeta potential at
the critical concentration. The critical chitosan concentration
for the spontaneous formation of aggregates depends on solu-
tion pH and chitosan molecular weight. Tables 1 and 2 show
that the critical chitosan concentration for LMW, MMW and
HMW chitosan is 0.65%, 0.25%, 0.15% (w/v) at pH 4.0, and
1.00%, 0.85%, 0.75% (w/v) at pH 5.0, respectively.

3.6. Morphological characteristics of chitosan–TPP

nanoparticles
Fig. 9. Kinetics of chitosan–TPP nanoparticle size growth. c = 0.15% (w/v),
The morphological characteristics of the LMW chitosan–
chitosan to TPP mass ratio = 5:1, T = 20 ± 1 ◦ C, pH 5.0.
TPP nanoparticles were examined using the TEM technique.
Table 1
Measured particles size and zeta potential at different chitosan molecular weight and concentration
LMW chitosan Size (nm) Zeta (mv) MMW chitosan Size (nm) Zeta (mv) HMW chitosan Size (nm) Zeta (mv)
(%) (w/v) (%) (w/v) (%) (w/v)
0.05 136.2 48.3 0.05 145.3 43.9 0.05 155.0 37.7
0.10 142.3 44.2 0.10 150.5 40.3 0.10 188.9 34.8
0.15 152.9 41.0 0.15 165.2 39.1 0.15 3884.2 17.4
0.20 171.2 39.7 0.20 182.3 37.2 0.20 5964.3 17.2
0.25 190.3 37.3 0.25 2175.4 16.2 0.25 6136.2 16.7
0.30 203.1 35.6 0.30 10016.2 15.2 0.30 7854.2 16.9
0.35 312.7 33.3 0.35 – – 0.35 – –
0.40 429.6 31.6 0.40 – – 0.40 – –
0.45 578.3 30.4 0.45 – – 0.45 – –
0.50 712.4 28.2 0.50 – – 0.50 – –
0.55 888.6 26.9 0.55 – – 0.55 – –
0.60 997.0 25.2 0.60 – – 0.60 – –
0.65 1628.6 14.3 0.65 – – 0.65 – –
Chitosan to TPP mass ratio = 4:1, T = 20 ± 1 ◦ C, pH 4.0.

Table 2
Measured particles size and zeta potential with different chitosan molecular weight and concentration
LMW chitosan Size (nm) Zeta (mv) MMW chitosan Size (nm) Zeta (mv) HMW chitosan Size (nm) Zeta (mv)
(%) (w/v) (%) (w/v) (%) (w/v)
0.05 143.2 49.2 0.05 156.1 46.8 0.05 162.7 41.3
0.10 152.1 46.8 0.10 163.7 44.4 0.10 176.1 37.4
0.15 159.2 45.6 0.15 170.7 40.3 0.15 208.9 36.9
0.20 172.8 44.3 0.20 181.5 39.8 0.20 216.8 35.1
0.25 181.9 42.7 0.25 192.2 37.8 0.25 234.2 34.1
0.30 189.6 40.7 0.30 209.8 35.3 0.30 257.0 33.2
0.35 273.2 39.4 0.35 310.2 33.8 0.35 362.3 32.6
0.40 387.2 37.1 0.40 427.5 32.7 0.40 478.0 31.8
0.45 519.6 36.8 0.45 546.3 32.1 0.45 566.6 31.0
0.50 604.3 34.2 0.50 654.3 30.4 0.50 697.3 29.5
0.55 692.1 33.3 0.55 721.5 29.0 0.55 748.5 27.8
0.60 726.6 32.7 0.60 783.2 28.6 0.60 828.0 26.3
0.65 740.6 32.1 0.65 848.3 27.5 0.65 898.5 25.9
0.70 846.7 30.8 0.70 881.0 25.3 0.70 976.8 23.2
0.75 858.9 30.0 0.75 936.8 25.1 0.75 1654.9 15.9
0.80 893.6 28.5 0.80 988.6 24.8 0.80 2400.5 10.6
0.85 908.6 26.7 0.85 2371.0 15.2 0.85 5116.8 8.0
0.90 938.7 26.2 0.90 2742.7 12.2 0.90 – –
0.95 998.6 24.3 0.95 4276.9 7.6 0.95 – –
1.00 1819.3 13.5 1.00 – – 1.00 – –
1.10 2059.0 11.2 1.10 – – 1.10 – –
1.20 2851.6 7.6 1.20 – – 1.20 – –
Chitosan to TPP mass ratio = 4:1, T = 20 ± 1 ◦ C, pH = 5.0.
72 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73

Fig. 10. TEM image of a single LMW chitosan–TPP nanoparticle.

