preparation of a visible light-reactive low molecular-o-carboxymethyl chitosan (lm-o-cmcs)...

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Macromolecular Research, Vol. 19, No. 9, pp 921-927 (2011) www.springer.com/13233

The Polymer Society of Korea

Preparation of a Visible Light-Reactive Low Molecular-O-Carboxymethyl Chitosan (LM-O-CMCS) Derivative and Applicability as an Anti-Adhesion Agent

Shin-hye Park1, Si-yoong Seo1, Ha-na Na1, Kwang-il Kim1, Jea-woo Lee1, Hee-dong Woo1, Jue-hee Lee2, Hyun-kwang Seok3, Jae-gwan Lee1, Sang-in Chung4, KyuHwan Chung5, DongKeun Han3, Yoshihiro Ito6,

Eui-chang Jang7, and Tae-il Son*,1

1Department of Biotechnology and Bio-Environmental Technology (BET) Research Institute, Chung-Ang University, Gyeonggi 456-756, Korea

2School of Korean Medicine, Pusan National University, Gyeongnam 626-870, Korea3Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, Korea

4Department of Microbiology, Collage of medicine, Chung-Ang University, Seoul 156-756, Korea5School of Bioresource and Bioscience, Chung-Ang University, Gyeonggi 456-756, Korea

6Nano Medical Engineering Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

7Department of Orthopaedic Surgery, School of Medicine, Chung-Ang University, Seoul 156-756, Korea

Received January 10, 2011; Revised May 2, 2011; Accepted May 10, 2011

Abstract: Photo-reactive low molecular-O-carboxymethyl chitosan (LM-O-CMCS) was synthesized by introduc-ing a furfuryl group to the amine groups of LM-O-CMCS. The prepared photo-reactive LM-O-CMCS was charac-terized by FTIR, 1H NMR, and a cross-linking test, whereas scanning Auger electron microscopy (SAM) and a MTTassay were used to confirm the cell viability. The water contact angle measurement and cell attachment test werecarried out to determine the applicability of LM-O-CMCS as an anti-adhesion agent.

Keywords: natural polymer, photo-reactive, anti-adhesion agent, carboxymethy chitsoan, water-soluble chitosan.

Introduction

Recently, studies of natural polymers have been con-ducted to assess their use and facilitate recycling of naturalresources. Some natural polymers like chitin and chitosanhave biological activities such as anti-tumor, immune enhance-ment, anti-inflammatory, anti-viral, hypoglycemic, and anti-coagulant effects. In particular, natural polymers containingchitin and algin are known having wound healing properties.1Among these natural polymers, chitin is a linear polysac-charide consisting of β-(1,4)-linked-2-acetamido-2-D-glu-cose. Chitosan, which is produced by N-deacetylation, is anatural hydrophilic biomolecule abundantly found in shrimp,crab, shells, and other crustaceans.2-6 Additionally, chitosanis very similar to human tissue because it is combined withglucosamine. Therefore, chitosan having biocompatibilityand biodegradability has been widely used for natural drug/gene delivery and tissue engineering. 7-9 Furthermore, non-toxicity and antibacterial activity of chitosan make thiscompound an effective wound healing agent.10-13 Chitosanhas also been examined for its pharmacological properties

including use as an anticoagulant, artificial skin, and medi-cal material because of its cholesterol-lowering effect andanti-tumor properties.

Naturally-occurring chitosan is insoluble in water. A varietyof chemical modification techniques such as PEG-grafting,N- and O-hydroxylation, sulfonation, and carboxymethyla-tion have been developed to produce water-soluble chito-san.14-18 Among the water-soluble chitosan derivatives thatare made by various methods, carboxymethylated chitosan(O-CMCS) in particular is an excellent bio-material becauseof its low toxicity, biocompatibility, and stability in bloodand cell.19-23 However, O-CMCS synthesized from naturally-occurring chitosan has high viscosity and loses its water-soluble properties when various hydrophobic functionalgroups are introduced. Therefore, it has difficulties to findpractical applications for this compound. Low molecular-O-CMCS (LM-O-CMCS), however, is more water-soluble andcan resolve the problem with viscosity, and thus may bewidely used.

In this study, a visible light reactive derivative and cross-linking methods were used for synthesizing water-solubleLM-O-CMCS. The chemical cross-linking method to mod-ify a surface by covalent bonding is limited to immobiliza-

DOI 10.1007/s13233-011-0914-9

*Corresponding Author. E-mail: [email protected]

S.-H. Park et al.

