Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers

Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers

Polymer 45 (2004) 7137–7142 www.elsevier.com/locate/polymer Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nan...

447KB Sizes 2 Downloads 74 Views

Polymer 45 (2004) 7137–7142 www.elsevier.com/locate/polymer

Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers Byung-Moo Mina, Sung Won Leeb, Jung Nam Limb, Young Youb, Taek Seung Leeb, Pil Hyun Kangc, Won Ho Parkb,* a

Department of Oral Biochemistry and Dental Research Institute, IBEC, and BK21 HLS, Seoul National University College of Dentistry, Seoul 110-749, South Korea b Department of Textile Engineering, Chungnam National University, 220 Gungdong, Yuseong-ku, Daejeon 305-764, South Korea c Radiation Application Research Division, Korea Atomic Energy Research Institute, Daejeon 305-600, South Korea Received 17 May 2004; received in revised form 11 August 2004; accepted 24 August 2004

Abstract An electrospinning method was used to fabricate chitin nanofibous matrix for wound dressings. Chitin was depolymerized by gamma irradiation to improve its solubility. The electrospinning of chitin was performed with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as a spinning solvent. Morphology of as-spun and deacetylated chitin (chitosan) nanofibers was investigated by scanning electron microscopy. Although as-spun chitin nanofibers had the broad fiber diameter distribution, most of the fiber diameters are less than 100 nm. From the image analysis, they had an average diameter of 110 nm and their diameters ranged from 40 to 640 nm. For deacetylation, as-spun chitin nanofibous matrix was chemically treated with a 40% aqueous NaOH solution at 60 or 100 8C. With the deacetylation for 150 min at 100 8C or for 1day at 60 8C, chitin matrix was transformed into chitosan matrix with degree of deacetylation (DD) w85% without dimensional change (shrinkage). This structural transformation from chitin to chitosan was confirmed by FT-IR and WAXD. q 2004 Elsevier Ltd. All rights reserved. Keywords: Electrospinning; Chitin; Chitosan

1. Introduction Chitin is the principal structural polysaccharide of the arthropods (for example, crabs and insects) and the second most abundant polysaccharide, next to cellulose. Chitin is a linear polysaccharide consisting of b-linked N-acetyl-Dglucosamine. Upon hydrolysis, chitin yields 2-amino-2deoxy-D-glucose (chitosan). It has been estimated that 1010–1012 tons of chitin are biosynthesized each year [1]. However, it has received much less industrial attention because of its poor solubility, while considerable interest has been devoted to biomaterials based on its aminoderivative chitosan. Until now, many researchers have investigated chitin or chitosan as one of candidate materials for biomedical * Corresponding author. Tel.: C82-42-821-6613; fax: C82-42-8233736. E-mail address: parkw[email protected] (W.H. Park). 0032-3861/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2004.08.048

applications because it has several distinctive biological properties including good biocompatibility, biodegradability, and wound healing effect [2–4]. In practice, it has been used in various fields, such as cosmetics, medical materials for human health, and food additives. Recently, much attention has been paid to electrospinning process as a unique technique because it can produce polymer nanofibers with diameter in the range from several micrometers down to tens of nanometers, depending on the polymer and processing conditions. In electrospinning, a high voltage is applied to create electrically charged jets of a polymer solution. These jets dry to form nanofibers, which are collected on a target as a nonwoven fabric. These nanofibers are of considerable interest for various kinds of applications, because they have several useful properties such as high specific surface area and high porosity. Examples are fiber membranes for filter applications [5], biomedical applications, such as wound dressings and scaffolds for tissue engineering [6–9], and sensing applications [10,11].

7138

B.-M. Min et al. / Polymer 45 (2004) 7137–7142

The ultimate aim of this study is to develop a nonwoventype wound dressings composed of the electrospun chitin (or chitosan) nanofibers. In this study, chitin nanofibrous matrix was fabricated via electrospinning of chitin in HFIP, and electrospinning conditions were investigated. The resulting chitin nanofibers were regenerated into chitosan nanofibers via heterogeneous deacetylation with aqueous NaOH solution. Chemical and structural changes according to the deacetylation were systematically investigated.