TEM image of single chitosan–TPP nanoparticles (Fig. 10)

reveals that the nanoparticles have a size range between 140 Fig. 12. TEM image of a large aggregate of LMW chitosan–TPP nanopar-
and 250 nm which conforms with the size measurement by ticles.
photon correlation spectroscopy using Zeasizer 3000HAS.
large aggregate formed with many distinctive single nanopar-
Fig. 11 shows the image of an aggregate of four distinc-
ticles, each still possessing a similar nano-metric dimension
tive single particles with clear joining boundaries formed
as being shown in Fig. 10.
alongside the regular geometry of the proximate polyhedron
Fig. 13 shows a TEM image of a chitosan–TPP particle
(pentagon and hexagon) shaped particles. Different to past
incorporating protein molecules of bovine serum albumin
reported works [9,23], the evidence of the formation of poly-
(BSA). 0.03 mg/ml BSA molecules were added to an equal
hydrons, instead of spheres, at nano-metric scale suggests a
volume of LMW chitosan solution (1.5%, w/v) in acidic acid
nucleation through ionic gelation followed by semi-crystal
and gently mixed for 1 h before TPP solution (0.5%, w/v) was
formation and growth.
added to make up chitosan to TPP mass ratio at 5:1. The BSA
As previously described in Section 3.5, the formation of
incorporated nanoparticles have a size range of 300–350 nm,
large nanoparticle aggregates depends on chitosan concen-
tration and solution pH. Fig. 12 shows the TEM image of a