922 Macromol. Res., Vol. 19, No. 9, 2011

tion using specific materials by specific methods. For example,β-glucosidase is immobilized on the cross-linked chitosanbeads.24 However, a cross-linking method using visible lightis not limited to immobilization and specific material, sothis technique can be used for various surface types. Addi-tionally, photochemistry has several advantages includingfast reaction times, use for manufacturing processes of vari-ous materials.25

Cross-linking of most recently-used photo-reactive poly-mers is accomplished by the generation of radical usingultraviolet (UV) radiation.26 However, UV radiation can leadto serious problems such as genetic variation, skin cancer,multiple sclerosis, insulin-dependent diabetes mellitus, andimmune system weakness; therefore, it is not suitable fordirect use on the human body.27-30 Due to these problems, aphotochemical method, which is harmless to human body,was used for synthesizing water-soluble LM-O-CMCS byintroducing a furfuryl group as a visible-light reactive deriv-ative in this study. This material was immobilized by oxy-gen obtained from rose bengal, a natural pigment. As a resultof immobilization, the compound became water-insolubleand was made into chitosan film. The object of this studywas to examine the curative and physical properties andtoxicity of the immobilized water-soluble and visible-lightreactive LM-O-CMCS, and to determine its possible use asan anti-adhesion barrier.

Experimental

Materials. Chitosan powder (degree of deacetylation: 88%)was supplied by Ja Kwang Co., Ltd. (Seoul, South Korea).Sodium nitrite, sodium hydroxide, hydrochloric acid, meth-anol, acetone, ether, ethanol, acetic acid, and isopropanolwere purchased from Duksan Pure Chemical Co., Ltd. (Seoul,South Korea). Ammonium hydroxide was purchased fromJunsei chemical Co., Ltd. (Tokyo, Japan). Sodium borohy-dride was purchased from Kanto Chemical Co., Ltd. (Tokyo,Japan) and used without further purification. Monochloro-acetic acid was purchased from Yakuri Pure Chemical Co.,Ltd. (Osaka, Japan) and used without further purification.Dodecyl sodium sulfate (99%) was purchased from AcrosOrganics (Pittsburge, PA, USA). Furfuryl glycidyl ether (96%),rose bengal, trypsin-EDTA, and MTT formazan were pur-chased from Sigma-Aldrich (Flanders, New Jersey, USA).Dimethylsulfoxide (DMSO) was purchased from DuchefaBiochemie (Haarlem, The Netherlands). For cell culturesand the MTT assay, 3T3-L1 (originating from Swiss mousemusculus fibroblasts) cells were purchased from KoreanCell Line Bank (Seoul, South Korea), DMEM (Dulbecco’sModified Eagle’s Medium) was purchased from WelGeneInc. (Deagu, South Korea), and FBS (fetal bovine serum)and penicillin-streptomycin were purchased from GIBCO(Eggenstein, Germarny). For every visible light irradiationexperiment in this study, the distance between the visible-

light lamp and sample was 5 cm.Preparation of Low Molecular Chitosan (LMCS) by

Using a Nitrous Acid Method. Chitosan (25 g) was dis-solved in an acetic acid solution (4% (v/v), 625 mL).NaNO2 (4.33 g) was dissolved in distilled water (43.3 mL)and added to the chitosan solution at 4 oC for 2 h with con-stant stirring. Afterwards, the solution was neutralized topH 7 by adding NH4OH. NaBH4 (4.27 g) was added over 90min and 1 N HCl was added to adjust the pH to 7. After thereaction, the depolymerized chitosan was filtered and con-centrated. The concentrated solution was precipitated byadding a five-fold volume of ice-cold methanol (concen-trated LMCS solution : methanol = 1 : 5). The precipitateswere collected by centrifugation and washed twice withmethanol, twice with acetone, and then once with etherbefore being vacuum dried at room temperature.

