Vol.2, No.7, 707-712 (2010) Natural Science
Copyright © 2010 SciRes. OPEN ACCESS
Synthesis and characterization of an amphiphilic
chitosan bearing octyl and methoxy polyethylene
glycol groups
Guanghua Liu, Jianqun Gan, Aimin Chen, Qian Liu, Xusheng Zhao*
Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou, China;
*Corresponding Author: zhao7503@yahoo.com
Received 14 April 2010; revised 18 May 2010; accepted 25 May 2010.
An amphiphilic N-octyl-O-methoxy poly (ethyl-
ene glycol) chitosan was successfully prepared
by grafting successively octyl groups onto
amino groups at chitosan’s C-2 position as hy-
drophobic moieties and methoxy polyethylene
glycol (MPEG) groups onto hydroxyl groups at
C-6, C-3 as hydrophilic ones. A certain amount
of -NH2 was retained in the structure of chitosan
derivatives through protection by phthalic an-
hydride. The chemical structures and degree of
N-and O-substitution of chitosan derivatives
were characterized by FTIR, 1H NMR, GPC and
elemental analysis, respectively. The amphiphi-
lic property for convenient self-assembly and
the preserved -NH2 groups for progressive
chemical cross-linking make the resultant N-
ocyl-O-MPEG chitosan soluble in water and po-
tentially applicable in preparing stable chitosan
hollow microspheres, a demanding drug-carrier
in medical and pharmaceutical sciences.
Keywords: Amphiphilic Groups; Chitosan
Derivatives; Protection; Hollow Microsperes
In recent years, micelles and hollow microspheres pre-
pared from self-assemblies of amphiphilic polymers
have attracted great attention due to a variety of applica-
tions in DNA, antigens, delivery carriers for drugs and
protection proteins or/and enzymes, especially for con-
trolled or sustained drug-delivery systems [1]. The
unique core-shell architecture is composed of hydropho-
bic segment, which acted as internal core, and hydro-
philic segment which acted as surrounding corona in
aqueous medium [2]. The hydrophobic core provides a
loading space for water-insoluble drugs and stabilizes
them, whereas the hydrophilic shell protects encapsu-
lated drugs [3]. Additionally, the modification of hydro-
philic shell affects pharmacokinetic behavior, such as
prolonged circulation time, target release [4] and con-
trolled drug release by using stimuli-sensitive copoly-
mers [5].
In comparison with common polymers, chitosan, the
second most abundant natural biopolymer only to cellu-
lose [6] and known as its excellent property in non-tox-
icity, biodegradability, good biocompatibility, etc. [7],
would exhibit special advantages in drug delivery sys-
tem. The active hydroxyl and amino groups within chi-
tosan could be easily modified to endow it with new or
improved properties, and at the same time, keeping its
original physiochemical and biochemical properties [6].
As a result, various amphiphilic chitosan derivatives and
hollow microspheres prepared from them have been ex-
tensively published [6]. For examples, highly N-methy-
lated modified chitosan possessing hydrophobic -N
(CH3)2, -NH(CH3) and hydrophilic -N+(CH3)3 groups
was reported by Sieval et al. which is soluble in water at
a broad pH value [8]. Through emulsion-crosslinking,
Peng et al. prepared hollow microspehres from N-me-
thylated chitosan with diameters range from 2-5 um [9].
Liu et al. synthesized an amphiphilic carboxymethyl-
hexanoyl chitosan [10] which formed hollow nanocap-
sules with 20-200 nm in diameter [11]. As a demanding
drug-carrier in medical and pharmaceutical sciences,
stable chitosan hollow microsphere is probably one of
the most valuable candidates.
Presently, the hydrophobic groups of amphiphilic chi-
tosan derivatives ever reported generally contain long
alkyl [12,13], long acyl [10] and aryl [14], while the
hydrophilic ones include carboxymethyl [10], sulfate
[12], phosphate [15,16], N-trimethyl [17] and polyeth-
ylene glycol [14,18]. To prepare amphiphilic chitosan
G. H. Liu et al. / Natural Science 2 (2010) 707-712
Copyright © 2010 SciRes. OPEN ACCESS
hollow microspheres as drug carriers, the substituents,
especially the hydrophilic groups, should be non-toxic
and biocompatible. It’s impossible to form stable core-
hell structure through nothing but self-assembly of am-
phiphilic chitosan. Amino groups within the backbone of
chitosan should be crosslinked chemically to form stable
and hard shell thereafter self-assembly process. That’s to
say, adequate primary amino groups (-NH2) existed in
amphiphilic chitosan are necessary for preparing hollow
microspheres. To the best of our knowledge, most re-
ports ever reported about amphiphilic chitosan deriva-
tives did not pay much attention to this problem.
