Engineering, 2012, 5, 114-117
doi:10.4236/eng.2012.410B029 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
O-Alkylation of Chitosan for Gene Delivery by Using Ionic
Liquid in an in- situ Reactor
Huiying Chen1, Shao hui Cui1, Yinan Zhao1, Bing Wang1, Shubiao Zhang1, Huiying Chen2, Xiaojun Peng2
1Key Laboratory of Bio-chemistry Engineering - The St ate Ethnic Affairs Commission-Minist r y of Education,
Dalian Nationalities University, Dal i a n, C hina
2State Key Laboratory of Fine Chemicals, Dalian Uni vers ity of Technology, Dalian, China
Received 2012
An in-situ reactor was elaborately designed for O-alkylation of chitosan in an ionic liquid ([BMIM]Cl) solvent, using N,
N'-carbonyldiimidazole as bonding agent. The original chitosan and the modified chitosan were characterized by FT-IR and XRD
analysis. FT-IR spectra revealed that the alkylation of chitosan selectively occurred at hydroxyl groups, with unprotected amino
groups untouched. It was proposed that the particular properties of the ionic liquid solvent should be responsible for the selectively
alkylation. The result from X-ray diffraction showed that the crystallinity of O-alkylation of chitosan decreases, most likely due to
the decomposition of CS in the ionic liquid. The solubility test of O-alkylated chitosan in aqueous HAc solution (w/w: 0.1%) con-
firmed that the product could be easily dissolved in aqueous HAc solution because of its abundant free amino groups. It was sug-
gested that the O -alkylated chitosan was suitable for the coming cell transfection test in vitro.
Keywords: Gene Delivery; Chitosan; Alkylation; Ionic Liquid; Iu-Situ Reactor
1. Introduction
Gene deliver y int o mammalian cells has b ecome an in disp ensa-
ble research tool in molecular and cell biology [1]. Despite
widespread use and numerous delivery systems available,
transfection is still a matter of compromise between accep table
toxicity and efficacy [2, 3]. Compared with viral vectors, non-
viral carriers are considerably safer and easy to produce, and
they possess large gene-carrying capacity and flexibility of
design [4]. Because lack of toxicity is a major demand in the
design of new gene delivery systems, the development of non-
viral vectors has been increasingly focused on biocompatible
systems and natural polymers. These include polysaccharides
such as schizophyllan, glycopolymers, or degradable synthetic
polymers [5].
Among the biopolymers, chitosans, a family of cationic and
linear polysaccharides derived from chitin, have received in-
creasing attention in biomedical research over the past decade
and show an attractive safety profile as well as a range of pos-
sibilities for further modifications. Recently, chitosan has
emerged as a p romising candi date for gene d elivery because o f
biocompatibility, biodegradability, low immunogenicity, low
cytotocixity, favorable physicochemical properties and ease of
chemical modification [6-9]. The advantage of chitosan-based
vectors lies not only in getting away from the cytotocixity
probl ems that are in herent in most syntheti c polymeric vehicles,
but also in its unique capability of transcellular transport. The
presence of positive charges from amine groups enables it to
transport plasmid DNA (pDNA) into cells via endocytosis and
membrane destability. However, as shown with other polyca-
tions/DNA complexes, chitosan/DNA complexes are formed by
electrostatic interaction between primary amino groups and
phosphate groups, which is strong enough to resist DNA un-
packing within cell to a certain degree. Okano, Sato, and Ka-
banov all reported that the incorporation of hydrophobic moie-
ties could considerably increase the transfection efficiency
[10-12]. Wen Guang Liu used N-alkylated chitosan to transfer
plasmid-encoding CAT into C2C12 cell lines, and the transfec-
tion efficiency is higher than that of chitosan. It is proposed that
the higher transfection efficiency of ACS is attributed to the
increasing entry into cells facilitated by hydrophobic interac-
tions and easier unpacking of DNA from N-alkylated chitosan
carriers due to the weakening of electrostatic attractions be-
tween DNA and N-alkylated chitosan [13].
In addition, by theoretical calculations, Kuhn and Levin
found that for sufficient hydrophobic amphiphilies, the neutra-
lization or even the inversion of charge of the DNA-amp hi-
phile complexes could achieve with rather low concentration of
cationic amphiphile [14].