Preparation of Depolymerized Low Molecular O-Car-boxymethyl Chitosan (LM-O-CMCS). LMCS (5 g) wasdissolved in a 60% (v/v) NaOH solution containing 0.2%dodecyl sodium sulfate, and kept ice-cold for 1 h then fro-zen. The frozen sample was pulverized by adding isopro-panol (700 mL) using a spatula and neutralized to pH 7using monochloroacetic acid. Ethanol was used to wash theprecipitate three times and the sample was vacuum dried.The dried powder was dissolved in distilled water (500 mL).After that, the solution was filtered to remove insolublecomponents and concentrated. The solution was subse-quently dialyzed to purify the LM-O-CMCS using a dialy-sis membrane (cut-off; 1,000 Da, Spectrum Laboratories,Inc., Rancho Dominguez, California) in distilled water for48 h. After that, the sample was evaporated and freeze dried.

Conjugation of Photo-Reactive Group and LM-O-CMCS.LM-O-CMCS (1 g) was dissolved in distilled water (100mL) and a NaOH solution was added to adjust the pH to 11.Furfuryl glycidyl ether (FG; 125 µL) was dissolved in DMSO(10 mL) and this solution was added to the LM-O-CMCSsolution at 4 oC. Subsequently, the resulting solution wasstirred at room temperature for 5 h at 60 oC for 24 h. Afterthat, the solution neutralized to pH 7 using 1N HCl. Theproduct was washed three times with acetone and once withether before being freeze dried.

Schematic of cross-linking between photo-reactive groupand LM-O-CMCS is shown in Scheme I.

FTIR Measurement. The Fourier Transform Infra-Red(FTIR) spectrum was confirmed using KBr pallets ofLMCS and LM-O-CMCS by an FTIR spectrometer (FTIR8400S, Shimadzu, Kyoto, Japan). Values of absorbance weredetermined between 4000 and 400 cm-1. These values wereused because they are proportional to the concentrationaccording to the Lambert-Beer law.31

1H NMR Measurement. 1H NMR spectra of LMC, LM-O-CMCS, and photo-reactive LM-O-CMCS were recordedon an NMR spectrometer (Gemini 2000, 300 MHz, Varian,Inc. California, USA) using D2O.

Preparation of a Visible Light-Reactive LM-O-CMCS Derivative and Applicability as an Anti-Adhesion Agent

Macromol. Res., Vol. 19, No. 9, 2011 923

Measurement of Cross-Linking. Various concentrationsof photo-reactive LM-O-CMCS solutions (5%, 10%, 15%,20%, and 25%) were prepared and spread on glass (Micro-scope slides, limpiados 75×25 mm, Marienfeld, Germany).The samples were exposed to visible light for various of time(1, 3, 5, and 7 min). The samples were then washed usingdistilled water and weighed. The degree of cross-linking wassubsequently calculated using the formula:

Degree of cross-linking (%) = ×100

Initial weight: (dried sample weight of cross-linked on glass)- (weight of the glass)

Washed weight: (washed sample weight of after visiblelight irradiation) - (weight of glass)

Measurement of Long-Term Cross-Linking Stability.Twelve samples of 25% photo-reactive LM-O-CMCS solu-tion were prepared and spread on cover glass (Microscopeslides, limpiados 18×18 mm, Marienfeld, Germany). Theglass specimen of each sample was exposed to visible lightfor 3 min. Thereafter, 3 mL of 1X phosphate buffer saline(PBS) and immobilized sample on the glass were added in6-well plate (Multiwell, Becton Dickinson Labware, USA)and the samples were kept in 37 oC in a 5% CO2 incubatorduring 2 weeks. After the incubation, the PBS was removedevery 24 h for 12 days and the cross-linking ratio of eachsample was calculated according to the degree of cross-link-ing (%).

Scanning Auger Electron Microscope (SAM). Photo-reactive polymer (25%) was cured by visible light for 3 min.The cross-linked surface (Thickness was 30.63 µm, FieldEmission Scanning Electron Microscope, JEOUL JSM-6700F,Japan) was then observed by a Scanning Auger ElectronMicroscope (S-4200, Hitachi, Kyoto, Japan).

