Applied Chemisσy ，

Vol. 12, No. I, May ;rna, 29-32

Methoxy poly(ethylene glycol)OI 수식흰 저톨짜량 수용성 키톨싼의

체죠및특성

깊호쿄 J 권중근 l 정영일 J 장미경 J 나재운 J.

l순천대학교 공과대학 고분자공학과

2조선이공대학 생명환경화꽁과

Preparation and spectroscopic characterization of methoxypoly(ethylene glycon-grafted low molecular weight

water-soluble chitosan

Don-Gon KimI. Joong-Kuen Kwon l. Young-II Jeong l

. Mi-Kyeong Jang l.

Jaw-Woon Nah l'

JDepartment of Polymer Science and Engineering. Sunchon National University. Suncheon. Jeonnam540-742. Korea

2Department of Bioenvironmental and Chemical Engineering. Chosun Collegeof Science & Technology. Gwang-ju 501-744. Korea

Abstract

The object of this study is to test solubility of methoxy poly (ethylene glycol)

(MPEG) -grafted chitosan copolymer in the organic solvents and aqueous solution.

Water-soluble chitosan with low molecular weight (LMWSC) was used to synthesis of

PEG-graft copolymerization. MPEG was conjugated to the water soluble chitosan using

4-dicyclohexylcarbodimide (DCC). and N-hydroxysuccimide (NHS), Introduction of

PEG was confirmed by JH NMR spectroscopy. Degree of substitution (DS) of MPEG into

chitosan was calculated from JH NMR results. From these results. Furthermore.

introduction of PEG into chitosan increases the solubility in aqueous solution with variousorganic solvent such as DMF. DMSO. ethanol. and acetone.

1. Introduction

To overcome drawbacks of chitosan , various kind of derivatives of chitosan have been

developed [1, 2]. Among them , PEGylation of chitosan was reported by several authors to

increase aqueous solubility of chitosan [1 -5]. Ouchi et a l. reported that PEGylation of

chitosan bring out increased solubility to aqueous solution or organic solvent, and

self-aggregation properties in aqueous solution. PEGylaion of chitosan derivatives is

favorable to biocompatibility improvementand increase cell viability compared to trimethyl

chitosan [4 J. Other candidates for modification of chitosan is to graft poly (L-lactide) or

polycaprolactone to give a amphiphilic properties, i.e. chitosan consisted of hydrophilic

domain and polyester composed of hydrophobic domain [6 ,71. Resultantly , nanoparticles of

chitosan-g-polyester has reduced particle size with sub-lOO nm and they showed

self-assembling properties of copolymed6,7J. In previous report , we showed that

water-soluble chitosan with low molecular weight (M.W.) (abbreviated as LMWSC) is a

distinguished candidates as a drug or DNA carriers due to their good aqueous-solubility and

elevated free-amine group in chitosan. Since LMWSC has high content of free amine group

in chitosan chain , it is distinguished from other kind of chitosan in the increased affinity to

DNA or anionic drug. Furthermore , PEG-graft LMWSC can be used as a excellent drug or

29

30 김동곤·권중근·정영일·장미경·나재운

gene carrier. Hu et a l. reported that PEG-grafted chitosan has increased solubility in

aqueous solution and organic solvent such as dimethylformamide (DMF) and

dimethylsulfoxide (DMSO). However , they did not showed aqueous solubility at various pH

and various kind of organic solven t. Increased solubility in aqueous solution with various pH

and organic solvent will give advantages to application of chitosan derivatives in biomedical

field.

In this study , we synthesized methoxy PEG-grafted LMWSC (abbreviated as ChitoPEG)

and characterized with I H- NMR. Furthermore , solubility potential of ChitoPEG was

investigated at organic solvent such as DMF , DMSO , acetone , acetonitrile , and ethano l.

Introduction of PEG to chitosan will increase processibility and application area by increase

solubility parameters.

2. Experimental

2. 1. MaterialsWater-soluble chitosan with low molecular weight (M.W. = 10,000 Da , deacetylation

degree = 97.0 %, abbreviated as LMWSC) was gift from Chittolife Co. Ltd. Seoul ,

Korea.Water-insoluble chitosan was purchased from Wako Pure Chemical , Co. Ltd. Japan

Cchitosan 10 , abbreviated as IS-chitosan). Methoxy poly(ethylene glyco l) (MPEG) (M.W. =2 ,000 g/mo l) was purchased from SunBio Co. Korea. , N,N’-dicyclohexy1carbodiimide

(DCC) , N- hydroxysuccinimide (NHS) , succinic anhydride , Dimethylformamide (DMF) ,

dimethylsulfoxide (DMSO) , diethylether , methylene chloride (DCM) , ethanol , acetonitrile

(AN) , and acetone as HPLC grade were purchased from Sigma Co. Ltd. USA. DMSO ,

chloroform , (D-form for NMR study) , deuterium oxide (020) , DCl and NaOD were

purchased from Aldrich Co. USA.

2.2. Synthesis of ChitoPEG copolymer

Synthesis procedure of ChitoPEG graft copolymer (as shown in Figure 1) was as follows:

100 mg of LMWSC was dissolved in 0.2 ml of deionized water and diluted with 9.8 ml of

DMSO. To this solution , MPEG-NHS dissolved in 2 ml of DMSO was added and reacted for

overnight at nitrogen atmosphere. After that , the resulting solution was dialyzed against a

plenty of deionized water for 2 days followed by its lyophilization. Lyophilized solid was

resuspended into a plenty of DCM to remove unreacted MPEG-NHS thee times and

fractionated into deionized water followed by its lyophilization.

2.3. lH NMR measurement

The I H NMR spectra of the copolymer and polymeric micelle were measured in 020 or

DMSO (d-form) using a 400 MHz NMR spectrometer (AVANCE 400FT-NMR 400MHz ,

Bruker) .