In this work, we report a novel amphiphilic N-octyl
O-methoxy polyethylene glycol (MPEG) chitosan pos-
sessing adequate primary amino groups. PEG chain has
been proved to be non-toxic, very flexible and highly
hydrated, soluble in water and majority organic solvents,
and is the most widely used biocompatible polymer for
chemical and biological applications [19]. Through the
effective protection of -NH2 groups within the backbone
of chitosan during the modification process, the chitosan
derivatives synthesized in this study preserve a certain
amount of -NH2 groups for latter chemical crosslinking
to prepare eventually sizecontrollable hollow micro-
2.1. Materials
Chitosan (Mr = 1 × 105, degree of deacetylation (DD) =
95%) was provided by Zhejiang Aoxing Biochemical Co.
Ltd (China). Octylaldehyde was obtained from Guang-
zhou Damo Chemical Co. Ltd (China). Methoxy poly-
ethylene glycol (MPEG, Mr = 1900) was purchased from
Alfa Aesar. Methyl iodide was ordered from Shanghai
Jingchun Chemical Reagent Factory (China). Dimethyl-
formamide (DMF) was distilled under reduced pressure
and stored over molecular sieves. Dialysis tube was
achieved from SPECTRUM (Canada) with a molecular
weight cut-off (MWCO) of 6000~8000. Ag2O was fresh-
ly prepared from AgNO3 and KOH in our laboratory. All
other materials used in this study were of analytical
grade and without further purification.
2.2. Synthetic Procedures
2.2.1. Synthesis of N-Octyl Chitosan (NOC)
N-octyl chitosan was prepared as reported previously
(Scheme 1). Briefly, chitosan (5.0 g, 31 mmol) was sus-
pended in 60 ml methanol with active stirring at room
temperature before the addition of octaldehyde (5.0 ml,
31 mmol). Then, KBH4 (2.5 g) dissolved in 10 ml water
was added dropwise to the mixture after 24 h. The reac-
tion solution was neutralized with hydrochloric acid and
O , rt
phthalic anhydride
DMF, 130, 6h
1) MPEGI, Ag
2) NH
90, 15h
Scheme 1. Synthetic procedure of N-octyl-O-MPEG chitosan.
the product was precipitated with methanol after a fur-
ther 24 h continuous stirring. The precipitate was filtered,
washed repeatedly with methanol and water and then
dried under vacuum at 60 overnight to give 7.0 g
N-octyl chitosan. Degree of N-substitution of octyl was
54.3% from element analysis.
2.2.2. Synthesis of N-Octyl-N-Phthaloyl
Chitosan (NONPC)
The synthesis of N-octyl-N-phthaloyl chitosan (NONPC)
is described briefly as follows. A mixture of N-octyl chi-
tosan (3.0 g, 13.8 mmol) and phthalic anhydride (PHA)
(2.8 g, 3 mol equiv to amino) in 30 ml DMF was heated to
130 under nitrogen with magnetic stirring. After 6 h,
the brown precipitate obtained by pouring the solution
into ice-water was collected by filtration, extracted with
ethanol in Soxhlet’s apparatus for 48 h, and dried under
vacuum at 50 to give 3.6 g NONPC products.
2.2.3. Synthesis of Methoxy Polyethylene Glycol
Lodide (MPEGI)
Methoxy polyethylene glycol iodide (MPEGI) was syn-
thesized according to the reports by Gorochovceva and
Makuska [19]. Briefly, MPEG-1900 (19 g, 10 mmol),
triphenylphosphite (8.0 ml, 30 mmol) and methyl iodide
(4.3 ml, 30 mmol) were mixed and stirred in a three-
neck flask at 120 for 6 h. Then, the reaction mixture
was cooled, dissolved in toluene, precipitated in 500 ml
diethyl ether, and then filtered, washed several times
with diethyl ether and dried in air to give pale yellow
product. The yield of the product was 92%.