Cytotoxicity Test. Cytotoxicity testing was performedusing an MTT assay. 3T3-L1 cells were cultured in DMEMcontaining 5% fetal bovine serum (FBS) and 2% penicillin-streptomycin. Cells were seeded (4×104 cells/mL) in a 96-well plate (DK-4000 Roskilde, Kamstrup Vej 90, Nunc A/S, Denmark), and incubated for 18 h. Photo-reactive LM-O-CMCS (5%, 10%, 15%, 20%, and 25%) was added to thecell cultured 96-well plate and the cells were incubated at

37 oC in a 5% CO2 incubator for 6, 12, 18, and 24 h. Eachmedia was removed and 1 mg/mL MTT solution in 1X PBSwas added. The plates were then incubated for 4 h. TheMTT solution was removed and DMSO was added to dis-solve formazan crystals that had formed in the live cells.The absorbance was measured at 595 nm by using micro-plate reader (Sepectramax190, Molecular Device, Sunny-vale, USA). Cell viability (%) was calculated by using theformula:

Cell viability (%) = (OD595(samples)/OD595(control))×100

OD595(samples) was the measurement taken from wells treatedwith photo-reactive LM-O-CMCS and OD595(control) was themeasurement taken from the well nontreated.

Water Contact Angle Measurement. LM-O-CMCS (25%),FG, and photo-reactive LM-O-CMCS (25%) solutions werespread on glass. Photo-reactive LM-O-CMCS film was irra-diated with visible light for 3 min. The properties of eachsample were then measured to determine whether they werehydrophilic or hydrophobic using a CA-W Automatic Con-tact Angle Meter (Kyowa Interfaces, Co., Saitama, Saitama,Japan).

Cell Attachment on the Coated Photo-Reactive LM-O-CMCS. 3T3-L1 cells were cultured on normal plates andones coated with photo-reactive LM-O-CMCS. First, half ofthe 12-well plate (tissue culture treated) was coated with25% photo-reactive LM-O-CMCS by visible-light irradia-tion. The plate was then washed with 1X PBS and 3T3-L1cells (4×104 cells/mL) were seeded in 2 mL DMEM mediacontaining 5% FBS and 2% penicillin-streptomycin. Finally,the 12-well plate was incubated in 37 oC in a 5% CO2 incu-bator for 18 h. After the incubation, the-well plate wasexamined with a microscope (CSB-IH5, Samwon Scien-tific, Ind., Seoul, South Korea).

Results and Discussion

FTIR and 1H NMR Analysis. The chemical structures ofchitosan, LMCS, and LM-O-CMCS were analyzed by usingFTIR spectrum (Figure 1). Common chitosan and LMCSshowed similar peaks at the same locations: 3454 cm-1 (O-Hstretch), 2866 cm-1 (C-H stretch), 1655 cm-1 (amide group),

Washed weight (g)Initial weight (g)

Scheme I. Schematic diagram of the photo-oxidation cross-linking (POC) mechanism. S (Dyes) got high energy by irradiation of visi-ble-light and then changed oxygen to singlet-oxygen. Singlet-oxygen led to cross-linking among LM-O-CMCS derivatives.

S.-H. Park et al.


1595 cm-1 (N-H bend), 1155 cm-1 (bridge-O stretch), and1080 cm-1 (C-O stretch). This confirmed that the two mate-rials had similar structures even though they had differentmolecular weight. However, LM-O-CMCS contained a car-boxymethyl group, so it produced peaks at 1603 cm-1 (C-Ostretch), 1711 cm-1 (COOH group), and 1595 cm-1 (amidegroup). LM-O-CMCS and LMCS have different peaks (Fig-ure 1). So, they proven the presence of carboxymethy groupconjugated to LMCS.

However the synthesis of photo-reactive LM-O-CMCSwas difficult to prove by FTIR spectrum analysis becausechitosan and furfuryl group have similar peaks on the FTIRspectrum. Therefore, the 1H NMR spectrum was used toconfirm whether the furfuryl group was properly introducedor not (Figure 2). The presence of furfuryl groups having afuran ring was confirmed between 6-8 ppm.

Measurement of Cross-Linking and Ratios. Cross-link-ing ratio of photo-reactive LM-O-CMCS was determinedby concentration and visible-light irradiation time. Increaseof LM-O-CMCS concentration and visible-light irradiationtime caused an increase in the cross-linking ratio (Table I).A 20% photo-reactive LM-O-CMCS solution showed 62%degree of cross-linking irradiated with visible light for 3min, and a 25% solution showed a 50% degree of cross-linking when irradiated with visible light for 1 min. Cross-linking in a 15% solution was primarily affected by irradia-tion time; however, increasing the irradiation time did notgreatly increase cross-linking in a 5% solution. A LM-O-CMCS concentration over 25% formed a harder film butthis high concentration is not suitable for in vivo use. Thus,the lower concentration (25%) and the shorter irradiationtime (3 min) to make the harder film were revealed by usingcross-linking testing.