Ionic liquids, combining good and tunable solubility proper-
ties with a negligible vapor pressure and excellent thermal sta-
bility, have recently been used for dissolving biological ma-
cromolecules including cellulose, wool keratin and silk fibroin
that are linked together by intermolecular hydrogen bonds
[15-17]. Early data showed that chitosan had a good solubility
in 1-butyl -3 -methylimidazolium chloride ([BMIM]Cl), and up
to 10 wt% of chitosan can dissolve in this media to form a
viscous solut ion [18 ].
To the best of our knowledge, to date, research work on the
O-alkylation of chitosan on the properties of chitosan/DNA
complexes and on the transfection efficiency of chitosan-based
vectors is unavailable in the literature. In this work, we synthe-
sized a novel O-alkylated chitosan (OACS) derivatives from
dodecanol in ionic liquid [BMIM]Cl in an in-situ r eactor, using
Copyright © 2012 SciRes. E NG
N, N'-carbonyldiimidazole (CDI) as bonding agent, then inves-
tigated the solubility of OACS in aqueous acetic acid solution
(w/w: 0.1%) , providing valuable information for the next
in-vitro cell transfection. The chemical structure of chitosan
(CS) and products were analyzed by FT-IR spectrometer and
x-ray diffract ion s pectro meter.
2. Experimental
2.1. Materials
Chitosan polymer (50 cP) with a degree of deacetylation (DD)
value over 8 5% was ob tained from XiaMen Sanl and Chemicals
Co. Ltd. (Xiamen, China). Ionic liquid [BMIM]Cl was prepared
as literature [19]. Dodecanol was purchased from J&K Chemi-
cals Co. Ltd. (Beijing, China) and was freshly distilled before
use. N, N'-carbonyldiimidazole (CDI) was supplied by Beijing
Chemicals Co. Ltd. (Beijing, China). Chloroform was from
Kaixin Fine Chemicals Co. Ltd. (Tianjin, China) and used after
dehydration. All other chemicals used were commercially
available and u s ed as received .
2.2. Synthesis of O-Alkylated CS (OACS)
Herein, an in-situ reactor was elaborately designed for the chi-
tosan alkylation in ionic liquid [BMIM]Cl. The alkylation of
CS was performed in the in-situ reactor. Firstly, 0.46 g of CDI
was dissolved by 10 ml of chloroform then added into a
three-necked bottle of 50ml. 0.42 g of dodecanol was dissolved
in 10 ml of chloroform and dropped into the three-necked bottle
under magnetic stirring and nitrogen atmosphere at 40 oC for 2
h. Then the chloroform was removed in vacuum in-situ. 0.2 g of
CS was dispersed in 15 g of ionic liquid [BMIM]Cl. After
heated at 80 oC for 4 h, the liquid was added into the
three-necked above bottle and heated to 80 oC and stirred for 8
h. The react ion mixture w as poured i nto ice water, fil tered, and
washed with dimethyl ether.
2.3. Analysis of OACS by FT-IR Spectrometer and
X-Ray Diffraction Spectrometer
The Fourier transform IR (FT-IR) spectra of the samples were
recorded with a Fourier transform IR spectrometer (IR Pres-
tige-21, Shimadzu, Japan) in the range of 400 4000 cm1 in
KBr pellets at ambient temperature. All spectra were recorded
with an accumulation of 32 scans and a resolution of 4 cm-1 in
the range from 4000 to 400 cm1. X-ray patterns of powdered
samples were obtained using a XRD-6000 X-ray diffract meter
(Shimadzu, Japan) with Cu Ka radiation at 0.15406 nm. The
relative intensity was recorded in the scattering range (2θ) of
5–50 o with steps of 0.1o per second.
3. Results and Discussions
3.1. O-Alkylation of CS
O-alkylation of CS was firstly prepared using CDI as bonding
agent. Alkyl was selectively anchored on hydroxyl groups of
chitosan without amino group protecting in ionic liquid in an
in-situ reactor . Firstly, CDI r eacted with dodecanol to produce a
reactive intermediate in chloroform. Then the solvent was re-
moved under vacuum in-situ to avoid the reaction system ex-
posing into atmosphere, rendering the intermediate deteriorat-
ing. The chloroform was necessarily removed lest the solvent
lead to insolubilization of CS in [BMIM]Cl, affecting the fol-
lowing reaction of CS with the intermediate in the ionic liquid.
