O-Alkylation of Chitosan for Gene Delivery by Using Ionic Liquid in an in- situ Reactor

doi:10.4236/eng.2012.410B029

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Engineering, 2012, 5, 114-117

doi:10.4236/eng.2012.410B029 Published Online October 2012 (http://www.SciRP.org/journal/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

Email: [email protected]

Received 2012

ABSTRACT

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

H. Y. CHEN ET AL.

115

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 cm−1 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 cm−1. 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 cm−1 [20]. After

alkylation with dodecanol using CDI as bonding reagent in

ionic liquid, the amino bands still exist. Two strong bands at

2925 cm−1 and 2856 cm−1, the C-H stretching vibration adsorp-

tion peaks, appear, and at 1750 cm−1, the carbonyl group of

carbonate band appears. Furthermore, bands at 1461 and 1260

cm−1 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

-1

a. u.

Figure 1. IR spectra of (a) unmodified chitosan, (b) modified chito-

san OACS.

H. Y. CHEN ET AL.

116

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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