Cross-linked photo-reactive LM-O-CMCS was water-insol-uble. It is also important to determine how long the cross-linked film can be maintained in vivo. Therefore, a 25%solution of photo-reactive LM-O-CMCS was irradiated withvisible light for 3 min, and then kept in 1X PBS at 37 oC in a5% CO2 incubator. Next, the cross-linking ratio was mea-sured every 24 h. After 5 days, the amount of remaining

Figure 1. FTIR spectra of (a) chitorsan, (b) low-molecular chito-san (LMCS), and (c) carboxymethyl low-molecular chitosan (LM-O-CMCS). LMCS’s chemical structure was similar to chitosan,but LM-O-CMCS was added carboxymethy group to LMCS. So,FTIR spectra was different to LMCS at 1500~1650 cm-1.

Figure 2. Photo reactive LM-O-CMCS was introduced furfuryl ring to NH2 group at LM-O-CMCS. So, every peaks at 1 to 5 ppm wereshown similar but photo reactive LM-O-CMCS had special peaks at 6 to 8 ppm. That peaks meant furfuryl ring. (a) was 1H NMR spec-trum of LM-O-CMCS dissolved in D2O. (b) was 1H NMR spectrum of photo-reactive LM-O-CMCS in D2O. Photo-reactive groups werefound at 6.4, 6.5, and 7.6 ppm.

Table I. Degree of Cross-Linking According to Visible-LightIrradiation Time and Concentration of Photo-Reactive LM-O-CMCS

Visible-Light Irradiation Time

(min)

Concentration (%)

5 10 15 20 25

1 7.3 10.5 9.8 40.1 46.2

3 13.2 26.8 37.5 62.2 76.2

5 16 25 61.1 58 83.1

7 40 37.8 68.7 85 92.9



film decreased to 60%; it continuously decreased in volumeover 1 week (Figure 3) and completely dissolved after 2weeks. Normally, a wound heals within 4 to 5 days, so photo-reactive LM-O-CMCS is suitable for use as an anti-adhe-sion agent for wound treatment.

Cross-linking in 25% photo-reactive LM-O-CMCS withand without exposure to visible light for 3 min was com-pared by using a scanning auger electron microscope (SAM).Figure 4(b) shows that surface of photo-reactive LM-O-CMCS treated with visible-light formed a film because ofcross-linking among its derivatives. Therefore, photo-reac-tive LM-O-CMCS exposed to visible-light was convertedinto a flat film. To use photo-reactive LM-O-CMCS as ananti-adhesion agent, it has to become a barrier that forms aflat film.

Cytotoxicity Testing. For use as an anti-adhesion agent,photo-reactive LM-O-CMCS should be non-cytotoxic asdetermined by performing an MTT assay (Figure 5). Theassay was performed with fibroblasts and photo-reactiveLM-O-CMCS (5%, 10%, 15%, 20%, and 25%). The sur-vival ratios of cells incubated with each photo-reactive LM-O-CMCS solution relative to the control were 100. Theseresults proved that photo-reactive LM-O-CMCS was non-

cytotoxic.Water Contact Angle and Cell Attachment Test. For

application as an anti-adhesion agent, it is very important dodetermine whether the photo-reactive LM-O-CMCS film ishydrophobic or not and whether cells can attach. The mostimportant reason of using an anti-adhesion barrier is to forma barrier between tissues. Hydrophilic LM-O-CMCS andhydrophobic FG showed 50.2o and 78.5o angles on the watercontact angle test, respectively. Photo-reactive LM-O-CMCSshowed a 59.7o angle (Figure 6). Thus, we concluded that

Figure 3. It was remaining cured film ratio of cross-linked photo-reactive LM-O-CMCS. For use anti-adhesion agent, cured photo-reactive LM-O-CMCS used have to be able to withstand in 1XPBS solution and 5% at 37 oC. CO2 incubator. During 6 days,cured photo-reactive LM-O-CMCS remained more than 50%,but after 8 days, cured film was few remained.

Figure 4. Photo-reactive LM-O-CMCS was cured by irradiatingvisible-light and cross-linked. Photo-reactive LM-O-CMCS beforevisible-light irradiation (a) was not made cross-linking and its formwas not film. But photo-reactive LM-O-CMCS after visible-lightirradiation (b) was made cross-linking and formed flat film.