2. Experimental 2.1. Materials Chitin powder (MwZ910,000, Degree of deacetylation (DD)Z8%) was supplied by Kumho Chemical Products Co. (Korea). All reagents were of analytical grade and used as received. 2.2. Radiation depolymerization of chitin Chitin powder (100w500 Nm) was packed in polyethylene bags and irradiated on Co60 gamma ray (Korea Atomic Energy Research Institute, Daejeon, Korea) with various dose from 50 to 200 kGy. The intrinsic viscosity and molecular weight of the depolymerized chitin sample were determined by dilute solution viscometry. The intrinsic viscosity ([h]) of chitin in 5% LiCl–DMAc solution at 25 8C was measured with a Ubbelohde type viscometer. The molecular weight (Mn) was determined according to the Mark–Houwink equation as follows [12]: ½h Z 2:1 !104 Mv0:88

(1)

2.3. Electrospinning of chitin

shaking water bath at 60 8C. The degree of deacetylation (DD) was calculated using integrals of the peak due to three protons of N-acetyl group at 2.0 ppm and of the peak due to proton at C2 at 3.5 ppm on 1H NMR spectrum [13]. 2.5. Measurement and characterization The viscosity of chitin solution in HFIP was determined with a Brookfield digital viscometer (model DV-E) at 25 8C. Fourier transform infrared spectroscopy (FT-IR) spectra of the samples were obtained with a Nicolet Magna-IR 560 spectrophotometer. The crystalline structure of the samples was analyzed on a wide-angle X-ray diffractometer (WAXD) (model Rigaku D/max-IIB, Rigaku International Corp.). Thermal properties were measured with a differential scanning calorimetry (DSC, PerkinElmer DSC-7) and a thermogravimetric analyzer (TGA, PerkinElmer TGA-7). The samples were heated to 300 and 500 8C at a heating rate of 10 8C/min under nitrogen flow for the DSC and TGA analyses, respectively. The morphology of electrospun chitin and its deacetylated fibers was observed on a scanning electron microscope (SEM) (Hitachi S-2350, Japan) after gold coating. The average fiber diameters were determined by analyzing the SEM images with a custom code imageanalysis program.

3. Results and discussion 3.1. Radiation depolymerization of chitin powder in the solid state Although chitin is the second most abundant polysaccharide in nature and has several distinctive biological properties including good biocompatibility, biodegradability, and wound healing effect [2–4], its applications are

Chitin solutions were prepared by dissolving the irradiated chitin powders (MwZ91,000) in HFIP (Aldrich) for 3 days. Concentrations of chitin solutions for electrospinning was in the range from 3w6% by weight. In the electrospinning process, a high electric potential was applied to a droplet of chitin solution at the tip (0.495 mm in internal diameter) of a syringe needle. The electrospun chitin nanofibers were collected on a target drum which was placed at a distance of w7 cm from the syringe tip. A voltage of 15 kV was applied to the collecting target by a high voltage power supply (Chungpa EMT Co., Korea). 2.4. Deacetylation of electrospun chitin fibrous matrix As-electrospun chitin fibers were refluxed in 40% NaOH aqueous solution for 30w150 min at 100 8C or for 1w3 days at 60 8C, and washed with distilled water, dried under vacuum for 24 h. Sample were heated in round bottom flask under reflux in a silicone oil bath at 100 8C or in vial in

Fig. 1. Effect of irradiation on molecular weight of chitin in dose range of 10–200 kGy.

B.-M. Min et al. / Polymer 45 (2004) 7137–7142

7139

3.2. Morphology and microstructure of electrospun chitin nanofibers

Fig. 2. Variation in viscosity with the concentration of chitin in HFIP.

limited due to its lack of solubility in major solvent. At first, we choose HFIP as an appropriate solvent for electrospinning of chitin. The maximum solubility of chitin in HFIP was only approximately 0.65 wt% because of its high molecular weight. To enhance the solubility, we conducted the depolymerization of chitin by irradiation, which can provide a useful tool for degradation of different polymers [14]. The irradiation of chitin in the solid state led to the reduction in molecular weight, as shown in Fig. 1. The viscosity average molecular weight of chitin decreased remarkably up to 50 kGy, and then gradually slowed down with increasing radiation dose. At radiation dose of 200 kGy, the viscosity average molecular weight (91,000) of chitin was only one tenth of original chitin. Hereafter, we used chitin sample with molecular weight of 91,000 for electrospinning.