2.2.4. Synthesis of N-Octyl-O-MPEG Chitosan
The synthetic procedure of N-octyl-O-MPEG chitosan
(NOOMC) is carried out by using NONPC, MPEGI and
Ag2O with different grams or molar ratios as the starting
materials (Table 1). Firstly, NONPC, MPEGI and Ag2O
were mixed and heated at 60 for 16 h with magnetic
stirring. Then, hydrazine monohydrate (80%, 40 ml or
20 ml) and distilled water (80 ml or 40 ml) were poured
G. H. Liu et al. / Natural Science 2 (2010) 707-712
Copyright © 2010 SciRes. OPEN ACCESS
into the mixture and the temperature was raised to 90
and maintained for 15 h before the mixture was filtered
to remove solid impurity. The filtrate solution was dia-
lyzed against water for 72 h and then concentrated
before the solid residue was dried in vacuum at 50 to
give final products of N-octyl-O-MPEG chitosan (NOO-
MC). Depending on different amount of hydrazine
monohydrate and distilled water used during the synthe-
sis, the products of NOOMC with two different degree
of substitutions (DS) of MPEG were labeled as NOOMC
(I) and NOOMC (II), respectively.
2.3. Characterization
1H NMR spectra were recorded at 400MHz using a
Bruker DRX-400 spectrometer. CF3COOD and D2O
were used as the solvents for chitosan and its derivatives,
respectively. Elemental analysis was determined using
an Elementar Vario EL III analyzer. FTIR spectra were
obtained from Fourier transformation infrared spec-
trometer (Analect RFX-65A) in KBr discs. Molecular
weight of the amphiphilic chitosan was determined using
Waters 515-410 gel permeation chromatography (GPC).
3.1. Synthesis of the Chitosan Derivatives
Generally, amphiphilic chitosan could be prepared
through introducing the hydrophilic moiety first and the
hydrophobic one later [12,13], or in the reverse order
[11,20]. The latter procedure was adopted in this inves-
tigation. As shown in Scheme 1, the amphiphilic chito-
san derivatives were synthesized through the following
three steps. At the first step, hydrophobic octyl groups
were introduced by Schiff reaction prior to MPEG. Oc-
tylaldhyde residue was found very easy to be removed
with heterogeneous reaction, and rather difficult to be
excluded when Schiff reaction is carried out at the last
step. Meanwhile, if MPEG was introduced onto hy-
droxyl firstly, the molecule weight of the resultant
O-MPEG chitosan would be much higher than chitosan,
and the mole ratio of octylaldehyde and O-MPEG chito-
san is difficult to calculate and control in Schiff reaction.
Table 1. Amounts of reactants in synthesizing NOOMC with
different degree of substitutions.
No. NONPC/g MPEGI/g Ag2O/g
m(Ag 2O) *
(I) 1.5 10.2 1.2 1:1:1 NOOMC()
(II) 0.8 10.8 1.3 1:2:2 NOOMC()
NONPC, N-octyl-N-phthaloyl chitosan; MPEGI, methoxy polyethyl-
ene glycol iodide; NOOMC, N-octyl-O-MPEG chitosan. * m repre-
sents mole ratio.
It has been estimated that amino groups at C-2 show
higher nucleophilic activity than hydroxyl groups (either
C-3 or C-6) in the main backbone of chitosan [21].
Thereby, to avoid the grafting of MPEG onto amino
groups which retained in N-octyl chitosan, it is necessary
to protect amino groups in advance by the use of phthalic
anhydride. This protection method was reported earlier in
1991 by Nishimura et al. [22], which has been applied to
various region-selective modifications of chitosan [21,23].
A key point for this pathway is the fact that chitosan is
insoluble in several organic solvents, such as DMF, while
N-phthaloyl derivative of chitosan is soluble [18,22]. N-
octyl chitosan, just like chitosan, has also a bad solubility
in organic solvent. It was found in our experiment that
N-octyl-N-phthaloyl chitosan is soluble in DMF, which is
of great significance for further reaction with MPEG io-
dide. Accordingly, N-phthaloyl group is the prerequisite
for both protection of active amino in chitosan and solubi-
lization of the product in DMF.