Figure 5. For use anti-adhesion agent, photo-reactive LM-O-CMCS was non-cytotocixity to 3T3-L1 fibroblast cell by MTTassay. The assay was performed using variety photo-reactiveLM-O-CMCS solution (5%, 10%, 15%, 20%, and 25%). Controlwas not added samples (0%). The cell viability was similarbetween cell viability of non-treated and treated.

Figure 6. For application as an anti-adhesion agent, cells werenot attached on the photo-reactive LM-O-CMCS film. Normally,3T3-L1 fibroblast cells were cultured on the hydrophilic plate.But anti-adhesion agent was not hydrophilic because it was per-formed barrier between tissues. Control was angle of non-treatedcell culture plate surface. LM-O-CMCS showed a 50.2o angleand FG showed a 78.5o angles. Photo-reactive LM-O-CMCs showeda 59.7o angle. It was middle of angle of LM-O-CMCS and FG.

S.-H. Park et al.


photo-reactive LM-O-CMCS was hydrophobic.Fibroblast can attach to and proliferate on hydrophilic

matrices or surfaces. However, an anti-adhesion barriershould be hydrophobic and not permit cell attachment. Ourstudy demonstrated that fibroblasts were able to attach tountreated surfaces of cell culture plates, but were unable toattach to surfaces coated with photo-reactive LM-O-CMCSin Figure 7, where (a) is nontreated control plate and (b) isphoto-reactive LM-O-CMCS coated plate. These observa-tions imply that photo-reactive LM-O-CMCS film can beused as an anti-adhesion agent.

Conclusions

These days anti-adhesion agent is sol or gel type. It isinconvenience to use because it need cutting before use andnot fit on the wound site. But photo-reactive LM-O-CMCSwas cured with visible light and became a flat water-insolu-ble film. This anti-adhesion agent is comfortable and easy touse on the wound site. For application of visible-light curedanti-adhesion agent, photo-reactive LM-O-CMCS was ana-lyzed that cross-liking ratio, cytotoxicity, cell attachment test,and so on.

1H NMR spectroscope proven introducing furan ring toLM-O-CMCS, successfully. The cross-liking ratio (used 25%solution, 3 min irradiation) of photo-reactive LM-O-CMCSwas closed to 90% and long-term cross-linking stability was60% in 1X PBS and CO2 incubator (37 oC, 5%) during 5days. So, cured photo-reactive LM-O-CMCS film was thoughtthat it had duration time of 5 days and it was effective anti-adhesion barrier between tissues during 5 days. SEM pic-tures was shown that photo-reactive LM-O-CMCS was

became insoluble flat film by cross-linking. Cell viabilityassociated with exposure to photo-reactive LM-O-CMCSwas similar to the control in the cytotoxicity test. Lastly,photo-reactive LM-O-CMCS was found to be hydrophobicaccording to the water contact angle, and cells could notattach to the cross-linked film. These results indicate thatvisible-light curable photo-reactive LM-O-CMCS can poten-tially be used as an anti-adhesion agent.

Acknowledgment. This research was supported by agrant from Fundamental R&D Program for Core Technol-ogy of Materials funded by the Ministry of KnowledgeEconomy, Republic of Korea (K00060-282).

References

(1) S. K. Hwang, K. C. Lee, and K. H. Rhim, J. Korean Ind. Eng.Chem., 10, 939 (1999).

(2) M. Zhang, A. Haga, H. Sekiguchi, and S. Hirono, Int. J. Biol.Macromol., 27, 99 (2000).

(3) J. R. Evans, W. G. Davids, and J. D. MacRae, Water Res., 36,3219 (2002).

(4) Y. Boonsongrit, B. W. Mueller, and A. Mitrevej, Eur. J.Pharm. Biopharm., 69, 388 (2008).

(5) A. P. Zhu, Z. Ming, and S. Jian, Appl. Surf. Sci., 241, 485(2005).

(6) T. Satoh, H. Kano, M. Nakatani, N. Sakairi, S. Shinkai, and T.Nagasaki, Carbohydr. Res., 341, 2406 (2006).

(7) A. P. Zhu, N. Fang, M. B. Chang-Park, and V. Chan, Bio-materials, 26, 6873 (2005).

(8) A. M. M. Sadeghi, M. Amini, M. R. Avadi, F. Siedi, M.Rafiee-Tehrani, and H. E. Junginger, J. Bioact. Compat.Polym., 23, 883 (2008).