In electrospinning, the morphology of the electrospun products was found to vary with concentration of the polymer solution. Fig. 2 showed the variation in viscosity with the concentration of chitin in HFIP. The SEM micrographs with magnification of 5000! of nanofibers electrospun from the chitin solutions with different concentrations or viscosities ranged from 1 to 6% by weight were shown in Fig. 3. At the concentration up to 3% by weight, large irregular beads or beaded fibers were generated by electrospinning (Fig. 3a and b). The continuous nanofibers can be obtained at the concentration above 4% by weight (Fig. 3c), and this concentration appears to correspond to the onset of significant chain entanglements in the viscosity data shown in Fig. 2. Therefore, we believe that extensive chain entanglements are necessary to produce the continuous fibers of chitin by electrospinning. However, at 6 wt% concentration, the continuous and uniform electrospinning was inhibited because chitin solution had very high viscosity (5310 cP) at that concentration. The resultant fibrous structure containing irregular, but small, beads was observed again. The as-spun nanofibrous chitin matrix consists of fibers with diameters ranging from 40 to 600 nm. Although it has the broad fiber diameter distribution, most of the fiber diameters are less than 100 nm, and the average diameter determined from the image analysis is 110 nm. The solution concentration of 5% by weight was chosen to fabricate matrices of randomly arranged chitin nanofibers. 3.3. Deacetylation of chitin nanofibrous matrix The physicochemical properties of chitin fibers strongly depend on its degree of deacetylation (DD), that is, amino

Fig. 3. SEM micrographs of chitin nanofibers electrospun at different concentrations.

7140

B.-M. Min et al. / Polymer 45 (2004) 7137–7142

Fig. 4. Changes in the degree of deacetylation (DD) with the reaction time at 60 and 100 8C.

group content. Deacetylation reaction of chitin can be easily induced by alkaline treatment, such as the concentrated NaOH aqueous solution. Therefore, the aqueous NaOH treatment (40 wt%) was performed to form chitosan nanofibrous matrix from chitin matrix. Fig. 4 shows the change of DD at two temperature conditions. At high temperature (100 8C), the DD rapidly increased up to 85% within 2 h. On the other hand, at low temperature (60 8C) the

DD reached at about 85% at the reaction time of 1 day, indicating that deacetylation reaction progresses very slowly. Fig. 5 shows the SEM micrographs and the corresponding diameter distributions of chitin and deacetylated chitin (chitosan) nanofibers, before and after deacetylation reaction for 150 min at 100 8C. No significant morphological changes were observed. The minor change in morphology after deacetylation reaction is also consistent with the low shrinkage (w10%) of the electrospun chitin matrix (Fig. 6). The shrinkage of electrospun polymer matrix often occurred during the thermal or chemical treatment. It was known that the major driving force for shrinkage is due to the thermally induced relaxation of stretched amorphous chains [15]. Therefore, we believe that the primary reason for low dimensional change of chitin or chitosan matrix during deacetylation at high temperature is due to its high crystallinity and/or high glass transition temperature. On the other hand, the chitosan is a cationic polysaccharide with amino groups at the C2 position which were ionizable under the acidic or neutral pH conditions. Until now, there are few reports on the nanofibers electrospun from ionic polymers or polyelectrolytes [16]. To our knowledge, the reason for that might be explained as followings. Under the high electric field, the repulsive force between ionic groups within polymer backbone is expected to inhibit the formation of continuous fiber during

Fig. 5. SEM micrographs of chitin and deacetylated chitin (chitosan) nanofibrous matrix, before and after deacetylation reaction for 150 min at 100 8C.

B.-M. Min et al. / Polymer 45 (2004) 7137–7142

7141

Fig. 6. Comparison of electrospun chitin matrix (a) before and (b) after deacetylation.

electrospinning process, especially during the jet stretching by whipping and bending. Therefore, the ionic polymer results in ultrafine particles, not ultrafine fibers, even at the optimum range of concentrations. We electrospun chitosan in several solvent systems containing formic acid across a broad range of concentrations, but we failed to electrospun chitosan into ultrafine fibers even at the concentrations in which the chitosan chains were extensively entangled. Therefore, it is notable that chitosan nanofibrous matrix can be obtained from the neutral nonionic form of chitin through the deacetylation of electrospun chitin matrix. Fig. 7 displays changes in the FT-IR spectra with the DD of chitin and its deacetylated matrices. With increasing the DD, the intensities of the characteristic adsorption peaks attributed to the vibrations of the amide group at 1700 cmK1 (nCZO) decreased. In addition, the absorption peaks at w3300 and w3100 cmK1 decreased with the DD. The decrease of peaks at w3300 cmK1 indicates the reduction of intermolecular C(2)NH–OaC(7) hydrogen bonds [17]. The decrease of peaks at w3100 cmK1 represents the reduction of intermolecular C(6)OH–HOC(6) hydrogen bonds, implying antiparallel arrangemnent of the chains was transformed

Fig. 7. Changes in the FT-IR spectra with the DD of chitin and its deacetylated matrices.