Fig. 11. TEM image of an aggregate of four single LMW chitosan–TPP Fig. 13. TEM image of a LMW chitosan–TPP nanoparticles incorporating
nanoparticles with distinctive polyhedron shapes. BSA molecules.
 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
doubling the size of chitosan–TPP particles. The BSA incor-
porated particles possessed a typical spherical shape and
smooth surface, which confirms the findings by Xu and Du
[23], and Janes et al. [1]. Future work will investigate the
encapsulation mechanism and efficiency of protein molecules
in the ionic initiated chitosan–TPP nanoparticle formation
process, and the release kinetics of protein molecules from
the particle.
4. Conclusion
The formation of high yield chitosan–TPP nanoparticles
with predetermined nano-metric size and surface charge den-
sity can be simply manipulated and controlled by varying the
key processing conditions of chitosan concentration, chitosan
to TPP weight ratio, and solution pH value. Within the tested
range of conditions, the increase in particle size and parti-
cle zeta potential showed a simple linear relationship with
increasing chitosan to TPP weight ratio, but the zeta poten-
tial at fixed chitosan to TPP ratio showed a linear decrease
with increasing chitosan concentration. Solution pH value
and chitosan concentration also had profound influence on the
stability of the nanoparticle system, and the critical chitosan
concentrations for spontaneous formation of large particle
aggregates at pH 5 were found to be 0.65%, 0.25%, 0.15%
(w/v) at pH 4.0, and 1.00%, 0.85%, 0.75% (w/v) at pH 5.0
for LMW, MMW and HMW chitosan, respectively. The iso-
electric point of the chitosan–TPP nanoparticles was found
at around pH 9.0.
Morphological study of the nanoparticles formed under
different conditions revealed the formation of regu-
larly shaped polyhydron particles, an indication of semi-
crystallisation mechanism during the particle formation and
growth, suggesting the particles were of compact structure
with orderly molecular arrangement, the discovery of which
bears important implications on gene/protein encapsulation
and release mechanisms.
Acknowledgement
To the European Social Funding programme for providing
a scholarship to Colette Cochrane for this project.
References
[1] K.A. Janes, P. Calvo, M.J. Alonso, Polysaccharide colloidal particles
as delivery systems for macromolecules, Adv. Drug Deliv. Rev. 47
(2001) 83–97.
[2] S.C.W. Richardson, H.V.J. Kolbe, R. Duncan, Potential of low molec-
ular mass chitosan as a DNA delivery system: biocompatibility, body
distribution and ability to complex and protect DNA, Int. J. Pharm.
178 (1999) 231–243.
73
[3] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for topical
genetic immunization, J. Control. Release 75 (2001) 409–419.
[4] L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A.N. Fisher, S.S. Davis,
Chitosan as a novel nasal delivery system for vaccines, Adv. Drug
Deliv. Rev. 51 (2001) 81–96.
[5] K.A. Mislick, J.D. Baldeschwieler, Evidence for the role of proteo-
glycans in cation-mediated gene transfer, Proc. Natl. Acad. Sci. USA
93 (1996) 12349–12354.
[6] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and
gene delivery to cells and tissue, Adv. Drug Deliv. Rev. 55 (2003)
329–347.
[7] K. Corsi, F. Chellat, L. Yahia, J.C. Fernandes, Mesenchymal stem
cells, MG63 and HEK293 transfection using chitosan–DNA nanopar-
ticles, Biomaterials 24 (2003) 1255–1264.
[8] K. Romoren, B.J. Thu, O. Evensen, Immersion delivery of plasmid
DNA. II. A study of the potentials of a chitosan based delivery sys-
tem in rainbow trout (Oncorhynchus mykiss) fry, J. Control. Release
85 (2002) 215–225.
[9] S.V. Vinagradov, T.K. Bronich, A.V. Kabanov, Nanosized cationic
hydrogels for drug delivery: preparation, properties and interactions
with cells, Adv. Drug Deliv. Rev. 54 (2002) 223–233.
[10] Y. Ohya, M. Shiratani, H. Kobayashi, T. Ouchi, Release behav-
ior of 5-fluorouracil from chitosan-gel nanospheres immobilizing
5-fluorouracil coated with polysaccharides and their cell specific
cytotoxicity, Pure Appl. Chem. A 31 (1994) 629–642.
[11] X.Z. Shu, K.J. Zhu, A new approach to prepare tripolyphos-
phate/chitosan complex beads for controlled drug delivery, Int. J.
Pharm. 201 (2000) 51–58.
[12] C. Song, V. Labhasetwar, X. Cui, T. Underwood, R.J. Levy, Arterial
uptake of biodegradable nanoparticles for intravascular local drug
delivery: results with an acute dog model, J. Control. Release 54
(1998) 201–211.
[13] M.P. Desai, V. Labhasetwar, G.L. Amidon, R.J. Levy, Gastrointesti-
nal uptake of biodegradable microparticles: effect of particle size,
Pharm. Res. 13 (1996) 1838–1845.
[14] S. Prabha, W.Z. Zhou, J. Panyam, V. Labhasetwar, Size depen-
dency of nanoparticles-mediated gene transfection: Studies with
fractionnated nanoparticles, Int. J. Pharm. 244 (2002) 105–
115.
[15] W. Paul, C. Sharma, Chitosan a drug carrier for the 21st century
S.T.P, Pharm. Sci. 10 (2000) 5–22.
[16] V. Dodane, D. Vilivalam, Pharmaceutical applications of chitosan,
Pharm. Sci. Technol. Today 16 (1998) 246–253.
[17] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Effect of lysozyme on the
stability of polyester nanocapsules and nanoparticles: stabilization
approaches, Biomaterials 18 (1997) 1305–1310.
[18] F.-L. Mi, H.-W. Sung, S.-S. Shyu, Synthesis and characterization of
biodegradable TPP/genipin co-crosslinked chitosan gel beads, Poly-
mer 44 (2003) 6521–6530.
[19] K.W. Leong, H.Q. Mao, V.L. Truong-Le, DNA-polycation
nanospheres as non-viral gene delivery vehicles, J. Control. Release
53 (1998) 183–193.
[20] J.A. Ko, H.J. Park, S.J. Hwang, Preparation and characterization
of chitosan microparticles intended for controlled drug delivery, J.
Pharm. 249 (2002) 165–174.
[21] X.Z. Shu, K.J. Zhu, Novel PH–sensitive citrate cross-linked chi-
tosan film for controlled drug release, Int. J. Pharm. 212 (2001)
19–28.
[22] K. Makino, H. Ohshima, T. Kondo, Transfer of protons from bulk
solution to the surface of poly(l-lactide) microcapsules, J. Microen-
capsul. 3 (1986) 195–202.
[23] Y. Xu, Y. Du, Effect of molecular structure of chitosan on pro-
tein delivery properties of chitosan nanoparticles, Int. J. Pharm. 250
(2003) 215–226.