(9) Y. Yeo, J. A. Burdick, C. B. Highley, R. Marini, R. Langer,and D. S. Kohane, J. Biomed. Mater. Res., 78A, 668 (2006).

(10) F.-L. Mi, Y.-B. Wu, S.-S. Shyu, J.-Y. Schoung, Y.-B. Huang,Y.-H. Tsai, and J.-Y. Hao, J. Biomed. Mater. Res., 59, 438(2002).

(11) T. I. Son, S. H. Park, H. S. Kang, and E. C. Jang, J. KoreanInd. Eng. Chem., 16, 226 (2005).

(12) N. V. Majeti and R. Kumar, React. Funct. Polym., 46, 1 (2002).(13) M. Lavertu, A. Xia, A. N. Serreqi, M. Berrada, A. Rodrigues,

D. Wang, M. D. Buschmann, and A. Gupta, J. Pharm.Biomed. Anal., 32, 1149 (2003).

(14) T. Ouchi, H. Nishizawa, and Y. Ohya, Polymer, 39, 21 (1998).(15) Y. Machida, T. Nagai, M. Abe, and T. Sannan, Drug Des.

Deliv., 1, 2 (1986).(16) K. R. Holme and A. S. Perlin, Carbohydr. Res., 302, 1 (1997).(17) T. J. Krause, N. K. Goldsmith, S. Ebner, G. A. Zazanis, and R.

D. McKinnon, J. Invest. Surg., 11, 2 (1998).(18) X.-G. Chem and H.-J. Park, Carbohydr. Polym., 53, 355

(2003).(19) H. Jung, J.-P. Nam, H.-S. Jang, S.-H. Heo, D.-G. Kim, Y.-I.

Jeong, J.-K. Park, M.-J. Jang, D.-H. Seo, M.-K. Jang, and J.-W. Nah, Appl. Chem., 11, 361 (2007).

(20) S. Cheng, S. Chen, T. Liu, X. G. Chang, and Y. Yin, Electro-chim. Acta, 52, 5932 (2007).

Figure 7. Fibroblast cell was used to cell attachment test in 12well plate. Half of the well (b) was coated with photo-reactiveLM-O-CMCS film and other was non-treated well (a). Cell wasnot attached on the film but non-treated surface showed attach-ment cells. It was meant that fibroblast cells were not attachedand growth on the photo-reactive LM-O-CMCS film.



(21) A. Zhu, W. Jin, L. Yuan, G. Yang, H. Yu, and H. Wu, Carbo-hydr. Polym., 68, 693 (2007).

(22) A. Zhu, M. B. Chang-Park, S. Dai, and L. Li, Colloids Surf.B: Biointerfaces, 43, 143 (2005).

(23) Y.-M. Li, G.-Y. Xu, D. Wu, and W.-P. Sui, Eur. Polym. J., 43,2690 (2007).

(24) R.-S. Juang, F.-C. Wu, and R.-L. Tseng, Adv. Environ. Res., 6,171 (2002).

(25) M. Sakuragi, S. Tsuzuki, S. Obuse, A. Wada, K. Matoba, I.Kubo, and Y. Ito, Mater. Sci. Eng. C, 30, 316 (2010).

(26) B. Hazer, S. I. Demirel, M. Borcakli, M. S. Eroglu, M. Cak-

mak, and B. Erman, Polym. Bull., 46, 389 (2001).(27) E. Kvam and R. M. Tyrrell, Carcinogenesis, 18, 2379 (1997).(28) A.-L. Ponsonby, A. McMichael, and I. van der Mei, Toxicol-

ogy, 71, 181 (2002).(29) T. W. Bochow, S. K. West, A. Azr, B. Munoz, A. Sommer,

and H. R. Taylor, Arch. Ophthalmol., 107, 369 (1989).(30) H.-N. Na, K.-I. Kim, J.-H. Han, J.-G. Lee, T.-I. Son, D.-K.

Han, Y. Ito, K.-S. Song, and E.-C. Jang, Macromol. Res., 18,10 (2010).

(31) Kathleen Van de Velde and Paul Kiekens, Carbohydr. Polym.,58, 409 (2004).

preparation of a visible light-reactive low molecular-o-carboxymethyl chitosan (lm-o-cmcs)...

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