Fig. 8. WAXD patterns with the DD of chitin and its deacetylated matrices.

into parallel arrangement. Fig. 8 shows the crystalline structure changes of chitin fibers with the DD. As the deacetylation proceeded, the WAXD intensity at 2qZ9.28 decreased. The WAXD pattern obtained after 150 min at 100 8C was almost the same as that of commercial chitosan with the corresponding degree of deacetylation. The heterogeneous deacetylation takes place preferentially in the amorphous regions then continues more slowly from surface to center of the crystalline region, thereby giving rise to blocky structure. Kurita et al. [18] reported that during heterogeneous deacetylation of chitin, the crystallinity as measured by X-ray diffraction decreased slightly up to a DD of 71% and then more rapidly, so that the crystalline peaks had almost disappeared by 81% deacetylation while by 89% deacetylation the sample was completely amorphous. Also, TGA revealed that their thermal properties were changed according to the deacetylation (Fig. 9).

Fig. 9. TGA thermograms with the DD of chitin and its deacetylated matrices.

7142

B.-M. Min et al. / Polymer 45 (2004) 7137–7142

The maximum thermal degradation temperatures decreased from w330 to w270 8C. These results confirmed that chitin was completely converted to chitosan.

Biointerface Engineering Center at Seoul National University.

4. Conclusions

References

In this study, the chitin nanofibrous matrix was produced by the elelctrospinning process. Before electrospinning, chitin was depolymerized by gamma irradiation to improve its solubility. The electrospinning of chitin was performed with 1,1,1,2,2,2-hexafluoro-2-propanol (HFIP) as a spinning solvent. Although as-spun chitin nanofibers had the broad fiber diameter distribution, most of the fiber diameters are less than 100 nm. From the image analysis, they had an average diameter of 110 nm and their diameters ranged from 40 to 640 nm. For deacetylation, as-spun chitin nanofibous matrix was chemically treated with a 40% aqueous NaOH solution at 60 or 100 8C. By the deacetylation for 120 min at 100 8C or for 1day at 60 8C, chitin matrix was transformed into chitosan matrix with DD w85% without dimensional change (shrinkage). This structural transformation from chitin to chitosan was confirmed by FT-IR and WAXD. The biodegradation and biocompatibility of chitin and chitosan nanofibrous matrices will be reported in near future.

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Intellectual

[16] [17] [18]

Percot A, Viton C, Domard A. Biomacromolecules 2003;4:12. Muzzarelli RAA. Carbohydr Polym 1993;3:52. Tomihata K, Ikada Y. Biomaterials 1997;18:567. Cho YW, Cho YN, Chung SH, Yoo G, Ko SW. Biomaterials 1999;20: 2139. Gibson P, Schreuder-Gibson H, Rivin D. Colloids Surf A 2001; 187–188:469. Li W, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. J Biomed Mater Res 2002;60:613. Kenawy E, Layman JM, Watkins JR, Bowlin GL, Matthews JA, Simpson DG, Wnek GE. Biomaterials 2003;24:907. Yoshimoto H, Shin YM, Terai H, Vacanti JP. Biomaterials 2003;24: 2077. Min BM, Lee G, Kim SH, Nam YS, Lee TS, Park WH. Biomaterials 2004;25:1289. Athreya SA, Martin DC. Sens Actuators 1999;72:203. Wang X, Lee S, Drew C, Senecal KJ, Kumar J, Samuelson LA. Nano Lett 2002;2:1273. Terbojevich M, Carraro C, Cosani A, Marsano E. Carbodydr Res 1988;180:73. Lavertu M, Xia Z, Serreqi AN, Berrada M, Rodrigues A, Wang D, Buschmann MD, Gupta A. J Pharm Biomed Anal 2003;32:1149. Woods RT, Pikaev AK, editors. Applied radiation chemistry: radiation processing; New York. Zong X, Ran S, Kim K, Fang D, Hsiao BS, Chu B. Biomacromolecules 2003;4:416. Duan B, Dong C, Yuan X, Yao K. J Biomater Sci Polym Ed 2004;15: 797. Roberts GAF, editor. Chitin chemistry. London: Macmillan; 1992. Kurita K, Sannan T, Iwakura Y. Makromol Chem 1977;178:3197.