2 .4. Solubility test

For solubility testof ChitoPEG copolymer , each 5 mg of LMWSC or ChitoPEG copolymer

was dissolved in 0.1 ml of deionized water and added 4.9 ml of organic solvent 0.0 mg/m l).

Orgarnic solvent is DMF , DMSO , ethanol , acetonitrile , acetone. To express solubility of

ChitoPEG graft copolymer , turbidity was measured using UV-spectrophotometer at 600 nm

(UV -160 I , Shimadzu Co. Ltd. Osaka , Japan).

용용화학， 제 12 권 제 l 호， 2008

Pre뼈ration and spectroscopic charac따ization of m에lOxy !X>ly(ethylene glyc이)-믿뼈ed low nlOlecular weight water-잉luble c에ωsan 31

3. Result and disccusion

3. 1. Characterization of ChitoPEG graft copolymer

ChitoPEG copolymer is composed of a non-ionic hydrophilic MPEG side chain and a

cationic chitosan main chain as shown in Figure 1.To characterize ChitoPEG copolymer.

MPEG, MPEG-NHS. LMWSC 10K, and ChitoPEG was dissolved in D20. As shown in Figure

2 , specific peaks of MPEG was shown at 3.5-3.7 ppm. At activated form of MPEG-NHS.

the peaks of carboxylic group and NHS group were shown between 2.5 - 3.0 (peak number

8 -11). In other words. specific peaks of chitosan (LMWSC 10K) was appeared between

1.8 - 5.0 ppm. At ChitoPEG copolymer. peak assign was as follows: Cl position. 4.6 ppm;

C2 position , 2.9-3.0 ppm; C3-C6 position. 3.5-4.0 ppm. The methyl group of MPEG was

also appeared about at 3.7 ppm.

빽vly 鍵찮~t'안/、경 、x떻3

灣露4鍵쐐\~t")“놨

iOm摩o$露A편/0/， ~'"’ X싱꺼。캔、。%

@뺨F꽉싫kt」뀐훨편랜많이 샘 _L. 싫[ 패μf藝繼

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Figure 1. Synthesis scheme of ChitoPEG copolymer

Figure 2. lH spectra of MPEG, MPEG-NHS , LMWSC 10K and ChitoPEG copolymer in D20.

of

solven t.

Increase

organic

theto

3.2. Solubility of ChitoPEG copolymer into water and organic solvent

Figure 8 showed solubility of chitosan and its derivatives at various organic solven t.

of IS-chitosan was lower than 60 % at all of theTransparency

Transparency of IS-chitosan was gradually decreased according

concentration of chitosan. Practically. IS-chitosan was insoluble at any other solvent and

thus aqueous solution with acid solution must required to dissolve it. Otherwise , LMWSC

10K was good transparency with DMSO at all of the concentration. Practically , no insoluble

fragments were observed at direct observation. Furthermore , DMF was expected to the nice

solvent at lower than 4 mg/ml concentration. However, transparency of LMWSC 10K was

significantly decreased according to the increased concentration at EtOH and acetone.

LMWSC 10 K did not practically insoluble in EtOH and acetone. Futhermore. LMWSC 10K

was insoluble to acetonitrile at any concentration.

Applied Chemistry, Vol. 12, No.1, 2ro3

32 김동곤·권중근·정영일·장미경·나재운

t。←、-、-←。-→'------..---.

혀꺼껴셔~→-→--→

‘ 、、

\ @

、\

、

’、..... ....--- 。

‘ “'~、 ~‘'"、‘

펙:뇨! ‘

。

、 \

\ 、 \、 ‘、 、‘‘、 、-----，-、

\..., , “‘-、←‘~、‘.‘:.:.::::R

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Cone. (mglml) Cone. (mglml)2 3 •

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Figure 8. Solubility of ChitoPEG copolymer in the organic solvent. IS-chitosan (a). LMWSC

10K (b) , ChitoPEG-5 (c) , ChitoPEG-10 (d) , ChitoPEG-15 (e) , and ChitoPEG-20 (f).

4. Conclusions

ChitoPEG copolymer was successfully synthesized and characterized. MPEG was

conjugated to LMWSC using DCC/NHS system. The NHS-activated MPEG and ChitoPEG

copolymer was confirmed by IH NMR spectroscopy. Four different kind of ChitoPEG

copolymer was synthesized with different feeding amount of MPEG- NHS. Degree of

substitution ratio of MPEG calculated from IH NMR. Furthermore , introduction of PEG into

LMWSC resulted in increased solubility organic solvent such as DMF , DMSO , ethaol , and

acetone.

References

1. Ouchi , T.; Nishizawa , H.; Ohya , Y. Polymer , 1998 , 39 , 5171-5175.

2. Jang , M. K.; Jeong , Y. I.; Cho , C. S.; Yang , S. H.; Kang , Y. E.; Nah , J. W. Bull.

Korean Chern. Soc. 2002 , 23 , 914-916.

3. Saito , H.; Wu , X.; Milton Harris , J.; Hoffman , A. S. Macromo l. Rapid Comm. 1997 , 18 ,

547-550.

4. Mao , S.; Shuai , X.; Unger , F.; Wittmar , M.; Xie , X.; Kissel , T. Biomaterials 2005 , 26 ,

6343-6356.

5. Kothe , P.; Kannan , R. M. Biomacromolecules , 2003. 4 , 173-180.

6. Feng , H.; Dong , C. M. Biomacromolecules , 2006 , 7 , 3069-3075.

7. Yu , H.; Wang , W.; Chen , X.; Deng , C.; Jing , X. Biopolymers , 2006 , 83 , 233-242.

옹용화학， 제 12 권 제 1 호，2(뼈