As mentioned above, a certain amount of primary
amino is indispensable for the preparing of amphiphilic
chitosan hollow microspheres. It is identified from
Scheme 1 that the content of primary amino of the final
product is determined by the first step reaction. Hence,
primary amino cannot be modified and replaced com-
pletely with octyl groups. In our study, to assure an in-
complete substitution reaction on amino groups, we
adopted an appropriate molar ratio of chitosan to octy-
laldehyde in Schiff reaction. It is expected that we could
get amphiphilic chitosan possessing different amount of
active amino groups. That is, a certain extent of primary
amino content could be preserved through controlling
the molar ratio of the starting materials. Further investi-
gations are currently under way in our laboratory.
Compared with Schiff reaction at the first step, the
amino groups retained in N-octyl chitosan should be modi-
fied completely with phthalimide groups to avoid their
participation in further reaction. Threefold excess of
phthalic anhydride was used in our experiment to protect
amino as complete as possible. The course of the etherifi-
cation of N-octyl-N-phthaloyl with MPEGI was similar to
that described by Natalijia Gorochovceva et al. [18] where
Ag2O is a suitable and effective catalyzer for etherification.
The reaction between chitosan hydroxyl groups and
MPEGI is irreversible and proceeds till the consumption of
active functional groups. In our study, the degree of sub-
stitution (DS) of MPEG is represented from the molar ratio
of MPEGI to N-octyl-N-phthaloyl chitosan. N-octyl-
O-MPEG chitosan with different DS of MPEG is illus-
trated from the molar ratio of the above two reagents.
3.2. Characterization of Chitosan Derivatives
The degree of substitution (DS) of chitosan derivatives
was calculated by comparing C and N molar ratio ob-
G. H. Liu et al. / Natural Science 2 (2010) 707-712
Copyright © 2010 SciRes. OPEN ACCESS
tained from the elemental analysis data (Table 1). The
DS (54.3%) of N-octyl chitosan (NOC) confirms the
incomplete modification by octylaldehyde with 54.3% of
amino groups substituted and about 45% of them re-
tained in NOC. According to the variation in m(C)/m(N)
of NOC and N-octyl-N-phthaloyl chitosan (NONPC), it
can be calculated that the DS of phthlimide groups is
66.3%, which exceeds the amino content (about 45%) of
NOC. This indicates that the retained amino groups were
entirely protected and only a few hydroxyl groups were
modified simultaneously, which is identical with the
report [23] that treatment of chitosan with phthalic an-
hydride generally results in partial O-phthaloylation in
addition to N-substitution. The DS of MPEG grafts on
the final product was obtained by comparing C/N of
NOC and N-octyl-O-MPEG chitosan. As can be seen
from Table 2, the DS of NOOMC (I) was only 41.8% as
the molar ratio m (NONPC)/m(MPEGI) equals to 1:1.
When the same molar ratio was increased up to 2:1, the
DS of NOOMC (II) reached 88.4%, more than twice as
much as the DS of NOOMC (I).
Structure changes of chitosan and its derivatives were
confirmed by FTIR spectra (Figure 1). The N-octyl-
N-phthaloyl chitosan (NONPC) shows new or intensi-
fied absorptions at 2927, 2858, 1464 cm-1 which attrib-
uted to octyl chains and 1776, 1716, 721 cm-1 which
assigned to the phthalimide groups. On the contrary, the
peak at about 1600 cm-1, which belongs to -NH2 in chi-
tosan, disappeared in the IR spectra of NONPC. The
above information from IR spectra indicates that octyl
groups were successfully introduced to chitosan and the
retained amino groups were protected completely by
phthalimide groups. The IR spectra (c, d in Figure 1) of
N-octyl-O-MPEG chitosan (NOOMC) also reveal the
absorption bands characteristic of chitosan and two mo-
dified groups. Distinctive absorption bands at 2889 cm-1
(C-H stretching) and 1110 cm-1 around belong to MPEG
grafts while those at 1676, 1646 cm-1 attribute to the
primary amino which retained in chitosan derivative.
Table 2. Elemental analysis (%) and the degree of substitution
(DS) of chitosan derivatives.
