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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 chitosanTPP (tripolyphosphate) nanoparticles in the size
range of 100250 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 chitosanTPP
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 chitosanTPP 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
Chitosan is a non-toxic biodegradable polycationic polymer 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 proteins [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 preparation, 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).
0927-7765/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2005.06.001
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Netherlands). One drop of dilute chitosanTPP nanoparticles 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.
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The purification study demonstrates convincingly that further purification of supplied chitosan materials is essential
especially with high molecular weight chitosans.
3.2. Modulation of particle size
Particle size is one of the most significant determinant
in mucosal and epithelial tissue uptake of nanoparticles and
in the intracellular trafficking of the particles [6]. Smaller
size nanoparticles (100 nm) demonstrated more than 3fold greater arterial uptake compared to larger nanoparticles
(275 nm) in an ex vivo canine carotid artery model [12,13]
as the smaller nanoparticles were able to penetrate throughout
the sub-mucosal layers while the larger size micron-particles
were predominantly localized in the epithelial lining. Prabha
et al. [14] investigated the gene transfection levels of different size fractions of PLGA nanoparticles and found that the
lower size nanoparticle fraction produced a 27-fold higher
transfection in COS-7 cells and 4-fold higher transfection in
HEK 293 cells for the same dose of nanoparticles. These studies also suggested that uniform particle size distribution are
important to enhance the nanoparticle-mediated gene expression.
Chitosans ability of quick gelling on contact with polyanions 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 distribution of the chitosanTPP 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 particles 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
the increase in size with concentration showed a linear relationship within the tested range.
Fig. 3. Effect of chitosan concentration from 0.05% to 0.30% (w/v) on particles size with three different chitosan molecular weight. Chitosan to TPP
mass ratio = 5:1, T = 20 1 C, pH 5.0.
Fig. 4. Effect of chitosan to TPP mass ratio from 3:1 to 7:1 on particles size with three different chitosan molecular weight. c = 0.50% (w/v),
T = 20 1 C, pH 5.0.
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.
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medium, the amine groups will be positively charged, conferring 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 chitosanTPP
nanoparticles is pH-responsive, providing opportunities to
modulate the formulation and properties of the chitosanTPP
nanoparticles.
To study the effects of changing environmental pH values on nanoparticle size and zeta potential, chitosanTPP
nanoparticles formulated with MMW chitosan at fixed concentration 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 values 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 nanoparticles 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 destabilisation of the cell membrane and escape of nanoparticles into
cytoplasmic compartment.
Low molecular weight chitosanTPP nanoparticles produced 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 predetermined value by titration with either 1 M NaOH or 1 M
HCl solutions. Fig. 8A and B showed that both measured par-
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Fig. 7. (A) Effect of solution pH on MMW chitosanTPP particle size at different chitosan to TPP mass ratio. c = 0.15% (w/v), T = 20 1 C. (B) Effect
of solution pH value on MMW chitosanTPP 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 solution 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 individual 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 electrostatic repulsion between the particles thereby increasing
the probability of particle aggregation. The idea of deprotonisation 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 chitosanTPP 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 residing solution pH values from 3.2 to 12.2. LMW chitosanTPP 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
chitosanTPP nanoparticles produced at conditions c = 0.50% (w/v), chitosan to TPP mass ratio = 5:1, T = 20 1 C, pH 5.5.
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Table 1
Measured particles size and zeta potential at different chitosan molecular weight and concentration
LMW chitosan
(%) (w/v)
Size (nm)
Zeta (mv)
MMW chitosan
(%) (w/v)
Size (nm)
Zeta (mv)
HMW chitosan
(%) (w/v)
Size (nm)
Zeta (mv)
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
136.2
142.3
152.9
171.2
190.3
203.1
312.7
429.6
578.3
712.4
888.6
997.0
1628.6
48.3
44.2
41.0
39.7
37.3
35.6
33.3
31.6
30.4
28.2
26.9
25.2
14.3
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
145.3
150.5
165.2
182.3
2175.4
10016.2
43.9
40.3
39.1
37.2
16.2
15.2
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
155.0
188.9
3884.2
5964.3
6136.2
7854.2
37.7
34.8
17.4
17.2
16.7
16.9
Size (nm)
Zeta (mv)
MMW chitosan
(%) (w/v)
Size (nm)
Zeta (mv)
HMW chitosan
(%) (w/v)
Size (nm)
Zeta (mv)
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
1.10
1.20
143.2
152.1
159.2
172.8
181.9
189.6
273.2
387.2
519.6
604.3
692.1
726.6
740.6
846.7
858.9
893.6
908.6
938.7
998.6
1819.3
2059.0
2851.6
49.2
46.8
45.6
44.3
42.7
40.7
39.4
37.1
36.8
34.2
33.3
32.7
32.1
30.8
30.0
28.5
26.7
26.2
24.3
13.5
11.2
7.6
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
1.10
1.20
156.1
163.7
170.7
181.5
192.2
209.8
310.2
427.5
546.3
654.3
721.5
783.2
848.3
881.0
936.8
988.6
2371.0
2742.7
4276.9
46.8
44.4
40.3
39.8
37.8
35.3
33.8
32.7
32.1
30.4
29.0
28.6
27.5
25.3
25.1
24.8
15.2
12.2
7.6
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
1.10
1.20
162.7
176.1
208.9
216.8
234.2
257.0
362.3
478.0
566.6
697.3
748.5
828.0
898.5
976.8
1654.9
2400.5
5116.8
41.3
37.4
36.9
35.1
34.1
33.2
32.6
31.8
31.0
29.5
27.8
26.3
25.9
23.2
15.9
10.6
8.0
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large aggregate formed with many distinctive single nanoparticles, each still possessing a similar nano-metric dimension
as being shown in Fig. 10.
Fig. 13 shows a TEM image of a chitosanTPP particle
incorporating protein molecules of bovine serum albumin
(BSA). 0.03 mg/ml BSA molecules were added to an equal
volume of LMW chitosan solution (1.5%, w/v) in acidic acid
and gently mixed for 1 h before TPP solution (0.5%, w/v) was
added to make up chitosan to TPP mass ratio at 5:1. The BSA
incorporated nanoparticles have a size range of 300350 nm,
doubling the size of chitosanTPP particles. The BSA incorporated 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 chitosanTPP nanoparticle formation
process, and the release kinetics of protein molecules from
the particle.
4. Conclusion
The formation of high yield chitosanTPP nanoparticles
with predetermined nano-metric size and surface charge density 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 particle zeta potential showed a simple linear relationship with
increasing chitosan to TPP weight ratio, but the zeta potential 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 isoelectric point of the chitosanTPP nanoparticles was found
at around pH 9.0.
Morphological study of the nanoparticles formed under
different conditions revealed the formation of regularly shaped polyhydron particles, an indication of semicrystallisation 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) 8397.
[2] S.C.W. Richardson, H.V.J. Kolbe, R. Duncan, Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body
distribution and ability to complex and protect DNA, Int. J. Pharm.
178 (1999) 231243.
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