J. Biomater. Sci. Polymer Edn, Vol. 17, No. 5, pp.

579– 589 (2006)

 VSP 2006.
Also available online - www.vsppub.com

Stabilization of gold nanoparticles by

hydrophobically-modified polycations

REMANT BAHADUR K. C. 1 , SANTOSH ARYAL 1 , SHANTA RAJ

BHATTARAI 1 , NARAYAN BHATTARAI 2 , CHI HUN KIM 3
and HAK YONG KIM 3,∗

1 Department of Bio-Nanosystem Engineering, Chonbuk National University,

Chonju 561-756, South Korea
2 Department of Materials Science and Engineering, University of Washington, Seattle,
WA 98195, USA
3 Department of Textile Engineering, Chonbuk National University, Chonju 561-756, South Korea

Received 31 May 2005; accepted 6 December 2005

Abstract—Surface-modified gold nanoparticles have pronounced benefits in the biomedical field due
to their significant interaction with delivery materials. In the present study we used hydrophobically-
modified polycations (i.e., N -acylated chitosan) to stabilize gold nanoparticles. Aliphatic hydropho-
bic groups, having carbon chains of different lengths, were first grafted onto the backbone of chitosan
by N -acylation with fatty-acid chlorides in order to increase its hydrophobicity. Gold nanoparticles
stabilized with native chitosan and N -acylated chitosan were prepared by the graft-onto approach.
Chemical modification and its quantification were studied by Fourier-transform infrared (FT-IR) spec-
troscopy. Further, the stabilized gold nanoparticles were characterized by different physico-chemical
techniques such as UV-Vis, FT-IR, TEM, TGA and DLS. Spectral studies of gold nanoparticles show
the backbone and the side chain functional groups of chitosan were not cleaved during the conjugation
process. TEM observations revealed that the modified chitosan gold nanoparticles were well dispersed
and spherical in shape with average size around 10–12 nm in triply-distilled water at pH 7.4, whereas
the native chitosan gold nanoparticles appeared as clusters with 9.9 nm as average diameter and were
dispersed only in dilute HCl. The size of modified chitosan gold nanoparticles varied depending on
the length of grafting molecules.

Key words: Polycation; gold nanoparticles; chitosan; self-assembly; biomaterial.

∗ Towhom correspondence should be addressed. Tel.: (82-63) 270-2351. Fax: (82-63) 270-2348.
E-mail: khy@moak.chonbuk.ac.kr
580 R. B. K. C. et al.

INTRODUCTION
These days preparation of metal nanoparticles with size homogeneity is a field of
extensive research [1]. Among the metal nanoparticles, gold nanoparticles are the
most desired ones, as they are more stable and possess most fascinating aspects such
as size-related electronic, magnetic and optical (quantum size effect) properties [2].
Some specific functional groups, like cyano ( CN), mercapto ( SH) and amino
( NH2 ), have a high affinity for gold [3, 4]. Hence, the capping agents with such
functional groups are expected to produce gold nanoparticles with narrow size dis-
tribution [5, 6]. Most of the studies were performed in non-polar organic solvents
using a tertiary ammonium ion as a phase-transfer agent [7, 8]. However, less atten-
tion has been paid to the preparation of discrete, well-dispersed gold nanoparticles
in aqueous medium. The specific interaction of such nanoparticles with biologi-
cal system and its stability under physiological conditions could provide a broad
application in biolabeling, drug delivery, catalysis [9], DNA detection [10], etc.
Chitosan is a transformed polysaccharide obtained by deacetylation of chitin, one
of the most important natural polymers constituting the shells of crustaceans and
cell walls of many fungi. The chemical structure of chitosan mainly composed
of 2-amino-2-deoxy-β-D-glucopyranose repeating units with a small amount of
2-acetamido-2-deoxy-β-D-glucopyranose units as a residue (Fig. 1). Recently,
much attention has been given to chitosan due to its excellent biological properties,
such as biodegradation [11], immunological, anti-bacterial [12] and wound-healing