Sample C N H
m (C)/
m (N)* DS (%)
Chitosan 39.68 7.59 7.85 6.10 —
NOC 53.50 5.98 9.19 10.44 54.3a
NONPC 55.74 4.13 6.75 15.75 66.3b
NOOMC (I) 52.01 1.17 8.89 51.86 41.8c
NOOMC (II) 52.62 0.71 9.47 86.46 88.4d
NOC, N-octyl chitosan; NONPC, N-octyl-N-phthaloyl chitosan;
NOOMC, N-octyl-O-MPEG chitosan. * m represents mole ratio. a DS
of N-octyl groups; b DS of N-phthalimide groups; c,d DS of O-MPEG
Figure 1. FTIR spectra of chitosan and its derivatives.
a-chitosan, b-NONPC, c-NOOMC (I), d-NOOMC (II).
NONPC, N-octyl-N-phthaloyl chitosan; NOOMC,
N-octyl -O-MPEG chitosan.
It should be noted that the absorptions of octyl groups
(2927, 2858, 1464 cm-1) were overlapped by the much
stronger absorptions of MPEG grafts (2889 cm-1, 1471
cm-1). The peak at 3458 cm-1 which attributed to -NH2
and -OH declined and that at 1776 cm-1 and 1716 cm-1
characteristic of phthalimide groups disappeared in the
FTIR spectra of the final product. It is thus concluded
that MPEG groups were introduced to hydroxyl and the
protecting groups were removed successfully.
More information about chitosan and its derivatives
can be obtained from 1H NMR analysis (Figures 2-3). In
comparison with the spectra of chitosan itself (Figure 2),
1H NMR spectra of NOOMC (II) (Figure 3) differ
greatly in that the signals at 3.50-3.60 and 3.25 which
assigned to the ethoxyl hydrogen (CH2-CH2-O) and
methoxyl hydrogen (-OCH3) of MPEG grafts, respec-
tively, and that the small peaks at 3.10, 2.80, 1.15 which
attributed to the methylene hydrogen around nitrogen
(N-CH2-(CH2)6-CH3), other six methylene hydrogens
(N-CH2-(CH2)6-CH3) and the methyl hydrogen (N-CH2-
(CH2)6-CH3), respectively. The signals of hydrogen
within chitosan backbone (3.60-3.80, H3 H4 H5 H6)
were covered partially by that of ethoxyl hydrogen. As
illustrated in Figure 3, the peaks of hydrogen in octyl
groups and chitosan backbone were not so obvious as
those of MPEG with a high DS of MPEG grafts and a
G. H. Liu et al. / Natural Science 2 (2010) 707-712
Copyright © 2010 SciRes. OPEN ACCESS
OH 1
H3 H4 H5 H6
7 6 5 4 3 2 1
Figure 2. 1H NMR spectra of chitosan.
Figure 3. 1H NMR spectra of N-octyl-O-MPEG chitosan
much large number of hydrogen it contained.
Molecular weight of the amphiphilic chitosan
(NOOMC (I)) was measured by gel permeation chro-
matography (GPC). The results showed that Mw of
NOOMC (I) is near 1 × 105 and Mz is about 1.6 × 105,
which is a little bit smaller than that calculated by
Element Analysis (2 × 105). It might be caused by the
existence of trace amount of unreacted MPEGI after
dialysis or cleavage of chitosan chains during modifi-
N-octyl-O-MPEG chitosan (NOOMC), was synthesized
for the first time with fair production yield and high de-
gree of substitution of amphiphilic groups. The degree of
substitution of octyl groups reached 54.3% while that of
MPEG grafts amounted to 41.8%-88.4% depending on
the variation of N-octyl-N-phthaloyl chitosan to meth-
oxy polyethylene glycol iodide molar ratio. With the
introduction of the amphiphilic groups, the chitosan de-
rivatives were endowed with good solubility in water
and potential self-assembly properties. Significantly, the
effective protection of amino groups was successfully
adopted to retain a certain amount of -NH2 in the deriva-
tives, which is indispensable for preparing hollow mi-
The authors express their gratitude to National Natural Science Foun-
dations of China and Natural Science Foundation of Guangdong Prov-
ince, China, for financial support.
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