To test the solubility of the product, 0.2 mg of OACS was dis-
persed in 1 ml chloroform, ethanol, water and 0.1 % (v/v) HAc
aqueous solution, respectively and observed by naked eyes. It
was confirmed that the obtained OACS was hardly dissolved in
water, slightly soluble in chloroform and ethanol, easily dis-
solved in aqueous HAc solution. The protonation of amino
groups of CS is responsible for the solubility in HAc aqueous
solution. Alkylation of CS improves the solubility of OACS in
organic solvent such as chloroform and ethanol. The results
show that OACS may be suitable for the cell transfeciton test
because o f its abu ndan t free amin o gro ups, which are cri tical in
compacting pDNA and delivering genes into cells.
3.2. IR Characterization of OACS
Figure 1 shows the IR spectra of unmodified CS and modified
chitosan products OACS. The primary amino group of chitosan
is recognized by two bands at 1650 and 1544 cm1 [20]. After
alkylation with dodecanol using CDI as bonding reagent in
ionic liquid, the amino bands still exist. Two strong bands at
2925 cm1 and 2856 cm1, the C-H stretching vibration adsorp-
tion peaks, appear, and at 1750 cm1, the carbonyl group of
carbonate band appears. Furthermore, bands at 1461 and 1260
cm1 also show alkyl and carboxylic groups, respectively. The
IR spectrum of OACS (b in figure 1) shows that the dodecanol
is anchored selectively on hydroxyl groups of CS, thus the
amino groups of CS remain untouched.
Ionic liquid [BMIM]Cl belongs to a class of non-aqueous but
polar solvents. This water free solvent is suitable for the reac-
tion using CDI as bonding agent, which is sensitive to water,
decomposing into carbon dioxide and imidazole in moist at-
mosphere. It is composed of cation and anion, displaying acidi-
ty, basicity or neutrality, depending on the component. Herein,
we found that the ionic liquid solvent can restrain the reaction
of amino group, render the alkylation selectively occurring on
hydroxyl groups of CS. It is proposed that the nitrogen atoms
be in some electron deficiency environment in the solvent,
w ave number cm
a. u.
Figure 1. IR spectra of (a) unmodified chitosan, (b) modified chito-
san OACS.
Copyright © 2012 SciRes. ENG
which is harmful for the nucleophilic reaction of amino groups.
So the nucleophilic reaction falls into hydroxyl groups of CS,
emerging as selectively alkylation of hydroxyl groups without
amin o groups protecting.
3.3. XRD Characterization of OACS
Figure 2 presents XRD patterns of powder CS and power
OACS, respectively. In Figure 2a, the original chitosan shows
two strong reflections at 2θ = 10.6˚ and 2θ =20.4˚, which are
coincided with the pattern of the form I crystal and the form II
crystal [21]. As shown in Figure 2b, the modified chitosan
OACS appears two more weak peaks compared with the origi-
nal chitosan and the crystallinity is 17.2% while it is 25.4% for
the original chitosan. The results show the amorphous region of
OACS increases and the crystalline structure partially changes
but maintains the form II crystal region in contrast to the origi-
nal chitosan. It was reported that the [BMIM]Cl, as an imidazo-
lium based ionic liquids, could depolymerize chitosan effec-
tively under mild conditions [22]. Therefore, the decrease in
crystallinity of OACS is most likely to arise from the decompo-
sition of CS to some extent under the reaction conditions.
4. Conclusions
O-alkylated chitosan was p repared in ionic liquid [BMIM]Cl in
an elaborately designed in-situ reactor, using N, N'-carbon-
yldiimidazole as bonding agent. The IR spectra characteriza-
tion results show that the dodecanol is anchored selectively on
hydroxyl groups of CS with a new carbonyl functional group,
leaving amino groups untouched. It is proposed that the partic-
ular properties of the ionic liquid solvent should be responsible
for the selective alkylation of hydroxyl groups of CS without
protecting amino groups of CS. The XRD patterns of CS and
O-alkylated chitosan show that the crystallinity of the latter
decreases perhaps due to the docomposition of CS in the ionic
liquid. The solubility test of O-alkylated chitosan confirmed
that it can be easily dissolved in aqueous HAc solution (w/w:
0.1%) because of its abundant free amino groups. The results
show that O-alkylated chitosan may be suitable for the follow-
ing gene delivery test.
05 10 15 20 25 30 35 40 45 50 55
2 Theta
a. u.
Fig ure 2. XRD patterns of power samples of (a) unmodified chito-
san, (b) modifie d chi tosan OACS.
5. Acknowledgements
The authors gratefully acknowledge the financial support from
the National Natural Science Foundation of China (21176046,
20876027), and the Fundamental Research Funds for the Cen-
tral Universities (DC10020103).
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