Figure 1. Chemical structures of native and N -acylated chitosan (subscripts m and n represent the
variable numbers 78 and 22, respectively).
 Stabilization of gold nanoparticles by hydrophobically-modified polycations 581

activity [13]. Furthermore, it has been found to be a good support material for gene
delivery [14], cell culture [15], tissue engineering [16], etc. Systematic exploration
of chitosan as a structural material has been done for designing functional layers on
the electrode surface [17]. In addition, many papers were already published on the
stabilization of gold nanoparticles using native chitosan as a capping agent [18 –20].
However, the applications of chitosan and chitosan-based materials are limited
by their solubility only in dilute acids. To overcome this limitation chemical
modification could be an easy way [21 –24].
Here, in order to provide a general means for the controlled self-assembly
of nanoparticles, we demonstrate the stabilization of gold nanoparticles using
acyl-modified chitosan. Two types of N -acylated chitosan (hexanoyl chitosan
(N ac-6) and octanoyl chitosan (N ac-8)) were used to stabilize gold nanoparticles.
The formation of gold nanoparticles was characterized by UV-Vis absorption
spectroscopy. FT-IR and thermo gravimetric analysis (TGA) were used to analyze
the nature of polymeric adsorption and its quantification on the surface of gold
nanoparticles, respectively. Transmission electron microscopy (TEM) and dynamic
light scattering (DLS) measurements were used to determine the morphology and
size distributions of the nanoparticles.

MATERIALS AND METHODS

Materials
Chitosan-10 (viscosity average molecular weight, Mv = 2.1 × 105 , degree of
deacetylation (fraction of free amino group) 78%) was purchased from Wako
(Japan). Viscosity average molecular weight of chitosan was determined accord-
ing to the previous report [25]. Fatty acyl chlorides (e.g., hexanoyl chloride and
octanoyl chloride), hydrogen tetrachloroaurate (HAuCl4 ) and sodium borohydride
were purchased from Sigma-Aldrich (USA) and used without any further purifica-
tion. All other chemicals were purchased from Showa (South Korea).

Preparation of N-acylated chitosan (Nac)

Chitosan-10 (0.83 g) was dissolved in 1.0% aqueous acetic acid (100 ml) by stirring
for 24 h. pH of the solution was adjusted to 7.0 with NaOH (0.1 M) under vigorous
stirring [24], followed by the addition of 0.2 mol fatty acid chloride (hexanoyl or
octanoyl chloride). The resultant solution was diluted 11 times with de-ionized
water. After 6 h of continuous stirring, it was neutralized (0.1 M NaOH) and
precipitated with acetone. The product was washed with excess methanol (50–
60◦ C) and dried under vacuum for 3 days at room temperature.

Stabilization of gold nanoparticles

Native and N -acylated-chitosan-stabilized gold nanoparticles were prepared as
described elsewhere [22]. Briefly, 1.0 ml of freshly prepared 10 mM HAuCl4
582 R. B. K. C. et al.

solution was added to 2.0 ml of a 0.33% solution of the respective polymer (native
chitosan solution in 1% aqueous acetic acid and N ac in 0.1 M HCl) and stirred for
1 h. To this solution, 0.4 ml (0.1 M) of freshly prepared ice-cold sodium borohydride
was added under stirring. Rapid color change to pink indicated the formation of gold
nanoparticles. Thus formed gold nanoparticles were collected by ultracentrifugation
at 35 000×g for 30 min at 4◦ C. A stock solution of native chitosan-Au nanoparticles
was made in dilute HCl, whereas that of modified chitosan-Au (N ac-6-Au and N ac-
8-Au) was in triply-distilled water at pH 7.4 for further characterization.

Characterization of modified chitosan

Measurement of degree of acylation. The fraction of substituted amino groups
(degree of substitution (DS)) and structural analysis were done by FT-IR spec-
troscopy [26]. DS was determined according to Moore et al. [27]. The intensity
ratio of the bands at 1656 cm−1 (amide I) and 3418 cm−1 ( OH) was used for
calculation of DS according to the following equation:
DS (%) = ((A1656 /A3418 ) − 0.22) × 100,
where 0.22 is the acetyl group specified in native chitosan.

Characterization of gold nanoparticles. UV-Vis absorption spectra of the sam-

ples were recorded using a Cary 500 UV-Vis-NIR spectrometer. FT-IR spectra were
recorded as KBr pellets using an ABB Bomen MB 100 spectrometer. TGA was car-
ried out using a 2050 thermogravimetric analyzer at a heating rate of 15◦ C/min from
room temperature to 1000◦ C, under a steady flow of nitrogen. Particle size and mor-
phology were observed using a Jeol JEM 2010 transmission electron microscope
operating at 200 kV. Samples for TEM were prepared by dipping a carbon-coated
copper grid in an aqueous dispersion of nanoparticles and dried at room temper-
ature. Particle size and its distribution were determined using a Malvern System
4700 dynamic light scattering (DLS) instrument equipped with vertically polarized
light supplied by an argon-ion laser (Cyonics) at 20 mW.

RESULTS AND DISCUSSION

Chemical modifications
FT-IR spectra of both native and modified chitosan are presented in Fig. 2. The
spectrum of native chitosan exhibited the characteristic amide bands at 1656 cm−1
(amide I), 1593 cm−1 (amide II) and 1373 cm−1 (amide III) (curve a). After
acylation, the intensity of the amide-I band was significantly increased as compared
to amide II, which shows the partial acylation of chitosan (curves b and c). If it
were fully acylated, the amide II band would have completely disappeared and
only one amide band (amide I) would appear. Furthermore, a shoulder peak at
 Stabilization of gold nanoparticles by hydrophobically-modified polycations 583

Figure 2. FT-IR spectra of (a) native chitosan, (b) N ac-8 and (c) N ac-6.

2895 cm−1 due to C H stretching confirms the alkyl substitution in chitosan

backbone. Intensity of this band increased linearly with the chain length of acid
chloride. A broad band in the region of 3200–3500 cm−1 in all the cases was
assigned to the H-bonded N H and O H stretching vibrations. The spectra of
the all the compounds showed a well-resolved band due to C O stretching of
saccharine moiety at 1075 cm−1 [28 –30]. Although the acylation was performed
under identical condition, the degree of acylation was different with types of fatty
acyl chlorides, i.e., 45.2% for hexanoyl chloride (N ac-6) and 41.2% for octanoyl
chloride (N ac-8).

Stabilization of gold nanoparticles

Pure gold nanoparticles, native chitosan-gold nanoparticles and modified chitosan-

gold nanoparticles (N ac-Au) were prepared by chemical reduction of gold salt.
Depending on the nature of capping agents, color of resulting dispersion was varied
from pink to dark purple. UV-Vis spectra of gold hydrosol, native chitosan-Au and
N ac-6-Au showed a characteristic surface plasmon band (SPB) at 512, 530 and
541 nm, respectively, suggesting the formation of gold nanoparticles by chemical
reduction [31] (Fig. 3). SPB is the collective oscillation of the electron cloud on the
surface of nanoparticles and can be taken in to account for the evaluation of particle
size [1]. A significant red shift in the SPB of chitosan-capped gold nanoparticles
584 R. B. K. C. et al.

Figure 3. UV-Vis absorption spectra of (a) pure gold nanoparticles, (b) native chitosan-Au and
(c) N ac-6-Au.

(curves b and c) compared to pure gold (curve a) suggests a linear increase in

particle size consequent to the surface modification of gold nano particle.
Figure 4 shows the FT-IR spectra of N ac-6-stabilized gold nanoparticles (curve a).
In these spectra, the characteristic bands, amide I, II and III, were shifted to 1635,
1525 and 1404 cm−1 , due to interaction with gold nanoparticles. Shifting of such
amide bands either higher or lower energies indicates the attachment of gold on
chitosan through nitrogen atom. Electrostatic interaction between nitrogen and gold
caused the diversion of surface electronic density from nitrogen to metal (gold). In
that case, bond strength between nitrogen and other atoms is increased; therefore,
the vibrational band is shifted toward shorter wavelength. Likewise, the broad band
(H-bonded N H and O H) at 3200–3500 cm−1 modified to an intense peak, which
is probably due to decrease in the extent of H-bonding and interaction with metallic
surface [32]. However, the above band shift and intensity variation were nearly
identical in all the cases, namely chitosan-Au, N ac-6-Au and N ac-8-Au.
Figure 5 shows the comparative TGA thermogram of pure gold nanoparticles, na-
tive chitosan, native chitosan-Au, modified chitosan (N ac-6) and modified chitosan-
Au (N ac-6-Au). A slight deviation was found in the TGA curve of pure gold
nanoparticles, which may be the cause of mineral impurities (curve a). From the
TGA, the inflection point temperatures (Td ) of native chitosan and N ac-6 were de-
termined at 333 and 316◦ C (curves d and e), respectively. Additionally, two new
Td points were found at 194 and 650◦ C after the grafting of native chitosan on
 Stabilization of gold nanoparticles by hydrophobically-modified polycations 585

Figure 4. FT-IR spectra of (a) N ac-6-Au, (b) native chitosan and (c) N ac-6.

Figure 5. TGA graph of (a) pure gold nanoparticles, (b) native chitosan-Au, (c) N ac-6-Au, (d) native
chitosan and (e) modified chitosan (N ac-6).
586 R. B. K. C. et al.

Table 1.
Characterization of gold nanoparticles

Sample Polymer/Au Polymer on the surface Mean diameterb

feed ratio (w/w) of nanoparticlesa (%) (nm)
Gold hydrosol — — 4.5 ± 0.1
Native chitosan-Au 2 22 9.9 ± 0.2
N ac-6-Au 2 39 12.9 ± 0.2
N ac-8-Au 2 54 11.0 ± 0.1
a Calculated from TGA data.
b Measured in TEM images.

Figure 6. TEM micrographs of nanoparticles. (a) Pure gold nanoparticles, (b) native chitosan-Au,
(c) N ac-6-Au.

gold nanoparticles (curve b). Interestingly, the modified chitosan-gold nanoparti-

cles (N ac-6-Au) showed four Td at 194, 243, 643 and 867◦ C (curve c). It implies
that the thermal stability of the native chitosan altered after modification and com-
plexation with gold. The concentration of ligand attached on the gold nanoparticles
was found to increase linearly with the hydrophobic modification and chain-length
modifier (Table 1).
TEM images show a distinct variation in size between pure gold nanoparticles,
native chitosan-Au and modified chitosan-Au (N ac-Au) (Fig. 6). Average particle
size of pure gold nanoparticles, native chitosan-Au and modified chitosan-Au (N ac-
6-Au and N ac-8-Au) were 4.5, 9.9, 12.9 (N ac-6-Au) and 11.0 (N ac-8-Au) nm,
respectively (Table 1). These variations may be probably due to the variation in
ionic strength of capping agents, since the inter-particle interaction in the colloidal
system increases with the ionic strength of stabilizing agent [33]. Figure 7 shows
the size distribution of the various nanoparticles. A small difference observed in the
size of nanoparticles between TEM and DLS measurements could be related to the
fact that DLS gives the hydrodynamic diameter rather than the actual diameter of
nanoparticles. Further, a difference in the solubility and stability of nanoparticles
stabilized by native chitosan and modified chitosan were found. N ac-Au is easily
soluble in triply-distilled water at pH 7.4, whereas the native chitosan-Au was
soluble only in aqueous mineral acidic.
 Stabilization of gold nanoparticles by hydrophobically-modified polycations 587

Figure 7. Particle size distribution of (a) pure gold nanoparticles, (b) native chitosan-Au and (c) N ac-
6-Au.
588 R. B. K. C. et al.

CONCLUSIONS

In this study, aliphatic hydrophobic chains of different lengths were grafted on to

the backbone of chitosan by N -acylation with fatty-acid chlorides. Native chi-
tosan and N -acylated chitosan (N ac-6 and N ac-8)-stabilized gold nanoparticles
were prepared by a graft-onto approach during the chemical reduction of gold tetra-
chloroaurate with sodium borohydride. FT-IR spectroscopic analysis of chitosan-
stabilized gold nanoparticles confirmed the attachment of polymer on the surface
of gold was through nitrogen atom. TEM photographs revealed that the modified
chitosan-stabilized gold nanoparticles (N ac-6-Au and N ac-8-Au) were discrete and
spherical with uniform size around 10–12 nm, whereas the native chitosan stabi-
lized gold nanoparticles were clusters with 9.9 nm as particle size. Both the par-
ticle size and concentration of ligand attached on the surface of gold nanoparticles
varied with the length of hydrophobic chain attached to chitosan molecule. From
the above results, it is clear that the gold nanoparticles, with interesting properties
such as increased dimensional stability, storage at ambient conditions and ease of
handling for applications in biological systems at physiological pH, were obtained
using hydrophobically-modified chitosan molecules. Hence, we believe that these
modified chitosan-stabilized gold nanoparticles could be suitable biomaterials for
the immobilization of DNA, proteins and drugs.

Acknowledgements
This work was supported by the Regional Research Centers Program of the Korean
Ministry of Educational and Human Resources Development through the Center for
Healthcare Technology Development.

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